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Studi Clinici

Sono presentati qui di seguito astratti per gli studi clinici su Madagascar Periwinkle.

  • Nome Botanico: Catharanthus Roseus

  • Nome Sanscrito: Vijaysaar

  • Nome Standardizzato: Madagascar Periwinkle

Catharanthus Roseus

Impianto Phytonutrient Profilo


1: Biochim Biophys Acta. 2007 Jan 30; [Epub ahead of print]

Promoter analysis of the Catharanthus roseus geraniol 10/​hydroxylase gene
involved in terpenoid indole alkaloid biosynthesis.

Suttipanta N, Pattanaik S, Gunjan S, Xie CH, Littleton J, Yuan L.

Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY
40546, USA.

Geraniol 10/​hydroxylase (G10H) is an important enzyme in the biosynthetic
pathway of monoterpenoid alkaloids found in diverse plant species. The
Catharanthus roseus G10H controls the first committed step in biosynthesis of
terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was
isolated by a PCR/​based genome walking method. Sequence analysis revealed that
the G10H promoter contains several potential eukaryotic regulatory elements
involved in regulation of gene expression. The major transcription start site of
the promoter was mapped to an adenine 31 bp downstream of the TATA/​box. For
functional characterization, transcriptional fusions between the G10H promoter
fragments with 5' or 3' deletions and the GUS reporter gene were generated and
their expressions were analyzed in a tobacco protoplast transient expression
assay. Deletion of the promoter down to /​318 bp had little effect on GUS
activity. However, further deletion of the promoter to position /​103 resulted in
approximately 5/​fold reduction of GUS activity. Gain/​of/​function experiments
revealed the presence of three potential transcriptional enhancers located in
regions between /​191 and /​147, /​266 and /​188, and /​318 and /​266, respectively.
The G10H promoter was capable of conferring stable GUS expression in transgenic
tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings GUS
expression was tissue/​specific, restricted to leaf and actively growing cells
around the root tip, and not detected in the hypocotyls, root cap and older
developing areas of the root. The GUS expression in both transgenic C. roseus
hairy roots and tobacco seedlings were responsive to fungal elicitor and
methyljasmonate. Compared to other known promoters of TIA pathway genes, the
G10H promoter contains unique binding sites for several transcription factors,
suggesting that the G10H promoter may be regulated by a different
transcriptional cascade.

PMID: 17321612 [PubMed /​ as supplied by publisher]

2: FEBS J. 2007 Mar;274(5):1290/​303.

Cloning, characterization and localization of a novel basic peroxidase gene from
Catharanthus roseus.

Kumar S, Dutta A, Sinha AK, Sen J.

National Centre for Plant Genome Research, JNU Campus, Aruna Asaf Ali Marg, New
Delhi, India.

Catharanthus roseus (L.) G. Don produces a number of biologically active
terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic
pathway. The final dimerization step of this pathway, leading to the synthesis
of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic
peroxidase. However, reports of the gene encoding this enzyme are scarce for C.
roseus. We report here for the first time the cloning, characterization and
localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp
partial peroxidase cDNA (CrInt1) was initially amplified from the internodal
stem tissue, using degenerate oligonucleotide primers, and cloned. The
full/​length coding region of CrPrx cDNA was isolated by screening a
leaf/​specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence
encodes a deduced translation product of 330 amino acids with a 21 amino acid
signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass
of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa,
and the pI value is 8.68. CrPrx was found to belong to a 'three intron' category
of gene that encodes a class III basic secretory peroxidase. CrPrx protein and
mRNA were found to be present in specific organs and were regulated by different
stress treatments. Using a beta/​glucuronidase/​green fluorescent protein fusion
of CrPrx protein, we demonstrated that the fused protein is localized in leaf
epidermal and guard cell walls of transiently transformed tobacco. We propose
that CrPrx is involved in cell wall synthesis, and also that the gene is induced
under methyl jasmonate treatment. Its potential involvement in the terpenoid
indole alkaloid biosynthetic pathway is discussed.

PMID: 17298442 [PubMed /​ in process]

3: Biotechnol Prog. 2007 Jan 27; [Epub ahead of print]

Effect of the Engineered Indole Pathway on Accumulation of Phenolic Compounds in
Catharanthus roseus Hairy Roots.

Chung IM, Hong SB, Peebles CA, Kim JA, San KY.

Department of Applied Life Science, Konkuk University, Seoul 143/​701, South
Korea, and Department of Bioengineering, Rice University, Houston, Texas, 77005.

Catharanthus roseus has been well/​known to contain indole alkaloids effective
for treatment of diverse cancers. We examined the intracellular accumulation
profiles of phenolic compounds in response to ectopic overexpression of
tryptophan feedback/​resistant anthranilate synthase holoenzyme (ASalphabeta) in
C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic
compounds were detected in late exponential phase ASalphabeta hairy roots.
Uninduced and induced ASalphabeta hairy roots accumulated up to 1.2 and 4.5 mg/g
DW over a 72/​h period, respectively. Upon induction, in parallel with a rapid
increase in tryptophan in the first 48 h, accumulation of phenolic compounds
tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which
phenolic levels decreased back to the uninduced level by 72 h. Naringin was a
predominant form that comprised about 72% and 36% of the total content of
phenolic compounds in the uninduced and induced lines, respectively. Upon
induction, accumulation of catechin drastically increased with the highest level
(3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic
acid showed no statistical difference. Catechin is a final product of the
flavonoid pathway, and thus metabolic flux into this pathway is transiently
increased by overexpression of AS. Like catechin, salicylic acid is very
sensitive to induction as it began to increase to 5/​fold within 4 h of
induction, but unlike catechin, no significant accumulation of salicylic acid
was noted after 4 h of induction. The results suggest differential regulation of
this particular biosynthesis branch within the phenolic pathway.

PMID: 17256967 [PubMed /​ as supplied by publisher]

4: Planta Med. 1998 May;64(4):390.

Rapid in vitro Multiplication of Plants from Mature Nodal Explants of
Catharanthus roseus.

Mitra A, Khan B, Rawal S.

Division of Plant Tissue Culture, National Chemical Laboratory, Pune, India.

PMID: 17253257 [PubMed /​ in process]

5: J Exp Bot. 2006 Dec 21; [Epub ahead of print]

Involvement of rapid nucleotide synthesis in recovery from phosphate starvation
of Catharanthus roseus cells.

Yin Y, Shimano F, Ashihara H.

Department of Advanced Bioscience, Graduate School of Humanities and Sciences,
Ochanomizu University, Tokyo, 112/​8610 Japan.

Growth of suspension/​cultured Catharanthus roseus cells ceased during phosphate
starvation, but the cells grew again upon addition of Pi even after long/​term
starvation. The metabolic fate of [(33)P]Pi was studied in 1/​week/​old stationary
phase cells in ordinary culture and in 1/​ or 2/​week/​old Pi/​starved cells.
Immediately after administration, the most heavily labelled organic compounds
are nucleotides, followed by sugar phosphates. Two weeks Pi starvation slowed
down the speed of incorporation of (33)P into nucleotides. The RNA, protein, and
free nucleotide content all decreased gradually during Pi starvation; however,
these compounds, especially nucleotides, increased markedly in the 24 h after
addition of Pi. These responses are found in all cells examined, although the
total amounts of these compounds were lower in the long/​term Pi/​deficient cells.
Of the nucleotides, a marked increase was observed in nucleoside triphosphates
and UDP/​glucose. The transcript level of phosphate transporter and the
activities of acid phosphatase, 5'/​ and 3'/​nucleotidase, and adenosine
nucleosidase were all reduced by the addition of Pi. In contrast, the activities
of adenine phosphoribosyltransferase, nicotinate phosphoribosyltransferase, and
nicotinamidase, which are salvage enzymes of purine and pyridine nucleotides,
were markedly increased in the Pi/​fed cells. Little or no increase was observed
in adenosine kinase. In the light of these results, the possible involvement of
net nucleotide synthesis in the initial metabolic events of recovery from Pi
deficiency are discussed.

PMID: 17185741 [PubMed /​ as supplied by publisher]

6: BMC Complement Altern Med. 2006 Dec 21;6:41.

Catharanthus roseus flower extract has wound/​healing activity in Sprague Dawley
rats.

Nayak BS, Pinto Pereira LM.

Department of Pre Clinical Sciences, Biochemistry Unit, Faculty of Medical
Sciences, The University of the West Indies, St, Augustine, Trinidad.
snayak@fms.uwi.tt

BACKGROUND: Catharanthus roseus L (C. roseus) has been used to treat a wide
assortment of diseases including diabetes. The objective of our study was to
evaluate the antimicrobial and wound healing activity of the flower extract of
Catharanthus in rats. METHODS: Wound healing activity was determined in rats,
after administration (100 mg kg/​1 day/​1) of the ethanol extract of C. roseus
flower, using excision, incision and dead space wounds models. The animals were
divided into two groups of 6 each in all the models. In the excision model,
group 1 animals were topically treated with carboxymethyl cellulose as placebo
control and group 2 received topical application of the ethanol extract of C.
roseus at a dose of 100 mg/kg body weight/day. In an incision and dead space
model group 1 animals were given normal saline and group 2 received the extract
orally at a dose of 100 mg kg/​1 day/​1. Healing was assessed by the rate of wound
contraction, period of epithelization, tensile strength (skin breaking
strength), granulation tissue weight, and hydoxyproline content. Antimicrobial
activity of the flower extract against four microorganisms was also assessed
RESULTS: The extract of C. roseus significantly increased the wound breaking
strength in the incision wound model compared with controls (P < 0.001). The
extract/​treated wounds were found to epithelialize faster, and the rate of wound
contraction was significantly increased in comparison to control wounds (P <
0.001), Wet and dry granulation tissue weights, and hydroxyproline content in a
dead space wound model increased significantly (p < 0.05). Pseudomonas
aeruginosa and Staphylococcus aureus demonstrated sensitivity to C. roseus
CONCLUSION: Increased wound contraction and tensile strength, augmented
hydroxyproline content along with antimicrobial activity support the use of C.
roseus in the topical management of wound healing.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 17184528 [PubMed /​ indexed for MEDLINE]

7: Se Pu. 2006 Sep;24(5):534.

[Determination of three endogenous hormones in catharanthus roseus (L.) G. Don
using solid/​phase extraction and high performance liquid chromatography]

[Article in Chinese]

Zhao X, Tang Z, Guo X, Zu Y, Jiao Y, Sun Y, Yang L.

zxj_daisy@163.com

PMID: 17165557 [PubMed /​ in process]

8: Metab Eng. 2007 Mar;9(2):125/​132. Epub 2006 Oct 21.

Transcription factor Agamous/​like 12 from Arabidopsis promotes tissue/​like
organization and alkaloid biosynthesis in Catharanthus roseus suspension cells.

Montiel G, Breton C, Thiersault M, Burlat V, Jay/​Allemand C, Gantet P.

Universite de Tours Unite sous Contrat reconnue par l'Institut National de la
Recherche Agronomique, Facteurs de Transcription et Ingenierie Metabolique
Vegetale, UFR des Sciences et Techniques et UFR des Sciences Pharmaceutiques,
Parc de Grandmont, 37200 Tours, France; Universite de Tours EA 2106,
Biomolecules et Biotechnologies Vegetales, UFR des Sciences et Techniques,
Laboratoire de Physiologie Vegetale, Parc de Grandmont, 37200 Tours, France;
Institut National de la Recherche Agronomique/​Orleans, Unite Amelioration,
Genetique et Physiologie Forestieres, BP 20619 ARDON, F/​45166 Olivet cedex,
France.

In Catharanthus roseus, monomeric terpenoid indole alkaloids (TIAs) are
biosynthesized in specific tissues, particularly in roots, but failed to be
produced by in vitro undifferentiated suspension cells. In this paper, we
describe the impact of the root/​specific MADS/​box transcription factor
Agamous/​like 12 (Agl12) from Arabidopsis thaliana on the differentiation of
suspension cells from C. roseus. The expression of Agl12 is sufficient to
promote an organization of suspension cells into globular parenchyma/​like
aggregates but is insufficient by itself to induce complete morphological root
differentiation. Agl12 expression selectively increases the expression of genes
encoding enzymes involved in the early biosynthesis steps of the terpenic
precursor of alkaloids. The transgenic cell lines expressing Agl12 produced
significant amounts of ajmalicine, an antihypertensive TIA that normally
accumulates in C. roseus roots. The present paper indicates that transcription
factors involved in tissue or organ differentiation may constitute new metabolic
engineering tools that could help to design in vitro cultured cells able to
produce specific valuable secondary metabolites.

PMID: 17157545 [PubMed /​ as supplied by publisher]

9: Pharmazie. 2006 Nov;61(11):952/​6.

Screening of herbal extracts for activation of the human peroxisome
proliferator/​activated receptor.

Rau O, Wurglics M, Dingermann T, Abdel/​Tawab M, Schubert/​Zsilavecz M.

Johann Wolfgang Goethe University Frankfurt, Institute of Pharmaceutical
Chemistry/ZAFES, Frankfurt/Main, Germany.

The peroxisome proliferator/​activated receptors play a pivotal role in metazoan
lipid and glucose homeostasis. Synthetic activators of PPARalpha (fibrates) and
PPARgamma (glitazones) are therefore widely used for treatment of dislipidemia
and diabetes, respectively. There is growing evidence for herbal compounds to
influence nuclear receptor signalling e.g. the PPARs. We recently reported
carnosic acid and carnosol, both being diterpenes found in the labiate herbs
sage and rosemary, to be activators of PPARgamma. The subsequent screening of a
variety of ethanolic extracts, obtained from traditionally used herbs, for PPAR
activation, led to an exceptionally high hit rate. Among 52 extracts nearly the
half significantly activated PPARgamma and 14 activated PPARalpha in addition,
whereas three of them were pan/​PPAR activators, which also activated PPARdelta.
The most active extracts, for which a concentration dependent effect could be
shown, were the extracts of Alisma plantago aquatica (ze xie/european
waterplantain), Catharanthus roseus (madagascar periwinkle), Acorus calamus
(sweet calamus), Euphorbia balsamifera (balsam spurge), Jatropha curcas
(barbados nut), Origanum majorana (marjoram), Zea mays (corn silk), Capsicum
frutescens (chilli) and Urtica dioica (stinging nettle). The results of the
present study provide a possible rationale for the traditional use of many herbs
as antidiabetics.

PMID: 17152989 [PubMed /​ indexed for MEDLINE]

10: Biotechnol Prog. 2006 Nov/​Dec;22(6):1659/​63.

Determination of biomass composition of Catharanthus roseus hairy roots for
metabolic flux analysis.

Sriram G, Gonzalez/​Rivera O, Shanks JV.

Department of Chemical and Biological Engineering, Iowa State University, 3031,
Sweeney Hall, Ames, Iowa 50011, USA.

Metabolic flux analysis is a powerful diagnostic tool in metabolic engineering,
and determination of biomass composition is indispensable to accurate flux
evaluation. We report the elemental and biomolecular composition of Catharanthus
roseus hairy roots, a pharmaceutically significant plant system and an important
metabolic engineering target. The molecular formula of the organic material in
the hairy roots was C12.0H22.7N0.4O7.6 during mid/​exponential growth. The
abundances of lipids, lignin, cellulose, hemicellulose, starch, protein,
proteinogenic amino acids, mineral ash, and moisture in the biomass were
quantified. Analysis of water/​soluble components of the biomass with 1/​D 13C and
2/​D [1H,1H] correlation (COSY) NMR spectroscopy revealed that the water/​soluble
components were composed almost entirely of /​glucans. Agropine, a frequently
reported hairy root biomass component, was not detected. Our measurements of the
biomass components quantified 83.6 +//​ 9.3% (w/w) of the biomass. Together with
previously reported abundances of indole alkaloids, we accounted for at least
85.9 +//​ 11.6% (w/w) of the carbon in the biomass, which enabled the precise
determination of 12 biomass synthesis fluxes.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 17137315 [PubMed /​ indexed for MEDLINE]

11: J Am Chem Soc. 2006 Nov 8;128(44):14276/​7.

Directed biosynthesis of alkaloid analogs in the medicinal plant Catharanthus
roseus.

McCoy E, O'Connor SE.

Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,
Massachusetts 02139, USA.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 17076499 [PubMed /​ in process]

12: J Exp Bot. 2006;57(14):3921/​32. Epub 2006 Oct 18.

Transcriptome analysis in Catharanthus roseus leaves and roots for comparative
terpenoid indole alkaloid profiles.

Shukla AK, Shasany AK, Gupta MM, Khanuja SP.

Genetic Resources and Biotechnology Division, Central Institute of Medicinal and
Aromatic Plants, Lucknow 226015, India.

In Catharanthus roseus (L.) G. Don each tissue is known to produce a distinct
spectrum of terpenoid indole alkaloids. Since the invaluable antineoplastic
bisindole alkaloids are restricted to the aerial parts of the plant and do not
occur in its underground tissues, identification of the structural and
regulatory factors operating distinctly in the shoot/leaf of the plant will be a
necessity for modulation of bisindole alkaloid biosynthesis. This study aimed at
elucidating the differential gene expression in the two main tissues (leaf and
root) of the plant, well known for their distinct terpenoid indole alkaloid
profiles. The leaf and root transcriptomes of C. roseus were comparatively
analysed using two different approaches: (i) indirectly through construction and
characterization of separate cDNA libraries; and (ii) directly through a
strategically designed suppression subtractive hybridization, using the leaf and
root cDNA populations as tester and driver, respectively. A total of 155 ESTs
(55 and 45 from the separate leaf and root cDNA libraries, respectively, and 55
from the subtracted leaf/​specific cDNA library) were subjected to homology/​based
classification and submitted to dbEST. The direct approach yielded an EST for
sgd (strictosidine beta/​D/​glucosidase) and 16 novel ESTs. Dat (acetyl/​CoA:
4/​O/​deacetylvindoline 4/​O/​acetyl/​transferase) and sgd transcripts could not be
detected in the root system of the plant (cv. 'Dhawal') at any developmental
stage (6 d, 6 weeks, or 6 months). The growth/​related decrease in shoot/leaf dat
and sgd transcript levels was paralleled by a concomitant decrease in shoot/leaf
vindoline content.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 17050644 [PubMed /​ indexed for MEDLINE]

13: J Ethnobiol Ethnomedicine. 2006 Oct 13;2:45.

Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes
mellitus.

Lans CA.

BCICS, University of Victoria, British Columbia, V8W 2Y2, Canada.
cher2lans@netscape.net.

ABSTRACT: BACKGROUND: This paper is based on ethnobotanical interviews conducted
from 1996/​2000 in Trinidad and Tobago with thirty male and female respondents.
METHODS: A non/​experimental validation was conducted on the plants used for
urinary problems and diabetes mellitus: This is a preliminary step to establish
that the plants used are safe or effective, to help direct clinical trials, and
to inform Caribbean physicians of the plants' known properties to avoid
counter/​prescribing. RESULTS: The following plants are used to treat diabetes:
Antigonon leptopus, Bidens alba, Bidens pilosa, Bixa orellana, Bontia
daphnoides, Carica papaya, Catharanthus roseus, Cocos nucifera, Gomphrena
globosa, Laportea aestuans, Momordica charantia, Morus alba, Phyllanthus
urinaria and Spiranthes acaulis. Apium graviolens is used as a heart tonic and
for low blood pressure. Bixa orellana, Bontia daphnoides, Cuscuta americana and
Gomphrena globosa are used for jaundice. The following plants are used for
hypertension: Aloe vera, Annona muricata, Artocarpus altilis, Bixa orellana,
Bidens alba, Bidens pilosa, Bonta daphnoides, Carica papaya, Cecropia peltata,
Citrus paradisi, Cola nitida, Crescentia cujete, Gomphrena globosa, Hibiscus
sabdariffa, Kalanchoe pinnata, Morus alba, Nopalea cochinellifera, Ocimum
campechianum, Passiflora quadrangularis, Persea americana and Tamarindus
indicus.The plants used for kidney problems are Theobroma cacao, Chamaesyce
hirta, Flemingia strobilifera, Peperomia rotundifolia, Petiveria alliacea,
Nopalea cochinellifera, Apium graveolens, Cynodon dactylon, Eleusine indica,
Gomphrena globosa, Pityrogramma calomelanos and Vetiveria zizanioides. Plants
are also used for gall stones and for cooling. CONCLUSION: Chamaesyce hirta,
Cissus verticillata, Kalanchoe pinnata, Peperomia spp., Portulaca oleraceae,
Scoparia dulcis, and Zea mays have sufficient evidence to support their
traditional use for urinary problems, "cooling" and high cholesterol.Eggplant
extract as a hypocholesterolemic agent has some support but needs more study.
The plants used for hypertension, jaundice and diabetes that may be safe and
justify more formal evaluation are Annona squamosa, Aloe vera, Apium graveolens,
Bidens alba, Carica papaya, Catharanthus roseus, Cecropia peltata, Citrus
paradisi, Hibsicus sabdariffa, Momordica charantia, Morus alba, Persea
americana, Phyllanthus urinaria, Tamarindus indicus and Tournefortia
hirsutissima. Several of the plants are used for more than one condition and
further trials should take this into account.

PMID: 17040567 [PubMed /​ in process]

14: Biotechnol Lett. 2006 Oct;28(20):1687/​94. Epub 2006 Aug 3.

Development of a quantitative method for determination of the optimal conditions
for protoplast isolation from cultured plant cells.

Aoyagi H.

Institute of Life Sciences and Bioengineering, Graduate School of Life and
Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305/​8572,
Japan. aoyagi@sakura.cc.tsukuba.ac.jp

An index [kv: average isolation rate of viable protoplast (number/ml min)] was
established to evaluate the optimal conditions for protoplast isolation from
cultured plant cells. The optimal conditions for protoplasts isolation from
Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv
[31.7 x 10(3) (number/ml min)]. The colony/​forming efficiency of the protoplasts
was about 46%. The optimal conditions for protoplasts isolation from
Catharanthus roseus [kv = 38.1 x 10(3) (number/ml min)] and Wasabia japonica [kv
= 14.2 x 10(3) (number/ml min)] cultured cells could also be determined.
Furthermore, a method for rapid regenerating cell wall of protoplast in liquid
culture using alginate gel containing locust bean gum was developed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16955360 [PubMed /​ indexed for MEDLINE]

15: Planta. 2007 Feb;225(3):711/​8. Epub 2006 Sep 6.

Inorganic phosphate uptake in intact vacuoles isolated from suspension/​cultured
cells of Catharanthus roseus (L.) G. Don under varying Pi status.

Ohnishi M, Mimura T, Tsujimura T, Mitsuhashi N, Washitani/​Nemoto S, Maeshima M,
Martinoia E.

Department of Biology, Faculty of Science, Kobe University, Rokkodai 1/​1, Nada,
Kobe, 678/​8501, Japan.

Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles
isolated from Catharanthus roseus suspension/​cultured cells was measured. Under
low Pi status, Pi uptake into the vacuole was strongly activated compared to
high Pi status. Since Pi uptake across the vacuolar membrane is correlated with
H(+) pumping, we examined the dependency of H(+) pumping on plant Pi status.
Both H(+) pumping and the activities of the vacuolar H(+)/​pumps, the V/​type
H(+)/​ATPase and the H(+)/​PPase were enhanced under low Pi status. Despite this
increase in H(+) pumping, Western blot analysis showed no distinct increase in
the amount of proton pump proteins. Possible mechanisms for the activation of Pi
uptake into the vacuole under low Pi status are discussed.

PMID: 16955272 [PubMed /​ in process]

16: Microbiology. 2006 Sep;152(Pt 9):2703/​16.

Plasmid pSci6 from Spiroplasma citri GII/​3 confers insect transmissibility to
the non/​transmissible strain S. citri 44.

Berho N, Duret S, Danet JL, Renaudin J.

UMR 1090 Genomique Developpement et Pouvoir Pathogene, INRA, Universite de
Bordeaux 2, Centre INRA de Bordeaux, 71 avenue Edouard Bourlaux, BP 81, 33883
Villenave d'Ornon Cedex, France.

The insect/​transmissible strain GII/​3 of Spiroplasma citri contains plasmids
pSci1/​6, five of which (pSci1/​5) encode adhesin/​like proteins and one (pSci6)
encodes protein P32, which has been associated with insect transmissibility. In
contrast, S. citri strains ASP/​1 and 44, which cannot be transmitted via
injection into the leafhopper vector Circulifer haematoceps, lack these proteins
and also do not carry plasmids pSci1/​6. To further study the apparent
relationship between the presence of plasmids and insect transmissibility,
plasmids from S. citri GII/​3 were introduced into the insect/​non/​transmissible
S. citri strain 44 by electrotransformation using the tetM gene as the selection
marker. Tetracycline/​resistant transformants were shown to carry one, two or
three distinct plasmids. Plasmids pSci1/​6 were all detected in the
transformants, pSci1 being the most frequently found, alone or together with
other plasmids. Selected S. citri 44 transformants having distinct plasmid
contents were submitted, separately or in combination, to experimental
transmission to periwinkle (Catharanthus roseus) plants via injection into the
leafhopper vector. The occurrence of symptomatic plants indicated that, in
contrast to S. citri 44, spiroplasmal transformants were transmitted to the host
plant, in which they multiplied. Spiroplasma cultures isolated from these
infected plants all contained pSci6, leading to the conclusion that, under the
experimental conditions used, transformation by pSci6 conferred insect
transmissibility to S. citri strain 44. This is believed to be the first report
of a phenotypic change associated with transformation of S. citri by natural
plasmids.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16946265 [PubMed /​ indexed for MEDLINE]

17: Biotechnol Lett. 2006 Oct;28(19):1567/​71. Epub 2006 Aug 2.

Preparation of mixed alginate elicitors with high activity for the efficient
production of 5'/​phosphodiesterase by Catharanthus roseus cells.

Aoyagi H, Akimoto/​Tomiyama C, Tanaka H.

Institute of Life Sciences and Bioengineering, Graduate School of Life and
Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305/​8572, Japan.
aoyagi@sakura.cc.tsukuba.ac.jp

When various autoclaved microbial cells suspensions (exogenous elicitors) were
added to Catharanthus roseus cell cultures, its growth was inhibited but
5'/​phosphodiesterase (PDase) production was stimulated. The greatest effect was
with autoclaved Alteromonas macleodii: the dry cell concentration decreased from
13 to 10.9 mg/ml while PDase production increased from 0.022 to 0.235 U/ml. A
combination of A. macleodii (as exogenous elicitor) and 0.1%(w/v) alginate
oligomers (AO: acting as both endogenous elicitor and scavenger of active oxygen
species) minimized the cell growth inhibition but enhanced PDase production
(0.474 U/ml) about 20 times higher than the control (no addition). The method
for the preparation of mixed alginate elicitors with high activities containing
exogenous elicitor (autoclaved A. macleodii), endogenous elicitor (AO), and
trans/​4,5/​dihydroxy/​2/​cyclopenten/​1/​one was developed. The mixed alginate
elicitors significantly promoted PDase production (2.67 U/ml) by C. roseus, and
the productivity was increased 120/​fold compared to the control without cell
growth inhibition.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16937247 [PubMed /​ indexed for MEDLINE]

18: Plant Cell Environ. 2006 Sep;29(9):1742/​50.

Predicting leaf/​level fluxes of O3 and NO2: the relative roles of diffusion and
biochemical processes.

Eller AS, Sparks JP.

Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY
14853/​2701, USA.

Pollutants like O(3) and NO(2) enter leaves through the stomata and cause damage
during reactions with components of biological cell membranes. The steady/​state
flux rates of these gases into the leaf are determined by a series of physical
and biochemical resistances including stomatal aperture, reactions occurring
within the cell wall and the ability of the leaf to remove the products of
apoplastic reactions. In the present study, multiple regression models
incorporating stomatal conductance, apoplastic and symplastic ascorbate
concentrations, and nitrate reductase (NR) activities were generated to explain
the observed variations in leaf/​level flux rates of O(3) and NO(2). These
measurements were made on the plant Catharanthus roseus (Madagascar periwinkle).
The best/​fit model explaining NO(2) flux included stomatal conductance,
apoplastic ascorbate and NR activity. This model explained 89% of the variation
in observed leaf fluxes and suggested physical resistances, reaction between
NO(2) and apoplastic ascorbate, and the removal rate of nitrate (generated by
reactions of NO(2) and water) from the apoplast all play controlling roles in
NO(2) flux to leaves. O(3) flux was best explained by stomatal conductance and
symplastic ascorbate explaining 66% of the total variation in leaf flux. Both
models demonstrate the importance of measuring processes other than stomatal
conductance to explain steady/​state leaf/​level fluxes of pollutant gases.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16913863 [PubMed /​ indexed for MEDLINE]

19: Plant Mol Biol. 2006 Jul;61(4/​5):719/​32.

Cloning, characterization and localization of three novel class III peroxidases
in lignifying xylem of Norway spruce (Picea abies).

Marjamaa K, Hilden K, Kukkola E, Lehtonen M, Holkeri H, Haapaniemi P, Koutaniemi
S, Teeri TH, Fagerstedt K, Lundell T.

Department of Biological and Environmental Sciences, University of Helsinki,
P.O. Box 65, 00014 Helsinki, Finland. kaisa.marjamaa@helsinki.fi

Plant class III peroxidases (POXs) take part in the formation of lignin and
maturation of plant cell walls. However, only a few examples of such peroxidases
from gymnosperm tree species with highly lignified xylem tracheids have been
implicated so far. We report here cDNA cloning of three xylem/​expressed class
III peroxidase encoding genes from Norway spruce (Picea abies). The translated
proteins, PX1, PX2 and PX3, contain the conserved amino acids required for
heme/​binding and peroxidase catalysis. They all begin with putative secretion
signal propeptide sequences but diverge substantially at phylogenetic level,
grouping to two subclusters when aligned with other class III plant peroxidases.
In situ hybridization analysis on expression of the three POXs in Norway spruce
seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm
of young, developing tracheids within the current growth ring where
lignification is occurring. Function of the putative N/​terminal secretion signal
peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions
with EGFP (enhanced green fluorescent protein) and expressing them in tobacco
protoplasts. Full/​length coding region of px1 was also heterologously expressed
in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1
peroxidase is processed via the endoplasmic reticulum (ER) most likely for
secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal
localization for participation in the maturation of the spruce tracheid
secondary cell wall.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16897487 [PubMed /​ indexed for MEDLINE]

20: FEBS Lett. 2006 Aug 7;580(18):4501/​7. Epub 2006 Jul 17.

Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus).

Murata J, Bienzle D, Brandle JE, Sensen CW, De Luca V.

Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St.
Catharines, Ont., Canada L2S3A1.

The Madagascar periwinkle (Catharanthus roseus) is well known to produce the
chemotherapeutic anticancer agents, vinblastine and vincristine. In spite of its
importance, no expressed sequence tag (EST) analysis of this plant has been
reported. Two cDNA libraries were generated from RNA isolated from the base part
of young leaves and from root tips to select 9,824 random clones for
unidirectional sequencing, to yield 3,327 related sequences and 1,696 singletons
by cluster analysis. Putative functions of 3,663 clones were assigned, from
5,023 non/​redundant ESTs to establish a resource for transcriptome analysis and
gene discovery in this medicinal plant.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16870181 [PubMed /​ indexed for MEDLINE]

21: Phytochemistry. 2006 Aug;67(16):1758/​64. Epub 2006 Jun 27.

Identification of a low vindoline accumulating cultivar of Catharanthus roseus
(L.) G. Don by alkaloid and enzymatic profiling.

Magnotta M, Murata J, Chen J, De Luca V.

Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St.
Catharines, Ont., Canada L2S 3A1.

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially
important horticultural flower species and is the only source of the
monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, key
pharmaceutical compounds used to combat a number of different cancers. The
present study uses high performance liquid chromatography for metabolic
profiling of the MIAs extracted from seedlings and young leaves of 50 different
flowering cultivars of C. roseus to show that, except for a single low vindoline
cultivar (Vinca Mediterranean DP Orchid), they accumulate similar levels of
MIAs. Further enzymatic studies with extracts from young leaves and from
developing seedlings show that the low vindoline cultivar has a 10/​fold lower
tabersonine/​16/​hydroxylase activity than those of C. roseus cv. Little Delicata.
It is concluded that rapid metabolic and more selective enzymatic profiling of
Catharanthus mutants could be useful for the identification of a range of
altered MIA biosynthesis lines.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16806326 [PubMed /​ indexed for MEDLINE]

22: Plant Biol (Stuttg). 2006 Sep;8(5):731/​6. Epub 2006 Jun 13.

Molecular cloning and expression of a cDNA encoding a hybrid histidine kinase
receptor in tropical periwinkle Catharanthus roseus.

Papon N, Bremer J, Vansiri A, Glevarec G, Rideau M, Creche J.

Laboratoire des Sciences Vegetales, Faculte des Sciences Pharmaceutiques et
Biologiques, Paris, France.

Signalling pathways involving histidine kinase receptors (HKRs) are widely used
by prokaryotes and fungi to regulate a large palette of biological processes. In
plants, HKRs are known to be implicated in cytokinin, ethylene, and osmosensing
transduction pathways. In this work, a full length cDNA named CRCIK was isolated
from the tropical species CATHARANTHUS ROSEUS (L.) G. Don. It encodes a 1205
amino acid protein that belongs to the hybrid HKR family. The deduced amino acid
sequence shows the highest homology with AtHK1, an osmosensing HKR in
ARABIDOPSIS THALIANA. In return, CrCIK protein shares very low identity with the
other 10 ARABIDOPSIS HKRs. Southern blot analysis indicates that the CRCIK
corresponding gene is either present in multiple copies or has very close
homologues in the genome of the tropical periwinkle. The gene is widely
expressed in the plant. In C. ROSEUS C20D cell suspension, it is slightly
induced after exposure to low temperature, pointing to a putative role in
cold/​shock signal transduction.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16773556 [PubMed /​ indexed for MEDLINE]

23: J Biosci Bioeng. 2006 Apr;101(4):287/​96.

Metabolic engineering of cell cultures versus whole plant complexity in
production of bioactive monoterpene indole alkaloids: recent progress related to
old dilemma.

Pasquali G, Porto DD, Fett/​Neto AG.

Programa de Pos/​graduacao em Biologia Celular e Molecular, Centro de
Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Goncalves
9500, Pr. 43.431, P.O. Box 15.005, CEP 91.501/​970, Porto Alegre, RS, Brazil.

Monoterpene indole alkaloids (MIAs) are a large class of plant alkaloids with
significant pharmacological interest. The sustained production of MIAs at high
yields is an important goal in biotechnology. Intensive effort has been expended
toward the isolation, cloning, characterization and transgenic modulation of
genes involved in MIA biosynthesis and in the control of the expression of these
biosynthesis/​related genes. At the same time, considerable progress has been
made in the detailed description of the subcellular/​, cellular/​, tissue/​ and
organ/​specific expressions of portions of the biosynthetic pathways leading to
the production of MIAs, revealing a complex picture of the transport of
biosynthetic intermediates among membrane compartments, cells and tissues. The
identification of the particular environmental and ontogenetic requirements for
maximum alkaloid yield in MIA/​producing plants has been useful in improving the
supply of bioactive molecules. The search for new bioactive MIAs, particularly
in tropical and subtropical regions, is continuously increasing the arsenal for
therapeutic, industrially and agriculturally useful molecules. In this review we
focus on recent progress in the production of MIAs in transgenic cell cultures
and organs (with emphasis on Catharanthus roseus and Rauvolfia serpentina
alkaloids), advances in the understanding of in planta spatial/​temporal
expression of MIA metabolic pathways, and on the identification of factors
capable of modulating bioactive alkaloid accumulation in nontransgenic
differentiated cultures and plants (with emphasis on new MIAs from Psychotria
species). The combined use of metabolic engineering and physiological modulation
in transgenic and wild/​type plants, although not fully exploited to date, is
likely to provide the sustainable and rational supply of bioactive MIAs needed
for human well being.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16716935 [PubMed /​ indexed for MEDLINE]

24: Can J Microbiol. 2006 May;52(5):419/​26.

Model plants for studying the interaction between Methylobacterium mesophilicum
and Xylella fastidiosa.

Andreote FD, Lacava PT, Gai CS, Araujo WL, Maccheroni W Jr, van Overbeek LS, van
Elsas JD, Azevedo JL.

Department of Genetics, Escola Superior de Agricultura Luiz de Queiroz,
University of Sao Paulo, ESALQ/USP, Piracicaba, Brazil.

Over the last few years, endophytic bacterial communities associated with citrus
have been studied as key components interacting with Xylella fastidiosa. In this
study, we investigated the possible interaction between the citrus endophyte
Methylobacterium mesophilicum SR1.6/6 and X. fastidiosa in model plants such as
Catharanthus roseus (Madagaskar periwinkle) and Nicotiana clevelandii
(Clevelands tobacco). The aim of this study was to establish the fate of M.
mesophilicum SR1.6/6 after inoculation of C. roseus and N. clevelandii plants,
using PCR/​DGGE (polymerase chain reaction/​/​denaturing gradient gel
electrophoresis) and plating techniques. Shifts in the indigenous endophytic
bacterial communities were observed in plants inoculated with strain SR1.6/6,
using specific primers targeting alpha/​ and beta/​Proteobacteria. Cells of strain
SR1.6/6 were observed in a biofilm structure on the root and hypocotyl surfaces
of in vitro seedlings inoculated with M. mesophilicum SR1.6/6. This emphasizes
the importance of these tissues as main points of entrance for this organism.
The results showed that C. roseus and N. clevelandii could be used as model
plants to study the interaction between M. mesophilicum and X. fastidiosa.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16699566 [PubMed /​ indexed for MEDLINE]

25: Methods Mol Biol. 2006;318:349/​55.

Catharanthus roseus shoot cultures for the production of monoterpenoid indole
alkaloids.

Hernandez/​Dominguez E, Campos/​Tamayo F, Carrillo/​Pech M, Vazquez/​Flota F.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Mexico.

A protocol for the establishment of in vitro shoot cultures of Catharanthus
roseus is described. Shoots can be maintained for more than 1 yr without
evidence of tissue vitrification, disaggregation, or callus formation. Vindoline
was the main alkaloid accumulated, reaching values similar to those found in
leaves from field/​grown plants, after a long period of culture. An induction
methodology to reduce such waiting time is also presented.

PMID: 16673929 [PubMed /​ indexed for MEDLINE]

26: Plant J. 2006 Apr;46(2):193/​205.

Methylation of sulfhydryl groups: a new function for a family of small molecule
plant O/​methyltransferases.

Coiner H, Schroder G, Wehinger E, Liu CJ, Noel JP, Schwab W, Schroder J.

TU Munchen, FG Biomolekulare Lebensmitteltechnologie, Lise/​Meitner/​Str. 34,
D/​85354 Freising, Germany.

In plants, type I and II S/​adenosyl/​l/​methionine/​dependent O/​methyltransferases
(OMTs) catalyze most hydroxyl group methylations of small molecules. A
homology/​based RT/​PCR strategy using Catharanthus roseus (Madagascar periwinkle)
RNA previously identified six new type I plant OMT family members. We now
describe the molecular and biochemical characterization of a seventh protein. It
shares 56/​58% identity with caffeic acid OMTs (COMTs), but it failed to
methylate COMT substrates, and had no activity with flavonoids. However, the in
vitro incubations revealed unusually high background levels without added
substrates. A search for the responsible component revealed that the enzyme
methylated dithiothreitol (DTT), the reducing agent added for enzyme
stabilization. Unexpectedly, product analysis revealed that the methylation
occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34
compounds indicated a broad substrate range, with a preference for small
hydrophobic molecules. Benzene thiol (Km 220 microm) and furfuryl thiol (Km 60
microm) were the best substrates (6/​7/​fold better than DTT). Small isosteric
hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also
methylated, but the activities were at least 5/​fold lower than with thiols. The
enzyme was named C. roseus S/​methyltransferase 1 (CrSMT1). Models based on the
COMT crystal structure suggest that S/​methylation is mechanistically identical
to O/​methylation. CrSMT1 so far is the only recognized example of an
S/​methyltransferase in this protein family. Its properties indicate that a few
changes in key residues are sufficient to convert an OMT into a
S/​methyltransferase (SMT). Future functional investigations of plant
methyltransferases should consider the possibility that the enzymes may direct
methylation at sulfhydryl groups.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16623883 [PubMed /​ indexed for MEDLINE]

27: Proc Natl Acad Sci U S A. 2006 Apr 4;103(14):5614/​9. Epub 2006 Mar 24.

Gene/​to/​metabolite networks for terpenoid indole alkaloid biosynthesis in
Catharanthus roseus cells.

Rischer H, Oresic M, Seppanen/​Laakso T, Katajamaa M, Lammertyn F, Ardiles/​Diaz
W, Van Montagu MC, Inze D, Oksman/​Caldentey KM, Goossens A.

VTT Technical Research Centre of Finland, Tietotie 2, FIN/​02044 VTT, Espoo,
Finland.

Rational engineering of complicated metabolic networks involved in the
production of biologically active plant compounds has been greatly impeded by
our poor understanding of the regulatory and metabolic pathways underlying the
biosynthesis of these compounds. Whereas comprehensive genome/​wide functional
genomics approaches can be successfully applied to analyze a select number of
model plants, these holistic approaches are not yet available for the study of
nonmodel plants that include most, if not all, medicinal plants. We report here
a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus
roseus), a source of the anticancer drugs vinblastine and vincristine.
Genome/​wide transcript profiling by cDNA/​amplified fragment/​length polymorphism
combined with metabolic profiling of elicited C. roseus cell cultures yielded a
collection of known and previously undescribed transcript tags and metabolites
associated with terpenoid indole alkaloids. Previously undescribed gene/​to/​gene
and gene/​to/​metabolite networks were drawn up by searching for correlations
between the expression profiles of 417 gene tags and the accumulation profiles
of 178 metabolite peaks. These networks revealed that the different branches of
terpenoid indole alkaloid biosynthesis and various other metabolic pathways are
subject to differing hormonal regulation. These networks also served to identify
a select number of genes and metabolites likely to be involved in the
biosynthesis of terpenoid indole alkaloids. This study provides the basis for a
better understanding of periwinkle secondary metabolism and increases the
practical potential of metabolic engineering of this important medicinal plant.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16565214 [PubMed /​ indexed for MEDLINE]

28: Curr Pharm Biotechnol. 2006 Feb;7(1):33/​49.

Micropropagation: a tool for the production of high quality plant/​based
medicines.

Debnath M, Malik CP, Bisen PS.

Tissue Culture Laboratory, Institute of Biotechnology and Allied Sciences,
Seedling Academy of Design, Technology And Management, Jaipur/​302025, India.

Medicinal plants are the most important source of life saving drugs for the
majority of the world's population. The biotechnological tools are important to
select, multiply and conserve the critical genotypes of medicinal plants. Plant
tissue culture techniques offer an integrated approach for the production of
standardized quality phytopharmaceutical through mass/​production of consistent
plant material for physiological characterization and analysis of active
ingredients. Micropropagation protocols for cloning of some medicinal plants
such as Catharanthus roseus (Apocynaceae), Chlorophytum borivilianum
(Liliaceae), Datura metel (Solanaceae), and Bacopa monnieri (Scrophulariaceae)
have been developed. Regeneration occurred via organogenesis and embryogenesis
in response to auxins and cytokinins. The integrated approaches of our culture
systems will provide the basis for the future development of novel, safe,
effective, and high/​quality products for consumers.

Publication Types:
Review

PMID: 16472132 [PubMed /​ indexed for MEDLINE]

29: Bioprocess Biosyst Eng. 2006 Apr;28(5):295/​313. Epub 2006 Feb 1.

Development of a kinetic metabolic model: application to Catharanthus roseus
hairy root.

Leduc M, Tikhomiroff C, Cloutier M, Perrier M, Jolicoeur M.

Department of Chemical Engineering, Ecole Polytechnique de Montreal, PO Box
6079, Centre/​ville Station, Montreal, Quebec, Canada, H3C 3A7.

A kinetic metabolic model describing Catharanthus roseus hairy root growth and
nutrition was developed. The metabolic network includes glycolysis,
pentose/​phosphate pathway, TCA cycle and the catabolic reactions leading to cell
building blocks such as amino acids, organic acids, organic phosphates, lipids
and structural hexoses. The central primary metabolic network was taken at
pseudo/​steady state and metabolic flux analysis technique allowed reducing from
31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate
was described as a function of intracellular concentration in cell building
blocks. Intracellular transport and accumulation kinetics for major nutrients
were included. The model uses intracellular nutrients as well as energy shuttles
to describe metabolic regulation. Model calibration was performed using
experimental data obtained from batch and medium exchange liquid cultures of C.
roseus hairy root using a minimal medium in Petri dish. The model is efficient
in estimating the growth rate.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16453114 [PubMed /​ indexed for MEDLINE]

30: Plant Cell Rep. 2006 Jun;25(6):607/​12. Epub 2006 Jan 24.

Precursor limitations in methyl jasmonate/​induced Catharanthus roseus cell
cultures.

Lee/​Parsons CW, Royce AJ.

Chemical Engineering Department, 342 Snell Engineering Center, Northeastern
University, 360 Huntington Avenue, Boston, MA 02115/​5000, USA. clee@coe.neu.edu

Jasmonates enhance the expression of various genes involved in terpenoid indole
alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding
to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the
precursor availability for TIA biosynthesis. C. roseus suspensions were induced
with MJ (100 microM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM),
loganin plus tryptamine, or geraniol (0.1/​1.0 mM) on day 7. While MJ increased
ajmalicine production by 3/​fold, induced cultures were still limited by
terpenoid precursors. However, both induced and non/​induced cultures became
tryptamine/​limited with excess loganin. Geraniol feeding also increased
ajmalicine production in non/​induced cultures. But MJ appeared to increase
geraniol availability in induced cultures, due presumably to the increased
expression of Dxs with MJ addition.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16432630 [PubMed /​ indexed for MEDLINE]

31: Phytomedicine. 2006 Jan;13(1/​2):67/​73. Epub 2005 Jun 29.

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants.

Usia T, Iwata H, Hiratsuka A, Watabe T, Kadota S, Tezuka Y.

Institute of Natural Medicine, Toyama Medical and Pharmaceutical University,
2630/​Sugitani, Toyama 930/​0194, Japan.

Thirty samples of Indonesian medicinal plants were analyzed for their capacity
to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6
with a radiometric assay. The MeOH/​soluble fractions of 25 samples, prepared
from water extracts, demonstrated inhibitory activity more than 50% on the
metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by
CYP2D6. Among the MeOH/​soluble fractions, Piper nigrum leaf showed the highest
inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6
(98.1%). The water extracts of which MeOH/​soluble fraction showed inhibitory
activity more than 70% were fractionated with EtOAc. From the EtOAc/​soluble
fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit
(84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated
inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only
Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory
activity against CYP2D6. For samples that showed more than 70% inhibition, their
IC(50) values were determined. The most potent inhibitory activity against
CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum
leaf, while that of Catharanthus roseus showed the most potent inhibitory effect
against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate
once more the possibility of potential medicinal plant/​drug interactions.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16360935 [PubMed /​ indexed for MEDLINE]

32: J Plant Physiol. 2006 Jan;163(1):11/​8. Epub 2005 Aug 10.

Effect of salinity and different nitrogen sources on the activity of antioxidant
enzymes and indole alkaloid content in Catharanthus roseus seedlings.

Misra N, Gupta AK.

Department of Biochemistry, Bundelkhand University, Jhansi (UP), India.
neelam_misra@rediffmail.com

The activities of antioxidant enzymes viz. glutathione reductase, GR; superoxide
dismutase, SOD; peroxidase, POD; catalase, CAT and glutathione/​S/​transferase,
GST and alkaloid accumulation were investigated in leaf pairs (apical, middle,
basal) and in roots of Catharanthus roseus seedlings under the conditions of
different nitrogen sources (20 mM KNO(3) and 2 mM NH(4)Cl) and salinity, in the
absence (non/​saline control) and in the presence of 100 mM NaCl in the nutrient
solution. Salinity caused a reduction in plant biomass. The biomass production
of ammonium/​fed plants was lower than that of nitrate/​fed plants. The
antioxidant enzymes exhibited higher activity in saline/​treated plants. Changes
in antioxidant enzyme activity caused by different nitrogen sources differed in
all leaf pairs, as well as in roots of C. roseus. Ammonium/​fed plants showed
higher CAT, GR and GST activity in leaf pairs as well as in roots, while POD and
SOD activity were higher in nitrate/​fed plants. Higher peroxidase activity
concomitant with the increased accumulation of alkaloid was found in all leaf
pairs, as well as in roots of C. roseus of NO(3)(/​) fed plants as compared to
NH(4)(+) fed plants.

PMID: 16360799 [PubMed /​ indexed for MEDLINE]

33: Cell Mol Biol Lett. 2005;10(4):649/​57.

Isolation of a cDNA encoding the alpha/​subunit of CAAX/​prenyltransferases from
Catharanthus roseus and the expression of the active recombinant protein
farnesyltransferase.

Courdavault V, Burlat V, St/​Pierre B, Gantet P, Giglioli/​Guivarc'h N.

Universite Francois/​Rabelais de Tours, EA 2106 Biomolecules et Biotechnologies
Vegetales, UFR Sciences et Techniques, Parc de Grandmont, 37200 Tours, France.
courdavault@univ/​tours.fr

Crfta/ggt_Ia (AF525030), a cDNA encoding the ?/​subunit of the two types of
CaaX/​prenyltransferase (CaaX/​PTase), i.e. protein farnesyltransferase (PFT) and
type I protein geranylgeranyltransferase, was cloned from Catharanthus roseus
via a PCR strategy. Crfta/ggt_Ia is 1381/​bp long and bears a 999/​bp open reading
frame encoding a protein of 332 residues (FTA) that shares 66% identity with its
Lycopersicon esculentum orthologue. Southern blot analysis revealed that FTA is
encoded by a single gene copy per haploid genome. Co/​expression of Crfta/ggt_Ia
and Crftb encoding the beta/​subunit of PFT yielded purified active recombinant
PFT. This enzyme is able to prenylate proteins from C. roseus, and could be used
as a potent tool for prenylated protein identification.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16341273 [PubMed /​ indexed for MEDLINE]

34: Planta. 2006 May;223(6):1191/​200. Epub 2005 Dec 2.

Epidermis is a pivotal site of at least four secondary metabolic pathways in
Catharanthus roseus aerial organs.

Mahroug S, Courdavault V, Thiersault M, St/​Pierre B, Burlat V.

Universite Francois/​Rabelais de Tours, EA 2106 Biomolecules et Biotechnologies
Vegetales UFR Sciences et Techniques, Parc de Grandmont, 37200 Tours, France.

Catharanthus roseus produces a wide range of secondary metabolites, some of
which present high therapeutic values such as antitumoral monoterpenoid indole
alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA,
ajmalicine. We have recently shown that a complex multicellular organisation of
the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular,
the final steps of both the secoiridoid/​monoterpene and indole pathways
specifically occurred in the epidermis of leaves and petals. Chorismate is the
common precursor of indole and phenylpropanoid pathways. In an attempt to better
map the spatio/​temporal organisation of diverse secondary metabolisms in
Catharanthus roseus aerial organs, we studied the expression pattern of genes
encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia/​lyase
[PAL, E.C. 4.3.1.5], cinnamate 4/​hydroxylase [C4H, E.C. 1.14.13.11] and chalcone
synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that
CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL,
CrC4H and CrCHS were specifically expressed in the flavonoid/​rich upper
epidermis. Interestingly, these three genes were co/​expressed in the epidermis
(at least the upper, adaxial one) together with three MIA/​related genes,
indicating that single epidermis cells were capable of concomitantly producing a
wide range of diverse secondary metabolites (e.g. flavonoids, indoles,
secoiridoid/​monoterpenes and MIAs). These results, and data showing
co/​accumulation of flavonoids and alkaloids in single cells of C. roseus cell
lines, indicated the spatio/​temporal feasibility of putative common regulation
mechanisms for the expression of these genes involved in at least four distinct
secondary metabolisms.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16322983 [PubMed /​ indexed for MEDLINE]

35: Phytochemistry. 2006 Jan;67(2):132/​41.

Effect of long/​term phosphate starvation on the levels and metabolism of purine
nucleotides in suspension/​cultured Catharanthus roseus cells.

Shimano F, Ashihara H.

Department of Molecular Biology and Biochemistry, Graduate Division of Life
Sciences, Graduate School of Humanities and Sciences, Ochanomizu University,
Bunkyo/​ku, Tokyo 112/​8610, Japan.

The effect of long/​term phosphate (Pi) starvation of up to 3 weeks on the levels
of purine nucleotides and related compounds was examined using
suspension/​cultured Catharanthus roseus cells. Levels of adenine and guanine
nucleotides, especially ATP and GTP, were markedly reduced during Pi/​starvation.
There was an increase in the activity of RNase, DNase, 5'/​ and 3'/​nucleotidases
and acid phosphatase, which may participate in the hydrolysis of nucleic acids
and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was
observed during the long/​term Pi starvation. Long/​term Pi starvation markedly
depressed the flux of transport of exogenously supplied [8/​(14)C]adenosine and
[8/​(14)C]adenine, but these labelled compounds which were taken up by the cells
were readily converted to adenine nucleotides even in Pi/​starved cells, in which
RNA synthesis from these precursors was significantly reduced. The activities of
adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase
were maintained at a high level in long/​term Pi starved cells.

PMID: 16321409 [PubMed /​ indexed for MEDLINE]

36: Bioelectromagnetics. 2006 Feb;27(2):98/​104.

Magnetic field exposure stiffens regenerating plant protoplast cell walls.

Haneda T, Fujimura Y, Iino M.

Department of Precision Engineering, Faculty of Engineering, Chiba Institute of
Technology, Chiba, Japan.

Single suspension/​cultured plant cells (Catharanthus roseus) and their
protoplasts were anchored to a glass plate and exposed to a magnetic field of
302 +//​ 8 mT for several hours. Compression forces required to produce constant
cell deformation were measured parallel to the magnetic field by means of a
cantilever/​type force sensor. Exposure of intact cells to the magnetic field did
not result in any changes within experimental error, while exposure of
regenerating protoplasts significantly increased the measured forces and
stiffened regenerating protoplasts. The diameters of intact cells or
regenerating protoplasts were not changed after exposure to the magnetic field.
Measured forces for regenerating protoplasts with and without exposure to the
magnetic field increased linearly with incubation time, with these forces being
divided into components based on the elasticity of synthesized cell walls and
cytoplasm. Cell wall synthesis was also measured using a cell wall/​specific
fluorescent dye, and no changes were noted after exposure to the magnetic field.
Analysis suggested that exposure to the magnetic field roughly tripled the
Young's modulus of the newly synthesized cell wall without any lag.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16304695 [PubMed /​ indexed for MEDLINE]

37: Plant J. 2005 Nov;44(4):581/​94.

Localization of tabersonine 16/​hydroxylase and 16/​OH
tabersonine/​16/​O/​methyltransferase to leaf epidermal cells defines them as a
major site of precursor biosynthesis in the vindoline pathway in Catharanthus
roseus.

Murata J, Luca VD.

Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St
Catharines, Ontario, L2S3A1 Canada.

The Madagascar periwinkle (Catharanthus roseus) produces the well known and
remarkably complex anticancer dimeric alkaloids vinblastine and vincristine,
which are derived by the coupling of vindoline and catharanthine monomers.
Recent data from in situ RNA hybridization and immunolocalization suggest that
combinatorial cell factories within the leaf are involved in vindoline
biosynthesis. In this study, the cell types responsible for vindoline
biosynthesis were identified by laser/​capture microdissection/RNA
isolation/RT/​PCR to show that geraniol hydroxylase, secologanin synthase,
tryptophan decarboxylase, strictosidine synthase, strictosidine ss/​glucosidase
and tabersonine 16/​hydroxylase can be detected preferentially in epidermal
cells. A new and complementary application of the carborundum abrasion (CA)
technique was developed to obtain epidermis/​enriched leaf extracts that can be
used to measure alkaloid metabolite levels, enzyme activities and gene
expression. The CA technique showed that tabersonine and 16/​methoxytabersonine,
together with 16/​hydroxytabersonine/​16/​O/​methyltransferase, are found
predominantly in Catharanthus leaf epidermis, in contrast to vindoline,
catharanthine and later enzymatic steps in vindoline biosynthesis. The results
show that leaf epidermal cells are biosynthetically competent to produce
tryptamine and secologanin precursors that are converted via many enzymatic
transformations to make 16/​methoxytabersonine. This alkaloid or its 2,3
dihydro/​derivative is then transported to cells (mesophyll/idioblast/laticifer)
within Catharanthus leaves to complete the last three or four enzymatic
transformations to make vindoline.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16262708 [PubMed /​ indexed for MEDLINE]

38: Biotechnol Bioeng. 2006 Feb 5;93(2):386/​90.

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced
by Agrobacterium tumefaciens harboring rol ABC genes.

Hong SB, Peebles CA, Shanks JV, San KY, Gibson SI.

Department of Biochemistry and Cell Biology, MS/​140, 6100 Main St., Rice
University, Houston, Texas 77005, USA.

We have established Catharanthus roseus hairy root cultures transgenic for the
rol ABC genes from T(L)/​DNA of the agropine/​type Agrobacterium rhizogenes strain
A4. The rol ABC hairy root lines exhibit a wild/​type hairy root syndrome in
terms of growth and morphology on solid medium. However, they differ from
wild/​type hairy root lines in that they more frequently have excellent
adaptability to liquid medium and do not appear to form calli during
cultivation. Moreover, they do not produce detectable levels of mannopine and
agropine which, in contrast, are often synthesized abundantly in wild/​type hairy
root lines. The absence of these opines does not appear to cause the rol ABC
lines to have higher levels of terpenoid indole alkaloids than wild/​type hairy
root lines. Unlike wild/​type lines, rol ABC lines produce very similar levels of
total alkaloids despite wide variations in individual alkaloid contents. This
work demonstrates that the three genes rol ABC are sufficient to induce
high/​quality hairy roots in Catharanthus roseus. (c) 2005 Wiley Periodicals,
Inc.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16261632 [PubMed /​ indexed for MEDLINE]

39: Biotechnol Bioeng. 2006 Feb 20;93(3):534/​40.

Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots
over/​expressing the alpha or the alpha and beta subunits of anthranilate
synthase.

Peebles CA, Hong SB, Gibson SI, Shanks JV, San KY.

Department of Bioengineering, Rice University, Houston, Texas 77005, USA.

Among the pharmacologically important terpenoid indole alkaloids produced by
Catharanthus roseus are the anti/​cancer drugs vinblastine and vincristine. These
two drugs are produced in small yields within the plant, which makes them
expensive to produce commercially. Metabolic engineering has focused on
increasing flux through this pathway by various means such as elicitation,
precursor feeding, and introduction of genes encoding specific metabolic enzymes
into the plant. Recently in our lab, a feedback/​resistant anthranilate synthase
alpha subunit was over/​expressed in C. roseus hairy roots under the control of a
glucocorticoid inducible promoter system. Upon induction we observed a large
increase in the indole precursors, tryptophan, and tryptamine. The current work
explores the effects of over/​expressing the anthranilate synthase alpha or alpha
and beta subunits in combination with feeding with the terpenoid precursors
1/​deoxy/​D/​xylulose, loganin, and secologanin. In feeding 1/​deoxy/​D/​xylulose to
the hairy root line expressing the anthranilate synthase alpha subunit, we
observed an increase of 125% in horhammericine levels in the induced samples,
while loganin feeding increased catharanthine by 45% in the induced samples.
Loganin feeding to the hairy root line expressing anthranilate synthase alpha
and beta subunits increases catharanthine by 26%, ajmalicine by 84%,
lochnericine by 119%, and tabersonine by 225% in the induced samples. These
results suggest that the terpenoid precursors to the terpenoid indole alkaloids
are important factors in terpenoid indole alkaloid production. Copyright 2005
Wiley Periodicals, Inc.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16240438 [PubMed /​ indexed for MEDLINE]

40: J Biosci Bioeng. 2005 Mar;99(3):208/​15.

Development of a novel system for producing ajmalicine and serpentine using
direct culture of leaves in Catharanthus roseus intact plant.

Iwase A, Aoyagi H, Ohme/​Takagi M, Tanaka H.

Life Science and Bioengineering, Graduate School of Life and Environmental
Sciences, University of Tsukuba, Tsukuba, Ibaraki 305/​8572, Japan.

Due to problems of production instability, the production of plant secondary
metabolites using dedifferentiated cells (callus) is not always feasible on an
industrial scale. To propose a new methodology, which does not use
dedifferentiated cells, a novel system for producing useful secondary
metabolites using the direct culture of intact plant leaves was developed.
Catharanthus roseus was used as a model medicinal plant to produce terpenoid
indole alkaloids (TIAs) by suspension culture of the leaves in the
phytohormone/​free MS liquid medium. Adjustment of the osmotic pressure (993 kPa
at 25 degrees C) in the medium, light irradiation (60 micromol m(/​2) s(/​1)) and
addition of glucose (10 g/l) were effective to promote the production of TIAs
such as ajmalicine (Aj) and serpentine (Sp). On the basis of semi/​quantitative
RT/​PCR analyses, it was revealed that the culture conditions promoted gene
expression of enzymes in the TIA pathway in the cultured leaves. By feeding
glucose (10 g/l) on day 10 of the culture period, Aj was produced at a
concentration of about 18 mg/l and Sp was produced at a concentration about
11/​fold that of the control. These results represent the first step in the
development of a novel production system for plant secondary metabolites.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16233779 [PubMed /​ indexed for MEDLINE]

41: J Biosci Bioeng. 2004;98(2):67/​70.

A novel hydroxylase from Catharanthus roseus participating in the hydroxylation
of 2/​hydroxybenzoic acid.

Shimoda K, Kubota N, Sano T, Hirakawa H, Hirata T.

Department of Pharmacology and Therapeutics, Faculty of Medicine, Oita
University, 1/​1 Hasama/​machi, Oita 879/​5593, Japan. shimoda@med.oita/​u.ac.jp

A novel 55/​kDa hydroxylase was isolated from cultured cells of Catharanthus
roseus by a three/​step procedure: anion exchange chromatography, affinity
chromatography and hydroxylapatite adsorption chromatography. The enzyme
specifically catalyzed the hydroxylation of 2/​hydroxybenzoic acid to give
2,5/​dihydroxybenzoic acid. The enzyme activity was optimal at pH 7.8 and was
completely inhibited by divalent cations, such as Cu(2+) and Hg(2+). The enzyme
showed sequence similarity to certain plant flavonoid 3'/​hydroxylases.

PMID: 16233668 [PubMed]

42: J Biosci Bioeng. 2002;94(2):154/​9.

Production of 5'/​phosphodiesterase by Catharanthus roseus cells promoted by
heat/​degraded products generated from uronic acid.

Akimoto/​Tomiyama C, Aoyagi H, Ozawa T, Tanaka H.

Institute of Applied Biochemistry, University of Tsukuba, Tsuhtba, Ibaraki
305/​8572, Japan.

Polyalginate was autoclaved at 121 degrees C for 20 min and its molecular weight
distribution was analyzed. The autoclaved alginate yielded alginate polymer,
oligomer and heat degraded products (HDPs). Each of the separated substances
promoted 5'/​phosphodiesterase (5'/​PDase) production in suspension culture of
Catharanthus roseus cells. HDPs could also be generated from other uronic acids
(galacturonic acid and glucuronic acid) by autoclave treatment. The most
effective substance in the HDPs was isolated and characterized as
trans/​4,5/​dihydroxy/​2/​cyclopenten/​1/​one (DHCP). The optimal conditions for DHCP
production were also established (autoclaving 1 mg/ml monogalacturonic acid [pH
2] at 121 degrees C for 2 h). A combination of oligo/​alginate (below 4 kDa) and
HDPs significantly promoted the production of 5'/​PDase in C. roseus. Based on
the above results, a novel alginate complex that gave a 44/​fold increase in
5'/​PDase production by C. roseus was developed.

PMID: 16233285 [PubMed]

43: J Biosci Bioeng. 1999;87(6):762/​8.

Development of an apparatus for monitoring protoplast isolation from plant
tissues based on both dielectric and optical methods.

Aoyagi H, Takayanagi T, Jitsufuchi T, Tanaka H.

Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki
305/​8572, Japan.

In order to develop a method allowing objective determination of the optimal
conditions for the isolation of protoplasts, the process of protoplast isolation
from plant tissues was quantitatively evaluated. First, a specialized
spectrophotometer cuvette (working volume = 2.0 ml) was designed for the
continuous monitoring of protoplast isolation from plant tissues based on the
optical method. Homogeneous mixing of tissue sections and the protoplast
suspension in the cuvette was accomplished by means of a magnetic bar. The
cuvette was divided into upper and lower parts by a nylon mesh. Since tissue
sections in the upper part could not pass through the mesh, they did not affect
the optical path in the lower part, and only isolated protoplasts were able to
move freely between the two parts. At the optimal agitation speed (200 rpm),
mechanical damage to protoplasts of Catharanthus roseus did not occur. Increases
in the protoplast concentration during their isolation from tissue sections
(leaf and petal) could be continuously monitored by measuring the optical
density (O.D.), making it possible to estimate the end of protoplast isolation.
Degassing treatment of the tissues markedly enhanced protoplast isolation. In
order to monitor the viable protoplast concentration, a larger specialized
spectrophotometer cuvette (working volume = 25 ml) was developed which enabled
simultaneous measurement of the permittivity and O.D. of the suspension to be
carried out during protoplast isolation. Permittivity is a measure of the viable
protoplast concentration, while the O.D. shows protoplast characteristics such
as color. Using this large cuvette, the time courses of protoplast isolation
from leaf and petal sections were monitored and large amounts of viable
protoplasts were obtained. The protoplast isolation process after degassing
treatment was described by a simple first/​order reaction model and the viable
protoplast isolation rate was quantitatively evaluated from the rate constant
(k) on the basis of permittivity changes.

PMID: 16232551 [PubMed]

44: Phytochem Anal. 2005 Sep/​Oct;16(5):328/​33.

Rapid identification of vinca alkaloids by direct/​injection electrospray
ionisation tandem mass spectrometry and confirmation by high/​performance liquid
chromatography/​mass spectrometry.

Zhou H, Tai Y, Sun C, Pan Y.

Department of Chemistry, Zhejiang University, Hangzhou, People's Republic of
China.

A simple and rapid method for the identification of Vinca alkaloids from a crude
extract of Catharanthus roseus G. Don (Apocynaceae) by direct/​injection
electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been
developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine
were evaluated in a commercial extract of C. roseus using this method.
Catharanthine and its isomers 19S/​vindolinine and vindolinine were detected in
the commercial product by direct injection ESI/MS/MS and confirmed by
preparation and by HPLC/​ESI/MS. For the characterisation of different fragment
fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which
to identify some constituents in complex and mixed plant extracts.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16223089 [PubMed /​ indexed for MEDLINE]

45: J Chromatogr Sci. 2005 Oct;43(9):450/​3.

Simultaneous determination of vincristine, vinblastine, catharanthine, and
vindoline in leaves of catharanthus roseus by high/​performance liquid
chromatography.

Gupta MM, Singh DV, Tripathi AK, Pandey R, Verma RK, Singh S, Shasany AK,
Khanuja SP.

Analytical Testing Laboratory, Central Institute of Medicinal and Aromatic
Plants, Lucknow 226 015, India. guptammg@rediffmail.com

A simple reversed/​phase liquid chromatographic method is developed for the
simultaneous quantitation of the anticancerous drugs vincristine, vinblastine,
and their precursors catharanthine and vindoline using a Merck Chromolith
Performance reversed/​phase high/​performance liquid chromatography column. A
better resolution is obtained in comparison with available particulate/​type C18
columns. The column provides good reproducibility and peak symmetry.
Chromatography is carried isocratically with a mobile phase of acetonitrile/​0.1M
phosphate buffer containing 0.5% glacial acetic acid (21:79, v/v; pH 3.5) at a
flow rate of 1.2 mL/min and UV detection at 254 nm. Parameters such as
linearity, limits of quantitation (LOQ) and detection (LOD), precision,
accuracy, recovery, and robustness are studied. The method is selective and
linear for alkaloid concentration in the range 0.25 microg/​25 microg/mL. The LOQ
and LOD are 25, 46, 56, and 32 microg/mL and 8, 14, 18, and 10 microg/mL,
respectively. The results of accuracy studies are good. Values for coefficient
of variation are 2.50, 1.82, 1.33, and 1.13, respectively. The percent recovery
of the alkaloids was found to be 96%, 97%, 98%, and 98%, respectively. Peak
purity and homogeneity of these compounds in plant extract is studied using a
photodiode/​array detector. This simple and rapid method of analysis is applied
for the determination of these alkaloids in a large number of leaf extracts of
Catharanthus roseus..

PMID: 16212789 [PubMed /​ indexed for MEDLINE]

46: Biotechnol Prog. 2005 Sep/​Oct;21(5):1572/​6.

Transient effects of overexpressing anthranilate synthase alpha and beta
subunits in Catharanthus roseus hairy roots.

Peebles CA, Hong SB, Gibson SI, Shanks JV, San KY.

Department of Bioengineering, Rice University, Houston, Texas 77005, USA.

Catharanthus roseus produces two economically valuable anticancer drugs,
vinblastine and vincristine. These drugs are members of the terpenoid indole
alkaloids and accumulate in small quantities within the plant; thus these two
drugs are expensive to produce. Metabolic engineering efforts have focused on
increasing the alkaloids in this pathway through various means such as
elicitation, precursor feeding, and gene overexpression. Recently we
successfully expressed Arabidopsis genes encoding a feedback/​insensitive
anthranilate synthase alpha subunit under the control of the
glucocorticoid/​inducible promoter system and the anthranilate synthase beta
subunit under the control of a constitutive promoter in C. roseus hairy roots.
In this work we look at the transient behaviors of terpenoid indole alkaloids
over a 72 h induction period in late exponential growth phase cultures. Upon
induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72
h. In contrast, the lochnericine, horhammericine, and tabersonine pools
decreased and leveled out over the 72 h induction period. Visible changes within
the individual compounds usually took from 4 to 12 h.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16209565 [PubMed /​ indexed for MEDLINE]

47: J Biotechnol. 2006 Mar 9;122(1):28/​38. Epub 2005 Sep 26.

Expression of the Arabidopsis feedback/​insensitive anthranilate synthase
holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy
roots.

Hong SB, Peebles CA, Shanks JV, San KY, Gibson SI.

Department of Biochemistry and Cell Biology, MS/​140, Rice University, Houston,
TX 77005, USA.

In plants, the indole pathway provides precursors for a variety of secondary
metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan,
tryptamine, is a building block for the biosynthesis of terpenoid indole
alkaloids. Previously, we manipulated the indole pathway by introducing an
Arabidopsis feedback/​insensitive anthranilate synthase (AS) alpha subunit (trp5)
cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a
glucocorticoid/​inducible promoter into C. roseus hairy roots [Hughes, E.H.,
Hong, S./​B., Gibson, S.I., Shanks, J.V., San, K./​Y. 2004a. Expression of a
feedback/​resistant anthranilate synthase in Catharanthus roseus hairy roots
provides evidence for tight regulation of terpenoid indole alkaloid levels.
Biotechnol. Bioeng. 86, 718/​727; Hughes, E.H., Hong, S./​B., Gibson, S.I.,
Shanks, J.V., San, K./​Y. 2004b. Metabolic engineering of the indole pathway in
Catharanthus roseus hairy roots and increased accumulation of tryptamine and
serpentine. Metabol. Eng. 6, 268/​276]. Inducible expression of either or both
transgenes did not lead to significant increases in overall alkaloid levels
despite the considerable accumulation of tryptophan and tryptamine. In an
attempt to more successfully engineer the indole pathway, a wild type
Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with
the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic
hairy roots expressing both trp5 and ASB1 show a significantly greater
resistance to feedback inhibition of AS activity by tryptophan than plants
expressing only trp5. In fact, a 4.5/​fold higher concentration of tryptophan is
required to achieve 50% inhibition of AS activity in plants overexpressing both
genes than in plants expressing only trp5. In addition, upon a 3 day induction
during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times
more tryptophan (specific yield ca. 3.0 mg g(/​1) dry weight) than the trp5 hairy
root line. Concurrently, tryptamine levels increase up to 9/​fold in the induced
trp5:ASB1 line (specific yield ca. 1.9 mg g(/​1) dry weight) as compared with
only a 4/​fold tryptamine increase in the induced trp5 line (specific yield ca.
0.3 mg g(/​1) dry weight). However, endogenous TDC activities of both trp5:ASB1
and trp5 lines remain unchanged irrespective of induction. When TDC is
ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC
hairy root line accumulates tryptamine up to 14/​fold higher than the uninduced
line. In parallel with the remarkable accumulation of tryptamine upon induction,
alkaloid accumulation levels were significantly changed depending on the
duration and dosage of induction.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16188339 [PubMed /​ indexed for MEDLINE]

48: Plant Cell Rep. 2005 Dec;24(11):677/​82. Epub 2005 Aug 11.

Ajmalicine production in methyl jasmonate/​induced Catharanthus roseus cell
cultures depends on Ca2+ level.

Lee/​Parsons CW, Erturk S.

Chemical Engineering Department, 342 Snell Engineering Center, 360 Huntington
Avenue, Northeastern University, Boston, MA 02115/​5000, USA. clee@coe.neu.edu

Cytosolic Ca(2+) and jasmonate mediate signals that induce defense responses in
plants. In this study, the interaction between Ca(2+) and methyl jasmonate (MJ)
in modulating defense responses was investigated by monitoring ajmalicine
production in Catharanthus roseus suspension cultures. C. roseus suspensions
were treated with nine combinations of CaCl(2) (3, 23, and 43 mM) and MJ (0, 10,
and 100 microM) on day 6 of growth. Increased Ca(2+) influx through the addition
of extracellular CaCl(2) suppressed ajmalicine production in MJ/​induced
cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells
were treated with a low level of calcium (3 mM) combined with a high level of MJ
(100 microM). In the presence of 3 mM CaCl(2) in the medium, the addition of
Ca(2+) chelator EGTA (1, 2.5, and 5 mM) or Ca(2+) channel blocker verapamil (1,
10, and 50 muM) to MJ/​induced (100 microM) cultures on day 6 also inhibited
ajmalicine production at higher levels of the Ca(2+) inhibitors. Hence,
ajmalicine production in MJ/​induced C. roseus cultures depended on the
intracellular Ca(2+) concentration and a low extracellular Ca(2+) concentration
(3 mM) enhanced MJ/​induced ajmalicine production.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 16094527 [PubMed /​ indexed for MEDLINE]

49: Biotechnol Lett. 2005 Jun;27(11):793/​7.

Biotransformation of alpha/​santonin by cell suspension cultures of five plants.

Yang L, Dai J, Sakai J, Ando M.

College of Life and Environmental Sciences, The Central University for
Nationalities, 100081, Beijing, P.R. China.

Cell suspension cultures of five plants (Catharanthus roseus, Ginkgo biloba,
Platycodon grandiflorum, Taxus cuspidata, Phytolacca asinosa) were employed to
bioconvert the eudesmanolide compound, alpha/​santonin. Reactions occurring were
hydroxylation (C/​1, C/​11 and C/​15), reduction of the double bond [1(2) or 3(4)],
rearrangment of the eudesmanolide skeleton to a guaianolide skeleton and
lactone/​ring hydrolysis. Four new compounds were identified.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 16086262 [PubMed /​ indexed for MEDLINE]

50: Zhongguo Zhong Yao Za Zhi. 2005 May;30(10):741/​3, 755.

[A study on the hairy root culture and antitumor alkaloids production of
Catharanthus roseus]

[Article in Chinese]

Sun M, Zeng JJ.

School of Life Science, Southwest Normal University, Chongqing 400715, China.

OBJECTIVE: To establish transformation system and obtain alkaloids from the
hairy root of Catharanthus roseus. METHOD: Hairy roots were obtained by
infecting the different explants of C. roseus. Culture conditions of hairy root
were optimized. RESULT: The best transformation condition was leaf infected by
two/​day's pre/​culture and two/​day's co/​culture and additional A(S)
(hydroxyacetosyringone) 100 mg x L(/​1). The inducing rate of hairy root was up
to 86.25%. The best condition of hairy root culture was MS medium with sucrose
as carbon material and lactalbumin as nitron material. The analysis result
showed that the contents of total alkaloids in hairy roots were higher than
explants and calli. CONCLUSION: Hairy root of C. roseus will be useful for the
production of active components in C. roseus.

Publication Types:
English Abstract
Research Support, Non/​U.S. Gov't

PMID: 16075710 [PubMed /​ indexed for MEDLINE]

51: Phytochemistry. 2005 Aug;66(15):1797/​803.

Fosmidomycin analogues as inhibitors of monoterpenoid indole alkaloid production
in Catharanthus roseus cells.

Mincheva Z, Courtois M, Andreu F, Rideau M, Viaud/​Massuard MC.

EA 3857, Laboratoire de Synthese et Physicochimie Organique et Therapeutique
(SPOT), 31 Avenue Monge, 37200 Tours, France.

Substituted 3/​[2/​(diethoxyphosphoryl)propyl]oxazolo[4,5/​b]pyridine/​2(3H)/​ones
were obtained by functionalization at 6/​position with various substituents
(aryl, vinyl, carbonyl chains) via reactions catalysed with palladium. We found
that these new fosmidomycin analogues inhibited the accumulation of ajmalicine,
a marker of monoterpenoid indole alkaloids production in plant cells. Some of
them have greater inhibitory effect than fosmidomycin and fully inhibit alkaloid
accumulation at the concentration of 100 microM.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16054176 [PubMed /​ indexed for MEDLINE]

52: BMC Bioinformatics. 2005 Jul 18;6:179.

Processing methods for differential analysis of LC/MS profile data.

Katajamaa M, Oresic M.

Turku Centre for Biotechnology, Tykistokatu 6, FIN/​20521, Turku, Finland.
mikko.katajamaa@btk.fi

BACKGROUND: Liquid chromatography coupled to mass spectrometry (LC/MS) has been
widely used in proteomics and metabolomics research. In this context, the
technology has been increasingly used for differential profiling, i.e. broad
screening of biomolecular components across multiple samples in order to
elucidate the observed phenotypes and discover biomarkers. One of the major
challenges in this domain remains development of better solutions for processing
of LC/MS data. RESULTS: We present a software package MZmine that enables
differential LC/MS analysis of metabolomics data. This software is a toolbox
containing methods for all data processing stages preceding differential
analysis: spectral filtering, peak detection, alignment and normalization.
Specifically, we developed and implemented a new recursive peak search algorithm
and a secondary peak picking method for improving already aligned results, as
well as a normalization tool that uses multiple internal standards.
Visualization tools enable comparative viewing of data across multiple samples.
Peak lists can be exported into other data analysis programs. The toolbox has
already been utilized in a wide range of applications. We demonstrate its
utility on an example of metabolic profiling of Catharanthus roseus cell
cultures. CONCLUSION: The software is freely available under the GNU General
Public License and it can be obtained from the project web page at:
http://mzmine.sourceforge.net/.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 16026613 [PubMed /​ indexed for MEDLINE]

53: Prikl Biokhim Mikrobiol. 2005 May/​Jun;41(3):340/​6.

[Role of elements and physiologically active compounds in the regulation of
synthesis and accumulation of indole alkaloids in Catharanthus roseus L]

[Article in Russian]

Lovkova MIa, Buzuk GN, Sokolova SM, Buzuk LN.

Effects of various elements (Co, Ni, Zn, W, Mn, Cr, B, Mo, Fe, and V), natural
and synthetic auxins, cytokinins, and gibberellin on biosynthesis and
accumulation of indole alkaloids was studied at increasing concentrations in the
model system of Madagascar periwinkle seedlings (Catharanthus roseus L.). The
main types of concentration dependences for the effect of physiologically active
compounds under study were evaluated. A possible mechanism of the influence of
Zn and auxin on this process was partly clarified. The compounds were shown to
modulate various stages in the biosynthesis of monomeric indole alkaloids
(catharanthine and vindoline).

Publication Types:
English Abstract

PMID: 15977796 [PubMed /​ indexed for MEDLINE]

54: Planta Med. 2005 Jun;71(6):572/​4.

Cytokinin and ethylene control indole alkaloid production at the level of the
MEP/terpenoid pathway in Catharanthus roseus suspension cells.

Papon N, Bremer J, Vansiri A, Andreu F, Rideau M, Creche J.

Biomolecules et Biotechnologies Vegetales EA 2106, Tours, France.

The Madagascar periwinkle Catharanthus roseus accumulates a number of terpenoid
indole alkaloids, some of which have high therapeutic interest. The
biotechnological approach with cells in vitro remains an alternative to the
field culture of periwinkle for the production of such compounds. We previously
reported that two phytohormones, cytokinin and ethylene, remarkably enhanced the
accumulation of alkaloids in periwinkle cell suspensions. In this work, we
investigated the effects of these hormones on the regulation of several genes of
the indole alkaloid biosynthetic pathway. We show that cytokinin and/or ethylene
greatly enhanced the expression of the geraniol 10/​hydroxylase gene. When given
together, these hormones also increased the expression of three genes belonging
to the methyl/​erythritol pathway. These results make it possible to consider
elements of cytokinin and ethylene signalling pathways as tools for improving
terpenoid indole alkaloid production through metabolic engineering.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15971133 [PubMed /​ indexed for MEDLINE]

55: Plant Physiol. 2005 Jul;138(3):1607/​14. Epub 2005 Jun 17.

Phytic acid synthesis and vacuolar accumulation in suspension/​cultured cells of
Catharanthus roseus induced by high concentration of inorganic phosphate and
cations.

Mitsuhashi N, Ohnishi M, Sekiguchi Y, Kwon YU, Chang YT, Chung SK, Inoue Y, Reid
RJ, Yagisawa H, Mimura T.

Japan Society for the Promotion of Science, Tokyo 102/​8471, Japan.

We have established a new system for studying phytic acid, myo/​inositol
hexakisphosphate (InsP(6)) synthesis in suspension/​cultured cells of
Catharanthus. InsP(6) and other intermediates of myo/​inositol (Ins) phosphate
metabolism were measured using an ion chromatography method. The detection limit
for InsP(6) was less than 50 nM, which was sufficient to analyze Ins phosphates
in living cells. Synthesis of Ins phosphates was induced by incubation in high
inorganic phosphate medium. InsP(6) was mainly accumulated in vacuoles and was
enhanced when cells were grown in high concentration of inorganic phosphates
with the cations K(+), Ca(2+), or Zn(2+). However, there was a strong tendency
for InsP(6) to accumulate in the vacuole in the presence of Ca(2+) and in
nonvacuolar compartments when supplied with Zn(2+), possibly due to
precipitation of InsP(6) with Zn(2+) in the cytosol. A vesicle transport
inhibitor, brefeldin A, stimulated InsP(6) accumulation. The amounts of both
Ins(3)P(1) myo/​inositol monophosphate synthase, a key enzyme for InsP(6)
synthesis, and Ins(1,4,5)P(3) kinase were unrelated to the level of accumulation
of InsP(6). The mechanisms for InsP(6) synthesis and localization into vacuoles
in plant cells are discussed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15965017 [PubMed /​ indexed for MEDLINE]

56: Plant Mol Biol. 2005 Apr;57(6):855/​70.

CaaX/​prenyltransferases are essential for expression of genes involvedin the
early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells.

Courdavault V, Thiersault M, Courtois M, Gantet P, Oudin A, Doireau P, St/​Pierre
B, Giglioli/​Guivarc'h N.

Biomolecules et Biotechnologies Vegetales, Labaratoire de Physiologie Vegetale,
UFR Science et Techniques, Universite Francois/​Rabelais de Tours, EA2106, 37200
, Parc de Grandmont, Tours, France.

CaaX/​prenyltransferases (CaaX/​PTases) catalyse the covalent attachment of
isoprenyl groups to conserved cysteine residues located at the C/​terminal CaaX
motif of a protein substrate. This post/​translational modification is required
for the function and/or subcellular localization of some transcription factors
and components of signal transduction and membrane trafficking machinery.
CaaX/​PTases, including protein farnesyltransferase (PFT) and type/​I protein
geranylgeranyltransferase (PGGT/​I), are heterodimeric enzymes composed of a
common alpha subunit and a specific beta subunit. We have established RNA
interference cell lines targeting the beta subunits of PFT and PGGT/​I,
respectively, in the Catharanthus roseus C20D cell line, which synthesizes
monoterpenoid indole alkaloids in response to auxin depletion from the culture
medium. In both types of RNAi cell lines, expression of a subset of genes
involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes),
including the MEP pathway, is strongly decreased. The role of CaaX/​PTases in
ESMB gene regulation was confirmed by using the general prenyltransferase
inhibitor s/​perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on
the wild type line. Furthermore, supplementation of SP inhibited cells with
monoterpenoid intermediates downstream of the steps encoded by the ESMB genes
restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein
targets for both PFT and PGGT/​I are required for the expression of ESMB genes
and monoterpenoid biosynthesis in C. roseus, this represents a non previously
described role for protein prenyltransferase in plants.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15952070 [PubMed /​ indexed for MEDLINE]

57: Biol Pharm Bull. 2005 Jun;28(6):1021/​4.

Cytochrome P450 2D6 (CYP2D6) inhibitory constituents of Catharanthus roseus.

Usia T, Watabe T, Kadota S, Tezuka Y.

Institute of Natural Medicine, Toyama Medical and Pharmaceutical University,
Japan.

The MeOH/​soluble fraction of the water extract of Catharanthus roseus from
Indonesia, having shown potent inhibitory activity on the metabolism mediated by
CYP2D6, was subjected to activity/​guided isolation to yield two triterpenes,
ursolic acid (1) and oleanolic acid (2), and three alkaloids, vindoline (3),
ajmalicine (4), and serpentine (5). The isolated compounds were tested for their
inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using
[N/​methyl/​14C]erythromycin or [O/​methyl/​14C]dextromethorphan as a substrate,
respectively. Ajmalicine (4) and serpentine (5) showed very potent inhibitory
activity against CYP2D6 with IC50 values of 0.0023 and 3.51 microM,
respectively. All isolated compounds showed weak or no inhibition against
CYP3A4. On time/​, concentration/​, and NADPH/​dependent assay, serpentine (5)
appear to be the mechanism/​based inhibitor for CYP2D6 enzyme in which the
inhibition was irreversible and driven by catalytic process. K(I) and k(inact)
values for serpentine (5) were 0.148 microM and 0.090 min/​1, respectively. On
the other hand, ajmalicine (4) showed no time/​dependent inhibition or reversible
inhibition, and thus appear to be not mechanism/​based inhibitor.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15930738 [PubMed /​ indexed for MEDLINE]

58: J Plant Physiol. 2005 Apr;162(4):393/​402.

Characterization of a polyclonal antiserum against the monoterpene
monooxygenase, geraniol 10/​hydroxylase from Catharanthus roseus.

Canto/​Canche BB, Meijer AH, Collu G, Verpoorte R, Loyola/​Vargas VM.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Calle 43 No. 130, CP 97200, Col. Chuburna de Hidalgo,
Merida, Yucatan CP 97200, Mexico.

Geraniol 10/​hydroxylase (G10H) is a P450 containing enzyme which is the first
committed step in the biosynthesis of monoterpene indole alkaloids (MIAs),
including the Catharanthus roseus/​anticancer drugs vinblastine and vincristine.
It is thought that G10H has a regulatory role in MIA production. In the present
paper, we report the characterization of a polyclonal serum raised against the
purified G10H polypeptide. Anti/​G10H IgG was able to inhibit the G10H activity
and also recognized the G10H polypeptide from C. roseus and other plants
producing MIAs. These results establish the usefulness of this antiserum as a
biochemical tool for the study of G10H regulation.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15900881 [PubMed /​ indexed for MEDLINE]

59: Biotechnol Prog. 2005 Mar/​Apr;21(2):580/​9.

Plant cell culture monitoring using an in situ multiwavelength fluorescence
probe.

Hisiger S, Jolicoeur M.

Development of Metabolic Engineering Tools, Bio/​P2, Department of Chemical
Engineering, Ecole Polytechnique de Montreal, P.O. Box 6079 Centre/​ville
Station, Montreal, Quebec, Canada.

A multiwavelength fluorescence probe is proposed for in situ monitoring of
Eschscholtzia californica and Catharanthus roseus plant cell cultures. The
potential of the probe as a tool for real/​time estimation of biomass and
production in secondary metabolites has been studied. The probe excitation range
is 270/​550 nm and the emission range is 310/​590 nm, with a step of 20 nm for
both excitation and emission filters. Many endogenous fluorophores such as
NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine
and tryptophan, and fluorescent secondary metabolites were analyzed
simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an
adequate signal for estimating cells activity. Riboflavins fluorescence signal
(450/530 nm) followed C. roseus cell concentration both for the growth phase and
after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered
with NAD(P)H signal during the production phase. For C. roseus, tryptophan,
tryptamine, ajmalicine and serpentine were monitored by the probe. For E.
californica, fluorescence from alkaloids overlapped with riboflavins preventing
from using the probe to follow cell growth but global alkaloids production could
be followed using the probe.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15801802 [PubMed /​ indexed for MEDLINE]

60: Curr Med Chem. 2005;12(6):703/​11.

Combinatorial natural products: from cloning to analysis.

Budworth P, Khandurina J, Guttman A.

Horvath Laboratory of Bioseparation Science, University of Innsbruck, Innsbruck,
Austria.

Medicinal compounds from plants represent one of the largest and most diverse
groups of plant secondary metabolites. The advent of advanced bioinformatics
tools and modern genetic technology allowed for manipulation of biosynthetic
pathways with the potential of generating novel chemical entities. First, public
databases of secondary metabolite related enzymes were interrogated to identify
relevant plant genes from vinca rosea (Catharanthus roseus) and other species.
Genes of interest were tested after cloning by transfection into tobacco cell
cultures using DNA viral vectors. The biosynthetic enzymes coded by these genes
were over/​expressed in the host. Automated solvent extraction procedure was
employed to extract secondary metabolites from plant leaf tissues and
transfected tobacco cell culture samples. The composition of the extracts was
analyzed by state of the art bioanalytical methods such as high performance
liquid chromatography and capillary electrophoresis to monitor changes in
secondary metabolite patterns.

PMID: 15790307 [PubMed /​ indexed for MEDLINE]

61: Biotechnol Bioeng. 2005 Feb 5;89(3):367/​71.

Effect of nitric oxide on catharanthine production and growth of Catharanthus
roseus suspension cells.

Xu M, Dong J, Zhu M.

State Key Laboratory of Plant Physiology and Biochemistry, College of Life
Sciences, Zhejiang University, Hangzhou 310012, China.

Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO) to
investigate its effect on catharanthine synthesis and the growth of Catharanthus
roseus suspension cells. The results showed that SNP at high concentrations
(10.0 and 20.0 mmol/L) stimulated catharanthine formation of C. roseus cells,
but inhibited growth of the cells. Low concentrations of SNP (0.1 and 0.5
mmol/L) enhanced the growth of C. roseus cells, but had no effect on
catharanthine synthesis. The maximum total catharanthine production was achieved
by the addition of 0.5 and 10.0 mmol/L SNP to the cultures at day 0 and day 10,
respectively, being about threefold of the control. NO/​induced catharanthine
production of C. roseus cells was strongly suppressed by jasmonic acid (JA)
biosynthesis inhibitor ibuprofen (IBU) and nordihydroguaiaretic (NDGA). The
result suggests that the stimulatory role of NO on catharanthine production is
partially JA/​dependent.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15744842 [PubMed /​ indexed for MEDLINE]

62: J Exp Bot. 2005 Apr;56(414):1221/​8. Epub 2005 Feb 28.

Purification, molecular cloning, and cell/​specific gene expression of the
alkaloid/​accumulation associated protein CrPS in Catharanthus roseus.

Lemenager D, Ouelhazi L, Mahroug S, Veau B, St/​Pierre B, Rideau M, Aguirreolea
J, Burlat V, Clastre M.

EA2106 Biomolecules et Biotechnologies Vegetales, Universite de Tours, 31 Avenue
Monge, F/​37200 Tours, France.

Identification of molecular markers of monoterpenoid indole alkaloid (MIA)
accumulation in cell/​suspension cultures of Madagascar periwinkle (Catharanthus
roseus (L.) G. Don) was performed by two/​dimensional polyacrylamide gel
electrophoresis. Comparison of the protein patterns from alkaloid/​producing and
non/​producing cells showed the specific occurrence of a 28 kDa polypeptide
restricted to cells accumulating MIAs. The polypeptide was purified by
preparative two/​dimensional gel electrophoresis, digested with trypsin, and
microsequenced by the Edman degradation method. Cloning of the corresponding
cDNA revealed that the protein which has been named CrPS (Catharanthus roseus
Protein S) is a member of the alpha/beta hydrolase superfamily. Time/​course
expression studies by northern blot analysis confirmed that CrPS gene expression
was associated with MIA accumulation in cell suspension cultures. In the whole
plant, multicellular compartmentation is required for alkaloid biosynthesis. In
situ mRNA hybridization on developing leaves revealed that CrPS mRNA and
transcripts encoding the first enzymes of the MIA pathway were co/​localized in
internal phloem parenchyma cells. The possible implication of the
alkaloid/​accumulation associated protein CrPS in the signal transduction pathway
leading to MIA production is discussed.

PMID: 15737982 [PubMed /​ indexed for MEDLINE]

63: Planta. 2005 Jan;220(3):376/​83. Epub 2004 Nov 10.

Expression of terpenoid indole alkaloid biosynthetic pathway genes corresponds
to accumulation of related alkaloids in Catharanthus roseus (L.) G. Don.

Dutta A, Batra J, Pandey/​Rai S, Singh D, Kumar S, Sen J.

National Centre for Plant Genome Research, Jawaharlal Nehru University Campus,
P.O. Box 10531, 110 067 New Delhi, India.

Madagascar periwinkle, Catharanthus roseus (L.) G. Don, a medicinally important
plant, produces anticancer dimeric alkaloids, vinblastine and vincristine, in
the leaves and accumulates antihypertensive alkaloids, ajmalicine and
serpentine, in the roots. This plant grows wild in distant tropical and
sub/​tropical geographical locations with different agro/​climates and shows wide
variations in morphological and alkaloid yield/​related traits. In order to
understand the correlation between the expression of terpenoid indole alkaloid
(TIA) pathway genes and accumulation of related alkaloids, six different genetic
resources of C. roseus, including the medicinal cultivars Nirmal, Prabal,
Dhawal, the mutants gsr/​3 and gsr/​6, and one horticultural variety, Pacifica
blush, were studied. The expression profiles of one early and two late TIA
biosynthetic pathway genes, namely, strictosidine synthase, desacetoxyvindoline
4/​hydroxylase and deacetyl vindoline 4/​O/​acetyl transferase were analyzed in
these plants. A positive correlation between transcript abundance and
accumulation of related alkaloids was observed in the different genetic
resources. The potential of these TIA biosynthetic pathway genes for use in
screening of high/​yielding C. roseus germplasm has been discussed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15714355 [PubMed /​ indexed for MEDLINE]

64: Planta. 2005 Jul;221(5):690/​704. Epub 2005 Jan 29.

Proteome analysis of the medicinal plant Catharanthus roseus.

Jacobs DI, Gaspari M, van der Greef J, van der Heijden R, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Gorlaeus
Laboratories, P.O. Box 9502, 2300 Leiden, The Netherlands.

A proteomic approach is undertaken aiming at the identification of novel
proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C.
roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids
strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and
alkaloid accumulation was monitored during this period. After a rapid increase
between day 3 and day 6, the alkaloid content reached a maximum on day 16.
Systematic analysis of the proteome was performed by two/​dimensional
polyacrylamide gel electrophoresis. After day 3, the proteome started to change
with an increasing number of protein spots. On day 13, the proteome changed back
to roughly the same as at the start of the growth cycle. 88 protein spots were
selected for identification by mass spectrometry (MALDI/​MS/MS). Of these, 58
were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2),
which catalyzes the formation of strictosidine in the alkaloid biosynthesis;
tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the
alkaloid precursor tryptamine; 12/​oxophytodienoate reductase, which is
indirectly involved in the alkaloid biosynthesis as it catalyzes the last step
in the biosynthesis of the regulator jasmonic acid. Unique sequences were found,
which may also relate to unidentified biosynthetic proteins.

PMID: 15682277 [PubMed /​ indexed for MEDLINE]

65: J AOAC Int. 2004 Nov/​Dec;87(6):1287/​96.

Simultaneous quantification of some pharmaceutical Catharanthus roseus leaf and
root terpenoid indole alkaloids and their precursors in single runs by
reversed/​phase liquid chromatography.

Digvijay S, Shashi PR, Suchi S, Kumar RS, Raghavendra M, Sushil K.

National Center for Plant Genome Research, Jawaharlal Nehru University Campus,
New Delhi/​110 067, India.

A new, rapid, sensitive, and reproducible reversed/​phase liquid chromatographic
(LC) method with photodiode array detection is described. It allows, in a single
run of 30 min, simultaneous separation of 6 pharmaceutically and biologically
important Catharanthus roseus leaf and root terpenoid indole alkaloids (TIAs)
and 3 of their precursors: TIA precursors tryptophan, tryptamine, and loganine;
and TIAs serpentine, catharanthine, ajmalicine, vincristine, vinblastine, and
vindoline. The method involves the use of a Phenomenex Luna 5 microm, C18 column
(250 mm x 4.6 mm id) and a linear binary gradient mobile phase profile.
Detection is performed at 220 and 254 nm, which provided good absorptivity for
all of the roseus compounds listed above and gave a minimum detection limit of
0.02 microg/mL. The extraction efficiency, peak purity, and homogeneity
parameters of the profiles could be validated using a photodiode array detector.
The method was successfully used to quantify major components of leaf and root
extracts of C. roseus accessions. The new method thus provides a reliable tool
for rapid screening of C. roseus samples in large numbers, which is needed in
breeding/genetic engineering and genetic mapping experiments and for monitoring
the reaction products, in the in vitro/in vivo conversions of precursors into
products, and vice versa.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15675438 [PubMed /​ indexed for MEDLINE]

66: Mol Plant Microbe Interact. 2005 Jan;18(1):33/​42.

Sugar import and phytopathogenicity of Spiroplasma citri: glucose and fructose
play distinct roles.

Andre A, Maucourt M, Moing A, Rolin D, Renaudin J.

UMR 1090 Genomique Developpement et Pouvoir Pathogene, INRA, Universite de
Bordeaux 2, Centre INRA de Bordeaux, B.P. 81, 33883 Villenave d'Ornon Cedex,
France.

We have shown previously that the glucose PTS (phosphotransferase system)
permease enzyme II of Spiroplasma citri is split into two distinct polypeptides,
which are encoded by two separate genes, crr and ptsG. A S. citri mutant was
obtained by disruption of ptsG through homologous recombination and was proved
unable to import glucose. The ptsG mutant (GII3/​glc1) was transmitted to
periwinkle (Catharanthus roseus) plants through injection to the leaf/​hopper
vector. In contrast to the previously characterized fructose operon mutant GMT
553, which was found virtually nonpathogenic, the ptsG mutant GII3/​glc1 induced
severe symptoms similar to those induced by the wild/​type strain GII/​3. These
results, indicating that fructose and glucose utilization were not equally
involved in pathogenicity, were consistent with biochemical data showing that,
in the presence of both sugars, S. citri used fructose preferentially. Proton
nuclear magnetic resonance analyses of carbohydrates in plant extracts revealed
the accumulation of soluble sugars, particularly glucose, in plants infected by
S. citri GII/​3 or GII3/​glc1 but not in those infected by GMT 553. From these
data, a hypothetical model was proposed to establish the relationship between
fructose utilization by the spiroplasmas present in the phloem sieve tubes and
glucose accumulation in the leaves of S. citri infected plants.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15672816 [PubMed /​ indexed for MEDLINE]

67: Fitoterapia. 2005 Jan;76(1):83/​90.

Methyljasmonate accelerates catabolism of monoterpenoid indole alkaloids in
Catharanthus roseus during leaf processing.

El/​Sayed M, Verpoorte R.

Department of Pharmacognosy, Section of Metabolomics, Institute of Biology
Leiden, Leiden University, Leiden, The Netherlands.
m.el/​sayed@chem.leidenuniv.nl

Variations in alkaloid pattern during drying of leaves (leaf processing) showed
that treatment with methyljasmonate can induce formation of bisindole alkaloids
as a result of catabolism of the monomeric alkaloids catharanthine and
vindoline. A two/​fold increase in 3',4'/​anhydrovinblastine was shown in treated
leaves especially from day 8 until day 21. Serpentine also increased in the same
period under the treatment as a catabolic product of ajmalicine. Basic
peroxidases that are responsible for the formation of anhydrovinblastine and
serpentine showed high activity at days 8 and 21 in treated leaves, causing the
increase in anhydrovinblastine and serpentine.

PMID: 15664467 [PubMed /​ indexed for MEDLINE]

68: J Enzyme Inhib Med Chem. 2004 Dec;19(6):559/​65.

Synthesis and biological evaluation with plant cells of new fosmidomycin
analogues containing a benzoxazolone or oxazolopyridinone ring.

Courtois M, Mincheva Z, Andreu F, Rideau M, Viaud/​Massuard MC.

EA 2106 Biomolecules et Biotechnologies Vegetales, Universite de Tours, UFR
Sciences Pharmaceutiques, 37200 Tours, France.

Fosmidomycin, 3/​(N/​formyl/​N/​hydroxyamido) propylphosphonic acid sodium salt, is
an efficient inhibitor of 1/​deoxy/​D/​xylulose/​5/​phosphate (DOXP)
reductoisomerase, the second enzyme of the 2C/​methyl/​D/​erythritol/​4/​phosphate
(MEP) pathway notably present in Plasmodium species. We have synthesized a new
series of analogues of fosmidomycin, containing a benzoxazolone,
benzoxazolethione or oxazolopyridinone ring. As the MEP pathway is involved in
the biosynthesis of all isoprenoids, accumulation of ajmalicine in Catharanthus
roseus cells was chosen as a marker of monoterpenoid indole alkaloid (MIA)
production. None of the twelve studied phosphonic esters 3 and phosphonic acids
4 affected periwinkle cell growth, but some of them (3c, 3e, 3g and 3h) showed a
significant inhibition of ajmalicine accumulation: 45/​85% at 125 microM.
Surprisingly, this effect disappeared by conversion of 3c and 3g into the
corresponding acids 4c and 4g, respectively.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15662959 [PubMed /​ indexed for MEDLINE]

69: Zhongguo Zhong Yao Za Zhi. 2003 Nov;28(11):1006/​9.

[Advances in the study of vincristine: an anticancer ingredient from
Catharanthus roseus]

[Article in Chinese]

Lu Y, Hou SX, Chen T.

College of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, China.
toLuYi@eyou.com

Vincristine is a dimer/​indo/​alkaloid which is extracted from the leaves of
Catharanthus roseus. It is effective to treat acute lymphocytic cell leukemia,
Hodgkin disease and non/​Hodgkin disease clinically. But the severe side effects,
such as neurotoxic and tissue damage, limit its application. In this paper, we
summarize physical, chemical, pharmacological and pharmacokinetical properties
of VCR and advances in decreasing its side effects. In clinic, association with
other medication is adopted. In pharmaceutics, people adopt some new methods and
technology such as conjugation with the antibody, encapsulation in liposomes or
controlled release films.

Publication Types:
English Abstract
Review

PMID: 15615402 [PubMed /​ indexed for MEDLINE]

70: Biotechnol Lett. 2004 Oct;26(20):1595/​9.

Enhancement of ajmalicine production in Catharanthus roseus cell cultures with
methyl jasmonate is dependent on timing and dosage of elicitation.

Lee/​Parsons CW, Erturk S, Tengtrakool J.

Chemical Engineering Department, 342 Snell Engineering Center, 360 Huntington
Avenue, Northeastern University, Boston, MA 02115/​5000, USA. clee@coe.neu.edu.

The optimum growth stage for enhancing ajmalicine production in Catharanthus
roseus cultures with methyl jasmonate (MJ) was after 6 d growth. MJ added at 10
or 100 microm on day 6 gave a maximum ajmalicine production of 10.2 mg l(/​1), a
300% increase over that of non/​elicited cultures.

Publication Types:
Comparative Study
Evaluation Studies
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 15604804 [PubMed /​ indexed for MEDLINE]

71: Plant Mol Biol. 2004 Aug;55(6):797/​805.

Activation of the oxidative burst by yeast elicitor in Catharanthus roseus cells
occurs independently of the activation of genes involved in alkaloid
biosynthesis.

Pauw B, van Duijn B, Kijne JW, Memelink J.

Institute of Biology, Leiden University, Wassenaarseweg 64, Leiden, 2333 AL, The
Netherlands.

In Catharanthus roseus cell suspensions, expression of several terpenoid indole
alkaloid (TIA) biosynthetic genes, including those encoding strictosidine
synthase and tryptophan decarboxylase, is coordinately induced by fungal
elicitors such as yeast extract (YE). This induction is mediated by several
signaling steps including the biosynthesis of jasmonic acid, and the activation
of the jasmonic acid/​responsive ORCA transcription factors. We investigated a
possible role of reactive oxygen species (ROS) as a second messenger in this
system. YE was shown to activate the production of ROS, which was dependent on
protein phosphorylation and calcium influx. However, ROS generation was neither
necessary for the induction of genes involved in TIA biosynthesis by YE nor by
itself sufficient to induce these genes. Therefore, we conclude that activation
of the oxidative burst by YE occurs independently of the activation of genes
involved in TIA biosynthesis.

PMID: 15604717 [PubMed /​ indexed for MEDLINE]

72: J Hered. 2005 Jan/​Feb;96(1):71/​7. Epub 2004 Dec 14.

Allelic differences at two loci govern different mechanisms of intraflower
self/​pollination in self/​pollinating strains of periwinkle.

Kulkarni RN, Sreevalli Y, Baskaran K.

Central Institute of Medicinal and Aromatic Plants, Field Station, Allalasandra,
Bangalore 560 065, India. krnpbg@yahoo.co.in

Periwinkle [Catharanthus roseus (L.) G. Don], an ornamental and medicinal plant,
is a self/​compatible, insect/​pollinated plant species in which intraflower
self/​pollination does not occur because of spatial separation of the stigma and
anthers. Recently three self/​pollinating strains/​MJ, VI, and OR/​were identified.
Self/​pollination in these strains was found to be brought about by continuous
increase in gynoecium length from anthesis to self/​pollination, in contrast to
non/​self/​pollinating strains, in which the stigma remained below the base of the
anthers from anthesis to flower drop. Self/​pollination in these strains was
found to be controlled by duplicate, recessive genes. Self/​pollination in
strains MJ and VI was brought about by an increase in gynoecium length resulting
from an increase in the length of the ovary, while in the strain OR, the
increase in gynoecium length was because of an increase in the length of the
style from anthesis to self/​pollination. The three strains were intercrossed to
determine the relationship between genes governing self/​pollination in these
strains. The F(1) plants and all plants of the F(2) generation of the cross MJ x
VI exhibited self/​pollination that was brought about by an increase in the
length of the ovary, indicating that the same genes were involved in these two
strains. The F(1) plants of crosses OR x MJ and OR x VI, exhibited
self/​pollination that was brought about by an increase in the length of the
ovary, indicating that self/​pollination brought about by an increase in the
length of the ovary was dominant over self/​pollination brought about by an
increase in the length of the style. In the F(2) and backcross [(OR x MJ) x OR
and (OR x VI) x OR] generations, both self/​pollinating and non/​self/​pollinating
plants were observed. The ratio of plants with self/​pollination brought about by
an increase in the length of the ovary, non/​self/​pollinating plants, and plants
with self/​pollination brought about by an increase in the length of the style in
the F(2) and backcross generations fit 9:6:1 and 1:2:1 ratios, respectively. All
plants of the backcrosses [(OR x MJ) x MJ and (OR x VI) x VI] exhibited
self/​pollination brought about by an increase in the length of the ovary. The
results thus supported the earlier finding that self/​pollination in the studied
strains was controlled by duplicate, recessive genes and suggested that three
alleles at two loci determine the occurrence or nonoccurrence of intraflower
self/​pollination in periwinkle.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15598715 [PubMed /​ indexed for MEDLINE]

73: Mol Plant Microbe Interact. 2004 Nov;17(11):1175/​84.

Distribution of phytoplasmas in infected plants as revealed by real/​time PCR and
bioimaging.

Christensen NM, Nicolaisen M, Hansen M, Schulz A.

Department of Plant Biology, The Royal Veterinary and Agricultural University,
Thorvaldsensvej 40, DK/​1871 Frederiksberg C, Copenhagen, Denmark.

Phytoplasmas are cell wall/​less bacteria inhabiting the phloem and utilizing it
for their spread. Infected plants often show changes in growth pattern and a
reduced crop yield. A quantitative real/​time polymerase chain reaction (Q/​PCR)
assay and a bioimaging method were developed to quantify and localize
phytoplasmas in situ. According to the Q/​PCR assay, phytoplasmas accumulated
disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser
extent, in petioles of source leaves and in stems. However, phytoplasma
accumulation was small or nondetectable in sink organs (roots and sink leaves).
For bioimaging, infected plant tissue was stained with vital fluorescence dyes
and examined using confocal laser scanning microscopy. With a DNA/​sensitive dye,
the pathogens were detected exclusively in the phloem, where they formed dense
masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by
counterstaining with aniline blue for callose and multiphoton excitation. With a
potentiometric dye, not all DNA/​positive material was stained, suggesting that
the dye stained metabolically active phytoplasmas only. Some highly infected
sieve tubes contained phytoplasmas that were either inactive or dead upon
staining.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15553243 [PubMed /​ indexed for MEDLINE]

74: Metab Eng. 2004 Oct;6(4):268/​76.

Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots
and increased accumulation of tryptamine and serpentine.

Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY.

Department of Chemical Engineering, Rice University, P.O. Box 1892, Houston, TX
77251/​1892, USA.

Transgenic hairy roots of Catharanthus roseus were established with
glucocorticoid inducible tryptophan decarboxylase (TDC) expression alone or in
combination with inducible expression of a feedback/​resistant anthranilate
synthase alpha subunit (ASalpha) from Arabidopsis. Northern blot analysis
confirmed transcription of the anthranilate synthase gene upon induction in the
double line (TDC+ASalpha) and in vitro enzyme assays confirmed increased
resistance to feedback inhibition by tryptophan. In TDC enzyme assays, increases
of 48% and 87% in the TDC and double lines, respectively, were noted. Although
the TDC line showed no significant increase in tryptamine levels on induction,
induction of the double line resulted in increases in tryptamine levels of as
much as six/​fold for a 3 day late exponential induction. Downstream effects on
alkaloids were noted in the TDC line where serpentine specific yields increased
as much as 129% on induction. No effects on measured alkaloids were noted in the
double line, but the two clones have very different basal alkaloid biosynthetic
capacities. Within this study, the engineering of the indole pathway in C.
roseus hairy roots is reported, and the role of the indole pathway in alkaloid
biosynthesis explored.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.
Research Support, U.S. Gov't, P.H.S.

PMID: 15491856 [PubMed /​ indexed for MEDLINE]

75: Appl Microbiol Biotechnol. 2005 Apr;67(1):40/​4. Epub 2004 Oct 5.

Elicitor/​induced nitric oxide burst is essential for triggering catharanthine
synthesis in Catharanthus roseus suspension cells.

Xu M, Dong J.

College of Food Sciences and Biotechnology and Environmental Engineering,
Hangzhou University of Commerce, Hangzhou 310035, China. maojunxu@163.com

Elicitor prepared from the cell walls of Penicillium citrinum induced multiple
responses in Catharanthus roseus suspension cells, including rapid generation of
nitric oxide (NO), sequentially followed by enhancement of catharanthine
production by C. roseus cells. Elicitor/​induced catharanthine biosynthesis was
blocked by NO/​specific scavenger
2/​4/​carboxyphenyl/​4,4,5,5/​tetramethylimidazoline/​1/​oxyl/​3/​oxide and nitric oxide
synthase (NOS) inhibitor S,S'/​1,3/​phenylene/​bis(1,2/​ethanediyl)/​bis/​isothiourea
(PBITU). PBITU also strongly inhibited elicitor/​induced NO generation by C.
roseus suspension cells. The inhibiting effect of PBITU on elicitor/​induced
catharanthine production was reversed by external application of NO via the
NO/​donor sodium nitroprusside. The results strongly suggested that NO, generated
by NOS or NOS/​like enzymes in C. roseus suspension cells when treated with the
fungal elicitor, was essential for triggering catharanthine synthesis.

PMID: 15480633 [PubMed /​ indexed for MEDLINE]

76: J Biol Chem. 2004 Dec 17;279(51):52940/​8. Epub 2004 Oct 1.

Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis
genes in Catharanthus roseus.

Pauw B, Hilliou FA, Martin VS, Chatel G, de Wolf CJ, Champion A, Pre M, van
Duijn B, Kijne JW, van der Fits L, Memelink J.

Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64,
2333 AL Leiden, The Netherlands.

In Catharanthus roseus cell suspensions, the expression of several terpenoid
indole alkaloid biosynthetic genes, including two genes encoding strictosidine
synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by
fungal elicitors such as yeast extract. To identify molecular mechanisms
regulating the expression of these genes, a yeast one/​hybrid screening was
performed with an elicitor/​responsive part of the TDC promoter. This screening
identified three members of the Cys(2)/His(2)/​type (transcription factor
IIIA/​type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3.
These proteins bind in a sequence/​specific manner to the TDC and STR promoters
in vitro and repress the activity of these promoters in trans/​activation assays.
In addition, the ZCT proteins can repress the activating activity of
APETALA2/ethylene response/​factor domain transcription factors, the ORCAs, on
the STR promoter. The expression of the ZCT genes is rapidly induced by yeast
extract and methyljasmonate. These results suggest that the ZCT proteins act as
repressors in the regulation of elicitor/​induced secondary metabolism in C.
roseus.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15465826 [PubMed /​ indexed for MEDLINE]

77: Plant Physiol Biochem. 2004 Jul/​Aug;42(7/​8):623/​8.

Alkaloid metabolism in wounded Catharanthus roseus seedlings.

Vazquez/​Flota F, Carrillo/​Pech M, Minero/​Garcia Y, De Lourdes Miranda/​Ham M.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Calle 43 n degrees. 130, Chuburna de Hidalgo, 97200
Merida, Yucatan, Mexico. felipe@cicy.mx

The effect of mechanical wounding on alkaloid metabolism was analyzed in
Catharanthus roseus seedlings. Wounding induced an increase in ajmalicine
accumulation, whereas catharanthine remained unaffected. A positive dual effect
on vindoline was noticed. Short and mid/​term effects were detected between 12
and 24 h after mechanical damage was inflicted, and apparently resulted from the
accelerated transformation of the tabersonine intermediaries to vindoline.
Long/​term effects involved a generalized increase in carbon flux towards
alkaloid synthesis. Exposure to ethylene (1 ppm) produced similar results to
those observed in wounded seedlings, suggesting that it might be mediating the
wound/​induced increase in alkaloid synthesis. No synergistic or additive effects
were observed when wounded seedlings were exposed to ethylene or jasmonate.
Copyright 2004 Elsevier SAS

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15331091 [PubMed /​ indexed for MEDLINE]

78: Plant Physiol. 2004 Aug;135(4):2398/​410. Epub 2004 Jul 30.

Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma
using 1H/​NMR spectroscopy and multivariate data analysis.

Choi YH, Tapias EC, Kim HK, Lefeber AW, Erkelens C, Verhoeven JT, Brzin J, Zel
J, Verpoorte R.

Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden
University, 2300 RA Leiden, The Netherlands.

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected
by 10 types of phytoplasmas was carried out using one/​dimensional and
two/​dimensional NMR spectroscopy followed by principal component analysis (PCA),
an unsupervised clustering method requiring no knowledge of the data set and
used to reduce the dimensionality of multivariate data while preserving most of
the variance within it. With a combination of these techniques, we were able to
identify those metabolites that were present in different levels in
phytoplasma/​infected C. roseus leaves than in healthy ones. The infection by
phytoplasma in C. roseus leaves causes an increase of metabolites related to the
biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids:
chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher
abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the
phytoplasma/​infected leaves. The PCA of the (1)H/​NMR signals of healthy and
phytoplasma/​infected C. roseus leaves shows that these metabolites are major
discriminating factors to characterize the phytoplasma/​infected C. roseus leaves
from healthy ones. Based on the NMR and PCA analysis, it might be suggested that
the biosynthetic pathway of terpenoid indole alkaloids, together with that of
phenylpropanoids, is stimulated by the infection of phytoplasma.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 15286294 [PubMed /​ indexed for MEDLINE]

79: Phytochemistry. 2004 Jul;65(13):1911/​7.

Salt/​induced lipid changes in Catharanthus roseus cultured cell suspensions.

Elkahoui S, Smaoui A, Zarrouk M, Ghrir R, Limam F.

Laboratoire Interactions Legumineuses/​Microorganismes, Institut National de
Recherche Scientifique et Technique, BP 95/​2050 Hammam/​Lif, Tunisia.

Salt treatment strongly affected cell growth by decreasing dry weight. Exposure
of Catharanthus roseus cell suspensions to increasing salinity significantly
enhanced total lipid (TL) content. The observed increase is mainly due to high
level of phospholipids (PL). Hundred mM NaCl treatment increased phospholipid
species phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas it
reduced glycolipid ones monogalactosyldiacylglycerol (MGDG) and
digalactosyldiacylglycerol (DGDG) but not sulfoquinovosyldiacylglycerol (SQDG).
Moreover, fatty acid composition was clearly modified when cells were cultured
in the presence of 100 mM NaCl, whereas only few changes occurred at 50 mM. Salt
treatment decreased palmitic acid (16:0) level and increased that of linolenic
acid (18:2). Such effect was observed in phospholipid species PC and PE and in
glycolipid DGDG. Double bond index (DBI) was enhanced more than 2/​fold in fatty
acids of either glycolipids or phospholipids from cells submitted to 100 mM
NaCl. Free sterol content was also significantly enhanced, especially at 100 mM
NaCl, whereas free sterols/phospholipids (St/PL) ratio was slightly decreased.
All these salt/​induced changes in membrane lipids suggest an increase in
membrane fluidity of C. roseus cells.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15279997 [PubMed /​ indexed for MEDLINE]

80: Biotechnol Lett. 2004 May;26(10):793/​8.

Alkaloid accumulation in Catharanthus roseus cell suspension cultures fed with
stemmadenine.

El/​Sayed M, Choi YH, Frederich M, Roytrakul S, Verpoorte R.

Department of Pharmacognosy, Section of Metabolomics, Institute of Biology
Leiden, Leiden University, The Netherlands.

Feeding stemmadenine to Catharanthus roseus cell suspension culture resulted in
the accumulation of catharanthine, tabersonine and condylocarpine.
Condylocarpine is not an intermediate in the pathway to catharanthine or
tabersonine when it is fed to the cultures. The results support the hypothesis
that stemmadenine is an intermediate in the pathway to catharanthine and
tabersonine.

PMID: 15269549 [PubMed /​ indexed for MEDLINE]

81: Plant Cell Rep. 2004 Sep;23(3):148/​54. Epub 2004 Jun 25.

Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy
root clones in relation to left/​ and right/​termini/​linked Ri T/​DNA gene
integration.

Batra J, Dutta A, Singh D, Kumar S, Sen J.

National Centre for Plant Genome Research, Jawaharlal Nehru University Campus,
P.O. Box 10531, 110 067, New Delhi, India.

Hairy root cultures of Catharanthus roseus var. Prabal were established by
infecting the leaves with Agrobacterium rhizogenes agropine/​type A4 strain. Two
hundred and fifty independent root clones were evaluated for growth, morphology,
number of integration of Ri T/​DNA genes and alkaloid contents. On the basis of
growth pattern, type of branching and number of lateral roots we were able to
separate the hairy root clones into four categories. However based on the
integration of the Ri T(L)/​DNA and T(R)/​DNA genes, there were only three
different categories of independent hairy root clones/​C1 (rolA&B(+)/ags(+)), C2
(rolA&B(/​)/ags(+)) and C3 (rolA&B(+)/ags(/​)). Southern hybridization analysis
revealed both single and multiple copies of T/​DNA integration in the root
clones. The accumulation of considerable amounts of the root/​specific alkaloids
ajmalicine and serpentine was observed in the presence of both the T(L)/​DNA and
T(R)/​DNA genes (C1) and the T(L)/​DNA gene (C3) alone. Two rolA&B(/​) but ags(+)
clones (C2) accumulated much less or only very negligible amounts of ajmalicine.
The possible role of the T(L)/​DNA and T(R)/​DNA genes on growth and alkaloid
accumulation in these root clones is discussed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15221274 [PubMed /​ indexed for MEDLINE]

82: Adv Exp Med Biol. 2003;527:643/​6.

Novel bisindole derivatives of Catharanthus alkaloids with potential cytotoxic
properties.

Ruszkowska J, Chrobak R, Wrobel JT, Czarnocki Z.

Faculty of Chemistry, Warsaw University, L. Pasteur Str. 1, 02/​093 Warsaw,
Poland. ruszko@chem.uw.edu.pl

Pantropical plant Catharanthus roseus (L) G. Don is known as a source of
valuable bisindole alkaloids: vinblastine (VBL) and vincristine (VCR),
oncolytics widely used as sulfates in therapy of malignant diseases. They are
biosynthesized in the plant from monoindolic vindoline and catharanthine, both
derived from L/​tryptophan and loganine units. In the course of phytochemical
screening of this plant cultivated in Poland and considered as a home source of
VBL and VCR we developed a new isolation method based on the solid phase
extraction. Mild conditions used during the isolation procedure enabled the
isolation of some labile compounds and so the monomeric alkaloids with high
yields. Vindoline and so its congeners and subjected to various oxidative
conditions gave 15,15'/​bisindolic derivatives in quite good yield. Biological
activities of the above mentioned bisindolic compounds are under study.

PMID: 15206784 [PubMed /​ indexed for MEDLINE]

83: Biotechnol Lett. 2004 Apr;26(8):671/​4.

Vindoline synthesis in in vitro shoot cultures of Catharanthus roseus.

Hernandez/​Dominguez E, Campos/​Tamayo F, Vazquez/​Flota F.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Calle 43 No. 130 Chuburna 97200, Merida Yucatan, Mexico.

Vindoline, the major alkaloid in cultures of Catharanthus roseus shoots, reached
2 mg g(/​1) dry wt after 27 d in culture. Maximal vindoline accumulation
coincided with maximum activities of deacetoxyvindoline 4/​hydroxylase,
deacetylvindoline acetyl/​CoA acetyl transferase and tryptophan decarboxylase.
Shoot exposure to jasmonate shortened the time required for the maximal
vindoline accumulation to 14 d.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15200179 [PubMed /​ indexed for MEDLINE]

84: FEBS Lett. 2004 Jun 4;567(2/​3):197/​202.

Molecular cloning and characterization of a glucosyltransferase catalyzing
glucosylation of curcumin in cultured Catharanthus roseus cells.

Kaminaga Y, Sahin FP, Mizukami H.

Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho/​ku,
Nagoya 467/​8603, Japan.

Catharanthus roseus cell suspension cultures are capable of converting
exogenously supplied curcumin to various glucosides. The glucosylation
efficiency is enhanced by addition of methyl jasmonate (MJ) to the cultures
prior to curcumin administration. Two cDNAs encoding UDP/​glucosyltransferases
(CaUGT1 and CaUGT2) were isolated from a cDNA library of cultured C. roseus
cells, using a PCR method directed at the conserved UDP/​binding domain of plant
glycosyltransferases. The sequence identity between their deduced amino acid
sequences was 27%. The expression of both genes was up/​regulated by addition of
MJ to the cell cultures although the mRNA level of CaUGT1 was much lower than
that of CaUGT2. The corresponding cDNAs were expressed in Escherichia coli as
fusion proteins with maltose/​binding protein. The recombinant CaUGT1 exhibited
no glucosylation activity with either curcumin or curcumin monoglucoside as
substrate, whereas the recombinant CaUGT2 catalyzed the formation of curcumin
monoglucoside from curcumin and also conversion of curcumin monoglucoside to
curcumin diglucoside. The use of the recombinant CaUGT2 may provide a useful new
route for the production of curcumin glucosides.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15178322 [PubMed /​ indexed for MEDLINE]

85: Zhongguo Zhong Yao Za Zhi. 2003 May;28(5):385/​90.

[Industrialization of medicinal plant tissue culture]

[Article in Chinese]

Gao WY, Jia W, Duan HQ, Xiao PG.

College of Pharmaceutical Sciences and Technology, Tianjin University, Tianjin
300072, China. biochemgao@hotmail.com

Compared with other countries, the industrialization of medicinal plant tissue
culture is more important for our country because China is consuming and
exporting the most amounts of herb materials in the world. Each year, many
papers and patents are published on cell cultures of popular medicinal plants,
such as Taxus sp., Catharanthus roseus, and Panax ginseng, and, meanwhile, the
research on organ cultures of medicinal plants is increasing very quickly, which
is deepening the study of medicinal plant tissue culture. During the past 30
years, Chinese scientists have cultured many medicinal plant cells, organs and
hairy roots. In addition, the large/​scale cultures have been tested on medicinal
plants, such as Catharanthus roseus, Panax notoginseng, Anisodus acutangulus,
Lithospermum erythrorhizon, and Taxus chinensis. However, the bioreactor size is
not big enough for the commercial cultivation and we have not mastered the
culture technique on a large scale. We should clearly understand the importance
and great potential benefit of medicinal plant tissue culture and develop the
tissue culture techniques for the modernization of TCM. To develop the technique
that we have the property right, the pioneering spirit is needed in our
research, and, meanwhile, it should be pointed out emphatically the
collaboration is indispensable among scientists from different research fields.

Publication Types:
English Abstract

PMID: 15139118 [PubMed /​ indexed for MEDLINE]

86: Biotechnol Bioeng. 2004 Jun 20;86(6):718/​27.

Expression of a feedback/​resistant anthranilate synthase in Catharanthus roseus
hairy roots provides evidence for tight regulation of terpenoid indole alkaloid
levels.

Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY.

Department of Chemical Engineering and Bioengineering, Rice University, Houston,
Texas 77251/​1892, USA.

Different plant species produce a variety of terpenoid indole alkaloids, which
are of interest as plant defensive secondary metabolites and as valuable
pharmaceuticals. Although significant progress has been made, the mechanisms
regulating the levels of this important class of compounds require continued
elucidation. Previous precursor feeding studies have indicated that alkaloid
accumulation can be improved during the exponential growth phase of hairy root
cultures through enhanced tryptophan availability. To test this relationship,
transgenic hairy root cultures of Catharanthus roseus were established with a
glucocorticoid/​inducible promoter controlling the expression of an Arabidopsis
feedback/​resistant anthranilate synthase alpha subunit. Enzyme assays
demonstrated that the Arabidopsis alpha subunit is compatible with the native
beta subunit and that anthranilate synthase activity is more resistant to
tryptophan inhibition in induced than in uninduced extracts. The metabolic
effects of expressing the feedback/​resistant anthranilate synthase alpha subunit
were also dramatic. Over a 6/​day induction period during the late exponential
growth phase, tryptophan and tryptamine specific yields increased from almost
undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g
dry weight, respectively. The greater than 300/​fold increase in tryptophan
levels observed in these studies under certain induction conditions compares
favorably with the fold increases obtained in previous constitutive expression
studies. Despite the large increases in tryptophan and tryptamine, the levels of
most terpenoid indole alkaloids were not significantly altered, with the
exception of lochnericine, which increased 81% after a 3/​day induction period.
These results suggest that terpenoid indole alkaloid levels are tightly
controlled. Copyright 2004 Wiley Periodicals, Inc.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.
Research Support, U.S. Gov't, P.H.S.

PMID: 15137084 [PubMed /​ indexed for MEDLINE]

87: Phytochemistry. 2004 Apr;65(8):1085/​94.

Flavonoid methylation: a novel 4'/​O/​methyltransferase from Catharanthus roseus,
and evidence that partially methylated flavanones are substrates of four
different flavonoid dioxygenases.

Schroder G, Wehinger E, Lukacin R, Wellmann F, Seefelder W, Schwab W, Schroder
J.

Institut fur Biologie II, Universitat Freiburg, D/​79104 Freiburg, Germany.
Joachim.Schroeder@biologie.uni/​freiburg.de

Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation
pattern. Characteristic are B/​ring 5' and 3' methylations and a methylation in
the position 7 of the A/​ring. The first two can be explained by a previously
identified unusual O/​methyltransferase (CrOMT2) that performs two sequential
methylations. We used a homology based RT/​PCR strategy to search for cDNAs
encoding the enzyme for the A/​ring 7 position. Full/​length cDNAs for three
proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides
shared 59/​66% identity among each other, with CrOMT2, and with CrOMT4 (a
previously characterized protein of unknown function). The five proteins formed
a cluster separate from all other OMTs in a relationship tree. Analysis of the
genes showed that all C. roseus OMTs had a single intron in a conserved
position, and a survey of OMT genes in other plants revealed that this intron
was highly conserved in evolution. The three cDNAs were cloned for expression of
His/​tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7
could be purified by affinity chromatography. CrOMT7 was inactive with all
compounds tested. The only substrates found for CrOMT6 were
3'/​O/​methyl/​eriodictyol (homoeriodictyol) and the corresponding flavones and
flavonols. The mass spectrometric analysis showed that the enzyme was not the
expected 7OMT, but a B/​ring 4'OMT. OMTs with this specificity had not been
described before, and 3',4'/​dimethylated flavonoids had not been found so far in
C. roseus, but they are well/​known from other plants. The identification of this
enzyme activity raised the question whether methylation could be a part of the
mechanisms channeling flavonoid biosynthesis. We investigated four purified
recombinant 2/​oxoglutarate/​dependent flavonoid dioxygenases: flavanone
3beta/​hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin
synthase. 3'/​O/​Methyl/​eriodictyol was a substrate for all four enzymes. The
activities were only slightly lower than with the standard substrate naringenin,
and in some cases much higher than with eriodictyol. Methylation in the A/​ring,
however, strongly reduced or abolished the activities with all four enzymes. The
results suggested that B/​ring 3' methylation is no hindrance for flavonoid
dioxygenases. These results characterized a new type of flavonoid
O/​methyltransferase, and also provided new insights into the catalytic
capacities of key dioxygenases in flavonoid biosynthesis.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15110688 [PubMed /​ indexed for MEDLINE]

88: Plant Physiol Biochem. 2004 Jan;42(1):65/​72.

MAP kinase/​like activity in transformed Catharanthus roseus hairy roots varies
with culture conditions such as temperature and hypo/​osmotic shock.

Islas/​Flores I, Zuniga/​Aguilar JJ, Rodriguez/​Zapata LC, Carrillo/​Pech M,
Baizabal/​Aguirre VM, Minero/​Garcia Y, Hernandez/​Sotomayor SM.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Calle 43 #130, Col. Chuburna de Hidalgo, Merida, Yucatan
97200, Mexico. islasign@cicy.mx

Mitogen activated protein (MAP) kinase/​like activity was determined in extracts
obtained from transformed Catharanthus roseus hairy roots by the ability to
phosphorylate myelin basic protein (MBP). Both in solution and in gel kinase
assays showed variation in activity, depending on root developmental stage. In
gel kinase assays, using the extract soluble fraction, revealed a 56 kDa
polypeptide with phosphorylation activity on MBP. In addition, another 75 kDa
polypeptide was observed in the particulate fraction. Immunodetection with
monoclonal antibodies against ERK/​1, a mammalian MAP kinase, and with
anti/​phosphotyrosine antibodies cross/​reacted with the 56 kDa polypeptide, named
SMK56, from the soluble fraction, suggesting that this polypeptide could be
related with members of the MAP kinase family. Antibodies against the dually
phosphorylated threonine/​tyrosine motif, characteristic of active forms of MAP
kinases, also cross/​reacted with this 56 kDa polypeptide. Changes in the levels
of SMK56 were detected within the first 30 min of root exposure to low
temperatures or hypo/​osmotic shock, suggesting that this protein may be involved
in the perception of environmental changes.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15061086 [PubMed /​ indexed for MEDLINE]

89: Plant J. 2004 Apr;38(1):131/​41.

Co/​expression of three MEP pathway genes and geraniol 10/​hydroxylase in internal
phloem parenchyma of Catharanthus roseus implicates multicellular translocation
of intermediates during the biosynthesis of monoterpene indole alkaloids and
isoprenoid/​derived primary metabolites.

Burlat V, Oudin A, Courtois M, Rideau M, St/​Pierre B.

EA 2106, Plant Biocompounds and Biotechnology, UFR des Sciences et Techniques,
Universite de Tours, 37200 Tours, France.

In higher plants, isopentenyl diphosphate (IPP) is synthesised both from the
plastidic 2/​C/​methyl/​d/​erythritol 4/​phosphate (MEP) and from the cytosolic
mevalonate (MVA) pathways. Primary metabolites, such as phytol group of
chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and
gibberellins (GAs) are derived directly from the MEP pathway. Many secondary
metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus,
are also synthesised from this source of IPP. Using Northern blot and in situ
hybridisation experiments, we show that three MEP pathway genes
(1/​deoxy/​d/​xylulose 5/​phosphate synthase (DXS), 1/​deoxy/​d/​xylulose 5/​phosphate
reductoisomerase (DXR) and 2C/​methyl/​d/​erythritol 2,4/​cyclodiphosphate synthase
(MECS)) and the gene encoding geraniol 10/​hydroxylase (G10H), a cytochrome P450
monooxygenase involved in the first committed step in the formation of iridoid
monoterpenoids display identical cell/​specific expression patterns. The
co/​localisation of these four transcripts to internal phloem parenchyma of young
aerial organs of C. roseus adds a new level of complexity to the multicellular
nature of MIA biosynthesis. We predict the translocation of pathway
intermediates from the internal phloem parenchyma to the epidermis and,
ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the
translocation of intermediates from the phloem parenchyma is probably also
required during the biosynthesis of hormones and photosynthetic primary
metabolites derived from the MEP pathway.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15053766 [PubMed /​ indexed for MEDLINE]

90: Physiol Plant. 2004 Jan;120(1):140/​151.

Interaction of spermine with a signal transduction pathway involving
phospholipase C, during the growth of Catharanthus roseus transformed roots.

Echevarria/​Machado I, Ku/​Gonzalez A, Loyola/​Vargas VM, Hernandez/​Sotomayor SM.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan. Calle 43 #130, Colonia Chuburna de Hidalgo. c.p 97200,
Merida, Yucatan, Mexico.

In Cantharanthus roseus transformed roots, the application of methylglyoxal
bis(guanylhydrazone) (MGBG), an inhibitor of S/​adenosylmethionine decarboxylase
(SAMDC; EC 4.1.1.50), inhibited the root growth in a dose/​dependent manner with
a DL(50) of about 300 micro m. Spermidine and spermine (Spm) levels and SAMDC
and phospholipase C (PLC; EC 3.1.4.3) activities were reduced in the presence of
the inhibitor. The inhibition was reversed by the addition of Spm. Radioactivity
from [(14)C]Spm was detected in an immunoprecipitated fraction with an antibody
anti/​PLC/​delta. To our knowledge, this is the first direct evidence that
demonstrates an interaction of Spm with the signal transduction cascade
phosphoinositide/​Ca(2+).

PMID: 15032886 [PubMed /​ as supplied by publisher]

91: Curr Med Chem. 2004 Mar;11(5):607/​28.

The Catharanthus alkaloids: pharmacognosy and biotechnology.

van Der Heijden R, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Gorlaeus
Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
heijden@lacdr.leidenuniv.nl

The Catharanthus (or Vinca) alkaloids comprise a group of about 130 terpenoid
indole alkaloids. Vinblastine is now marketed for more than 40 years as an
anticancer drug and became a true lead compound for drug development. Due to the
pharmaceutical importance and the low content in the plant of vinblastine and
the related alkaloid vincristine, Catharanthus roseus became one of the
best/​studied medicinal plants. Consequently it developed as a model system for
biotechnological studies on plant secondary metabolism. The aim of this review
is to acquaint a broader audience with the recent progress in this research and
with its exciting perspectives. The pharmacognostical aspects of the
Catharanthus alkaloids cover botanical (including some historical),
phytochemical and analytical data. An up/​to/​date view on the biosynthesis of the
alkaloids is given. The pharmacological aspects of these alkaloids and their
semi/​synthetic derivatives are only discussed briefly. The biotechnological part
focuses on alternative production systems for these alkaloids, for example by in
vitro culture of C. roseus cells. Subsequently it will be discussed to what
extent the alkaloid biosynthetic pathway can be manipulated genetically
("metabolic engineering"), aiming at higher production levels of the alkaloids.
Another approach is to produce the alkaloids (or their precursors) in other
organisms such as yeast. Despite the availability of only a limited number of
biosynthetic genes, the research on C. roseus has already led to a broad
scientific spin/​off. It is clear that many interesting results can be expected
when more genes become available.

Publication Types:
Research Support, Non/​U.S. Gov't
Review

PMID: 15032608 [PubMed /​ indexed for MEDLINE]

92: Life Sci. 2004 Apr 2;74(20):2467/​78.

Angiogenesis and anti/​angiogenesis activity of Chinese medicinal herbal
extracts.

Wang S, Zheng Z, Weng Y, Yu Y, Zhang D, Fan W, Dai R, Hu Z.

Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese
Medicine, Shanghai 201203, China.

The aqueous extracts of 24 herbs traditionally used as curing ischemic heart
disease in clinic in China were screened for their in vitro angiogenic activity,
another twenty/​four traditionally used as anti/​tumor or anti/​inflammatory
remedies in China were screened for their in vitro anti/​angiogenic activity. The
activity of angiogenesis was determined by quantitation of vessels on chick
embryo chorioallantoic membrane (CAM) model and cell proliferation of cultured
bovine aortic endothelial cells (BAECs). Among the herbal extracts examined, the
aqueous extracts of Epimedium sagittatum, Trichosanthes kirilowii and Dalbergia
odorifera showed the strong angiogenetic activity both in CAM and BAECs models;
and the aqueous extracts of Berberis paraspecta, Catharanthus roseus, Coptis
chinensis, Taxus chinensis, Scutellaria baicalensis, Polygonum cuspidatum and
Scrophularia ningpoensis elicited significant inhibition at a concentration of
1g dry herb /ml.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15010258 [PubMed /​ indexed for MEDLINE]

93: J Asian Nat Prod Res. 2004 Jun;6(2):93/​7.

Biotransformation of triptolide and triptonide by cell suspension cultures of
Catharanthus roseus.

Ning LL, Han J, Zhang XY, Guo HZ, Bi KS, Guo DA.

The State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing 100083, China.

Catharanthus roseus cell suspension cultures were used to bioconvert both
triptolide (1) and triptonide (2). The same reaction path was followed in both
biotransformations. Two biotransformed products were obtained and their
structures identified as triptriolide (3) and 12beta,13alpha/​dihydroxytriptonide
(4), respectively, from 1 and 2. Product 4 is a new compound.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 15008455 [PubMed /​ indexed for MEDLINE]

94: Protoplasma. 2004 Mar;223(1):45/​51. Epub 2004 Mar 4.

Catharanthus roseus L. plants and explants infected with phytoplasmas: alkaloid
production and structural observations.

Favali MA, Musetti R, Benvenuti S, Bianchi A, Pressacco L.

Dipartimento di Biologia Evolutiva e Funzionale, Universita degli Studi di
Parma, Parco Area delle Scienze 11A, 43100 Parma, Italy. favali@biol.unipr.it

The results of several experiments concerning the presence and composition of
alkaloids in different tissues (stems, leaves, roots) of Catharanthus roseus L.
plants and explants, healthy and infected by clover phyllody phytoplasmas, are
reported. The alkaloids extracted and determined by the reverse phase
high/​pressure liquid chromatography were vindoline, ajmalicine, serpentine,
vinblastine, and vincristine. The total alkaloid concentration was higher in
infected plants than in the controls, in particular the increase of vinblastine
in infected roots was very significant. The ultrastructural observations of
infected roots showed alterations of the cell walls and of the nuclei. These
results demonstrate that phytoplasmas, detected in all infected tissues by light
fluorescence and transmission electron microscopy, play an important role on
secondary metabolism of the diseased plants, modifying both the total content of
alkaloids and their ratio.

PMID: 15004742 [PubMed /​ indexed for MEDLINE]

95: Plant Cell Rep. 2004 Jun;22(11):828/​31. Epub 2004 Feb 13.

Plant regeneration from hairy/​root cultures transformed by infection with
Agrobacterium rhizogenes in Catharanthus roseus.

Choi PS, Kim YD, Choi KM, Chung HJ, Choi DW, Liu JR.

Laboratory of Functional Genomics for Plant Secondary Metabolism (National
Research Laboratory), Eugentech Inc., P.O. Box 115, Yuseong, 305/​333 Daejeon,
Korea.

Hypocotyl explants of Catharanthus roseus produced hairy roots when cultured on
Murashige and Skoog (MS) basal medium after infection by Agrobacterium
rhizogenes. Explants gave rise to adventitious shoots at a frequency of up to
80% when cultured on MS medium supplemented with 31.1 microM 6/​benzyladenine and
5.4 microM alpha/​naphthaleneacetic acid. There was a significant difference in
the frequency of adventitious shoot formation for each hairy/​root line derived
from a different cultivar. Plants derived from hairy roots exhibited prolific
rooting and had shortened internodes. Approximately half of the plants had
wrinkled leaves and an abundant root mass with extensive lateral branching, but
otherwise appeared morphologically normal. Plants with hairy roots that were
derived from the cultivar Cooler Apricot developed flowers with petals that were
white in the proximal region, whereas the wild/​type flower petals are red. PCR
and Southern blot analyses revealed that plants derived from hairy roots
retained the Ri TL/​DNA. Copyright 2004 Springer/​Verlag

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 14963692 [PubMed /​ indexed for MEDLINE]

96: FEBS Lett. 2004 Jan 30;558(1/​3):85/​8.

Histidine/​containing phosphotransfer domain extinction by RNA interference turns
off a cytokinin signalling circuitry in Catharanthus roseus suspension cells.

Papon N, Vansiri A, Gantet P, Chenieux JC, Rideau M, Creche J.

EA 2106, Plant Molecular Biology and Biochemistry Department, Faculty of
Pharmacy, 31 avenue Monge, F/​37200 Tours, France.

We previously reported that cytokinins (CK) induce the fast and specific
transcription of CrRR1, a gene encoding a type A response regulator in
Catharanthus roseus cell cultures. Here, we characterized the CrHPt1 gene that
encodes a histidine/​containing phosphotransfer domain. CrHPt1 was silenced
through RNA interference (RNAi) to test its possible implication in the CK
signalling pathway. In transgenic lines stably transformed with an
intron/​spliced construct, the degradation of CrHPt1 transcripts abolishes the CK
inductive effect on CrRR1 transcription. These result give a new in vivo
functional argument for the crucial role of HPt proteins in the CK signalling
pathway leading to the expression of the genes encoding type A response
regulators. They also show that RNAi is a powerful strategy to turn off the CK
signalling circuitry.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 14759521 [PubMed /​ indexed for MEDLINE]

97: Micron. 2003;34(8):387/​93.

Cytochemical localization of calcium and X/​ray microanalysis of Catharanthus
roseus L. infected with phytoplasmas.

Musetti R, Favali MA.

Dipartimento di Biologia Applicata alla Difesa delle Piante, Universita di
Udine, Via delle Scienze 208, Udine 33100, Italy. rita.musetti@pdef.uniud.it

The potassium pyroantimonate (KPA) Ca(2+) precipitation technique, X/​ray
microanalysis and Electron Energy Loss Spectroscopy carried out by transmission
electron microscopy were used to analyze the Ca(2+) distribution in Catharanthus
roseus L. leaves infected with phytoplasmas belonging to different taxonomic
groups, and in phytoplasma cells. The analysis revealed that the distribution of
Ca(2+) was different in healthy and diseased plants (where the KPA deposits were
numerous) and no differences were observed in the tissues of the three types of
infected C. roseus L. Since no KPA precipitates were visible in the phloem and
on phytoplasma cells, it is likely that Ca(2+) ions are not directly involved in
phytoplasma replication, but, in infected cells is a response to the pathogen
indicative of a higher Ca(2+) in the plasmalemma.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 14680925 [PubMed /​ indexed for MEDLINE]

98: FEBS Lett. 2003 Dec 4;555(2):311/​6.

Production of unnatural glucosides of curcumin with drastically enhanced water
solubility by cell suspension cultures of Catharanthus roseus.

Kaminaga Y, Nagatsu A, Akiyama T, Sugimoto N, Yamazaki T, Maitani T, Mizukami H.

Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe/​dori
3/​1, Mizuho/​ku, Nagoya 467/​8603, Japan.

Catharanthus roseus cell suspension cultures converted exogenously supplied
curcumin to a series of glucosides, none of which has been found in nature so
far. The efficiency of glucosylation was dependent on culture stage of the cells
and medium sucrose concentration. Methyl jasmonate and salicylic acid enhanced
the glucoside formation only when they were added to the cultures prior to the
addition of curcumin. The glucoside yield was 2.5 micromol/g fresh weight of the
cells at an optimal culture condition. The water solubility of
curcumin/​4',4"/​O/​beta/​D/​digentiobioside was 0.65 mmol/ml, which was 20
million/​fold higher than that of curcumin.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 14644434 [PubMed /​ indexed for MEDLINE]

99: Biotechnol Lett. 2003 Oct;25(20):1687/​93.

Production of cell wall accumulative enzymes using immobilized protoplasts of
Catharanthus roseus in agarose gel.

Mera N, Aoyagi H, DiCosmo F, Tanaka H.

Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki
305/​8572, Japan.

Catharanthus roseus cells and protoplasts were used for production of peroxidase
and alpha/​galactosidase which are accumulated in the cell wall. Only 4% (0.026 U
ml(/​1)) of the total peroxidase was secreted into the broth by cultured cells
while in cultured protoplasts, 45% (0.12 U ml(/​1)) was secreted. Protoplasts
were protected against the physical and osmotic stresses by immobilizing them in
3% agarose gel (high mass transfer, non/​electric charge, low gelation
temperature). In order to increase peroxidase production, the immobilized
protoplasts were cultivated in shake cultures at low osmotic pressure (12.3 to
16.4 atm) without disruption. During batch peroxidase production, the total
activities obtained with free cells at 4.9 atm, free protoplasts at 19.3 atm,
and immobilized protoplasts at 12.3 atm were 0.17, 2.54, and 5.16 U,
respectively. When four repeated/​batch cultures of the immobilized protoplasts
were performed at 16.4 atm, 11.8 U of peroxidase was obtained. This system was
also useful for the production of alpha/​galactosidase.

Publication Types:
Comparative Study
Evaluation Studies
Research Support, Non/​U.S. Gov't

PMID: 14626409 [PubMed /​ indexed for MEDLINE]

100: Biotechnol Lett. 2003 Aug;25(16):1345/​9.

Increase in the indole alkaloid production and its excretion into the culture
medium by calcium antagonists in Catharanthus roseus hairy roots.

Moreno/​Valenzuela OA, Minero/​Garcia Y, Chan W, Mayer/​Geraldo E, Carbajal E,
Loyola/​Vargas VM.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientificas de Yucatan, Calle 43 No. 130, Col. Chuburna de Hidalgo, CP 97200,
Merida, Yucatan, Mexico.

Treatment of Catharanthus roseus hairy roots with antagonists, like verapamil
and CdCl2, that block the Ca2+ flux across the plasma membrane enhanced the
total alkaloid content by 25% and their secretion 10 times. The specific Ca2+
chelator, EGTA, stimulated 90% of the total alkaloid secretion. Treatment with
inhibitors of intracellular Ca2/​ movement, like TMB/​8 and trapsigargin, enhanced
the total alkaloid content by 74% and their secretion into the culture media by
4/​ to 6/​fold. The results suggest that an inhibition of external and internal
Ca2+ fluxes induces an increase in the indole alkaloid accumulation and
secretion in C. roseus hairy roots.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 14514063 [PubMed /​ indexed for MEDLINE]

101: Protoplasma. 2003 Sep;222(1/​2):97/​105.

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of
alpha/​3',4'/​anhydrovinblastine: a specific role for a multifunctional enzyme.

Sottomayor M, Ros Barcelo A.

Department of Botany, Faculty of Sciences, and Institute for Molecular and Cell
Biology, University of Porto, Porto, Portugal. msottoma@ibmc.up.pt

We have characterized a basic peroxidase with alpha/​3',4'/​anhydrovinblastine
(AVLB) synthase activity, which was purified from Catharanthus roseus leaves.
This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves,
and the single AVLB synthase activity detected in C. roseus extracts. It was
observed that the monomeric substrates of AVLB, vindoline and catharanthine, are
both suitable electron donors for the oxidizing intermediates of the basic
peroxidase, compounds I and II. Results also showed that the reaction proceeds
by a radical/​propagated mechanism. Substrate specificity studies of the enzyme
revealed that it was also able to oxidize several common peroxidase substrates,
indicating a broad range of substrate specificity that is characteristic of
class III plant peroxidases. Cytochemical studies showed that the enzyme is
localized in C. roseus mesophyll vacuoles, in individual spots at the inner
surface of the tonoplast. This particular location suggests a meaningful spatial
organization that led to the proposal of a metabolic channeling model for the
peroxidase/​mediated synthesis of AVLB. The importance of this type of mechanism
in the regulation of peroxidase isoenzyme functions in vivo is discussed. In
view of the results obtained it is concluded that the basic peroxidase present
in C. roseus leaves fulfills all the requirements to be considered as an AVLB
synthase, and it is proposed that this specific function of this multifunctional
enzyme is determined by metabolic channeling resulting from specific
protein/​protein interactions.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 14513315 [PubMed /​ indexed for MEDLINE]

102: J Exp Bot. 2003 Nov;54(392):2587/​8. Epub 2003 Sep 9.

CrMYC1, a Catharanthus roseus elicitor/​ and jasmonate/​responsive bHLH
transcription factor that binds the G/​box element of the strictosidine synthase
gene promoter.

Chatel G, Montiel G, Pre M, Memelink J, Thiersault M, Saint/​Pierre B, Doireau P,
Gantet P.

EA 2106, Biomolecules et Biotechologies Vegetale, Universite de Tours, UFR des
Sciences et Techniques, Laboratoire de Physiologie Vegetale, France.

A cDNA encoding a bHLH transcription factor was isolated by the yeast one/​hybrid
system from a Catharanthus roseus cDNA library using the G/​box element of the
Strictosidine synthase gene promoter as bait. The corresponding protein (named
CrMYC1) was shown to bind specifically to the G/​box in yeast. In C. roseus
suspension cells CrMYC1 mRNA levels are induced by fungal elicitor and jasmonate
suggesting that CrMYC1 may be involved in the regulation of gene expression in
response to these signals.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12966042 [PubMed /​ indexed for MEDLINE]

103: BMC Complement Altern Med. 2003 Sep 2;3:4. Epub 2003 Sep 2.

The juice of fresh leaves of Catharanthus roseus Linn. reduces blood glucose in
normal and alloxan diabetic rabbits.

Nammi S, Boini MK, Lodagala SD, Behara RB.

Pharmacology Division, Department of Pharmaceutical Sciences, Andhra University,
Visakhapatnam/​530 003, Andhra Pradesh, India. nammi@rediffmail.com

BACKGROUND: The leaf juice or water decoction of Catharanthus roseus L.
(Apocyanaceae) is used as a folk medicine for the treatment of diabetes all over
the world. In the present investigation, the leaf juice of C. roseus has been
evaluated for its hypoglycemic activity in normal and alloxan/​induced diabetic
rabbits. METHODS: The blood glucose lowering activity of the leaf juice was
studied in normal and alloxan/​induced (100 mg/kg, i.v.) diabetic rabbits, after
oral administration at doses of 0.5, 0.75 and 1.0 ml/kg body weight. Blood
samples were collected from the marginal ear vein before and also at 4, 6, 8,
10, 12, 16, 18, 20 & 24 h after drug administration and blood glucose was
analyzed by Nelson/​Somogyi's method using a visible spectrophotometer. The data
was compared statistically by using Student's t/​test. RESULTS: The leaf juice of
C. roseus produced dose/​dependent reduction in blood glucose of both normal and
diabetic rabbits and comparable with that of the standard drug, glibenclamide.
The results indicate a prolonged action in reduction of blood glucose by C.
roseus and the mode of action of the active compound(s) of C. roseus is probably
mediated through enhance secretion of insulin from the beta/​cells of Langerhans
or through extrapancreatic mechanism. CONCLUSIONS: The present study clearly
indicated a significant antidiabetic activity with the leaf juice of
Catharanthus roseus and supports the traditional usage of the fresh leaves by
Ayurvedic physicians for the control of diabetes.

Publication Types:
Comparative Study

PMID: 12950994 [PubMed /​ indexed for MEDLINE]

104: Phytochemistry. 2003 Sep;64(2):401/​9.

Jasmonate/​induced epoxidation of tabersonine by a cytochrome P/​450 in hairy root
cultures of Catharanthus roseus.

Rodriguez S, Compagnon V, Crouch NP, St/​Pierre B, De Luca V.

Service de l'environnement et de l'energie, Les Croisettes, CP 33 1066
Epalinges, Switzerland.

Methyl jasmonate, a chemical inducer of secondary metabolism, was shown to
promote tabersonine 2 biosynthesis in hairy root cultures of Catharanthus
roseus. Tabersonine 6,7/​epoxidase activity was detected in total protein extract
of jasmonate/​induced hairy root cultures using labeled 14C/​tabersonine 2. This
enzyme converted tabersonine 2 to lochnericine 3 by selective epoxidation at
positions 6 and 7 via a reaction dependent on NADPH and molecular oxygen. Carbon
monoxide, clotrimazole, miconazole, and cytochrome C were shown to be strong
inhibitors of the enzyme. The activity was found in microsomes, indicating that
tabersonine 6,7/​epoxidase was a cytochrome P/​450/​dependent monooxygenase.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12943756 [PubMed /​ indexed for MEDLINE]

105: J Exp Bot. 2003 Jul;54(388):1793/​5.

Differential expression of two type/​A response regulators in plants and cell
cultures of Catharanthus roseus (L.) G. Don.

Papon N, Oudin A, Vansiri A, Rideau M, Chenieux JC, Creche J.

EA 2106, Biomolecules et Biotechnologies Vegetales, Universite de Tours, 31
Avenue Monge, F/​37200 Tours, France.

Two full/​length cDNAs named CrRR2 and CrRR3 have been isolated by PCR from a C.
roseus cDNA library. The first one encodes a 154 amino acid putative protein
with a high percentage of identity with the Arabidopsis thaliana response
regulators ARR16 and ARR17, and is not expressed in C. roseus organs and cell
cultures. The second one encodes a 188 amino acid ORF sharing the highest
homologies with the A. thaliana ARR8 and ARR9 response regulators. Its
expression is root/​specific and the transcripts are transiently up/​regulated
after trans/​zeatin treatment in C. roseus suspension cells. CrRR3 protein might
be involved in the cytokinin/​enhanced alkaloid production in C. roseus cell
cultures.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12810857 [PubMed /​ indexed for MEDLINE]

106: Drug News Perspect. 2001 Oct;14(8):465/​82.

Erratum in:
Drug News Perspect. 2001 Nov;14(9):534.

Natural products of plant origin as anticancer agents.

Ram VJ, Kumari S.

Medicinal Chemistry Division, Central Drug Research Institute, Lucknow, India.

Natural products have been used as effective remedies for the treatment of
various ailments. Numerous plant products in the form of decoction, tincture,
tablets and capsules have been clinically used for the treatment of different
kinds of cancer. This review covers some of the important plants with clinically
proven anticancer activity, including Catharanthus roseus, Podophyllum peltatum,
Taxus brevifolia, Camptothecin acuminata, Cephalotaxus harringtonia, Viscum
album, Onchrosia elliptica, Annona bullata, Asmina triloba and Rhizoma
zedoariae. Synthetic analogues in some cases have also been prepared to improve
the efficacy and decrease the side effects of parent compounds. The modes of
action of clinically used drugs are also delineated. (c) 2001 Prous Science. All
rights reserved.

PMID: 12806432 [PubMed]

107: Biotechnol Prog. 2003 May/​Jun;19(3):1105/​8.

Role of the non/​mevalonate pathway in indole alkaloid production by Catharanthus
roseus hairy roots.

Hong SB, Hughes EH, Shanks JV, San KY, Gibson SI.

Department of Biochemistry and Cell Biology, Rice University, MS/​140, 6100 Main
St., Houston, Texas 77005, USA.

The 1/​deoxy/​D/​xylulose/​5/​phosphate (DXP) pathway (non/​mevalonate pathway)
leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur
in most bacteria and in all higher plants. Treatment with the antibiotic
fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably
inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and
lochnericine by Catharanthus roseus hairy root cultures in the exponential
growth phase. However, fosmidomycin did not significantly affect alkaloid levels
in stationary phase hairy root cultures. Feeding with 1/​deoxy/​D/​xylulose,
10/​hydroxygeraniol, or loganin resulted in significant increases in alkaloid
production by exponential phase hairy root cultures. These results suggest that
the DXP pathway is a major provider of carbon for the monoterpenoid pathway
leading to the formation of indole alkaloids in C. roseus hairy roots in the
exponential phase.

Publication Types:
Comparative Study

PMID: 12790690 [PubMed /​ indexed for MEDLINE]

108: Biotechnol Prog. 2003 May/​Jun;19(3):1071/​5.

Studies on production of ajmalicine in shake flasks by multiple shoot cultures
of Catharanthus roseus.

Satdive RK, Fulzele DP, Eapen S.

Plant Biotechnology and Secondary Products Section, Nuclear Agriculture and
Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085,
India.

The effects of different concentrations of indole/​3/​acetic acid (IAA) and
benzyladenine (BA) on production of ajmalicine by multiple shoot cultures of
Catharanthus roseus (C. roseus) were studied. By supplementing Murashige and
Skoog's (MS) medium with a high concentration of IAA (11.42 microM) and a low
concentration of BA (2.22 microM), shoot cultures accumulated high levels of
ajmalicine. When culture medium was fortified with a low concentration of IAA
(2.85 microM) and a high concentration of BA (8.90 microM), shoots released high
levels of ajmalicine into the culture medium. Quantification of ajmalicine was
performed by high performance liquid chromatography (HPLC). The highest
concentration of ajmalicine production (0.166% dry wt) was obtained by shoot
cultures grown in MS medium containing IAA (11.42 microM) on 20 days of
cultivation. Shoot cultures accumulated ajmalicine 4.2/​fold more in IAA (11.42
microM) supplemented medium compared with the high concentration of BA (8.90
microM). The content of ajmalicine concentration in the medium was quantified.
Shoot cultures grown in BA (8.90 microM) supplemented medium released the
maximum production of ajmalicine (0.853 g/L) into the culture medium after 15
days of cultivation. The experimental data show that the secretion of ajmalicine
was 2/​fold more into the culture medium supplemented with a high concentration
of BA compared to that with a low concentration of BA. Data presented here show
that production of ajmalicine by shoot cultures is not correlated with growth
rate. Dimeric indole alkaloids vincristine and vinblastine were not present in
shoot cultures. Ajmalicine production by shoot cultures was 2.4/​fold higher
compared to leaves of 1/​year/​old naturally grown plants.

Publication Types:
Comparative Study
Evaluation Studies

PMID: 12790683 [PubMed /​ indexed for MEDLINE]

109: Toxicol Lett. 2003 Jul 20;143(2):195/​207.

Screening of medicinal plants used in South African traditional medicine for
genotoxic effects.

Elgorashi EE, Taylor JL, Maes A, van Staden J, De Kimpe N, Verschaeve L.

Division of Environmental Toxicology, Flemish Institute for Technological
Research (VITO), Boeretang 200, B/​2400, Mol, Belgium.

Dichloromethane and 90% methanol extracts from 51 South African medicinal plants
were evaluated for potential genotoxic effects using the bacterial Ames and
VITOTOX tests with and without metabolic activation. Dichloromethane extracts
from bulbs of Crinum macowanii showed mutagenicity in strain TA98 with and
without metabolic activation, whereas extracts from leaves of Chaetacme aristata
and foliage of Plumbago auriculata showed mutagenicity and/or toxicity. Extracts
from the leaves of Catharanthus roseus and twigs of Combretum mkhzense were
mutagenic with metabolic activation only. The only 90% methanol extracts that
were mutagenic in strain TA98 were from the leaves of C. roseus and Ziziphus
mucronata in the presence of metabolic activation. No genotoxic effects were
found in strain TA100 or in the VITOTOX test.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12749823 [PubMed /​ indexed for MEDLINE]

110: Rev Neurol. 2003 May 1/​15;36(9):860/​71.

[Neurological syndromes linked with the intake of plants and fungi containing a
toxic component (I). Neurotoxic syndromes caused by the ingestion of plants,
seeds and fruits]

[Article in Spanish]

Carod/​Artal FJ.

Servicio de Neurologia, Hospital Sarah, Brasilia DF, Brasil. javier@bsb.sarah.br

INTRODUCTION: A wide range of plants, seeds and fruits used for nutritional and
medicinal purposes can give rise to neurotoxic symptoms. DEVELOPMENT: We review
the neurological pathology associated with the acute or chronic consumption of
plants, seeds and fruits in human beings and in animals. Of the plants that can
trigger acute neurotoxic syndromes in humans, some of the most notable include
Mandragora officinalis, Datura stramonium, Conium maculatum (hemlock), Coriaria
myrtifolia (redoul), Ricinus communis, Gloriosa superba, Catharanthus roseus,
Karwinskia humboldtiana and Podophyllum pelatum. We also survey different
neurological syndromes linked with the ingestion of vegetable foodstuffs that
are rich in cyanogenic glycosides, Jamaican vomiting sickness caused by Blighia
sapida, Parkinson dementia ALS of Guam island and exposition to Cycas
circinalis, Guadeloupean parkinsonism and exposition to Annonaceae, konzo caused
by ingestion of wild manioc and neurolathyrism from ingestion of Lathyrus
sativus, the last two being models of motor neurone disease. Locoism is a
chronic disease that develops in livestock feeding on plants belonging to
Astragalus and Oxytropis sp., Sida carpinifolia and Ipomea carnea, which are
rich in swainsonine, a toxin that inhibits the enzyme alpha mannosidase and
induces a cerebellar syndrome. CONCLUSIONS: The ingestion of neurotoxic seeds,
fruits and plants included in the diet and acute poisoning by certain plants can
give rise to different neurological syndromes, some of which are irreversible.

Publication Types:
English Abstract
Review

PMID: 12717675 [PubMed /​ indexed for MEDLINE]

111: Biochem J. 2003 Jul 1;373(Pt 1):261/​9.

Molecular characterization of recombinant T1, a non/​allergenic periwinkle
(Catharanthus roseus) protein, with sequence similarity to the Bet v 1 plant
allergen family.

Laffer S, Hamdi S, Lupinek C, Sperr WR, Valent P, Verdino P, Keller W, Grote M,
Hoffmann/​Sommergruber K, Scheiner O, Kraft D, Rideau M, Valenta R.

Department of Pathophysiology, University of Vienna, Wahringer Gurtel 18/​20,
A/​1090 Vienna, Austria.

More than 25% of the population suffer from Type I allergy, an IgE/​mediated
hypersensitivity disease. Allergens with homology to the major birch ( Betula
verrucosa ) pollen allergen, Bet v 1, belong to the most potent elicitors of
IgE/​mediated allergies. T1, a cytokinin/​inducible cytoplasmic periwinkle (
Catharanthus roseus ) protein, with significant sequence similarity to members
of the Bet v 1 plant allergen family, was expressed in Escherichia coli.
Recombinant T1 (rT1) did not react with IgE antibodies from allergic patients,
and failed to induce basophil histamine release and immediate/​type skin
reactions in Bet v 1/​allergic patients. Antibodies raised against purified rT1
could be used for in situ localization of natural T1 by immunogold electron
microscopy, but did not cross/​react with most of the Bet v 1/​related allergens.
CD analysis showed significant differences regarding secondary structure and
thermal denaturation behaviour between rT1 and recombinant Bet v 1, suggesting
that these structural differences are responsible for the different
allergenicity of the proteins. T1 represents a non/​allergenic member of the Bet
v 1 family that may be used to study structural requirements of allergenicity
and to engineer hypo/​allergenic plants by replacing Bet v 1/​related allergens
for primary prevention of allergy.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 12656672 [PubMed /​ indexed for MEDLINE]

112: Fitoterapia. 2003 Feb;74(1/​2):62/​7.

Production of anthocyanins by Catharanthus roseus.

Filippini R, Caniato R, Piovan A, Cappelletti EM.

Dipartimento di Biologia, Via U. Bassi 58/B, Padova I/​35131, Italy.
raffilip@civ.bio.unipd.it

A stable cell suspension line of Catharanthus roseus producing anthocyanin was
obtained. In this strain it was found that approximately 30% of cells regularly
accumulated these metabolites and that anthocyanin accumulation occurred between
the second half of log phase and the stationary phase of the culture growth
cycle. The anthocyanins in the suspension cultures were compared with those
biosynthetized in the flowers both of regenerated by somatic embryogenesis and
field/​grown plants. Six anthocyanins were identified in all the examined
samples, three 3/​O/​glucosides and three 3/​O/​(6/​O/​p/​coumaroyl) glucosides of
petunidin, malvidin and hirsutidin. The hirsutidin coumaroyl glucoside has not
been reported previously, and was predominat in all samples. The anthocyanin
relative content was similar for cell suspensions and flowers from regenerated
plants but different from field/​grown plant flowers; instead, the total content
was almost the same for the two flower types and higher compared to suspension
culture content.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12628396 [PubMed /​ indexed for MEDLINE]

113: Phytochemistry. 2003 Mar;62(5):715/​21.

Phytotoxicity of the tetramic acid metabolite trichosetin.

Marfori EC, Kajiyama S, Fukusaki E, Kobayashi A.

Department of Biotechnology, Graduate School of Engineering, Osaka University,
2/​1 Yamada/​oka, Suita/​shi, Osaka 565/​0871, Japan.

Trichosetin, a tetramic acid/​containing metabolite produced in the dual culture
of Trichoderma harzianum and Catharanthus roseus (L.) G. Don callus, was
subjected to phytotoxicity assays. In seedling growth assays, trichosetin
inhibited root and shoot growth of all five plant species tested by damaging the
cell membrane, as evidenced by the dose/​dependent increase in electrolyte
leakage and lipid peroxidation. Vital staining of trichosetin/​treated Nicotiana
tabacum BY/​2 cells, with rhodamine 123, showed a weaker green fluorescence
compared to controls indicating damaging effects on mitochondria. FDA/​PI
staining, to determine cell viability, indicated that cells of the
trichosetin/​treated roots were mostly dead.

PMID: 12620323 [PubMed /​ indexed for MEDLINE]

114: Metab Eng. 2002 Jul;4(3):257/​62.

Quantification of metabolic flux in plant secondary metabolism by a biogenetic
organizational approach.

Morgan JA, Shanks JV.

Department of Bioengineering, Rice University, 6100 S. Main St., Houston, Texas
77005/​1892, USA.

Metabolic engineering represents a promising approach to enhance the yield of
valuable natural products from plants. A method to quantify flux through
metabolite measurements is necessary for the analysis of native and modified
pathways. Rather than focusing only on the accumulation of the final products,
analyzing a wide range of secondary metabolites has significant advantages. We
propose a model that organizes the flux analysis by grouping metabolites of
similar biosynthetic origin. To this end, we have quantified temporal profiles
of metabolites from several branches of the indole alkaloid pathway in
Catharanthus roseus hairy root cultures. By analyzing these data, we are able to
examine the distribution of flux around key branchpoints. Furthermore, this
analysis provides crucial information such as an estimate of total flux to
secondary metabolism.

Publication Types:
Comparative Study
Research Support, U.S. Gov't, Non/​P.H.S.
Research Support, U.S. Gov't, P.H.S.

PMID: 12616695 [PubMed /​ indexed for MEDLINE]

115: Mol Biotechnol. 2003 Jan;23(1):11/​8.

Immunocytolocalization of tryptophan decarboxylase in Catharanthus roseus hairy
roots.

Moreno/​Valenzuela OA, Minero/​Garcia Y, Brito/​Argaez L, Carbajal/​Mora E,
Echeverria O, Vazquez/​Nin G, Loyola/​Vargas VM.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de investigacion
Cientifica de Yucatan, Calle 43, numero 130, CP 97200, Merida, Yucatan, Mexico.

We investigated the intracellular distribution of tryptophan decarboxylase (TDC)
(EC 4.1.1.28) in Catharanthus roseus hairy roots using immunofluorescence and
immunogold techniques. TDC was detected by immunofluorescence localization in
the cytosol and in the apoplastic region of the meristematic cells of the roots,
with a slight enrichment in the epidermal cells of the root cap and in the
meristematic region. In the enlargement zone, TDC was localized only in the
first three layers of the cortex. In the maturation zone, the enzyme was not
present. Immunogold studies confirmed that the enzyme was localized in the
cytosol of the meristematic region, and intense gold labeling was found in the
apoplastic zone. A protein fraction isolated from the apoplastic zone and
assayed for TDC activity showed high activity.

PMID: 12611265 [PubMed /​ indexed for MEDLINE]

116: FEBS Lett. 2003 Feb 27;537(1/​3):101/​5.

Inhibition of the plant cytokinin transduction pathway by bacterial histidine
kinase inhibitors in Catharanthus roseus cell cultures.

Papon N, Clastre M, Gantet P, Rideau M, Chenieux JC, Creche J.

Plant Molecular Biology and Biochemistry Department, EA 2106, Plant Biocompounds
and Biotechnology, Faculty of Pharmacy, Universite de Tours, 31 avenue Monge,
France.

We describe the isolation of two Catharanthus roseus cDNAs encoding proteins
putatively involved in the final steps of a 'histidine/​to/​aspartate'
phosphorelay in cytokinin (CK) signaling. The expression of one of these genes,
CrRR1, was specifically up/​regulated by CKs in C. roseus cell suspensions. We
used this system as a biological model to test the activity of bacterial
histidine kinase inhibitors. Our data demonstrate that these inhibitors are
active on the CK transduction pathway and represent powerful chemical tools to
study hormone signal transduction in plants. Moreover, these data suggest a
strong conservation of functional features between prokaryotic and plant
signaling pathways utilizing histidine kinases.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 12606039 [PubMed /​ indexed for MEDLINE]

117: Theor Appl Genet. 2003 Jan;106(2):221/​30. Epub 2002 Sep 13.

Salt/​tolerant mutants in glycophytic salinity response (GSR) genes in
Catharanthus roseus.

Rai SP, Luthra R, Kumar S.

Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow/​226 015,
India.

The periwinkle Catharanthus roseus shares glycophytic properties of crop plants.
To contribute towards an understanding of the glycophytic response to salinity,
large populations of M(2) seeds having an origin in nitroso/​methyl urea and
ethyl methane sulphonate treatments were screened for germination with 250 mM of
NaCl. Out of the nine mutant lines so recovered, which tolerated salt stress due
to loss of the normal glycophytic salinity response ( GSR), the characteristics
of six gsr mutants are reported here. All six, gsr/​1 to gsr/​6, differed from the
wild/​type in both seedling and adult/​plant morphological characters beside being
salt tolerant. The mutations in them were inherited as monogenic recessive
alleles at the corresponding wild/​type loci. The trans/​complementation tests
revealed that the gsr/​1 to gsr/​6 mutants specified six complementation groups.
The mutant seedlings generally accumulated more proline and glycine betaine,
constitutively, than the wild/​type. The mutant plants transpired lower amounts
of water and accumulated higher amounts of proline under drought stress. It was
inferred that the products of the six GSR genes defined here are involved in the
regulation of salt stress, as well as cell division, developmental and/or
morphogenetic pathway(s), in C. roseus.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 12582847 [PubMed /​ indexed for MEDLINE]

118: Planta Med. 2002 Dec;68(12):1113/​7.

Biotransformation of 2alpha,5alpha,10beta,14beta/​tetraacetoxy/​4(20),11/​taxadiene
by cell suspension cultures of Catharanthus roseus.

Dai J, Cui Y, Zhu W, Guo H, Ye M, Hu Q, Zhang D, Zheng J, Guo D.

The State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing 100083, P.R.China.

Catharanthus roseus cell suspension cultures were employed for the
biotransformation of 2alpha,5alpha,10beta,14beta/​tetraacetoxy/​4(20),11/​taxadiene
( 1), and four metabolites were obtained. Based on their physical and chemical
data, the structures of the four metabolites were respectively elucidated as
10beta/​hydroxy/​2alpha,5alpha,14beta/​triacetoxy/​4(20),11/​taxadiene (2),
5alpha/​hydroxy/​2alpha,10beta, 14beta/​triacetoxy/​4(20),11/​taxadiene (3),
6alpha,10beta/​dihydroxy/​2alpha,5alpha,14beta/​triacetoxy/​4(20),11/​taxadiene (4),
and
6alpha,9alpha,10beta/​trihydroxy/​2alpha,5alpha,14beta/​triacetoxy/​4(20),11/​taxadie
ne (5), among which 3 and 5 were characterized as new taxoids. The effects of
the stages of substrate addition on the biotransformation were also
investigated. The results revealed that the biotransformation rate for 1 reached
85.3 % and the yield of 2 70 % when 1 was administered during the mid/​linear
phase (9 /​ 12 th day) of the cell growth cycle. On the other hand, the yield for
4 reached the highest level of 11.8 % when 1 was added in the early linear phase
(6 th day).

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12494340 [PubMed /​ indexed for MEDLINE]

119: Phytochemistry. 2003 Jan;62(2):127/​37.

Erratum in:
Phytochemistry. 2003 May;63(2):237.

A flavonol O/​methyltransferase from Catharanthus roseus performing two
sequential methylations.

Cacace S, Schroder G, Wehinger E, Strack D, Schmidt J, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Schanzlestr. 1, D/​79104
Freiburg, Germany.

Protein extracts from dark/​grown cell suspension cultures of Catharanthus roseus
(Madagascar periwinkle) contained several O/​methyltransferase (OMT) activities,
including the 16/​hydroxytabersonine O/​methyltransferase (16HT/​OMT) in indole
alkaloid biosynthesis. This enzyme was enriched through several purification
steps, including affinity chromatography on adenosine agarose. SDS/​PAGE of the
purified protein preparation revealed a protein band at the size expected for
plant OMTs (38/​43 kDa). Mass spectrometry indicated two dominant protein species
of similar mass in this band, and sequences of tryptic peptides showed
similarities to known OMTs. Homology/​based RT/​PCR identified cDNAs for four new
OMTs. Two of these cDNAs (CrOMT2 and CrOMT4) encoded the proteins dominant in
the preparation enriched for 16HT/​OMT. The proteins were closely related (73%
identity), but both shared only 48/​53% identity with the closest relatives found
in the public databases. The enzyme functions were investigated with purified
recombinant proteins after cDNA expression in Escherichia coli. Unexpectedly,
both proteins had no detectable 16HT/​OMT activity, and CrOMT4 was inactive with
all substrates investigated. CrOMT2 was identified as a flavonoid OMT that was
expressed in dark/​grown cell cultures and copurified with 16HT/​OMT. It
represented a new type of OMT that performs two sequential methylations at the
3'/​ and 5'/​positions of the B/​ring in myricetin (flavonol) and dihydromyricetin
(dihydroflavonol). The resulting methylation pattern is characteristic for C.
roseus flavonol glycosides and anthocyanins, and it is proposed that CrOMT2 is
involved in their biosynthesis.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12482447 [PubMed /​ indexed for MEDLINE]

120: Biotechnol Prog. 2002 Nov/​Dec;18(6):1183/​6.

Characterization of an inducible promoter system in Catharanthus roseus hairy
roots.

Hughes EH, Hong SB, Shanks JV, San KY, Gibson SI.

Department of Chemical Engineering, Rice University, PO Box 1892, Houston, Texas
77251, USA.

Transgenic hairy root cultures of Catharanthus roseus were established with a
glucocorticoid/​inducible promoter controlling the expression of green
fluorescent protein (GFP), and GFP expression was characterized. The inducible
system shows a tightly controlled, reversible, and dosage/​dependent response to
the glucocorticoid dexamethasone in C. roseus hairy roots. Full induction was
noted after 12/​18 h in the mature regions of the root tips and after 6 h in the
meristem tissue. Upon removal of the inducing agent, GFP expression declined to
undetectable levels in the mature tissues after 24 h and in the meristem after
48 h. Although no dosage/​dependent response was noted in the meristem region,
such a response was apparent in the mature region of the tip and verified by
quantitative GFP analysis. The inducible promoter system allowed quantitative
control of GFP expression between 0.01 and 10 microM dexamethasone with
saturation occurring at higher levels. Using GFP as a model system allowed
demonstration of the ability to control temporal and quantitative gene
expression with the glucocorticoid/​inducible promoter in transgenic C. roseus
hairy roots.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.
Research Support, U.S. Gov't, P.H.S.

PMID: 12467449 [PubMed /​ indexed for MEDLINE]

121: Trends Pharmacol Sci. 2002 Dec;23(12):563/​9.

Transcription factors: tools to engineer the production of pharmacologically
active plant metabolites.

Gantet P, Memelink J.

Universite de Tours, EA 2106, Biomolecules et Biotechnologies Vegetales, UFR des
Sciences et Techniques, Laboratoire de Physiologie Vegetale, Parc de Grandmont,
France. gantet@univ/​tours.fr

Plants produce a variety of secondary metabolites, some of which are used as
pharmaceuticals or are health promoting as food components. Recent genetic
studies on the flavonoid biosynthetic pathway show that transcription factors
are efficient new molecular tools for plant metabolic engineering to increase
the production of valuable compounds. The use of specific transcription factors
would avoid the time/​consuming step of acquiring knowledge about all enzymatic
steps of a poorly characterized biosynthetic pathway. Although genetic
approaches are difficult for most plant species, promoter studies of
single/​pathway genes and T/​DNA activation tagging are feasible alternative
approaches for isolating transcription factors, as illustrated for terpenoid
indole alkaloid biosynthesis in Catharanthus roseus.

Publication Types:
Research Support, Non/​U.S. Gov't
Review

PMID: 12457774 [PubMed /​ indexed for MEDLINE]

122: J Biotechnol. 2003 Jan 9;100(1):13/​22.

Plant/​cell bioreactors with simultaneous electropermeabilization and
electrophoresis.

Yang RY, Bayraktar O, Pu HT.

Bioreaction Engineering Laboratory, Department of Chemical Engineering, West
Virginia University, Morgantown 26506/​6102, USA. ryang@mail.wvu.edu

Experimental investigations on using low/​level electric currents and voltages to
extract, transport, and collect intracellular secondary metabolites from plant
cells while maintaining their viabilities were conducted focusing on the
production of: (1) ionic betalains, mainly negatively/​charged betanin, from Beta
vulgaris cells, and (2) ionic alkaloids, particularly positively/​charged
ajmalicine and yohimbine, from Catharanthus roseus cells. Three versions of
tubular membrane reactors in which electropermeabilization of cell membranes and
electrophoresis and diffusion of ionic products take place simultaneously, with
or without convective flow, to achieve desirable extraction were developed.
Concentrations of secondary metabolites produced from these plant/​cell reactors
under steady and oscillatory electrical forcings were recorded and the
viabilities of treated cells examined. Oscillatory application of electrical
field appears to produce more products while retaining higher cell viability.

Publication Types:
Comparative Study
Evaluation Studies
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.
Validation Studies

PMID: 12413782 [PubMed /​ indexed for MEDLINE]

123: Plant Physiol. 2002 Oct;130(2):930/​9.

An early C/​22 oxidation branch in the brassinosteroid biosynthetic pathway.

Fujioka S, Takatsuto S, Yoshida S.

RIKEN (The Institute of Physical and Chemical Research), Wako/​shi, Saitama
351/​0198, Japan. sfujioka@postman.riken.go.jp

The natural occurrence of 22/​hydroxylated steroids in cultured Catharanthus
roseus cells and in Arabidopsis seedlings was investigated. Using full/​scan gas
chromatography/​mass spectrometry analysis, (22S)/​22/​hydroxycampesterol
(22/​OHCR), (22S,24R)/​22/​hydroxyergost/​4/​en/​3/​one (22/​OH/​4/​en/​3/​one),
(22S,24R)/​22/​hydroxy/​5alpha/​ergostan/​3/​one (22/​OH/​3/​one), 6/​deoxocathasterone
(6/​deoxoCT), 3/​epi/​6/​deoxoCT, 28/​nor/​22/​OHCR, 28/​nor/​22/​OH/​4/​en/​3/​one,
28/​nor/​22/​OH/​3/​one, 28/​nor/​6/​deoxoCT, and 3/​epi/​28/​nor/​6/​deoxoCT were
identified. Metabolic experiments with deuterium/​labeled 22/​OHCR were performed
in cultured C. roseus cells and Arabidopsis seedlings (wild type and det2), and
the metabolites were analyzed by gas chromatography/​mass spectrometry. In both
C. roseus cells and wild/​type Arabidopsis seedlings, [(2)H(6)]22/​OH/​4/​en/​3/​one,
[(2)H(6)]22/​OH/​3/​one, [(2)H(6)]6/​deoxoCT, and [(2)H(6)]3/​epi/​6/​deoxoCT were
identified as metabolites of [(2)H(6)]22/​OHCR, whereas the major metabolite in
det2 seedlings was [(2)H(6)]22/​OH/​4/​en/​3/​one. Analysis of endogenous levels of
these brassinosteroids revealed that det2 accumulates 22/​OH/​4/​en/​3/​one. The
levels of downstream compounds were remarkably reduced compared with the wild
type. Exogenously applied 22/​OH/​3/​one and 6/​deoxoCT were found to rescue det2
mutant phenotypes, whereas 22/​OHCR and 22/​OH/​4/​en/​3/​one did not. These results
substantiate the existence of a new subpathway (22/​OHCR /​/​> 22/​OH/​4/​en/​3/​one /​/​>
22/​OH/​3/​one /​/​> 6/​deoxoCT) and reveal that the det2 mutant is defective in the
conversion of 22/​OH/​4/​en/​3/​one to 22/​OH/​3/​one, which leads to brassinolide
biosynthesis.

Publication Types:
Comparative Study

PMID: 12376657 [PubMed /​ indexed for MEDLINE]

124: Biotechnol Prog. 2002 Sep/​Oct;18(5):1003/​9.

Continuous selective extraction of secondary metabolites from Catharanthus
roseus hairy roots with silicon oil in a two/​liquid/​phase bioreactor.

Tikhomiroff C, Allais S, Klvana M, Hisiger S, Jolicoeur M.

Bio/​P(2) Research Unit, Department of Chemical Engineering, Ecole Polytechnique
de Montreal, PO Box 6079, Centre/​ville Station, Montreal, Quebec, Canada H3C
3A7.

A two/​liquid/​phase bioreactor was designed to extract indole alkaloids from
Catharanthus roseus hairy roots with silicon oil. Partition studies between
silicon oil and culture medium showed that the silicon oil did not alter the
availability of nutrients. The affinity of tabersonine and lochnericine for
silicon oil is nine times higher than for the aqueous phase. Cultures were
elicited with 25 mg/L of jasmonic acid. The growth of the hairy roots was not
significantly modified by the presence of silicon oil. The overall specific
yields of tabersonine and lochnericine were increased by 100/​400% and 14/​200%,
respectively, with the use of silicon oil in nonelicited control cultures. In
elicited cultures, these values were 10/​55% for tabersonine and 20/​65% for
lochnericine. Serpentine was never found in the silicon oil. All measured
alkaloids' specific yields were higher using silicon oil and elicitation,
suggesting that the silicon oil, while acting as a metabolic sink for
tabersonine and lochnericine, was efficient in increasing metabolic fluxes of
the secondary metabolism pathways.

Publication Types:
Comparative Study
Evaluation Studies
Research Support, Non/​U.S. Gov't

PMID: 12363351 [PubMed /​ indexed for MEDLINE]

125: Mol Biotechnol. 2002 Sep;22(1):1/​8.

Vindoline biosynthesis is transcriptionally blocked in Catharanthus roseus cell
suspension cultures.

Vazquez/​Flota F, De Luca V, Carrillo/​Pech M, Canto/​Flick A, de Lourdes
Miranda/​Ham M.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Merida Yucatan, Mexico. felipe@cicy.mx

Catharanthus roseus cell cultures were exposed to different conditions in order
to induce alkaloid metabolism. The exposure to jasmonate and fungal elicitors
resulted in the transcriptional activation of tryptophan decarboxylase and in
the accumulation of the monoterpenoid indole alkaloids ajmalicine and
catharanthine, but not of vindoline. The inability of the cell cultures to
produce vindoline was related to a lack of expression of the desacetoxyvindoline
4/​hydroxylase (D4h) gene. Southern blot analysis revealed that D4h gene was not
lost in the cell cultures.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 12353909 [PubMed /​ indexed for MEDLINE]

126: Chem Pharm Bull (Tokyo). 2002 Sep;50(9):1294/​6.

Supercritical fluid extraction and liquid chromatography/​electrospray mass
analysis of vinblastine from Catharanthus roseus.

Choi YH, Yoo KP, Kim J.

GreenTek21 Co., Ltd., Mapo/​gu, Seoul, Korea.

Supercritical fluid extraction using carbon dioxide modified with methanol,
methanol/​diethylamine, or methanol/​triethylamine was used to extract vinblastine
from the aerial portions of Catharanthus roseus. An HPLC/​electrospray ionization
(ESI)/MS analysis method was also developed to quantify the alkaloids in these
extracts. Of the supercritical solvents evaluated, carbon
dioxide/​methanol/​triethylamine (80 : 18 : 2) at 80 degrees C and 34.0 MPa
greatly improved the supercritical fluid extraction (SFE) yield of vinblastine
by as much as 76.4% over methanol extraction, while the other solvent conditions
extracted the compound at yields less than 25% that of a methanol extraction.
These results were confirmed by the robust HPLC/​ESI/MS analytical method
developed in this study.

PMID: 12237558 [PubMed /​ indexed for MEDLINE]

127: J Exp Bot. 2002 Sep;53(376):1989/​90.

Expression analysis in plant and cell suspensions of CrCKR1, a cDNA encoding a
histidine kinase receptor homologue in Catharanthus roseus (L.) G. Don.

Papon N, Clastre M, Andreu F, Gantet P, Rideau M, Creche J.

EA 2106, Biomolecules et Biotechnologies Vegetales, Universite de Tours, 31
Avenue Monge, F/​37200 Tours, France.

A full length cDNA (CrCKR1) encoding a hybrid histidine kinase was isolated from
a Catharanthus roseus cDNA library. The kinase belongs to the subfamily of
cytokinin receptors represented by CRE1/AHK4/WOL in Arabidopsis thaliana. In
cell suspensions, the expression of CrCKR1 is not affected by various stress and
hormonal treatments but is stimulated in cells continuously exposed to
cytokinin. In plants, CrCKR1 is strongly expressed only in the petals of mature
flowers. These data suggest that CrCKR1 could take part in the mechanisms
leading to the production of secondary metabolites in C. roseus.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12177139 [PubMed /​ indexed for MEDLINE]

128: Z Naturforsch [C]. 2002 May/​Jun;57(5/​6):465/​70.

Trichosetin, a novel tetramic acid antibiotic produced in dual culture of
Trichoderma harzianum and Catharanthus roseus Callus.

Marfori EC, Kajiyama S, Fukusaki E, Kobayashi A.

Department of Biotechnology, Graduate School of Engineering, Osaka University,
Japan.

The dual culture of Trichoderma harzianum and Catharanthus roseus callus
produced an antimicrobial compound with a remarkable activity against the
Gram/​positive bacteria Staphylococcus aureus and Bacillus subtilis. Structural
elucidation revealed that this compound, which we have named trichosetin, is a
novel tetramic acid (2,4/​pyrrolidinedione) antibiotic and a homolog of the
fungal metabolite equisetin. This compound however, was not produced in the
individual culture of T. harzianum or C. roseus callus.

Publication Types:
Comparative Study

PMID: 12132686 [PubMed /​ indexed for MEDLINE]

129: Biotechnol Bioeng. 2002 Aug 20;79(4):408/​15.

The effect of ajmalicine spiking and resin addition timing on the production of
indole alkaloids from Catharanthus roseus cell cultures.

Lee/​Parsons CW, Shuler ML.

School of Chemical Engineering, Cornell University, 120 Olin Hall, Ithaca, New
York 14853/​5201, USA.

The potential for the feedback inhibition of indole alkaloid synthesis was
investigated by spiking suspension cultures of Catharanthus roseus with 0, 9, or
18 mg/L ajmalicine on day 0. The production of ajmalicine, catharanthine, and
serpentine were inhibited in a dose/​dependent manner. The inhibition was
transient as the exogenous ajmalicine was ultimately either metabolized in the
medium or within the cell. The addition of neutral resin has previously been
shown to enhance ajmalicine production. To minimize product inhibition and
product metabolism, Amberlite XAD/​7 resin was added to immobilized cultures of
C. roseus starting on either day 0, 5, or 15, and fresh resin was exchanged for
spent resin every 5 days. The addition of resin did not decrease the viability
of the culture. Growth was reduced only in cultures with resin added on day 0.
Alkaloid production was enhanced to different extents by the timing of resin
addition, suggesting that feedback inhibition or product metabolism was present
throughout the culture period. Ajmalicine recovery was nearly 100% when the
resin was added initially either on day 0 or day 5. Ajmalicine recovery was
reduced to 55% when the resin was added later in the culture period starting on
day 15, presumably because of resin saturation or the inaccessibility of
alkaloids trapped in the vacuole. Delaying the addition of XAD/​7 resin until 5
days after the start of the culture resulted in the highest improvement in
ajmalicine production, i.e approximately 70% and also resulted in the complete
recovery of ajmalicine from the cell. Copyright 2002 Wiley Periodicals, Inc.

Publication Types:
Comparative Study
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 12115404 [PubMed /​ indexed for MEDLINE]

130: Mol Biotechnol. 2002 Jul;21(3):211/​6.

Tryptophan decarboxylase from transformed roots of Catharanthus roseus.

Islas/​Flores I, Moreno/​Valenzuela O, Minero/​Garcia Y, Loyola/​Vargas VM,
Miranda/​Ham Mde L.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Mexico.

Tryptophan decarboxylase (TDC, EC 4.1.1.28) from Catharanthus roseus hairy roots
was purified 80/​fold. Antibodies against TDC were obtained and they recognized
only one protein of 55 kDa in crude extracts from hairy root cultures.
Elicitation of transformed root cultures with macerozyme yielded a marked
increase in TDC activity, which was accompanied by a similar increase in the
amount of immunoreactive TDC protein. These results suggest that the alkaloid
accumulation, produced by elicitation, requires the synthesis of new TDC
polypeptide in C. roseus root cultures and establishes important differences in
the regulatory control of this enzyme in root cultures compared to developing
seedlings, where the posttranslational regulation apparently plays a major role.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12102544 [PubMed /​ indexed for MEDLINE]

131: Biol Pharm Bull. 2002 Jun;25(6):753/​60.

Antiproliferative activity of Vietnamese medicinal plants.

Ueda JY, Tezuka Y, Banskota AH, Le Tran Q, Tran QK, Harimaya Y, Saiki I, Kadota
S.

Institute of Natural Medicine, Toyama Medical and Pharmaceutical University,
Japan.

Methanol, methanol/​water (1:1) and water extracts were prepared from
seventy/​seven Vietnamese medicinal plants and tested for their antiproliferative
activities against human HT/​1080 fibrosarcoma cells. Among them, fifteen
extracts including seven methanol extracts of Caesalpinia sappan, Catharanthus
roseus, Coscinium fenestratum, Eurycoma longifolia, Hydnophytum formicarum and
Streptocaulon juventas (collected at two areas), six methanol/​water (1:1)
extracts of Cae. sappan, Cat. roseus, Co. fenestratum, H. formicarum and S.
juventas (at two areas), and two water extracts of Cae. sappan and S. juventas
exhibited antiproliferative activities in a concentration/​dependent manner.
Their antiproliferative activities against human cervix HeLa adenocarcinoma,
human lung A549 adenocarcinoma, murine colon 26/​L5 carcinoma, murine Lewis lung
carcinoma (LLC) and murine B16/​BL6 melanoma cells were then examined. Co.
fenestratum showed selective activity against lung carcinoma and/or lung
metastatic cell lines, A549, LLC and B16/​BL6, while H. formicarum and S.
juventas showed selective activity against human tumor cell lines, HeLa and
A549. Characteristic morphological change and DNA fragmentation indicated the
antiproliferative activity to be due to the induction of apoptosis.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12081142 [PubMed /​ indexed for MEDLINE]

132: J Chromatogr A. 2002 Apr 26;955(1):87/​93.

Screening of Catharanthus roseus secondary metabolites by high/​performance
liquid chromatography.

Tikhomiroff C, Jolicoeur M.

Department of Chemical Engineering, Ecole Polytechnique de Montreal, Quebec,
Canada.

Two direct HPLC analytical methods for the screening of the major indole
alkaloids of Catharanthus roseus hairy roots and their iridoid precursors have
been developed. Photodiode array and fluorescence detection were performed. The
separation was achieved on a reversed/​phase C18 column. The first method allowed
the separation of catharanthine, serpentine, tabersonine, vindoline,
vinblastine, and vincristine in 20 min. Ajmalicine, tryptophan, tryptamine and
secologanine were separated using the second method in 13 min. The
identification of the compounds was based on the retention time and the
comparison of UV spectra with those of authentic standards. A simplified
alkaloid extraction method was developed in order to accelerate sample
preparation. The assays were successfully used to quantify major compounds of
the secondary metabolism of hairy root cultures of C. roseus, thus providing a
reliable tool for rapid screening of C. roseus secondary metabolite samples. In
these cultures, ajmalicine, serpentine, catharanthine, tabersonine, and
tryptamine were detected, but tryptophan, vindoline, vinblastine and vincristine
were not.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12061566 [PubMed /​ indexed for MEDLINE]

133: J Biotechnol. 2002 Jun 26;96(2):193/​203.

Effect of precursor feeding on alkaloid accumulation by a tryptophan
decarboxylase over/​expressing transgenic cell line T22 of Catharanthus roseus.

Whitmer S, van der Heijden R, Verpoorte R.

Gorlaeus Laboratories, Division of Pharmacognosy, LACDR, Leiden/Amsterdam Center
for Drug Research, Leiden University, Einsteinweg 55, PO Box 9502, 2300 RA
Leiden, The Netherlands.

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA)
biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly
to identify possible rate limiting steps, a transgenic cell line over/​expressing
tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was
fed with various amounts of precursors (tryptophan, tryptamine, loganin and
secologanin) in different time schedules and analyzed for TIA production. When
these precursors were added to this culture it was found that the optimal time
for supplying the precursors was at inoculation of the cells into the production
medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or
secologanin; however, the secologanin feeding was less effective. Tryptamine or
tryptophan alone had no effect on TIA accumulation. The over/​expression of Tdc
causes this cell line to produce quite large quantities of alkaloids after
feeding loganin or secologanin. However, in combination with tryptophan or
tryptamine, feeding of these precursors resulted in an even further increase of
alkaloid accumulation and under optimal conditions line T22 accumulated around
1200 micromol l(/​1) of TIAs whereas the control cultures accumulated less than
10 micromol l(/​1) TIAs.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 12039535 [PubMed /​ indexed for MEDLINE]

134: Phytochemistry. 2002 Jun;60(3):275/​9.

Biosynthesis of cholestanol in higher plants.

Nakajima N, Fujioka S, Tanaka T, Takatsuto S, Yoshida S.

RIKEN (The Institute of Physical and Chemical Research), Wako/​shi, Saitama
351/​0198, Japan.

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic
experiments were performed with Arabidopsis thaliana and Nicotiana tabacum
seedlings, and with cultured Catharanthus roseus cells. [26,
28/​2H(6)]Campestanol, [26/​2H(3)]cholesterol, and [26/​2H(3)]cholestanol were
administered to each plant, and the resulting metabolites were analyzed by gas
chromatography/​mass spectrometry. In all the species examined,
[2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and
[2H(3)]cholest/​4/​en/​3/​one and [2H(3)]cholestanol were identified as metabolites
of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was
biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28)
sterol). It was also demonstrated that cholestanol was converted to
6/​oxocholestanol, and campestanol was converted to 6/​oxocampestanol.

PMID: 12031446 [PubMed /​ indexed for MEDLINE]

135: J Hered. 2002 Jan/​Feb;93(1):55/​8.

Inheritance of flower color in periwinkle: orange/​red corolla and white eye.

Sreevalli Y, Kulkarni RN, Baskaran K.

Central Institute of Medicinal and Aromatic Plants, Field Station, Allalasandra,
Bangalore 560 065, India.

The commonly found flower colors in periwinkle (Catharanthus roseus)/​/​pink,
white, red/​eyed, and pale pink center/​/​are reported to be governed by the
epistatic interaction between four genes/​/​A, R, W, and I. The mode of
inheritance of an uncommon flower color, orange/​red corolla and white eye, was
studied by crossing an accession possessing this corolla color with a white
flowered variety (Nirmal). The phenotype of the F(1) plants and segregation data
of F(2) and backcross generations suggested the involvement of two more
interacting and independently inherited genes, one (proposed symbol E)
determining the presence or absence of red eye and another (proposed symbol O)
determining orange/​red corolla.

PMID: 12011178 [PubMed /​ indexed for MEDLINE]

136: Appl Biochem Biotechnol. 2002 Feb;97(2):135/​45.

Overexpression in Catharanthus roseus hairy roots of a truncated hamster
3/​hydroxy/​3/​methylglutaryl/​CoA reductase gene.

Ayora/​Talavera T, Chappell J, Lozoya/​Gloria E, Loyola/​Vargas VM.

Unidad de Bioquimica y Biologia Molecular de Plantas, Centro de Investigacion
Cientifica de Yucatan, Merida Yucatan, Mexico.

Catharanthus roseus (L.) G. Don hairy roots harboring hamster
3/​hydroxy/​3/​methylglutaryl/​CoA reductase (HMGR) (EC 1.1.1.88) cDNA without
membrane/​binding domain were evaluated by quantifying the levels of sterols and
some indol/​alkaloids. Clone 236, with the highest hybridization signal, had the
lowest soluble and microsomal HMGR activity and produced more ajmalicine and
catharanthine than the control but had reduced campesterol concentration. Clone
19, with low hybridization signal, had high soluble HMGR activity and produced
high levels of campesterol and five to seven times more serpentine than the
control but a low level of ajmalicine and no accumulation of catharanthine.
These results suggest a possible role for HMGR in indole alkaloid biosynthesis
and a possible cosuppression of both the endogenous and foreign HMGR genes in
clone 236.

PMID: 11996224 [PubMed /​ indexed for MEDLINE]

137: Eur J Biochem. 2002 Apr;269(8):2204/​13.

Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase,
a biosynthetic key to over 2000 monoterpenoid indole alkaloids.

Gerasimenko I, Sheludko Y, Ma X, Stockigt J.

Lehrstuhl fur Pharmazeutische Biologie, Institut fur Pharmazie, Johannes
Gutenberg/​Universitat Mainz, Germany.

Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in
the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on
the comparison of cDNA sequences of SG from Catharanthus roseus and
raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT/​PCR
were designed and the cDNA encoding SG was cloned from R. serpentina cell
suspension cultures. The active enzyme was expressed in Escherichia coli and
purified to homogeneity. Analysis of its deduced amino/​acid sequence assigned
the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the
SG from C. roseus, the enzyme from R. serpentina is predicted to lack an
uncleavable N/​terminal signal sequence, which is believed to direct proteins to
the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic
parameters and substrate specificity of the heterologously expressed SG were
studied and compared to those of the C. roseus enzyme, revealing some
differences between the two glucosidases. In vitro deglucosylation of
strictosidine by R. serpentina SG proceeds by the same mechanism as has been
shown for the C. roseus enzyme preparation. The reaction gives rise to the end
product cathenamine and involves 4,21/​dehydrocorynantheine aldehyde as an
intermediate. The enzymatic hydrolysis of dolichantoside
(Nbeta/​methylstrictosidine) leads to several products. One of them was
identified as a new compound, 3/​isocorreantine A. From the data it can be
concluded that the divergence of the biosynthetic pathways leading to different
classes of indole alkaloids formed in R. serpentina and C. roseus cell
suspension cultures occurs at a later stage than strictosidine deglucosylation.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11985599 [PubMed /​ indexed for MEDLINE]

138: Proteomics. 2001 Nov;1(11):1345/​50.

Sequential solubilization of proteins precipitated with trichloroacetic acid in
acetone from cultured Catharanthus roseus cells yields 52% more spots after
two/​dimensional electrophoresis.

Jacobs DI, van Rijssen MS, van der Heijden R, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center of Drug Research, The
Netherlands. d.jacobs@lacdr.leidenuniv.nl

Sample preparation is still the most critical step in two/​dimensional gel
electrophoresis (2/​DE), and needs to be optimized for each type of sample. To
analyze the proteome of the medicinal plant Catharanthus roseus, we developed
and evaluated a sequential solubilization procedure for the solubilization of
proteins after precipitation in trichloroacetic acid and acetone. The procedure
includes solubilization with a conventional urea buffer followed by a stronger
solubilizing buffer containing thiourea. The sequential solubilization of the
precipitated proteins results in very different spot patterns following 2/​DE.
The number of protein spots which could be detected in both samples of the
sequential solubilization was only about 10% of the total number of spots.
Compared to solubilization in a single step, the total number of spots that
could be detected in the sequential solubilization procedure was increased by
52%. The method described is simple and is applicable to different types of
plant tissue.

PMID: 11922593 [PubMed /​ indexed for MEDLINE]

139: Microsc Res Tech. 2002 Mar 15;56(6):462/​4.

Application of immunoelectron microscopy techniques in the diagnosis of
phytoplasma diseases.

Musetti R, Loi N, Carraro L, Ermacora P.

Dipartimento di Biologia Applicata alla Difesa delle Piante, Universita di
Udine, 33100 Udine, Italy. Rita.Musetti@pldef.uniud.it

An immunoelectron microscopy technique was applied to label Chrysanthemum
leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum
leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal
antibodies at different dilutions and secondary antimouse antibody conjugated
with colloidal gold particles of different sizes were used. The monoclonal
antibodies demonstrated their specificity against the antigen; immunocytological
methods permitted the precise localization and identification of phytoplasmas in
thin sections from infected tissues. Copyright 2002 Wiley/​Liss, Inc.

Publication Types:
Evaluation Studies

PMID: 11921348 [PubMed /​ indexed for MEDLINE]

140: Biotechnol Prog. 2002 Jan/​Feb;18(1):159/​62.

Influence of fungal elicitors on production of ajmalicine by cell cultures of
Catharanthus roseus.

Namdeo A, Patil S, Fulzele DP.

Pharma/​Biotech Division, Kabra Drugs Ltd., 26, Sector A, Sanwer Road, Indore 421
305, India.

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal
cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F.
moniliforme), and Trichoderma viride (T. viride). The effects of elicitor
dosage, exposures time, and age of subculture on ajmalicine accumulation were
studied. A higher concentration of elicitor extract responded positively to C.
roseus suspension cultures. Ajmalicine accumulation increased by about 3/​fold
when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum
ajmalicine production (75 microg g(/​1) dry weight (DW)) was observed in cells
treated with T. viride. Cell cultures were elicited with 5% preparation of A.
niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for
elicitation. Suspension cultures elicited with T. viride for 48 h showed a
3/​fold increase (87 microg g(/​1) DW) in ajmalicine contents, whereas A. niger
and F. moniliforme synthesized a 2/​fold increase in alkaloid and yielded 52 and
56 microg g(/​1) DW ajmalicine, respectively. C. roseus cells of different age
(5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F.
moniliforme, and T. viride and investigated elicitors activity at different age
of cell cultures. Maximum yield 166 microg g(/​1) DW of ajmalicine was
synthesized in 20 day old suspension cultures treated with T. viride. A longer
period of incubation of cell cultures with elicitors adversely affected the
ajmalicine synthesis.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 11822914 [PubMed /​ indexed for MEDLINE]

141: Transgenic Res. 2001 Dec;10(6):513/​21.

T/​DNA activation tagging as a tool to isolate regulators of a metabolic pathway
from a genetically non/​tractable plant species.

van der Fits L, Hilliou F, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
The Netherlands.

T/​DNA activation tagging is a method used to generate dominant mutations in
plants or plant cells by the insertion of a T/​DNA which carries constitutive
enhancer elements that can cause transcriptional activation of flanking plant
genes. We applied this approach to the species Catharanthus roseus (L.) G. Don
(Madagascar periwinkle), in an attempt to isolate regulators of genes that are
involved in the biosynthesis of secondary metabolites of the terpenoid indole
alkaloid (TIA) class. Several TIAs have pharmaceutically interesting activities,
including the anti/​tumour agents vincristine and vinblastine. The use of
suspension/​cultured cells enabled us to screen in a relatively easy way hundreds
of thousands of T/​DNA/​tagged cells for resistance to a toxic substrate of one of
the TIA biosynthetic enzymes: tryptophan decarboxylase. This screening yielded
several interesting tagged cell lines. Further characterisation of one of the
tagged cell lines led to the isolation of Orca3, a gene encoding an
AP2/ERF/​domain transcription factor that acts as a master regulator of primary
and secondary metabolism. The T/​DNA activation tagging results described in
detail in this paper illustrate the usefulness of this approach to isolate
regulators of a complex metabolic pathway from a genetically non/​tractable plant
species.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11817539 [PubMed /​ indexed for MEDLINE]

142: Phytochemistry. 2002 Jan;59(1):1/​8.

Predicting the substrates of cloned plant O/​methyltransferases.

Schroder G, Wehinger E, Schroder J.

Universitat Freiburg, Institut fur Biologie II, Schanzlestr. 1, D/​79104,
Freiburg, Germany.

Plant O/​methyltransferases (OMTs) have important roles in secondary metabolite
biosynthesis. Sequencing projects and homology/​based cloning strategies yield
sequences for proteins with similarities to known OMTs, but the identification
of the physiological substrates is not trivial. We investigated with a cDNA
cloned from Catharanthus roseus the possibilities for predicting the substrates
of OMTs, using the information from previous work and two newly identified
motifs that were based on information from the crystal structures of two plant
OMTs. The results, confirmed by functional analysis of the recombinant protein,
indicated that a careful analysis of the deduced protein sequence can provide
clues for predicting the substrates of cloned OMTs.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11754938 [PubMed /​ indexed for MEDLINE]

143: J Exp Bot. 2002 Jan;53(366):149/​50.

Cloning of a cDNA encoding an E2 ubiquitin/​conjugating enzyme from Catharanthus
roseus: expression analysis in plant organs and in response to hormones in cell
suspensions.

Siberil Y, Thiersault M, Nepumoceno G, Doireau P, Gantet P.

EA 2106, Biomolecules et Biotechnologies Vegetales, Universite de Tours, UFR des
Sciences et Techniques, Laboratoire de Physiologie Vegetale, Parc de Grandmont,
37200 Tours, France.

A novel cDNA (Crubie2) encoding ubiquitin/​conjugating enzyme E2 was isolated
from a Catharanthus roseus cDNA library. Sequence comparison with Arabidopsis
thaliana E2 sequences revealed that CrUBIE2 is a member of a new plant E2
sub/​family. Expression of Crubie2 is repressed in developing organs and
down/​regulated by cytokinin suggesting that a decrease in the
ubiquitin/​dependent proteolytic pathway may take part in the regulation of
alkaloid biosynthesis in C. roseus cell suspensions.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11741052 [PubMed /​ indexed for MEDLINE]

144: Planta. 2001 Oct;213(6):898/​906.

Isolation and partial characterization of norcoclaurine synthase, the first
committed step in benzylisoquinoline alkaloid biosynthesis, from opium poppy.

Samanani N, Facchini PJ.

Department of Biological Sciences, University of Calgary, Alberta, Canada.

Norcoclaurine synthase (NCS) catalyzes the condensation of dopamine and
4/​hydroxyphenylacetaldehyde (4/​HPAA) to yield norcoclaurine, the common
precursor to all benzylisoquinoline alkaloids produced in plants. In opium poppy
(Papaver somniferum L.), NCS activity was detected in germinating seeds, young
seedlings, and all mature plant organs, especially stems and roots. However, the
highest levels of activity were found in cell/​suspension cultures treated with a
fungal elicitor. NCS activity was induced more than 20/​fold over an 80/​h period
in response to elicitor treatment. Compared to opium poppy. basal NCS activity
was 3/​and 5/​fold higher in benzylisoquinoline alkaloid/​producing cell cultures
of Eschscholzia californica and Thalictrum flavum ssp. glaucum, respectively. In
contrast, NCS activity was not detected in cultured cells of Nicotiana tabacum
and Catharanthus roseus, which do not produce benzylisoquinoline alkaloids. NCS
displayed maximum activity between pH 6.5 and 7.0, and a broad temperature
optimum between 42 and 55 degrees C. Enzyme activity was not affected by Ca2+ or
Mg2+, and was not inhibited by a variety of benzylisoquinoline alkaloids. NCS
showed hyperbolic saturation kinetics for 4/​HPAA, with an apparent Km of 1.0 mM.
However, the enzyme exhibited sigmoidal saturation kinetics for dopamine with a
Hill coefficient of 1.84. NCS enzymes from E. californica and T. flavum
displayed similar properties. These data indicate that NCS exhibits positive
cooperativity between substrate/​binding sites. Enzymes of this type catalyze
regulatory, or rate/​limiting, steps in metabolism, suggesting that NCS plays a
role in controlling the rate of pathway flux in benzylisoquinoline alkaloid
biosynthesis.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 11722126 [PubMed /​ indexed for MEDLINE]

145: FEBS Lett. 2001 Nov 16;508(2):215/​20.

Geraniol 10/​hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole
alkaloid biosynthesis.

Collu G, Unver N, Peltenburg/​Looman AM, van der Heijden R, Verpoorte R, Memelink
J.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden
University, The Netherlands.

Geraniol 10/​hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in
the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid
alkaloids found in a diverse range of plant species. Catharanthus roseus
(Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of
which are pharmaceutically important. Vinblastine and vincristine, for example,
find widespread use as anti/​cancer drugs. G10H is thought to play a key
regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from
C. roseus cells. Using degenerate PCR primers based on amino acid sequence
information we cloned the corresponding cDNA. The encoded CYP76B6 protein has
G10H activity when expressed in C. roseus and yeast cells. The stress hormone
methyljasmonate strongly induced G10h gene expression coordinately with other
terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11718718 [PubMed /​ indexed for MEDLINE]

146: J Agric Food Chem. 2001 Nov;49(11):5165/​70.

Antioxidant activity and phenolic compounds in selected herbs.

Zheng W, Wang SY.

Fruit Laboratory, Beltsville Agricultural Research Center, Agricultural Research
Service, U. S. Department of Agriculture, Beltsville, Maryland 20705, USA.

The antioxidant capacities (oxygen radical absorbance capacity, ORAC) and total
phenolic contents in extracts of 27 culinary herbs and 12 medicinal herbs were
determined. The ORAC values and total phenolic contents for the medicinal herbs
ranged from 1.88 to 22.30 micromol of Trolox equivalents (TE)/g of fresh weight
and 0.23 to 2.85 mg of gallic acid equivalents (GAE)/g of fresh weight,
respectively. Origanum x majoricum, O. vulgare ssp. hirtum, and Poliomintha
longiflora have higher ORAC and phenolic contents as compared to other culinary
herbs. The ORAC values and total phenolic content for the culinary herbs ranged
from 2.35 to 92.18 micromol of TE/g of fresh weight and 0.26 to 17.51 mg of
GAE/g of fresh weight, respectively. These also were much higher than values
found in the medicinal herbs. The medicinal herbs with the highest ORAC values
were Catharanthus roseus, Thymus vulgaris, Hypericum perforatum, and Artemisia
annua. A linear relationship existed between ORAC values and total phenolic
contents of the medicinal herbs (R = 0.919) and culinary herbs (R = 0.986).
High/​performance liquid chromatography (HPLC) coupled with diode/​array detection
was used to identify and quantify the phenolic compounds in selected herbs.
Among the identified phenolic compounds, rosmarinic acid was the predominant
phenolic compound in Salvia officinalis, Thymus vulgaris, Origanum x majoricum,
and P. longiflora, whereas quercetin/​3/​O/​rhamnosyl/​(1 /​/​> 2)/​rhamnosyl/​(1 /​/​>
6)/​glucoside and kaempferol/​3/​O/​rhamnosyl/​(1 /​/​> 2)/​rhamnosyl/​(1 /​/​>
6)/​glucoside were predominant phenolic compounds in Ginkgo biloba leaves.

PMID: 11714298 [PubMed /​ indexed for MEDLINE]

147: Phytochemistry. 1998 Nov 20;49(6):1627/​1629.

Deracemization of racemic 4/​pyridyl/​1/​ethanol by Catharanthus roseus cell
cultures.

Takemoto M, Achiwa K.

School of Pharmaceutical Sciences, University of Shizuoka, 52/​1 Yada, 422/​8526,
Shizuoka, Japan

Immobilized cells of Catharanthus roseus synthesized (R)/​4/​pyridyl/​1/​ethanol
from the corresponding racemate in 100% yield by stereoinversion of the
(S)/​alcohol in the racemate to the (R)/​alcohol. Furthermore, in the reduction of
4/​acetylpyridine, we could synthesize both (R) and (S)/​4/​pyridyl/​1/​ethanol
during a short or a long incubation.

PMID: 11711075 [PubMed /​ as supplied by publisher]

148: Phytochem Anal. 2001 May/​Jun;12(3):206/​10.

Symmetry C18 column: a better choice for the analysis of indole alkaloids of
Catharanthus roseus.

Uniyal GC, Bala S, Mathur AK, Kulkarni RN.

Central Institute of Medicinal and Aromatic Plants (CIMAP), PO CIMAP, Lucknow
226015, India. root@cimap.sirnetd.ernet.in

By virtue of the different elution patterns of vindoline, catharanthine,
vincristine and vinblastine present in the leaves of Catharanthus roseus plants,
a Symmetry C18 column provided a better resolution for all of these compounds as
compared with other C18 columns. The binary gradient system with a linear
gradient profile, employed in a method developed using a Symmetry C18 column,
gave excellent peak symmetry, resolution and reproducibility. Detection was
performed at 220 nm which presented better absorptivity for these indole
alkaloids giving a minimum detection limit of 0.5 microgram. Photodiode array
detection was used to determine the homogeneity and purity of each compound. The
described analysis is rapid and economical to perform as small amounts of
solvent are consumed per analysis, and the method will find application where a
large number of samples are to be analysed as, for example, in crop improvement
studies where plants need to be selected regularly on the basis of alkaloid
production.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11705028 [PubMed /​ indexed for MEDLINE]

149: J Org Chem. 1998 Oct 16;63(21):7162/​7167.

A New Structural Class of Bisindole Alkaloids from the Seeds of Catharanthus
roseus: Vingramine and Methylvingramine.

Jossang A, Fodor P, Bodo B.

Laboratoire de Chimie, CNRS URA 401, Museum National d'Histoire Naturelle, 63
Rue Buffon 75005 Paris, France, and Centre de Recherches Helena Rubinstein, 188
Rue P. Hochart, Chevilly/​Larue BP 553, 94152 Rungis Cedex.

Two new bisindole alkaloids, vingramine (1) and methylvingramine (2), were
isolated from the seeds of Catharanthus roseus, Apocynaceae. The structures were
determined by HRFABMS as well as one/​ and two/​dimensional NMR experiments. They
possess a new bisindole skeleton involving an indole alkaloid part B with loss
of 5',6'/​ethylene, a C7'/​C16' linkage, a 14'/​O/​19'/​tetrahydrofuran, and a
N/​4'/​isobutyramide group. The 12/​methyl vincorine part A and part B are
connected via an 11,10'/​biphenyl linkage. The relative configuration was
determined by NMR analysis. Biogenetic considerations suggested a rearrangement
and a double fragmentation at C6'/C7' and N4'/C5' for the formation of 1 from
strictamine, further allowing deduction of the absolute configuration of 10
stereocenters: 2S, 7R, 15R, 16R, 3'R, 14'S, 15'S, 16'R, 19'S, and 20'R. The
alkaloids 1 and 2 display, in vitro, cytotoxic activity against nasopharynx
carcinoma KB cells, IC(50) 5 and 6 &mgr;M (4 and 5 &mgr;g/mL).

PMID: 11672355 [PubMed /​ as supplied by publisher]

150: J Chromatogr A. 2001 Aug 24;927(1/​2):39/​45.

Assay of 2,3/​dihydroxybenzoic acid and related compounds in plant materials by
high/​performance liquid chromatography.

Muljono RA, Darsono FL, Scheffer JJ, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden
University, The Netherlands.

Salicylic acid and its putative biosynthetic precursors were assayed
isocratically by RP/​HPLC with UV detection at 280 nm. Optimum resolution was
provided by an HPLC mobile phase consisting of MeOH/​1% aqueous HOAc (40:60,
v/v), at pH 4. Furthermore, for the analysis of 2,3/​dihydroxybenzoic acid
(2,3/​DHBA) in Catharanthus roseus cell cultures after elicitation, a mobile
phase consisting of acetonitrile/​1% aqueous HCOOH containing 0.25%
trichloroacetic acid (1:5, v/v), at pH 2, was used. The recovery for the free
form of 2,3/​DHBA was about 80% after a one/​step extraction of the cells. The
detection limit of 2,3/​DHBA was 3 microg by using saligenin as an internal
standard.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11572396 [PubMed /​ indexed for MEDLINE]

151: Curr Med Chem. 2001 Sep;8(11):1363/​81.

Aryltetralin lignans: chemistry, pharmacology and biotransformations.

Botta B, Delle Monache G, Misiti D, Vitali A, Zappia G.

Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente
Attive, Universita "La Sapienza", P.le Aldo Moro 5, 00185, Roma, Italy.
Bruno.Botta@uniroma1.it

Podophyllotoxin derivatives like etoposide 7a, etophos 7b, and teniposide 7c are
used clinically as potent chemotherapeutic agents for a variety of tumors
including small cell lung carcinoma, testicular cancer, and malignant lymphoma.
These compounds derived from a series of modifications which converted
podophyllotoxin 1a from an entity that interacted with tubulin and blocks
mitosis to one that induced a block in late S or early G2 by interacting with
topoisomerase II. Synthetic studies on podophyllotoxin derivatives can be
divided in four general approaches (the oxo/​ester route, the digydroxy acid
route, the tandem conjugate addition route and the Diels/​Alder route). Albeit a
number of synthetic sequences afforded products with excellent enantiopurities,
the low overall yields still disqualify synthesis as an alternative for
naturally produced materials. An alternative route based on the enzyme/​catalyzed
cyclization of synthetic intermediates to analogues of the podophyllotoxin
family is being explored. Synthetic dibenzylbutanolides, which were revealed by
biosynthetic studies to be the precursors of aryltetralin lignans, have been
treated with enzymes derived from cell cultures of Podophyllum peltatum,
Catharanthus roseus, Nicotiana sylvestris and Cassia didymobotrya. The
ciclyzation process afforded however compounds with a different stereochemistry
in the C ring. The obtainment of a novel compound with a
bynzylidenebenzylbutirolactone structure still leaves considerable scope for
exploring biotransformations in order to obtain podophyllotoxin analogues via a
combination of synthetic chemistry and biotechnological methods.

Publication Types:
Review

PMID: 11562272 [PubMed /​ indexed for MEDLINE]

152: Cardiovasc Res. 2001 Oct;52(1):95/​102.

Activation and inactivation of cAMP/​response element/​mediated gene transcription
in cardiac myocytes.

Muller FU, Boknik P, Knapp J, Linck B, Luss H, Neumann J, Schmitz W.

The Institute for Pharmacology and Toxicology, University of Munster, Domagkstr.
12, D/​48149, Munster, Germany. mullerf@uni/​muenster.de

OBJECTIVE: Chronic beta/​adrenergic stimulation of the cAMP/​dependent signalling
pathway is implicated in functionally relevant expressional changes in
congestive heart failure. We studied activation and inactivation of the cardiac
gene transcription mediated by the cAMP/​response element (CRE) and the
CRE/​binding protein (CREB) as an important mechanism of a cAMP/​dependent gene
regulation. METHODS: We investigated the transcriptional activation by
forskolin, an activator of the adenylyl cyclase, in chick embryonic
cardiomyocytes transfected with a CRE/​controlled luciferase construct in
comparison to the phosphorylation and expression of CREB determined on
immunoblots. RESULTS: Forskolin (10 micromol/l; 8 h) increased CRE/​mediated
transcription and phosphorylation of CREB 13/​ and 1.5/​fold, respectively. The
phosphorylation was further elevated in combination with cantharidin, an
inhibitor of type 1+2A protein phosphatases. The transcriptional response to
forskolin was desensitized by pretreatment with forskolin (1 micromol/l; 24 h)
while CREB phosphorylation was increased. In forskolin/​pretreated cells, total
CREB protein levels were decreased. Cantharidin did not restore the attenuated
transcriptional response. CONCLUSIONS: In cardiomyocytes, there is an activation
of the CRE/​mediated gene transcription by forskolin that is attenuated after
prolonged stimulation, and this attenuation is not dependent from a
dephosphorylation of CREB. We suggest that attenuation of the CRE/​mediated
transcription through chronic stimulation of the cAMP/​pathway, e.g. by elevated
catecholamines, contributes to the altered expressional regulation in congestive
heart failure.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11557237 [PubMed /​ indexed for MEDLINE]

153: Appl Microbiol Biotechnol. 2001 Aug;56(3/​4):420/​4.

Biotransformation of tryptamine and secologanin into plant terpenoid indole
alkaloids by transgenic yeast.

Geerlings A, Redondo FJ, Contin A, Memelink J, van der Heijden R, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden
University, The Netherlands.

A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs
coding for strictosidine synthase (STR) and strictosidine beta/​glucosidase (SGD)
from the medicinal plant Catharanthus roseus. Both enzymes are involved in the
biosynthesis of terpenoid indole alkaloids. The yeast culture was found to
express high levels of both enzymes. STR activity was found both inside the
cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium),
whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh
weight). Upon feeding of tryptamine and secologanin, this transgenic yeast
culture produced high levels of strictosidine in the medium; levels up to 2 g/l
were measured. Inside the yeast cells strictosidine was also detected, although
in much lower amounts (0.2 mg/g cells). This was due to the low permeability of
the cells towards the substrates, secologanin and tryptamine. However, the
strictosidine present in the medium was completely hydrolyzed to cathenamine,
after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was
able to grow on an extract of Symphoricarpus albus berries serving as a source
for secologanin and carbohydrates. Under these conditions, the addition of
tryptamine was sufficient for the transgenic yeast culture to produce indole
alkaloids. Our results show that transgenic yeast cultures are an interesting
alternative for the production of plant alkaloids.

Publication Types:
Evaluation Studies

PMID: 11549013 [PubMed /​ indexed for MEDLINE]

154: Appl Microbiol Biotechnol. 2001 Jun;55(6):693/​8.

Effects of stress factors, bioregulators, and synthetic precursors on indole
alkaloid production in compact callus clusters cultures of Catharanthus roseus.

Zhao J, Hu Q, Guo YQ, Zhu WH.

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing. jianzhao@agr.kyushu/​u.ac.jp

Compact callus cluster (CCC) cultures established from Catharanthus roseus
consist of cohesive callus aggregates displaying certain levels of cellular or
tissue differentiation. CCC cultures synthesize about two/​fold more indole
alkaloids than normal dispersed/​cell cultures. Our studies here show that
additions of KCl, mannitol, and a variety of synthetic precursors and
bioregulators to the CCC cultures markedly improved indole alkaloid production
and release of these alkaloids into the medium. Treatment with 250 mM mannitol
and 4 g/l KCl yielded 42.3 mg l(/​1) and 33.6 mg l(/​1)of ajmalicine,
respectively; these amounts were about four/​fold higher than the control.
Succinic acid, tryptamine, and tryptophan feedings also significantly increased
ajmalicine (41.5 mg l(/​1), 36.9 mg l(/​1), and 31.8 mg l(/​1), respectively) and
catharanthine (21.1 mg l(/​1), 17.2 mg l(/​1), and 18 mg l(/​1), respectively)
production by the CCC cultures, while geraniol feeding inhibited biomass and
alkaloid accumulation. We also found that tetramethyl ammonium bromide could
significantly improve ajmalicine production (49.3 mg l(/​1)) and catharanthine
production (18.3 mg l(/​1)) in C. roseus CCC cultures. The mechanisms responsible
for these treatment effects are discussed herein.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11525616 [PubMed /​ indexed for MEDLINE]

155: Chemosphere. 2001 Aug;44(5):1259/​64.

Studies on plant/​mediated fate of the explosives RDX and HMX.

Bhadra R, Wayment DG, Williams RK, Barman SN, Stone MB, Hughes JB, Shanks JV.

Department of Bioengineering, Rice University, Houston, TX 77005/​1892, USA.

The fate of the explosives RDX and HMX on exposure to plants was investigated in
'natural' aquatic systems of Myriophyllum aquaticum for 16 days, and in axenic
hairy root cultures of Catharanthus roseus for > or = 9 weeks. Exposure levels
were: HMX, 5 mg/l; and RDX, approximately 8 mg/l. Exposure outcomes observed
include: HMX, no transformation by aquatic plants, and minimal biological
activity by axenic roots; and RDX, removal by both plant systems. In the case of
RDX exposure to axenic roots, since 14C/​RDX was included, removal was confirmed
by the accumulation of 14C/​label in the biomass. The intracellular 14C/​label in
these RDX studies was detected in two forms: intact RDX and bound unknown(s).

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 11513416 [PubMed /​ indexed for MEDLINE]

156: Fitoterapia. 2000 Sep;71(5):547/​52.

Evaluation of antitumor activity of some medicinal plants of Bangladesh by
potato disk bioassay.

Haque N, Chowdhury SA, Nutan MT, Rahman GM, Rahman KM, Rashid MA.

Department of Pharmacology, Bangabandhu Seikh Mujib Medical University,
Dhaka/​1000, Bangladesh.

The antitumor activity of the ethanolic extracts of 12 medicinal plants of
Bangladesh, including the vincristine/​vinblastine producing Catharanthus roseus
was studied using the potato disk bioassay technique. Among these, 10 plant
extracts at 25.0/​microgram/disc exhibited significant inhibition of crown gall
tumors caused by Agrobacterium tumefaciens.

PMID: 11449504 [PubMed /​ indexed for MEDLINE]

157: J Ethnopharmacol. 2001 Aug;76(3):269/​77.

Effect of an antidiabetic extract of Catharanthus roseus on enzymic activities
in streptozotocin induced diabetic rats.

Singh SN, Vats P, Suri S, Shyam R, Kumria MM, Ranganathan S, Sridharan K.

Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur,
Delhi 110054, India. shoba72@yahoo.com

Hypoglycemic activity was detected in dichloromethane:methanol extract (1:1) of
leaves and twigs of Catharanthus roseus (family Apocynaceae), a traditionally
used medicinal plant, using streptozotocin (STZ) induced diabetic rat model.
Extract at dose 500 mg/kg given orally for 7 and 15 days showed 48.6 and 57.6%
hypoglycemic activity, respectively. Prior treatment at the same dose for 30
days provided complete protection against STZ challenge (75 mg/kg/i.p.x1).
Enzymic activities of glycogen synthase, glucose 6/​phosphate/​dehydrogenase,
succinate dehydrogenase and malate dehydrogenase were decreased in liver of
diabetic animals in comparison to normal and were significantly improved after
treatment with extract at dose 500 mg/kg p.o. for 7 days. Results indicate
increased metabolization of glucose in treated rats. Increased levels of lipid
peroxidation measured as 2/​thiobarbituric acid reactive substances (TBARS)
indicative of oxidative stress in diabetic rats were also normalized by
treatment with the extract.

PMID: 11448549 [PubMed /​ indexed for MEDLINE]

158: J Biol Chem. 2001 Aug 17;276(33):30717/​23. Epub 2001 Jun 12.

Molecular characterization of the salutaridinol 7/​O/​acetyltransferase involved
in morphine biosynthesis in opium poppy Papaver somniferum.

Grothe T, Lenz R, Kutchan TM.

Leibniz/​Institut fur Pflanzenbiochemie, Weinberg 3, 06120 Halle/Saale, Germany.

Salutaridinol 7/​O/​acetyltransferase (EC ) catalyzes the conversion of the
phenanthrene alkaloid salutaridinol to salutaridinol/​7/​O/​acetate, the immediate
precursor of thebaine along the morphine biosynthetic pathway. We have isolated
a cDNA clone that corresponds to the internal amino acid sequences of the native
enzyme purified from a cell suspension culture of opium poppy Papaver
somniferum. The recombinant enzyme acetylated the 7/​hydroxyl moiety of
salutaridinol in the presence of acetyl/​CoA. The apparent K(m) value for
salutaridinol was determined to be 9 microm and 54 microm for acetyl/​CoA. The
gene transcript was detected in extracts from Papaver orientale and Papaver
bracteatum in addition to P. somniferum. Genomic DNA gel blot analysis indicated
that there is likely a single copy of this gene in the P. somniferum genome. The
amino acid sequence of salutaridinol 7/​O/​acetyltransferase is most similar (37%
identity) to that of deacetylvindoline acetyltransferase of Catharanthus roseus.
Salutaridinol 7/​O/​acetyltransferase is the second enzyme specific to morphine
biosynthesis for which we have isolated a cDNA. Taken together with the other
cDNAs cloned encoding norcoclaurine 6/​O/​methyltransferase,
(S)/​N/​methylcoclaurine 3'/​hydroxylase, the cytochrome P/​450 reductase, and
codeinone reductase, significant progress has been made toward accumulating
genes of this pathway to enable the end goal of a biotechnological production of
morphinan alkaloids.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11404355 [PubMed /​ indexed for MEDLINE]

159: Phytochemistry. 2001 Jul;57(5):669/​73.

Biotransformation of (+)/​ and (/​)/​camphorquinones by plant cultured cells.

Chai W, Hamada H, Suhara J, Horiuchi CA.

Department of Chemistry, Rikkyo (St. Paul's) University, Nishi/​Ikebukuro,
Toshima/​ku, Tokyo 171/​8501, Japan.

Biotransformation of (+)/​ and (/​)/​camphorquinones with suspension plant cultured
cells of Nicotiana tabacum and Catharanthus roseus was investigated. It was
found that the plant cultured cells of N. tabacum and C. roseus reduce
stereoselectively the carbonyl group of (+)/​ and (/​)/​camphorquinones to the
corresponding alpha/​keto alcohols.

PMID: 11397432 [PubMed /​ indexed for MEDLINE]

160: Plant Mol Biol. 2001 Mar;45(4):477/​88.

Catharanthus roseus G/​box binding factors 1 and 2 act as repressors of
strictosidine synthase gene expression in cell cultures.

Siberil Y, Benhamron S, Memelink J, Giglioli/​Guivarc'h N, Thiersault M, Boisson
B, Doireau P, Gantet P.

Universite de Tours, EA 2106, Biomolecules et Biotechnologies Vegetales, UFR des
Sciences et Techniques, Laboratoire de Physiologie Vegetale, France.

The enzyme encoded by the strictosidine synthase (Str) gene catalyses a key step
in the biosynthesis of therapeutically valuable terpenoid indole alkaloids. In
Catharanthus roseus the Str gene was shown to be regulated by a wide variety of
signals including auxin, methyl jasmonate and fungal elicitors in cell
suspension cultures and by tissue/​specific control in plant organs. The Str
promoter contains a functional G/​box (CACGTG) cis/​regulatory sequence. In order
to understand better the mechanisms involved in the regulation of Str gene
expression, we isolated the C. roseus cDNAs encoding G/​box binding factors
Crgbf1 and Crgbf2. The binding specificity of their protein products CrGBF1 and
CrGBF2 was analysed by competitive electrophoresis mobility and saturation
binding assays. CrGBF1 had a high binding specificity for class I G/​boxes
including the Str G/​box. CrGBF1 showed a lower affinity for class II G/​boxes and
for the G/​box/​like element (AACGTG) found in the tryptophan decarboxylase (Tdc)
gene which encodes another enzyme involved in TIA biosynthesis. CrGBF2 showed a
high affinity for all types of G/​boxes tested and to a lesser extent for the Tdc
G/​box/​like element. Transient bombardment experiments demonstrated that both
CrGBF1 and CrGBF2 can act in vivo as transcriptional repressors of the Str
promoter via direct interaction with the G/​box. These data indicate that GBFs
may play functional role in the regulation of expression of the terpenoid indole
alkaloid biosynthetic gene Str.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11352466 [PubMed /​ indexed for MEDLINE]

161: Prostaglandins Other Lipid Mediat. 2001 May;65(1):45/​56.

Phospholipase C activity from Catharanthus roseus transformed roots: aluminum
effect.

Pina/​Chable ML, Hernandez/​Sotomayor SM.

Unidad de Biologia, Experimental, Centro de Investigacion Cientifica de Yucatan
Apdo, Cordemex, Mexico.

The effect of aluminum on the activity of PLC was examined in transformed roots
from Catharanthus roseus (L) G. Don. When added in vitro to the reaction
mixture, Al inhibited the enzymatic activity in a concentration and
time/​dependent fashion. This effect is very similar for both activities (soluble
and membrane/​associated). When roots were treated in vivo with Al 0.1 mM for
short periods (0/​4 h), PLC activity was also inhibited. Aluminum (1 mM)
diminished root growth in approximately 50% when added on the first day of the
culture cycle conditions in which PLC activity is also affected. Other enzymatic
activities (NAD+/​GDH, NADH/​GDH, NADH/​GOGAT and HMGR) were not affected when
roots were treated with Al (0.1 mM) for short periods of time (1 h). Results
obtained in this work suggest that the Al can affect PLC activity as a specific
target. Enzymes: Phospholipase C (EC 3.1.4.10).

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11352226 [PubMed /​ indexed for MEDLINE]

162: Enzyme Microb Technol. 2001 May 7;28(7/​8):673/​681.

Enhanced catharanthine production in catharanthus roseus cell cultures by
combined elicitor treatment in shake flasks and bioreactors.

Zhao J, Zhu W, Hu Q.

Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union
Medical College, 100050, Beijing, China

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension
cultures so as to improve the production of indole alkaloids. A synergistic
effect on alkaloid accumulation was observed in C. roseus cell cultures when
treated with some combined elicitors of fungal preparations and chemicals. Among
them, the combination of tetramethyl amminium bromide and Aspergillum niger
mycelial homogenate gave the highest ajmalicine yield (63 mg l(/​1)) and an
improved catharanthine accumulation (17 mg l(/​1)). The combined elicitors of
malate and sodium alginate resulted in the highest catharanthine yield (26 mg
l(/​1)) and a high ajmalicine accumulation (41 mg l(/​1)) in the cell cultures.
Based on the synergistic effect of malate and sodium alginate, a process with
enhanced catharanthine production in Catharanthus roseus cell cultures was
developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg
l(/​1), 32 mg l(/​1) and 22 mg l(/​1) catharanthine yield were obtained in 500/​ml
flasks, 1000/​ml flasks and in a 20/​l airlift bioreactor, respectively. Upon
malate/​alginate combining treatments, peroxidase, catalase and superoxide
dismutase activities decreased in elicited cells but phenylalanine ammonia lyase
and lipoxygenase activities increased dramatically. That suggests a typical
defense responses took place in the combined elicitors/​treated cell cultures.
Furthermore, the combined elicitors also caused a significant increase of
malondialdehyde level in cell cultures, which suggests a serious lipid
peroxidation occurred in the elicited cell cultures. Comparison of these results
suggests that malate and alginate combining treatment also stimulates defense
responses, such as lipid peroxidation, in all C. roseus culture processes and
this may mediate the indole alkaloid production via jasmonate pathway.

PMID: 11339952 [PubMed /​ as supplied by publisher]

163: Enzyme Microb Technol. 2001 May 7;28(7/​8):666/​672.

Selection of fungal elicitors to increase indole alkaloid accumulation in
catharanthus roseus suspension cell culture.

Zhao J, Zhu W, Hu Q.

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College, Peking, P.R., China

Various fungal elicitors derived from 12 fungi were tested to improve indole
alkaloid production in Catharanthus roseus cell suspension cultures. Results
show that different fungal mycelium homogenates stimulate different kinds of
indole alkaloid (ajmalicine, serpentine and catharanthine) accumulation, which
ranged from 2/​ to 5/​fold higher than the control. Some fungal culture filtrates
also efficiently elicited the biosynthesis of different indole alkaloids. The
optimal elicitor addition and exposure time for the maximal alkaloid production
were on day 7 after subculture and for 3 days of treatment but different fungal
elicitors showed the different optimal treatment dosages. Additions of elicitor
at the doses ranging from 5 mg/l to 30 mg/l of carbon hydrate equivalent
resulted in varieous amounts and kinds of indole alkaloid accumulation. Exposed
to a same fungal elicitor, several different cell lines generated the different
responses regarding as growth rate, culture color and alkaloid production.

PMID: 11339951 [PubMed /​ as supplied by publisher]

164: Prostaglandins. 2001 May;65(1):45/​56.

Phospholipase C activity from Catharanthus roseus transformed roots: aluminum
effect.

Pina/​Chable ML, Hernandez/​Sotomayor SM.

Unidad de Biologia, Experimental, Centro de Investigacion Cientifica de Yucatan
Apdo, Postal 87, 97310, Cordemex, Yucatan, Mexico

The effect of aluminum on the activity of PLC was examined in transformed roots
from Catharanthus roseus (L) G. Don. When added in vitro to the reaction
mixture, Al inhibited the enzymatic activity in a concentration and
time/​dependent fashion. This effect is very similar for both activities (soluble
and membrane/​associated). When roots were treated in vivo with Al 0.1 mM for
short periods (0/​4 h), PLC activity was also inhibited. Aluminum (1 mM)
diminished root growth in approximately 50% when added on the first day of the
culture cycle conditions in which PLC activity is also affected. Other enzymatic
activities (NAD(+)/​GDH, NADH/​GDH, NADH/​GOGAT and HMGR) were not affected when
roots were treated with Al (0.1 mM) for short periods of time (1 h). Results
obtained in this work suggest that the Al can affect PLC activity as a specific
target.Enzymes: Phospholipase C (EC 3.1.4.10)

PMID: 11334641 [PubMed /​ as supplied by publisher]

165: J Biol Chem. 2001 Jul 13;276(28):25687/​91. Epub 2001 Apr 23.

Selective interaction of triazole derivatives with DWF4, a cytochrome P450
monooxygenase of the brassinosteroid biosynthetic pathway, correlates with
brassinosteroid deficiency in planta.

Asami T, Mizutani M, Fujioka S, Goda H, Min YK, Shimada Y, Nakano T, Takatsuto
S, Matsuyama T, Nagata N, Sakata K, Yoshida S.

RIKEN, 2/​1 Hirosawa, Wako, Saitama 351/​0198, Japan. tasami@postman.riken.go.jp

Brassinazole, a synthetic chemical developed in our laboratory, is a
triazole/​type brassinosteroid biosynthesis inhibitor that induces dwarfism in
various plant species. The target sites of brassinazole were investigated by
chemical analyses of endogenous brassinosteroids (BRs) in brassinazole/​treated
Catharanthus roseus cells. The levels of castasterone and brassinolide in
brassinazole/​treated plant cells were less than 6% of the levels in untreated
cells. In contrast, campestanol and 6/​oxocampestanol levels were increased, and
levels of BR intermediates with hydroxy groups on the side chains were reduced,
suggesting that brassinazole treatment reduced BR levels by inhibiting the
hydroxylation of the C/​22 position. DWF4, which is an Arabidopsis thaliana
cytochrome P450 isolated as a putative steroid 22/​hydroxylase, was expressed in
Escherichia coli, and the binding affinity of brassinazole and its derivatives
to the recombinant DWF4 were analyzed. Among several triazole derivatives,
brassinazole had both the highest binding affinity to DWF4 and the highest
growth inhibitory activity. The binding affinity and the activity for inhibiting
hypocotyl growth were well correlated among the derivatives. In
brassinazole/​treated A. thaliana, the CPD gene involved in BR biosynthesis was
induced within 3 h, most likely because of feedback activation caused by the
reduced levels of active BRs. These results indicate that brassinazole inhibits
the hydroxylation of the C/​22 position of the side chain in BRs by direct
binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11319239 [PubMed /​ indexed for MEDLINE]

166: J Biosci. 2001 Mar;26(1):57/​70.

Pleiotropic morphological and abiotic stress resistance phenotypes of the
hyper/​abscisic acid producing Abo/​ mutant in the periwinkle Catharanthus roseus.

Rai SP, Luthra R, Gupta MM, Kumar S.

Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226 015,
India.

The pleiotropic properties of a abo abo (Abo/​) gamma/​ray induced mutant of
Catharanthus roseus cv. Nirmal, selected among the M2 generation seeds for
ability to germinate at 45 degrees C, are described. The mutant produced seeds
possessing tricotyledonous embryos, unlike the typically dicotyledonous embryos
present in the wild type Abo+ seeds. In comparison to Abo+ adults, the mutant
plants had short stature and lanceolate leaves. The vascular bundles in the
leaves and stem were poorly developed. Leaf surfaces were highly trichomatous,
epidermal, cortex and mesophyll cells were small sized and a large majority of
stomata were closed. Besides high temperature, the mutant was salinity and
water/​stress tolerant. The abscisic acid (ABA) content in the leaves was about
500/​fold higher. The genetic lesion abo responsible for the above pleiotropy was
recessive and inherited in Mendelian fashion. The seedlings and adult plants of
the mutant accumulated higher proline than Abo+ plants. The phenotypes of abo
abo mutants permitted the conclusions that (i) the mutant synthesizes ABA
constitutively, (ii) both ABA/​dependent and ABA independent pathways for proline
and betaine accumulation are functional in the mutant, and (iii) cell division,
elongation and differentiation processes in embryo and adult plant stages are
affected in the mutant

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11255514 [PubMed /​ indexed for MEDLINE]

167: Rapid Commun Mass Spectrom. 2001;15(5):364/​9.

Monitoring the production yields of vincristine and vinblastine in Catharanthus
roseus from somatic embryogenesis. Semiquantitative determination by
flow/​injection electrospray ionization mass spectrometry.

Favretto D, Piovan A, Filippini R, Caniato R.

Centro di Studio sulla Chimica e Tecnologia dei Composti Organometallici degli
Elementi di Transizione, Via Marzolo 9, I/​35100 Padua, Italy.
favretto@pdadr1.pd.cnr.it

A semiquantitative determination of two bis/​indole antitumor alkaloids,
vincristine and vinblastine, has been performed by flow/​injection electrospray
ionization mass spectrometric analysis of the extracts of Catharanthus roseus.
Leaves and flowers of two different phenotypes (pink flower and white flower)
obtained from somatic embryogenesis were thus examined and compared with the
field/​grown mother plant. Different amounts of vincristine and vinblastine were
detected depending on the examined samples. Copyright 2001 John Wiley & Sons,
Ltd.

PMID: 11241768 [PubMed /​ indexed for MEDLINE]

168: Yi Chuan Xue Bao. 2001;28(2):144/​51.

[Differential accumulation of the new high/​affinity phosphate transporter
candidated gene fragment in rice roots in response to phosphorus deficiency
stress]

[Article in Chinese]

Yu FT, Zhang AM, Chen SY, Zhang FS.

Key Laboratory of Plant Nutrition, Ministry of Agricultures, Department of Plant
Nutrition, China Agricultural University, Beijing 100094, China.

Phosphate is a major constraint to crop production, and phosphate uptake in
plant is mainly by high/​affinity phosphate transporter under phosphate
deficiency condition. Using RT/​PCR, a 1,178 bp phosphate transporter gene
fragment OjPT1 was cloned from roots of Jingxi17 (Oryza sativa L. ssp. japanica)
supplied with no phosphate. The comparison of this sequence with ones in GenBank
indicated that it shared about 70% similarity at amino acid level with other
phosphate transporters in higher plants, such as Arabidopsis thaliana, potate,
tamato, Medicago truncatula and Catharanthus roseus, and high similarity with
phosphate transporters in Saccharomyces cerevisiae and Neurospora crassa. RT/​PCR
assay showed that the OjPT1 transcripts were induced under phosphate deficiency
condition. This gene fragment OjPT1 has been deposited in GenBank (accession No.
AF249619).

Publication Types:
English Abstract
Research Support, Non/​U.S. Gov't

PMID: 11233258 [PubMed /​ indexed for MEDLINE]

169: Mol Plant Microbe Interact. 2001 Feb;14(2):225/​33.

Catharanthus roseus genes regulated differentially by mollicute infections.

Jagoueix/​Eveillard S, Tarendeau F, Guolter K, Danet JL, Bove JM, Garnier M.

Laboratoire de Biologie Cellulaire et Moleculaire, Institut de Biologie Vegetale
Moleculaire INRA & Universite Victor Segalen Bordeaux 2, Villenave d'Ornon,
France.

A differential display of mRNAs was used to isolate periwinkle cDNAs
differentially expressed following infection with one of three mollicutes:
Spiroplasma citri, Candidatus Phytoplasma aurantifolia, and stolbur phytoplasma.
Twenty/​four differentially expressed cDNAs were characterized by Northern blots
and sequence analysis. Eight of them had homologies with genes in databanks
coding for proteins involved in photosynthesis, sugar transport, response to
stress, or pathways of phytosterol synthesis. The regulation of these genes in
periwinkle plants infected by additional phloem/​restricted bacteria showed that
they were not specific to a given mollicute, but correlations with particular
symptoms could be established. Expression of transketolase was down regulated
following infection with a pathogenic strain of S. citri. No down regulation was
observed for the nonphytopathogenic mutant GMT553, which is deficient for
fructose utilization.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11204786 [PubMed /​ indexed for MEDLINE]

170: Plant Mol Biol. 2000 Nov;44(5):675/​85.

A Catharanthus roseus BPF/​1 homologue interacts with an elicitor/​responsive
region of the secondary metabolite biosynthetic gene Str and is induced by
elicitor via a JA/​independent signal transduction pathway.

van der Fits L, Zhang H, Menke FL, Deneka M, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
Netherlands.

Plants respond to pathogen attack by induction of various defence responses,
including the biosynthesis of protective secondary metabolites. In Catharanthus
roseus, the elicitor/​induced expression of the terpenoid indole alkaloid
biosynthetic gene Strictosidine synthase (Str) is mediated via the plant stress
hormonejasmonate. In the promoters of several defence/​related genes, cis/​acting
elements have been identified that are important for transcriptional regulation
upon stress signals. Here we show that an upstream region in the Str promoter
confers responsiveness to partially purified yeast elicitor and jasmonate. Yeast
one/​hybrid screening with this element as a bait identified a MYB/​like protein,
which shows high homology to parsley box P/​binding factor/​1 (PcBPF/​1). In vitro
analyses showed that the Str promoter fragment contained a novel binding site
for BPF/​1/​like proteins with higher binding affinity than the previously
described box P. CrBPF/​1 mRNA accumulated rapidly in elicitor/​treated C. roseus
suspension cells, whereas no induction was observed with jasmonate. Inhibitor
studies indicated that CrBPF/​1 plays a role in an elicitor/​responsive but
jasmonate/​independent signal transduction pathway, acting downstream of protein
phosphorylation and calcium influx.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11198427 [PubMed /​ indexed for MEDLINE]

171: Plant J. 2001 Jan;25(1):43/​53.

The jasmonate/​inducible AP2/ERF/​domain transcription factor ORCA3 activates gene
expression via interaction with a jasmonate/​responsive promoter element.

van der Fits L, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.

The AP2/ERF/​domain transcription factor ORCA3 is a master regulator of primary
and secondary metabolism in Catharanthus roseus (periwinkle). Here we
demonstrate that ORCA3 specifically binds to and activates gene expression via a
previously characterized jasmonate/​ and elicitor/​responsive element (JERE) in
the promoter of the terpenoid indole alkaloid biosynthetic gene Strictosidine
synthase (Str). Functional characterization of different domains in the ORCA3
protein in yeast and plant cells revealed the presence of an N/​terminal acidic
activation domain and a serine/​rich C/​terminal domain with a negative regulatory
function. Orca3 mRNA accumulation was rapidly induced by the plant stress
hormone methyljasmonate with biphasic kinetics. A precursor and an intermediate
of the jasmonate biosynthetic pathway also induced Orca3 gene expression,
further substantiating the role for ORCA3 in jasmonate signaling. The protein
synthesis inhibitor cycloheximide did not inhibit jasmonate/​responsive
expression of Orca3, nor of its target genes Str and Tryptophan decarboxylase
(Tdc). In conclusion, ORCA3 regulates jasmonate/​responsive expression of the Str
gene via direct interaction with the JERE. The activating activities of ORCA
proteins do not seem to depend on jasmonate/​induced de novo protein synthesis,
but presumably occur via modification of pre/​existing ORCA protein.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 11169181 [PubMed /​ indexed for MEDLINE]

172: Plant Physiol. 2001 Jan;125(1):189/​98.

Molecular and biochemical analysis of a Madagascar periwinkle root/​specific
minovincinine/​19/​hydroxy/​O/​acetyltransferase.

Laflamme P, St/​Pierre B, De Luca V.

Institut de Recherche en Biologie Vegetale, Departement de Sciences Biologiques,
Universite de Montreal, 4101 rue Sherbrooke est, Montreal, Quebec, Canada H1X
2B2.

The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids
vindoline and minovincinine are catalyzed by separate acetyl coenzyme
A/​dependent O/​acetyltransferases in Madagascar periwinkle (Catharanthus roseus
G. Don). Two genes were isolated that had 63% nucleic acid identity and whose
deduced amino acid sequences were 78% identical. Active enzymes that were
expressed as recombinant His/​tagged proteins in Escherichia coli were named
minovincinine/​19/​O/​acetyltransferase (MAT) and
deacetylvindoline/​4/​O/​acetyltransferase (DAT) because they catalyzed the
19/​O/​acetylation of indole alkaloids such as minovincinine and horhammericine
and the 4/​O/​acetylation of deacetylvindoline, respectively. Kinetic studies
showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor
compared with that of recombinant DAT (rDAT), whose turnover rates for
Acetyl/​coenzyme A and deacetylvindoline were approximately 240/​ and 10,000/​fold
greater than those of rMAT. Northern/​blot analyses showed that MAT is expressed
in cortical cells of the root tip, whereas DAT is only expressed in specialized
idioblast and laticifer cells within light exposed tissues like leaves and
stems. The coincident expression of trytophan decarboxylase, strictosidine
synthase, and MAT within root cortical cells suggests that the entire pathway
for the biosynthesis of tabersonine and its substituted analogs occurs within
these cells. The ability of MAT to catalyze the 4/​O/​acetylation of
deacetylvindoline with low efficiency suggests that this enzyme, rather than
DAT, is involved in vindoline biosynthesis within transformed cell and root
cultures, which accumulate low levels of this alkaloid under certain
circumstances.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11154328 [PubMed /​ indexed for MEDLINE]

173: Plant J. 2000 Dec;24(6):797/​804.

Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and
identification of cytochrome P450 CYP72A1 as secologanin synthase.

Irmler S, Schroder G, St/​Pierre B, Crouch NP, Hotze M, Schmidt J, Strack D,
Matern U, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Schanzlestrasse 1, D/​79104
Freiburg, Germany.

The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar
periwinkle) was described nearly a decade ago, but the enzyme function remained
unknown. We now show by in situ hybridization and immunohistochemistry that the
expression in immature leaves is epidermis/​specific. It thus follows the pattern
previously established for early enzymes in the pathway to indole alkaloids,
suggesting that CYP72A1 may be involved in their biosynthesis. The early
reactions in that pathway, i.e. from geraniol to strictosidine, contain several
candidates for P450 activities. We investigated in this work two reactions, the
conversion of 7/​deoxyloganin to loganin (deoxyloganin 7/​hydroxylase, DL7H) and
the oxidative ring cleavage converting loganin into secologanin (secologanin
synthase, SLS). The action of DL7H has not been demonstrated in vitro
previously, and SLS has only recently been identified as P450 activity in one
other plant. We show for the first time that both enzyme activities are present
in microsomes from C. roseus cell cultures. We then tested whether CYP72A1
expressed in E. coli as a translational fusion with the C. roseus P450 reductase
(P450Red) has one or both of these activities. The results show that CYP72A1
converts loganin into secologanin.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11135113 [PubMed /​ indexed for MEDLINE]

174: Phytochemistry. 2000 Nov;55(6):531/​6.

Light activation of vindoline biosynthesis does not require cytomorphogenesis in
Catharanthus roseus seedlings.

Vazquez/​Flota FA, St/​Pierre B, De Luca V.

Institut de Recherche en Biologie Vegetale, Departement des Sciences
Biologiques, Universite de Montreal, Quebec, Canada.

Upon illumination, the cotyledons of Catharanthus roseus seedlings readily
synthesise vindoline from late biosynthetic intermediates, which accumulate in
etiolated seedlings. The cellular localisation of tryptophan decarboxylase (TDC)
and desacetoxyvindoline 4/​hydroxylase (D4H), which catalyse the first and
penultimate reactions of vindoline biosynthesis, was identified by
immunocytochemistry in developing seedlings. The expression of TDC was
restricted to the upper epidermis of cotyledons, whereas that of D4H was
confined to laticifer cells. Light exposure of etiolated seedlings significantly
induced D4H enzyme activity without changing the steady/​state levels of D4H
immunoreactive protein or modifying the cellular distribution of D4H expression
in dark/​grown seedlings. These results suggest that the early and late stages of
vindoline biosynthesis occupy different cellular compartments, even in the early
phases of etiolated seedling development. The role of light in activating the
late stages of vindoline biosynthesis does not, therefore, seem to be related to
the formation of the laticifer and idioblast cell types. It is concluded that
light is not required for formation of these cell types, whereas regulatory
factors, restricted to idioblasts and laticifers, may respond to light to
activate localised expression of the late stages of vindoline biosynthesis.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11130662 [PubMed /​ indexed for MEDLINE]

175: Biochim Biophys Acta. 2000 Dec 15;1517(1):159/​63.

Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in
Catharanthus roseus.

Veau B, Courtois M, Oudin A, Chenieux JC, Rideau M, Clastre M.

Laboratoire de Biologie Moleculaire et Biochimie vegetale, EA2106, Faculte de
Pharmacie, Universite de Tours, 31 avenue Monge, 37200 Tours, France.

Two periwinkle cDNAs (crdxr and crmecs) encoding enzymes of the non/​mevalonate
terpenoid pathway were characterized using reverse transcription/​PCR strategy
based on the design of degenerated oligonucleotides. The deduced amino acid
sequence of crdxr is homologue to 1/​deoxy/​D/​xylulose 5/​phosphate
reductoisomerases. Crmecs represents the first plant cDNA encoding a protein
similar to the 2C/​methyl/​D/​erythritol 2,4/​cyclodiphosphate synthase from
Escherichia coli. Expression of crdxr and crmecs genes was up/​regulated in
periwinkle cells producing monoterpenoid indole alkaloids. Involvement of the
2C/​methyl/​D/​erythritol 4/​phosphate pathway in alkaloid biosynthesis is
discussed.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 11118631 [PubMed /​ indexed for MEDLINE]

176: Yakugaku Zasshi. 2000 Oct;120(10):863/​73.

[Studies on the biosynthesis of sterol side chain in higher plants]

[Article in Japanese]

Fujimoto Y, Morisaki M, Ikekawa N.

Department of Chemistry and Materials Science, Tokyo Institute of Technology,
Japan.

Campesterol (3) and dihydrobrassicasterol (4), typical C28/​sterols in higher
plants, are biosynthesized from a steroidal 24/​ene precursor (desmosterol 1) via
24/​methylenecholesterol (2) and 24/​methyldesmosterol (5). A typical plant
C29/​sterol, sitosterol (6), is produced from 24/​methylenecholesterol via
isofucosterol (7) and 24/​ethyldesmosterol (8). The biosynthetic mechanism,
focussing stereochemical features, of these side/​chain transformations has been
studied in detail by feeding regio/​ and stereoselectively 13C/​ or 2H/​labeled
steroidal substrates to cell cultures of higher plants such as Oryza sativa,
Catharanthus roseus and Morus alba. These studies allowed to correlate the
metabolic origin of C/​26 and C/​27 of the intermediate sterols. It has been
established that the 1st methylation leading to 24/​methylenecholesterol from
desmosterol involves a Re/​face hydrogen migration from C/​24 to C/​25 based on
unambiguous assignment of the isopropyl pro/​R/​Me and pro/​S/​Me of
24/​methylenecholesterol. The 2nd methylation leading to isofucosterol was
revealed to proceed in a trans/​mechanism in which addition of the methyl group
and elimination of the C/​28 hydrogen occur on opposite faces of the original
delta 24(28) plane. The double bond isomerization from delta 24(28) to delta
24(25) was found to proceed in a syn/​SE2' mechanism with the pro/​S/​methyl group
of isofucosterol becoming the (E)/​methyl of 24/​ethyldesmosterol. Finally,
feeding studies of [E/​Me/​13C]/​ and [Z/​Me/​13C]/​24/​methyldesmosterols established
that an anti/​mode of hydrogen addition is operating in the conversion of
24/​methyldesmosterol to campesterol and dihydrobrassicasterol. Similar studies
established that 24/​ethyldesmosterol is converted to sitosterol in an anti/​mode
of hydrogen addition. In addition, the mechanism of sterol side/​chain formation
in hairy roots of Ajuga reptans var. atropurpurea is briefly described.

Publication Types:
English Abstract
Review

PMID: 11082699 [PubMed /​ indexed for MEDLINE]

177: Phytochemistry. 2000 Sep;55(2):97/​101.

28/​Norcastasterone is biosynthesized from castasterone.

Fujioka S, Noguchi T, Sekimoto M, Takatsuto S, Yoshida S.

RIKEN, The Institute of Physical and Chemical Research, Saitama, Japan.
sfujioka@postman.riken.go.jp

Metabolic experiments with deuterium/​labeled castasterone in seedlings of
Arabidopsis thaliana, Oryza saliva and Lycopersicon esculentum, and cultured
cells of Catharanthus roseus were performed, and the metabolites were analyzed
by GC/​MS. In all the plant species examined, [2H3]28/​norcastasterone was
identified as a metabolite of [26,28/​2H6]castasterone, indicating that
castasterone is the biosynthetic origin of 28/​norcastasterone. Moreover, the
natural occurrence of 28/​norcastasterone and 28/​nortyphasterol in seedlings of
A. thaliana has been demonstrated. This is the first report of the natural
occurrence of 28/​nortyphasterol in plants.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11065283 [PubMed /​ indexed for MEDLINE]

178: Plant Mol Biol. 2000 Jul;43(4):495/​502.

The ternary transformation system: constitutive virG on a compatible plasmid
dramatically increases Agrobacterium/​mediated plant transformation.

van der Fits L, Deakin EA, Hoge JH, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
The Netherlands.

This paper describes a so/​called ternary transformation system for plant cells.
We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a
constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of
very efficient T/​DNA transfer to a diverse range of plant species. For the plant
species Catharanthus roseus it is shown that increased T/​DNA transfer results in
increased stable transformation frequencies. Analysis of stably transformed C.
roseus cell lines showed that, although the T/​DNA transfer frequency is greatly
enhanced by addition of virGN54D, only one or a few T/​DNA copies are stably
integrated into the plant genome. Thus, high transformation frequencies of
different plant species can be achieved by introduction of a ternary plasmid
carrying a constitutive virG mutant into existing A. tumefaciens strains in
combination with standard binary vectors.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 11052201 [PubMed /​ indexed for MEDLINE]

179: Biol Chem. 2000 Aug;381(8):695/​703.

Plant methionine synthase: new insights into properties and expression.

Eckermann C, Eichel J, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

We investigated the enzyme methionine synthase (MSY) in Catharanthus roseus. The
properties were characterized with purified protein isolated either from plant
cell cultures or after heterologous expression in Escherichia coli. The protein
was a monomer and accepted both the triglutamate (CH3/​H4PteGlu3, apparent Km =
80 microM) and the monoglutamate (CH3/​H4PteGlu1, apparent Km = 350 microM) of
methyl/​5,6,7,8/​tetrahydropteroate as methyl donor, with a ratio of approximately
90:1 in favor of the triglutamate. Both activities required inorganic phosphate,
but with different kinetics, and both were dependent on reducing agents. The
activity required zinc, as shown by depletion and reconstitution experiments.
Mg2+ had no effect on the activity. Two MSY isoforms purified from parsley cell
cultures revealed the same properties as the C. roseus enzyme, however, the
parsley proteins had no detectable activity with the monoglutamate substrate.
The second part of the work compared the expression of the three enzymes of the
methyl cycle (MSY, S/​adenosyl/​L/​methionine synthetase, S/​adenosyl/​L/​homocysteine
hydrolase). In cell cultures, all three enzymes were present under all
conditions investigated, with small changes at the protein level and more
pronounced changes at the RNA level. Studies with seedlings revealed a low
expression of all three enzymes in cotyledons, when compared to hypocotyls and
radiculas. Immunohistochemical experiments indicated that MSY expression in
cotyledons is cell/​type specific, with the strongest signals detected in the
upper epidermis.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 11030427 [PubMed /​ indexed for MEDLINE]

180: Planta. 2000 Aug;211(3):390/​5.

Phosphate uptake across the tonoplast of intact vacuoles isolated from
suspension/​cultured cells of Catharanthus roseus (L.) G. Don.

Massonneau A, Martinoia E, Dietz KJ, Mimura T.

Institut de Botanique, Lab. de Physiologie Vegetale, Universite de Neuchatel,
Switzerland.

Transport of inorganic orthophosphate (Pi) across the tonoplast membrane was
studied using intact vacuoles isolated from suspension/​cultured cells of
Catharanthus roseus. Orthophosphate uptake was strongly stimulated in the
presence of Mg/​ATP and Mg/​pyrophosphate and inhibited by bafilomycin and
concanamycin which are potent inhibitors of the vacuolar H+/​ATPase. These
results indicated that the build/​up of an electrochemical gradient by the H /​
pumps was essential for the uptake of Pi. Potassium thiocyanate, which
dissipates the membrane potential across the tonoplast, strongly inhibited the
Mg/​ATP/​stimulated uptake of Pi, while only a weak inhibition was observed in the
presence of NH4Cl, which dissipates the pH gradient. These results indicate
that, as observed for other anions like malate or chloride, the electrical
component is the driving force of Pi uptake, whereas the deltapH plays only a
minor role. Possible competitive inhibitors of Pi, MoO4(2/​) , VO4(3/​) and
CrO4(2/​) were tested. Among them, CrO4(2/​) strongly inhibited Pi uptake into the
vacuoles. Various inhibitors of anion transport were also tested. Only
4,4/​diisothiocyanostilbene/​2,2'/​disulfonic acid strongly inhibited Pi uptake
into the vacuoles. The function of the vacuolar Pi transporters for cytoplasmic
Pi homeostasis is discussed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10987558 [PubMed /​ indexed for MEDLINE]

181: Cytobios. 2000;102(401):133/​47.

Histopathology and polyphenol content in plants infected by phytoplasmas.

Musetti R, Favali MA, Pressacco L.

Dipartimento di Biologia Applicata alla Difesa delle Piante, Universita' di
Udine, Italy.

The alterations of cell walls and the localization of several compounds such as
polyphenols, suberin, lignin, in plum and apple plants infected with plum
leptonecrosis (PLN) and apple proliferation (AP) phytoplasmas respectively, were
investigated. Catharanthus roseus plants, infected with AP or PLN were also
studied. The 4,6/​diamidino/​2/​phenylindole (DAPI) test and transmission electron
microscopy showed the presence of phytoplasmas in all infected plants. Specific
histological stainings for cutinized/suberinized cell walls, tannin deposits and
vacuolar polyphenol inclusions, performed on leaf and stem tissues, revealed an
increase of these substances in infected plum and apple plants. No differences
occurred in C. roseus. Total polyphenol analysis confirmed a strong increase
(3/​fold) in the polyphenol content in infected tissues, particularly in plum
leaves. From the data obtained it appears that polyphenols can be considered as
defence/​related metabolites in plum and apple plants infected by phytoplasmas.
Further investigations are necessary to determine whether these compounds play a
specific role in the development of all phytoplasma/host interactions and in the
defence/​related processes.

Publication Types:
Comparative Study

PMID: 10969878 [PubMed /​ indexed for MEDLINE]

182: Metab Eng. 1999 Jan;1(1):12/​25.

In vivo 31P and multilabel 13C NMR measurements for evaluation of plant
metabolic pathways.

Rijhwani SK, Ho CH, Shanks JV.

Department of Bioengineering, Rice University, Houston, Texas 77251/​1892, USA.

Reliable measurements of intracellular metabolites are useful for effective
plant metabolic engineering. This study explored the application of in situ 31P
and 13C NMR spectroscopy for long/​term measurements of intracellular pH and
concentrations of several metabolites in glycolysis, glucan synthesis, and
central carbon metabolic pathways in plant tissues. An NMR perfusion reactor
system was designed to allow Catharanthus roseus hairy root cultures to grow for
3/​6 weeks, during which time NMR spectroscopy was performed. Constant
cytoplasmic pH (7.40+//​0.06), observed during the entire experiment, indicated
adequate oxygenation. 13C NMR spectroscopy was performed on hairy root cultures
grown in solutions containing 1/​13C/​, 2/​13C/​, and 3/​13C/​labeled glucose in
separate experiments and the flow of label was monitored. Activities of pentose
phosphate pathways, nonphotosynthetic CO2 fixation, and glucan synthesis
pathways were evident from the experimental results. Scrambling of label in
glucans also indicated recycling of triose phosphate and their subsequent
conversion to hexose phosphates.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.
Research Support, U.S. Gov't, P.H.S.

PMID: 10935751 [PubMed /​ indexed for MEDLINE]

183: Science. 2000 Jul 14;289(5477):295/​7.

ORCA3, a jasmonate/​responsive transcriptional regulator of plant primary and
secondary metabolism.

van der Fits L, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
Wassenaarseweg 64, 2333 AL, Leiden, Netherlands.

Biosynthesis of many classes of secondary metabolites in plants is induced by
the stress hormone jasmonate. The gene for ORCA3, a jasmonate/​responsive
APETALA2 (AP2)/​domain transcription factor from Catharanthus roseus, was
isolated by transferred DNA activation tagging. Orca3 overexpression resulted in
enhanced expression of several metabolite biosynthetic genes and, consequently,
in increased accumulation of terpenoid indole alkaloids. Regulation of
metabolite biosynthetic genes by jasmonate/​responsive AP2/​domain transcription
factors may link plant stress responses to changes in metabolism.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10894776 [PubMed /​ indexed for MEDLINE]

184: Arch Biochem Biophys. 2000 Jun 15;378(2):287/​98.

Purification and characterization of mevalonate kinase from suspension/​cultured
cells of Catharanthus roseus (L.) G. Don.

Schulte AE, van der Heijden R, Verpoorte R.

Division of Pharmacognosy, Gorlaeus Laboratories, Leiden, 2300 RA, The
Netherlands. Annelies.Schulte@DeRuiterSeeds.com

Mevalonate kinase was purified to homogeneity from Catharanthus roseus (L.) G.
Don suspension/​cultured cells. The purified enzyme had an M(r) of 104,600 and a
subunit size of about 41,500. Kinetic studies indicated an ordered sequential
mechanism of action, in which mevalonate was the first substrate to bind and ADP
was the last product to leave the enzyme. True values for the kinetic constants
were determined for mevalonate, with K(ma) = 76 microM and K(ia) = 74 microM,
and for ATP, with K(mb) = 0.13 mM and K(ib) = 0. 13 mM; the true V(max) was
calculated to be 138.7 nkat/mg of protein. Product inhibition was only
detectable at rather high concentrations: above 0.7 mM for 5/​phosphomevalonate
and above 2 mM for ADP, with an ADP/ATP ratio of at least 1. Mevalonate kinase
activity was shown to be strongly inhibited by farnesyl diphosphate. Farnesyl
diphosphate acted as a competitive inhibitor toward ATP, with a K(i) value of
0.1 microM. Mevalonate kinase activity was dependent on the presence of divalent
ions. At a concentration of 2 mM, Mg(2+) and Mn(2+) were best and equally
effective in sustaining activity; compared to Mg(2+) and Mn(2+), relative
activities of 35, 30, 16, 4.8, and 3.4% were detected at equimolar
concentrations of Zn(2+), Fe(2+), Co(2+), Ca(2+), and Ni(2+), respectively. The
pH/​dependent activity profile of mevalonate kinase showed a broad pH optimum
between pH 7 and 10, with a maximum at about pH 8.9. Copyright 2000 Academic
Press.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10860546 [PubMed /​ indexed for MEDLINE]

185: Plant Cell Physiol. 2000 Apr;41(4):424/​31.

Regulation of cytoplasmic pH under extreme acid conditions in suspension
cultured cells of Catharanthus roseus: a possible role of inorganic phosphate.

Mimura T, Shindo C, Kato M, Yokota E, Sakano K, Ashihara H, Shimmen T.

Biological Laboratory, Hitotsubashi University, Kunitachi, Tokyo, Japan.

Changes in cytoplasmic pH of suspension/​cultured cells of Catharanthus roseus
under extreme acid conditions were measured with the pH/​dependent fluorescence
dye; 2',7'/​bis/​(2/​carboxyethyl)/​5 (and/​6) carboxyfluorescein
(/​acetoxymethylester) (BCECF). When cells were treated with 1 mM HCl (pH 3
solution), the cytoplasmic pH first decreased then returned to the original
level. Treatment with 10 mM HCl (pH 2 solution) acidified the cytoplasm to a
greater extent, and the acidification continued at a constant level throughout
the measurement. Treatment with a pH 2 solution resulted in a gradual decrease
of the malate content, indicating the operation of biochemical pH regulation
mechanism. The pH 2 treatment also caused a sudden decrease of the intracellular
level of Pi. The cellular content of total phosphorus did not change during the
acidification. The Pi was converted to the organic phosphate form. The ATP level
was not increased by the pH 2 treatment, but slightly decreased. The role of Pi,
which might be functioning as a regulatory factor of cytoplasmic pH, a
non/​competitive inhibitor of the H+/​pumps of both the plasma membrane and
tonoplast is discussed.

PMID: 10845455 [PubMed /​ indexed for MEDLINE]

186: J Biotechnol. 2000 Apr 28;79(2):137/​45.

Determination of metabolic rate/​limitations by precursor feeding in Catharanthus
roseus hairy root cultures.

Morgan JA, Shanks JV.

Department of Bioengineering and Chemical Engineering, Rice University, MS/​142,
6100 South Main Street, Houston, Texas 77005/​1892, USA.

Precursors from the terpenoid and tryptophan branches were fed to Catharanthus
roseus to determine which of the two branches limits metabolic flux to indole
alkaloids. The feeding of tryptophan at 17 days of the culture cycle produced
auxin/​like effects. Addition of low levels of auxin or tryptophan resulted in
significant increases in flux to the indole alkaloids. Conversely, feeding
higher levels of auxin or tryptophan resulted in increased branching and
thickening of the hairy root cultures. A dramatic reduction in flux to the
alkaloids was also observed. However, feeding tryptamine or terpenoid precursors
had no effect. Therefore, neither pathway tested revealed to be rate/​limiting
during the late growth phase. Feeding of either geraniol, 10/​hydroxygeraniol, or
loganin at 21 days each resulted in significant increases in the accumulation of
tabersonine. The addition of tryptophan or tryptamine had no effect during the
stationary phase of the growth cycle. Thus, during the early stationary phase of
growth the terpenoid pathway appears to be rate/​limiting. Combined elicitation
with jasmonic acid and feeding either loganin or tryptamine did not further
enhance the accumulation of indole alkaloids.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.
Research Support, U.S. Gov't, P.H.S.

PMID: 10812182 [PubMed /​ indexed for MEDLINE]

187: Appl Microbiol Biotechnol. 2000 Mar;53(3):262/​5.

Effects of buffered media upon growth and alkaloid production of Catharanthus
roseus hairy roots.

Morgan JA, Barney CS, Penn AH, Shanks JV.

Department of Chemical Engineering, Iowa State University, Ames 50011/​2230, USA.

The influence of buffered media upon the growth and alkaloid productivity of
Catharanthus roseus hairy root culture was examined. As expected, the buffers
minimized shifts in the pH of the media and had slightly negative effects upon
growth. The growth of the hairy roots remained optimal in unbuffered media. The
specific yield of lochnericine was significantly lower in response to the
addition of buffers, while tabersonine was significantly higher. In contrast,
the specific yields of ajmalicine, serpentine, and horhammericine remained
unchanged.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.
Research Support, U.S. Gov't, P.H.S.

PMID: 10772463 [PubMed /​ indexed for MEDLINE]

188: Phytochemistry. 2000 Mar;53(5):549/​53.

Biosynthesis of brassinosteroids in cultured cells of Catharanthus roseus.

Fujioka S, Noguchi T, Watanabe T, Takatsuto S, Yoshida S.

The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
sfujioka@postman.riken.go.jp

Precursor administration experiments with 2H/​labeled 6/​oxocampestanol,
6/​deoxocastasterone and 6alpha/​hydroxycastasterone in cultured cells of
Catharanthus roseus were performed and the metabolites were analyzed by GC/​MS.
[2H6]Cathasterone was identified as a metabolite of [2H6]6/​oxocampestanol,
whereas [2H6]6alpha/​hydroxycastasterone and [2H6]castasterone were identified as
metabolites of [2H6]6/​deoxocastasterone, and [2H6]castasterone was identified as
a metabolite of [2H6]6alpha/​hydroxycastasterone, indicating that
6/​deoxocastasterone is converted to castasterone via 6alpha/​hydroxycastasterone.
In addition, 6/​deoxocathasterone, a putative biosynthetic intermediate in the
late C6/​oxidation pathway, was identified as an endogenous brassinosteroid.
These studies provide further evidence supporting our proposed biosynthetic
pathways for brassinolide.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10724179 [PubMed /​ indexed for MEDLINE]

189: J Biol Chem. 2000 Feb 4;275(5):3051/​6.

Molecular cloning and analysis of strictosidine beta/​D/​glucosidase, an enzyme in
terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Geerlings A, Ibanez MM, Memelink J, van Der Heijden R, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden
University, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands.

Strictosidine beta/​D/​glucosidase (SGD) is an enzyme involved in the biosynthesis
of terpenoid indole alkaloids (TIAs) by converting strictosidine to cathenamine.
The biosynthetic pathway toward strictosidine is thought to be similar in all
TIA/​producing plants. Somewhere downstream of strictosidine formation, however,
the biosynthesis diverges to give rise to the different TIAs found. SGD may play
a role in creating this biosynthetic diversity. We have studied SGD at both the
molecular and enzymatic levels. Based on the homology between different plant
beta/​glucosidases, degenerate polymerase chain reaction primers were designed
and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A
full/​length clone gave rise to SGD activity when expressed in Saccharomyces
cerevisiae. SGD shows approximately 60% homology at the amino acid level to
other beta/​glucosidases from plants and is encoded by a single/​copy gene. Sgd
expression is induced by methyl jasmonate with kinetics similar to those of two
other genes acting prior to Sgd in TIA biosynthesis. These results show that
coordinate induction of the biosynthetic genes forms at least part of the
mechanism for the methyl jasmonate/​induced increase in TIA production. Using a
novel in vivo staining method, subcellular localization studies of SGD were
performed. This showed that SGD is most likely associated with the endoplasmic
reticulum, which is in accordance with the presence of a putative signal
sequence, but in contrast to previous localization studies. This new insight in
SGD localization has significant implications for our understanding of the
complex intracellular trafficking of metabolic intermediates during TIA
biosynthesis.

PMID: 10652285 [PubMed /​ indexed for MEDLINE]

190: Cytobios. 1999;100(394):119/​26.

Subcellular localization of the coat protein in tobacco cells infected by
cucumber mosaic virus isolated from Catharanthus roseus.

Espinha LM, Gaspar JO.

Departamento de Botanica, IBILCE/UNESP, Sao Jose do Rio Preto, SP, Brasil.

Electron microscopy and immunolabelling with antiserum specific to cucumber
mosaic virus coat protein were used to examine tobacco leaf cells infected by
cucumber mosaic virus isolated from Catharanthus roseus (CMV/​Cr). Crystalline
and amorphous inclusions in the vacuoles were the most obvious cytological
modifications seen. Immunogold labelling indicated that the crystalline
inclusion was made up of virus particles and amorphous inclusions contained coat
protein. Rows of CMV/​Cr particles were found between membranes of dictyosomes,
but membranous bodies and tonoplast/​associated vesicles were not evident. Virus
particles and/or free coat protein were easily detected in the cytoplasm by
immunolabelling. No gold labelling was found within nuclei, chloroplasts and
mitochondria.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10643647 [PubMed /​ indexed for MEDLINE]

191: J Ethnopharmacol. 1999 Nov 30;67(3):367/​72.

A comparative evaluation of some blood sugar lowering agents of plant origin.

Chattopadhyay RR.

Biometry Research Unit, Indian Statistical Institute, Calcutta.

A comparison of blood sugar lowering activity of four important medicinal plants
(Azadirachta indica, Gymnema sylvestre, Catharanthus roseus and Ocimum sanctum)
were carried out against normal and streptozotocin/​induced diabetic rat models.
The plant extracts decreased the blood sugar level in varying degrees. Blood
sugar lowering unit (BLU) of activity of each leaf extract and tolbutamide was
calculated by ED50 values. Statistical analysis revealed significant (P < 0.05)
variation among the treatments as well as doses with regard to their blood sugar
lowering capacity. A. indica leaf extract was found to have the most potent
blood sugar/​lowering activity followed by C. roseus, G. sylvestre and O.
sanctum.

Publication Types:
Comparative Study

PMID: 10617074 [PubMed /​ indexed for MEDLINE]

192: Plant Mol Biol. 1999 Nov;41(4):491/​503.

Identification of UV/​B light/​responsive regions in the promoter of the
tryptophan decarboxylase gene from Catharanthus roseus.

Ouwerkerk PB, Hallard D, Verpoorte R, Memelink J.

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory,
The Netherlands.

The tryptophan decarboxylase (Tdc) gene encodes a key enzyme in the biosynthesis
of terpenoid indole alkaloids (TIAs) in Catharanthus roseus. TIAs absorb
ultraviolet light (UV) and putative functions in plants include a role as UV
protectants. In support of this possible function we demonstrate here that UV
light induces accumulation of several TIAs as well as expression of the Tdc gene
in C. roseus. In addition, in tobacco a Tdc/​gusA construct was found to be
specifically induced by UV/​B light. Lack of induction by UV/​A or other
wavelengths of light indicate that Tdc expression is regulated by a specific
UV/​B receptor and corresponding signal transduction pathway. To identify
UV/​responsive Tdc promoter elements, a loss/​of/​function analysis was performed,
in which deletion derivatives were fused to the gusA reporter gene and analysed
in transgenic tobacco plants. Truncation of the Tdc promoter from /​1818
(relative to the start of transcription) to /​160 reduced expression levels
two/​fold without affecting the qualitative UV response. Deletion to /​37 further
reduced expression levels five/​fold, but the delta37 promoter also remained
UV/​responsive. Subsequently, the /​160 to /​37 region was further studied by
gain/​of/​function experiments, in which the transcriptional activities of
tetramerized subfragments fused to truncated promoters were analysed.
Combination of the data identified several functional regions in the /​160 to
+198 promoter. The /​ 160 to /​99 region acts as the main transcriptional
enhancer. UV/​responsive elements appeared to be redundant in the /​160 Tdc
promoter and to reside between /​99 and /​37 and between /​37 and + 198.

PMID: 10608659 [PubMed /​ indexed for MEDLINE]

193: J Bacteriol. 1999 Dec;181(24):7449/​56.

Gene disruption through homologous recombination in Spiroplasma citri: an
scm1/​disrupted motility mutant is pathogenic.

Duret S, Danet JL, Garnier M, Renaudin J.

Laboratoire de Biologie Cellulaire et Moleculaire, INRA et Universite Victor
Segalen Bordeaux 2, 33883 Villenave d'Ornon Cedex, France.

To determine whether homologous recombination could be used to inactivate
selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt
the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid
pKT1, which replicates in Escherichia coli but not in S. citri, and into the S.
citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E.
coli. Electrotransformation of S. citri with the nonreplicative, recombinant
plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants
were obtained with the replicative, pBOT1/​derived plasmid pCJ32. During
passaging of the transformants, the plasmid was found to integrate into the
chromosome by homologous recombination either at the oriC region or at the scm1
gene. In the latter case, plasmid integration by a single crossover between the
scm1 gene fragment carried by the plasmid and the full/​length scm1 gene carried
by the chromosome led to a nonmotile phenotype. Transmission of the
scm1/​disrupted mutant to periwinkle (Catharanthus roseus) plants through
injection into the leafhopper vector (Circulifer haematoceps) showed that the
motility mutant multiplied in the insects and was efficiently transmitted to
plants, in which it induced symptoms similarly to the wild/​type S. citri strain.
These results suggest that the spiroplasmal motility may not be essential for
pathogenicity and that, more broadly, the S. citri oriC plasmids can be
considered promising tools for specific gene disruption by promoting homologous
recombination in S. citri, a mollicute which probably lacks a functional RecA
protein.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10601200 [PubMed /​ indexed for MEDLINE]

194: Biotechnol Bioeng. 2000 Jan 5;67(1):61/​71.

The effect of inoculum density and conditioned medium on the production of
ajmalicine and catharanthine from immobilized Catharanthus roseus cells.

Lee CW, Shuler ML.

120 Olin Hall, School of Chemical Engineering; Cornell University, Ithaca, New
York 14853/​5201, USA.

The effect of the cell/​inoculum size and the addition of conditioned medium on
ajmalicine and catharanthine production were studied using immobilized
Catharanthus roseus cells. Higher specific/​uptake rates of ammonium, nitrate,
and sugars were observed in the low/​inoculum/​density cultures (50 g FW/L)
compared to the high/​inoculum/​density cultures (100 g FW/L). Alkaloid production
was not correlated with the exhaustion of a particular nutrient from the medium.
The high/​inoculum/​density cultures produced higher ajmalicine concentrations
throughout the experiment. Catharanthine production was similar between the two
inoculum/​density cultures. The addition of conditioned medium to MS/​production
medium dramatically improved the production of ajmalicine and catharanthine. The
addition of conditioned medium enhanced ajmalicine production from immobilized
Catharanthus roseus cultures on day 15 by at least two/​ to fourfold compared to
media without the conditioning factors. Catharanthine production was increased
by nearly fivefold in cultures with conditioned medium compared to those without
conditioned medium. The enhancing effects of conditioned medium on alkaloid
production were attributed to an unidentified factor produced and secreted by
suspension cultures of C. roseus. The presence of conditioned medium also
decreased the sucrose hydrolysis rate. The ajmalicine concentration in these
immobilized cell cultures was found to be a function of the fresh/​weight
concentration, irrespective of the inoculum density or the culture medium. The
medium choice and the inoculum density determined how rapidly fresh weight was
accumulated and thus, how quickly ajmalicine was produced. Ajmalicine production
correlated positively with fresh/​weight concentration, but catharanthine
production was not correlated with fresh/​weight concentration. Copyright 2000
John Wiley & Sons, Inc.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 10581436 [PubMed /​ indexed for MEDLINE]

195: Biotechnol Bioeng. 1999;66(3):147/​57.

A simple structured model for maintenance, biomass formation, and ajmalicine
production by nondividing Catharanthus roseus cells.

Schlatmann JE, ten Hoopen HJ, Heijnen JJ.

Biotechnological Sciences Delft Leiden, Sector Industrial Plant Biotechnology,
Department of Biochemical Engineering, Delft University of Technology,
Julianalaan 67, 2628 BC Delft, The Netherlands.

The stoichiometry of maintenance and carbohydrate storage as well as ajmalicine
production kinetics of non/​dividing Catharanthus roseus cells in the second
stage of a two/​stage batch process were investigated. For the mathematical
description of these processes, a simple structured model with 5 parameters is
proposed. In the model the biomass is divided in two compartments: active
biomass and storage carbohydrates. In induction medium (standard medium without
phosphate, nitrogen and hormones), biomass formation, glucose consumption, and
CO(2) production appeared to be constant in time. Therefore, it is assumed that
the active biomass level is constant. The maintenance coefficient m(S), and the
yield of storage carbohydrates on glucose Y(SC) were optimized by fitting the
model on experimental data: 0.003 C/​mol/C/​mol/h and 0.82 C/​mol/C/​mol,
respectively. Production kinetics were incorporated in this model and related to
the active biomass fraction. The maximum specific ajmalicine production rate
q(p)(max) was fitted on the data: 7.5 micromol/C/​mol/h. The model was tested at
several different experimental conditions, and proved to describe the
experimental results adequately. An independent experiment at a very high cell
density in order to obtain maximum product formation was used to validate the
model. It provided a satisfactory description of the results, but the final
ajmalicine concentration (198 micromol/L after 18 days) was lower than the
calculated maximum, due to accumulation of inhibiting gaseous metabolites.
Copyright 1999 John Wiley & Sons, Inc.

PMID: 10577468 [PubMed /​ indexed for MEDLINE]

196: FEBS Lett. 1999 Sep 17;458(2):97/​102.

Light/​induced cytochrome P450/​dependent enzyme in indole alkaloid biosynthesis:
tabersonine 16/​hydroxylase.

Schroder G, Unterbusch E, Kaltenbach M, Schmidt J, Strack D, De Luca V, Schroder
J.

Institut fur Biologie II, Universitat Freiburg, Germany.

Vinblastine and vincristine are two medically important bisindole alkaloids from
Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell
cultures failed because a part of the complex pathway was not active, i.e. from
tabersonine to vindoline. It starts with tabersonine 16/​hydroxylase (T16H), a
cytochrome P450/​dependent enzyme. We now show that T16H is induced in the
suspension culture by light and we report the cloning of the cDNA. The enzyme
was expressed in Escherichia coli as translational fusion with the P450
reductase from C. roseus, and the reaction product was identified by mass
spectrometry. The protein (CYP71D12) shares 47/​52% identity with other members
of the CYP71D subfamily with unknown function. The induction by light was
strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose).
We discuss the possibility that the entire pathway to bisindoles can be
expressed in suspension cultures.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10481044 [PubMed /​ indexed for MEDLINE]

197: Plant J. 1999 Jul;19(2):183/​93.

Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression,
enzyme properties and cell/​type specific expression in plants.

Kaltenbach M, Schroder G, Schmelzer E, Lutz V, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

We investigated the P450 dependent flavonoid hydroxylase from the ornamental
plant Catharanthus roseus. cDNAs were obtained by heterologous screening with
the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68/​78%
identity with other CYP75s, and genomic blots suggested one or two genes. The
protein was expressed in Escherichia coli as translational fusion with the P450
reductase from C. roseus. Enzyme assays showed that it was a flavonoid 3',
5'/​hydroxylase, but 3'/​hydroxylated products were also detected. The substrate
specificity was investigated with the C. roseus enzyme and a fusion protein of
the Petunia hybrida CYP75 with the C. roseus P450 reductase. Both enzymes
accepted flavanones as well as flavones, dihydroflavonols and flavonols, and
both performed 3'/​ as well as 3'5'/​hydroxylation. Kinetics with C. roseus
cultures on the level of enzyme activity, protein and RNA showed that the F3'5'H
was present in dark/​grown cells and was induced by irradiation. The same results
were obtained for cinnamic acid 4/​hydroxylase and flavanone 3beta/​hydroxylase.
In contrast, CHS expression was strictly dependent on light, although CHS is
necessary in the synthesis of the F3'5'H substrates. Immunohistochemical
localization of F3'5'H had not been performed before. A comparison of CHS and
F3'5'H in cotyledons and flower buds from C. roseus identified CHS expression
preferentially in the epidermis, while F3'5'H was only detected in the phloem.
The cell/​type specific expression suggests that intercellular transport may play
an important role in the compartmentation of the pathways to the different
flavonoids.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10476065 [PubMed /​ indexed for MEDLINE]

198: J Chromatogr A. 1999 Jul 23;849(2):421/​31.

Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition
chromatography in the pH/​zone refining mode.

Renault JH, Nuzillard JM, Le Crouerour G, Thepenier P, Zeches/​Hanrot M, Le
Men/​Olivier L.

Laboratoire de Pharmacognosie, UPRES/​A CNRS 6013, CPCBAI, Reims, France.

Centrifugal partition chromatography (CPC) in the pH/​zone refining mode allowed
a preparative and efficient isolation of vindoline, vindolinine, catharanthine
and vincaleukoblastine from a crude mixture of Catharanthus roseus alkaloids.
The separation protocol was tested with a synthetic mixture of vindoline,
catharanthine and vincaleukoblastine. The fraction content was analyzed and the
results compared with theoretical chromatograms obtained by numerical
simulation. The increase in injected sample mass results in an improvement of
the purity of the isolated compounds. This observation, confirmed by theory, is
of prime importance for the development of preparative pH/​zone refining CPC as a
preparative separation method.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10457440 [PubMed /​ indexed for MEDLINE]

199: EMBO J. 1999 Aug 16;18(16):4455/​63.

A novel jasmonate/​ and elicitor/​responsive element in the periwinkle secondary
metabolite biosynthetic gene Str interacts with a jasmonate/​ and
elicitor/​inducible AP2/​domain transcription factor, ORCA2.

Menke FL, Champion A, Kijne JW, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.

Jasmonate (JA) is an important plant stress hormone that induces various plant
defense responses, including the biosynthesis of protective secondary
metabolites. The induction of the secondary metabolite biosynthetic gene
Strictosidine synthase (Str) in Catharanthus roseus (periwinkle) cells by
elicitor requires JA as a second messenger. A 42 bp region in the Str promoter
is both necessary and sufficient for JA/​ and elicitor/​responsive expression.
This region is unlike other previously identified JA/​responsive regions, and
contains a GCC/​box/​like element. Yeast one/​hybrid screening identified cDNAs
encoding two AP2/​domain proteins. These octadecanoid/​derivative responsive
Catharanthus AP2/​domain (ORCA) proteins bind in a sequence/​specific manner the
JA/​ and elicitor/​responsive element. ORCA2 trans/​activates the Str promoter and
its expression is rapidly inducible with JA and elicitor, whereas Orca1 is
expressed constitutively. The results indicate that a GCC/​box/​like element and
ORCA2 play key roles in JA/​ and elicitor/​responsive expression of the terpenoid
indole alkaloid biosynthetic gene Str.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10449411 [PubMed /​ indexed for MEDLINE]

200: Plant Physiol. 1999 Aug;120(4):1075/​82.

Kinetic analysis of phospholipase C from catharanthus roseus transformed roots
using different assays

Hernandez/​Sotomayor SM, De Los Santos/​Briones C, Munoz/​Sanchez JA, Loyola/​Vargas
VM.

Unidad de Biologia Experimental, Centro de Investigacion Cientifica de Yucatan,
Apartado Postal 87 Cordemex 97310, Merida, Yucatan, Mexico.

The properties of phospholipase C (PLC) partially purified from Catharanthus
roseus transformed roots were analyzed using substrate lipids dispersed in
phospholipid vesicles, phospholipid/​detergent mixed micelles, and phospholipid
monolayers spread at an air/​water interface. Using [(33)P]phosphatidylinositol
4,5/​bisphosphate (PIP(2)) of high specific radioactivity, PLC activity was
monitored directly by measuring the loss of radioactivity from monolayers as a
result of the release of inositol phosphate and its subsequent dissolution on
quenching in the subphase. PLC activity was markedly affected by the surface
pressure of the monolayer, with reduced activity at extremes of initial
pressure. The optimum surface pressure for PIP(2) hydrolysis was 20 mN/m.
Depletion of PLC from solution by incubation with sucrose/​loaded PIP(2) vesicles
followed by ultracentrifugation demonstrated stable attachment of PLC to the
vesicles. A mixed micellar system was established to assay PLC activity using
deoxycholate. Kinetic analyses were performed to determine whether PLC activity
was dependent on both bulk PIP(2) and PIP(2) surface concentrations in the
micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol
fraction, and the equilibrium dissociation constant of PLC for the lipid was
45.5 &mgr;M. These findings will add to our understanding of the mechanisms of
regulation of plant PLC.

PMID: 10444091 [PubMed /​ as supplied by publisher]

201: Protein Expr Purif. 1999 Jul;16(2):231/​5.

Purification of xyloglucan endotransglycosylase based on affinity sorption of
the active glycosyl/​enzyme intermediate complex to cellulose.

Sulova Z, Farkas V.

Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9,
Bratislava, 84238, Slovakia.

Xyloglucan endotransglycosylase (XET) catalyzes the cleavage of xyloglucan (XG)
molecules by a transglycosylation mechanism involving two steps: (a)
endocleavage of the beta/​(1,4)/​linked polyglucosyl backbone of the xyloglucan
molecule with formation of a glycosyl/​enzyme intermediate; (b) transfer of the
glycosyl residue from the intermediate to the C/​4 position of the nonreducing
end glucosyl unit of another molecule of XG or an XG/​derived oligosaccharide
with liberation of the enzyme (Z. Sulova et al., 1998, Biochem. J. 330,
1475/​1480). The formation of a relatively stable active complex of XET with XG
and the tendency of xyloglucan to bind tightly via hydrogen bonds to cellulose
were exploited in the present method of purification of XET. Crude extracts from
nasturtium (Tropaeolum majus) cotyledons and other plant sources containing the
enzyme were mixed with XG in order to form the XET:XG complex, which was applied
onto cellulose. Unadsorbed proteins were removed by washing and the XET was
released from the adsorbed XET:XG complex by transglycosylation of its glycosyl
moiety to added XG/​derived oligosaccharides. The described procedure resulted in
an over 100/​fold increase in specific activity of XET in a single step. Further
purification of the enzyme to homogeneity was achieved by gel/​permeation
chromatography on Bio/​Gel P30. Similar procedure could be used for purification
of XET from other plant sources, such as lentil (Lens culinaris) seeds, pea
(Pisum sativum) epicotyls, and supernatant of suspension/​cultured Catharanthus
roseus cells. Copyright 1999 Academic Press.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10419819 [PubMed /​ indexed for MEDLINE]

202: Plant Physiol. 1999 Jul;120(3):833/​40.

Arabidopsis det2 is defective in the conversion of
(24R)/​24/​methylcholest/​4/​En/​3/​one to (24R)/​24/​methyl/​5alpha/​cholestan/​3/​one in
brassinosteroid biosynthesis.

Noguchi T, Fujioka S, Takatsuto S, Sakurai A, Yoshida S, Li J, Chory J.

The Institute of Physical and Chemical Research (RIKEN), Wako/​shi, Saitama
351/​0198, Japan.

Previously, we have shown that the Arabidopsis det2 (deetiolated2) mutant is
defective in the biosynthesis of brassinosteroids (BR) and that DET2 (a steroid
5alpha/​reductase) acts early in the proposed BR biosynthetic pathway. In this
paper we present further biochemical characterization of det2. We have
undertaken metabolic experiments with 2H/​labeled substrates of intermediates
involved in the formation of campestanol from campesterol, and quantitative
analysis of intermediates in Arabidopsis wild type and det2. The results of
these studies indicate the early operating steps of BR biosynthesis as:
campesterol /​/​> 4/​en/​3beta/​ol /​/​> 4/​en/​3/​one /​/​> 3/​one /​/​> campestanol in
Arabidopsis, with det2 deficient in the conversion of 4/​en/​3/​one to 3/​one. We
have also detected these intermediates in the formation of campestanol from
campesterol and their metabolic conversions using cultured cells of Catharanthus
roseus. These studies confirmed the biosynthetic sequence of events from
campesterol to campestanol as was found in Arabidopsis. As such, the originally
proposed biosynthetic pathway should be modified.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10398719 [PubMed /​ indexed for MEDLINE]

203: Mol Gen Genet. 1999 Jun;261(4/​5):635/​43.

A G/​box element from the Catharanthus roseus strictosidine synthase (Str) gene
promoter confers seed/​specific expression in transgenic tobacco plants.

Ouwerkerk PB, Memelink J.

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory,
The Netherlands.

The enzyme encoded by the strictosidine synthase (Str) gene from Catharanthus
roseus catalyses a key step in the biosynthesis of the pharmaceutically
important terpenoid indole alkaloids. Str cDNA and genomic clones have already
been isolated, allowing us to study the regulation of Str gene expression. Here
we focus on the role of a putative cis/​acting element, CACGTG, in the Str
promoter. This sequence is known as a G/​box, and functions as a
transcription/​regulating sequence in a number of other promoters. By means of
electrophoretic mobility shift assays it was demonstrated that the Str G/​box is
capable of interacting with nuclear factors in tobacco and with the cloned
tobacco G/​box/​binding factor TAF/​1. Disruption of the Str G/​box sequence by two
single/​nucleotide mutations prevented binding of factors, thereby demonstrating
the specificity of the observed interactions. Functional analysis in transgenic
tobacco plants demonstrated that these mutations also reduced the
transcriptional activity of constructs containing tetramers of the Str G/​box
sequence. Expression directed by a tetramer of the Str G/​box fused to a
truncated promoter containing only a TATA box was confined to seeds and was
found to increase during seed maturation. Thus, the Str G/​box tetramer is able
to direct seed/​specific expression independently of other regulatory sequences.
G/​box/​directed expression in leaves required the presence of an enhancer region
from the cauliflower mosaic virus (CaMV) 35S promoter. The results indicate that
the G/​box needs to interact with other elements to drive expression in leaf, and
that it can by itself confer seed/​specific expression as a multimer. The fact
that only some of the G/​boxes found in different promoters serve as
seed/​specific elements indicates that sequences flanking the G/​box determine the
transcriptional activity in different tissues. Based on sequence comparisons we
propose that the nucleotides at positions /​4, /​3, /​2 and/or +4 are important in
determining seed/​specific expression.

PMID: 10394900 [PubMed /​ indexed for MEDLINE]

204: Mol Gen Genet. 1999 Jun;261(4/​5):610/​22.

Nuclear factors GT/​1 and 3AF1 interact with multiple sequences within the
promoter of the Tdc gene from Madagascar periwinkle: GT/​1 is involved in UV
light/​induced expression.

Ouwerkerk PB, Trimborn TO, Hilliou F, Memelink J.

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory,
The Netherlands.

Plant secondary metabolites of the terpenoid indole alkaloid (TIA) class
comprise several compounds with pharmaceutical applications. A key step in the
TIA biosynthetic pathway is catalysed by the enzyme tryptophan decarboxylase
(TDC), which channels the primary metabolite tryptophan into TIA metabolism. In
Catharanthus roseus (Madagascar periwinkle), the Tdc gene is expressed
throughout plant development. Moreover, Tdc gene expression is induced by
external stress signals, such as fungal elicitor and UV light. In a previous
study of Tdc promoter architecture in transgenic tobacco it was shown that the
/​538 to /​112 region is a quantitative determinant for the expression level in
different plant organs. Within this sequence one particular region (/​160 to /​99)
was identified as the major contributor to basal expression and another region
(/​99 to /​37) was shown to be required for induction by fungal elicitor. Here,
the in vitro binding of nuclear factors to the /​572 to /​37 region is described.
In extracts from tobacco and C. roseus, two binding activities were detected
that could be identified as the previously described nuclear factors GT/​1 and
3AF1, based on their mobility and binding characteristics. Both factors appeared
to interact with multiple regions in the Tdc promoter. Mutagenesis of GT/​1
binding sites in the Tdc promoter did not affect the basal or elicitor/​induced
expression levels. However, induction of the Tdc promoter constructs by UV light
was significantly lower, thereby demonstrating a functional role for GT/​1 in the
induction of Tdc expression by UV light.

PMID: 10394897 [PubMed /​ indexed for MEDLINE]

205: Plant Mol Biol. 1999 Apr;39(6):1299/​310.

The promoter of the strictosidine synthase gene from periwinkle confers
elicitor/​inducible expression in transgenic tobacco and binds nuclear factors
GT/​1 and GBF.

Pasquali G, Erven AS, Ouwerkerk PB, Menke FL, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden, Netherlands.

Strictosidine synthase (STR) is a key enzyme in the biosynthesis of terpenoid
indole alkaloids. This class of secondary metabolites harbours several
pharmaceutically important compounds used, among other applications, in cancer
treatment. Terpenoid indole alkaloid biosynthesis and expression of biosynthetic
genes including Str1 is induced by fungal elicitors. To identify
elicitor/​responsive regulatory promoter elements and trans/​acting factors, the
single/​copy Str1 gene was isolated from the subtropical plant species
Catharanthus roseus (Madagascar periwinkle). Str1 upstream sequences conferred
elicitor/​responsive expression to the beta/​glucuronidase (gusA) reporter gene in
transgenic tobacco plants. Main enhancer sequences within the Str1 promoter
region studied were shown to be located between /​339 and /​145. This region and
two other regions of the promoter bound the tobacco nuclear protein factor GT/​1.
A G/​box located around position /​105 bound nuclear and cloned G/​box/​binding
factors (GBFs). A mutation that knocked out GBF binding had no measurable effect
on expression, which indicates that the G/​box is not essential for the elicitor
responsiveness of the Str1 promoter. No obvious homologies with promoter
elements identified in other elicitor/​responsive genes were observed, suggesting
that the Str1 gene may depend on novel regulatory mechanisms.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10380815 [PubMed /​ indexed for MEDLINE]

206: Plant Cell. 1999 May;11(5):887/​900.

Multicellular compartmentation of catharanthus roseus alkaloid biosynthesis
predicts intercellular translocation of a pathway intermediate

St/​Pierre B, Vazquez/​Flota FA, De Luca V.

Institut de Recherche en Biologie Vegetale, Departement de Sciences Biologiques,
Universite de Montreal, 4101 rue Sherbrooke est, Montreal, Quebec H1X 2B2,
Canada.

In situ RNA hybridization and immunocytochemistry were used to establish the
cellular distribution of monoterpenoid indole alkaloid biosynthesis in
Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and
strictosidine synthase (STR1), which are involved in the biosynthesis of the
central intermediate strictosidine, and desacetoxyvindoline 4/​hydroxylase (D4H)
and deacetylvindoline 4/​O/​acetyltransferase (DAT), which are involved in the
terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs
were present in the epidermis of stems, leaves, and flower buds, whereas they
appeared in most protoderm and cortical cells around the apical meristem of root
tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer
and idioblast cells of leaves, stems, and flower buds. Immunocytochemical
localization for TDC, D4H, and DAT proteins confirmed the differential
localization of early and late stages of vindoline biosynthesis. Therefore, we
concluded that the elaboration of the major leaf alkaloids involves the
participation of at least two cell types and requires the intercellular
translocation of a pathway intermediate. A basipetal gradient of expression in
maturing leaves also was shown for all four genes by in situ RNA hybridization
studies and by complementary studies with dissected leaves, suggesting that
expression of the vindoline pathway occurs transiently during early leaf
development. These results partially explain why attempts to produce vindoline
by cell culture technology have failed.

PMID: 10330473 [PubMed /​ as supplied by publisher]

207: Anal Biochem. 1999 May 1;269(2):245/​54.

Microplate enzyme/​coupled assays of mevalonate and phosphomevalonate kinase from
Catharanthus roseus suspension cultured cells.

Schulte AE, van der Heijden R, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Gorlaeus
Laboratories, Leiden, 2300 RA, The Netherlands. A.E.Schulte@STM.TUDelft.nl

A microplate assay for mevalonate and 5/​phosphomevalonate kinase activities has
been developed using an enzyme/​coupled system of pyruvate kinase and lactate
dehydrogenase. Mevalonate and 5/​phosphomevalonate kinase activities were
measured in crude and partially purified enzyme preparations from Catharanthus
roseus suspension/​cultured plant cells. The assay was validated with respect to
protein and substrate concentration. Mevalonate and 5/​phosphomevalonate kinase
showed Michaelis/​Menten kinetics with respect to ATP and their specific
substrates; the apparent Km values of mevalonate kinase for ATP and mevalonate
were 210 and 65 microM, respectively, and of 5/​phosphomevalonate kinase for ATP
and 5/​phosphomevalonate were 0.41 and 0.4 mM, respectively. Considering
mevalonate kinase, the relative standard deviation of enzyme activity within a
determination (n = 3) is always less than 2.5% and in between determinations (n
= 9) is less than 2%. The method can be used in a continuous assay as well as in
a discontinuous assay. Copyright 1999 Academic Press.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 10221996 [PubMed /​ indexed for MEDLINE]

208: Plant Physiol. 1999 Apr;119(4):1289/​96.

Involvement of the octadecanoid pathway and protein phosphorylation in fungal
elicitor/​induced expression of terpenoid indole alkaloid biosynthetic genes in
catharanthus roseus

Menke FL, Parchmann S, Mueller MJ, Kijne JW, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
Wassenaarseweg 64, 2333 AL Leiden, The Netherlands (F.L. H.M., J.W.K., J.M.).

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding
tryptophan decarboxylase and strictosidine synthase, respectively, are
coordinately induced by fungal elicitors in suspension/​cultured Catharanthus
roseus cells. We have studied the roles of the jasmonate biosynthetic pathway
and of protein phosphorylation in signal transduction initiated by a partially
purified elicitor from yeast extract. In addition to activating Tdc and Str gene
expression, the elicitor also induced the biosynthesis of jasmonic acid. The
jasmonate precursor alpha/​linolenic acid or methyl jasmonate (MeJA) itself
induced Tdc and Str gene expression when added exogenously.
Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both
the elicitor/​induced formation of jasmonic acid and the activation of terpenoid
indole alkaloid biosynthetic genes. The protein kinase inhibitor K/​252a
abolished both elicitor/​induced jasmonate biosynthesis and MeJA/​induced Tdc and
Str gene expression. Analysis of the expression of Str promoter/gusA fusions in
transgenic C. roseus cells showed that the elicitor and MeJA act at the
transcriptional level. These results demonstrate that the jasmonate biosynthetic
pathway is an integral part of the elicitor/​triggered signal transduction
pathway that results in the coordinate expression of the Tdc and Str genes and
that protein kinases act both upstream and downstream of jasmonates.

PMID: 10198087 [PubMed /​ as supplied by publisher]

209: Biotechnol Bioeng. 1998 Apr 5;58(2/​3):333/​8.

Quantification of metabolites in the indole alkaloid pathways of catharanthus
roseus: implications for metabolic engineering

Shanks JV, Bhadra R, Morgan J, Rijhwani S, Vani S.

Institute of Biosciences and Bioengineering, Department of Chemical Engineering,
MS/​362, Rice University, Houston, Texas 77005/​1892, USA.

In this article, we present a review of the current state of metabolic
engineering in Catharanthus roseus. A significant amount of research has
contributed to characterization of several individual steps in the biosynthetic
pathway of medicinally valuable alkaloids. However, knowledge of the regulation
of these pathways is still sparse. Using hairy root cultures, we studied the
responses of alkaloid metabolism to environmental stimulation such as light and
elicitation. Through precursor feeding studies, the putative rate/​limiting steps
of the terpenoid pathway in hairy root cultures also have been examined.
Relating this knowledge to specific events at the molecular level, and the
cloning of corresponding genes are the next key steps in metabolic engineering
of the C. roseus alkaloids. Copyright 1998 John Wiley & Sons, Inc.

PMID: 10191413 [PubMed /​ as supplied by publisher]

210: Biotechnol Bioeng. 1998 Dec 20;60(6):670/​8.

Transient studies of light/​adapted cultures of hairy roots of Catharanthus
roseus: growth and indole alkaloid accumulation.

Bhadra R, Morgan JA, Shanks JV.

Department of Bioengineering, MS/​142, Rice University, 6100 Main Street,
Houston, Texas 77005/​1892, USA.

Cultures of C. roseus transgenic ("hairy") root clones LBE/​6/​1 and LBE/​4/​2 were
adapted with periodic daily illumination to investigate the effect of light on
growth and nutrient utilization, and the accumulation of the indole alkaloids.
Light/​adapted roots appeared green and had radially thickened morphology
compared with dark/​grown controls. Their growth rates were higher than
dark/​grown controls, with 45% lower doubling times: LBE/​6/​1, 3.6 days; LBE/​4/​2,
2.8 days. Relative to dark/​grown controls, light/​adapted growth increased the
biomass (DW) of LBE/​6/​1 by 25%, but had no effect on the DW of LBE/​4/​2. The
macronutrients NH4+, NO3/​, Pi, and sugars, were depleted completely by
light/​adapted root cultures in that order. The specific and total levels of the
indole alkaloid serpentine was enhanced and of tabersonine was lowered in both
root clones, while the overall trends of growth and non/​growth association of
tabersonine and serpentine, respectively, remained unaltered by light
adaptation. Ajmalicine accumulation was enhanced in LBE/​6/​1, but lowered in
LBE/​4/​2; its accumulation was growth/​associated in dark/​grown LBE/​6/​1, but
appeared non/​growth associated in light/​adapted cultures. The accumulation of
tabersonine/​related compounds, lochnericine, and horhammericine exhibited
growth/​associated trends, and were either negatively affected or unaffected by
light adaptation of LBE/​6/​1. Neither vindoline nor its precursor,
deacetylvindoline, was detected. Copyright 1998 John Wiley & Sons, Inc.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 10099477 [PubMed /​ indexed for MEDLINE]

211: Biochim Biophys Acta. 1999 Feb 10;1430(1):25/​38.

Structural and kinetic properties of adenylyl sulfate reductase from
Catharanthus roseus cell cultures.

Prior A, Uhrig JF, Heins L, Wiesmann A, Lillig CH, Stoltze C, Soll J, Schwenn
JD.

Lehrstuhl Biochemie der Pflanzen, Ruhr/​Universitat/​Bochum, Bochum ND 2/​149,
44780, Bochum, Germany.

A cDNA encoding a plant/​type APS reductase was isolated from an axenic cell
suspension culture of Catharanthus roseus (Genbank/EMBL/​databank accession
number U63784). The open reading frame of 1392 bp (termed par) encoded for a
protein (Mr=51394) consisting of a N/​terminal transit peptide, a PAPS
reductase/​like core and a C/​terminal extension with homology to the
thioredoxin/​like domain of protein disulfide isomerase. The APS reductase
precursor was imported into pea chloroplasts in vitro and processed to give a
mature protein of approximately 45 kDa. The homologous protein from pea
chloroplast stroma was detected using anti:par polyclonal antibodies. To
investigate the catalytical function of the different domains deleted par
proteins were purified. ParDelta1 lacking the transit sequence liberated sulfite
from APS (Km 2.5+//​0.23 microM) in vitro with glutathione (Km 3+//​0.64 mM) as
reductant (Vmax 2.6+//​0.14 U mg/​1, molecular activity 126 min/​1). ParDelta2
lacking the transit sequence and C/​terminal domain had to be reconstituted with
exogenous thioredoxin as reductant (Km 15. 3+//​1.27 microM, Vmax 0.6+//​0.014 U
mg/​1). Glutaredoxin, GSH or DTT were ineffective substitutes. ParDelta1 (35.4%)
and parDelta2 (21. 8%) both exhibited insulin reductase activity comparable to
thioredoxin (100%). Protein disulfide isomerase activity was observed for
parDelta1.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 10082930 [PubMed /​ indexed for MEDLINE]

212: Methods Mol Biol. 1999;111:393/​402.

Alkaloid accumulation in Catharanthus roseus suspension cultures.

Scragg AH.

Department of Environmental Health and Science, University of the West of
England, Frenchay, Bristol, UK.

Publication Types:
Review

PMID: 10081005 [PubMed /​ indexed for MEDLINE]

213: Plant Mol Biol. 1999 Jan;39(1):129/​36.

Elicitor/​responsive promoter regions in the tryptophan decarboxylase gene from
Catharanthus roseus.

Ouwerkerk PB, Memelink J.

Institute of Molecular Plant Sciences, Leiden University, The Netherlands.

The tryptophan decarboxylase (Tdc) gene from Catharanthus roseus (Madagascar
periwinkle) encodes a key enzyme in biosynthesis of terpenoid indole alkaloids.
The expression of the Tdc gene is transcriptionally induced by fungal elicitors.
Tdc upstream sequences from /​1818 to +198 relative to the transcriptional start
site were functionally analysed to identify cis/​acting elements that determine
basal expression or respond to elicitor. In a loss/​of/​function analysis promoter
derivatives with 5' or internal deletions fused to the gusA reporter gene were
analysed in transgenic tobacco plants. Whereas promoter activity dropped
considerably following deletion down to /​160, this short promoter derivative was
still elicitor/​responsive. Subsequently, the /​160 to /​37 region was further
studied by gain/​of/​function experiments, in which subfragments were tested as
tetramers cloned on two different truncated promoters. Combination of the data
resulted in the identification of three functional regions in the /​160 promoter.
The region between /​160 to /​99 was shown to act as the main transcriptional
enhancer. Two separable elicitor/​responsive elements were found to reside
between /​99 and /​87 and between /​87 and /​37. These two elements are not
redundant in the Tdc promoter, since their combination gave a distinct elicitor
response.

PMID: 10080715 [PubMed /​ indexed for MEDLINE]

214: Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1309/​14.

Dimethylallyl pyrophosphate is not the committed precursor of isopentenyl
pyrophosphate during terpenoid biosynthesis from 1/​deoxyxylulose in higher
plants

Arigoni D, Eisenreich W, Latzel C, Sagner S, Radykewicz T, Zenk MH, Bacher A.

Laboratorium fur Organische Chemie, Eidgenossische Technische Hochschule,
Universitatsstrasse 16, CH/​8092 Zurich, Switzerland.

Cell cultures of Catharanthus roseus were supplied with [2/​13C,
3/​2H]/​deoxyxylulose or [2/​13C,4/​2H]1/​deoxyxylulose. Lutein and chlorophylls were
isolated from the cell mass, and hydrolysis of the chlorophyll mixtures afforded
phytol. Isotope labeling patterns of phytol and lutein were determined by 2H NMR
and 1H,2H/​decoupled 13C NMR. From the data it must be concluded that the
deuterium atom in position 3 of deoxyxylulose was incorporated into both
isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate with a rate of
75% (with respect to the internal 13C label). The detected stereochemical
signature implies that the label is located preferentially in the (E)/​hydrogen
atom of IPP. This preferential labeling, in turn, rules out dimethylallyl
pyrophosphate as the compulsory precursor of IPP. In the experiment with [2/​13C,
4/​2H]1/​deoxyxylulose, the 13C label was efficiently transferred to the
terpenoids whereas the 2H label was completely washed out, most probably after
IPP formation as a consequence of the isomerization and elongation process. In
addition, the data cast light on the stereochemical course of the
dehydrogenation and cyclization steps involved in the biosynthesis of lutein.

PMID: 9990020 [PubMed /​ as supplied by publisher]

215: Plant Physiol. 1999 Feb;119(2):705/​12.

Purification and cDNA cloning of isochorismate synthase from elicited cell
cultures of Catharanthus roseus.

van Tegelen LJ, Moreno PR, Croes AF, Verpoorte R, Wullems GJ.

Department of Experimental Botany, University of Nijmegen, Toernooiveld 1, 6525
ED Nijmegen, The Netherlands.

Isochorismate is an important metabolite formed at the end of the shikimate
pathway, which is involved in the synthesis of both primary and secondary
metabolites. It is synthesized from chorismate in a reaction catalyzed by the
enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to
homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with
an apparent molecular mass of 64 kD were purified and characterized. The Km
values for chorismate were 558 and 319 microM for isoforms I and II,
respectively. The isoforms were not inhibited by aromatic amino acids and
required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library
from elicited C. roseus cells with a degenerated primer based on the sequence of
an internal peptide from isoform II resulted in an amplification product that
was used to screen the cDNA library. This led to the first isolation, to our
knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an
N/​terminal chloroplast/​targeting signal. The deduced amino acid sequence shares
homology with bacterial ICS and also with anthranilate synthases from plants.
Southern analysis indicates the existence of only one ICS gene in C. roseus.

PMID: 9952467 [PubMed /​ indexed for MEDLINE]

216: Plant Mol Biol. 1998 Nov;38(5):765/​74.

Expression of two consecutive genes of a secondary metabolic pathway in
transgenic tobacco: molecular diversity influences levels of expression and
product accumulation.

Leech MJ, May K, Hallard D, Verpoorte R, De Luca V, Christou P.

John Innes Centre, Norwich Research Park, Colney, UK.

We have created a population of transgenic tobacco plants carrying cDNAs
encoding two consecutive enzymes from early stages in monoterpenoid alkaloid
biosynthesis in Catharanthus roseus. The cDNAs, encoding tryptophan
decarboxylase (tdc) and strictosidine synthase (str1) together with a selectable
marker gene, were introduced on a single transforming plasmid into tobacco
leaves by particle bombardment. Analysis of 150 independent transgenic plants at
the DNA and RNA levels demonstrated a range of integration events and
steady/​state transcript levels for the tdc and str1 transgenes. Southern blot
analysis indicated that the tdc and str1 transgenes were integrated at least
once in all 150 transformants giving a 100% co/​integration frequency of the two
unselected genes carried on the same plasmid. A comparison of Southern and
northern data suggested that in 26% of the plants, both tdc and str1 transgenes
were silenced, 41% demonstrated a preferential silencing of either the tdc or
the str1 transgene, with the remaining 33% of the plants expressing both
transgenes. We observed no clear correlation between the number of integration
events of a specific transgene and the levels of accumulated transcript. Twenty
plants representing the range of molecular diversity in the transgenic
population were selected for further analysis. Seeds were collected from
self/​fertilised transformants and germinated on medium containing kanamycin.
Seedlings were harvested after 7 weeks and TDC and STR1 enzymatic assays were
carried out. We observed a 24/​ and 110/​fold variation in levels of TDC and STR1
activities, respectively. Our data correlate molecular diversity with
biochemistry and accumulation of end/​product and provide a detailed molecular
and biochemical characterization of transgenic plants transformed with a single
plasmid carrying two genes of secondary metabolism.

PMID: 9862494 [PubMed /​ indexed for MEDLINE]

217: Phytochemistry. 1998 Nov;49(5):1219/​25.

Phytohormone regulation of isoperoxidases in Catharanthus roseus suspension
cultures.

Limam F, Chahed K, Ouelhazi N, Ghrir R, Ouelhazi L.

Laboratoire de Biochimie Vegetale et Symbiotes, Institut National de la
Recherche Scientifique et Technique BP 95, Hammam/​Lif, Tunisie.

Peroxidase (POD) activity was investigated in Catharanthus roseus cell
suspensions cultured under different hormonal conditions. Depletion of
2,4/​dichlorophenoxyacetic acid (2,4/​D) from the culture medium enhanced POD
activity in cells and spent medium. Addition of phytohormones, in particular the
auxin 2,4/​D, reduced POD activity in medium and cellular compartments and
enhanced ionically cell/​wall bound POD. The differential modulation of POD is
due to hormone effects on synthesis and/or accumulation of POD, rather than on
the secretion process. Qualitative analysis showed that 2,4/​D, but not
cytokinins, regulated the synthesis of a basic isoform. The cytokinin treatment
seemed to affect acidic rather than basic isoforms. The presence of basic POD is
correlated with the capacity of cells to produce indole alkaloids. The major
extracellular basic isoperoxidase was purified to homogeneity from culture
medium of Catharanthus roseus cell suspensions. The isolated peroxidase is a
haem protein with a M(r) of 33,000 and a pI close to 9. The effect of pH on
peroxidase activity was studied using guaiacol as substrate and the optimum pH
determined at 25 degrees was 6.0. This enzyme acted on guaiacol,
2,2'/​azino/​bis/​(3/​ethylbenzthiazoline/​6/​sulfonic acid) (ABTS), o/​dianisidine,
o/​phenylenediamine (o/​PD) and pyrogallol, but had no effect on syringaldazine or
coniferyl alcohol substrates.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9842727 [PubMed /​ indexed for MEDLINE]

218: Phytochemistry. 1998 Sep;49(2):395/​402.

Jasmonate modulates development/​ and light/​regulated alkaloid biosynthesis in
Catharanthus roseus.

Vazquez/​Flota FA, De Luca V.

Departement de Sciences Biologiques, Universite de Montreal, Sherbrooke Est,
Quebec, Canada.

Methyl jasmonate, a chemical inducer of secondary metabolism, has been shown to
promote vindoline biosynthesis in developing seedlings, as a result of induction
of tryptophan decarboxylase (TDC) and desacetylvindoline 4/​hydroxylase (D4H).
The present studies suggest that jasmonate/​based induction of TDC and D4H
activities involves modulation of transcriptional, post/​transcriptional and
post/​translational controls. The effects of jasmonate on both enzymes were
transient with maximum TDC activity appearing 12 h earlier than that of D4H.
Jasmonate treatment of etiolated seedlings neither enhanced TDC activity nor
could it replace the light requirement for D4H induction. Jasmonate, therefore,
appears to modulate events which are already triggered by developmental and
environmental specific controls. Salicylic acid, another chemical inducer of
secondary metabolism, was ineffective in activating either TDC or D4H under the
experimental conditions used.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9747537 [PubMed /​ indexed for MEDLINE]

219: FEBS Lett. 1998 Sep 4;434(3):413/​6.

The iridoid glucoside secologanin is derived from the novel triose
phosphate/pyruvate pathway in a Catharanthus roseus cell culture.

Contin A, van der Heijden R, Lefeber AW, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Gorlaeus
Laboratories, The Netherlands. contin_a@chem.leidenuniv.nl

Secologanin is the iridoid building block of the majority of the terpenoid
indole alkaloids. In the biosynthesis of secologanin, mevalonate was considered
to be the exclusive precursor of isopentenyl diphosphate. After [1/​(13)C]glucose
feeding to a cell culture of Catharanthus roseus, its incorporation into
secologanin was studied by 13C NMR spectroscopy. The data showed that the novel
triose phosphate/pyruvate and not the mevalonate pathway was the major route for
the biosynthesis of secologanin.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9742965 [PubMed /​ indexed for MEDLINE]

220: Plant Physiol. 1998 Aug;117(4):1351/​61.

Developmental and light regulation of desacetoxyvindoline 4/​hydroxylase in
catharanthus roseus (L.) G. Don. . Evidence Of a multilevel regulatory mechanism

Vazquez/​Flota FA, De Luca V.

Institut de Recherche en Biologie Vegetale, Departement de Sciences Biologiques,
Universite de Montreal, 4101 Rue Sherbrooke est, Montreal, Quebec, Canada H1X
2B2.

The expression of desacetoxyvindoline 4/​hydroxylase (D4H), which catalyzes the
second to the last reaction in vindoline biosynthesis in Catharanthus roseus,
appears to be under complex, multilevel developmental and light regulation.
Developmental studies with etiolated and light/​treated seedlings suggested that
although light had variable effects on the levels of d4h transcripts, those of
D4H protein and enzyme activity could be increased, depending on seedling
development, up to 9/​ and 8/​fold, respectively, compared with etiolated
seedlings. However, light treatment of etiolated seedlings could stop and
reverse the decline of d4h transcripts at later stages of seedling development.
Repeated exposure of seedlings to light was also required to maintain the full
spectrum of enzyme activity observed during seedling development. Further
studies showed that a photoreversible phytochrome appeared to be involved in the
activation of D4H, since red/​light treatment of etiolated seedlings increased
the detectable levels of d4h transcripts, D4H protein, and D4H enzyme activity,
whereas far/​red/​light treatment completely reversed this process. Additional
studies also confirmed that different major isoforms of D4H protein exist in
etiolated (isoelectric point, 4.7) and light/​grown (isoelectric point, 4.6)
seedlings, suggesting that a component of the light/​mediated activation of D4H
may involve an undetermined posttranslational modification. The biological
reasons for this complex control of vindoline biosynthesis may be related to the
need to produce structures that could sequester away from cellular activities
the cytotoxic vinblastine and vincristine dimers that are derived partially from
vindoline.

PMID: 9701591 [PubMed /​ as supplied by publisher]

221: Prostaglandins Other Lipid Mediat. 1998 May;56(1):19/​31.

Effect of different inhibitors on phospholipase C activity in Catharanthus
roseus transformed roots.

Pina/​Chable ML, de los Santos/​Briones C, Munoz/​Sanchez JA, Echevarria Machado I,
Hernandez/​Sotomayor SM.

Unidad de Biologia Experimental, Centro de Investigacion Cientifica de Yucatan,
Merida, Mexico.

We have previously reported that Catharanthus roseus transformed roots contain
at least two phosphatidylinositol 4,5/​bisphosphate/​phospholipase C (PLC)
activities, one soluble and one membrane associated. In this paper, the effect
of neomycin and several divalent cations was analyzed, both in the soluble and
the membrane/​associated PLC activity in C. roseus transformed roots. In this
system, neomycin, an aminoglycoside antibiotic, inhibited PLC in a
concentration/​dependent fashion. The neomycin IC50 (100 microM) was the same for
the inhibition of the soluble and the membrane associated PLC activity. The
effect of different divalent cations such as Ni2+, Cu2+, and Zn2+ was studied as
well. In order to see the effect of these cations on PLC activity, we selected
two conditions: a) in the presence of and b) in the absence of calcium. In the
presence of calcium, these three divalent cations were able to inhibit PLC
activity in both fractions in a concentration/​dependent manner; however, the
IC50s were different for the membrane and the soluble activities. For the
soluble activity, the inhibition due to the three cations was very similar
(IC50s between 0.2 and 0.3 mM). For the membrane associated PLC activity, Cu2+
was the most potent inhibitor (IC50 3.6 microM), then Ni2+ and then Zn2+. In the
absence of calcium, higher concentrations of Cu2+ and Zn2+ demonstrated some
inhibitory effect. We discuss the possible physiological role of these
inhibitors on PLC activity.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9674018 [PubMed /​ indexed for MEDLINE]

222: Phytochemistry. 1998 Jun;48(4):595/​9.

Brassinosteroids in Arabidopsis thaliana.

Fujioka S, Noguchi T, Yokota T, Takatsuto S, Yoshida S.

Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.

From the seeds and siliques of Arabidopsis thaliana, six brassinosteroids,
brassinolide, castasterone, typhasterol, 6/​deoxocastasterone, 6/​deoxotyphasterol
and 6/​deoxoteasterone, were identified by GC/​mass spectrometry or GC/​selected
ion monitoring. As the occurrence of castasterone, typhasterol,
6/​deoxocastasterone and 6/​deoxotyphasterol in the shoots of A. thaliana has
already been reported, this study provides evidence for the occurrence of the
above four brassinosteroids in different organs, seeds and siliques, and the
first evidence for the occurrence of brassinolide and 6/​deoxoteasterone in A.
thaliana. All brassinosteroids identified in this study belong to important
components of both the early and late C/​6 oxidation pathways, which were
established in the cultured cells of Catharanthus roseus. This suggests that
both pathways are operating in A. thaliana to produce the most biologically
active brassinosteroid, brassinolide, which is responsible for growth and
development of the plant.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9664702 [PubMed /​ indexed for MEDLINE]

223: FEBS Lett. 1998 May 29;428(3):299/​303.

Purification and characterization of alpha/​3',4'/​anhydrovinblastine synthase
(peroxidase/​like) from Catharanthus roseus (L.) G. Don.

Sottomayor M, Lopez/​Serrano M, DiCosmo F, Ros Barcelo A.

Department of Botany and Institute for Molecular and Cell Biology, University of
Porto, Portugal.

An H2O2/​dependent enzyme capable of coupling catharanthine and vindoline into
alpha/​3',4'/​anhydrovinblastine (AVLB) was purified to apparent homogeneity from
Catharanthus roseus leaves. The enzyme shows a specific AVLB synthase activity
of 1.8 nkat/mg, and a molecular weight of 45.40 kDa (SDS/​PAGE). In addition to
AVLB synthase activity, the purified enzyme shows peroxidase activity, and the
VIS spectrum of the protein presents maxima at 404, 501 and 633 nm, indicating
that it is a high spin ferric heme protein, belonging to the plant peroxidase
superfamily. Kinetic studies revealed that both catharanthine and vindoline were
substrates of the enzyme, AVLB being the major coupling product.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9654153 [PubMed /​ indexed for MEDLINE]

224: Planta. 1998 Jul;205(3):414/​9.

Effects of over/​expression of strictosidine synthase and tryptophan
decarboxylase on alkaloid production by cell cultures of Catharanthus roseus.

Canel C, Lopes/​Cardoso MI, Whitmer S, van der Fits L, Pasquali G, van der
Heijden R, Hoge JH, Verpoorte R.

Division of Pharmacognosy, Gorlaeus Laboratories, Leiden University, The
Netherlands. ccanel@olemiss.edu

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to
over/​express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan
decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis
of terpenoid indole alkaloids (TIAs). The cultures established after
Agrobacterium/​mediated transformation showed wide phenotypic diversity,
reflecting the complexity of the biosynthetic pathway. Cultures transgenic for
Str consistently showed tenfold higher STR activity than wild/​type cultures,
which favored biosynthetic activity through the pathway. Two such lines
accumulated over 200 mg.L/​1 of the glucoalkaloid strictosidine and/or
strictosidine/​derived TIAs, including ajmalicine, catharanthine, serpentine, and
tabersonine, while maintaining wild/​type levels of TDC activity. Alkaloid
accumulation by highly productive transgenic lines showed considerable
instability and was strongly influenced by culture conditions, such as the
hormonal composition of the medium and the availability of precursors. High
transgene/​encoded TDC activity was not only unnecessary for increased
productivity, but also detrimental to the normal growth of the cultures. In
contrast, high STR activity was tolerated by the cultures and appeared to be
necessary, albeit not sufficient, to sustain high rates of alkaloid
biosynthesis. We conclude that constitutive over/​expression of Str is highly
desirable for increased TIA production. However, given its complexity, limited
intervention in the TIA pathway will yield positive results only in the presence
of a favorable epigenetic environment.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 9640666 [PubMed /​ indexed for MEDLINE]

225: Biotechnol Prog. 1998 May/​Jun;14(3):442/​9.

Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids
by Catharanthus roseus hairy root cultures.

Rijhwani SK, Shanks JV.

Department of Chemical Engineering, MS/​362, 6100 South Main, Rice University,
Houston, Texas 77005/​1892, USA.

Late exponential phase hairy root cultures of Catharanthus roseus were elicited
with pectinase and jasmonic acid. The effects of elicitor concentration and
exposure time on growth and levels of several compounds in the indole alkaloid
biosynthetic pathway were monitored. Pectinase decreased the fresh weight to dry
weight ratio of the roots, while addition of jasmonic acid had no significant
effect. Selective effects on indole alkaloid yields were observed upon addition
of elicitors. An increase of 150% in tabersonine specific yield was observed
upon addition of 72 units of pectinase. Transient studies at the same level
demonstrated possible catabolism as serpentine, tabersonine, and lochnericine
levels decreased immediately after elicitation. The levels of these compounds
recovered back to control levels or were higher than the control levels after
some time. Jasmonic acid was found to be a unique elicitor leading to an
enhancement in flux to several branches in the indole alkaloid pathway. Jasmonic
acid addition caused an increase in the specific yields of ajmalicine (80%),
serpentine (60%), lochnericine (150%), and horhammericine (500%) in dosage
studies. Tabersonine, the likely precursor of lochnericine and horhammericine,
decreased at lower levels of jasmonic acid and then increased with increasing
jasmonic acid concentration. Transient studies showed that lochnericine and
tabersonine levels go through a maxima, then decrease back to control levels and
reduce below control levels, respectively. The yields of ajmalicine, serpentine,
and horhammericine increased continuously after the addition of jasmonic acid.
The methods described in this article could generally be used in devising
strategies for enhancement in productivity of secondary metabolites and for
probing and studying the complex secondary metabolite pathways in plant tissue
cultures.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 9622525 [PubMed /​ indexed for MEDLINE]

226: Acta Pharm Hung. 1998 Mar;68(2):87/​93.

[New antitumor derivatives of vinblastine]

[Article in Hungarian]

Bolcskei H, Szantay C Jr, Mak M, Balazs M, Szantay C.

Richter Gedeon Vegyeszeti Gyar Rt., Budapest.

The alkaloids of Catharanthus roseus vincristine 1 and vinblastine 2 are widely
used in the chemotherapy of cancer. Their nitro/​derivatives have also antitumour
activity [2]. The dinitro/​derivatives 4, 6/​9 were prepared for pharmacological
investigation. Some new hydroxymethyl derivatives 12/​14, 17/​18, 23/​24 of the
antitumour indole/​indoline alkaloids vinblastine 2 and leurosine 5 were
synthetised in two different ways a) by the selective reduction of 2, 5 or
deacetoxy/​vinblastine 16, b) by the ferric chloride mediated coupling reaction
of catharanthine 19 and the corresponding vindoline derivatives 21, 22.
Synthetising 24 a new bisvindoline 25 was the main product. 13 and 14 showed
cytotoxic activity for small cell lung cancer LXFS 650.

Publication Types:
English Abstract

PMID: 9592933 [PubMed /​ indexed for MEDLINE]

227: Plant Cell. 1998 Mar;10(3):331/​41.

A novel cis/​acting element in promoters of plant B/​type cyclin genes activates M
phase/​specific transcription.

Ito M, Iwase M, Kodama H, Lavisse P, Komamine A, Nishihama R, Machida Y,
Watanabe A.

Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Hongo, Tokyo 113, Japan. masakito@biol.s.u/​tokyo.ac.jp

Plant B/​type cyclin genes are expressed late in the G2 and M phases of the cell
cycle. Previously, we showed that the promoter of a Catharanthus roseus B/​type
cyclin, CYM, could direct M phase/​specific transcription of a beta/​glucuronidase
reporter gene in synchronously dividing BY2 tobacco cells. In this study, we
determined the regulatory elements contained within the CYM promoter by using a
luciferase reporter gene. Mutational analysis showed that a 9/​bp element is
essential for M phase/​specific promoter activity in synchronized BY2 cells. The
CYM promoter contains three other sequences similar to this element. A
gain/​of/​function assay demonstrated that when fused to a heterologous promoter,
these elements are sufficient for M phase/​specific expression; therefore, we
named these elements M/​specific activators (MSAs). We found MSA/​like sequences
in B/​type cyclin promoters from tobacco, soybean, and Arabidopsis as well as in
the promoters of two M phase/​specific genes, NACK1 and NACK2, which encode
tobacco kinesin/​like proteins. Thus, MSA may be a common cis/​acting promoter
element that controls M phase/​specific expression of cell cycle/​related genes in
plants.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9501108 [PubMed /​ indexed for MEDLINE]

228: Biochem Biophys Res Commun. 1998 Mar 6;244(1):110/​4.

Glyphosate is an inhibitor of plant cytochrome P450: functional expression of
Thlaspi arvensae cytochrome P45071B1/reductase fusion protein in Escherichia
coli.

Lamb DC, Kelly DE, Hanley SZ, Mehmood Z, Kelly SL.

Institute of Biological Sciences, University of Wales Aberystwyth, United
Kingdom.

Glyphosate (Roundup) is an herbicide used extensively worldwide which acts as an
inhibitor of 5'enolpyruvylshikimate/​3/​phosphate synthase and for which
transgenic herbicide resistant plants have been developed. Here we report for
the first time that glyphosate is an inhibitor of cytochrome P450 using a
functional expression system for Thlaspi arvensae CYP71B1 in Escherichia coli.
CYP71B1 was fused to the soluble domain of a plant cytochrome P450 reductase
(CPR) from Catharanthus roseus. CYP71B1 could obtain reducing equivalents in
this fusion construct and metabolised the polycyclic aromatic hydrocarbon,
benzo(a)pyrene. The fusion protein retained normal spectral characteristics
having a Soret peak at 448 nm in the reduced carbon monoxide difference
spectrum. Addition of the herbicide resulted in a Type II spectrum indicative of
binding via the nitrogen group to haem as a sixth ligand. A Ks of 60 microM was
observed and an IC50 of 12 microM was observed for glyphosate inhibition of
CYP71B1 activity. The implications of these results are discussed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9514851 [PubMed /​ indexed for MEDLINE]

229: Plant Physiol. 1998 Feb 1;116(2):853/​7.

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell
Line of Catharanthus roseus

Whitmer S, Canel C, Hallard D, Goncalves C, Verpoorte R.

Department of Pharmacognosy, Gorlaeus Laboratories, Leiden University, P.O. Box
9502, 2300 RA Leiden, The Netherlands

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study
the relative importance of the supply of biosynthetic precursors for the
synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant,
constitutively overexpressed version of the endogenous strictosidine synthase
(Str) gene. Various concentrations and combinations of the substrate tryptamine
and of loganin, the immediate precursor of secologanin, were added to suspension
cultures of S10. Our results indicate that high rates of tryptamine synthesis
can take place under conditions of low tryptophan decarboxylase activity, and
that high rates of strictosidine synthesis are possible in the presence of a
small tryptamine pool. It appears that the utilization of tryptamine for
alkaloid biosynthesis enhances metabolic flux through the indole pathway.
However, a deficiency in the supply of either the iridoid or the indole
precursor can limit flux through the step catalyzed by strictosidine synthase.
Precursor utilization for the synthesis of strictosidine depends on the
availability of the cosubstrate; the relative abundance of these precursors is a
cell/​line/​specific trait that reflects the metabolic status of the cultures.

PMID: 9490777 [PubMed /​ as supplied by publisher]

230: Plant Mol Biol. 1998 Feb;36(3):343/​51.

Expression of extensin genes is dependent on the stage of the cell cycle and
cell proliferation in suspension/​cultured Catharanthus roseus cells.

Ito M, Kodama H, Komamine A, Watanabe A.

Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Japan.

To isolate cDNAs expressed at a specific phase of the cell cycle in a higher
plant, we performed differential screening of a cDNA library prepared from the
S/​phase cells of synchronized cultures of Catharanthus roseus. Sequence analysis
shows that two of the identified cDNAs, cyc15 and cyc17, encode extensins that
represent a family of cell wall hydroxyproline/​rich glycoproteins. Protein
sequences deduced from the two cDNAs contain the characteristic pentapeptide
repeat sequence, Ser/​Pro/​Pro/​Pro/​Pro, which is commonly observed in extensins.
The protein sequences also share several other extensin characteristics such as
the presence of a N/​terminal signal peptide and a high content of Tyr and Lys
residues. When C. roseus cell suspension cultures were synchronized by phosphate
starvation, the mRNAs of both cyc15 and cyc17 were transiently expressed during
the S and G2 phases of the cell cycle. However, significant amounts of the mRNAs
also accumulated in phosphate/​starved cells arrested in the G1 phase. In
asynchronous cultures, both genes were expressed during the stationary phase,
when cell proliferation ceased. The observed patterns of expression suggest that
the extensin genes, cyc15 and cyc17, are under two types of regulation: one that
depends on the stage of the cell cycle and another that is induced during the
growth arrest. Thus, the products of these genes may function both during the
progression through the cell cycle and in the strengthening of the cell wall
after cell division.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9484475 [PubMed /​ indexed for MEDLINE]

231: Allerg Immunol (Paris). 1997 Oct;29(8):242/​3.

[Characterization of a protein related to Bet v 1, the major birch pollen
antigen, from cells of the Madagascar periwinkle. Universal presence of proteins
of this type among higher plants]

[Article in French]

Carpin S, Hamdi S, Rideau M.

Laboratoire de Biologie Moleculaire et Biochimie Vegetale, Faculte de Pharmacie
de Tours.

In this work we present the characterisation of a gene that codes for a protein
of 17 kDa, in in vitro cultures of a plant with ornamental and pharmaceutical
properties, the Madagascan periwinkle, (Catharanthus roseus [L] G. DON). This
protein is very close to the principal allergen of birch and also to allergens
isolated from or demonstrated in some foods such as celery, parsley, apple tree,
peas, asparagus and potato, but it has no allergenic characteristics.

Publication Types:
Comparative Study
English Abstract

PMID: 9453736 [PubMed /​ indexed for MEDLINE]

232: Anal Biochem. 1998 Jan 1;255(1):39/​46.

Direct fluorometry of phase/​extracted tryptamine/​based fast quantitative assay
of L/​tryptophan decarboxylase from Catharanthus roseus leaf.

Sangwan RS, Mishra S, Kumar S.

Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India.

An assay for the enzyme L/​tryptophan decarboxylase (TDC; EC 4.1.1.28) is
described. It is based on direct fluorometry of the enzymatic reaction product
(tryptamine) selectively recovered in ethyl acetate from the reaction mixture.
Catalytically formed tryptamine from tryptophan in the incubation mixture is
selectively (free from tryptophan) physically separated as ethyl acetate
solution under basic (pH > or = 11) conditions and subjected to direct
fluorescence measurement in the organic solvent using a spectrofluorometer with
excitation and emission wavelengths of 280 and 350 nm, respectively. Tryptamine
production rate was quantitated from the luminescence response curve of
tryptamine drawn under similar extraction and measurement conditions.
Luminescence calibration curves were drawn for tryptamine in aqueous (water or
buffer system) as well as in organic solvent as recovered from the varied
aqueous solution conditions including those similar to the enzyme incubation
mixture. The luminescence calibration graphs were linear for at least 0.5 to 10
microM tryptamine. The examination of interassay variations and the comparative
magnitude of fluorescence response allowed to infer that a satisfactory and
sufficient sample luminescence response was retained under the varied conditions
including those akin to the enzymatic assay mixture, allowing adaptation of the
fluorometry for the TDC activity quantitation. The assay was found to follow the
proportionality principle of product formation with respect to catalytic
reaction time as well as protein concentration in the assay mixture using
Catharanthus roseus leaf crude homogenate as well as the enzyme preparation at
different states of purity. The rate of tryptamine formation under the catalytic
conditions was linear for at least 1 h at 30 degrees C. Though the assay has
been demonstrated to use the C. roseus leaf as the enzyme source, it should be
equally applicable to other plant and nonplant sources. The merits and
precautions of the protocol have been discussed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9448840 [PubMed /​ indexed for MEDLINE]

233: Planta. 1998 Jan;204(1):70/​7.

Evidence of protein/​tyrosine kinase activity in Catharanthus roseus roots
transformed by Agrobacterium rhizogenes.

Rodriguez/​Zapata LC, Hernandez/​Sotomayor SM.

Unidad de Biologia Experimental, Centro de Investigacion Cientifica de Yucatan
Apdo., Mexico.

Homogenate fractions (soluble and particulate) from transformed roots of
Catharanthus roseus (L.) G. Don showed several phosphorylated proteins when
incubated with gamma/​[32P]ATP. The phosphorylation in the proteins of 55, 40,
25, 18 and 10 kDa in the particulate fraction and 63 kDa in the soluble fraction
was resistant to alkali treatment. Several proteins in both fractions gave a
positive signal with monoclonal antiphosphotyrosine antibodies. In/​situ
phosphorylation in both fractions showed several proteins that cross/​reacted
with the antiphosphotyrosine antibodies. Tyrosine kinase activity was detected
using an exogenous substrate RR/​SRC, a synthetic peptide derived from the amino
acid sequence surrounding the phosphorylation site in pp60src. This activity was
inhibited by genistein, a tyrosine kinase inhibitor. These results indicate, for
the first time, the presence of protein/​tyrosine kinase (EC 2.7.1.112) activity
in transformed plant tissues.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

PMID: 9443385 [PubMed /​ indexed for MEDLINE]

234: Mol Gen Genet. 1997 Nov;256(6):674/​81.

A promoter region that controls basal and elicitor/​inducible expression levels
of the NADPH:cytochrome P450 reductase gene (Cpr) from Catharanthus roseus binds
nuclear factor GT/​1.

Cardoso MI, Meijer AH, Rueb S, Machado JA, Memelink J, Hoge JH.

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory,
The Netherlands.

NADPH:cytochrome P450 reductase (CPR) is essential for the activation of
cytochrome P450 enzymes, which are involved in a wide variety of metabolic
pathways in plants, including those related to defence responses. In the
subtropical plant Catharanthus roseus several cytochrome P450 enzymes operate in
the biosynthesis of defence/​related terpenoid indole alkaloids (TIAs). In
agreement with the importance of CPR in defence, Cpr mRNA levels in C. roseus
were found to be enhanced by fungal elicitor preparations that also induce TIA
biosynthesis and P450 gene expression. Here we describe the isolation of a C.
roseus genomic DNA clone covering the 5' part of the Cpr gene and 1.6/​kb of
upstream sequences. Mapping of the transcription start site showed the
untranslated leader sequence is approximately 280 bp long. To study the control
of gene expression by the Cpr promoter, transcriptional fusions between Cpr
promoter fragments and the gusA reporter gene were generated and their
expression was analyzed in stably transformed tobacco plants. The Cpr promoter
fragment extending from /​1510 to /​8, with respect to the ATG start codon,
conferred basal and elicitor/​inducible expression on the gusA reporter gene,
strongly indicating that the Cpr gene of C. roseus is indeed controlled by this
promoter region. Progressive deletion from the 5' end of the promoter to
position /​632 had little effect on gusA expression. However, deletion to
position /​366 resulted in a complete loss of basal activity and largely
eliminated elicitor/​induced expression, indicating that the region from /​632 to
/​366 contains the main transcription/​enhancing cis/​regulatory sequences.
Electrophoretic mobility shift assays with tobacco nuclear extracts showed that
binding sites for nuclear factor GT/​1 are redundant in the Cpr promoter, but
absent from the downstream part of the leader sequence. The presence of strong
GT/​1 binding sites in the main enhancer region (/​632 to /​366), is suggestive of
a functional role for this factor in basal expression and elicitor
responsiveness of the Cpr promoter.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9435792 [PubMed /​ indexed for MEDLINE]

235: Anal Biochem. 1997 Dec 15;254(2):249/​53.

Determination of inorganic phosphate by coupling thymidine phosphorylase and
reversed/​phase high/​performance liquid chromatography: application to tonoplast
pyrophosphatase activity.

Hill M, Arrio B.

ERS 571 du CNRS, Bioenergetique Membranaire, Universite de Paris Sud, Centre
d'Orsay, France.

We developed an HPLC method for the measurement of inorganic phosphate using
thymidine phosphorylase (EC 2.4.2.4). This enzyme catalyzes the phosphorolysis
of thymidine to 2/​deoxyribose 1/​phosphate and thymine. Thymine release was
measured at 265 nm after separation by reverse/​phase HPLC. The assay was
sensitive enough to detect as little as 10 pmol of Pi. The response to the
phosphate concentration was linear from 1 to 100 microM. The value of this
method was demonstrated in an analysis of the kinetics of Pi release from PPi in
the presence of Catharanthus roseus tonoplast pyrophosphatase.

PMID: 9417785 [PubMed /​ indexed for MEDLINE]

236: Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10600/​5.

Terpenoid biosynthesis from 1/​deoxy/​D/​xylulose in higher plants by
intramolecular skeletal rearrangement.

Arigoni D, Sagner S, Latzel C, Eisenreich W, Bacher A, Zenk MH.

Laboratorium fur Organische Chemie, Eidgenossische Technische Hochschule,
Universitatsstrasse 16, CH/​8092 Zurich, Switzerland.

The incorporation of [1/​13C]/​ and [2,3,4,5/​13C4]1/​deoxy/​D/​xylulose into
beta/​carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus
roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene
precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were
obtained from the terpenes by a retrobiosynthetic approach. 13C Enrichment and
13C13C coupling patterns showed conclusively that 1/​deoxy/​D/​xylulose and not
mevalonate is the predominant isoprenoid precursor of phytol, beta/​carotene, and
lutein. Label from 1/​deoxyxylulose was also diverted to phytosterols to a minor
extent (6% relative to carotene and phytol formation). The data demonstrate that
the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an
intramolecular rearrangement process.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9380681 [PubMed /​ indexed for MEDLINE]

237: Biochim Biophys Acta. 1997 Aug 29;1336(2):323/​30.

Theophylline metabolism in higher plants.

Ito E, Crozier A, Ashihara H.

Department of Biology, Ochanomizu University, Faculty of Science, Tokyo, Japan.

Metabolism of [8/​(14)C]theophylline was investigated in leaf segments from
Camellia sinensis (tea), Camellia irrawadiensis, Ilex paraguariensis (mate) and
Avena sativa, root segments of Vigna mungo seedlings and cell suspension
cultures of Catharanthus roseus. There was extensive uptake and metabolism of
[8/​(14)C]theophylline by leaves of tea and Camellia irrawadiensis and, to a
lesser extent, mate. These purine alkaloid/​containing species converted
[8/​(14)C]theophylline into 3/​methylxanthine, xanthine, the ureides allantoin and
allantoic acid, and CO2. With the other test systems, which were from species
that do not produce purine alkaloids, there were low levels of
[8/​(14)C]theophylline uptake which were accompanied by incorporation of
relatively small amounts of label into 3/​methylxanthine, xanthine and CO2. None
of the higher plants converted [8/​(14)C]theophylline to either 1/​methyluric acid
or 1,3/​dimethyluric acid, which are the main catabolites of theophylline in
mammals. The data indicate that the main route of theophylline degradation in
higher plants involves a theophylline /​/​> 3/​methylxanthine /​/​> xanthine /​/​> uric
acid /​/​> allantoin /​/​> allantoic acid /​/​> /​/​> CO2 + NH3 pathway. In tea and
mate, large amounts of [8/​(14)C]theophylline were also converted to theobromine
and caffeine via a theophylline /​/​> 3/​methylxanthine /​/​> theobromine /​/​>
caffeine salvage pathway. The diversity of theophylline metabolism in higher
plants and mammals is discussed.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9305805 [PubMed /​ indexed for MEDLINE]

238: Plant Mol Biol. 1997 Aug;34(6):935/​48.

Molecular cloning and characterization of desacetoxyvindoline/​4/​hydroxylase, a
2/​oxoglutarate dependent/​dioxygenase involved in the biosynthesis of vindoline
in Catharanthus roseus (L.) G. Don.

Vazquez/​Flota F, De Carolis E, Alarco AM, De Luca V.

Departement de Sciences Biologiques, Universite de Montreal, Quebec, Canada.

A 2/​oxoglutarate/​dependent dioxygenase (EC 1.14.11.11) which catalyzes the
4/​hydroxylation of desacetoxyvindoline was purified to homogeneity. Three
oligopeptides isolated from a tryptic digest of the purified protein were
microsequenced and one oligopeptide showed significant homology to hyoscyamine 6
beta/​hydroxylase from Hyoscyamus niger. A 36/​mer degenerate oligonucleotide
based on this peptide sequence was used to screen a Catharanthus roseus cDNA
library and three clones, cD4H/​1 to /​3, were isolated. Although none of the
three clones were full/​length, the open reading frame on each clone encoded a
putative protein containing the sequence of all three peptides. Primer extension
analysis suggested that cD4H/​3, the longest cDNA clone, was missing 156 bp at
the 5' end of the clone and sequencing of the genomic clone, gD4H/​8, confirmed
these results. Southern blot analysis suggested that d4h is present as a
single/​copy gene in C. roseus which is a diploid plant, and the significant
differences in the sequence of the 3'/​UTR between cD4H/​1 and /​3 suggest that
they represent dimorphic alleles of the same hydroxylase. The identity of the
clone was further confirmed when extracts of transformed Escherichia coli
expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401
amino acids with a calculated molecular mass of 45.5 kDa and the amino acid
sequence showed a high degree of similarity with those of a growing family of
2/​oxoglutarate/​dependent dioxygenases of plant and fungal origin. The similarity
was not restricted to the dioxygenase protein sequences but was also extended to
the gene structure and organization since the 205 and 1720 bp introns of d4h
were inserted around the same highly conserved amino acid consensus sequences as
those for e8 protein, hyoscyamine/​6 beta/​hydroxylase and ethylene/​forming
enzyme. These results provide further support that a common ancestral gene is
responsible for the appearance of this family of dioxygenases. Hydroxylase
assays and RNA blot hybridization studies showed that enzyme activity followed
closely the levels of d4h transcripts, occurring predominantly in young leaves
and in much lower levels in stems and fruits. In contrast, etiolated seedlings
which contained considerable levels of d4h transcripts had almost undetectable
hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid
increase of enzyme activity without a significant further increase in d4h
transcripts over those detected in dark/​grown seedlings. These results suggest
that the activating effect of light may occur at a point downstream of
transcription which remains to be elucidated.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 9290645 [PubMed /​ indexed for MEDLINE]

239: Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):7982/​6.

An important developmental role for oligosaccharides during early embryogenesis
of cyprinid fish.

Bakkers J, Semino CE, Stroband H, Kijne JW, Robbins PW, Spaink HP.

Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64,
2333 AL Leiden, The Netherlands.

Derivatives of chitin oligosaccharides have been shown to play a role in plant
organogenesis at nanomolar concentrations. Here we present data which indicate
that chitin oligosaccharides are important for embryogenesis in vertebrates. We
characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp
embryos in the late gastrulation stage by incorporation of radiolabeled
N/​acetyl/​D/​[U14C]glucosamine and by HPLC in combination with enzymatic
conversion using the Bradyrhizobium NodZ alpha/​1, 6/​fucosyltransferase and
chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also
used employing suspension/​cultured plant cells of Catharanthus roseus. We show
that chitin oligosaccharide synthase activity is apparent only during late
gastrulation and can be inhibited by antiserum raised against the Xenopus DG42
protein. The DG42 protein, a glycosyltransferase, is transiently expressed
between midblastula and neurulation in Xenopus and zebrafish embryogenesis.
Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in
fertilized eggs of zebrafish led to severe defects in trunk and tail
development.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

PMID: 9223299 [PubMed /​ indexed for MEDLINE]

240: Plant J. 1997 May;11(5):983/​92.

Cell/​cycle/​regulated transcription of A/​ and B/​type plant cyclin genes in
synchronous cultures.

Ito M, Marie/​Claire C, Sakabe M, Ohno T, Hata S, Kouchi H, Hashimoto J, Fukuda
H, Komamine A, Watanabe A.

Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Japan. masakito@uts2.s.u/​tokyo.ac.jp

Synchronously dividing cell cultures of Catharanthus roseus were used to isolate
cDNAs for two mitotic cyclins, named CYS and CYM. The deduced protein sequence
of CYS is similar to that of A/​type cyclins, and CYM belongs to the group of
B/​type cyclins. In a fashion similar to the pattern of expression seen for
A/​type and B/​type cyclins in mammalian cells, CYS is expressed before CYM in C.
roseus cells during the cell cycle. CYS mRNA accumulated at the onset of S phase
and disappeared early in the G2 phase, whereas CYM mRNA was detected in the G2
and M phases of the cell cycle. Tobacco homologs of the two genes showed similar
cell/​cycle dependent expression patterns in synchronous cultures of tobacco BY2
cells. In both systems, CYS was expressed much earlier in the cell cycle than
most other plant A/​type cyclins, and hence CYS along with the soybean cyc1GM can
be classified into a distinct subclass. The activities of CYM and CYS promoters
during the cell cycle were analyzed in stably transformed tobacco BY2 cells.
Cyclin promoter sequences of 0.5 kb could confer the typical
cell/​cycle/​dependent expression to the beta/​glucuronidase (GUS) reporter gene:
the CYS promoter directed S/​phase/​specific expression, whereas the CYM promoter
drove M/​phase/​specific expression. These results indicate the important role of
transcriptional regulation in the oscillations of cyclin mRNA levels during the
cell cycle.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9193070 [PubMed /​ indexed for MEDLINE]

241: Biosci Biotechnol Biochem. 1997 May;61(5):757/​62.

Studies on biosynthesis of brassinosteroids.

Sakurai A, Fujioka S.

Institute of Physical and Chemical Research (RIKEN), Saitama 351/​01, Japan.

Biosynthesis of steroidal plant hormones, brassinosteroids, was studied using
the cell culture system of Catharanthus roseus. Feeding labeled compounds of
possible intermediates to the cultured cells, followed by analyzing the
metabolites by gas chromatography/​mass spectrometry disclosed the pathways from
a plant sterol, campesterol, to brassinolide. There are two pathways, named the
early C6/​oxidation pathway and late C6/​oxidation pathway, both of which would be
operating in a wide variety of plants. Recent findings of
brassinosteroid/​deficient mutants of Arabidopsis and the garden pea by several
groups, and the possible blocked steps of the mutants in the biosynthetic
pathways are also introduced.

Publication Types:
Research Support, Non/​U.S. Gov't
Review

PMID: 9178548 [PubMed /​ indexed for MEDLINE]

242: Plant Mol Biol. 1997 Mar;33(5):943/​6.

Comparison of the activities of CaMV 35S and FMV 34S promoter derivatives in
Catharanthus roseus cells transiently and stably transformed by particle
bombardment.

van der Fits L, Memelink J.

Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
the Netherlands.

Activities of several CaMV 35S and FMV 34S promoter derivatives fused to the
gusA reporter gene were compared in suspension/​cultured Catharanthus roseus
cells that were transiently and stably transformed using particle bombardment.
Our data demonstrate that the 35S and a deletion derivative of the 34S promoter
combined with particle bombardment form useful tools for genetic engineering of
C. roseus cells. Our results disagree on several points with activities of 35S
and 34S promoter derivatives reported for tobacco, indicating that absolute and
relative promoter activities can differ between plant species.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 9106518 [PubMed /​ indexed for MEDLINE]

243: Plant Mol Biol. 1997 Jan;33(2):211/​22.

Three differentially expressed S/​adenosylmethionine synthetases from
Catharanthus roseus: molecular and functional characterization.

Schroder G, Eichel J, Breinig S, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

We describe the molecular and functional characterization of three closely
related S/​adenosyl/​L/​methionine synthetase (SAMS) isoenzymes from Catharanthus
roseus (Madagascar periwinkle). The genes are differentially expressed in cell
cultures during growth of the culture and after application of various stresses
(elicitor, nutritional down/​shift, increased NaCl). Seedlings revealed
organ/​specific expression and differential gene regulation after salt stress. A
relationship analysis indicated that plant SAMS group in two main clusters
distinguished by characteristic amino acid exchanges at specific positions, and
this suggested differences in the enzyme properties or the regulation. SAMS1 and
SAMS2 are of type I and SAMS3 is of type II. The properties of the isoenzymes
were compared after heterologous expression of the individual enzymes, but no
significant differences were detected in a) optima for temperature (37 to 45
degrees C) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+,
Co2+; monovalent: K+, NH4+, Na+); c) K(m)s for ATP and L/​methionine; d)
inhibition by reaction products (S/​adenosyl/​L/​methionine, PPi, Pi), by the
reaction intermediate tripolyphosphate, and by the substrate analogues ethionine
and cycloleucine; e) response to metabolites from the methyl cycle
(L/​homocysteine) or from related pathways (L/​ornithine, putrescine, spermidine,
spermine); f) native protein size (gel permeation chromatography). The results
represent the first characterization of plant SAMS isoenzyme properties with
individually expressed proteins. The possibility is discussed that the isoenzyme
differences reflect specificities in the association with enzymes that use
S/​adenosyl/​L/​methionine.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 9037140 [PubMed /​ indexed for MEDLINE]

244: Biochem Biophys Res Commun. 1996 Nov 1;228(1):141/​7.

The primary structures of rat ribosomal proteins S3a (the V/​Fos transformation
effector) and of S3b.

Chan YL, Olvera J, Paz V, Wool IG.

Department of Biochemistry and Molecular Biology, University of Chicago,
Illinois 60637, USA.

The amino acid sequence of the rat 40S ribosomal subunit protein S3a was deduced
from the sequence of nucleotides in two recombinant cDNAs and confirmed by the
determination of the NH2/​terminal sequence by Edman degradation. Ribosomal
protein S3a has 263 amino acids (the NH2/​terminal methionine is removed after
translation of the mRNA) and the molecular weight is 29,794. The protein
designated S3b has the same amino acid sequence as S3a except that it lacks the
carboxyl/​terminal 12 residues. We are unable to determine whether there are
separate genes for S3a and S3b, or whether there is a single gene and alternate
splicing of the precursor to yield separate mRNAs for S3a and S3b, or whether
there is a single gene and a single mRNA whose translation yields S3a which is
converted by proteolysis, either physiological or fortuitous, to S3b. The mRNA
for S3a is about 1000 nucleotides in length. Hybridization of cDNA to digests of
nuclear DNA suggests that there are 8/​13 copies of the S3a gene. Rat ribosomal
protein S3a is identical to the product of the rat Fte/​1 gene which encodes the
V/​Fos transformation effector; S3a is also related to the plant protein cyc07,
which is encoded by a cell cycle S/​phase specific gene.

Publication Types:
Research Support, U.S. Gov't, P.H.S.

PMID: 8912649 [PubMed /​ indexed for MEDLINE]

245: J Biol Chem. 1996 Oct 25;271(43):26684/​9.

Novel type of receptor/​like protein kinase from a higher plant (Catharanthus
roseus). cDNA, gene, intramolecular autophosphorylation, and identification of a
threonine important for auto/​ and substrate phosphorylation.

Schulze/​Muth P, Irmler S, Schroder G, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Schanzlestrasse 1, D/​79104
Freiburg, Federal Republic of Germany.

We characterize CrRLK1, a novel type of receptor/​like kinase (RLK), from the
plant Catharanthus roseus (Madagascar periwinkle). The protein (90.2 kDa)
deduced from the complete genomic and cDNA sequences is a RLK by predicting a
N/​terminal signal peptide, a large extracytoplasmic domain, a membrane/​spanning
hydrophobic region followed by a transfer/​stop signal, and a C/​terminal
cytoplasmic protein kinase with all 11 conserved subdomains. It is a novel RLK
type because the predicted extracytoplasmic region shares no similarity with
other RLKs. The autophosphorylation was investigated with affinity/​purified
proteins expressed in Escherichia coli. The activity was higher with Mn2+ than
with Mg2+ and achieved half/​maximal rates at 2/​2.5 microM ATP. The
phosphorylation was predominantly on Thr, less on Ser, and not on Tyr. In
contrast to other plant RLK, the kinase used an intra/​ rather than an
intermolecular phosphorylation mechanism. After protein cleavage with formic
acid, most of the radioactivity was in a 14.1/​kDa peptide located at the end of
the kinase domain. Mutagenesis of the four Thr residues in this peptide
identified Thr/​720 in the subdomain XI as important for autophosphorylation and
for phosphorylation of beta/​casein. This Thr is conserved in other related
kinases, suggesting a subfamily sharing common autophosphorylation mechanisms.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8900145 [PubMed /​ indexed for MEDLINE]

246: Indian J Biochem Biophys. 1996 Aug;33(4):261/​73.

Enzymology of indole alkaloid biosynthesis in Catharanthus roseus.

Misra N, Luthra R, Kumar S.

Department of Biochemistry and Molecular Biology, CIMAP, Lucknow, India.

Indole alkaloids in Catharanthus roseus have been in focus because of their
medicinal value. These alkaloids consist of an indole moiety provided by
tryptamine and a terpenoid portion provided by the secologanin. The most
important catharanthus alkaloids are vinblastine (VLB), vincristine (VCR) and
ajmalicine. VLB and VCR are clinically useful anticancer agents whereas
ajmalicine is used for the treatment of circulatory diseases. VCR and VLB are
the most expensive because of their low abundance in the plant, and are formed
by the coupling of monomeric indole alkaloids vindoline and catharanthine,
catalysed by peroxidases. The pathway that lead to monomeric indole alkaloids
involves more than 20 enzymes of which 16 enzymes have been isolated and
characterized biochemically, and only three at the molecular level. The present
state of knowledge on enzymes and genes involved in indole alkaloid biosynthesis
and various aspects of their regulation has been discussed.

Publication Types:
Review

PMID: 8936815 [PubMed /​ indexed for MEDLINE]

247: Planta Med. 1996 Jun;62(3):278/​80.

Analysis of several iridoid and indole precursors of terpenoid indole alkaloids
with a single HPLC run.

Dagnino D, Schripsema J, Verpoorte R.

Division of Pharmacognosy, Center for Biopharmaceutical Sciences, Leiden
University, P.O. Box 9502, NL/​2300 RA Leiden, The Netherlands.

An isocratic HPLC system is described which allows the separation of the iridoid
and indole precursors of terpenoid indole alkaloids, which are present in a
single crude extract. The system consists of a column of LiChrospher 60 RP
select B 5 microm, 250 x 4 mm (Merck) with an eluent of 1% formic
acid/​acetonitrile/​trichloroacetic acid (100:10:0.25, v:v:w) at a flow of 1.2
ml/min. In the suspension cultures of CATHARANTHUS ROSEUS secologanin and
tryptophan were detected. In the cultures of TABERNAEMONTANA DIVARICATA loganin,
tryptophan, and tryptamine accumulated.

PMID: 17252445 [PubMed /​ in process]

248: FEBS Lett. 1996 Feb 12;380(1/​2):188/​93.

UV/​B/​ and oxidative stress/​induced increase in nicotinamide and trigonelline and
inhibition of defensive metabolism induction by poly(ADP/​ribose)polymerase
inhibitor in plant tissue.

Berglund T, Kalbin G, Strid A, Rydstrom J, Ohlsson AB.

Department of Biochemistry and Biotechnology, Royal Institute of Technology,
Stockholm, Sweden.

Nicotinamide and trigonelline contents increased in Catharanthus roseus tissue
culture after exposure to 2,2'/​azobis(2/​amidinopropane)dihydrochloride (AAPH) or
vanadylsulfate and in Pisum sativum leaves after exposure to UV/​B radiation.
Vanadylsulfate increased phenylalanine ammonia/​lyase (PAL) activity and the
content of reduced and oxidized gluthathione in C. roseus tissue culture. The
increases in PAL activity caused by 2 mM AAPH or 0.2mM vanadylsulfate were
prevented by 0.1 mM 3/​aminobenzamide (3/​AB), an inhibitor of
poly(ADP/​ribose)polymerase. Present results support the hypothesis [Berglund,
T., FEBS Lett. (1994) 351, 145/​149] that nicotinamide and/or its metabolites may
function as signal transmittors in the response to oxidative stress in plants
and that poly(ADP/​ribose)polymerase has a function in the induction of defensive
metabolism.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8603735 [PubMed /​ indexed for MEDLINE]

249: Hindustan Antibiot Bull. 1996 Feb/​Nov;38(1/​4):53/​6.

In vitro evaluation of medicinal plant extracts against Pestalotiopsis
mangiferae.

Rai MK.

Department of Botany, Danielson College, Chhindwara, India.

A serious leaf/​spot disease of Mangifera indica was noted during the last 10
years in Satpura plateau of India. On the basis of characteristic symptoms and
cultural characters, the pathogen was identified as Pestalotiopsis mangiferae
which is hitherto not reported from Satpura plateau of India. Screening of
17/​medicinal plants against the test pathogen revealed 14 antimycotic whereas
3/​plants, viz., Argemone mexicana, Caesalpinia bonducella, and Casia fistula
acclerated the growth of the pathogen. The maximum activity was shown by
Eucalyptus globulus (88%) and Catharanthus roseus (88%) followed by Ocimum
sanctum (85.50%), Azadirachta indica (84.66%), Ricinus communis (75%) and
Lawsonia inermis (74.33%) while the minimum activity was exhibited by Jatropha
curcas (10%).

PMID: 9676046 [PubMed /​ indexed for MEDLINE]

250: FEBS Lett. 1995 Nov 6;374(3):345/​50.

Cinnamate 4/​hydroxylase from Catharanthus roseus, and a strategy for the
functional expression of plant cytochrome P450 proteins as translational fusions
with P450 reductase in Escherichia coli.

Hotze M, Schroder G, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

A PCR/​based approach was used to isolate cDNAs for cinnamate 4/​hydroxylase (C4H)
from Catharanthus roseus cell cultures. The protein shared 75.9% identity with
C4H from other plants, and the transcription was induced under various stress
conditions. The cloned protein was used to investigate the functional expression
of plant P450/P450/​reductase fusions in E. coli. Fusions containing a modified
N/​terminal membrane anchor were located in the membrane and possessed C4H
activity without solubilization or addition of other factors. The results
indicate that the fusion protein strategy provides a useful tool to analyze the
activities encoded in the rapidly increasing number of plant P450 sequences of
uncertain or unknown function. We also discuss critical elements of the
strategy: the choice of the E. coli host strain, the N/​terminal membrane anchor,
and the conditions for protein expression.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 7589568 [PubMed /​ indexed for MEDLINE]

251: Plant Cell. 1995 Nov;7(11):1787/​1799.

Creation of a Metabolic Sink for Tryptophan Alters the Phenylpropanoid Pathway
and the Susceptibility of Potato to Phytophthora infestans.

Yao K, De Luca V, Brisson N.

Department of Biochemistry, Universite de Montreal, Montreal, Quebec, H3C 3J7
Canada.

The creation of artificial metabolic sinks in plants by genetic engineering of
key branch points may have serious consequences for the metabolic pathways being
modified. The introduction into potato of a gene encoding tryptophan
decarboxylase (TDC) isolated from Catharanthus roseus drastically altered the
balance of key substrate and product pools involved in the shikimate and
phenylpropanoid pathways. Transgenic potato tubers expressing the TDC gene
accumulated tryptamine, the immediate decarboxylation product of the TDC
reaction. The redirection of tryptophan into tryptamine also resulted in a
dramatic decrease in the levels of tryptophan, phenylalanine, and
phenylalanine/​derived phenolic compounds in transgenic tubers compared with
nontransformed controls. In particular, wound/​induced accumulation of
chlorogenic acid, the major soluble phenolic ester in potato tubers, was found
to be two/​ to threefold lower in transgenic tubers. Thus, the synthesis of
polyphenolic compounds, such as lignin, was reduced due to the limited
availability of phenolic monomers. Treatment of tuber discs with arachidonic
acid, an elicitor of the defense response, led to a dramatic accumulation of
soluble and cell wall/​bound phenolics in tubers of untransformed potato plants
but not in transgenic tubers. The transgenic tubers were also more susceptible
to infection after inoculation with zoospores of Phytophthora infestans, which
could be attributed to the modified cell wall of these plants. This study
provides strong evidence that the synthesis and accumulation of phenolic
compounds, including lignin, could be regulated by altering substrate
availability through the introduction of a single gene outside the pathway
involved in substrate supply. This study also indicates that phenolics, such as
chlorogenic acid, play a critical role in defense responses of plants to fungal
attack.

PMID: 12242360 [PubMed /​ as supplied by publisher]

252: Plant Physiol. 1995 Oct;109(2):717/​720.

Tryptophan Decarboxylase, Tryptamine, and Reproduction of the Whitefly.

Thomas JC, Adams DG, Nessler CL, Brown JK, Bohnert HJ.

Department of Biochemistry (J.C.T., D.G.A., H.J.B.) and Department of Plant
Sciences (J.K.B., H.J.B.), University of Arizona, Tucson, Arizona 85721.

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts
tryptophan to the indole/​alkaloid tryptamine. When the TDC gene was expressed in
transgenic tobacco, the 55/​kD TDC enzyme and tryptamine accumulated. Bemisia
tabaci (sweetpotato whitefly) reproduction on transgenic plants decreased up to
97% relative to controls. Production of tryptamine, its derivatives, or other
products resulting from TDC activity may discourage whitefly reproduction and
provide a single/​gene/​based plant protection strategy.

PMID: 12228625 [PubMed /​ as supplied by publisher]

253: Plant Physiol. 1995 Sep;109(1):131/​139.

A Cytochrome P/​450 Monooxygenase Catalyzes the First Step in the Conversion of
Tabersonine to Vindoline in Catharanthus roseus.

St/​Pierre B, De Luca V.

Institut de Recherche en Biologie Vegetale, Departement de Sciences Biologiques,
Universite de Montreal, Montreal, Quebec, Canada H1X 2B2.

Hydroxylation at the C/​16 position of the indole alkaloid tabersonine has been
suggested as the first step toward vindoline biosynthesis in Catharanthus
roseus. Tabersonine 16/​hydroxylase (16/​OH) activity was detected in total
protein extracts from young leaves of C. roseus using a novel coupled assay
system. Enzyme activity was dependent on NADPH and molecular oxygen and was
inhibited by CO, clotrimazole, miconazole, and cytochrome c. 16/​OH was localized
to the endoplasmic reticulum by linear sucrose density gradient centrifugation.
These data suggest that 16/​OH is a cytochrome P/​450/​dependent monooxygenase. The
activity of 16/​OH reached a maximum in seedlings 9 d postimbibition and was
induced by light. The leaf/​specific distribution of 16/​OH in the mature plant is
consistent with the localization of other enzymes in the tabersonine to
vindoline pathway. However, in contrast to enzymes that catalyze the last four
steps of vindoline biosynthesis, enzymes responsible for the first two steps
from tabersonine (16/​OH and 16/​O/​methyltransfersase) were detected in C. roseus
cell/​suspension cultures. These data complement the complex model of vindoline
biosynthesis that has evolved with respect to enzyme compartmentalization,
metabolic transport, and control mechanisms.

PMID: 12228585 [PubMed /​ as supplied by publisher]

254: Transgenic Res. 1995 Sep;4(5):315/​23.

Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown
gall calluses results in increased tryptamine levels but not in increased
terpenoid indole alkaloid production.

Goddijn OJ, Pennings EJ, van der Helm P, Schilperoort RA, Verpoorte R, Hoge JH.

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory,
Netherlands.

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in
the biosynthesis of terpenoid indole alkaloids in C. roseus by converting
tryptophan into tryptamine. Hardly any tdc mRNA could be detected in
hormone/​independent callus and cell suspension cultures transformed by the
oncogenic T/​DNA of Agrobacterium tumefaciens. Supply of tryptamine may therefore
represent a limiting factor in the biosynthesis of alkaloids by such cultures.
To investigate this possibility, chimaeric gene constructs, in which a tdc cDNA
is linked in the sense or antisense orientation to the cauliflower mosaic virus
35S promoter and terminator, were introduced in C. roseus cells by infecting
seedlings with an oncogenic A. tumefaciens strain. In the resulting crown gall
tumour calluses harbouring the tdc sense construct, an increased TDC protein
level, TDC activity and tryptamine content but no significant increase in
terpenoid indole alkaloid production were observed compared to
empty/​vector/​transformed tumour calluses. In tumour calluses containing the tdc
antisense construct, decreased levels of TDC activity were measured. Factors
which might be responsible for the lack in increased terpenoid indole alkaloid
production in the tdc cDNA overexpressing crown gall calluses are discussed.

PMID: 8589734 [PubMed /​ indexed for MEDLINE]

255: Free Radic Biol Med. 1995 Sep;19(3):319/​27.

Plant defense metabolism is increased by the free radical/​generating compound
AAPH.

Ohlsson AB, Berglund T, Komlos P, Rydstrom J.

Department of Biochemistry and Biotechnology, Royal Institute of Technology,
Stockholm, Sweden.

Effects of the free radical/​generating substance 2,2'/​azobis(2/​amidinopropane)
dihydrochloride (AAPH) on defense systems in plant tissue cultures were
investigated. Exposure of Catharanthus roseus, C. tricophyllus, and Pisum
sativum cultures to AAPH caused altered levels of reduced and oxidized
glutathione. An increased total glutathione content in C. roseus was prevented
by the glutathione biosynthesis inhibitor buthionine/​sulfoximine. The specific
phenylalanine ammonia/​lyase activity in a C. roseus culture was increased from 4
to 34 mukat(kg protein)/​1 by 1 mM AAPH. 5 mM AAPH increased the excretion of
phenolic substances into the culture medium of a Pisum sativum culture, from 18
to 67 micrograms ml/​1. The level of thiobarbituric acid reactants in a C.
tricophyllus culture was increased from 46 to 93 nmol(g fresh weight)/​1 by 0.4
mM AAPH. The present results, which constitute the first report on effects of
the radical/​generator AAPH on plant tissue, were achieved with cultures of
various plant species and various types of tissue differentiation and
demonstrate that AAPH is a suitable agent for the stimulation of the defensive
and secondary metabolism in plant tissue cultures. It is proposed that the
effects caused by AAPH are mediated by the generation of free radicals and
oxidative stress, and that this agent may be used as a model substance for ozone
and UV/​B exposure.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 7557546 [PubMed /​ indexed for MEDLINE]

256: Gene. 1995 Aug 19;161(2):295/​6.

A cDNA encoding a plant homologue to animal HMG box proteins involved in
structure/​specific recognition of DNA (SSRP family).

Hotze M, Lurz G, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

We report a cDNA encoding a 71/​kDa protein with a single high/​mobility group
(HMG) box and two nuclear localization signals from the higher plant
Catharanthus roseus (Madagascar periwinkle). The protein had 40% amino acid
identity with animal DNA/​binding proteins of the SSRP (structure/​specific
recognition protein) family that recognize bent, unwound DNA structures. Genomic
Southern analysis suggested the presence of two genes.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 7665097 [PubMed /​ indexed for MEDLINE]

257: Eur J Biochem. 1995 Jun 15;230(3):1053/​8.

Vitamin/​B12/​independent methionine synthase from a higher plant (Catharanthus
roseus). Molecular characterization, regulation, heterologous expression, and
enzyme properties.

Eichel J, Gonzalez JC, Hotze M, Matthews RG, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

Methionine synthases catalyze the formation of methionine by the transfer of a
methyl group from 5/​methyltetrahydrofolate to homocysteine. This reaction is the
last step in L/​methionine biosynthesis, and it also serves to regenerate the
methyl group of S/​adenosylmethionine, a cofactor required for biological
methylation reactions. We describe the cloning, expression and characterization
of a methionine synthase from the higher plant Catharanthus roseus. cDNAs were
identified that encoded a protein of 85 kDa sharing 50% identify with the
cobalamin/​independent methionine synthase from Escherichia coli (MetE) and 41%
identity with a partial sequence of a yeast homolog of MetE. The C. roseus
protein was expressed at high levels in E. coli. The enzyme accepts the
triglutamate form of methyltetrahydrofolate as a methyl donor but not the
monoglutamate form, and it does not require S/​adenosylmethionine or cobalamin
for activity. The properties indicate that the enzyme is a cobalamin/​independent
methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant
protein does not require phosphate or magnesium ions for activity. Immunoblots
of plants extracts showed that the protein was localized in the cytosol, and was
present in a variety of plant species. A nutritional downshift of the C. roseus
cell culture revealed a strong, transient transcriptional activation, but no
significant increment in the total level of the protein. The availability of the
protein and the cDNA now provide tools to investigate the complexities of
methionine biosynthesis in plants.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

PMID: 7601135 [PubMed /​ indexed for MEDLINE]

258: Plant Physiol. 1995 May;108(1):295/​302.

Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don
Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast
Inorganic Phosphate Transport System?).

Sakano K, Yazaki Y, Okihara K, Mimura T, Kiyota S.

National Institute of Agrobiological Resources, Department of Applied
Physiology, 2/​1/​2 Kannondai, Tsukuba, Ibaraki 305, Japan (K.S., Y.Y., K.O.,
S.K.).

Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was
studied in relation to its apparent uncontrolled uptake using 31P/​nuclear
magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated
that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g/​1 fresh weight h/​1,
respectively. Pi uptake in Murashige/​Skoog medium under different Pi
concentrations and different initial cell densities followed basically the same
kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate
(Vm) for the first hours and accumulated it in the vacuole. As the endogenous
pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi
accumulation was 100 to 120 [mu]mol g/​1 fresh weight if cell swelling during Pi
uptake (about 2/​fold in cell volume) was not considered. Results indicated that
(a) the rate of Pi uptake by Catharanthus cells was independent of initial cell
density and was constant over a wide range of Pi concentrations (2 mM to about
10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no
apparent feedback control over the Pi uptake process in the plasma membrane to
avoid Pi toxicity. The importance of the tonoplast Pi transport system in
cytoplasmic Pi homeostasis is discussed.

PMID: 12228474 [PubMed /​ as supplied by publisher]

259: Biochem J. 1995 Mar 1;306 ( Pt 2):571/​80.

Strictosidine synthase from Catharanthus roseus: purification and
characterization of multiple forms.

de Waal A, Meijer AH, Verpoorte R.

Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden
University, Gorlaeus Laboratories, The Netherlands.

Multiple (six) forms of strictosidine synthase from Catharanthus roseus cell
suspension cultures were purified and characterized. A purification protocol is
presented composed of hydrophobic/​interaction, gel/​permeation and ion/​exchange
chromatography and chromatofocusing. Four of six isoforms were purified to
apparent homogeneity, whereas two others were nearly homogeneous. All
strictosidine synthase isoforms were found to be glycoproteins. The isoforms
were also found in leaves and roots of the plant, in seedlings and in hairy root
cultures. The ratio of the different isoforms differed slightly between these
sources. The kinetic parameters of the isoforms showed no significant
differences. The maximal velocity (300/​400 nkat/mg of protein) is the highest
reported so far. It was demonstrated that the apparent Michaelis constant for
tryptamine (approx. 9 microM) is much lower than values reported previously. The
presence of weak product inhibition (Kp approx. 35 times Km) was established,
whereas substrate inhibition was not detected.

Publication Types:
Comparative Study

PMID: 7887913 [PubMed /​ indexed for MEDLINE]

260: Eur J Biochem. 1995 Feb 15;228(1):74/​8.

cDNAs for S/​adenosyl/​L/​methionine decarboxylase from Catharanthus roseus,
heterologous expression, identification of the proenzyme/​processing site,
evidence for the presence of both subunits in the active enzyme, and a conserved
region in the 5' mRNA leader.

Schroder G, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

S/​Adenosyl/​L/​methionine decarboxylases (AdoMetDC) are pyruvoyl/​dependent enzymes
producing the aminopropyl group for spermidine biosynthesis, and this reaction
is the rate/​limiting step in polyamine biosynthesis. We characterized cDNAs from
Catharanthus roseus (Madagascar periwinkle) and investigated the enzyme after
heterologous expression. The largest cDNA (1842 bp) had an 5' leader of 469 bp
and encoded a protein of 357 residues and 30/​35% identity with mammalian
AdoMetDC. The proenzyme expressed in Escherichia coli was processed into active
enzyme, and the processing site was identified by site/​directed mutagenesis as
the second Ser in the sequence Leu/​Ser/​Glu/​Ser/​Ser. The analysis of
affinity/​purified proteins indicated that the active enzyme contained both
subunits. The Km for S/​adenosyl/​L/​methionine was 35/​40 microM, and the enzyme
activity was not stimulated by putrescine. The 5' leader of the mRNA contained
start and stop codons for a polypeptide of 51 amino acids, and this region was
conserved in the 5' leaders of other plant AdoMetDC mRNAs. The putative
polypeptide had no similarity with the hexapeptide responsible for modulation of
AdoMetDC mRNA translation in mammals. The possibility is discussed that plants
evolved a different type of translational regulation.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 7883014 [PubMed /​ indexed for MEDLINE]

261: Drug Metabol Drug Interact. 1995;12(3/​4):369/​82.

Cytochrome P/​450 in plant/insect interactions: geraniol 10/​hydroxylase and the
biosynthesis of iridoid monoterpenoids.

Hallahan DL, West JM.

Biochemistry & Physiology Department, IACR Rothamsted Experimental Station,
Harpenden, Herts, U.K.

The interactions between plant secondary metabolites (particularly monoterpenes)
and insects are discussed. Such metabolites are likely to have influenced the
evolution of cyt P450/​linked detoxification systems in animals, through
animal/plant coevolution. The biosynthesis of many classes of plant secondary
metabolites involves cyt P450 enzymes. Of these, one of the best characterised
is the geraniol/nerol 10/​hydroxylase which catalyses a key step in the
biosynthesis of the iridoid class of plant terpenes. It would appear that these
monoterpenoids are synthesised (via cyt P450 hydroxylation) from different
precursors in different plant species, namely geraniol, its isomer nerol, or the
related monoterpenoid, citronellol. We show that cyt P450 from the plants
Catharanthus roseus and Nepeta racemosa are capable of hydroxylating geraniol,
nerol and citronellol, and thus do not impose precursor specificity on iridoid
biosynthesis in plants.

Publication Types:
Review

PMID: 8820862 [PubMed /​ indexed for MEDLINE]

262: Bull Environ Contam Toxicol. 1994 Nov;53(5):779/​86.

Effects of mercury (II) species on cell suspension cultures of Catharanthus
roseus.

Zhu L, Cullen WR.

Department of Environmental Science, Hangzhou University, China.

Publication Types:
Comparative Study

PMID: 7833617 [PubMed /​ indexed for MEDLINE]

263: J Chromatogr B Biomed Appl. 1994 Oct 14;660(2):405/​8.

Application of high/​performance liquid chromatography to the determination of
vinblastine in Catharanthus roseus.

Volkov SK, Grodnitskaya EI.

All/​Russia Institute of Medicinal and Aromatic Plants, Moscow.

A method for the determination of vinblastine in Catharanthus roseus by HPLC is
described. A crude alkaloid extract, obtained by extraction of leaves with
toluene, 2% citric or tartaric acid and benzene, was separated by TLC. The
vinblastine fraction was cut out and vinblastine was eluted from the sorbent.
The amount of vinblastine was determined by HPLC with peak/​height measurement.
The standard deviation is 0.2 microgram/ml. The detection limit is 0.05 ng of
vinblastine in a sample applied to the HPLC column.

PMID: 7866534 [PubMed /​ indexed for MEDLINE]

264: Phytochemistry. 1994 Sep;37(2):401/​3.

Introduction of oxygenated functional groups into 3/​carene and 2/​pinene by
cultured cells.

Hirata T, Ikeda Y, Izumi S, Shimoda K, Hamada H, Kawamura T.

Department of Chemistry, Faculty of Science, Hiroshima University, Japan.

The biotransformation of the monoterpene hydrocarbons 3/​carene and 2/​pinene by
cell suspension cultures of Nicotiana tabacum and Catharanthus roseus was
investigated. The cultures have the ability to regio/​ and enantioselectively
introduce the oxygenated functional groups into the C = C double bond and the
allylic positions of the substrates.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 7765622 [PubMed /​ indexed for MEDLINE]

265: Plant Physiol. 1994 Jul;105(3):903/​909.

Phosphatidate Kinase, A Novel Enzyme in Phospholipid Metabolism
(Characterization of the Enzyme from Suspension/​Cultured Catharanthus roseus
Cells).

Wissing JB, Kornak B, Funke A, Riedel B.

Enzymologie, Gesellschaft fur Biotechnologische Forschung, D/​38124 Braunschweig,
Germany.

Phosphatidate kinase (adenosine 5[prime]/​triphosphate:phosphatidic acid
phosphotransferase), a novel enzyme of phospholipid metabolism, was detected
recently in the plasma membranes of suspension/​cultured Catharanthus roseus
cells and purified (J.B. Wissing, H. Behrbohm [1993] Plant Physiol 102:
1243/​1249). In the present work the properties of phosphatidate kinase are
described. The enzyme showed a pH optimum of 6.1 and an isoelectric point of
4.8, and was rather stable in the presence of its substrates. Although the
kinase accepted both ATP and GTP, with Km values of about 12 and 18 [mu]M,
respectively, the only lipid substrate was phosphatidic acid; neither
lysophosphatidic acid nor any other lipid tested was phosphorylated. With 32P/​
and 14C/​labeled diacylglycerol pyrophosphate, the product of the enzyme, it was
shown that the kinase catalyzes a reversible reaction. The activity of the
extracted enzyme depended on the presence of surfactants such as Triton X/​100 or
[beta]/​octylglucoside, whereas deoxycholate was strongly inhibitory. Kinetic
analysis with Triton X/​100/phosphatidate mixed micelles performed according to
the "surface dilution" kinetic model showed saturation kinetics with respect to
both bulk and surface concentration of phosphatidate. The interfacial Michaelis
constant for phosphatidate was determined as 0.6 mol %.

PMID: 12232252 [PubMed /​ as supplied by publisher]

266: J Biotechnol. 1994 Jun 15;35(1):1/​7.

Large/​scale cultivation of Catharanthus roseus cells: production of ajmalicine
in a 20/​l airlift bioreactor.

Fulzele DP, Heble MR.

Plant Biotechnology Section, Bhabha Atomic Research Centre, Trombay, Bombay,
India.

Bioreactor systems have been developed for the production of ajmalicine, an
alkaloid used in the treatment of hypertension. Cell cultures of Catharanthus
roseus produced higher levels of ajmalicine (323 micrograms g/​1 dry weight) in a
production medium enriched with tryptophan. The cell cultures were grown in
medium prepared in tap water and market sugar with a view to minimise the costs
of production. Large/​scale cultivation of cell suspension was performed in a
20/​l airlift bioreactor under controlled conditions. An ajmalicine production of
315 micrograms g/​1 dry weight was achieved in the bioreactor after 14 d of
cultivation.

PMID: 7765026 [PubMed /​ indexed for MEDLINE]

267: Int J Dev Biol. 1994 Jun;38(2):287/​99.

Mechanisms of the proliferation and differentiation of plant cells in cell
culture systems.

Fukuda H, Ito M, Sugiyama M, Komamine A.

Biological Institute, Faculty of Science, Tohoku University, Sendai, Japan.

Plant cell functions have been investigated in various cell culture systems. In
this review, we summarize results obtained from investigations of gene
expression during the cell cycle in synchronized cultures of Catharanthus roseus
during somatic embryogenesis in suspension cultures of Daucus carota, during
organogenesis in tissue cultures of Arabidopsis thaliana and during the
transdifferentiation of isolated mesophyll cells to tracheary elements in
single/​cell cultures of Zinnia elegans.

Publication Types:
Review

PMID: 7981037 [PubMed /​ indexed for MEDLINE]

268: Planta Med. 1994 Apr;60(2):149/​52.

Long/​Term Effect of a Pythium Elicitor Treatment on the Growth and Alkaloid
Production of Catharanthus roseus Cell Suspensions.

Nef/​Campa C, Trouslot MF, Trouslet P, Chrestin H.

Laboratoire de Microbiologie, ORSTOM, Centre de Bel/​Air, BP 1386. Dakar,
Republique du Senegal.

The treatment of a CATHARANTHUS ROSEUS cell suspension culture with a low
concentration of PYTHIUM elicitor stimulated the alkaloid production. When these
pretreated cells were resuspended in a medium that did not contain the fungal
extract, the positive effects of the treatment on alkaloid synthesis and
excretion were lost and, moreover, the standard level of production was not
recovered. A second treatment of these cells with PYTHIUM elicitor at day 5 of
the second culture cycle greatly impaired growth kinetics, but did not stimulate
the alkaloid production observed with standard cultures. Repeated treatments
with a low concentration of fungal elicitor seemed to have a negative long/​term
effect on both growth and alkaloid synthesis and did not appear to be a useful
process for production purposes.

PMID: 17236032 [PubMed /​ in process]

269: Plant Mol Biol. 1994 Mar;24(6):863/​78.

Meristem/​specific gene expression directed by the promoter of the
S/​phase/​specific gene, cyc07, in transgenic Arabidopsis.

Ito M, Sato T, Fukuda H, Komamine A.

Nagoya University BioScience Center, Japan.

A genomic clone for the cyc07 gene, which is expressed specifically at the S
phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus
roseus) cells, was isolated. Determination of the nucleotide sequence of the
clone revealed that the cyc07 gene consists of seven exons separated by six
introns. Genomic Southern analysis indicated that the cyc07 gene is present as a
single copy per haploid genome in periwinkle. Expression of related genes was
detected in a wide range of other plants. Transgenic Arabidopsis plants were
generated that expressed the gene for beta/​glucuronidase (GUS) under the control
of the promoter of the cyc07 gene. The tissue/​specific pattern of expression
directed by the promoter was investigated by analysis of GUS activity.
Histochemical tests demonstrated that 589 bp of the 5'/​upstream sequence of the
cyc07 gene could direct specifical expression of the GUS reporter gene in
meristematic tissues in transgenic plants. The spatial pattern of expression
directed by the promoter was closely correlated with meristematic activity and
cell proliferation, suggesting an association between the function of the cyc07
gene and cell proliferation.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8204824 [PubMed /​ indexed for MEDLINE]

270: Plant Physiol. 1994 Mar;104(3):1099/​100.

cDNA for S/​adenosyl/​L/​homocysteine hydrolase from Catharanthus roseus.

Schroder G, Waitz A, Hotze M, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8165255 [PubMed /​ indexed for MEDLINE]

271: Plant Physiol. 1994 Mar;104(3):1097/​8.

cDNA for a 14/​kilodalton polypeptide from Madagascar periwinkle (Catharanthus
roseus). Eight conserved cysteines in six related proteins from different plants
suggest common functional elements.

Hotze M, Waitz A, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 8165254 [PubMed /​ indexed for MEDLINE]

272: Plant Mol Biol. 1994 Feb;24(3):545/​7.

Isolation and characterization of a rice cDNA similar to the S/​phase/​specific
cyc07 gene.

Kidou S, Umeda M, Tsuge T, Kato A, Uchimiya H.

Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.

We isolated a rice cDNA clone (T151) which encodes an open reading frame of 262
amino acids. This clone is similar to the S/​phase/​specific cyc07 gene of
Catharanthus roseus. Expression of this gene is much higher in callus than in
seedlings and regulated by external stresses such as high osmotic pressure,
salinity, low temperature and submergence.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 7510136 [PubMed /​ indexed for MEDLINE]

273: Mol Gen Genet. 1994 Jan;242(2):217/​25.

Nucleotide sequence of the tryptophan decarboxylase gene of Catharanthus roseus
and expression of tdc/​gusA gene fusions in Nicotiana tabacum.

Goddijn OJ, Lohman FP, de Kam RJ, Schilperoort RA, Hoge JH.

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory,
The Netherlands.

The enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) converts tryptophan into
tryptamine. In Catharanthus roseus and other plants capable of producing
terpenoid indole alkaloids (TIAs) TDC links primary metabolism to the secondary
metabolic pathway involved in the biosynthesis of these compounds. The
accumulation of tdc mRNA in C. roseus cells is developmentally regulated and
transcriptionally influenced by elicitors (induction) and auxins (repression).
Here we report that TDC is encoded by a single copy gene in the C. roseus
genome. No introns were observed upon isolation and sequencing of this gene. To
study gene expression controlled by the tdc promoter, a 2 kb promoter fragment
and a number of 5' deleted promoter derivatives were joined in translational
fusion to a beta/​D/​glucuronidase reporter gene (gusA). Expression of the
chimaeric constructs was monitored in stably transformed tobacco plants and in
transiently transfected tobacco protoplasts. Histochemical and fluorimetric
analysis of transgenic plants revealed that 1938 bp of the tdc promoter (with
respect to the translational start codon) give rise to GUS activity in roots,
stems and leaves. No tissue or cell type specificity was noted. Promoter
deletions up to nucleotide /​398 directed lower levels of gusA expression but
conferred the same pattern of staining for GUS activity as the /​1938 construct.
Further deletion of the tdc promoter up to nucleotide /​232 resulted in
drastically reduced GUS activity levels and loss of GUS staining in all parts of
the transgenic plants. In contrast to stable transformation, the /​232 tdc/​gusA
construct gave rise to GUS activity levels comparable to those of the /​398
construct in an assay system for transient expression in protoplasts.(ABSTRACT
TRUNCATED AT 250 WORDS)

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8159173 [PubMed /​ indexed for MEDLINE]

274: Chin J Biotechnol. 1994;10(3):203/​9.

Suspension culture of Catharanthus roseus crown gall cell induced by
Agrobacterium C58.

Wang N, Wang S, Tian J, Li X, Zhu L.

Biochemistry and Molecular Biology Department of Nankai University, Tianjing,
China.

In comparison with calli from leaf or stem, Catharanthus roseus crown gall cell
cultured on MS basic medium was superior in growth, total indole alkaloids and
ajmalicine contents. The effects of illumination, cultural temperature, sucrose
level of the medium and exogenous L/​Trp on the growth, total indole alkaloids
and ajmalicine contents of C. roseus crown gall cell cultures were studied. The
results will provide a theoretical basis for the attempt of using suspension
cultures of C. roseus crown gall cells to produce indole alkaloids.

PMID: 7893941 [PubMed /​ indexed for MEDLINE]

275: Biochimie. 1994;76(5):410/​6.

2,4/​D and alkaloid accumulation in periwinkle cell suspensions.

Arvy MP, Imbault N, Naudascher F, Thiersault M, Doireau P.

Laboratoire de Physiologie Vegetale, Faculte des Sciences, Tours, France.

Omission of 2,4/​D from culture medium during one subculture of Catharanthus
roseus cells, strain C20, resulted in an increased alkaloid accumulation,
without effect on growth. Alkaloid accumulation, rather than growth, seemed to
be more sensitive to 2,4/​D. 2,4/​D inhibited alkaloid accumulation essentially
during growth phase, but its inhibitory effect during this period was partially
reversible. As this reversibility was underlined only during the stationary
phase, this suggested that this action could be situated upstream in a terpenoid
non/​specific pathway. 2,4/​D feeding showed that inhibition is weaker and weaker
as the alkaloid accumulation period proceeds. Auxin action during this period
could take place downstream in specific alkaloid pathways. The lower alkaloid
accumulation obtained after loganic acid feeding compared to that obtained with
secologanin and loganin could indicate that loganic acid methylation should be
one of the 2,4/​D target(s).

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 7849107 [PubMed /​ indexed for MEDLINE]

276: Biochem Biophys Res Commun. 1993 Dec 15;197(2):518/​22.

Purification and partial characterization of microsomal NADH/​cytochrome b5
reductase from higher plant Catharanthus roseus.

Madyastha KM, Chary NK, Holla R, Karegowdar TB.

Department of Organic Chemistry, Indian Institute of Science, Bangalore.

A simple three step procedure was used to purify microsomal NADH/​cytochrome b5
(ferricyanide) reductase to homogeneity from the higher plant C. roseus. The
microsomal bound reductase was solubilized using zwitterionic detergent/​CHAPS.
The solubilized reductase was subjected to affinity chromatography on octylamino
Sepharose 4B, blue 2/​Sepharose CL/​6B and NAD(+)/​Agarose. The homogeneous enzyme
has an apparent molecular weight of 33,000 as estimated by SDS/​PAGE. The
purified enzyme catalyzes the reduction of purified cytochrome b5 from C. roseus
in the presence of NADH. The reductase also readily transfers electrons from
NADH to ferricyanide (Km 56 microM), 2, 6/​dichlorophenolindophenol (Km 65
microM) and cytochrome c via cytochrome b5 but not to menadione.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8267585 [PubMed /​ indexed for MEDLINE]

277: Plant Mol Biol. 1993 Nov;23(3):583/​94.

HSP90 homologue from Madagascar periwinkle (Catharanthus roseus): cDNA sequence,
regulation of protein expression and location in the endoplasmic reticulum.

Schroder G, Beck M, Eichel J, Vetter HP, Schroder J.

Institut fur Biologie II, Universitat Freiburg, Germany.

We describe cDNAs for a HSP90 homologue from Catharanthus roseus and studies on
the regulation of expression. The largest cDNA (2670 bp) coded for a protein of
817 amino acids with a calculated size of 93,491 Da and a pI of 4.61. It
contained a eucaryotic secretory signal, the endoplasmic reticulum (ER)
targeting and retention signal (Lys/​Asp/​Glu/​Leu), and the HSP90 protein family
signature with one conservative exchange (Asn/​Lys/​Asp/​Ile/​Phe/​Leu instead of
Asn/​Lys/​Glu/​Ile/​Phe/​Leu). RNA blots revealed a transcript of 2.8/​2.9 kb, and
genomic DNA blots suggested a single gene. The expression was analysed with
antiserum against a fusion protein expressed in Escherichia coli. Immunoblots
revealed a protein of 93 +//​ 1.5 kDa (often a doublet) only in the membrane
fraction, and sucrose density gradients suggested association with the ER. The
protein was constitutively expressed in C. roseus cell cultures grown at 25
degrees C, and expression was apparently unaffected by various stress
conditions, such as heat, high sucrose, elicitor from Phytophthora megasperma or
yeast extract. It was not detectable in young C. roseus plants at room
temperature, and heat shock for several hours at 37 degrees C was necessary to
obtain detectable expression. In maize (Zea mays), a cross/​reacting protein was
detectable in cell cultures, but not in young plants. The results suggested that
the cloned protein is not a major component in the heat shock response. We
propose a chaperone role in the assembly and processing of cell wall components
and other secreted proteins, i.e. functions that are very active in cells with a
high rate of growth and division.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8106014 [PubMed /​ indexed for MEDLINE]

278: Plant Physiol. 1993 Aug;102(4):1243/​1249.

Phosphatidate Kinase, a Novel Enzyme in Phospholipid Metabolism (Purification,
Subcellular Localization, and Occurrence in the Plant Kingdom).

Wissing JB, Behrbohm H.

Enzymologie, Gesellschaft fur Biotechnologische Forschung, D/​3300 Braunschweig,
Germany.

Microsomal membranes from suspension/​cultured Catharanthus roseus cells possess
an enzymic activity that catalyzes the ATP/​dependent phosphorylation of
phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B.
Wissing [1993] FEBS Lett 315: 95/​99). This enzyme activity, PA kinase, was
purified and characterized. Plasma membranes, obtained from C. roseus microsomes
by aqueous two/​phase partitioning, were extracted, and PA kinase was purified
3200/​fold by applying different chromatographic steps that resulted in a
specific activity of about 10 [mu]mol min/​1 mg/​1. Sodium dodecyl sulfate/​gel
electrophoresis of the fractions obtained from the final chromatographic step
revealed a 39/​kD protein that correlated with the enzyme activity; PA kinase
activity could be eluted from this protein band. Subcellular localization,
investigated with C. roseus cells, showed that the activity was confined to
membrane fractions, and at least 80% was associated with plasma membranes. The
data revealed the same distribution within the cellular membranes of PA kinase
as reported for diacylglycerol kinase, which is a typical plasma
membrane/​located enzyme. Furthermore, PA kinase activity was detected in the
calli of 16 different plant species and in the different organs of C. roseus
plants and obviously occurs ubiquitously in the plant kingdom.

PMID: 12231900 [PubMed /​ as supplied by publisher]

279: J R Soc Health. 1993 Aug;113(4):190/​4.

Exploration of the frontiers of tradomedical practices: basis for development of
alternative medical healthcare services in developing countries.

Osujih M.

Rivers State College of Education, Port Harcourt, Nigeria.

The study is a brief exploration of the functions and roles of the traditional
healers in the total health care delivery system as a basis for tapping the
salient features of this age old art: for the purpose of refining, and
establishing it as an alternative medical health/​care service. The investigation
is considered relevant particularly in the developing countries where, in
addition to the dearth of orthodox medical services, institutions and personnel,
it is relatively cheaper, socio/​culturally accessible and acceptable. Refining
and developing some aspects of the traditional healers' services will serve the
interest of the health consumers whose main concern is with service and not the
source. Furthermore, it is hoped that the study will stimulate purposeful
discussions on the need for an unbiased examination of the materials, methods
and techniques of the traditional healers including, eventually, compiling a
native pharmacopoeia. A more comprehensive account of the traditional healers
contributions to the battle against diseases and maintenance of health and well
being is envisaged.

PIP: In traditional healing, practitioners use barks, leaves, nuts, fruit juices
and roots, and parts of domestic animals. They practice their craft mostly in
Africa, Asia, and other Third World countries, and they are variously called
juju priests, diviners, herbalists, and witch doctors. Cases of achievements in
their contributions to preventive and curative health have been documented. In
Nigeria, clients regularly patronize both orthodox and traditional medical
practitioners. Their remedies include healing the bite of the very poisonous
carpet viper, chronic bronchitis, peptic ulcer, and heart problems, as well as
performing uvulectomy and tonsillectomy. Quinine, the cure for malaria, was
originally the ritual medicine of the Incas of Peru. It was confirmed that
Azadirachta Indica (Meliaceae), the neem tree, used against malaria in Nigeria,
India, and Asia, had a potent antiplasmodial activity. The plant Streblus
asper, Linn (Shakhotoha Siora) is well known in Indian Ayurvedic medicine to
treat fever, filariasis, dysentery, and diarrhea. The alkaloids derived from
the Madagascan periwinkle Catharanthus roseus (Apocynaceae), used in a West
Indian remedy for diabetes mellitus, have antitumor activity. The drug
Maytensine, obtained from Mytenus ovatus Loes (Celastraceae), was found to be a
powerful antitumor agent in animals. Tea made from the leaves of Osyris
wightiana stimulated the flow of breast milk and also acted as a labor/​inducing
agent. Saponaria officinalis and Enterobbium cyclocarpum are both used in Egypt
and Tanzania as spermicide contraceptives. A 1985 survey in Cross River State,
Nigeria, demonstrated that 165 (61%) of respondents went to traditional healers
for treatment. Part of their continued popularity is the person/​centered
approach that is virtually lacking in orthodox hospitals, although this
humanistic approach to therapy is gradually gaining inroads into Western medical
education. The services of both kinds of medicine could be harmonized by
open/​minded appraisal, identification of positive aspects, and acceptance of
their complimentary nature.

Publication Types:
Comparative Study

PMID: 8410912 [PubMed /​ indexed for MEDLINE]

280: Plant Mol Biol. 1993 Aug;22(5):907/​12.

A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant
cells.

Goddijn OJ, van der Duyn Schouten PM, Schilperoort RA, Hoge JH.

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory,
Netherlands.

A novel selection system for plant genetic transformation was developed based on
the enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus.
This enzyme converts the toxic tryptophan analogue 4/​methyl tryptophan (4/​mT)
into the non/​toxic compound 4/​methyl tryptamine. Expression of tdc in transgenic
plants that have no endogenous TDC/​activity allows selection on 4/​mT. A vector
was constructed containing a tdc cDNA clone under control of the constitutively
expressed cauliflower mosaic virus 35S promoter. This vector was used in
Agrobacterium/​mediated tobacco leaf disc transformation experiments. The optimal
concentration for selection with 4/​mT was found to be 0.1 mM. The transformed
nature of shoots obtained after tdc gene transfer and subsequent selection on
0.1 mM 4/​mT was confirmed by northern blot analysis.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8358036 [PubMed /​ indexed for MEDLINE]

281: Plant J. 1993 Jul;4(1):47/​60.

Isolation and characterization of a cDNA clone from Catharanthus roseus encoding
NADPH:cytochrome P/​450 reductase, an enzyme essential for reactions catalysed by
cytochrome P/​450 mono/​oxygenases in plants.

Meijer AH, Lopes Cardoso MI, Voskuilen JT, de Waal A, Verpoorte R, Hoge JH.

Division of Pharmacognosy, Leiden University, Gorlaeus Laboratories, The
Netherlands.

The membrane/​bound flavoprotein NADPH:cytochrome P/​450 (cytochrome c) reductase,
that functions in electron transfer to cytochrome P/​450 monooxygenases, was
purified from a cell suspension culture of the higher plant Catharanthus roseus.
Anti/​serum raised against the purified protein was found to inhibit
NADPH:cytochrome c reductase activity as well as the activities of the
cytochrome P/​450 enzymes geraniol 10/​hydroxylase and trans/​cinnamate
4/​hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid
biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression
library resulted in the isolation of a partial NADPH: cytochrome P/​450 reductase
cDNA clone, which was identified on the basis of sequence homology with
NADPH:cytochrome P/​450 reductases from yeast and animal species. The identify of
the cDNA was confirmed by expression in Escherichia coli as a functional protein
capable of NADPH/​dependent reduction of cytochrome c and neotetrazolium, two in
vitro substrates for the reductase. The N/​terminal sequence of the reductase,
which was not present in the cDNA clone, was determined from a genomic NADPH:
cytochrome P/​450 reductase clone. It was demonstrated that the reductase
probably is encoded by a single copy gene. A sequence comparison of this plant
NADPH:cytochrome P/​450 reductase with the corresponding enzymes from yeast and
animals species showed that functional domains involved in binding of the
cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C.
roseus cell cultures a rapid increase of the reductase steady state mRNA level
was observed after the addition of fungal elicitor preparations that are known
to induce cytochrome P/​450/​dependent biosynthetic pathways.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 8220474 [PubMed /​ indexed for MEDLINE]

282: Plant Mol Biol. 1993 May;22(2):379/​83.

Isolation of cytochrome P/​450 cDNA clones from the higher plant Catharanthus
roseus by a PCR strategy.

Meijer AH, Souer E, Verpoorte R, Hoge JH.

Centre for Bio/​Pharmaceutical Sciences, Leiden University, Gorlaeus
Laboratories, Netherlands.

Cytochrome P/​450 monooxygenases are membrane/​bound enzymes involved in a wide
range of biosynthetic pathways in plants. An efficient PCR strategy for
isolating cytochrome P/​450 cDNA clones from plant cDNA libraries is described. A
set of degenerate primers for PCR amplification was designed to recognize
nucleotide sequences specifying the highly conserved haembinding region of
cytochrome P/​450 proteins. Using this primer set and a non/​specific primer,
complementary to either the poly(A) tail of the cDNA clones or a phage vector
sequence, we isolated 16 different cytochrome P/​450 cDNA sequences from a cDNA
library of Catharanthus roseus.

PMID: 8507838 [PubMed /​ indexed for MEDLINE]

283: Appl Microbiol Biotechnol. 1993 Apr;39(1):42/​7.

Induction of ajmalicine formation and related enzyme activities in Catharanthus
roseus cells: effect of inoculum density.

Moreno PR, Schlatmann JE, van der Heijden R, van Gulik WM, ten Hoopen HJ,
Verpoorte R, Heijnen JJ.

Division of Pharmacognosy, Leiden University, The Netherlands.

In Catharanthus roseus cell cultures the time courses of four enzyme activities,
tryptophan decarboxylase (TDC), strictosidine synthase (SSS),
geraniol/​10/​hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid
accumulation were compared under two different culture conditions (low/​inoculum
density and high/​inoculum density on induction medium) and a control on growth
medium. In growth medium a transient increase in TDC activity was first observed
after which G10H reached its maximum activity; only tryptamine accumulated, no
ajmalicine could be detected. Apparently, a concerted induction of enzyme
activities is required for ajmalicine formation. Cells inoculated in induction
medium showed such a concerted induction of AS, TDC and G10H activities. After
30 days the low/​density culture had accumulated six times more ajmalicine (in
mumoles/g) than the high/​density culture. Thus, increase in biomass
concentration (high/​density cultures) did not enhance the total alkaloid
production. The major differences observed in enzyme levels between high/​ and
low/​density cultures were in the AS and TDC activities, which were two to three
times higher in the low/​density culture, indicating that there is a positive
correlation between ajmalicine formation and AS and TDC activities.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 7763550 [PubMed /​ indexed for MEDLINE]

284: J Biol Chem. 1993 Mar 15;268(8):5504/​11.

Purification, characterization, and kinetic analysis of a
2/​oxoglutarate/​dependent dioxygenase involved in vindoline biosynthesis from
Catharanthus roseus.

De Carolis E, De Luca V.

Departement de Sciences Biologiques, Universite de Montreal, Quebec, Canada.

A 2/​oxoglutarate/​dependent dioxygenase (EC 1.14.11.11) which catalyzes the
hydroxylation at position 4 of the indole alkaloid, desacetoxyvindoline has been
purified to near homogeneity from Catharanthus roseus. The purification
procedure combined conventional chromatographic methods and cosubstrate affinity
chromatography on alpha/​ketoglutarate/​Sepharose. The specific activity of the
4/​hydroxylase was enriched over 2000/​fold compared to the crude homogenate with
a recovery of 1.6%. The molecular mass of the native and denatured 4/​hydroxylase
was found to be 45 and 44.7 kDa, respectively, suggesting that the native enzyme
is a monomer. Two/​dimensional isoelectric focusing under denaturing conditions
resolved the purified 4/​hydroxylase into three charge isoforms of pI values 4.6,
4.7, and 4.8. The enzyme did not require most divalent cations, but inactive
enzyme was reactivated in a time/​dependent manner by incubation with ferrous
ions. The mechanism of action of desacetoxyvindoline 4/​hydroxylase was
investigated. The results of substrate interaction kinetics and product
inhibition studies suggest an Ordered Ter Ter mechanism where 2/​oxoglutarate is
the first substrate to bind followed by the binding of O2 and
desacetoxyvindoline. The first product to be released was deacetylvindoline
followed by CO2 and succinate, respectively.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8449913 [PubMed /​ indexed for MEDLINE]

285: Eur J Biochem. 1993 Mar 1;212(2):431/​40.

Purification and characterization of anthranilate synthase from Catharanthus
roseus.

Poulsen C, Bongaerts RJ, Verpoorte R.

Center for Bio/​Pharmaceutical Sciences, University of Leiden, The Netherlands.

Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of
Catharanthus roseus by poly(ethylene glycol) precipitation/fractionation and
subsequent separation by anion exchange on Q/​Sepharose, Orange A dye
chromatography, Mono Q anion/​exchange chromatography and Superose 6 gel
filtration. By analogy to anthranilate synthases from other sources it does look
like the enzyme is a tetramer composed of two large and two small subunits, with
molecular mass 67 and 25.5 +//​ 0.5 kDa, respectively. The molecular mass
determined by gel filtration was 143 +//​ 5 kDa. The enzyme had a pI of 5.1
determined by chromatofocusing. The pH optimum was between pH 7.5 and pH 8.3,
but the type of buffer used affected the results. The enzyme could utilize NH4+
as ammonium donor instead of glutamine. The enzyme showed normal
Michaelis/​Menten kinetics with respect to the substrates L/​glutamine and
chorismate, and the cofactor Mg2+, Km values for L/​glutamine was determined to
be 0.37 +//​ 0.05 mM, for chorismate 67 +//​ 3 microM, and for MgCl2 0.26 +//​ 0.03
mM respectively. Anthranilate synthase was inhibited by L/​tryptophan, tryptamine
and D/​tryptophan (with L/​tryptophan being the best inhibitor). The enzyme was
allosterically regulated showing positive cooperatively of chorismate binding at
higher concentrations of tryptophan. For a tryptophan concentration of 20 microM
the Hill coefficient was determined to be 2. The tryptophan binding sites showed
positive cooperatively for higher concentrations of chorismate. The purified
enzyme did not contain anthranilate/​5/​phosphoribosylpyrophosphate
phosphoribosyltransferase activity and is thus not of the same type as the well
characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl
pyrophosphate transferase bifunctional type.

PMID: 8444181 [PubMed /​ indexed for MEDLINE]

286: Plant Physiol. 1993 Mar;101(3):809/​17.

Isolation and characterization of a cDNA clone for plant nuclear antigen 21D7
associated with cell division.

Smith MW, Ito M, Yamada T, Suzuki T, Komamine A.

Biological Institute, Faculty of Science, Tohoku University, Sendai, Japan.

A cDNA clone was isolated from a carrot (Daucus carota L.) cDNA expression
library using monoclonal antibody 21D7, which recognizes a nuclear antigen
associated with cell division in plant cells. To show that the isolated cDNA
encodes the 21D7 antigen, a polyclonal antiserum was raised against a
recombinant fusion protein specified by the cDNA. Both the polyclonal antiserum
and the monoclonal antibody 21D7 recognized the same plant protein on
immunoblots, in immunoprecipitation experiments, and in peptide mapping.
Analysis of the cDNA revealed that the deduced amino acid sequence has 45%
identity to the predicted sequence of the mouse transplantation antigen P91A
from mutant tumor cells that is responsible for the immune rejection of the
corresponding cell clone in a syngeneic mouse. The expression of the plant cDNA
at the mRNA level was highly correlated with cell proliferation. In suspension
cultures of Catharanthus roseus (L.) G Don. cells, the highest level of
expression was observed during the midlogarithmic phase of growth. When auxin
was added to stimulate cell division of auxin/​starved cells arrested in the G1
phase, transcription was immediately enhanced, and the level of expression
remained high throughout the G1 and S phases and dropped dramatically at the end
of DNA replication.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 8310059 [PubMed /​ indexed for MEDLINE]

287: Planta Med. 1993 Feb;59(1):46/​50.

Effects of Near/​Ultraviolet Light on Alkaloid Production in Catharanthus roseus
Plants.

Hirata K, Asada M, Yatani E, Miyamoto K, Miura Y.

Department of Biochemical Engineering, Faculty of Pharmaceutical Sciences, Osaka
University, 1/​6, Yamadaoka, Suita, 565, Japan.

Artificial near/​ultraviolet light with a peak at 370 nm and the light of natural
radiation with wavelengths between 290 and 380 nm stimulated the synthesis of
dimeric indole alkaloids in intact plants of CATHARANTHUS ROSEUS. The artificial
light also specifically stimulated an IN VITRO FMN/​mediated, non/​enzymatic
coupling of vindoline and catharanthine to synthesize an iminium intermediate,
the IN VIVO precursor of dimeric alkaloid synthesis. These results suggest that
near/​ultraviolet light is necessary for catharanthine oxidation as a trigger
reaction of dimer synthesis in the plants.

PMID: 17230336 [PubMed /​ in process]

288: Plant Physiol. 1992 Nov;100(3):1613/​1614.

Nucleotide Sequence of a cDNA Encoding 3/​Hydroxy/​3/​Methylglutaryl Coenzyme A
Reductase from Catharanthus roseus.

Maldonado/​Mendoza IE, Burnett RJ, Nessler CL.

Department of Biology, Texas A&M University, College Station, Texas 77843/​3258.

PMID: 16653173 [PubMed /​ as supplied by publisher]

289: Biotechnol Prog. 1992 Nov/​Dec;8(6):583/​6.

Isolation of vindoline from Catharanthus roseus by supercritical fluid
extraction.

Song KM, Park SW, Hong WH, Lee H, Kwak SS, Liu JR.

Department of Chemical Engineering, Korea Advanced Institute of Science and
Technology, Daejon.

Vindoline was extracted from the leaves of Catharanthus roseus over the ranges
of 35/​70 degrees C and 100/​300 bar using supercritical carbon dioxide with and
without the addition of 3 wt % ethanol as a cosolvent. The vindoline contents in
the extracts were determined by HPLC and identified by LC/MS. The remarkable
highest vindoline concentration, 58 wt %, was obtained at the lowest
temperature, 35 degrees C, and the highest pressure, 300 bar, of this study. The
use of a cosolvent only slightly improved the extraction yields or selectivities
at some experimental conditions.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 1369042 [PubMed /​ indexed for MEDLINE]

290: Plant Physiol. 1992 Oct;100(2):998/​1007.

Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P/​450
Protein from Periwinkle (Catharanthus roseus L.).

Vetter HP, Mangold U, Schroder G, Marner FJ, Werck/​Reichhart D, Schroder J.

Institut fur Biologie II, Lehrstuhl fur Biochemie der Pflanzen, Universitat
Freiburg, Schanzlestrasse 1, D/​7800 Freiburg, Federal Republic of Germany.

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures
induced for indole alkaloid synthesis and selected clones for induced cytochrome
P/​450 (P/​450) proteins by differential hybridization, size of the hybridizing
mRNA, and presence of amino acid motifs conserved in many P/​450 families. Four
cDNAs satisfying these criteria were analyzed in detail. They were grouped in
two classes (pCros1, pCros2) that represented two closely related genes of a new
P/​450 family designated CYP72. Antiserum against a cDNA fusion protein
overexpressed in Escherichia coli recognized in C. roseus a protein band of 56
kD. Quantification of western blots showed that it represented 1.5 +//​ 0.5 and 6
+//​ 1 mug/mg of protein in the membranes from noninduced and induced cells,
respectively, and analysis of the total P/​450 content suggested that the
cDNA/​encoded protein was one of the dominant P/​450 proteins. The pathway to
indole alkaloids contains two known P/​450 enzymes, geraniol/​10/​hydroxylase
(GE10H) and nerol/​10/​hydroxylase (NE10H). The induction kinetics of the cloned
P/​450 protein and of GE10H activity were similar, but those of NE10H were
different. Western blots with membranes from other plants suggested that P/​450
CYP72 is specific for C. roseus and other plants with GE10H activity. A
tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for
expression in Saccharomyces cerevisiae, and the presence of the protein was
demonstrated by western blots. Assays for GE10H failed to detect enzyme
activity, and the same negative result was obtained for NE10H and other P/​450
enzymes that are present in C. roseus.

PMID: 16653087 [PubMed /​ as supplied by publisher]

291: Plant Physiol. 1992 Oct;100(2):1029/​1032.

Phytochrome Is Involved in the Light/​Regulation of Vindoline Biosynthesis in
Catharanthus.

Aerts RJ, De Luca V.

Institut de Recherche en Biologie Vegetale, Universite de Montreal, 4101
Sherbrooke est, Montreal, Quebec, Canada, H1X 2B2.

The enzyme acetylcoenzyme A:deacetylvindoline 4/​O/​acetyl/​transferase (DAT)
catalyzes the final step in the biosynthesis of the monoterpenoid indole
alkaloid, vindoline. Previous studies have shown that the appearance of DAT
activity in etiolated seedlings of Catharanthus roseus is induced by exposure of
seedlings to light and that enzyme activity is restricted principally to the
cotyledons. Evidence is now presented that phytochrome is involved in the
light/​mediated induction of DAT activity in Catharanthus cotyledons.

PMID: 16653011 [PubMed /​ as supplied by publisher]

292: Plant Physiol. 1992 Oct;100(2):1014/​1019.

Auxins Induce Tryptophan Decarboxylase Activity in Radicles of Catharanthus
Seedlings.

Aerts RJ, Alarco AM, De Luca V.

Institut de Recherche en Biologie Vegetale, Universite de Montreal, 4101 rue
Sherbrooke est, Montreal, Quebec, H1X 2B2 Canada.

Germinating seedlings of Catharanthus roseus produce monoterpenoid indole
alkaloids as a result of a transient increase of tryptophan decarboxylase (TDC)
activity. The influence of auxins on this transient rise of TDC activity was
studied. External application of indolebutyric acid or 2,4/​dichlorophenoxyacetic
acid at a concentration of 20 to 40 mum enhanced and prolonged the rise in TDC
activity in developing seedlings. Auxin treatment also influenced the morphology
of the seedlings; it induced a shortening and thickening of the hypocotyl and
the radicle and promoted the initiation of lateral roots in the radicle. During
development, the radicles of auxin/​treated seedlings displayed a gradual
increase in TDC activity that was absent in the radicles of untreated controls.
Examination of immunoblots revealed anti/​TDC reactive proteins in extracts from
radicles of auxin/​treated seedlings, but none in extracts from radicles of
control seedlings. In contrast, TDC activity and immunoreactive protein levels
in the aerial parts of controls and auxin/​treated seedlings were comparable. Our
results indicate that externally applied auxins induce both abnormal development
and TDC activity in the radicles of Catharanthus seedlings. Although auxins
slightly delayed the light/​mediated induction of the cotyledon/​specific last
step in vindoline biosynthesis (i.e. acetylcoenzyme A:
deacetylvindolin/​O/​acetyltransferase activity), seedlings still synthesized
vindoline, one of the major alkaloid end products.

PMID: 16653009 [PubMed /​ as supplied by publisher]

293: Phytochemistry. 1992 Sep;31(9):3065/​8.

Biotransformation of tabersonine in cell suspension cultures of Catharanthus
roseus.

Furuya T, Sakamoto K, Iida K, Asada Y, Yoshikawa T, Sakai S, Aimi N.

School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.

To investigate the reactions involved in the biosynthesis of vindoline from
tabersonine, the bioconversion products formed when the latter compound was fed
to cell suspension cultures of Catharanthus roseus were isolated and
characterized. Two biotransformation products of tabersonine were isolated and
shown to be lochnericine, which is formed by epoxidation of tabersonine at
positions 14, 15, and lochnerinine, the 11/​methoxylation product of
lochnericine. The bioconversion ratio of the main biotransformation product,
lochnericine, reached a value of 80.6% within three days.

PMID: 1368411 [PubMed /​ indexed for MEDLINE]

294: Biochem Biophys Res Commun. 1992 Aug 14;186(3):1503/​9.

Sensitive detection and identification of mycoplasma/​like organisms in plants by
polymerase chain reactions.

Schaff D, Lee IM, Davis RE.

Microbiology and Plant Pathology Laboratory, Agricultural Research Service,
Beltsville, MD 20705.

DNA amplification by polymerase chain reactions (PCR) was employed to detect
host plant infection by several mycoplasmalike organisms (MLOs), including the
aster yellows (AY), dwarf aster yellows (DAY), and periwinkle little leaf (0/​1)
MLOs. For PCR, two pairs of oligonucleotide primers, designated AY18pm and
AY19pm, respectively, were synthesized on the basis of partial sequences of
cloned AY MLO DNA fragments AY18 and AY19. Reaction mixtures containing primer
pair AY18pm yielded a DNA product of 1.6Kbp, when template consisted of DNA
extracted from AY MLO/​ or DAY MLO/​infected Catharanthus roseus (periwinkle). A
DNA product of 1.0Kbp was obtained with primer pair AY19pm, when template
consisted of DNA extracted from C. roseus infected by AY MLO, DAY MLO, or
periwinkle little leaf (strain O/​1) MLO. MLO/​specific bands were observed when
reaction mixtures contained as little as 5 pg total nucleic acid from infected
plants. No PCR product was observed when reaction mixtures contained only DNA
from healthy plants or DNA from plants infected by western X MLO or by tomato
big bud MLO. The findings indicated that the PCR system is useful for sensitive
detection and differentiation of MLOs in infected hosts.

PMID: 1510676 [PubMed /​ indexed for MEDLINE]

295: J Ethnopharmacol. 1992 Aug;37(1):1/​11.

The pharmacology of extinction.

Huxtable RJ.

Department of Pharmacology, College of Medicine, University of Arizona, Tucson
85724.

It is impossible to predict what compounds of pharmacological interest may be
present in an unexamined species. The extinction of such species may result,
therefore, in the loss of therapeutically significant compounds. The fact that
science will never know what has been lost does not lessen the significance of
the loss. A number of species are discussed to exemplify the potential loss.
Ginkgo biloba is an ancient plant, apparently saved from a natural extinction by
human intervention. From this tree, the ginkgolides have been isolated. These
are potent inhibitors of platelet activating factor and hold promise in the
treatment of cerebral ischemia and brain edema. Two species, the tree Taxus
brevifolia and the leech Hirudo medicinalis, are threatened as a result of human
activity. Both have recently yielded complex compounds of therapeutic
importance. The antitumor agent, taxol, is obtained from T. brevifolia and the
thrombin inhibitor, hirudin, is found in H. medicinalis. Catharanthus roseus,
source of the anticancer agents vincristine and vinblastine, although not
threatened, derives from a largely unexamined but severely stressed ecosystem of
some 5000 plant species. In other examples, ethnobotanical knowledge of certain
plants may be lost while the species survive, as exemplified by the suppression
of the Aztec ethnobotany of Mesoamerica by the invading Spanish. Finally, the
fallacy of the 'snail darter syndrome', where species may be viewed as too
insignificant to worry about, is exposed by consideration of the pharmacological
activities of a sea hare (a shell/​less marine mollusc) and various leeches.

Publication Types:
Review

PMID: 1453701 [PubMed /​ indexed for MEDLINE]

296: J Membr Biol. 1992 Aug;129(2):137/​43.

Identification of an essential histidine residue at the active site of the
tonoplast malate carrier in Catharanthus roseus cells.

Dietz KJ, Canut H, Marigo G.

Signaux et Messages Cellulaires chez les Vegetaux, URA CNRS n.1457, Universite
Paul Sabatier, Toulouse, France.

The involvement of a histidyl residue in the binding or translocation step was
investigated in the malate carrier at the tonoplast of Catharanthus roseus
cells. The transport rate was strongly stimulated when the pH of the incubation
medium was decreased from pH 7.0 to 5.0. The histidine/​specific reagent
diethylpyrocarbonate (DEPC) efficiently inhibited the activity of the malate
carrier. Inhibition developed rapidly and was completed after 5 min at a
concentration of 2 mM DEPC. The original substrate, malate, partially protected
the carrier from inactivation by DEPC. Other organic acids (citrate, quinate)
which are known to affect the malate transport of isolated vacuoles or tonoplast
vesicles also showed protective properties. Inhibition of malate transport on
tonoplast vesicles can also be achieved by photooxidation in the presence of the
dye Rose Bengal. Malate also proved to protect against inactivation. The results
strongly support the notion that a histidyl residue(s) is involved either in the
binding or translocation of malate and that the protonation of the histidyl
residue is essential to provide a high rate of malate transport.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 1433274 [PubMed /​ indexed for MEDLINE]

297: Protein Expr Purif. 1992 Aug;3(4):295/​300.

Purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase,
from a recombinant strain of Escherichia coli.

Roessner CA, Devagupta R, Hasan M, Williams HJ, Scott AI.

Chemistry Department, Texas A&M University, College Station 77843/​3255.

The gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of
Catharanthus roseus has been cloned into an inducible Escherichia coli
expression vector using an expression cassette polymerase chain reaction
technique. Induction of the gene resulted in overexpression of the enzyme which
accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of
the insoluble protein resulted in the ability to purify up to 6 mg of active
enzyme from a single liter of cell culture. The recombinant enzyme has good
activity (approximately 30 nkat/mg).

Publication Types:
Research Support, U.S. Gov't, P.H.S.

PMID: 1330135 [PubMed /​ indexed for MEDLINE]

298: Plant Physiol. 1992 Jun;99(2):672/​680.

Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension
Cultured Catharanthus roseus Cells: Measurement with Fluorescent pH Indicator
and P/​Nuclear Magnetic Resonance.

Sakano K, Yazaki Y, Mimura T.

Department of Applied Physiology, National Institute of Agrobiological
Resources, Tsukuba, Ibaraki 305, Japan.

Cytoplasmic acidification during inorganic phosphate (Pi) absorption by
Catharanthus roseus cells were studied by means of a fluorescent pH indicator,
2',7'/​bis/​(2/​carboxyethyl)/​5 carboxyfluorescein (acetomethylester) (BCECF), and
(31)P/​nuclear magnetic resonance spectroscopy. Cytoplasmic acidification
measured by decrease in the fluorescence intensity started immediately after Pi
application. Within a minute or so, a stable state was attained and no further
acidification occurred, whereas Pi absorption was still proceeding. As soon as
Pi in the medium was exhausted, cytoplasmic pH started to recover.
Coincidentally, the medium pH started to recover toward the original acidic pH.
The Pi/​induced changes in the cytoplasmic pH were confirmed by (31)P/​nuclear
magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7
millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some
experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during
Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton
pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms
that could consume extra protons in the cytoplasm are suggested.

PMID: 16668939 [PubMed /​ as supplied by publisher]

299: FEBS Lett. 1992 Apr 13;301(1):29/​33.

A gene family homologous to the S/​phase specific gene in higher plants is
essential for cell proliferation in Saccharomyces cerevisiae.

Ito M, Yasui A, Komamine A.

Biological Institute, Faculty of Science, Tohoku University, Sendai, Japan.

Previously we reported the isolation and characterization of the gene, cyc07,
which was specifically expressed in the S phase during the cell cycle in
synchronous cell division cultures of the higher plant, Catharanthus roseus. We
found that the yeast Saccharomyces cerevisiae contains two closely related genes
which show a high degree of similarity (about 64% at the amino acid level) to
cyc07 of C. roseus. Site/​directed disruption mutations demonstrated that the two
yeast genes, homologous to cyc07, constitute an essential gene family for cell
proliferation in yeast cells. Furthermore, the rate of cell proliferation varied
with the gene copy number.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 1451783 [PubMed /​ indexed for MEDLINE]

300: Plant Mol Biol. 1992 Apr;18(6):1121/​31.

Coordinated regulation of two indole alkaloid biosynthetic genes from
Catharanthus roseus by auxin and elicitors.

Pasquali G, Goddijn OJ, de Waal A, Verpoorte R, Schilperoort RA, Hoge JH,
Memelink J.

Biotechnology Delft/​Leiden, Project Group Plant Cell Biotechnology, Leiden
University, Netherlands.

Catharanthus roseus (periwinkle) produces a wide range of terpenoid indole
alkaloids, including several pharmaceutically important compounds, from the
intermediate strictosidine. The complete mRNA sequence for the enzyme
strictosidine synthase (SSS) was determined. Comparison of the primary structure
of the encoded protein with the amino/​terminal sequence of purified SSS
indicated the presence of a signal peptide of 31 amino acids in the putative
primary translation product. SSS is encoded by a single/​copy gene indicating
that isoenzymes reported by others are formed post/​translationally from a single
precursor. The sss gene and the tryptophan decarboxylase gene (tdc), encoding
another enzyme essential for indole alkaloid biosynthesis, are coordinately
regulated. In plants steady/​state mRNA levels are highest in roots. In cell
suspension cultures the genes are rapidly down/​regulated by auxin. In contrast,
both genes are strongly induced by fungal elicitors such as Pythium
aphanidermatum culture filtrate or yeast extract. Induction is a rapid,
transcriptional event occurring independent of de novo protein synthesis. These
results show that a first important regulatory step in the complex process
leading to indole alkaloid accumulation in C. roseus suspension cells is
transcription of the biosynthetic genes.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 1600148 [PubMed /​ indexed for MEDLINE]

301: Plant Mol Biol. 1992 Apr;18(6):1113/​20.

Auxin rapidly down/​regulates transcription of the tryptophan decarboxylase gene
from Catharanthus roseus.

Goddijn OJ, de Kam RJ, Zanetti A, Schilperoort RA, Hoge JH.

Biotechnology Delft/​Leiden, Project Group Plant Cell Biotechnology, Leiden
University, Netherlands.

The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) converts tryptophan into
tryptamine, and thereby channels primary metabolites into indole alkaloid
biosynthesis. The production of these secondary metabolites in suspension cells
of Catharanthus roseus depends on medium composition. Of the possible variables,
we investigated the effect of hormones on the expression of the tdc gene in cell
cultures. Omission of NAA from the growth medium resulted in accumulation of tdc
mRNA. The addition of 1/​naphthaleneacetic acid (NAA), indoleacetic acid (IAA) or
2,4/​dichlorophenoxyacetic acid (2,4/​D) rapidly reduced the enhanced tdc
transcript level. Cytokinin was unable to suppress the enhanced transcript
level. Hairy roots transformed by Agrobacterium rhizogenes also showed a
reduction of the tdc mRNA level after NAA addition. Run/​off transcription
experiments showed that the down/​regulation takes place at the transcriptional
level within 15 minutes and independent of de novo protein synthesis. Thus one
of the mechanisms which control the activity of terpenoid indole alkaloid
biosynthesis in C. roseus cell cultures is the negative regulation by auxin of
the gene involved in the first committed step.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 1600147 [PubMed /​ indexed for MEDLINE]

302: Plant Physiol. 1992 Mar;98(3):1148/​1153.

Diacylglycerol Kinase from Suspension Cultured Plant Cells : Characterization
and Subcellular Localization.

Wissing JB, Wagner KG.

Enzymologie, Gesellschaft fur Biotechnologische Forschung, D/​3300 Braunschweig,
Federal Republic of Germany.

Diacylglycerol kinase (adenosine 5'/​triphosphate:1,2/​diacylglycerol
3/​phosphotransferase, EC 2.7.1.107), purified from suspension cultured
Catharanthus roseus cells (J Wissing, S Heim, KG Wagner [1989] Plant Physiol 90:
1546/​1551), was further characterized and its subcellular location was
investigated. The enzyme revealed a complex dependency on lipids and
surfactants; its activity was stimulated by certain phospholipids, with
phosphatidylinositol and phosphatidylglycerol as the most effective species, and
by deoxycholate. In the presence of Triton X/​100, used for its purification, a
biphasic dependency upon diacylglycerol was observed and the apparent Michaelis
constant values for diacylglycerol decreased with decreasing Triton
concentration. The enzyme accepted both adenosine 5'/​triphosphate and guanosine
5'/​triphosphate as substrate and showed rather low apparent inhibition constant
values for all nucleoside diphosphates tested. Diacylglycerol kinase is an
intrinsic membrane protein and no activity was found in the cytosol. An
investigation of different cellular membrane fractions confirmed its location in
the plasma membrane.

PMID: 16668739 [PubMed /​ as supplied by publisher]

303: Appl Environ Microbiol. 1991 Dec;57(12):3565/​3569.

Genetic Interrelatedness among Clover Proliferation Mycoplasmalike Organisms
(MLOs) and Other MLOs Investigated by Nucleic Acid Hybridization and Restriction
Fragment Length Polymorphism Analyses.

Lee IM, Davis RE, Hiruki C.

Microbiology and Plant Pathology Laboratory, Agricultural Research Service, U.S.
Department of Agriculture, Beltsville, Maryland 20705, and Department of Plant
Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5.

DNA was isolated from clover proliferation (CP) mycoplasmalike organism
(MLO)/​diseased periwinkle plants (Catharanthus roseus (L.) G. Don.) and cloned
into pSP6 plasmid vectors. CP MLO/​specific recombinant DNA clones were biotin
labeled and used as probes in dot hybridization and restriction fragment length
polymorphism analyses to study the genetic interrelatedness among CP MLO and
other MLOs, including potato witches'/​broom (PWB) MLO. Results from dot
hybridization analyses indicated that both a Maryland strain of aster yellows
and a California strain of aster yellows are distantly related to CP MLO. Elm
yellows, paulownia witches'/​broom, peanut witches'/​broom, loofah witches'/​broom,
and sweet potato witches'/​broom may be very distantly related, if at all, to CP
MLO. A new Jersey strain of aster yellows MLO, tomato big bud MLO, clover
phyllody MLO, beet leafhopper/​transmitted virescence MLO, and ash yellows MLO
are related to CP MLO, but PWB MLO is the most closely related. Similarity
coefficients derived from restriction fragment length polymorphism analyses
revealed that PWB and CP MLOs are closely related strains and thus provided
direct evidence of their relatedness in contrast to reliance solely on
biological characterization.

PMID: 16348604 [PubMed /​ as supplied by publisher]

304: Virology. 1991 Dec;185(2):896/​900.

First field isolation of wound tumor virus from a plant host: minimal sequence
divergence from the type strain isolated from an insect vector.

Hillman BI, Anzola JV, Halpern BT, Cavileer TD, Nuss DL.

Department of Plant Pathology, Rutgers University, New Brunswick, New Jersey
08903.

A new strain of wound tumor virus (WTV) has been isolated from a periwinkle
plant (Catharanthus roseus) that was among several used as bait plants in a
blueberry field. The 12 segments of double/​stranded RNA of the viral genome were
isolated directly from infected tissue and found to have mobilities through
agarose gels that were identical to those of the type strain WTV. Coupled
complementary DNA (cDNA) and polymerase chain reactions (PCR) primed with
oligonucleotides complementary to the termini of segments 4/​12 of the type
strain of WTV successfully amplified those segments. Amplification products of
the 9 segments were of the size expected for the full/​length segment, with no
shorter than full/​length products representing defective RNAs detected. PCR
products representing segments 7, 11, and 12 were cloned and sequenced in their
entirety. The sequence of each segment varied only slightly from the homologous
segment of the type strain. Variation ranged from less than 1% for segment 12 to
approximately 3% for segment 7, but even these low levels of variation were much
greater than the variation found in WTV isolates maintained in the laboratory.
Most of the variation in each of the three segments was confined to the coding
regions, and most of the differences were third position transitions. The new
WTV strain has been designated WTVNJ.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 1962460 [PubMed /​ indexed for MEDLINE]

305: Enzyme Microb Technol. 1991 Nov;13(11):882/​92.

Large/​scale (20 l) culture of surface/​immobilized Catharanthus roseus cells.

Archambault J.

Biotechnology Research Institute, National Research Council of Canada, Montreal.

Surface/​immobilized C. roseus cell cultures were grown in a 20/​l modified
airlift bioreactor operated at 0.51 vvm (kLa approximately 8 h/​1) under various
gassing regimes [air, 2% (v/v) and 5% CO2]. Extracellular ammonium, phosphate,
and nitrate ions as well as carbohydrate uptake and pH value of the medium were
monitored together with on/​line dissolved oxygen concentration, conductivity of
the medium, and carbon dioxide production rate (CPR) of the cultures. Cultures
supplemented with 2% CO2 showed higher nitrate (5.0/​7.0 mM d/​1) and carbohydrate
(3.3 g l/​1 d/​1) uptake rates and biomass production (mu approximately 0.24 d/​1,
yield approximately 0.33 g dw g CHO/​1 and 7.4 g dw L/​1) as compared to air (3.6
mM d/​1, 2.1 g l/​1 d/​1; 0.20 d/​1, 0.25 g dw g CHO/​1 and 5 g dw l/​1) and 5% CO2
(2.0/​3.6 mM d/​1, 2.0 g l/​1 d/​1; 0.11 d/​1, 0.20 g dw g CHO/​1 and 5 g dw l/​1)
cultures and as reported previously for suspension cultures. In addition, air
and 5% CO2 cultures displayed incomplete carbohydrate uptake and, more
important, phosphate and ammonium ion release into the medium at the end, which
was ascribed to loss of viability. This was not observed for 2% CO2 immobilized
bioreactor as well as shake flask control suspension cultures, which suggests
that sparged C. roseus surface/​immobilized cell cultures require 2% CO2
supplementation of the gas phase for both maximum growth and retained viability.
The maximum CPRs of all cultures were in the same range (2.1/​2.8 mM CO2 l/​1
h/​1). However, the estimated maximum specific CO2 production rates of 2% CO2 and
5% CO2 immobilized cultures (0.6 mM g dw/​1 h/​1) were lower than those found for
air/​sparged immobilized cultures (1.0/​1.3 mM g dw/​1 h/​1). These rates are
significantly higher than those reported in the literature for C. roseus cell
suspension cultures performed in bioreactors gassed with air (approximately
0.2/​0.55 mM g dw/​1 h/​1).

PMID: 1367999 [PubMed /​ indexed for MEDLINE]

306: J Biotechnol. 1991 Nov;21(1/​2):43/​62.

Stimulated indole alkaloid release from Catharanthus roseus immobilized
cultures. Initial studies.

Jardin B, Tom R, Chavarie C, Rho D, Archambault J.

Ecole Polytechnique of Montreal, Chemical Engineering Department, Montreal,
Canada.

Vacuolar sequestration of valuable secondary metabolites remains the major
limitation to the use of immobilization technology for large scale
plant/​cell/​based bioprocesses, which otherwise may be a more efficient culture
system than suspension for this biomass. In this initial study, the release of
indole alkaloids produced by immobilized Catharanthus roseus cells cultured in
Zenk's Alkaloid Production Medium was evaluated. Unstimulated alkaloid release
in immobilized cultures reached levels of 10 to 50% of total production or 3 to
100% of known alkaloid content (30 to 4700 micrograms l/​1), which was higher
than that found for suspension cultures of the cell line used (10 to 25% of
total production) without apparent cell lysis. Modifications of the medium pH
value of immobilized cultures were explored in order to improve this release.
Periodical additions of acid (HCl 0.1 N) or base (KOH 0.1 N) solutions (2% v/v)
to different cultures resulted in rapid (less than 3 h) and transient variations
in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both
cases, these variations provoked significant increase in total alkaloid (from
approximately 5/​10 mg l/​1 to 15 mg l/​1), ajmalicine (from 0 to approximately
0.29 mg l/​1) and serpentine (from 0 to approximately 0.20 mg l/​1) release,
without apparent cell lysis or decrease in the culture viability. This product
release was estimated to represent 100% of alkaloids produced.

PMID: 1367690 [PubMed /​ indexed for MEDLINE]

307: J Biotechnol. 1991 Nov;21(1/​2):21/​42.

Effect of culture process on alkaloid production by Catharanthus roseus cells.
II. Immobilized cultures.

Tom R, Jardin B, Chavarie C, Rho D, Archambault J.

Ecole Polytechnique of Montreal, Chemical Engineering Department, Montreal,
Canada.

Two processes for the production of indole alkaloids 2 l surface/​immobilized
bioreactor cultures of Catharanthus roseus cells using Zenk's Alkaloid
Production Medium (APM) were evaluated. The 1/​stage process consisted of
inoculating APM containing bioreactors and incubating for 15 d. The 2/​stage
process involved inoculating growth medium/​containing bioreactors, growing the
immobilized cultures for a certain period of time and subsequently replacing
this medium with APM. The production stage which lasted for 15 d. High
production in 2/​stage cultures required the replacement of the growth regulator
2,4/​dichlorophenoxyacetic acid by indole/​3/​acetic acid in the growth medium and
a growth stage of 6 d (late exponential phase) before production initiation.
Growth, main nutrient consumption and alkaloid production were monitored. Both
culture regimes resulted in similar biomass production, dw (10/​13 g l/​1). The
2/​stage cultures yielded biomass richer in organic nutrients (200/​300%) and with
higher respiratory activity (approximately 250%), indicated by their lower
biomass/​to/​carbohydrate yields (31% and 26%), as compared to 1/​stage cultures
(41%). Two/​stage cultures produced more known products (10 as compared to 6) at
yields (5 to 4800 micrograms g/​1) 3 to 5 times higher than 1/​stage cultures.
More alkaloids were alkaloids released in the medium of 2/​stage cultures, under
non/​lysing conditions, (20 to 4700 micrograms l/​1) than in 1/​stage cultures (20
to 460 micrograms l/​1). These results were compared to those obtained from shake
flask cultures performed at the same time, with the same C. roseus cell line and
under similar regimes and reported previously. Suspension and immobilized
cultures performed according to the 1/​stage regime showed similar total
production. However, release of known alkaloids was 2 to 3 times higher in
immobilized than in suspension cultures. Total alkaloid production of 2/​stage
suspension cultures was 3.8/​fold higher than 2/​stage immobilized cultures. Two
stage immobilized cultures released 4 more known alkaloids than the 2/​stage
suspensions. Lower oxygen availability in the 2 l immobilized cultures may
explain lower specific growth rates (0.15/​0.22 d/​1) and total alkaloid
production levels, compared to 200 ml suspension cultures (0.2/​0.4 d/​1) reported
in our previous paper.

PMID: 1367689 [PubMed /​ indexed for MEDLINE]

308: J Biotechnol. 1991 Nov;21(1/​2):1/​19.

Effect of culture process on alkaloid production by Catharanthus roseus cells.
I. Suspension cultures.

Tom R, Jardin B, Chavarie C, Archambault J.

Biotechnology Research Institute, National Research Council Canada, Montreal.

The processes for production of indole alkaloids in shake flask suspension
cultures of Catharanthus roseus cells using Zenk's alkaloid production medium
(APM) were evaluated. The 1/​stage process consisted of inoculating APM and
incubating for 15 days. The 2/​stage process involved 6 d of cultivation in
growth medium followed by 15 d of incubation in APM. Growth, main nutrient
consumption and alkaloid production were monitored. Both culture processes
produced approximately 20 g dw per 1 biomass. However, 2/​stage cultures yielded
an inorganic nutrient richer and more active plant cell biomass, richer in
inorganic nutrients, as indicated by higher (greater than 70%) nutrient
availability and consumption. Total and individual indole alkaloid production
were 10 times higher (740 mg l/​1 and 25 to 4000 micrograms per g dw,
respectively) for 2/​stage than for 1/​stage cultures. For both processes, highest
alkaloid productivity coincided with complete extracellular consumption of major
inorganic nutrients, especially nitrate, by the cells. Complete carbohydrate
consumption in 2/​stage cultures resulted in a 40% decline in production. Small
but significant (approximately 10%) product release was observed for both
culture regimes, which seemed not to be related to cell lysis.

PMID: 1367684 [PubMed /​ indexed for MEDLINE]

309: Planta Med. 1991 Oct;57(7 Suppl):S27/​35.

Strategies for the genetic manipulation of alkaloid/​producing pathways in
plants.

Robins RJ, Waltons NJ, Hamill JD, Parr AJ, Rhodes MJ.

Plant Biotechnology Group, Genetics and Microbiology Department, Institute of
Food Research (Norwich Laboratory), Norwich Science Park, Colney, Norwich NR4
7UA, U.K.

Increasingly, as a result of recent biochemical work, there exists a realistic
possibility of taking a molecular genetic approach to the manipulation of
alkaloid/​producing pathways in plant tissue cultures. In the pathways forming
indole alkaloids in CATHARANTHUS ROSEUS, tropane alkaloids in DATURA and
HYOSCYAMUS species, and nicotine in NICOTIANA species, recent studies have
identified a number of key enzymes and at least some of the factors that
regulate their levels of activity. Such knowledge contributes the basis upon
which it has become feasible to design a strategy by which the flux in these
pathways may be enhanced at the genomic level. This review presents a summary of
the state/​of/​the/​art pertaining to these pathways and discusses the strategy to
be adopted for a molecular approach to their manipulation, together with some of
the pitfalls that may arise when trying to alter their natural regulation.

PMID: 17226220 [PubMed /​ in process]

310: Planta Med. 1991 Oct;57(5):499/​500.

Effect of Near/​Ultraviolet Light on Alkaloid Production in Multiple Shoot
Cultures of Catharanthus roseus.

Hirata K, Horiuchi M, Ando T, Asada M, Miyamoto K, Miura Y.

Department of Biochemical Engineering, Faculty of Pharmaceutical Sciences, Osaka
University, 1/​6, Yamadaoka, Suita, 565, Japan.

PMID: 17226190 [PubMed /​ in process]

311: Plant J. 1991 Sep;1(2):141/​8.

Identification of a novel S/​phase/​specific gene during the cell cycle in
synchronous cultures of Catharanthus roseus cells.

Ito M, Kodama H, Komamine A.

Biological Institute, Faculty of Science, Tohoku University, Sendai, Japan.

The cell/​cycle specific cDNAs were isolated from a cDNA library prepared from
cells in the S phase in the synchronous cultures of Catharanthus roseus. One of
the isolated genes, which we refer to as cyc07, was analyzed in detail. The
full/​length cDNA of cyc07 contains an open reading frame of 735 nucleotides,
encoding a protein of 245 amino acids with a molecular weight of 28,356 Da. The
protein predicted from the nucleotide sequence is highly basic, as are mammalian
histones. cyc07 mRNA was detected specifically in cells at the S phase in
synchronous cultures. The induction and accumulation of mRNA in the S phase were
suppressed when DNA synthesis was inhibited by aphidicolin. In the intact plant,
cyc07 mRNA was found preferentially in root tips that contained meristematic
tissue. A databank search revealed that a sequence homologous to the nucleotide
sequence of cyc07 cDNA is present in the downstream region of the SIR3 gene in
the yeast genome. The amino acid sequence predicted from the corresponding
region of the yeast genome exhibited significant homology with that of cyc07
protein. These similarities between cyc07 and the corresponding region in yeast
suggest that the homologous sequence in yeast is a novel gene that is
functionally homologous to cyc07. Our results presented here suggest the
possibility that cyc07 may play a role in the proliferation of higher plant
cells, in particular in the entry into or progression of the S phase of the cell
cycle.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 1844881 [PubMed /​ indexed for MEDLINE]

312: Plant Physiol. 1991 Aug;96(4):1008/​1013.

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture.

Maki H, Ando S, Kodama H, Komamine A.

Biological Institute, Faculty of Science, Tohoku University, Sendai, 980, Japan.

Investigation was made on the effect of partial depletion of polyamines (PAs),
induced by treatment with inhibitors of the biosynthesis of PAs, on the
distribution of cells at each phase of the cell cycle in Catharanthus roseus
(L.) G. Don. cells in suspension cultures, using flow cytometry. More cells
treated with inhibitors of arginine decarboxylase (ADC) and ornithine
decarboxylase (ODC) were accumulated in the G(1) phase than those in the
control, while the treatment with an inhibitor of spermidine (SPD) synthase
showed no effect on the distribution of cells. The endogenous levels of the PAs,
putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle
in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in
particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs
increased markedly prior to synthesis of DNA in the S phase and prior to
cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle.
Activities of ADC was much higher than that of ODC throughout the cell cycle,
but both activities of ODC and ADC changed in concert with changes in levels of
PAs. Therefore, it is suggested that these enzymes may regulate PA levels during
the cell cycle. These results indicate that inhibitors of PUT biosynthesis
caused the suppression of cell proliferation by prevention of the progression of
the cell cycle, probably from the G(1) to the S phase, and PUT may play more
important roles in the progression of the cell cycle than other PAs.

PMID: 16668290 [PubMed /​ as supplied by publisher]

313: Appl Microbiol Biotechnol. 1991 Jun;35(3):382/​92.

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.)
G. Don immobilized by adhesion to glass fibres.

Facchini PJ, DiCosmo F.

Department of Botany, University of Toronto, Ontario, Canada.

Suspension/​cultured cells of Catharanthus roseus (L.) G. Don were immobilized on
glass fibre mats and cultivated in shake flasks. The highly/​aggregated
immobilized cells exhibited a slower growth rate and accumulated reduced levels
of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine,
in comparison to cells in suspension. The increased total protein synthesis in
immobilized cells suggests a diversion of the primary metabolic flux toward
protein biosynthetic pathways and away from other growth processes. In/​vitro
assays for the specific activity of tryptophan decarboxylase (TDC) and
tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine
in immobilized cells was due to reduced tryptophan biosynthesis. The specific
activity of TDC was similar in immobilized and suspension/​cultured cells.
However, the expression of TS activity in immobilized cells was reduced to less
than 25% of the maximum level in suspension/​cultured cells. The reduced
availability of a free tryptophan pool in immobilized cells is consistent with
the reduced TS activity. Reduced tryptamine accumulation, however, was not
responsible for the decreased accumulation of indole alkaloids in immobilized
cells. Indole alkaloid accumulation increased to a similar level in immobilized
and suspension/​cultured cells only after the addition of exogenous secologanin
to the culture medium. The addition of tryptophan resulted in increased
accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced
biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in
immobilized cells is suggested. Immobilization does not appear to alter the
activity of indole alkaloid biosynthetic enzymes in our system beyond, and
including, strictosidine synthase.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 1367318 [PubMed /​ indexed for MEDLINE]

314: Eur J Biochem. 1991 Apr 23;197(2):495/​503.

Molecular cloning of the gene for plant proliferating/​cell nuclear antigen and
expression of this gene during the cell cycle in synchronized cultures of
Catharanthus roseus cells.

Kodama H, Ito M, Ohnishi N, Suzuka I, Komamine A.

Biological Institute, Faculty of Science, Tohoku University, Japan.

A cDNA library was screened for plant proliferating/​cell nuclear antigen (PCNA)
from Catharanthus roseus (periwinkle). A lambda gt11 cDNA library was
constructed using poly(A)/​rich RNA isolated from the cells in the S phase. A
cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ/​1, which
contains PCNA/​related gene sequences. The cDNA contains an open reading frame of
804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of
29,765 Da. When conservative substitutions were included, a high degree of
similarity (about 85%) was observed between the predicted amino acid sequence of
periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA
was undetectable or very weak in quiescent cells, such as phosphate/​starved
cells, auxin/​starved cells and cells in the stationary phase. In the synchronous
progression of the cell cycle induced by the addition of phosphate or auxin, the
active accumulation of periwinkle PCNA mRNA was observed preferentially in the S
phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells
at the G1 phase, an increase in the level of PCNA mRNA was observed. The partial
inhibition of protein synthesis at the G1 phase by a protein inhibitor,
anisomycin, caused the arrest of cells in the G1 phase. No increase of the level
of periwinkle PCNA mRNA was observed in cells arrested at the G1 phase by the
inhibition of protein synthesis. These results indicate that the induction of
mRNA for periwinkle PCNA occurred independently of the initiation of DNA
replication, but that synthesis of certain proteins at the G1 phase was required
for the induction of periwinkle PCNA mRNA at the S phase.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 1902790 [PubMed /​ indexed for MEDLINE]

315: Plant Physiol. 1991 Feb;95(2):406/​411.

Isolation of Genes that Are Preferentially Expressed at the G(1)/S Boundary
during the Cell Cycle in Synchronized Cultures of Catharanthus roseus Cells.

Kodama H, Ito M, Hattori T, Nakamura K, Komamine A.

Biological Institute, Faculty of Science, Tohoku University, Sendai 980, Japan.

A cDNA library was screened for genes that may be involved in the progression of
the cell cycle of cells of higher plants. The Catharanthus roseus L. (G) Don.
cells were synchronized by the double phosphate starvation method, and a
lambdagt11 cDNA library was prepared using poly(A)(+) RNA from cells in the S
phase of the cell cycle. Two independent sequences, cyc02 and cyc07, were
identified by differential screening. The levels of cyc02 and cyc07 mRNAs
increased dramatically, but transiently, at the G(1)/S boundary of the cell
cycle. High levels of cyc02 mRNA, but not of cyc07 mRNA, were also present in
cells arrested at the G(1) phase by phosphate starvation. In an asynchronous
batch culture, cyc02 and cyc07 mRNAs accumulated transiently at different stages
of the growth cycle, cyc02 mRNA early in the stationary phase, and cyc07 mRNA in
the midlogarithmic phase. When the proliferation of cells was arrested by
nutrient starvation, i.e. by sucrose or nitrogen starvation, the relative
amounts of the cyc02 and cyc07 mRNAs decreased. These results indicate that
cyc02 and cyc07 contain nucleotide sequences from growth/​related genes. The
analysis of nucleotide sequence of cyc02 shows that the predicted product of
this gene is basic and is composed of 101 amino acids. No significant homology
to other known proteins was detected.

PMID: 16667998 [PubMed /​ as supplied by publisher]

316: Biochem Cell Biol. 1990 Dec;68(12):1344/​51.

The discovery of the vinca alkaloids/​/​chemotherapeutic agents against cancer.

Noble RL.

Department of Cancer Endocrinology, Cancer Control Agency of British Columbia,
Vancouver, Canada.

In folklore medicine, extracts of the leaves of the subtropical plant
Catharanthus roseus (L.) G. Don (sometimes known as Madagascar periwinkle) were
reputed to be useful in the treatment of diabetes. This review describes how
attempts to verify the antidiabetic properties of the extracts led instead to
the discovery and isolation of two complex indole alkaloids, vinblastine and
vincristine, which are used in the clinical treatment of a variety of cancers.
The two alkaloids, although structurally almost identical, nevertheless differ
markedly in the type of tumors that they affect and in their toxic properties.
These and related alkaloids have been the subject of many pharmacological and
biochemical investigations both in vivo and in vitro in the search for improved
cancer treatments. A model system used in these studies, a transplantable
lymphoma in Noble strain rats designated Nb2 node, has serendipitously led to
the development of a highly sensitive and specific bioassay for lactogenic
hormones.

Publication Types:
Biography
Historical Article

Personal Name as Subject:
Noble RL

PMID: 2085431 [PubMed /​ indexed for MEDLINE]

317: FEBS Lett. 1990 Nov 26;275(1/​2):73/​6.

A reversible carrier mediates the transport of malate at the tonoplast of
Catharanthus roseus cells.

Bouyssou H, Canut H, Marigo G.

Centre de Physiologie Vegetale, Universite Paul Sabatier, URA CNRS 241,
Toulouse, France.

The conditions of malate transport were defined in tonoplast vesicles purified
from a microsomal homogenate of Catharanthus roseus cells by preparative
free/​flow electrophoresis. Isolated vesicles exhibited malate transport when the
membranes were prepared by grinding the cells in a homogenisation medium only
buffered in the acidic pH range. By using vesicles energized artificially by an
imposed pH gradient (acid interior), it was shown that malate is actively
accumulated in response to the generation of a proton/​motive force. Several
lines of evidence (saturation kinetics, action of malate analogs and protein
modifiers) support the concept that malate transport is mediated by a protein
carrier which could be implicated in the uptake process as its protonated form.
The malate transported in the vesicles was released by lowering the external
malate concentration. The release was prevented by the anion transport inhibitor
DIDS indicating the reversibility of the carrier.

Publication Types:
In Vitro

PMID: 2262004 [PubMed /​ indexed for MEDLINE]

318: Biochim Biophys Acta. 1990 Nov 9;1036(2):138/​42.

Identification of non/​equilibrium glycolytic reactions in suspension/​cultured
plant cells.

Kubota K, Ashihara H.

Department of Biology, Faculty of Science, Ochanomizu University, Tokyo, Japan.

Levels of all enzymes and metabolites involved in glycolysis were determined in
suspension/​cultured Catharanthus roseus cells. From both the maximum catalytic
activities of the enzymes and comparisons of mass/​action ratios with the
apparent equilibrium constants for the reactions, it is concluded that the
reactions catalysed by hexokinase, fructokinase, phosphofructokinase and
pyruvate kinase are far from equilibrium, whereas the other reactions including
that catalysed by pyrophosphate/​fructose/​6/​phosphate 1/​phosphotransferase, are
close to equilibrium in vivo.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2223831 [PubMed /​ indexed for MEDLINE]

319: Plant Physiol. 1990 Nov;94(3):1410/​1413.

High Levels of Tryptamine Accumulation in Transgenic Tobacco Expressing
Tryptophan Decarboxylase.

Songstad DD, De Luca V, Brisson N, Kurz WG, Nessler CL.

Plant Biotechnology Institute, National Research Council of Canada, Saskatoon,
Saskatchewan S7N 0W9, Canada.

A full/​length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC
4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc
Natl Acad Sci USA 86: 2582/​2586) driven by the CaMV 35S promoter was introduced
into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid
tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV
35S/​TDC transformed plants had from four to 45 times greater TDC activity than
did controls. Tryptamine accumulated in transgenic plants to levels that were
directly proportional to their TDC specific activity. Despite their increased
tryptamine content, the growth and development of the CaMV 35S/​TDC plants
appeared normal with no significant differences in indole/​3/​acetic acid levels
between high tryptamine and control plants. Plants with the highest TDC activity
contained more than 1 milligram of tryptamine per gram fresh weight, a 260/​fold
increase over controls.

PMID: 16667846 [PubMed /​ as supplied by publisher]

320: Plant Physiol. 1990 Nov;94(3):1323/​1329.

Isolation and Characterization of a 2/​Oxoglutarate Dependent Dioxygenase
Involved in the Second/​to/​Last Step in Vindoline Biosynthesis.

De Carolis E, Chan F, Balsevich J, De Luca V.

Departement de Sciences Biologique, Universite de Montreal, Saskatoon,
Saskatchewan Canada S7N 0W9.

Young leaves from Catharanthus roseus plants contain the enzymes which convert
the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two
methylations, and one acetylation step to vindoline. A novel direct enzyme assay
has been developed for a hydroxylase involved in vindoline biosynthesis, which
catalyzes the C4/​hydroxylation of 2,3/​dihydro/​3/​hydroxy/​N(1)/​methyltabersonine
to the 3,4/​dihydroxy derivative. The enzyme showed an absolute requirement for
2/​oxoglutarate and enzymatic activity was enhanced by ascorbate, establishing it
as a 2/​oxoglutarate/​dependent dioxygenase (EC 1.14.11./​). The hydroxylase
exhibited specificity for position 4 of various alkaloid substrates. The enzyme
exhibited a pH optima between 7 and 8 and an apparent molecular weight of
45,000. The appearance of 4/​hydroxylase activity was developmentally regulated
and was shown to be inducible by light treatment of seedlings. Substrate
specificity studies of this enzyme for indole alkaloid substrate suggested that
hydroxylation at position 3 and N/​methylation occur prior to hydroxylation at
position 4. This is in agreement with previous studies which suggest that
C4/​hydroxylation is the second to last step in vindoline biosynthesis in
Catharanthus roseus.

PMID: 16667836 [PubMed /​ as supplied by publisher]

321: Nucleic Acids Res. 1990 Aug 25;18(16):4939.

Nucleotide sequence of a cDNA encoding the vacuolar protein strictosidine
synthase from Catharanthus roseus.

McKnight TD, Roessner CA, Devagupta R, Scott AI, Nessler CL.

Department of Biology, Texas A & M University, College Station 77843.

PMID: 2395663 [PubMed /​ indexed for MEDLINE]

322: Biomed Environ Mass Spectrom. 1990 Jul;19(7):400/​4.

Identification of indole alkaloids of Catharanthus roseus with liquid
chromatography/mass spectrometry using collision/​induced dissociation with the
thermospray ion repeller.

Auriola S, Naaranlahti T, Kostiainen R, Lapinjoki SP.

Department of Pharmaceutical Chemistry, University of Kuopio, Finland.

Fragmentation of protonated molecular ions produced from catharanthine,
tabersonine, ajmalicine and serpentine in a high/​performance liquid
chromatography (HPLC) thermospray ion source was studied. Collision/​induced
dissociation (CID) of the compounds was achieved by merely increasing the
repeller potential; i.e. no discharge current was applied and filament was not
on. Proportion of fragments to the protonated molecular ions increased with the
potential at the 180/​350 V range studied, but overall yield of detected ions
decreased at the higher voltages in all cases. Comparison of fragments produced
in the thermospray CID with those derived from the protonated molecular ions by
colliding with argon in a triple/​stage quadrupole instrument showed that the
fragmentation is very similar in both cases. An analytical application for
identifying the alkaloids from plant cell culture material by HPLC/mass
spectrometry using the thermospray CID is described.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2400851 [PubMed /​ indexed for MEDLINE]

323: Biotechnol Prog. 1990 Jul/​Aug;6(4):243/​8.

Indole alkaloid formation by Catharanthus roseus cells in a biofilm reactor.

Kargi F, Ganapathi B, Maricic K.

Department of Chemical Engineering, Washington University, St. Louis, Missouri
63130.

Catharanthus roseus cells producing indole alkaloids were grown in the form of a
biofilm. Production medium was circulated through the reactor parallel to the
upper surface of the horizontal biofilm. Sugar consumption and indole alkaloid
formation were followed to compare the performance of cultures with different
biofilm thicknesses. Dissolved oxygen concentrations gradients within the
biofilms were determined at the end of each run. RNA and protein content of the
cells in the upper and lower layers of the the biofilms were compared. Results
obtained in the biofilm experiments were compared to those obtained with
suspension cultures. At optimized biofilm thicknesses, the biofilm reactor was
more effective than suspension cultures in maximizing indole alkaloid titers.
This is thought to be due to better cell/​cell contact within the biofilm and
nutrient concentration gradients, which resulted in low growth rates.

Publication Types:
Comparative Study
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 1370003 [PubMed /​ indexed for MEDLINE]

324: Plant Physiol. 1990 Jun;93(2):479/​483.

Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured
Cells of Catharanthus roseus (L.) G. Don.

Sakano K.

Department of Applied Physiology, National Institute of Agrobiological
Resources, Tsukuba, Ibaraki 305, Japan.

Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don
caused a rapid alkalinization of the medium in which they were suspended. The
alkalinization continued until the added phosphate was completely exhausted from
the medium, at which time the pH of the medium started to drop sharply toward
the original pH value. Phosphate exposure caused the pH of the medium to
increase from pH 3.5 to values as high as 5.8, while the rate of phosphate
uptake was constant throughout (10/​17 micromoles per hour per gram fresh
weight). This indicates that no apparent pH optimum exists for the phosphate
uptake by the cultured cells. The amount of protons cotransported with phosphate
was calculated from the observed pH change up to the maximum alkalinization and
the titration curve of the cell suspension. Proton/phosphate transport
stoichiometry ranged from less than unity to 4 according to the amount of
phosphate applied. At low phosphate doses, the stoichiometries were close to 4,
while at high phosphate doses, smaller stoichiometries were observed. This
suggests that, at high phosphate doses, activation of the proton pump is induced
by the longer lasting proton influx acidifying the cytoplasm. The increased H(+)
efflux due to the proton pump could partially compensate protons taken up via
the proton/​phosphate cotransport system. Thus, the H(+)/H(2)PO(4) (/​)
stoichiometry of the cotransport is most likely to be 4.

PMID: 16667491 [PubMed /​ as supplied by publisher]

325: Bull Environ Contam Toxicol. 1990 Jun;44(6):865/​70.

Effect of thermal power plant emissions on Catharanthus roseus L.

Khan AM, Pandey V, Shukla J, Singh N, Yunus M, Singh SN, Ahmad KJ.

Environmental Botany Laboratory, National Botanical Research Institute, Lucknow,
India.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2354262 [PubMed /​ indexed for MEDLINE]

326: Arch Biochem Biophys. 1990 Jun;279(2):370/​6.

Purification and characterization of acetylcoenzyme A: deacetylvindoline
4/​O/​acetyltransferase from Catharanthus roseus.

Power R, Kurz WG, De Luca V.

Plant Biotechnology Institute, National Research Council of Canada, Saskatoon,
Saskatchewan.

The enzyme acetylcoenzyme A:deacetylvindoline 4/​O/​acetyltransferase (EC 2.3.1./​)
(DAT), which catalyzes the final step in vindoline biosynthesis in Catharanthus
roseus, was purified 3300/​fold using ammonium sulfate precipitation followed by
gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies.
Sodium dodecyl sulfate/​polyacrylamide gel electrophoresis (SDS/​PAGE) of the
purified DAT showed the presence of two major proteins having Mr values of
33,000 and 21,000, whereas native PAGE showed three protein bands, and
isoelectric focusing/​PAGE one diffuse protein band (pI = 4.7/​5.3) plus two minor
protein bands (pI = 5.7 and 6.1). Purified DAT possessed Km values of 6.5 microM
and 1.3 microM for acetylcoenzyme A and deacetylvindoline, respectively, and
Vmax values of 12.6 pkat/microgram protein (acetylcoenzyme A) and 10.1
pkat/micrograms protein (deacetylvindoline). Inhibition of DAT by tabersonine,
coenzyme A, and cations (K+, Mg2+, and Mn2+) was observed, while the pH optimum
of this enzyme was determined to be 7.5 to 9.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2350183 [PubMed /​ indexed for MEDLINE]

327: Brain Res Mol Brain Res. 1990 Jun;8(1):83/​7.

Deduced amino acid sequence of bovine aromatic L/​amino acid decarboxylase:
homology to other decarboxylases.

Kang UJ, Joh TH.

Laboratory of Molecular Neurobiology, Burke Rehabilitation Center, Cornell
University Medical College, White Plains, NY 10605.

Nucleotide sequence of cDNA for bovine aromatic L/​amino acid decarboxylase
(AADC) was analyzed. The deduced amino acid sequence of bovine AADC shows 57%
identity to drosophila AADC and 37% to plant Catharanthus roseus AADC. The
7/​amino acid sequence of the pyridoxal phosphate binding site is completely
conserved among drosophila, pig and bovine AADC. AADC primary structure also
shows high homology to that of feline glutamic acid decarboxylase.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

PMID: 2166204 [PubMed /​ indexed for MEDLINE]

328: J Chromatogr. 1990 May 4;505(2):429/​34.

Gas chromatographic/​mass spectrometric analysis of major indole alkaloids of
Catharanthus roseus.

Ylinen M, Suhonen P, Naaranlahti T, Lapinjoki SP, Huhtikangas A.

Department of Pharmaceutical Chemistry, University of Kuopio, Finland.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2355069 [PubMed /​ indexed for MEDLINE]

329: Appl Environ Microbiol. 1990 May;56(5):1471/​1475.

Nonradioactive Screening Method for Isolation of Disease/​Specific Probes To
Diagnose Plant Diseases Caused by Mycoplasmalike Organisms.

Lee IM, Davis RE, Dewitt ND.

Microbiology and Plant Pathology Laboratory, Agricultural Research Service, U.S.
Department of Agriculture, Beltsville, Maryland 20705.

DNA fragments of tomato big bud (BB) mycoplasmalike organism (MLO) in diseased
periwinkle plants (Catharanthus roseus L.) were cloned to pSP6 plasmid vectors
and amplified in Escherichia coli JM83. A nonradioactive method was developed
and used to screen for MLO/​specific recombinants. Cloned DNA probes were
prepared by nick translation of the MLO recombinant plasmids by using
biotinylated nucleotides. The probes all hybridized with nucleic acid from BB
MLO/​infected, but not healthy, plants. Results from dot hybridization analyses
indicated that several MLOs, e.g., those of Italian tomato big bud, periwinkle
little leaf, and clover phyllody, are closely related to BB MLO. The Maryland
strain of aster yellows and maize bushy stunt MLOs are also related to BB MLO.
Among the remaining MLOs used in this study, Vinca virescence and elm yellows
MLOs may be very distantly related, if at all, to BB MLO. Potato witches' broom,
clover proliferation, ash yellows, western X, and Canada X MLOs are distantly
related to BB MLO. Southern hybridization analyses revealed that BB MLO contains
extrachromosomal DNA that shares sequence homologies with extrachromosomal DNAs
from aster yellows and periwinkle little leaf MLOs.

PMID: 16348195 [PubMed /​ as supplied by publisher]

330: Photochem Photobiol. 1990 May;51(5):515/​8.

Photochemical one/​pot synthesis of vinblastine and vincristine.

Pennanen S, Huhtikangas A.

Helsinki University of Technology, Department of Chemistry, Espoo, Finland.

The naturally occurring cytostatic dimer alkaloids vinblastine 1 and vincristine
2 were photochemically synthesized in slightly acidic aqueous solution from the
monomer alkaloids catharanthine 3 and vindoline 4. Multi/​centre reactions should
obviously be involved and some of the principal photochemistry/​associated
phenomena here discussed are quite likely to be characteristic even to the
biosynthetic reactions yielding vinblastine 1 and vincristine 2 in the cells of
Catharanthus roseus.

PMID: 2367548 [PubMed /​ indexed for MEDLINE]

331: Arch Biochem Biophys. 1990 May 1;278(2):392/​7.

Characterization of tonoplast/​enriched vesicles isolated by gradient density
fractionation of suspension/​cultured cells.

Dupaix A, Guyen L, Hill M, Arrio B.

U.R.A. 1116 du C.N.R.S., Universite de Paris/​Sud, Centre d'Orsay, France.

A tonoplast/​enriched microsomal fraction was isolated from Catharanthus roseus
cells. It was characterized by structural and functional criteria. This fraction
presented a homogeneous size distribution as shown by quasi/​elastic light
scattering and a homogeneous density on self/​generated gradients of Percoll. The
mean diameter of the vesicles was estimated to be 0.30 micron and the buoyant
density around 1.04 g/ml. Sodium dodecyl sulfate/​polyacrylamide gel
electrophoresis analysis of its polypeptide pattern was in good agreement with
the one obtained for tonoplast purified from isolated vacuoles. According to
enzymatic assays and inhibition tests, this fraction possessed pyrophosphate and
ATP/​dependent proton pumps and very low contamination by submitochondrial
particles, endoplasmic reticula and Golgi membranes. In light of our previous
published results on tonoplast purified from isolated vacuoles, the very low
extent of AMP hydrolysis by the microsomal fraction is interpreted as
supplementary proof in favor of a tonoplast/​enriched fraction.

PMID: 2139317 [PubMed /​ indexed for MEDLINE]

332: J Chromatogr Sci. 1990 Apr;28(4):173/​4.

Isolation of Catharanthus alkaloids by solid/​phase extraction and
semipreparative HPLC.

Naaranlahti T, Nordstrom M, Lapinjoki SP, Huhtikangas A, Lounasmaa M.

Department of Pharmaceutical Chemistry, University of Kuopio, Finland.

A combination of moderate/​pressure chromatography on C18 sorbent and preparative
HPLC is developed for rapid isolation of alkaloids from Catharanthus roseus. The
procedure is optimized for vindoline and catharanthine with respective yields of
3 and 2 mg per 1 g of dried leaves of the plant. The methodology is also applied
for identification of the above and other alkaloids from cultured plant cells.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2079546 [PubMed /​ indexed for MEDLINE]

333: Phytochemistry. 1990;29(7):2109/​14.

Monoclonal antibodies to bis/​indole alkaloids of Catharanthus roseus and their
use in enzyme/​linked immuno/​sorbent/​assays.

Cibotti MC, Freier C, Andrieux J, Plat M, Cosson L, Bohuon C.

Laboratoire de Botanique et Phytochimie, Faculte de Pharmacie, Chatenay/​Malabry,
France.

High affinity monoclonal antibodies directed against bis/​indole alkaloids from
Catharanthus roseus were produced. Once characterized, they were used to develop
sensitive enzyme/​linked/​immuno/​sorbent/​assays, enabling us to determine minute
quantities of alkaloids related to vinblastine in plant material.

PMID: 1366691 [PubMed /​ indexed for MEDLINE]

334: Planta Med. 1989 Dec;55(6):525/​530.

Homogeneous Strictosidine Synthase Isoenzymes from Cell Suspension Cultures of
Catharanthus roseus.

Pfitzner U, Zenk MH.

Lehrstuhl fur Pharmazeutische Biologie der Universitat Munchen, Karlstrasse 29,
D/​8000 Munchen 2, Federal Republic of Germany.

Four separable isoforms of Strictosidine synthase, which catalyze condensation
of tryptamine with secologanin to form Strictosidine, were purified to
homogeneity from cultured cells of CATHARANTHUS ROSEUS and from leaves of C.
ROSEUS plants. These enzymes are distinguished by their isoelectric points as
well as by their catalytic properties. The specific activity of the main form
(isoform III) is 104 nkat/mg, K (cat) = 3.2 s (/​1). The relative molecular mass
is 31 kDa as estimated by gel filtration, and 41.5 kDa as estimated by SDS gel
electrophoresis. The pH/​optimum is observed at pH 6.7. The apparent K (M)/​value
for tryptamine is 1.9 mM (isoform III). Secologanin shows a positive
cooperativity with an n (H) of 2.2 (isoform III). Polyclonal antibodies raised
against isoform III show cross reactivity against all four isoforms.
Furthermore, these antibodies also react with Strictosidine synthases from cell
cultures of other species of the family Apocynaceae but not with those of the
family Rubiaceae.

PMID: 17262473 [PubMed /​ as supplied by publisher]

335: Plant Physiol. 1989 Dec;91(4):1317/​1322.

Carrier/​Mediated Uptake of 1/​(Malonylamino)cyclopropane/​1/​Carboxylic Acid in
Vacuoles Isolated from Catharanthus roseus Cells.

Bouzayen M, Latche A, Pech JC, Marigo G.

Ecole Nationale Superieure Agronomique, 145, Avenue de Muret, F/​31076 Toulouse
Cedex, France.

The uptake of 1/​(malonylamino)cyclopropane/​1/​carboxylic acid (MACC), the
conjugated form of the ethylene precursor, into vacuoles isolated from
Catharanthus roseus cells has been studied by silicone layer floatation
filtering. The transport across the tonoplast of MACC is stimulated fourfold by
5 millimolar MgATP, has a K(m) of about 2 millimolar, an optimum pH around 7,
and an optimum temperature at 30 degrees C. Several effectors known to inhibit
ATPase (N,N'/​dicyclohexylcarbodiimide) and to collapse the transtonoplastic H(+)
electrochemical gradient (carbonylcyanide m/​chlorophenylhydrazone, gramicidin,
and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with
SCN(/​) and valinomycin also greatly inhibited MACC transport. Our data
demonstrate that MACC accumulates in the vacuole against a concentration
gradient by means of a proton motive force generated by a tonoplastic ATPase.
The involvement of a protein carrier is suggested by the strong inhibition of
uptake by compounds known to block SH/​, OH/​, and NH(2)/​ groups. MACC uptake is
antagonized competitively by malonyl/​d/​tryptophan, indicating that the carrier
also accepts malonyl/​d/​amino acids. Neither the moities of these compounds taken
separately [1/​aminocyclopropane/​1/​carboxylic acid, malonate, d/​tryptophan or
d/​phenylalanine] nor malate act as inhibitors of MACC transport. The absence of
inhibition of malate uptake by MACC suggests that MACC and malate are taken up
by two different carriers. We propose that the carrier identified here plays an
important physiological role in withdrawing from the cytosol MACC and
malonyl/​d/​amino acids generated under stress conditions.

PMID: 16667182 [PubMed /​ as supplied by publisher]

336: Analyst. 1989 Oct;114(10):1229/​31.

Electrochemical detection of indole alkaloids of Catharanthus roseus in
high/​performance liquid chromatography.

Naaranlahti T, Ranta VP, Jarho P, Nordstrom M, Lapinjoki SP.

Hydrodynamic voltammograms for indole and five indole alkaloids with different
amino functions were obtained in order to evaluate the applicability of
high/​performance liquid chromatography (HPLC) with coulometric detection to
these compounds. With the exception of serpentine, which has a quaternary
nitrogen in its structure, all the compounds were oxidised and gave net signals
of greater than 25 nA pmol(/​1) at potentials of between +0.2 and +0.85 V versus
a solid Pd electrode in an acetate/​buffered (pH 6.5) water/​methanol/​acetonitrile
system. An HPLC assay for quantifying picomole amounts of catharanthine and
ajmalicine in Catharanthus roseus cell culture samples is described.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2619069 [PubMed /​ indexed for MEDLINE]

337: Plant Physiol. 1989 Sep;91(1):79/​84.

Immunological Detection and Quantitation of Tryptophan Decarboxylase in
Developing Catharanthus roseus Seedlings.

Fernandez JA, Owen TG, Kurz WG, De Luca V.

Helix Biotechnology Corporation, Richmond, Vancouver, British Columbia, Canada
V6X 1X5.

l/​Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in
cell suspension cultures of Catharanthus roseus after treatment with a Pythium
aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized
cells containing high levels of TDC and the protein was purified to homogeneity.
The pure protein was used to produce highly specific polyclonal antibodies, and
an enzyme/​linked immunosorbent assay (ELISA) was developed to quantitate the
level of TDC antigen during seedling development and in leaves of the mature
plant. Western immunoblotting of proteins after SDS/​PAGE with anti/​TDC
antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67
kilodaltons) which appeared at different stages during seedling development and
in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic
proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of
seedling development, respectively. The 54.8 kilodalton protein was devoid of
TDC enzyme activity, whereas the appearance of the 55 kilodalton protein
coincided with the appearance of this decarboxylase activity. The minor
immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of
seedling development and in older leaves of the mature plant, and their
relationship, if any, to TDC is presently unknown. Results suggest that the
synthesis and degradation of TDC protein is highly regulated in Catharanthus
roseus and that this regulation follows a preset developmental program.

PMID: 16667047 [PubMed /​ as supplied by publisher]

338: Plant Physiol. 1989 Aug;90(4):1546/​1551.

Diacylglycerol Kinase from Suspension Cultured Plant Cells : Purification and
Properties.

Wissing J, Heim S, Wagner KG.

Enzymologie, Gesellschaft fur Biotechnologische Forschung (GBF), D/​3300
Braunschweig, Federal Republic of Germany.

Diacylglycerol kinase (ATP:1,2/​diacylglycerol 3/​phosphotransferase, EC
2.7.1.107) from suspension/​cultured Catharanthus roseus cells was extracted from
a membrane fraction with 0.6% Triton X/​100 and 150 millimolar NaCl and was
purified about 900/​fold by DEAE/​cellulose, blue Sepharose, gel permeation, and
phenyl/​Sepharose chromatography. The enzyme is obviously membrane bound as
activity in the cytosol could not be detected. In the presence of detergents
such as Triton X/​100 (3/​[3/​cholamidopropyl]dimethylamino)/​1/​propanesulfonate
(Chaps), or deoxycholate, a molecular weight of about 250,000 was determined by
gel filtration. In glycerol density gradients, the enzyme sedimented slightly
more slowly than bovine serum albumin, indicating a molecular weight of less
than 68,000. On sodium dodecyl sulfate/​polyacrylamide gel electrophoresis enzyme
activity could be assigned to a protein of 51,000 daltons. As found previously
for bacterial and animal diacylglycerol kinases, the purified enzyme was
completely devoid of activity without the addition of phospholipids or
deoxycholate. Cardiolipin was found to be most effective, whereas higher amounts
of detergent were inhibitory. The enzyme needs divalent cations for activity,
with Mg(2+) ions being the most effective. Apparent K(m) values for ATP and
diacylglycerol were determined as 100 and 250 micromolar, respectively.

PMID: 16666963 [PubMed /​ as supplied by publisher]

339: Planta Med. 1989 Aug;55(4):364/​6.

Modification of Catharanthus roseus alkaloids: a lactone derived from
17/​deacetylvinblastine.

De Bruyn A, Verzele M, Dejonghe JP, Bhushana Rao KS, Collard MP, Trouet A,
Hannart J.

A spirolactone with anti/​tumor activity is formed during the reaction of
paraformaldehyde with 17/​deacetylvinblastine. The compound was identified by NMR
techniques. The chemotherapeutic activity was assessed.

PMID: 2813571 [PubMed /​ indexed for MEDLINE]

340: Biomaterials. 1989 Jul;10(5):318/​24.

Adhesion of suspension/​cultured Catharanthus roseus cells to surfaces: effect of
pH, ionic strength, and cation valency.

Facchini PJ, Neumann AW, DiCosmo F.

Department of Botany, University of Toronto, Ontario, Canada.

The correlation between the effects of pH, ionic strength and cation valency on
the electrophoretic mobility and the extent of adhesion of suspension/​cultured
Catharanthus roseus cells to various polymer substrates is presented. The
electrophoretic mobility of cells was unaltered in the pH range of 6/​8, but
decreased from approximately /​2.2 x 10(/​8) m V/​1 s/​1 and approached zero as the
pH of the suspending liquid was decreased from 6 to 2. Similarly, the value of
electrophoretic mobility decreased continuously as the ionic strength was
increased from 0 to 1.0 M when cells were suspended in salt solutions of sodium
chloride, calcium chloride, and aluminum chloride. However, using equimolar
concentrations, the slope of the decrease in electrophoretic mobility increased
following the sequence sodium chloride less than calcium chloride less than
aluminium chloride. The electrophoretic mobility was near zero for suspensions
containing 1.0 M calcium chloride or 0.1 M aluminium chloride. The extent of
adhesion of the cells to the polymers sulphonated polystyrene less than
polyethylene terephthalate less than polystyrene less than fluorinated
ethylene/​propylene followed this sequence. These results agree with a
thermodynamic model of plant cell adhesion that implicates the importance of
interfacial tensions in the adhesion process. However, higher levels of adhesion
were generally observed when the electrophoretic mobility for the cells in the
corresponding test liquid was at a minimum absolute value. These results can be
explained by considering the effects of the electrolytic properties of the
suspending liquid on the electrostatic repulsive interactions between the cells
and the polymer surface in terms of a double/​layer phenomenon and the DLVO
theory.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2765628 [PubMed /​ indexed for MEDLINE]

341: Planta Med. 1989 Jun;55(3):262/​4.

Quantitative Determination of Vinblastine in Tissue Cultures of Catharanthus
roseus by Radioimmunoassay.

Hirata K, Kobayashi M, Miyamoto K, Hoshi T, Okazaki M, Miura Y.

Department of Biochemical Engineering, Faculty of Pharmaceutical Sciences, Osaka
University, 1/​6, Yamadaoka, Suita, 565, Japan.

A radioimmunoassay has been developed for the quantitative determination of
small amounts of vinblastine in tissue cultures of CATHARANTHUS ROSEUS (L.) G.
Don. Antibody was obtained by the immunization of rabbits against a conjugate of
vinblastine with bovine serum albumin. The antibody had a high affinity (Ka =
1.2 x 10 (9)l/mol) and specificity for vinblastine. The usable range of standard
curve for assay was 0.5/​10 ng/ml. Crude alkaloid extracts of tissue cultures
could be assayed and many samples could be processed in one time. It was found
that the vinblastine contents of multiple shoot cultures were lower than that of
intact plants but much higher than that of callus cultures.

PMID: 17262413 [PubMed /​ in process]

342: Planta Med. 1989 Apr;55(2):155/​7.

Mass Spectral Evidence of the Occurrence of Vindoline in Heterotrophic Cultures
of Catharanthus roseus Cells.

Naaranlahti T, Lapinjoki SP, Huhtikangas A, Toivonen L, Kurten U, Kauppinen V,
Lounasmaa M.

Department of Pharmaceutical Chemistry, University of Kuopio, FOB 6, SF/​70211
Kuopio, Finland.

Vindoline concentrations in the leaves of 70 CATHARANTHUS ROSEUS of 3 cultivars
were analyzed by HPLC, and 3 plants were selected for starting callus cultures
on different media. When the initial calli were analyzed using a
vindoline/​specific RIA, the assay suggested a vindoline content of about 10
(/​5)% dry weight for one/​third of the first 60 cultures examined. Due to the
unexpectedly high incidence of vindoline/​positive calli, the screening programme
was discontinued and efforts were concentrated on verifying the existence of
this alkaloid in the cells. Suspension cultures derived from the 5 most
immunopositive calli in an alkaloid production medium were analyzed by HPLC and
GC/MS. Comparison with reference material showed that the heterotrophic
suspension cultures contained a compound identical to vindoline.

PMID: 17262331 [PubMed /​ in process]

343: Proc Natl Acad Sci U S A. 1989 Apr;86(8):2582/​6.

Molecular cloning and analysis of cDNA encoding a plant tryptophan
decarboxylase: comparison with animal dopa decarboxylases.

De Luca V, Marineau C, Brisson N.

Plant Biotechnology Institute, National Research Council of Canada, Saskatoon.

The sequence of a cDNA clone that includes the complete coding region of
tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle
(Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated
by antibody screening of a cDNA expression library produced from poly(A)+ RNA
found in developing seedlings of C. roseus. The clone hybridized to a
1.8/​kilobase mRNA from developing seedlings and from young leaves of mature
plants. The identity of the clone was confirmed when extracts of transformed
Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme
activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino
acids with a calculated molecular mass of 56,142 Da. The amino acid sequence
shows a high degree of similarity with the aromatic L/​amino acid decarboxylase
(dopa decarboxylase) and the alpha/​methyldopa/​hypersensitive protein of
Drosophila melanogaster. The tryptophan decarboxylase sequence also showed
significant similarity to feline glutamate decarboxylase and mouse ornithine
decarboxylase, suggesting a possible evolutionary link between these amino acid
decarboxylases.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 2704736 [PubMed /​ indexed for MEDLINE]

344: Plant Physiol. 1989 Mar;89(3):910/​917.

Phase/​Specific Polypeptides and Poly(A) RNAs during the Cell Cycle in
Synchronous Cultures of Catharanthus roseus Cells.

Kodama H, Kawakami N, Watanabe A, Komamine A.

Biological Institute, Faculty of Science, Tohoku University, Sendai, 980, Japan.

This study shows an overall analysis of gene expression during the cell cycle in
synchronous suspension cultures of Catharanthus roseus cells. First, the
cellular cytoplasmic proteins were fractionated by two/​dimensional gel
electrophoresis and visualized by staining with silver. Seventeen polypeptides
showed qualitative or quantitative changes during the cell cycle. Second, the
rates of synthesis of cytoplasmic proteins were also investigated by
autoradiography by labeling cells with [(35)S]methionine at each phase of the
cell cycle. The rates of synthesis of 13 polypeptides were found to vary during
the cell cycle. The silverstained electrophoretic pattern of proteins in the
G(2) phase in particular showed characteristic changes in levels of
polypeptides, while the rates of synthesis of polypeptides synthesized during
the G(2) phase did not show such phase/​specific changes. This result suggests
that posttranslational processing of polypeptides occurs during or prior to the
G(2) phase. In the G(1) and S phases and during cytokinesis, several other
polypeptides were specifically synthesized. Finally, the variation of mRNAs was
analyzed from the autoradiograms of in vitro translation products of poly(A)(+)
RNA isolated at each phase. Three poly(A)(+) RNAs increased in amount from the
G(1) to the S phase and one poly (A)(+) RNA increased preferentially from the
G(2) phase to cytokinesis.

PMID: 16666641 [PubMed /​ as supplied by publisher]

345: Diabetes Res. 1989 Feb;10(2):69/​73.

Glycaemic effects of traditional European plant treatments for diabetes. Studies
in normal and streptozotocin diabetic mice.

Swanston/​Flatt SK, Day C, Flatt PR, Gould BJ, Bailey CJ.

Department of Biochemistry, University of Surrey, Guildford, UK.

Twelve plants used for the traditional treatment of diabetes mellitus in
northern Europe were studied using normal and streptozotocin diabetic mice to
evaluate effects on glucose homeostasis. The plants were administered in the
diet (6.25% by weight) and/or as decoctions or infusions in place of drinking
water, to coincide with the traditional method of preparation. Treatment for 28
days with preparations of burdock (Arctium lappa), cashew (Anacardium
occidentale), dandelion (Taraxacum officinale), elder (Sambucus nigra),
fenugreek (Trigonella foenum/​graecum), guayusa (Ilex guayusa), hop (Humulus
lupulus), nettle (Urtica dioica), cultivated mushroom (Agaricus bisporus),
periwinkle (Catharanthus roseus), sage (Salvia officinale), and wild carrot
(Daucus carrota) did not affect the parameters of glucose homeostasis examined
in normal mice (basal plasma glucose and insulin, glucose tolerance,
insulin/​induced hypoglycaemia and glycated haemoglobin). After administration of
streptozotocin (200 mg/kg) burdock and nettle aggravated the diabetic condition,
while cashew, dandelion, elder, fenugreek, hop, periwinkle, sage and wild carrot
did not significantly affect the parameters of glucose homeostasis studied
(basal glucose and insulin, insulin/​induced hypoglycaemia, glycated haemoglobin
and pancreatic insulin concentration). Guayusa and mushroom retarded the
development of hyperglycaemia in streptozotocin diabetes and reduced the
hyperphagia, polydipsia, body weight loss, and glycated haemoglobin. Mushroom
also countered the initial reduction in plasma insulin and the reduction in
pancreatic insulin concentration, and improved the hypoglycaemic effect of
exogenous insulin. These studies suggest the presence of potentially useful
antidiabetic agents in guayusa and mushroom.

Publication Types:
Comparative Study
Research Support, Non/​U.S. Gov't

PMID: 2743711 [PubMed /​ indexed for MEDLINE]

346: Anal Biochem. 1989 Feb 1;176(2):412/​5.

Assay of strictosidine synthase from plant cell cultures by high/​performance
liquid chromatography.

Pennings EJ, van den Bosch RA, van der Heijden R, Stevens LH, Duine JA,
Verpoorte R.

Center for Bio/​Pharmaceutical Sciences, Division of Pharmacognosy, Leiden
University, The Netherlands.

An HPLC assay is described for the enzyme strictosidine synthase in which the
formation of strictosidine and the decrease of tryptamine can be followed at the
same time. In cell cultures of Catharanthus roseus significant amounts of
strictosidine glucosidase activity were detected. In crude preparations, the
strictosidine synthase reaction is therefore best measured by the
secologanin/​dependent decrease of tryptamine. In this way, the specific synthase
activity in a cell free extract was found to be 56 pkat/mg of protein. Inclusion
of 100 mM D(+)/​gluconic acid/​delta/​lactone in the incubation mixture inhibited
75% of the glucosidase activity, without inhibiting the synthase activity. The
synthase activity was readily separated from the glucosidase activity by gel
filtration on Sephadex G/​75 or Ultrogel AcA/​44. Cell cultures of Tabernaemontana
orientalis did not contain measurable amounts of strictosidine glucosidine
activity. The specific strictosidine synthase activity was 130/​200 pkat/mg of
protein during the growth of this cell culture. Strictosidine synthase is stable
at /​20 degrees C for at least 2 months.

PMID: 2742131 [PubMed /​ indexed for MEDLINE]

347: Plant Physiol. 1989 Jan;89(1):27/​36.

Regulation of Vacuolar pH of Plant Cells: II. A P NMR Study of the Modifications
of Vacuolar pH in Isolated Vacuoles Induced by Proton Pumping and Cation/H
Exchanges.

Guern J, Mathieu Y, Kurkdjian A, Manigault P, Manigault J, Gillet B, Beloeil JC,
Lallemand JY.

Laboratoire de Physiologie Cellulaire Vegetale, CNRS, 91198, Gif/​sur/​Yvette
cedex, France.

The vacuolar pH and the trans/​tonoplast DeltapH modifications induced by the
activity of the two proton pumps H(+)/​ATPase and H(+)/​PPase and by the proton
exchanges catalyzed by the Na(+)/H(+) and Ca(2+)/H(+) antiports at the tonoplast
of isolated intact vacuoles prepared from Catharanthus roseus cells enriched in
inorganic phosphate (Y Mathieu et al 1988 Plant Physiol [in press]) were
measured using the (31)P NMR technique. The H(+)/​ATPase induced an intravacuolar
acidification as large as 0.8 pH unit, building a trans/​tonoplast DeltapH up to
2.2 pH units. The hydrolysis of the phosphorylated substrate and the vacuolar
acidification were monitored simultaneously to estimate kinetically the apparent
stoichiometry between the vectorial proton pumping and the hydrolytic activity
of the H(+)/​ATPase. A ratio of H(+) translocated/ATP hydrolyzed of 1.97 +//​ 0.06
(mean +//​ standard error) was calculated. Pyrophosphate/​treated vacuoles were
also acidified to a significant extent. The H(+)/​PPase at 2 millimolar PPi
displayed hydrolytic and vectorial activities comparable to those of the
H(+)/​ATPase, building a steady state DeltapH of 2.1 pH units. Vacuoles incubated
in the presence of 10 millimolar Na(+) were alkalinized by 0.4 to 0.8 pH unit.
It has been shown by using (23)Na NMR that sodium uptake was coupled to the H(+)
efflux and occurred against rather large concentration gradients. For the first
time, the activity of the Ca(2+)/H(+) antiport has been measured on isolated
intact vacuoles. Ca(2+) uptake was strongly inhibited by NH(4)Cl or gramicidin.
Vacuoles incubated with 1 millimolar Ca(2+) were alkalinized by about 0.6 pH
unit and this H(+) efflux was associated to a Ca(2+) uptake as demonstrated by
measuring the external Ca(2+) concentration with a calcium specific electrode.
Steady state accumulation ratios of Ca(2+) as high as 100 were reached for
steady state external concentrations about 200 micromolar. The rate of Ca(2+)
uptake appeared markedly amplified in intact vacuoles when compared to tonoplast
vesicles but the antiport displayed a much lower affinity for calcium. The
different behavior of intact vacuoles compared to vesicles appears mainly to be
due to differences in the surface to volume ratio and in the rates of
dissipation of the pH gradient. Despite its low affinity, the Ca(2+)/H(+)
antiport has a high potential capacity to regulate cytoplasmic concentration of
calcium.

PMID: 16666525 [PubMed /​ as supplied by publisher]

348: Plant Physiol. 1989 Jan;89(1):19/​26.

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of
Vacuoles Suitable for P NMR Studies.

Mathieu Y, Guern J, Kurkdjian A, Manigault P, Manigault J, Zielinska T, Gillet
B, Beloeil JC, Lallemand JY.

Laboratoire de Physiologie Cellulaire Vegetale, CNRS, 91198 Gif/​sur/​Yvette
Cedex, France.

For the first time, the (31)P nuclear magnetic resonance technique has been used
to study the properties of isolated vacuoles of plant cells, namely the vacuolar
pH and the inorganic phosphate content. Catharanthus roseus cells incubated for
15 hours on a culture medium enriched with 10 millimolar inorganic phosphate
accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar
phosphate ions were largely retained in the vacuoles when protoplasts were
prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar
inorganic phosphate concentrations up to 150 millimolar were routinely obtained.
Suspensions prepared with 2 to 3 x 10(6) vacuoles per milliliter from the
enriched C. roseus cells have an internal pH value of 5.50 +//​ 0.06 and a mean
trans/​tonoplast DeltapH of 1.56 +//​ 0.07. Reliable determinations of vacuolar
and external pH could be made by using accumulation times as low as 2 minutes.
These conditions are suitable to follow the kinetics of H(+) exchanges at the
tonoplast. The (31)P nuclear magnetic resonance technique also offered the
possibility of monitoring simultaneously the stability of the trans/​tonoplast pH
and phosphate gradients. Both appeared to be reasonably stable over several
hours. The buffering capacity of the vacuolar sap around pH 5.5 has been
estimated by several procedures to be 36 +//​ 2 microequivalents per milliliter
per pH unit. The increase of the buffering capacity due to the accumulation of
phosphate in the vacuoles is, in large part, compensated by a decrease of the
intravacuolar malate content.

PMID: 16666513 [PubMed /​ as supplied by publisher]

349: Planta Med. 1988 Aug;54(4):356/​9.

Resistance to Pythium aphanidermatum in Diploids and Induced Autotetraploids of
Catharanthus roseus.

Kulkarni RN, Ravindra NS.

Central Institute of Medicinal and Aromatic Plants, Regional Centre,
Bangalore/​560037, India.

Resistance to PYTHIUM APHANIDERMATUM (which causes die/​back and collar and root
rot) in seven diploid and four induced autotetraploid lines of CATHARANTHUS
ROSEUS was studied in the field during 1983 to 1985. The tetraploid lines were,
depending on the year of evaluation, about 13 to 155, 18 to 218, and 17 to 366
times more resistant than diploid lines, when resistance was measured in terms
of plant mortality, infection rate, and area under disease curve, respectively.
On an average over three years, the tetraploid lines yielded about four and five
times more leaf and root total alkaloids, respectively, than the diploid lines.
The relative levels of resistance of diploid and tetraploid lines to PYTHIUM
APHANIDERMATUM determined in the laboratory were similar to those observed in
the field.

PMID: 17265286 [PubMed /​ in process]

350: Plant Physiol. 1988 Jun;87(2):402/​408.

Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don
Protoplasts.

McCaskill DG, Martin DL, Scott AI.

Department of Biochemistry and Biophysics, Texas A&M University, College
Station, Texas 77843.

The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don
var. Little Bright Eye was studied to determine the specificity of uptake and
the role of ion trapping in the storage of alkaloids. Accumulation of the indole
alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be
biphasic, with an initial burst of uptake followed by a slow, prolonged phase of
accumulation. The concentration and pH dependence of the initial burst of uptake
for vindoline suggested that uptake occurred by simple diffusion. Uptake of
nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio
(Ci/Ce) for nicotine at steady state and for the initial burst of uptake for
vindoline and ajmalicine suggested that accumulation was driven by the pH
gradient between the vacuole and the external assay medium. The second,
sustained phase of uptake of vindoline was sensitive to inhibition by either 20
millimolar NaN(3) or 0.5 millimolar Cu(2+). In azide/​treated protoplasts, the
uptake for vindoline conformed to the kinetics of simple diffusion, with a half
life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive
to inhibition by Cu(2+), was insensitive to inhibition by NaN(3). The biphasic
uptake of the indole alkaloids was not due to any significant metabolism. It is
concluded that accumulation and storage of the indole alkaloids is due only
partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In
the case of vindoline, there appears to be a specific energy/​requiring uptake
that is not seen with nicotine (which is not endogenous to Catharanthus).
Accumulation of ajmalicine appears to involve both ion trapping and an
azide/​insensitive component, which may be due to complexation with organic
counterions and phenolics.

PMID: 16666154 [PubMed /​ as supplied by publisher]

351: Planta Med. 1988 Apr;54(2):149/​52.

Stimulation of Indole Alkaloid Content in Vanadium/​Treated Catharanthus roseus
Suspension Cultures.

Tallevi SG, Dicosmo F.

Centre for Plant Biotechnology, Department of Botany, University of Toronto,
Ontario M5S 1A1, Canada.

Addition of vanadyl sulphate to cell suspensions of CATHARANTHUS ROSEUS was
found to increase ajmalicine, catharanthine, and tryptamine levels. Up to 500
microg/g dry weight catharanthine and 131.0 microg/g dry weight ajmalicine were
detected in vanadyl sulphate/​treated cells. This represents an approximate
increase of 50% over control levels. This stimulation was found to be dependent
upon the concentration of vanadyl sulphate administered and upon the cell age.
High tryptamine levels were not correlated with increased tryptophan
decarboxylase activity. Vanadium content of the cells was found to reach a
maximum within 1 min following vanadyl sulphate treatment as measured by
Instrumental Neutron Activation Analysis.

PMID: 17265225 [PubMed /​ in process]

352: Plant Physiol. 1988 Feb;86(2):447/​450.

Developmental Regulation of Enzymes of Indole Alkaloid Biosynthesis in
Catharanthus roseus.

De Luca V, Fernandez JA, Campbell D, Kurz WG.

Plant Biotechnology Institute, National Research Council, Saskatoon,
Saskatchewan, Canada, S7N 0W9.

Developing seedlings of Catharanthus roseus were analyzed for appearance of
tryptophan decarboxylase (TDC), strictosidine synthase (SS), N/​methyltransferase
(NMT) and O/​acetyltransferase (DAT) enzyme activities. SS enzyme activity
appeared early after germination and was present throughout most of the
developmental study. TDC activity was highly regulated and peaked over a 48 hour
period achieving a maximum by day of 5 of seedling development. Both TDC and SS
were present in all tissues of the seedling. NMT and DAT enzyme activities were
induced after TDC and SS had peaked and these activities could only be found in
hypocotyls and cotyledons. TDC, SS, and NMT did not require light for induction
whereas DAT enzyme activity was increased approximately 10/​fold after light
treatment of dark grown seedlings.

PMID: 16665928 [PubMed /​ as supplied by publisher]

353: Planta Med. 1988 Feb;54(1):18/​20.

Formation of vinblastine in multiple shoot culture of Catharanthus roseus.

Miura Y, Hirata K, Kurano N, Miyamoto K, Uchida K.

PMID: 3375331 [PubMed /​ indexed for MEDLINE]

354: Nature. 1988 Jan 14;331(6152):176/​8.

Auxin induces rapid changes in phosphatidylinositol metabolites.

Ettlinger C, Lehle L.

Fakultat fur Biologie, Universitat Regensburg, FRG.

The plant growth/​hormone auxin (indole/​3/​acetic acid, IAA) is involved in
regulating such diverse processes as cell elongation, cell division and
differentiation. The sequence of events leading to the various phenomena is
still poorly understood. Both changes in extra/​ and intracellular pH (refs 1/​4)
and selective transcription are known to be induced by auxin. Evidence for auxin
receptors at the plasmalemma membrane has been reported, but the signal
transduction pathway is not known, for this nor for other plant hormones. In
animal cells, hydrolysis of inositolphospholipids is a major mechanism for
transmembrane signalling in response to external stimuli such as hormones,
growth factors, neurotransmitters, antigens or light (reviewed in refs 8/​11).
Here we report that auxin can generate transient changes in
inositol/​1,4,5/​trisphosphate (Ins(1,4,5)P3) and inositol bisphosphate (InsP2)
within minutes in Catharanthus roseus cells arrested in G1. These changes are
accompanied by a redistribution within the polyphosphoinositide fraction. As the
physiological response to auxin addition is to relieve the arrest in G1, we
suggest that these effects are an element in the signal transduction of this
plant hormone.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 3257546 [PubMed /​ indexed for MEDLINE]

355: Plant Physiol. 1987 Dec;85(4):1099/​1102.

Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in
Catharanthus roseus.

De Luca V, Cutler AJ.

Plant Biotechnology Institute, National Research Council of Canada, Saskatoon,
Saskatchewan, Canada, S7N 0W9.

The subcellular localization of enzymes involved in indole alkaloid biosynthesis
in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase
and strictosidine synthase which together produce strictosidine, the first
indole alkaloid of this pathway, are both cytoplasmic enzymes.
S/​Adenosyl/​l/​methionine:
16/​methoxy/​2,3/​dihydro/​3/​hydroxytabersonine/​N/​methyltransferase which catalyses
the third to last step in vindoline biosynthesis could be localized in the
chloroplasts of Catharanthus leaves and is specifically associated with
thylakoids. Acetyl/​coenzyme/​A/​deacetylvindoline/​O/​acetyltransferase which
catalyses the last step in vindoline biosynthesis could also be localized in the
cytoplasm. The participation of the chloroplast in this pathway suggests that
indole alkaloid intermediates enter and exit this compartment during the
biosynthesis of vindoline.

PMID: 16665811 [PubMed /​ as supplied by publisher]

356: Planta Med. 1987 Dec;53(6):565/​7.

Immunoanalytical methods for screening vindoline from Catharanthus roseus cell
cultures.

Lapinjoki S, Verajankorva H, Heiskanen J, Niskanen M, Huhtikangas A, Lounasmaa
M.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 3444865 [PubMed /​ indexed for MEDLINE]

357: Planta Med. 1987 Oct;53(5):479/​82.

Alkaloid Production in Root and Shoot Cultures of Catharanthus roseus.

Endo T, Goodbody A, Misawa M.

Allelix Inc. 6850 Goreway Drive, Mississauga, Ontario, Canada L4V 1P1.

Root and shoot cultures were induced from seedlings of CATHARANTHUS ROSEUS. Root
cultures and cell suspension cultures derived from the root cultures showed
similar alkaloid spectra to the roots of intact plants. The alkaloid spectra of
shoot cultures and the aerial parts of intact plants were similar to each other
but different from those of root and suspension cultures. Illumination affected
the alkaloid spectra of shoot cultures. Shoot cultures produced
3',4'anhydrovinblastine, an immediate precursor of vinblastine.

PMID: 17269072 [PubMed /​ in process]

358: Planta Med. 1987 Oct;53(5):470/​4.

Stimulation of Indole Alkaloid Production in Cell Suspension Cultures of
Catharanthus roseus by Abscisic Acid.

Smith JI, Smart NJ, Kurz WG, Misawa M.

Allelix Inc., 6850 Goreway Drive, Mississauga, Ontario, L4V 1P1, Canada.

When added to suspension cultures of CATHARANTHUS ROSEUS, abscisic acid (ABA)
stimulated intracellular accumulation of the indole alkaloids catharanthine and
ajmalicine in both flask and 30 litre fermenter/​scale systems. The response
varied, and depended upon the cell line, the concentration and source of the
ABA, and the growth phase at which the cells were treated. Precise timing of ABA
addition to cells in a 301 fermenter resulted in a catharanthine yield of 85.25
mg/l after 10 days of cultivation. We propose that ABA may be useful for
increasing the yield and reducing the production time for commercially useful
secondary plant metabolites.

PMID: 17269070 [PubMed /​ in process]

359: Anal Biochem. 1987 Aug 15;165(1):133/​6.

Assay of tryptophan decarboxylase from Catharanthus roseus plant cell cultures
by high/​performance liquid chromatography.

Pennings EJ, Hegger I, van der Heijden R, Duine JA, Verpoorte R.

Center for Bio/​Pharmaceutical Sciences, State University of Leiden, The
Netherlands.

An assay is described for the enzyme tryptophan decarboxylase from plant cell
suspension cultures. It is based on the fluorometric detection of tryptamine by
HPLC on a LiChrosorb RP/​8 Select B column. Tryptophan decarboxylase from
Catharanthus roseus was induced by transferring 14/​day/​old cells into an
induction medium. Optimum activity was found 2 days after transfer, the increase
being 5/​ to 10/​fold. When kept at /​15 degrees C the crude enzyme lost half its
activity in about 7 days. The rate of the decarboxylation reaction was linear
for at least 3 h at 35 degrees C.

PMID: 3688427 [PubMed /​ indexed for MEDLINE]

360: Planta Med. 1987 Aug;53(4):373/​6.

Ajmalicine, Serpentine, and Catharanthine Accumulation in Catharanthus roseus
Bioreactor Cultures.

Drapeau D, Blanch HW, Wilke CR.

Department of Chemical Engineering, University of California, Berkeley,
California 94720, U.S.A.

A 141, stirred/​tank bioreactor was used to investigate the effect of 7% glucose
solutions on CATHARANTHUS ROSEUS suspension cultures. Measurement of oxygen
uptake rate indicated that alkaloid accumulation occurred primarily during a
20/​day transition period between growth/​oriented metabolism and
maintenance/​oriented metabolism. Exposure of the cells to light during this
period stimulated catharanthine accumulation and triggered a switch from
ajmalicine accumulation to serpentine accumulation. In addition, it suppressed
the secretion of both ajmalicine and serpentine; without light nearly 80% of the
ajmalicine and serpentine was found in the medium, whereas with light less than
20% was secreted. Alkaloid accumulation was found to be adversely affected by
increasing the volume of inoculum culture transferred to a given volume of fresh
glucose solution, apparently due to the entry of 2,4/​D with the inoculum.

PMID: 17269046 [PubMed /​ in process]

361: Planta Med. 1987 Aug;53(4):364/​7.

Time/​Course Studies in Indole Alkaloid Accumulation and Changes in Tryptophan
Decarboxylase and Strictosidine Synthase Activities: A Comparison in Three
Strains of Catharanthus roseus Cells.

Doireau P, Meriollon JM, Guillot A, Rideau M, Chenieux JC, Brillard M.

Laboratoire de Physiologie Vegetale, Faculte des Sciences, Universite de Tours,
F/​37032 Tours Cedex, France.

Three different strains of CATHARANTHUS ROSEUS cells were compared during one
subculture with regard to tryptophan, tryptamine, ajmalicine, serpentine
contents and tryptophane decarboxylase (TDC) (4) and Strictosidine synthase
activities. The strains differed greatly in their accumulation of tryptamine and
alkaloid. The TDC of all three strains showed the highest activity during the
growth phase and declined sharply at the end of this phase. On the contrary,
strictosidine synthase activity was the lowest during the growth phase and
increased distinctly at the same time when the alkaloids were accumulating. By
comparing the three strains with each other, no correlation was observed between
the values of enzymatic activities and the contents of accumulated alkaloids.

PMID: 17269044 [PubMed /​ in process]

362: Arch Biochem Biophys. 1987 May 1;254(2):491/​7.

Elicitor/​mediated induction of tryptophan decarboxylase and strictosidine
synthase activities in cell suspension cultures of Catharanthus roseus.

Eilert U, De Luca V, Constabel F, Kurz WG.

Treatment of one cell line (No. 615) of Catharanthus roseus c.v. Little Delicata
with an elicitor preparation of autoclaved and homogenized Pythium
aphanidermatum culture resulted in rapid accumulation of indole alkaloids.
Alkaloid formation was preceded by rapid transient increases in the extractable
activities of the enzymes tryptophan decarboxylase and strictosidine synthase.
The induction of these two enzyme activities occurred when cells were
transferred to alkaloid production medium or treatment with fungal elicitors.
Treatment of this cell line with translational or transcriptional inhibitors
prevented the Pythium/​induced increases of enzyme activity as well as alkaloid
accumulation. When cells were transferred to alkaloid production medium the
induction of strictosidine synthase activity preceded that of tryptophan
decarboxylase by many hours even when cells were also treated with Pythium
elicitor. Results suggested that tryptophan decarboxylase induction proceeds
only when endogenous tryptamine levels were decreased by two/​third. The internal
cellular level of tryptamine, therefore, could regulate expression of tryptophan
decarboxylase, whereas induction of strictosidine synthase or of another enzyme
in the biosynthetic pathway could control channeling of tryptamine into
alkaloids. The results demonstrate that fungal elicitors can be used to
facilitate studies of the factors which regulate expression of indole alkaloid
pathway enzymes and their ultimate pathway products.

PMID: 3579315 [PubMed /​ indexed for MEDLINE]

363: Appl Biochem Biotechnol. 1987 Mar;14(2):101/​6.

Increased synthesis of ajmalicine and catharanthine by cell suspension cultures
of Catharanthus roseus in response to fungal culture/​filtrates.

DiCosmo F, Quesnel A, Misawa M, Tallevi SG.

The ammonium sulfate/​precipitated fraction from mycelia and culture/​filtrates
and the crude, cell/​free culture filtrates from the growth medium of the fungi
Chrysosporium palmorum, Eurotium rubrum, Micromucor isabellina, and Pythium
aphanidermatum when aseptically added to cell suspensions of Cantharanthus
roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. Up
to 400 micrograms/L ajmalicine and 600 micrograms/L catharanthine were detected
in C. roseus cell suspension grown in the presence of the M. isabellina fungal
culture filtrate for 3 d. Untreated cells produced only trace levels of
ajmalicine and catharanthine per liter of cell suspension after 15 d of culture.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 3619437 [PubMed /​ indexed for MEDLINE]

364: Eksp Onkol. 1987;9(5):76/​7.

[Antitumor and toxic effects of amotin]

[Article in Russian]

Sedakova LA, Bukharova IK, Treshchalin ID, Syrkin AB.

An indole alkaloid isolated from Catharanthus roseus in the Soviet Union and
named amotin differs in terpenoid moiety structure of the indole part of the
molecule from the foreign preparation vinblastine and its Soviet analog
rosevine. In experimental study amotin has exhibited a high antileukemic
activity which was more expressed than that of vinblastine. Tolerant doses of
amotin cause moderate reversible morphological alterations in hematopoietic
organs, gastrointestinal mucosa, liver, kidney, adrenals, testes, ovaries.

Publication Types:
English Abstract

PMID: 3691399 [PubMed /​ indexed for MEDLINE]

365: Biochimie. 1986 Dec;68(12):1299/​302.

The proton/​pumps at the plasmalemma of Catharanthus roseus cells.

Belkoura M, Marigo G.

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide (FIC)
reduction which is associated with a proton translocation and a decrease in the
ATP content of the cells. The H+ efflux and the ATP consumption may be
counteracted by vanadate, a specific inhibitor of the ATPase activity, and by
Na2WO4 which prevents FIC reduction. From these data it is concluded that the
redox chain could be coupled with ATP hydrolysis for electrogenic proton
extrusion which may involve a redox control mechanism for the plasmamembrane
ATPase.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 2878688 [PubMed /​ indexed for MEDLINE]

366: Biochimie. 1986 Dec;68(12):1293/​8.

Does a proton/​pumping ATPase exist in the tonoplast?

Dupaix A, Hill M, Volfin P, Arrio B.

In order to account for the accumulation of metabolites in plant vacuoles, the
existence of a proton/​pumping ATPase has been widely suggested in the
literature. The demonstration of such a tonoplast/​bound ATPase was merely based
on the characterization of a nitrate/​sensitive microsomal fraction. In some
examples, this ATPase activity has been evidenced on vacuole preparations
obtained under conditions which were criticized by Boller. The application of
the reverse phase high/​performance liquid chromatography method (RP/​HPLC) to the
simultaneous separation of adenine nucleotides, in the presence of tonoplast
vesicles isolated from Catharanthus roseus, showed results not necessarily
correlated with the ATPase hypothesis. Moreover, in light of the H+/​quenching of
quinacrine fluorescence observed during ATP hydrolysis by vacuoles or tonoplast
vesicles, the existence of a proton/​pumping ATPase may be questioned.

PMID: 2878687 [PubMed /​ indexed for MEDLINE]

367: Plant Physiol. 1986 Nov;82(3):840/​845.

Cytoplasmic pH Regulation in Acer pseudoplatanus Cells: I. A P NMR Description
of Acid/​Load Effects.

Guern J, Mathieu Y, Pean M, Pasquier C, Beloeil JC, Lallemand JY.

Laboratoire de Physiologie Cellulaire Vegetale, Central National de la Recherche
Scientifique, 91190 Gif/​sur/​Yvette, France.

Modifications of cytoplasmic pH in Acer pseudoplatanus L. cells cultivated in
suspension have been induced by acid/​loads and studied by using (31)P nuclear
magnetic resonance spectroscopy. The initial drop of cytoplasmic pH, observed in
the first minutes of exposure to weak lipophilic acids, was followed by a slow
recovery to reach a plateau phase with a pH value lower than the initial one.
Conversely, removal of the acid led to a sharp increase of cytoplasmic pH with
in most cases an overshoot toward more alkaline values than the initial one and
a subsequent decrease to more acidic values. This shows that A. pseudoplatanus
cells powerfully regulate their cytoplasmic pH both on the acid side of their
normal pH, during the acid/​load, and on the alkaline side, after removal of
acid. Similar results were obtained with different types of acid/​loads, i.e.
treatments with propionic or benzoic acid or bubbling with CO(2)/​enriched air.
This indicates that the occurrence of pH regulation does not depend upon the
method used to acid/​load the cells. The time courses of cytoplasmic pH observed
for A. pseudoplatanus and also Catharanthus roseus cells are similar to those
recorded for animal cells but different from those described for other plant
materials for which no recovery phase was observed. This can be explained by
different balances between the initial rate of proton influx brought in by the
acids, and the capacity of proton consumption by the regulatory mechanisms. The
existence of the recovery phase offers a unique possibility to study the
regulation of the cytoplasmic pH of plant cells, as it has been done in animal
systems.

PMID: 16665119 [PubMed /​ as supplied by publisher]

368: Biochem Biophys Res Commun. 1986 Apr 29;136(2):570/​6.

Purification and partial characterization of microsomal cytochrome b555 from the
higher plant Catharanthus roseus.

Madyastha KM, Krishnamachary N.

Microsomal b/​type hemoprotein designated, cytochrome b555 of C.roseus seedlings
was solubilized using detergents and purified by a combination of ion exchange
chromatography and gel filtration to a specific content of 18.5 nmol per mg of
protein. The purified cytochrome b555 was homogeneous and estimated to have an
apparent molecular weight of 16500 on SDS/​PAGE. The absorption spectrum of the
reduced form has major peaks at 424, 525 and 555 nm. The alpha/​band of the
reduced form is asymmetric with a pronounced shoulder at 559 nm. The spectrum of
the pyridine ferrohemochrome shows absorption peaks at 557, 524 and 418 nm
indicating that the cytochrome has protoheme prosthetic group. The purified
cytochrome is autoxidizable and does not combine with carbon monoxide, azide or
cyanide. It is reducible by NADH in the presence of NADH/​cytochrome b555
reductase partially purified from C.roseus microsomes.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 3754748 [PubMed /​ indexed for MEDLINE]

369: Biochem Biophys Res Commun. 1986 Feb 13;134(3):1175/​81.

Evidence of phosphorylated phosphatidylinositols in the growth cycle of
suspension cultured plant cells.

Heim S, Wagner KG.

Suspension cultured cells of Catharanthus roseus rapidly consume the inorganic
phosphate of the medium and incorporate about 25% of it into their
phospholipids. By three different methods of analysis it was shown that from
these plant cells phosphorylated phosphatidylinositol species can be extracted;
the mono/​ and diphosphorylated species were detected in amounts of about 7 and
1%, respectively, of the phosphatidylinositol fraction. Autoradiography of
32P/​labeled phospholipids showed that especially the amount of diphosphorylated
species varies with the growth cycle of the suspension culture indicating that
also in the higher plant cell this phospholipid turnover may play a role in the
regulation of proliferation.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 3947362 [PubMed /​ indexed for MEDLINE]

370: Acta Eur Fertil. 1985 May/​Jun;16(3):203/​5.

Antifertility efficacy of Catharanthus roseus Linn: a biochemical and
histological study.

Mathur R, Chaudan S.

Oral administration of Catharanthus roseus Linn, leaf extract caused widespread
testicular necrosis, hyalinization of tubules and Sertoli cell/​only/​Syndrome.
Biochemical studies revealing notable reduction in glycogen and fructose levels
in reproductive tissues support the histological observations and confirm the
antifertility properties of C. roseus extract.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 4036518 [PubMed /​ indexed for MEDLINE]

371: J Biol Chem. 1985 Mar 25;260(6):3556/​60.

A phosphorus/​31 nuclear magnetic resonance study of phosphate uptake and storage
in cultured Catharanthus roseus and Daucus carota plant cells.

Brodelius P, Vogel HJ.

High resolution 31P NMR spectra (103.2 MHz) of oxygenated Catharanthus roseus
and Daucus carota cells grown in suspension cultures were obtained using a
solenoidal perfusion probe. The spectra showed resonances for various
phosphorylated metabolites such as ATP, ADP, NAD(P)(H), nucleoside
diphosphoglucose, and sugar phosphates. The relative levels of the
phosphorylated metabolites remained constant throughout the growth curve. No
resonances for storage compounds such as polyphosphates, pyrophosphate, or
phytates were observed. Two resolved resonances for Pi indicated an
intracellular pH of 7.3 and 5.7 (or below) for the cytoplasm and vacuoles,
respectively. The time course of Pi uptake and storage during growth in fresh
culture medium was followed by studying the level of vacuolar Pi with 31P NMR
(145.7 MHz). Simultaneously, the level of Pi in the culture medium was followed
with radioactive 32P. C. roseus quickly takes up all the Pi from the culture
medium (maximum rate 1.7 mumol min/​1 g/​1 (dry weight of cells]. The Pi is first
stored in the vacuoles; subsequently, one part of this pool is used to keep a
constant cytoplasmic Pi level while another part is apparently accumulated as an
NMR invisible Pi store, probably in another cell organelle. In contrast, D.
carota does not accumulate Pi in the vacuoles and consequently it takes up Pi
from the medium at a much slower rate (0.05 mumol min/​1 g/​1 (dry weight of
cells].

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 3972837 [PubMed /​ indexed for MEDLINE]

372: Plant Physiol. 1984 Jul;75(3):726/​731.

Cryopreservation of Alkaloid/​Producing Cell Cultures of Periwinkle (Catharanthus
roseus).

Chen TH, Kartha KK, Leung NL, Kurz WG, Chatson KB, Constabel F.

Plant Biotechnology Institute, National Research Council of Canada, Saskatoon,
Saskatchewan S7N 0W9 Canada.

A procedure for cryogenic storage of alkaloid producing cell lines of
periwinkle, Catharanthus roseus (L.) G. Don., has been developed. The procedure
differs from established cryopreservation protocols in several aspects.
Specifically, 4/​day/​old suspension subcultures of three cell lines were
precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20
hours. The cells were then incubated in nutrient media with 1 molar sorbitol
plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this
solution at a cooling rate of 0.5 degrees C per minute to /​40 degrees C prior to
immersion in liquid nitrogen (LN). After rapid thawing in a 40 degrees C water
bath, the regrowth of LN stored cells was achieved by transferring them without
washing onto filter paper discs over nutrient media solidified with agar for a
period of 4 to 5 hours. The filter paper discs with the cells were then
transferred to fresh media of the same composition for regrowth. The viability
immediately after thawing as evaluated by the 2,3,5/​triphenyl tetrazolium
chloride method was about 60% of controls. Suspension cultures established from
LN stored cells retained the capability for alkaloid synthesis and accumulation.

PMID: 16663695 [PubMed /​ as supplied by publisher]

373: Plant Physiol. 1984 Jul;75(3):720/​725.

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells
Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation.

Chen TH, Kartha KK, Constabel F, Gusta LV.

Plant Biotechnology Institute, National Research Council of Canada, Saskatoon,
Saskatchewan, S7N 0W9 Canada.

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and
periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and
in combination was examined by nuclear magnetic resonance and differential
thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth
medium had a significant effect in the temperature range for initiation to
completion of ice crystallization. Compared to the control, less water
crystallized at temperatures below /​30 degrees C in DMSO/​treated cells. Similar
results were obtained with sorbitol/​treated cells, except sorbitol had less
effect on the amount of water crystallized at temperatures below /​25 degrees C.
There was a close association between the per cent unfrozen water at /​40 degrees
C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It
appears that, in periwinkle suspension cultures, the amount of liquid water at
/​40 degrees C is critical for a successful cryopreservation. The combination of
DMSO and sorbitol was the most effective in preventing water from freezing. The
results obtained may explain the cryoprotective properties of DMSO and sorbitol
and why DMSO and sorbitol in combination are more effective as cryoprotectants
than when used alone.

PMID: 16663694 [PubMed /​ as supplied by publisher]

374: Anal Biochem. 1984 Jun;139(2):482/​6.

Quantitation of the delivery of liposome contents into plant protoplasts.

Cutler AJ, Constabel F, Kurz WG, Shargool PD.

A procedure for the quantitation of the delivery of liposome contents into
Catharanthus roseus protoplasts has been developed. The method is based on the
uptake of liposome encapsulated methylumbelliferyl beta/​D/​glucoside and its
enzymatic hydrolysis to yield fluorescent methylumbelliferone. Since the free
glucoside is not taken up by the protoplasts to a significant extent, the
delivery of material in the nanomole range can be measured with ease.

Publication Types:
Research Support, Non/​U.S. Gov't

PMID: 6476383 [PubMed /​ indexed for MEDLINE]

375: J Nat Prod. 1984 May/​Jun;47(3):554/​5.

Isolation and structural studies on the alkaloids in flowers of Catharanthus
roseus.

Atta/​ur/​Rahman, Ali I, Bashir M.

PMID: 6481367 [PubMed /​ indexed for MEDLINE]

376: J Gen Microbiol. 1984 Apr;130(4):927/​33.

Polycationic polypeptides: a possible model for the penetration/​enhancing factor
in the invasion of host cells by Toxoplasma gondii.

Werk R, Dunker R, Fischer S.

The effect of polycationic polypeptides (polylysine, polyarginine and
polyhistidine) on the invasion of mammalian cells and plant protoplasts by
Toxoplasma gondii was studied. In JM cells, a human lymphoblastoid cell line
with T cell characteristics, all polycationic polypeptides used increased the
invasion rate in a concentration/​dependent manner. This effect and the
morphological changes revealed by electron microscopy resembled the action of
the penetration/​enhancing factor previously described by E. Lycke and
co/​workers. Plant protoplasts of Catharanthus roseus, which are resistant to T.
gondii invasion, showed the same morphological changes in the presence of
polycationic polypeptides as observed for JM cells, but were not invaded.

PMID: 6736923 [PubMed /​ indexed for MEDLINE]

377: J Nat Prod. 1983 Jul/​Aug;46(4):517/​27.

Catharanthus alkaloids, XXXVIII. Confirming structural evidence and
antineoplastic activity of the bisindole alkaloids leurosine/​N'b/​oxide
(pleurosine), roseadine and vindolicine from Catharanthus roseus.

El/​Sayed A, Handy GA, Cordell GA.

Additional and confirming chemical and spectroscopic evidence for vindolicine
(4), roseadine (5), and leurosine/​N'b/​oxide (6) is presented.
Leurosine/​N'b/​oxide (6) was found to be exceptionally active in the B/​16
melanoma test system in vivo. Roseadine (5), a new isolate of Catharanthus
roseus, and 6 displayed significant activity in the P/​388 lymphocytic leukemia
test system in vivo. Preliminary spectral studies on the new alkaloid roseamine
are also described.

Publication Types:
Research Support, Non/​U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

PMID: 6631435 [PubMed /​ indexed for MEDLINE]

378: J Nat Prod. 1983 May/​Jun;46(3):409/​13.

Catharanthus alkaloids XXXVII. 16/​Epi/​Z/​isositsirikine, a monomeric indole
alkaloid with antineoplastic activity from Catharanthus roseus and Rhazya
stricta.

Mukhopadhyay S, El/​Sayed A, Handy GA, Cordell GA.

16/​Epi/​Z/​isositsirikine (1) has been isolated from the leaves of Catharanthus
roseus and Rhazya stricta and identified through a combination of spectral
interpretation and chemical correlation. The compound displayed antineoplastic
activity in the KB test system in vitro and the P/​388 test system in vivo.

Publication Types:
Research Support, U.S. Gov't, P.H.S.

PMID: 6619887 [PubMed /​ indexed for MEDLINE]

379: Plant Physiol. 1982 Oct;70(4):1156/​1161.

A P Nuclear Magnetic Resonance Study of Intracellular pH of Plant Cells
Cultivated in Liquid Medium.

Martin JB, Bligny R, Rebeille F, Douce R, Leguay JJ, Mathieu Y, Guern J.

Laboratoire de Chimie, CENG/​DRF, 38041 Grenoble, France.

(31)P nuclear magnetic resonance has been used to study the vacuolar and
cytoplasmic pH of Acer pseudoplatanus, Catharanthus roseus, and Glycine max
cells grown as cell suspensions. The adaptation of this technique to plant cells
grown in liquid medium is described with emphasis on the removal of Mn(2+) and
phosphate from the extracellular medium and on providing the O(2) supply of the
cells in the nuclear magnetic resonance tube and the various problems of
calibration. Aerobic and anaerobic cells show large differences in their
glucose/​6/​phosphate, their cytoplasmic inorganic phosphate pools, and their
cytoplasmic pH. Differences in the relative sizes of the cytoplasmic and
vacuolar inorganic phosphate pools have been observed for the three cell strains
studied.

PMID: 16662631 [PubMed /​ as supplied by publisher]

380: Eur J Biochem. 1982 Sep;127(1):67/​70.

Quinone reductases of higher plants.

Spitsberg VL, Coscia CJ.

NAD(P)H: quinone oxidoreductase (DT/​diaphorase) was detected in 100000 x g
supernatant fractions of extracts of a wide variety of higher plants. Smaller
amounts were also found in microsomes and chloroplast fractions. The enzyme was
partially purified from soluble extracts of several plants and the quinone
reductase from Catharanthus roseus was enriched 25/​fold. Plant quinone
reductases have molecular weights in the range of 38000/​53000 as determined by
gel filtration. The plant enzyme is far less sensitive to dicoumarol than its
mammalian counterpart and it is inhibited by superoxide dismutase. Quinone
reductase is capable of reducing simple p/​benzoquinone and naphthoquinone
including vitamins K3 and K1. These results indicate that, although the plant
enzyme exhibits a similar substrate specificity, it is distinguishable from
mammalian DT/​diaphorase particularly with respect to its mechanism of reduction.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 7140760 [PubMed /​ indexed for MEDLINE]

381: Res Front Fertil Regul. 1982 Jun;2(1):1/​16.

Current status of plant products reported to inhibit sperm.

Farnsworth NR, Waller DP.

PIP: This report reviews research on plant/​derived agents that prevent sperm
production if taken orally by the male or that incapacitate or kill sperm on
contact if used vaginally by the female. It would be of great value to develop
fertility inhibitors that are totally selective for reproductive systems and
enzymes, and there is a possibility that a plant/​derived drug may have this
effect. Plants that have been studied for their fertility inhibiting effects in
the male include: Aristolochia indica L. (Aristolochiaceae); Azadirachta indica
A. Juss (Meliaceae); Balanites roxburghii Planch. (Zygophyllaceae); Calotropis
procera (Ait) R.Br. (Asclepiadaceae); Carica papaya L. (Caricaceae);
Catharanthus roseus (L.) G. Don (Apocynaceae); Dieffenbachia seguine (Jacquin)
Schott. (Araceae); Ecaballium elaterium A. Richard (Cucurbitaceae); Gossypium
species (Malvaceae); Hibiscus rosa/​sinensis L. (Malvaceae); Hippophae
salicifolia D. Don (Elaeagnaceae); Leucaena glauca (L.) Benth. (Leguminosae);
Lonicera ciliosa Poir. (Caprifoliaceae); Lupinus termis Forsk. (Leguminosae);
Malvaviscus conzattii Greenm. (Malvaceae); Momordica charantia L.
(Curcurbitaceae); Ocimum sanctum L. (Labiatae); Prunus emarginata Walp.
(Rosaceae); and Withania somnifera (L.) Dunal (Solanaceae). A large number of
plants have been randomly selected and screened for spermicidal activity "in
vitro" and several seem promising. Those species found to be active and the
nature of the active principle(s), when known, are presented in a table as are
plant/​derived chemical substances of known or partially known structure reported
to be spermicidal "in vitro." Plants warrant systematic study as potential
sources of sperm/​agglutinating compounds. Of 1600 Indian plants tested, 90
showed positive semen coagulating properties. There seems to be a lack of
correlation among experimental results obtained by different groups of
investigators, between data obtained "in vitro" and "in vivo," and between
experimental results and information found in folklore. Factors complicating
the adequate assessment of plants affecting male fertility are inadequate
numbers of vehicle/​treated controls, poor experimental design, problems related
to insolubility of crude plant extracts, variation in routes of administration,
diversity in reproductive function and control among various laboratory species,
and problems in identifying plant names consistently.

PMID: 12179631 [PubMed /​ indexed for MEDLINE]

382: J Nat Prod. 1981 Sep/​Oct;44(5):611/​3.

Catharanthus alkaloids. XXXV. Isolation of leurosidine N'b/​oxide from
Catharanthus roseus.

Mukhopadhyay S, Cordell GA.

Publication Types:
In Vitro
Research Support, U.S. Gov't, P.H.S.

PMID: 7320741 [PubMed /​ indexed for MEDLINE]

383: J Nat Prod. 1981 May/​Jun;44(3):335/​9.

Catharanthus alkaloids. XXXVI. Isolation of vincaleukoblastine (VLB) and
periformyline from Catharanthus trichophyllus and pericyclivine from
Catharanthus roseus.

Mukhopadhyay S, Cordell GA.

Publication Types:
Research Support, U.S. Gov't, P.H.S.

PMID: 7264682 [PubMed /​ indexed for MEDLINE]

384: J Nat Prod. 1981 May/​Jun;44(3):289/​93.

Catharanthus alkaloids. XXXIV. Catharanthamine, a new antitumor bisindole
alkaloid from Catharanthus roseus.

El/​Sayed A, Cordell GA.

Publication Types:
Research Support, U.S. Gov't, P.H.S.

PMID: 7264679 [PubMed /​ indexed for MEDLINE]

385: J Nat Prod. 1980 Jan/​Feb;43(1):157/​61.

Catharanthus alkaloids XXXIII. 21'Oxo/​leurosine from Catharanthus roseus
(apocynaceae).

El/​Sayed A, Handy GA, Cordell GA.

Publication Types:
In Vitro
Research Support, U.S. Gov't, P.H.S.

PMID: 7400819 [PubMed /​ indexed for MEDLINE]

386: Eur J Biochem. 1979 Nov 1;101(1):225/​33.

Purification and properties of strictosidine synthase, the key enzyme in indole
alkaloid formation.

Treimer JF, Zenk MH.

A new enzyme, strictosidine synthase, which catalyzes the synthesis of
3/​alpha(S)/​strictosidine from tryptamine and secologanin was isolated from the
soluble protein extract of Catharanthus roseus cell suspension cultures and was
purified approximately 50/​fold by ammonium sulfate fractionation, column
chromatography on DEAE/​cellulose. Ultrogel AcA34 and isoelectric focusing. The
apparent molecular weight of the enzyme was 34000. The pH optimum was 6.8,
apparent Km values for tryptamine and secologanin were 2.3 mM and 3.4 mM
respectively for the enzyme to synthesize strictosidine. Strictosidine synthase
shows high substrate specificity. No apparent cofactor requirement could be
demonstrated. Of several enzyme inhibitors tested, only p/​chloromercuribenzoate
inhibited the enzyme. The enzyme was relatively stable and could be stored at
/​20 degrees C for periods of up to 1 year without appreciable loss of catalytic
activity. The enzyme was demonstrated to occur in suspension cultures of 15
different species belonging to 9 different genera of the
indole/​alkaloid/​producing subfamily Plumerioideae of the Apocynaceae family.
This enzyme is responsible for the synthesis of strictosidine the key
intermediate in the formation of the majority of monoterpenoid indole alkaloids
occurring in the plant kingdom.

Publication Types:
Comparative Study

PMID: 510306 [PubMed /​ indexed for MEDLINE]

387: Rev Cubana Med Trop. 1979 Sep/​Dec;31(3):199/​204.

[Evaluation of antimicrobial activity of indol alkaloids]

[Article in Spanish]

Rojas Hernandez NM.

In pursuing the study of the antimicrobial properties of alkaloids prepared from
Cuban plants the activity of 10 indol alkaloids and 4 semisynthetic variables
obtained from three plants/​/​Catharanthus roseus G. Don., Vallesia antillana Wood
and Ervatamia coronaria Staph, of the family Apocynaceae/​/​growing in Cuba was
assessed in vitro. The alkaloids and the variables used were catharantine,
vindoline, vindolinine, perivine, reserpine, tabernaemontanine,
tetrahydroalstonine, aparicine, vindolinic acid, reserpic acid and vindolininol.
These were faced to 40 bacterial strains from the genera Salmonella, Shigella,
Proteus, Escherichia, Pseudomonas, Staphylococcus and Corynebacterium as well as
to fungi and yeasts from the genera Aspergillus, kCunnighamella, kCandida and
Saccharomyces. The method involving cylindric sections in a double agar layer
was applied and lectures were obtained at 24/​48 hours of incubation at 25
degrees C for fungi and yeasts and 37 degrees C for bacteria. Inhibition zones
are reported in millimeters.

Publication Types:
Comparative Study
English Abstract

PMID: 399043 [PubMed /​ indexed for MEDLINE]

388: Biochemistry. 1979 Aug 21;18(17):3760/​3.

Purification and properties of strictosidine synthetase (an enzyme condensing
tryptamine and secologanin) from Catharanthus roseus cultured cells.

Mizukami H, Nordlov H, Lee SL, Scott AI.

Strictosidine synthetase, which catalyzes the condensation of tryptamine with
secologanin to form strictosidine (isovincoside), was purified 740/​fold to
homogeneity from cultured cells of Catharanthus roseus in 10% yield. The
specific activity is 5.85 nkat/mg. The molecular weight as estimated by gel
filtration is 38,000. The isoelectric point is 4.6. Apparent Km values for
tryptamine and secologanin are 0.83 and 0.46 mM, respectively. The enzyme shows
a broad pH optimum between 5.0 and 7.5. The product of the enzymic reaction is
exclusively strictosidine, while no trace of its epimer vincoside can be
detected. Sulfhydryl inhibitors have no effect on the enzyme. End products in
the biosynthetic pathway of indole alkaloids such as ajmalicine, vindoline, and
catharanthine do not inhibit the activity of strictosidine synthetase.

Publication Types:
Research Support, U.S. Gov't, P.H.S.

PMID: 476085 [PubMed /​ indexed for MEDLINE]

389: Z Naturforsch [C]. 1979 Aug;34(7/​8):541/​5.

5/​Methyltryptophan resistant cells of Catharanthus roseus.

Schallenberg J, Berlin J.

Several cell lines resistant to 5/​methyltryptophan were selected from wild type
cells of different Catharanthus roseus suspension cultures. The resistant cells
had up to 30 times the normal levels of free tryptophan. Despite the increased
pool size of tryptophan anthranilate synthetase activity of resistant cells was
as sensitive to inhibition by L/​tryptophan as wild type cells. The
overproduction of tryptophan did not lead to intensified accumulation of
tryptamine nor of indole alkaloids. This was supported by a low conversion of
tryptophan to tryptamine in vivo and in vitro. The overproduction of one of the
primary precursors was evidently not sufficient to stimulate the rate of indole
alkaloid synthesis in Catharanthus cells.

PMID: 158896 [PubMed /​ indexed for MEDLINE]

390: Planta Med. 1979 May;36(1):87/​90.

A bioassay of antimitotic alkaloids of Catharanthus roseus.

El/​Merzabani MM, El/​Aaser AA, El/​Duweini AK, El/​Masry AM.

PMID: 461560 [PubMed /​ indexed for MEDLINE]

391: J Biol Chem. 1979 Apr 10;254(7):2419/​27.

Detergent/​solubilized NADPH/​cytochrome c(P/​450) reductase from the higher plant,
Catharanthus roseus. Purification and characterization.

Madyastha KM, Coscia CJ.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 107158 [PubMed /​ indexed for MEDLINE]

392: Ann Pharm Fr. 1979;37(5/​6):217/​23.

[Ajmalicine assay in Catharanthus roseus G. Don. A comparison between
densitometric, spectrometric and hight performance liquid chromatographic
methods (author's transl)]

[Article in French]

Gleye J, Lavergne de Cerval E, Stanislas E, Leverd E, Beziat D, Hatinguais P.

Publication Types:
Comparative Study
English Abstract

PMID: 543596 [PubMed /​ indexed for MEDLINE]

393: Pharmazie. 1978 Apr;33(4):233/​4.

Distribution and accumulation of alkaloids in Catharanthus roseus G. Don during
development.

Reda F.

Pattern of the total alkaloids distribution and accumulation in Catharanthus
roseus G. Don was investigated during six different stages of flowering and
fruiting. The highest concentration of alkaloids (mg perivine/g dry weight) was
found in the roots at the start of flowering and the minimum in the stems during
full fruiting stage; the rate of alkaloidal accumulation in the leaves, stems
and roots tended to decrease during fruit maturation. The most active stage of
alkaloidal biosynthesis was at the start of flowering in all vegetative organs.
The maximum dry weights of leaves, stems and roots were obtained during the
start of fruiting. The weights of flowers and fruits were relatively very small
during different developmental stages. Harvesting Catharanthus roseus should
best be carried out at the full/​flowering stage to obtain the optimum yield of
total alkaloidal content of leaves, stems and roots (as mg perivine/total dry
weight of each organ per plant).

PMID: 674322 [PubMed /​ indexed for MEDLINE]

394: Rev Cubana Med Trop. 1977 Sep/​Dec;29(3):147/​52.

[Fungal activity of various alkaloids isolated from Catharanthus roseus G. Don]

[Article in Spanish]

Rojas Hernandez NM, Diaz Perez C.

Evaluation is made of the fungal activity of ajmalicine, aparacine, catarantine,
reserpine, tetrahydroalstonine, vincubine, vindoline and vindolinina/​/​alkaloids
isolated from C. roseus growing in Cuba/​/​on new fungi strains and yeasts which
include some of human clinical interest. The method employed was the diffusion
in an agar mean with sections or cylinders containing solutions of the alkaloids
at 2% and 3% concentrations. The best results are obtained with an aparicine
base, while tetrahydroalstonine, vincubine and vindolinine solutions did not
inhibit the growth in any of the germs tested.

Publication Types:
Comparative Study
English Abstract

PMID: 98170 [PubMed /​ indexed for MEDLINE]

395: Lloydia. 1977 Jul/​Aug;40(4):326/​36.

Catharanthus roseus tissue culture: the effects of medium modifications on
growth and alkaloid production.

Carew DP, Krueger RJ.

A number of nutritional factors as well as the growth factors 2,4/​D and IAA were
studied to determine their influence on growth and alkaloid formation in
Catharanthus roseus suspension cultures. The optimal 2,4/​D concentration for
growth and alkaloid production was 1 mg/liter. With IAA, both 0.5 and 2.0
mg/liter in media produced tissue growth comparable to tissue receiving 1
mg/liter 2,4/​D; however, qualitative and quantitative differences in alkaloid
production were observed. Media formulations containing 2,4 and 6% sucrose
showed proportionate increases in cell yield with increased sucrose, but
concomitant decreases in alkaloid production. Suspension cultures in media
containing 2, 5, and 10 times the normal level of phosphate exhibited little
change in growth or alkaloid production. When thiamin HCl, yridoxine nicotinic
acid and inositol were deleted from the medium, the tissue continued to grow
well through the 24 month duration of the experiment; however, alkaloid
production was altered quantitatively and qualitatively.

PMID: 895391 [PubMed /​ indexed for MEDLINE]

396: Biochem Biophys Res Commun. 1977 Apr 25;75(4):1004/​9.

Indole aldaloid biosynthesis: partial purification of "ajmalicine synthetase"
from Catharanthus roseus.

Scott AI, Lee SL, Wan W.

Publication Types:
Research Support, U.S. Gov't, P.H.S.

PMID: 861021 [PubMed /​ indexed for MEDLINE]

397: J Med Chem. 1977 Mar;20(3):409/​13.

Alkaloids of Vinca rosea L. (Catharanthus roseus G. Don). 38. 4'/​Dehydrated
derivatives.

Miller JC, Gutowski GE, Poore GA, Boder GB.

A series of 4'/​dehydrated derivatives of various dimeric Vinca alkaloids has
been synthesized to further define the structure/​activity relationships of Vinca
alkaloids with onolytic potency. The concentrated sulfuric acid dehydration in
most cases gave mixtures of the 3',4'/​and two isomeric 4',20'/​alkenes, which
were isolated and characterized primarily by proton and 13C NMR. Compound tested
for antitumor activity include the three dehydro isomers of
4'/​deacetylvinblastine, 4/​deacetylvincristine, and
4/​deacetylvinblastine/​23/​amide and some4'/​dehydrated derivatives epimeric at
C/​18'. Generally, the decrease in toxicity imparted by the new double bond was
accompained by a decrease in potency. An exception was
3',4'/​dehydro/​4/​deacetylvincristine, which showed a decrease in toxicity and
increase in potency against at least one tumor in which vincristine itself has
little effect.

Publication Types:
Comparative Study

PMID: 576619 [PubMed /​ indexed for MEDLINE]

398: J Cell Biol. 1977 Feb;72(2):302/​13.

Subcellular localization of a cytochrome P/​450/​dependent monogenase in vesicles
of the higher plant Catharanthus roseus.

Madyastha KM, Ridgway JE, Dwyer JG, Coscia CJ.

The intracellular location of a cytochrome P/​450/​dependent monoterpene
hydroxylase from the higher plant, Catharanthus roseus, has been investigated.
By differential and sucrose density gradient centrifugation, utilizing marker
enzymes and electron microscopy, the monooxygenase was demonstrated to be
associated with vesicles having a membrane thickness of 40/​60 nm. The vesicles
could be distinguished from endoplasmic reticulum, Golgi apparatus,
mitochondria, and plasma membrane and were found in light membrane fractions
containing provacuoles. Most definitive results were obtained when seedlings
were ground in the presence of sand and in a medium containing sorbitol. Upon
subjection of the 20,000/​g pellet preparation to linear sucrose density gradient
centrifugation, a threefold enrichment in hydroxylase activity was afforded in a
yellow band having vesicles varying in size from 0.1 to 0.8 mum in diam and
having a density of 1.09 to 1.10 g/cm3. Since the monooxygenase has been
implicated in indole alkaloid biosynthesis in this plant, the data suggest the
compartmentalization of at least a part of this pathway.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.
Research Support, U.S. Gov't, P.H.S.

PMID: 833200 [PubMed /​ indexed for MEDLINE]

399: FEBS Lett. 1976 Nov;70(1):267/​70.

Synthesis of ajmalicine and related indole alkaloids by cell free extracts of
Catharanthus roseus cell suspension cultures.

Stockigt J, Treimer J, Zenk MH.

PMID: 11128 [PubMed /​ indexed for MEDLINE]

400: Planta Med. 1976 Aug;30(1):14/​20.

[Production of indole alkaloids in tissue cultures of Catharanthus roseus
(author's transl)]

[Article in German]

Doller G, Alfermann AW, Reinhard E.

Publication Types:
English Abstract

PMID: 959385 [PubMed /​ indexed for MEDLINE]

401: J Org Chem. 1976 Mar 19;41(6):1001/​5.

Alkaloids of Vinca rosea L. (Catharanthus roseus G. Don). XXXVII. Structure of
vincathicine.

Tafur SS, Occolowitz JL, Elzey TK, Paschal JW, Dorman DE.

PMID: 3633 [PubMed /​ indexed for MEDLINE]

402: Lloydia. 1976 Mar/​Jun;39(2/​3):147/​9.

Biotransformations with plant tissue cultures.

Carew DP, Bainbridge T.

Suspension cultures of Catharanthus roseus, Apocynum cannabinum and Conium
maculatum were examined for their capacity to transform aniline, anisole,
acetanilide, benzoic acid and coumarin. None of the cultures transformed
acetanilide but each produced acetanilide when fed aniline. All three cultures
converted benzoic acid to its para/​hydroxy derivative. Coumarin was selectively
hydroxylated at the 7/​position by Catharanthus and Conium and anisole was
O/​demethylated only by older Catharanthus tissue.

Publication Types:
Research Support, U.S. Gov't, Non/​P.H.S.

PMID: 1084950 [PubMed /​ indexed for MEDLINE]

403: Rev Cubana Med Trop. 1976 Jan/​Apr;28(1):45/​51.

[Microbiological analysis of vindolinina (an alkaloid isolated from Catharanthus
roseus) and some of its structural changes]

[Article in Spanish]

Rojas Hernandez NM, Cuellar Cuellar A.

Bacteriostatic properties of vindolinina (an alkaloid isolated from Catharanthus
roseus that has an ester group within its molecule) as well as of its
alkaline/​hydrolysis product (vindolininic acid) crystallized as chlorhydrate,
and of the product of its reduction with lithium and aluminum hydrides
(vindolininol) are compared. Several strains of bacteria pathogenic to man
(Proteus, Escherichia, Shigella, Staphylococcus, and Pseudomonas) cultured in
nutritive/​agar dishes containing disks and incubated at 37 degrees C were used
and results were assessed 24 hours later. Data obtained show that bacterial
growth inhibition is closely related to the structure of the compound as well as
to its type of grouping.

Publication Types:
English Abstract

PMID: 802793 [PubMed /​ indexed for MEDLINE]

404: J Pharm Sci. 1975 Dec;64(12):1953/​7.

Alkaloids of Vinca rosea L. (Catharanthus roseus G. Don) XXXVI: Isolation and
characterization of new dimeric alkaloids.

Tafur S, Jones WE, Dorman DE, Logsdon EE, Svoboda GH.

In a continuing effort to study thoroughly the alkaloids of Catharanthus roseus,
new dimeric alkaloids were isolated and characterized. Structures are proposed
for leurocolombine and vinamidine based on UV, IR, PMR, high/​resolution mass
spectrometry, and CMR. Pseudovincaleukoblastine diol was identified by PMR and
mass spectrometry. Leurocolombine exhibited antimitotic activity and marginal
antitumor activity against the Ridgeway osteogenic sarcoma.

PMID: 1206487 [PubMed /​ indexed for MEDLINE]

405: J Am Chem Soc. 1975 Nov 12;97(23):6906/​8.

Letter: Biosynthesis of the indole alkaloids. A cell/​free system from
Catharanthus roseus.

Scott AI, Lee SL.

Publication Types:
Research Support, U.S. Gov't, P.H.S.

PMID: 1184890 [PubMed /​ indexed for MEDLINE]

406: Experientia. 1975 Jan 15;31(1):18/​20.

Vinca alkaloids XXXV.1 Desacetoxyvinblastine a new minor alkaloid from Vinca
rosea L. (Catharanthus roseus G. Don).

Neuss N, Barnes AJ, Huckstep LL.

PMID: 1116520 [PubMed /​ indexed for MEDLINE]

407: Haematologica. 1973;53(1):51/​64.

[Place of Vinca rosea alkaloids (Catharanthus roseus) in the treatment of
Hodgkin's disease]

[Article in Italian]

Cardinali G.

Publication Types:
Clinical Trial

PMID: 4198540 [PubMed /​ indexed for MEDLINE]

408: Lloydia. 1970 Jun;33(2):275/​7.

The effect of antibiotics on the growth of Catharanthus roseus tissue cultures.

Carew DP, Patterson BD.

PMID: 5495518 [PubMed /​ indexed for MEDLINE]

409: Lloydia. 1969 Jun;32(2):131/​40.

Growth and alkaloid formation in Catharanthus roseus tissue cultures.

Patterson BD, Carew DP.

PMID: 5812244 [PubMed /​ indexed for MEDLINE]

410: Pharm Weekbl. 1969 Apr 18;104(16):321/​30.

[Alkaloids with tumor inhibiting action from Catharanthus roseus]

[Article in Dutch]

Uffelie OF.

PMID: 5769032 [PubMed /​ indexed for MEDLINE]

411: J Pharm Sci. 1968 Apr;57(4):589/​93.

Effect of gibberellic acid on the growth, alkaloid production, and VLB content
of Catharanthus roseus.

Masoud AN, Sciuchetti LA, Farnsworth NR, Blomster RN, Meer WA.

PMID: 5652148 [PubMed /​ indexed for MEDLINE]

412: Acta Genet Med Gemellol (Roma). 1968 Jan;17(1):197/​208.

The Catharanthus roseus (Vinca rosea) alkaloids: a new class of stathmokinetic
agents.

Cardinali G, Centurelli G.

PMID: 5659730 [PubMed /​ indexed for MEDLINE]

413: Postepy Biochem. 1968;14(2):209/​32.

[Alkaloids of Catharanthus roseus G. Don./​/​new group of biologically active
compounds]

[Article in Polish]

Kohlmunzer S.

Publication Types:
Review

PMID: 4872670 [PubMed /​ indexed for MEDLINE]

414: Yao Xue Xue Bao. 1965 Dec;12(12):772/​7.

[The antitumour action and toxicity of the alkaloidal fraction AC/​875 from
Catharanthus roseus]

[Article in Chinese]

Chang SY, Mao BY, Hsu B.

Publication Types:
In Vitro

PMID: 5898964 [PubMed /​ indexed for MEDLINE]

415: Chem Ind. 1965 Jul 10;28:1260.

Catharosine, a new alkaloid from Catharanthus roseus G.Don.

Moza BK, Trojanek J.

PMID: 5842019 [PubMed /​ indexed for MEDLINE]

416: Naturwissenschaften. 1965 Jun;52:305/​6.

[ON ALKALOID FORMATION IN TISSUE CULTURE BY CATHARANTHUS ROSEUS G. DON.]

[Article in German]

RICHTER I, STOLLE K, GROEGER D, MOTHES K.

PMID: 14331116 [PubMed /​ OLDMEDLINE]

417: Indian J Pathol Bacteriol. 1965 Apr;39:98/​102.

EFFECT OF COLCHICINE AND CATHARANTHUS ROSEUS (VINCA ROSEA) ALKALOIDS ON MITOSIS
OF SYRIAN HAMSTER BONE MARROW CELLS.

MEHROTRA TN, CARDINALI G.

PMID: 14300954 [PubMed /​ OLDMEDLINE]

418: J Pharm Sci. 1964 Oct;53:1227/​31.

ALKALOIDS OF VINCA ROSEA LINN. (CATHARANTHUS ROSEUS G. DON). XXIV. VINASPINE,
VINCATHICINE, ROVIDINE, DESACETYL VLB, AND VINAPHAMINE.

SVOBODA GH, BARNES AJ Jr.

PMID: 14249445 [PubMed /​ OLDMEDLINE]

419: Therapie. 1964 Jul/​Aug;19:1037/​46.

[THERAPEUTIC ACTION OF THE ALKALOIDS OF CATHARANTHUS ROSEUS (VINCA ROSEA) ON
HODGKIN'S DISEASE AND ACUTE LEUKOSIS.]

[Article in French]

CHASSAGNE P, CRUVEILLER J, GEORGES/​JANET L, MARY F.

PMID: 14190930 [PubMed /​ indexed for MEDLINE]

420: J Pharm Sci. 1963 Jul;52:688/​92.

Alkaloids of Vinca rosea Linn. (Catharanthus roseus G. Don). XV. Analysis of
vinca alkaloids by thin/​layer chromatography.

CONE NJ, MILLER R, NEUSS N.

PMID: 14022560 [PubMed /​ indexed for MEDLINE]

421: J Pharm Sci. 1962 Aug;51:707/​20.

Current status of research on the alkaloids of Vinca rosea Linn. (Catharanthus
roseus G. Don).

SVOBODA GH, JOHNSON IS, GORMAN M, NEUSS N.

PMID: 13918740 [PubMed /​ indexed for MEDLINE]

422: J Pharm Sci. 1962 Jun;51:518/​23.

Alkaloids of Vinca rosea Linn. (Catharanthus roseus G. Don.) XII. Preparation
and characterization of trace alkaloids.

SVOBODA GH, GORMAN M, BARNES AJ Jr, OLIVER AT.

PMID: 13918739 [PubMed /​ indexed for MEDLINE]

423: J Am Pharm Assoc Am Pharm Assoc. 1959 Nov;48:659/​66.

Alkaloids of Vinca rosea Linn. (Catharanthus roseus G. Don.). V. Preparation and
characterization of alkaloids.

SVOBODA GH, NEUSS N, GORMAN M.

PMID: 13854990 [PubMed /​ indexed for MEDLINE]tha

424: J Am Pharm Assoc Am Pharm Assoc (Baltim). 1959 Apr;48(4):256/​7.

A note on the alkaloids of Vinca rosea Linn. (Catharanthus roseus G. Don.). II.
Catharanthine, lochnericine, vindolinine, and vindoline.

GORMAN M, NEUSS N, SVOBODA GH, BARNES AJ Jr, CONE NJ.

PMID: 13641069 [PubMed /​ indexed for MEDLINE]


 

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