jeanflop/post-ocr-correction huggingface dataset:
puisqu'un clone apparaît différent. Ce dernier possède pourtant toutes les caractéristiques botaniques de l'espèce : fleurs bleu lilas, bractées sèches avant la floraison et de couleur écrue, feuillage vert glauque. Il s'en distingue cependant par une taille du feuillage inférieure d'environ 10 cm, mais pas significativement différente des autres plantes, par des couleurs légèrement plus prononcées ABRIDGED VERSION The rhizome of some iris species (such as Iris pallida Lam. or Iris germanica L.) is a source of irones (cis-a, trans-a, B, and cis-y), ketonic compounds used in perfumes for their scent of violets. Rhizomes are usually harvested after three years
scent of violets. Rhizomes are usually harvested after three years of plant growth. Extracts from freshly harvested rhizomes contain only small traces of irones; these are released only after about 3 years of storage, by an unknown mechanism at about 0.5 g per kg of dried matter of rhizome. Therefore, until now, no plant selection to improve the production of irones was done within an iris species, and the cultivation of iris is decreasing. In this paper, a study of the intraclonal stability and the plant selection on 19 different clones is presented. For an early selection of high producing
clones is presented. For an early selection of high producing plants, a recently developed technique which releases irones directly from fresh rhizomes was used. The intraclonal stability was studied with 2 clones, 1 with 10 plants issued (by vegetative splitting) from one Iris pallida plant, another with 7 plants from one Iris germanica plant. After one year of cultivation in the fields, the mean concentrations were 1686 and 1134 mg of irones per kg of dried matter for Iris pallida and Iris germanica clones respectively. The variance analysis has shown that, for both species, the irone contents of the rhizomes
that, for both species, the irone contents of the rhizomes of different plants derived from one single clone were not statistically different. This means that further differences observed among different clones will not be due to an eventual heterogeneity of plants issued from a single clone. The actual selection was performed in two locations: 19 Iris pallida clones in Notre-Dame-d’Oë, France (oceanic climate) and 6 clones in Allemagne-en-Provence, France (Mediterranean climate). In both locations, 5 repetitions were done with 3 plants per clone and per repetition. After one growth cycle in the fields, all 3 plants per clone and per
in the fields, all 3 plants per clone and per repetition were harvested, mixed together and then submitted to a microbiological treatment for the irone determination. The results have shown that at Notre-Dame-d’Oë the irone content (mg irones per kg of dry matter) and the irone yields (mg irones per plant) were significantly different among the clones. For example, one clone (P1) has a high irone content (1720 versus 1284 mg/kg dried matter for the mean value) and another (B2) has a high yield (85 versus 58 mg/plant for the mean value). No significant differences concerning growth have been noticed.
mean value). No significant differences concerning growth have been noticed. At Allemagne-en-Provence, the yields were also significantly different between clones, but this was due to differences of growth, the irone contents being very similar. For example, 2 clones (M1 and K1) presented a high biomass (80 versus 63 g fresh matter per plant for the mean value) and another has a high yield (49 versus 38 mg per plant for the mean value). The comparison between the clones grown at both locations led to the following conclusions: the growth and the irone yield was higher in Notre-Dame-d’Oë whereas the irone
the irone yield was higher in Notre-Dame-d’Oë whereas the irone content was higher in Allemagne-en-Provence. These preliminary results require a confirmation with a longer experimental time (more than one year of cultivation) and an enlarged number of tested clones (only 4 clones were common to both locations). It has been demonstrated for the first time that an important variability in terms of growth, irone concentration or yield can be observed. New field trials are in progress to check the reproducibility of the results, mainly on high producing clones already tested and other clones from the established collection. REFERENCES 1. Naves
and other clones from the established collection. REFERENCES 1. Naves Y.-R., Grampoloff A. V., Bachmann P. 1947. Études sur les matières végétales volatiles. L) Etudes dans les séries des méthyl-3-linalols, des méthyl-3-citrals et des méthyl-6-ionones. Helv. Chim. Acta 30: 1599-613. 2. Schinz H., Ruzicka L., Seidel C.F., Tavel C.F. 1947. Produits à odeur de violette. Synthèse de la d, l-irone. Helv. Chim. Acta 30: 1810-4. Kolheim F. 1986. Iris. In : Iris. London : C. Helm. 200-303. Crabalona J. 1959. Élaboration des irones dans le rhizome d'iris au cours de sa conservation. La France et ses parfums 3: 22-8. Galfré
sa conservation. La France et ses parfums 3: 22-8. Galfré A., Martin P., Petrzilka M. 1993. Direct Enantioselective separation and olfactory evaluation of all irone isomers. J. Essent. Oil Res. 5: 265-77. Bretin M., Abrial M. 1922. Culture de l'Iris à parfum. In : Les irises cultivés. Paris : Actes et Comptes rendus de la Conférence Internationale des Iris, 185-96. Garnero J., Joulain D., Buil P. 1978. De l'influence du stockage des rhizomes d'iris sur la composition de l'huile essentielle ou beurre d'Iris et quelques constituants inédits. Riv. Int. E.P.P.O.S. 40: 568-90. Guenther E. 1952. Oil of Orris root. In
40: 568-90. Guenther E. 1952. Oil of Orris root. In : The Essential Oils, vol. 4. New York : Van Nostrand, 79-89. Belcour B., Courtois D., Ehret C, Pétiard V. 1993. Rapid production of irones by maturation of iris rhizomes with two bacterial strains. Phytochemistry 34: 1313-5. Rodonienko G.I. 1961. The genus Iris. Leningrad : Moskau. Belcour B., Courtois D., Ehret C. 1989. A process for the preparation of y-irone by bioconversion, comprising treating iris rhizomes, parts such as rhizomes, iris extracts, iris extraction wastes or plant cell cultures of iris, with bacteria in the presence of a plant cell
iris, with bacteria in the presence of a plant cell culture medium, is described. Brevet EP 0353 683 A2. Murashige T., Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiol. Plant 15: 473-97. Likens S.T., Nickerson G.B. 1964. Detection of certain hop oil constituents in brewing products. A.S.B.C. Proceedings : 5-13. Steel R.G., Torrie J.H. 1980. Principles and procedures of statistics. A biometrical approach. New York : McGraw Hill Book Company, 633 p. Jaenicke L., Marner F.J. 1985. Of irones, dihydroirones and their precursors. In : Berger R.G., Nitz S., Schreier P., eds.
precursors. In : Berger R.G., Nitz S., Schreier P., eds. Topics in flavor research. Marzling-Hangenham : H. Eichhorn, 253-76. Gabe M. 1968. Techniques histologiques. Paris : Masson, 1113p. Simonet M. 1932. Recherches cytologiques et génétiques chez les Iris. Bull. Biol. France Belgique 105: 255-444. C.R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317 : 424-9 429 C.R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317 : 430-6 Pharmacologie/Pharmacology Potentiation of 2',3'-dideoxycytidine (ddC) by hydroxyurea and thymidine on the Moloney murine leukemia virus (MoMLV) early replicative steps Potentialisation de l'effet du 2',3'-dideoxycytidine (ddC) par hydroxyurée et la
Potentialisation de l'effet du 2',3'-dideoxycytidine (ddC) par hydroxyurée et la thymidine sur les étapes précoces du cycle replicatif du virus de la leucémie murine de Moloney (MoMLV) HÉLÈNE GOULAOUIC, FRÉDÉRIC SUBRA, JEAN-FRANÇOIS MOUSCADET, SANDRINE CARTEAU CHRISTIAN AUCLAIR Laboratoire de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA 147, Institut Gustave-Roussy, 39, rue Camille-Desmoulins, 94805 Villejuif, France. Tirés à part : C. Auclair RÉSUMÉ Le ddC est un analogue nucléosidique inhibiteur de la transcription inverse et présentant une activité antivirale contre le virus de l'immunodéficience humaine (VIH). L'efficacité du ddC dépend étroitement du métabolisme nucléotidique endogène. Des lignées cellulaires murines comme
étroitement du métabolisme nucléotidique endogène. Des lignées cellulaires murines comme les fibroblastes 3T3 peuvent être considérées comme un modèle expérimental reproduisant des conditions phénotypiques défavorables à l'action antivirale du ddC. Dans ces cellules, le ddC présente en effet une activité antivirale extrêmement faible avec une IC50 voisine de 100 pM, soit une efficacité environ 30000 fois plus faible que celle de l'AZT. Cela provient essentiellement du fait que dans les lignées murines, la phosphorylation du ddC en ddCTP est peu efficace alors que la synthèse de dCTP est élevée. Une stratégie possible, visant à augmenter l'efficacité du ddC, consiste à
stratégie possible, visant à augmenter l'efficacité du ddC, consiste à élaborer un traitement combinatoire avec des composés comme l'hydroxyurée (HU) ou la thymidine réduisant in fine la synthèse intracellulaire de dCTP. Nous avons donc étudié dans des fibroblastes 3T3 l'effet du ddC utilisé en combinaison avec des concentrations non cytotoxiques d'HU ou de thymidine sur les étapes précoces d'un rétrovirus recombinant défectif (psi neo) de la leucémie murine de Moloney (MoMLV). Le traitement des cellules infectées par le ddC en association avec 50 pM d'HU réduit la IC50 du ddC d'un facteur 10. Un effet synergique est également obtenu par
d'un facteur 10. Un effet synergique est également obtenu par l'association du ddC avec la thymidine. Le traitement combinatoire avec 100 pM de thymidine diminue la IC50 du ddC d'un facteur 50. Enfin, un traitement associant 40 μM de ddC à 100 pM de thymidine se traduit par une inhibition pratiquement totale de la replication virale. Ces résultats montrent que dans un modèle expérimental dans lequel les conditions métaboliques sont très défavorables à l'action antivirale du ddC, un traitement combinatoire avec des composés conduisant à la diminution de la synthèse endogène du dCTP restaure une activité antivirale voisine de celle
endogène du dCTP restaure une activité antivirale voisine de celle observée dans des lignées cellulaires présentant un phénotype favorable à l'action du ddC. Mots clés : ddC, transcription inverse, MoMLV, hydroxyurée, thymidine. Note présentée par Roger Monier. Note remise le 15 avril 1994, acceptée le 19 avril 1994. 430 Effect of hydroxyurea and thymidine on ddC activity ABSTRACT Combinations of ddC with either the ribonucleotide reductase inhibitor hydroxyurea (HU) or with the natural nucleoside thymidine have been investigated on the cycle of a defective (psi neo) Moloney Leukemia Virus (MoMLV) using 3T3 fibroblasts as host cells. In this experimental model,
using 3T3 fibroblasts as host cells. In this experimental model, ddC displayed very poor antiviral action which was obvious given an IC50 value close to 100 pM, i.e. an efficiency about thirty thousand fold lower than that of AZT. Both HU and thymidine alone resulted in significant inhibition of MoMLV replication with IC50 values of 40 pM and 100 pM respectively. The combination of ddC with 50 pM HU lowered the IC50 of ddC by a factor of 10. A similar but more pronounced effect was obtained by combining ddC and 100 pM thymidine, which decreases the IC50 value of
and 100 pM thymidine, which decreases the IC50 value of ddC by a factor of 50. Combining 40 pM ddC and 100 pM thymidine resulted in the quite complete inhibition of viral replication. These results show that in cell types with strongly restricted ddC action, combination treatment with compounds known to ultimately decrease dCTP biosynthesis leads to the restoration of efficient antiviral activity. A Key words: ddC, reverse transcription, MoMLV, hydroxyurea, thymidine. Voir Version abrégée (p. 434) The retrovirus replicative cycle appears to be mainly governed by host cell multiplication [1-3]. This control is particularly effective at the early stages
[1-3]. This control is particularly effective at the early stages of viral replication including reverse transcription, translocation of the nucleoprotein complex and integration [4-6]. We have previously shown that, in quiescent fibroblasts (Balb/c 3T3), the parameter limiting the completion of reverse transcription of the Moloney murine leukemia virus (MoMLV) was the availability of the nucleotide pool [7]. This observation strongly suggests that reverse transcription in cells is strictly dependent on deoxynucleotides synthesis including de novo and salvage pathways. Accordingly, the ribonucleotide reductase inhibitor hydroxyurea markedly impaired MoMLV [7] and HIV-1 [8, 9] reverse transcription. This metabolic control may imply that
[8, 9] reverse transcription. This metabolic control may imply that antitumor drugs known to inhibit deoxyribonucleotide synthesis such as hydroxyurea and natural nucleosides such as thymidine which exert a feedback regulatory effect on pyrimidine reduction through an increase of dTTP synthesis [10] may have additive or synergistic effects when combined with antiviral nucleoside analogs acting at the reverse transcription step. Furthermore, exogenous supply of low thymidine concentrations results in a quite selective decrease of dCTP [11-13]. This hypothesis is reinforced by the fact that the obligatory phosphorylation steps of these drugs compete with the phosphorylation of endogenous deoxynucleosides [14-18]. Experimental
drugs compete with the phosphorylation of endogenous deoxynucleosides [14-18]. Experimental models with nucleoside analogs exhibiting low efficiency due to poor intracellular phosphorylation are needed to test this hypothesis. Such conditions are present in murine cell lines treated by the anti-HIV drug ddC [15-18]. We therefore decided to investigate the effect of hydroxyurea and thymidine on the efficiency of ddC in 3T3 fibroblasts after acute infection by a recombinant defective MoMLV (psi neo). Materials and methods Chemicals Polybrene, ddC, hydroxyurea and thymidine were obtained from Sigma Chemical Co (USA). Geneticin G418 was purchased from Gibco and AZT from Wellcome laboratories (USA).
was purchased from Gibco and AZT from Wellcome laboratories (USA). Polybrene and G418 were diluted in sterile isotonic NaCl at a concentration of 10 mg/ml and 30 mg/ml respectively and stored either at 4 °C or -20 °C. ddC and AZT were dissolved in distilled sterile water or isotonic NaCl at a concentration of 50 mM or 10 mM respectively and stored at -20 °C. Hydroxyurea and thymidine were extemporaneously dissolved in isotonic NaCl and in culture medium containing 5 % FCS respectively. Cells and virus • Chronically infected cells (Psi2 neo) The psi2 cell lines were derived from 3T3
(Psi2 neo) The psi2 cell lines were derived from 3T3 cells and contain, integrated in their cellular genome, pMov-Psi DNA which provides all the trans functions (gag pol env genes) required for encapsidation of recombinant viral RNA [19]. The psi2 neo cell line was obtained by transfection of the SVX shuttle vector [20] into psi2 cells. SVX DNA contains the MoMLV transcriptional unit, the cis acting region needed to package viral RNA (i.e. the psi sequence) and encodes G418 resistance in mammalian cells. This genetic information leads to the replication and production of infectious defective viral particles containing the psi
and production of infectious defective viral particles containing the psi sequence and the neo gene. The Psi2 neo cell lines used in the present work yielded a titer of recombinant viruses of 104 CFU/ml. The NIH 3T3 and the Psi2 neo cells were maintained in Dulbecco modified Eagle medium containing 5 % fetal calf serum, 100 μg/ml streptomycin and 100 U/ml penicillin. MoMLV retroviral cycle assay • Production of the recombinant defective MoMLV Psi2 neo cells (2.5 x 106) were plated in 100 mm petri dishes and incubated at 37 °C. Twenty-four hours after plating, the supernatants containing the defective
°C. Twenty-four hours after plating, the supernatants containing the defective viral particles were harvested and filtered through a 0.45 μm Nalgene HA filter. The viral suspension was stored at -70°C. The number of infectious recombinant viral particles was estimated using the geneticin resistance assay, as described below. Infection of the 3T3 cells by the recombinant defective MoMLV 2.5 x 105 NIH 3T3 cells were plated in 60 mm petri dishes and incubated at 37 °C. Twenty-four hours after plating, 2 ml of an appropriate dilution of a viral suspension containing 8 μg/ml polybrene were added to the petri dishes containing
8 μg/ml polybrene were added to the petri dishes containing the 3T3 cells. In the standard operating conditions, the drugs to be tested were added to the viral suspension used to infect the 3T3 cells. Twenty-four hours after infection, geneticin G418 was added at a concentration of 0.6 mg/ml. Seven days later the resistant clones were stained using violet crystal dye and counted. In these conditions, the number of resistant clones is a linear function of the number of infectious viral particles added. The possible cytostatic or cytotoxic effect of the drugs tested was estimated as follows: cytotoxicity control dishes,
the drugs tested was estimated as follows: cytotoxicity control dishes, in which geneticin was omitted, were monitored in parallel. Twenty-four hours after the infection, the cells were trypsinized and counted. Aliquots of 800 cells were then replated and the colonies counted 7 days later. Results To confirm the relevance of the experimental model, we comparatively investigated the effect of AZT and ddC on the formation of 3T3 colonies resistant to the antibiotic G418. Figure 1 shows, first of all, that the addition of non-cytotoxic concentrations of AZT with the infectious viral particles results in a strong decrease in the formation
viral particles results in a strong decrease in the formation of resistant colonies with an IC50 close to 3 nM. In this experimental model, this effect is due to the impairment of reverse transcription of the recombinant virus and a subsequent decrease in integrated viral DNA in the genome of host cells [21]. CR. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 430-6 431 H. Goulaouic et al. In contrast to AZT, there was no significant effect of ddC up to 1 µM and the IC50 value was close to 100 µM indicating that the drug was not
close to 100 µM indicating that the drug was not very efficient. These observations are in agreement with previous studies which reported that ddC is poorly phosphorylated by the cellular enzymes deoxycytidine kinase, nucleoside mono- and diphosphate kinases, in murine cell lines [15, 18]. This results in a limited production of ddCTP and reverse-transcriptase-mediated incorporation of nucleotides in viral DNA maintains a preference for dCTP. In these conditions, any action leading to a decrease in dCTP synthesis should significantly increase ddC incorporation and antiviral activity. In this respect, the reduction of ribonucleotides, catalyzed by ribonucleotide reductase (RR), is considered a
of ribonucleotides, catalyzed by ribonucleotide reductase (RR), is considered a key step in dNTP synthesis and the modulation of dCTP synthesis in cells can be therefore achieved at the RR level. Inhibition of overall RR catalytic activity can be obtained with the antitumor drug hydroxyurea (HU) whereas selective inhibition of the reduction of pyrimidine nucleotides can be accomplished with dTTP, whose binding to specific control sites of the enzyme enhances the reduction of GDP and also inhibits further reduction of TDP and CDP. Treatment of cells with the natural nucleoside precursor thymidine results in the same effect by increasing dTTP
precursor thymidine results in the same effect by increasing dTTP biosynthesis via a salvage pathway. We therefore investigated the effect of HU and thymidine, both of which producing ultimately a decrease in dCTP synthesis as explained above. Figure 2A shows the effect of increasing concentrations of HU alone on the establishment of G418 resistant colonies as well as the cytotoxic effect on 3T3 cells. The data clearly show that at non-cytotoxic concentrations, HU significantly decreases the replication of the virus. This is in agreement with recent reports which demonstrated that HU impairs reverse transcription of viral RNA via dNTP pool
HU impairs reverse transcription of viral RNA via dNTP pool depletion [7-9]. Figure 2B shows the dose response of ddC in the absence and in the presence of 50 µM HU. When adjusted for the effect of HU alone, the IC50 values of ddC decrease by a factor of 10 in the presence of 50 µM HU. Combination treatment with 40 µM ddC and 50 µM HU resulted in about 95% inhibition of virus replication. As stated above, thymidine, which is readily converted into dTTP in cells, selectively inhibits the reduction of pyrimidine diphosphate occasioning a marked decrease in dCTP
reduction of pyrimidine diphosphate occasioning a marked decrease in dCTP biosynthesis. As already observed in HU experiments, the treatment of cells with non-cytotoxic concentrations of thymidine gave rise to a significant decrease in G418 resistant colonies (Fig. 3A) although the IC50 value was close to 100 µM. Figure 3B shows that the presence of 50 µM thymidine leads to marked enhancement of ddC efficiency which is obvious given the decrease in the IC50 value of one order of magnitude. This effect is markedly increased in the presence of 100 µM thymidine which reduced the IC50 value by a factor close
thymidine which reduced the IC50 value by a factor close to 50. Figures 3C and 3D illustrate respectively the effect of treatment with a combination of ddC and thymidine on 3T3 cloning efficiency (toxic effect) and on the establishment of G418 resistant colonies (antiviral effect). These figures clearly show that the association of non-cytotoxic concentrations of thymidine and ddC strongly decreases G418 colony formation. The combination of 100 µM ddC and 100 µM thymidine, which are non-toxic concentrations and thus relevant in terms of pharmacology, results in the quite complete disappearance of G418 resistant clones. It should be pointed out
disappearance of G418 resistant clones. It should be pointed out that thymidine has a more highly selective synergistic effect on ddC than HU. This may stem from the fact that, besides its inhibitory action on CDP reduction, dTTP enhances the deoxycytidine kinase-mediated phosphorylation reaction leading to increased phosphorylation of ddC to ddCMP and a subsequent increase in ddCTP biosynthesis. Previous reports dealing with similar experiments performed in HIV-1 infected ATH8 cells have provided disappointing results [22]. This is probably because human T lymphocytes ATH8 offer highly favorable metabolic conditions for ddC activity which cannot be significantly improved with combination treatments.
ddC activity which cannot be significantly improved with combination treatments. Conclusion In agreement with recent reports, our results confirm first of all that alteration of dNTP levels could represent a possible strategy to selectively inhibit retrovirus replication. Along this line, we clearly showed that the dNTP modulators hydroxyurea and thymidine strongly decrease the viral replication at non-cytotoxic concentrations. Both compounds have been used in cancer therapy and display toxic side effects less pronounced than other anticancer agents. Accordingly, when administered at dosages not greater than 40 mg/kg/day, toxic effects including marrow depression produced by hydroxyurea are readily reversible whereas it