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M Robert-Gero

Publications and source records attributed to M Robert-Gero.

At least 19 recordsLinked to original sources
PubMed1992

Leishmania donovani: antagonistic effect of S-adenosyl methionine on ultrastructural changes and growth inhibition induced by sinefungin.

Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent in vitro and in vivo (Bachrach et al. 1980, FEBS Letters 121, 287-291; Neal et al. 1985, Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 85-122). It was previously shown that this molecule is a competitive inhibitor of AdoMet for transmethylases (Paolantonacci et al. 1986, Molecular and Biochemical Parasitology 21, 47-54; Avila et al. 1987, Molecular and Biochemical Parasitology 26, 69-76) and that it induces shape changes of Leishmania donovani promastigotes as observed by light microscopy (Lawrence and Robert-Gero 1990; Bulletin de la Societé Française de Parasitologie 8, 13-18). In the present work the effect of the antibiotic on the ultrastructure was analyzed by electron microscopy. The main changes induced at sublethal concentrations (0.26 microM sinefungin for 16 hr) were progressive rounding, decreased motility, enlargement of the flagellar pocket, and shortening and loss of the external part of the flagellum. The comparison with control cells showed shorter Golgi saccules and fragmentation of the trans-Golgi network into vesicles, indicating a stimulated Golgi apparatus activity. This result, associated with the enlarged flagellar pocket, suggests an unbalanced cytoplasmic exchange between exocytosis and endocytosis. These effects are quite different from those induced by tunicamycin (Dagger et al. 1984, Biology of the Cell 50; 173-180) or paromomycin. In addition, other nucleoside and nonnucleoside growth inhibitors failed to induce similar changes. AdoMet antagonized the sinefungin-induced shape changes and ultrastructural modifications but had no effect with respect to other growth inhibitors. This suggests that the sinefungin activity at the cellular level is specifically related to competition with AdoMet. A comparative study of N-methylation and carboxylmethylation of proteins in sinefungin-treated promastigotes showed that the antibiotic preferentially inhibits the latter, catalyzed by protein-O-methyltransferases. These enzymes are known to regulate the function of various proteins involved in secretion. Overall the results suggest that one of the main targets of sinefungin in exponentially growing cells is the protein carboxylmethylation involved in membrane transport.

Adenosine↗
PubMed1992

Total synthesis of uracil analogues of sinefungin.

Analogues of sinefungin derivatives 18a and 18b have been prepared from uridine and L-aspartic acid. The key step in the synthesis was the coupling of the radical derived from 14 with the unsaturated amide 13. The latter was produced from the known N-hydroxy-2-thiopyridone ester of L-aspartic acid 12 with the olefin 11. Thus, the essential carbon skeleton was constructed by way of two radical coupling reactions. These analogues as well as 1a and 1b synthesized previously were tested for their antileishmanial effect in vivo and for their inhibitory activity of protein carboxymethylase (protein methylase II). The replacement of the adenine moiety by uracil or dihydrouracil considerably decreases the antiparasitic activity and the affinity for protein methylase II. The synthetic (S)-sinefungin was as active as the natural one. Interestingly, the C-6' epimer 1b was 50% less active in vitro than the natural sinefungin, but both had identical affinities for the target enzyme.

Adenosine↗
PubMed1992

Characterization of an 8.7-kilobase thiostrepton resistance-encoding plasmid (pGIF3) of Streptomyces incarnatus.

The low-copy-number 8.7-kb plasmid pGIF3 of Streptomyces incarnatus was studied after cloning in the Escherichia coli vector pBR322; a restriction map was constructed. Southern blot analysis showed that pGIF3 in S. incarnatus occurs predominantly as integrated in a larger replicon. The plasmid carries a gene for thiostrepton resistance having no homology with the known thiostrepton resistance gene from Streptomyces azureus.

Blotting, Southern↗
PubMed1991

Synthesis and biological activity of sinefungin analogues.

A series of nucleosides (2-4) that derive from adenosine by chain extension at the 5'-end have been synthesized starting from the known phosphonate 7. The latter was first combined with 4-pentenal to give 8, which underwent chemical manipulations to provide triacetate 11, which was found suitable for the adenylation step. Further transformations, among them the Hofmann degradation of the amide group of compound 13, and final deprotection gave nucleosides 2-4. They were considered as analogues of sinefungin (1) and tested for their antileishmanial activity together with compounds 5 and 6, which were obtained independently. All the modifications with respect to sinefungin resulted in nearly complete loss of growth inhibitory activity. These results indicate that the 9' terminal amino and carboxyl groups are necessary for the activity and that the presence of the amino group at C-6' is not sufficient to maintain the antileishmanial effect. Some of the analogues however could antagonize or reverse the inhibitory activity of sinefungin (1).

Adenosine↗
PubMed1990

Involvement of plasmids in Streptomyces incarnatus phenotype.

The sinefungin producing, pock forming strain Streptomyces incarnatus was shown to be thiostrepton resistant. However, it does not produce thiostrepton and structurally related antibiotics. In this strain, five low copy plasmids of variable sizes were detected with electron microscopy. The strain S. lividans TK24 became thiostrepton resistant upon transformation by one of the plasmids of S. incarnatus.

Anti-Bacterial Agents↗
PubMed1990

Effect of sinefungin on macromolecular biosynthesis and cell cycle of Plasmodium falciparum.

The growth of the malarial parasite P. falciparum was arrested by the adenine containing nucleoside sinefungin at the trophozoite stage. The synthesis of DNA, of polyamines and the specific proteins of the schizont stage were completely blocked by the drug. The inhibition of DNA synthesis was not due to a decrease in the amount of DNA polymerase, but to the depletion of polyamines which are required for DNA synthesis.

Adenosine↗
PubMed1988

Regulation of sinefungin biosynthesis by the wild-type strain and mutants of Streptomyces incarnatus.

Sinefungin, an antifungal and antiparasitic antibiotic, is produced efficiently from ammonium citrate by prototrophic strains of Streptomyces incarnatus. The regulation of the biosynthesis of this nucleoside, composed of adenosine and ornithine, was studied by using auxotrophic mutants and a resting-cell system. Mutants blocked in arginine synthesis were not able to produce sinefungin. A uridine-negative mutant produced sinefungin in the presence of ATP, but this production was strongly inhibited when amino acids of the urea cycle were added. The same mutant produced sinefungin from aspartic acid, and this production was enhanced by ornithine. Our results show that the ornithine part of the molecule originates from arginine, liberated by either anabolic or catabolic processes.

Adenosine↗
PubMed1987

Biosynthesis of sinefungin by cell-free extract of Streptomyces incarnatus NRRL 8089.

The formation of sinefungin an antifungal and anti-parasitic nucleoside antibiotic was demonstrated in cell-free extracts from Streptomyces incarnatus. Incorporation studies as well as HPLC analysis showed that the immediate biosynthetic precursors of the antibiotic are L-arginine and ATP. The biosynthesis was optimal when the cell-free extract was prepared from 72 hours mycelium and incubated at least 80 minutes with arginine and ATP. The presence of dithiothreitol, pyridoxal phosphate and Mg2+ in the incubation mixture was also necessary. When a membrane fraction was incubated under the same conditions with ATP and L-arginine, sinefungin production was not observed. A working hypothesis is put forward to explain the biosynthesis of this antibiotic.

Adenosine↗
PubMed1986

Protein methylation and protein methylases in Leishmania donovani and Leishmania tropica promastigotes.

We studied the content of acid-stable methylated amino acids of soluble proteins in promastigotes of Leishmania donovani and L. tropica. epsilon-N-Trimethyllysine and NG,NG-dimethylarginine were found in both Leishmania species after culture in the presence of [methyl-14C]methionine. In addition, 3-N-methylhistidine was found only in L. tropica and epsilon-N-dimethyllysine only in proteins of L. donovani. As sinefungin, an antileishmanial nucleoside antibiotic, is a known transmethylase inhibitor, its effect on protein methylation was studied, in whole cells and in vitro. In the first case the drug had no effect on the content of methylated amino acid residues of soluble proteins. In vitro, histone methylation by crude extracts was studied at pH 7.2 and 9.0, known in other organisms as optimum pH values for arginine and lysine methylation, respectively. Surprisingly, arginine methylation by extracts of L. donovani was the same at both pH values while lysine residues were more efficiently methylated at pH 7.2 than at pH 9 by the extracts of the two species. These results indicate that the properties of protein methylases I and III of these parasites are different from those of other organisms hitherto studied. The inhibition constants of sinefungin for the leishmanial protein methylases were weak in comparison with those for enzymes from other sources, with the exception of the constant of L. donovani enzyme at pH 9.

Adenosine↗
PubMed1985

Induction of heat shock and stress proteins in promastigotes of three Leishmania species.

The induction of heat shock proteins in three species of Leishmania, L. tropica, L. enrietti, and L. donovani is reported. When cultures of promastigotes are shifted from 26 degrees C to 37 degrees C or 40 degrees C, the synthesis of proteins with apparent molecular weights of 88,000, 74,000, and 54,000 is stimulated. Actinomycin D added just prior to the shift prevented the appearance of these proteins but had no effect when present 30 min after the transfer onward, suggesting that the regulation of leishmanial heat shock proteins occurs at the transcriptional level. Exposure of L. tropica promastigotes to sodium arsenite elicits the synthesis of three major and four minor polypeptides. Their apparent molecular weights are, respectively, 94,000, 78,000, and 56,000 and 70,000, 45,000, 22,000, and 18,000. The response of Leishmania organisms to heat shock and to sodium arsenite is similar to that of other organisms, but some of the proteins identified as stress proteins in the parasite differ in size. The heat shock proteins might play a role in cytodifferentiation during the life cycle of the parasite and also in cellular adaptation to higher temperatures.

Animals↗
PubMed1985

Enhanced sinefungin production by medium improvement, mutagenesis and protoplast regeneration of Streptomyces incarnatus NRRL 8089.

Increased production of sinefungin, a very potent antifungal and antiparasitic nucleoside antibiotic was achieved by medium and strain improvement. When soybean-meal, dextrin and yeast extract were added as carbon and nitrogen sources to the fermentation medium, instead of corn steep liquor, soya-oil and glucose; the antibiotic yield increased from 40 micrograms/ml to 126 micrograms/ml with low biomass production. Strain improvement was attempted by two methods. The mean antibiotic yield of the variants after multistep mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine and ethyleneimine was 466 micrograms/ml. Protoplasts of the parental strain were prepared by lysozyme digestion from mycelia grown in a medium containing 0.7% glycine. The mean activity of the regenerated protoplasts was 664 micrograms/ml. Thus, the overall sinefungin production could be increased 16-fold.

Adenosine↗
PubMed1985

[Decrease in the transforming action of Rous sarcoma virus produced by avian cells grown in the presence of 5'-deoxy-5'-S-isobutyladenosine (SIBA)].

Treatment of Rous Sarcoma virus transformed chick embryo fibroblasts with 1 mM 5'-deoxy-5'-S-isobutyladenosine for 24 hrs. leads to the inhibition of transforming virus production. A kinetic analysis of the inhibition of active virion production revealed that the effect of the drug was time and concentration dependent. After 24 hrs. with 1 mM SIBA, the production of transforming virus was inhibited 165 fold. However, under these conditions there was only a 2 fold inhibition in viral particle production. Thus, these viral particles were either non infective (non adsorbed on cell membrane) or non transforming. The majority of viral particles produced by cells cultured with the drug have a decreased density. Analysis of these virions showed a decrease of protein P19 and an accumulation of proteins with high molecular weight.

Animals↗
PubMed1979

Relationship between inhibition of protein methylase I and inhibition of Rous sarcoma virus-induced cell transformation.

A correlation was found between inhibition of protein methylase I and inhibition of virus-induced cell transformation by structural analogs of S-adenosylhomocysteine; all good inhibitors of this enzyme are also good inhibitors of Rous sarcoma virus-induced chicke embryo fibroblast transformation. The inhibitory effect of these analogs was similar on enzymes from normal and transformed cells; no significant variation of the inhibition constants was observed after purification of protein methylase I. From the kinetic constants obtained, a structure-activity relationship can be established for protein methylase I.

Animals↗
PubMed1978

Identification of some metabolic products of 5' -deoxy-5' -S-isobutylthioadenosine, an inhibitor of virus-induced cell transformation.

5' -Deoxy-5' -S-isobutylthioadenosine (iBuS)5' Ado has been shown to be rapidly degraded to 5-deoxy-5-S-isobutylthioribose and adenine in procaryotes. In chick embryo fibroblasts there are two metabolic pathways for (iBuS)5' Ado degradation: (a) oxidative deamination into 5' -deoxy-5'-S-isobutylthioinosine (the main product) and (b) hydrolysis into 5-deoxy-5-S-isobutylthioribose plus adenine. The latter reaction is not due to bacterial contamination, since the same results were obtained under sterile conditions and in chick embryo fibroblasts in culture. The inhibition of the virus-induced cell transformation reported by us previously was due to (iBuS)5' Ado rather than to the main metabolic product of this molecule in chick embryo fibroblasts.

Animals↗