Isolement et sélection de souches d'actinomycètes productrices de substances antifongiques de structure non-polyénique

Résumé Dans le but d'étudier des souches d'actinomycètes productrices de substances antifongiques de structure non-polyénique, 13, échantillons de sol prélevés dans le sud de la France ont été examinés. L'utilisation de milieux sélectifs a permis d'isoler 486 souches d'actinomycètes qui ont été testées vis-à-vis de quatre espèces de champignons et de levures: 18% des souches isolées sont actives sur au moins l'une des espèces utilisées. Parmi celles-ci 14 souches, productrices de substances de structure non-polyénique, ont été sélectionnées après étude des spectres d'absorption en UV des surnageants de culture, des extraits butanoliques de ces surnageants ou des extraits méthanoliques de mycélium. L'utilisation d'un test bactérien de toxicité à court terme (SOS Chromotest) a permis de montrer que 10 souches sur 14 présentent aussi une activité génotoxique.

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Summary In order to study actinomycete strains producing non-polyenic antifungal substances 13 soil samples were collected in S. France. By using selective media 486 strains of actinomycetes were isolated and tested on four species of moulds and yeasts: 18% of the isolated strains were active against one or more of the test organisms. From these isolates 14 producers of non-polyenic antifungal substances were selected by means of u.v. absorption spectra of culture supernatant fractions, butanol extracts of these fractions, or methanol extracts of mycelium. A rapid bacterial toxicity assay (SOS Chromotest) demonstrated that 10 of the 14 selected strains had genotoxic activity.

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Resumen Para el estudio de cepas de actinomicetes productores de sustancias antifúngicas no poliénicas, se recogieron 13 muestras de suelo en el S. de Francia. Utilizando medios selectivos se aislaron 486 cepas de actinomicetes que se ensayaron frente a cuatro especies de mohos y levaduras: 18% de las cepas aisladas mostraron actividad frente a uno o más de los organismos utilizados en el test. De estas cepas se seleccionaron 14 que eran productoras de sustancias antifúngicas no poliénicas mediante el espectro de absorción al U.V. de las fracciones sobrenadantes del cultivo, de extractos butanólicos de dichas fracciones o de extractos metanólicos del micelio. Un ensayo rápido de toxicidad bacteriana (SOS Chromotest) mostró que de las 14 cepas seleccionadas lo tenían actividad genotóxica.

     

Identification of the glucose transporter in rat skeletal muscle

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [³H]cytochalasin B in the presence of l- and d-glucose. [³H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.     

The structural nif genes of the cyanobacteria Gloeothece sp. and Calothrix sp. share homology with those of Anabaena sp., but the Gloeothece genes have a different arrangement.

Probes carrying the Anabaena sp. strain PCC 7120 nitrogenase reductase (nifH) and nitrogenase (nifK and nifD) genes were hybridized to Southern blots of DNA from the unicellular, aerobic nitrogen-fixing cyanobacterium Gloeothece sp. strain PCC 6909 and from the filamentous cyanobacterium Calothrix sp. strain PCC 7601. These data suggest that the Gloeothece sp. nif structural proteins must be similar to those of other diazotrophs and that the ability for aerobic nitrogen fixation does not reside in the nif protein complex. We also found that the nif structural genes of Gloeothece sp. are clustered, whereas those of Calothrix sp. are arranged more like those of Anabaena sp.     

Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12.

Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101. The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound. Both effects were shown to be dependent upon excision repair. Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E. coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid. With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101. These results suggest that pKM101-induced repair differs from normal SOS repair.     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.     

Leishmania mexicana amazonensis: Surface charge of amastigote and promastigote forms

The surface charge of Leishmania mexicana amazonensis was evaluated by means of the binding of colloidal iron hydroxyde particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, visualizated by electron microscopy and by direct measurements of the electrophoretic mobility of cells suspended in solutions of different pH. The following forms of the parasite were analysed: amastigotes (surrounded or not by the membrane of the endocytic vacuole, isolated from lesions), transitional forms, and infective (5 passages) and noninfective (176 passages) promastigotes. The results obtained indicate that the surface of L. m. amazonensis contains both negatively and positively charged dissociating groups and that changes occur in the surface charge during amastigote-promastigote transformation. Treatment of the parasite with neuraminidase significantly reduced the electrophoretic mobility of the cells. Neuraminidase-treated cells recovered their normal electrophoretic mobility when incubated for 8 hr in fresh culture medium by a process that is inhibited by puromycin.     

Comparative studies of seed proteins of the genusArtocarpus with respect to lectins

Proteins from two species of the genusArtocarpus (A. integrifolia L. andA. incisa L.) were compared by ammonium sulphate fractionation, molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis, with special attention to the lectins. The protein content and hemagglutinating activity were markedly different in the two seeds. The protein pattern obtained by both molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis were quite different. The only similarities found were the elution volume of the lectins in the Sephadex G-100 column and the lectin bands (11 500 and 15 000 daltons) in SDS-polyacrylamide gel electrophoresis.     

Simple computer method for evaluation of lateral diffusion coefficients from fluorescence photobleaching recovery kinetics.

A method is presented for the analysis of fluorescence photobleaching recovery curves. Based on the simplified kinetic expression of Yguerabide, J., J.A. Schmidt, and E.E. Yguerabide (1982, Biophys. J., 40:69-75), a linearization procedure is described that permits unequivocal determination of all diffusion parameters. The presence of additional membrane flow or multiple diffusion coefficients can easily be detected by this method, and simple corrections for the presence of these alternative recovery processes can be made by the use of a regular mini-computer. The validity of the method is tested on simulated recovery curves, varying the contribution of flow, multiple diffusion coefficients, and statistical noise due to counting error.     

Mixtures of gramicidin and lysophosphatidylcholine from lamellar structures

It is shown by ³¹P-NMR and freeze-fracture electron microscopy that in aqueous dispersions of mixtures of gramicidin and palmitoyllysophosphatidylcholine lamellar structures are formed which contain four lysophosphatidylcholine molecules per gramicidin monomer.