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Biomedical subjects

G Tiraby

Publications and source records attributed to G Tiraby.

At least 37 records · Page 2Linked to original sources
PubMed1991

A screening method to identify antibiotics of the aminoglycoside family.

A simple and selective screening method was developed for detecting new aminoglycoside (AMG) antibiotics from actinomycetes strains propagated on solid culture media. The first phase of the screening program is designed to isolate AMG-type activities using: 1) two Serratia marcescens strains, one susceptible and one resistant to AMGs, 2) the high tolerance of AMGs to heat in acidic solutions and 3) the specific resistance of a streptothricin producing strain of Streptomyces lavendulae to streptothricin antibiotics. The second phase of the screening program identifies already known AMG antibiotics through the characteristic spectrum of action which each AMG shows toward a group of bacterial strains synthesizing various AMG-inactivating enzymes.

Actinomyces↗
PubMed1990

Cloning of Saccharomyces cerevisiae promoters using a probe vector based on phleomycin resistance.

Vectors that confer high levels of phleomycin (Ph) resistance to Saccharomyces cerevisiae have been constructed with the TEF1 and ENO1 promoters, the Tn5 ble gene and the CYC1 terminator. They are able to transform yeast cells grown on rich glucose medium containing a moderate level of Ph (10 micrograms/ml, corresponding to 100-fold the minimal inhibitory concentration). Frequencies of transformation are identical to those obtained with the URA3 marker on a defined medium. A promoter probe vector, based on the same ble marker, enabled us to isolate sequences from chromosomal yeast DNA that had promoter activities. These DNA fragments have been sequenced and those which promote the highest levels of Ph resistance have been found to be either A + T-rich or have a potentially new and more efficient translation start site.

Amino Acid Sequence↗
PubMed1990

A screening method for antifungal substances using Saccharomyces cerevisiae strains resistant to polyene macrolides.

Strains of Saccharomyces cerevisiae FL200 capable of growing on a solid medium containing a mixture of polyene macrolide antibiotics (nystatin, 40 micrograms/ml, amphotericin B, 40 micrograms/ml, pimaricin, 150 micrograms/ml and RP9971 antibiotic, 10 micrograms/ml) have been isolated after successive selection steps. The mutant strains, PR13 and PRC24, are 10 to 100 times more resistant than the polyene macrolide antibiotics. When 4% Tween 80 is added to the medium, resistance to these antifungal drugs is further increased. In addition, strain PRC24, derived from strain PR13, is resistant to a non-polyene macrolide antifungal antibiotic, cycloheximide. In contrast, PR13 and PRC24 are both highly susceptible to a large range of compounds, including non-polyenic antifungal, antitumor and antibacterial agents. These particular characteristics make these strains useful for the rapid detection of antifungal compounds of the polyene macrolide and cycloheximide types, as well as for the recognition of antimitotic substances.

Anti-Bacterial Agents↗
PubMed1989

Crystallographic studies of the mechanism of xylose isomerase.

The mechanism of xylose isomerase (EC 5.3.1.5) has been studied with X-ray crystallography. Four refined crystal structures are reported at 3-A resolution: native enzyme, enzyme + glucose, enzyme + glucose + Mg2+, and enzyme + glucose + Mn2+. One of these structures (E.G.Mg) was determined in a crystal mounted in a flow cell. The other structures were equilibrium experiments carried out by soaking crystals in substrate containing solution. These structures and other studies suggest that, contrary to expectation, xylose isomerase may not use the generally expected base-catalyzed enolization mechanism. A mechanism involving a hydride shift is consistent with the structures presented here and warrants further investigation. Additional evidence in support of a hydride shift comes from comparing xylose isomerase with triosephosphate isomerase which is known to catalyze an analogous reaction via an enediol intermediate. Evidence is presented that suggests that aldose-ketose isomerases can be divided into two groups. Phospho sugar isomerases generally do not require a metal ion for activity and show exchange of substrate protons with solvent. In contrast, simple sugar isomerases all require a metal ion and show very low solvent exchange. These observations are rationalized on the basis of the need for stereospecific sugar binding.

Aldose-Ketose Isomerases↗
PubMed1989

Phleomycin resistance as a dominant selectable marker for plant cell transformation.

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance ('Ble') genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however, the highest frequency of rooted plants was obtained when transformation was carried out with the 'Sh Ble' gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.

Bleomycin↗
PubMed1988

Characterization of crystals of xylose isomerase from Streptomyces violaceoniger.

Crystals of the tetrameric xylose isomerase from Streptomyces violaceoniger have been examined by x-ray analysis. Octahedral crystals with a maximum dimension of 0.7 mm were grown from ammonium sulfate solution. They possess the symmetry of P4(1)2(1)2 or P4(3)2(1)2 space groups, which are crystallographically indistinguishable. The unit cell dimensions are a = b = 140 A and c = 134 A. There is one tetramer of molecular weight 160,000 per asymmetric unit. The crystals diffract to 2.2 A.

Aldose-Ketose Isomerases↗
PubMed1988

Bleomycin resistance conferred by a drug-binding protein.

The protein coded by a bleomycin-resistance gene (ble) cloned from producing actinomycetes was purified from a culture of a recombinant E. coli strain and its action on bleomycin was determined by in vitro assays. The protein binds reversibly in a one to one ratio to bleomycin which can no longer cleave DNA. The bleomycin resistance of cells harboring a ble gene could be accounted for by a sequestering effect of the bleomycin-binding protein.

Actinomycetales↗
PubMed1988

Phleomycin resistance as a dominant selectable marker in CHO cells.

The Tn5 and the Streptoalloteichus hindustanus (Sh) ble genes conferring resistance to bleomycin-phleomycin antibiotics have been cloned into a mammalian vector under the RSV-LTR promoter. The resulting plasmids, pUT506 and pUT507 respectively, were used to transfect CHO cells by either the calcium phosphate or the recently described polybrene-DMSO method. Phleomycin- or bleomycin-resistant clones arose with a higher frequency after transfection with pUT507, and pUT507 transfectants were more resistant to both antibiotics than pUT506 transfectants. Phleomycin resistance in pUT507 transfectants was stable and associated with integration of plasmid sequences in genomic DNA. The Sh ble gene, which confers a dominant phleomycin-resistance phenotype, should provide a useful transferable selectable marker in CHO cells as well as in other animal cell lines.

Animals↗
PubMed1988

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus.

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.

Amino Acid Sequence↗
PubMed1988

A simple screening method for insecticidal substances from actinomycetes.

A simple and selective assay system was developed in the search for new insecticidal substances from Actinomycetales strains propagated on solid culture media. The strains were first tested for their ability to produce antimicrobial compounds. Only strains displaying weak or no activity were retained and screened in the insecticidal bioassay. Microbial solid cultures were given as food to larvae and to adults of Musca domestica to detect insecticide producers. A second phase, after extraction of the active compounds, consisted of an evaluation of the insecticidal potency and a primarily biological identification of the products synthesized by the selected strain. Of 6,280 actinomycete strains which were screened, 47 were active but only 30 of these were finally chosen in the second phase of screening. All these strains, except one, produced known metabolites such as piericidins, avermectins or valinomycin. The one strain, CL307-24, and its insecticide products appeared novel and will be the topic of further study.

Actinomycetales↗
PubMed1987

Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae.

Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells. The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E. coli-yeast shuttle plasmid. Higher resistance levels are obtained in S. cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene. The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions. Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements. Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.

Bleomycin↗
PubMed1987

A bacteriological study of rampant caries in children.

We undertook a microbiological study, in children, of dental plaque from sound surfaces or associated with rampant caries, both black-colored and unstained. Improved selective media allowed for the enumeration of bacteria belonging to specific genera or species present in plaque samples. A nearly similar bacterial distribution was found in both types of rampant caries. Aciduric flora, Streptococcus mutans, Veillonella, and Lactobacillus predominated in plaque over the lesions, whereas extracellular polysaccharide-producing streptococci other than S. mutans, as well as Actinomyces, were more abundant in plaque from sound surfaces. However, more lactobacilli and Actinomyces were recovered from pigmented lesions than from the unstained ones. These findings suggest that the microbial flora associated with black-pigmented lesions did not strongly differ from that observed over unstained caries lesions.

Actinomyces↗
PubMed1978

Chloramphenicol resistance in Streptococcus pneumoniae: enzymatic acetylation and possible plasmid linkage.

Clinical isolates of Streptococcus pneumoniae resistant to chloramphenicol were observed in France for the first time in 1973. During a 4-year survey, these strains were found to represent 6% of a total of 564 isolates of S. pneumoniae in a general hospital and to belong to 13 different serotypes. One such strain, referred to as BM 6001, was shown to inactivate chloramphenicol, and the process was found to be inducible. The inactivated products were demonstrated to be O-acetoxy esters of chloramphenicol. The synthesis of an inducible chloramphenicol acetyltransferase was shown to be responsible for the inactivation of the drug. The resistant strain was able to transfer the chloramphenicol marker by transformation to competent strains of pneumococci at a frequency of 1% of that observed for control chromosomal markers. The loss of resistance was enhanced by ethidium bromide treatment, but no chloramphenicol-resistant mutant was isolated by mutagenesis of a "cured" clone or naturally susceptible isolates. All attempts to isolate plasmid deoxyribonucleic acid as covalently closed circular molecules from strain BM 6001 have been unsuccessful, but epidemiological evidence and the fact that the genes specifying chloramphenicol acetyltransferase synthesis are usually located on plasmids suggest that this marker may be plasmid-borne in S. pneumoniae.

Acetylation↗