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Biomedical subjects

V Fontaine

Publications and source records attributed to V Fontaine.

28 records · Page 2Linked to original sources
PubMed1996

Simplified ganglioside composition of photoreceptors compared to other retinal neurons.

PURPOSE: The quantitative and qualitative ganglioside composition of retinal photoreceptor cells is unknown. The aim of this study was to analyze the lipid, especially ganglioside, make-up of photoreceptors compared to other retinal cells. METHODS: Retinas from adult normal rats were mechanically separated into outer (photoreceptors) and inner (other retinal neurons and glia) halves be planar vibratome sectioning. Total lipids were extracted, and each fraction (neural, phospholipids, and glycosphingolipids) was eluted sequentially by column chromatography and quantitated through high-performance thin layer chromatogram analysis. Similar analyses were performed on entire retinas from adult normal rats, adult dystrophic rats lacking photoreceptors (RCS-rdy-p+ strain), and isolated photoreceptor outer segments. RESULTS: Whereas phospholipids were distributed equally between the two halves, inner retina contained significantly more cholesterol (68% total) and gangliosides (74% total) than outer retina on a unit protein basis. The distribution on a percent molar basis of specific gangliosides also was significantly different between the two halves: Outer retina was dominated by GD3 (45% total ganglioside) and contained only trace amounts (<4%) of complex species (GT1b and GQ1b); inner retina was more typical of mature brain tissue exhibiting substantial amounts (approximately 25%) of more complex species. These data were supported by lipid compositional analyses of mutant photoreceptor-less retina. However, isolated outer segments resembled whole retina in containing higher levels of complex gangliosides. CONCLUSIONS: These data indicate that, compared to other central nervous system-derived neurons, photoreceptor cell body membranes exhibit a highly unusual simplified ganglioside composition. Such an unusual neuronal lipid composition may reflect structural adaptations to their specialized function.

Animals↗
PubMed1994

Development of a human interleukin-6 receptor antagonist.

Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.

Animals↗
PubMed1994

Mutagenesis of the human interleukin-6 fourth predicted alpha-helix: involvement of the Arg168 in the binding site.

Random substitutions of amino acid 161-184 of human interleukin-6 (hIL-6) have been generated at the cDNA level using oligonucleotide-directed mutagenesis. Among the majority of the mutant proteins showing a reduced biological activity on murine hybridoma cells, only those having a substitution of Met161, Arg168, Arg179 or Met184, retained a tertiary structure similar to the IL-6 folding. These residues are thus probably involved in the interaction with the IL-6 receptor. However, the contacts established by Arg168 and Arg179 seem far more important for the biological activity. According to Bazan's model of cytokine folding and the receptor binding site on the fourth alpha-helix, based on growth hormone similarity, we propose that Arg168 and Arg179 are located on the exposed surface of this presumed helix.

Amino Acid Sequence↗
PubMed1993

Involvement of the Arg179 in the active site of human IL-6.

Three internal-amino acid deletions of amino acids 171-179 of human interleukin 6 (IL-6) were introduced at the cDNA level. While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation-specific anti-(IL-6) monoclonal antibodies showed that only mutant delta 177-179 does not present major alterations in folding. This finding, together with the observation that delta 177-179 is not able to compete with IL-6 for binding to the soluble human IL-6 receptor, suggested that some or all of these three residues participate to the composition of the receptor-binding site of human IL-6. A large number of single-amino-acid-substitution mutants were generated in residues 177, 178 and 179. Their detailed analysis revealed that Arg179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding. The only substitution which preserved some residual activity was the conservative Arg to Lys change. This demonstrates the absolute requirement for a positive charge in position 179 for the interaction of human IL-6 with its receptor.

Arginine↗
PubMed1991

Internal deletions of amino acids 29-42 of human interleukin-6 (IL-6) differentially affect bioactivity and folding.

Internal deletions of the human interleukin-6 (IL-6) cDNA have been generated in the region encoding residues 29 to 42. Mutant proteins were produced by in vitro transcription-translation or in Escherichia coli and tested for their biological activity using the hybridoma growth factor (HGF) assay or a transcriptional activation assay on human hepatoma cells. The folding of the mutants was also checked by immunoprecipitation with conformation-specific monoclonal antibodies. The results show that only residues 29 to 34 are crucial for IL-6 activity and that the first two amino acids are probably involved in the definition of the IL-6 active site.

Amino Acid Sequence↗
PubMed1991

Internal deletions in human interleukin-6: structure-function analysis.

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector.

Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.

Amino Acid Sequence↗
PubMed1988

In-vitro activity of fleroxacin against urinary tract and genital tract pathogens.

The activity of fleroxacin was compared to those of nalidixic acid, ampicillin, trimethoprim-sulphamethoxazole, tetracycline and penicillin against a total of 452 strains isolated from urinary tract and genital tract infections. These included strains resistant to gentamicin, methicillin-resistant Staphylococcus aureus and beta-lactamase producing strains. MICs were determined by a standard agar dilution procedure using Mueller-Hinton agar. Fleroxacin inhibited 90% of Escherichia coli, Klebsiella pneumoniae, Proteus spp., and Enterobacter spp. at less than or equal to 1 mg/l. All the gentamicin-resistant Enterobacteriaceae strains were highly susceptible to fleroxacin (MIC90 less than or equal to 0.5 mg/l). Against Pseudomonas, fleroxacin was the most active agent tested (MIC90 2 mg/l). All nalidixic acid-resistant strains were susceptible to fleroxacin (MIC90 less than or equal to 1 mg/l). The activity of fleroxacin was similar against methicillin-resistant and methicillin-susceptible strains of Staph. aureus (MIC90 less than or equal to 0.5 mg/l). Against Neisseria gonorrhoeae, fleroxacin (MIC90 0.12 mg/l) was as active as penicillin and tetracycline. However against N. gonorrhoeae strains producing beta-lactamase, fleroxacin (MIC90 0.5 mg/l) was at least twice as active as tetracycline. Fleroxacin is a potent quinolone with in-vitro activity against a variety of resistant bacterial species.

Ampicillin↗
PubMedDate not supplied

Ganglioside and neurotrophic growth factor interactions in retinal neuronal and glial cells.

Ganglioside (GG) and neurotrophic growth factor (GF) interactions in retinal neuronal and glial cells have been very little studied. Rat retinas were mechanically separated into outer (photoreceptor or PR) and inner (other neurons, IR) halves by planar vibratome sectioning and retinal Müller glial (RMG) cells were isolated and cultured according to previously published methods. The distribution on a percent molar basis of individual GG was different between the two halves: PR were dominated by GD3 (48% total GG) and contained only trace amounts (< 4%) of complex species (GT1b, GQ); IR was more typical of mature brain tissue, exhibiting substantial amounts (approximately 25%) of more complex GG. The GG profile of RMG cells was also simple, dominated by GM3 (60%) and GD1a (20%). A single addition to the medium of 500 pM bFGF or EGF for 48 hr to cultured RMG cells led to significant increases in total GG levels of 30-40%. Such treatments by both growth factors induced increases in GM3, whereas longer exposure (96 hr) of confluent RMG to these factors additionally stimulated synthesis of more complex GG. Incubations of RMG with [3H]-glucosamine showed that GG synthesis was 2-fold stimulated by growth factors. We also tested the effect of GM3 on one of the bFGF receptor transduction pathways, namely PI-3 kinase activation. To our knowledge these data constitute the first demonstration of neurotrophic factor stimulation of GG levels in cells of CNS in vitro. Such complex interactions may have particularly important consequences for neural physiopathology.

Animals↗