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C Philippe

Publications and source records attributed to C Philippe.

At least 55 records · Page 3Linked to original sources
PubMed1995

A high-resolution interval map of the q21 region of the human X chromosome.

In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome (Philippe et al., Genomics 17: 147-152, 1993). Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region.

Chromosome Mapping↗
PubMed1995

Molecular dissection of the pseudoknot governing the translational regulation of Escherichia coli ribosomal protein S15.

The ribosomal protein S15 controls its own translation by binding to a mRNA region overlapping the ribosome binding site. That region of the mRNA can fold in two mutually exclusive conformations that are in dynamic equilibrium: a structure with two hairpins and a pseudoknot. A mutational analysis provided evidence for the existence and requirement of the pseudoknot for translational control in vivo and S15 recognition in vitro. In this study, we used chemical probing to analyze the structural consequences of mutations and their effect on the stem-loop/pseudoknot equilibrium. Interactions between S15 and the pseudoknot structure were further investigated by footprinting experiments. These data, combined with computer modelling and the previously published data on S15 binding and in vivo control, provide important clues on pseudoknot formation and S15 recognition. An unexpected result is that the relevant control element, here the pseudoknot form, can exist in a variety of topologically equivalent structures recognizable and shapable by S15. S15 sits on the deep groove of the co-axial stack and makes contacts with both stems, shielding the bridging adenine. The only specific sequence determinants are found in the helix common to the pseudoknot and the hairpin structures.

Base Sequence↗
PubMed1995

Role of CCK in the regulation of secretion and adaptation in the pig pancreas.

The effect of cholecystokinin (CCK) on the pancreas was investigated in the pig in two experiments. Fifteen pigs were fed a diet containing 17 or 48% protein with or without MK329 (4.5 mg per meal). MK329 enhanced the postprandial peak of plasma CCK during the first 30 min, but pancreas adaptation to high protein was not affected. Sixteen pigs were divided into two groups: 12 pigs were infused with CCK-8 + secretin for 1 h and four pigs received a standard meal. In both groups, pancreatic secretion tests were performed under infusion of the vehicle alone or with MK329. After CCK + secretin, MK329 (65-500 micrograms/kg/h) did not alter CCK plasma levels and reduced the early pancreatic protein response by about 30%. Enzyme outputs in pancreatic juice were modestly affected by MK329. After the meal, MK329 (500 micrograms/kg/h) doubled the postprandial peak of plasma CCK and lowered the pancreatic protein output by 35-40% for the first 30 min. We suggest that (a) pancreatic adaptation to high dietary protein is not mediated via CCK-A receptors and (b) the stimulation of pancreatic protein secretion by a meal or by exogenous CCK-8 is mediated partly by CCK-A receptors.

Adaptation, Physiological↗
PubMed1995

Monosomy 21q: two cases of del(21q) and review of the literature.

We report on two cases of partial monosomy 21 and review cases with a partial or an apparently full monosomy 21. In situ hybridization and/or molecular studies appear to be necessary tools to study imbalance in such a small chromosome and to perform further genotype-phenotype correlations. The segregation mode in cases with a translocation is adjacent 1, adjacent 2, and 3:1 in about 1/4, 1/4 and 1/2 of the cases, respectively.

Child, Preschool↗
PubMed1995

Mesangial cell-derived interleukin-10 modulates mesangial cell response to lipopolysaccharide.

Interleukin (IL)-10 is a novel cytokine produced by a variety of cells, including monocytes/macrophages, upon exposure to lipopolysaccharide (LPS). Recent observations indicate that, in turn, IL-10 exerts suppressive effects on macrophage response to LPS. Because mesangial cells are also a target for LPS, we have examined the potential role of IL-10 in the regulation of mesangial cell response to LPS. To this aim, we have studied the synthesis and the autocrine/paracrine function of IL-10 in cultured mouse mesangial cells. IL-10 mRNA expression and IL-10 protein secretion were determined by a reverse transcription polymerase chain reaction technique and a specific enzyme-linked immunosorbent assay, respectively. No IL-10 mRNA expression was detectable in unactivated cells. LPS induced IL-10 mRNA expression in a dose-dependent fashion (1 to 100 micrograms/ml). In addition, LPS induced IL-10 protein release that was both dose dependent (1 to 100 micrograms/ml) and time dependent (24 to 72 hours). We have also studied the effect of IL-10 on the production of inflammatory mediators by LPS-activated mouse mesangial cells. Whereas recombinant IL-10 inhibited the generation of tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta by 90 and 60%, respectively, it did not affect the formation of nitric oxide-derived nitrite (NO2-) and nitrate (NO3-). As shown by the use of anti-IL-10 monoclonal antibody, endogenously produced IL-10 affected the generation of TNF-alpha but neither that of IL-1 beta nor that of NO2- and NO3-. Finally, we have examined whether conditions known to also reduce the generation of TNF-alpha modified the expression of IL-10. Of all the conditions tested, only the addition of desferrioxamine and transforming growth factor-beta were found to increase IL-10 release. Together, these data demonstrate that mesangial cell-derived IL-10 has important regulatory effects on the inflammatory response of these cells to LPS because of its capacity to blunt TNF-alpha generation.

Animals↗
PubMed1994

Additional case of female monozygotic twins discordant for the clinical manifestations of Duchenne muscular dystrophy due to opposite X-chromosome inactivation.

A pair of female monozygotic (MZ) twins, heterozygous carriers for a deletion in the DMD gene and discordant for the clinical manifestations of Duchenne muscular dystrophy, were analyzed by molecular studies, in situ hybridization, and methylation pattern of X chromosomes to search for opposite X inactivation as an explanation of their clinical discordance. Results in lymphocytes and skin fibroblast cell lines suggest a partial mirror inactivation with the normal X chromosome preferentially active in the unaffected twin, and the maternal deleted X chromosome preferentially active in the affected twin. A review shows that MZ female twins discordant for X-linked diseases are not uncommon. Twinning and X inactivation may be interrelated and could explain the female twins discordant for X-linked traits.

Base Sequence↗
PubMed1994

Structural elements of rps0 mRNA involved in the modulation of translational initiation and regulation of E. coli ribosomal protein S15.

Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state. In this study, we investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA(fMet) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex. The results were compared to in vivo expression and repression rates. The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.

Base Sequence↗
PubMed1994

Hypomagnesemia with secondary hypocalcemia in a female with balanced X;9 translocation: mapping of the Xp22 chromosome breakpoint.

Magnesium-dependent hypocalcaemia (HSH), a rare inherited disease, is caused by selective disorders of magnesium absorption. Both X-linked and autosomal recessive modes of inheritance have been reported for HSH; this suggests a genetically heterogeneous condition. A balanced de novo t(X;9)(p22;q12) translocation has been reported in a female manifesting hypomagnesemia with secondary hypocalcemia. In a lymphoblastoid cell line, derived from this patient, the normal X chromosome is preferentially inactivated, suggesting that the patient's phenotype is caused by disruption of an HSH gene in Xp22. In an attempt to define more precisely the position of the X breakpoint, we have constructed a hybrid cell line retaining the der(X)(Xqter-Xp22.2::9q12-9qter) in the absence of the der(9) and the normal X chromosome. Southern blot analysis of this hybrid and in situ hybridization on metaphase chromosomes have localized the breakpoint between DXS16 and the cluster (DXS207, DXS43), in Xp22.2. Thus, if a gene involved in HSH residues at or near the translocation breakpoint, our findings should greatly facilitate its isolation.

Blotting, Southern↗
PubMed1994

Chromosome rearrangements and human gene mapping.

The successful mapping of numerous mendelian disorders by chromosome rearrangements turned out to be a key method for positional location of disease genes. We present some personal observations and comments on the interest of cytogenetic studies in human gene mapping.

Child↗
PubMed1994

Fish oil supplementation and essential fatty acid deficiency reduce nitric oxide synthesis by rat macrophages.

Both fish oil-derived omega-3 polyunsaturated fatty acid (omega 3 PUFA) supplementation and essential fatty acid (EFA) deficiency have been shown to exert anti-inflammatory effects and, hence, to ameliorate immune-mediated glomerulonephritis. The mechanisms underlying these effects include alterations in the production of eicosanoids, cytokines (that is, tumor necrosis factor, TNF-alpha) and reactive oxygen species by blood borne cells. Because, in addition to these mediators nitric oxide (NO) is also implicated in glomerular injury, we have examined if both diets affected macrophage NO production as well. Rats were fed a standard chow, an omega 3 PUFA-supplemented diet, or an EFA-deficient diet for six weeks before resident peritoneal macrophages were isolated. These cells were exposed to lipopolysaccharide (LPS) and the NO metabolite, nitrite (NO2-), was measured in the medium using the Griess reagent. Release of NO2- was enhanced by LPS in a dose-dependent manner. With 10 ng/ml LPS challenge, NO2- release was reduced by 37% and 57% by omega 3 PUFA supplementation and EFA deficiency, respectively. NO2- returned to control levels two weeks after the end of diet. Macrophage production of TNF-alpha responded in a similar manner. Diet-induced reduction of NO2- release was neither attributable to a reduction of inducible NO synthase mRNA levels as shown by Northern blot analysis, nor to an increased competition of NO synthase and arginase for the substrate (L-arginine). Indeed, arginase activity of macrophages was even slightly reduced by both omega 3 PUFA-supplemented diet and EFA-deficient diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1994

Cloning and characterization of the human choroideremia gene.

Positional cloning has previously resulted in the identification of a gene which is disrupted by deletions in patients with the classic choroideremia (CHM) phenotype. More subtle mutations had been identified in 4 exons of the 3' portion but not elsewhere in the CHM gene. We have now isolated and characterized the complete open reading frame of the CHM gene and determined its exon-intron structure. The CHM gene encodes a protein of 653 amino acids, which is highly homologous to the mouse and rat CHM proteins, and, to a slightly lesser extent, to the human CHM-like (CHML) protein. The open reading frame (ORF) of the human CHM gene consists of 15 exons, spanning at least 150 kb of Xq21.2, and it is possible that there is an additional exon corresponding to the 5' non-coding region of the gene. Cloning of the 5' end of the CHM gene and the elucidation of its intron-exon structure enabled us to localize the X-chromosomal breakpoint in a CHM female with an X;7 translocation between exons 3 and 4.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli.

Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site. By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.

Bacteriophage lambda↗
PubMed1993

Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site.

From genetic and biochemical evidence, we previously proposed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops. Here, we use "toeprint" experiments with Moloney murine leukemia virus reverse transcriptase to analyze the effect of S15 on the formation of the ternary mRNA-30S-tRNA(fMet) complex. We show that the binding of the 30S subunit on the mRNA stops reverse transcriptase near position +10, corresponding to the 3' terminus of the pseudoknot, most likely by stabilizing the pseudoknot conformation. Furthermore, S15 is found to stabilize the binary 30S-mRNA complex. When the ternary 30S-mRNA-tRNA(fMet) complex is formed, a toeprint is observed at position +17. This toeprint progressively disappears when the ternary complex is formed in the presence of increasing concentrations of S15, while a shift from position +17 to position +10 is observed. Beside, RNase T1 footprinting experiments reveal the simultaneous binding of S15 and 30S subunit on the mRNA. Otherwise, we show by filter binding assays that initiator tRNA remains bound to the 30S subunit even in the presence of S15. Our results indicate that S15 prevents the formation of a functional ternary 30S-mRNA-tRNA(fMet) complex, the ribosome being trapped in a preternary 30S-mRNA-tRNA(fMet) complex.

Base Sequence↗
PubMed1993

Physical mapping of DNA markers in the q13-q22 region of the human X chromosome.

DNA probe screening of somatic cell hybrids derived from females with X;autosome translocations has enabled us to define eight new breakpoints within the Xq13-q22 region. Together with other X-chromosome rearrangements that have been described earlier, these breakpoints subdivide the Xq21-q22 region into 20 intervals. Our panel refines the physical assignment of 40 probes in the Xq21-q22 segment. Thus, these X-chromosome rearrangements are useful tools for ordering X-linked markers and genes on the proximal long arm of the human X chromosome.

Amenorrhea↗
PubMed1993

Rearrangements between irradiated chromosomes in three-species radiation hybrid cell lines revealed by two-color in situ hybridization.

A human-hamster hybrid cell line containing only the human X chromosome (GM06318B) was exposed to 6,000-7,000 rad of X-rays and fused with a mouse cell line (CL1D,TK-). Three radiation hybrids, LXKC40, LXKC50, and LXKC56, were selected among 39 independent clones containing human material. Two-color in situ hybridization with total genomic DNA probes (cot1 human DNA and hamster total genomic DNA) was used to analyse the irradiated chromosome rearrangements. With this three-species model system (human-hamster-mouse) and the chromosome painting process it was possible to determine the origin of each chromosomal fragment in metaphase and interphase. The results obtained indicate preferential rearrangement between irradiated human and hamster chromosomes. Whole, apparently intact hamster chromosomes were observed in all the mitoses. We suggest that these chromosomes could be neoformated from random fragments after irradiation. Hamster and human "minichromosomes" were also detected. While the integration of human material into the mouse genome was exceptional, the integration of hamster material into mouse chromosomes was more frequent. During interphase the irradiated chromosome domains were often at the periphery of the nucleus. Irradiated material protruded at the periphery of the nuclei. Micronuclei containing hamster material were detected in the vicinity of these protrusions.

Animals↗
PubMed1993

Increased expression and occupancy of receptors for tumour necrosis factor on blood monocytes from tuberculosis patients.

Blood monocytes from tuberculosis patients release high amounts of tumour necrosis factor-alpha (TNF-alpha). Because the biological efficiency of TNF-alpha would depend on the expression of TNF-alpha receptors on target cells, we thought to analyse the capacity of blood monocytes from a group of patients with pulmonary tuberculosis to bind 125I-TNF-alpha. We report a slight but not significant enhancement in specific binding of 125I-TNF-alpha on monocytes of 15 consecutively studied patients compared with 10 controls. Per cent cell surface bound and internalized 125I-TNF-alpha was identical in the two groups. To evaluate the receptor occupancy by endogenously generated TNF-alpha, similar experiments were performed after cell exposure to low-pH glycine buffer. Under these conditions, specific binding of 125I-TNF-alpha was significantly higher on tuberculosis monocytes compared with control monocytes. Moreover, the occupancy of TNF-alpha receptors by endogenously generated TNF-alpha that was found to be significantly higher on tuberculosis monocytes than on control monocytes, was directly related to the enhanced capacity of mononuclear cells to generate TNF-alpha in vitro. It normalized after 3 months of antituberculous therapy. Scatchard analysis of the binding data revealed that tuberculosis infection caused a significant increase in high affinity 125I-TNF-alpha binding to monocytes without any significant change in the dissociation constant. Collectively, these results indicate an up-regulation of TNF-alpha generation and binding to blood monocytes in patients with pulmonary tuberculosis. They provide support to the hypothesis that TNF-alpha is of critical importance in the pathogenesis of this infection.

Adult↗
PubMed1993

Cytokine formation within rat glomeruli during experimental endotoxemia.

Increasing evidence supports a role of cytokines, tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and IL-6 in the development of endotoxin-induced acute renal failure. Several activities of these cytokines require a local rather than a systemic production and function. Thus, this study investigates the chronology of cytokine expression in glomeruli isolated from normal rats or rats given iv lipopolysaccharide injections. Detectable levels of TNF alpha could be found in glomeruli isolated from normal rats as assessed by L-929 fibroblast lytic assay and ELISA. Glomeruli isolated from rats given lipopolysaccharide transiently released increased amounts of TNF alpha in relation to the dose of lipopolysaccharide (10 to 500 micrograms/kg body wt) and the lag period between lipopolysaccharide injection and glomerular isolation (20 to 120 min). TNF alpha was released in similar amounts by glomeruli from normal rats that were exposed in vitro to lipopolysaccharide challenge (0.01 to 10 micrograms/mL), indicating that lipopolysaccharide had direct effects on the release of TNF alpha from glomerular cells. These cells consisted mainly of resident cells because reduction of glomerular infiltration by bone marrow-derived cells after the irradiation of normal rats did not affect TNF alpha release. Glomerular IL-1 and IL-6 production was evaluated by specific bioassays under identical conditions. No IL-1 activity could be detected in the medium or within the glomerular cells at any time within 120 min after lipopolysaccharide injection. By contrast, glomerular IL-6 production was induced after lipopolysaccharide challenge both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗