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Biomedical subjects

M Rivière

Publications and source records attributed to M Rivière.

At least 19 recordsLinked to original sources
PubMed2005

Chromosome evolution of MMU16 and RNO11: conserved synteny associated with gene order rearrangements explicable by intrachromosomal recombinations and neocentromere emergence.

Comparative mapping between the rat and mouse genomes has shown that some chromosomes are entirely or almost entirely conserved with respect to gene content. Such is the case of rat chromosome 11 (RNO11) and mouse chromosome 16 (MMU16). We determined to what extent such an extensive conservation of synteny is associated with a conserved gene order. Therefore, we regionally localized several genes on RNO11. The comparison of the gene map of RNO11 and MMU16 unambiguously shows that the gene order has not been conserved in the Murinae lineage, thereby implying the occurrence of intrachromosomal evolutionary rearrangements. The transition from one chromosome configuration to the other one can be explained either by two intrachromosomal recombinations or by a single intrachromosomal recombination accompanied by neocentromere emergence.

Animals↗
PubMed2003

Importance of the lid and cap domains for the catalytic activity of gastric lipases.

Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

Antibodies, Monoclonal↗
PubMed2001

Genetic identification of multiple susceptibility genes involved in the development of endometrial carcinoma in a rat model.

There are clear indications that inheritance plays an essential role in certain cases of human endometrial cancer, and there are at least 2 forms of early-onset heritable endometrial adenocarcinomas (EACs). Females of the BDII inbred rat strain are known to be genetically predisposed to endometrial carcinoma, and we have performed a genetic analysis of susceptibility to endometrial cancer in this strain. F(2) populations were generated by crossing BDII females with males from 2 different strains with a low incidence of EAC, and the occurrence of endometrial cancer was studied. Three chromosome regions associated to EAC susceptibility were identified, and the susceptibility genes in these regions were designated Ecs1, Ecs2 and Ecs3. Our results indicate that the genes affecting susceptibility to EAC are different in the 2 crosses, suggesting that the genes behind the susceptibility in BDII animals may interact with different genes in different genetic backgrounds.

Adenocarcinoma↗
PubMed2001

Crystallization and preliminary crystallographic study of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp).

The plant phospholipase D (PLD) is considered to be a key enzyme involved in various physiological processes such as signal transduction and membrane metabolism. Crystals of the PLD protein from Vigna unguiculata have been produced from the recombinant 768 amino-acid protein. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 157.7, b = 65.6, c = 90.2 A, beta = 111.5 degrees. There is one molecule in the asymmetric unit. Frozen crystals diffract to at least 1.94 A resolution using synchrotron radiation. A search for heavy-atom derivatives using ytterbium and tungstate is currently under way in order to solve the three-dimensional structure.

Crystallization↗
PubMed2001

Mapping of the rat glypican genes.

The glypicans compose a family of glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycans that play a role in the control of cell division and growth regulation. So far, six members (GPC1-6) of this family are known in vertebrates. The rat glypican gene 3 (Gpc3) was previously assigned to chromosome Xq36 (Shen et al., 1997). Using standard and radiation cell hybrids, we localized the five other rat glypican genes.

Animals↗
PubMed2000

Identification of KLF13 and KLF14 (SP6), novel members of the SP/XKLF transcription factor family.

Using the sequence of the SP1 zinc-finger DNA-binding domain as a probe to screen a mouse EST database, we identified two novel members of the SP/XKLF transcription factor family, KLF13 and KLF14. The mouse Klf13 cDNA (1310 bp in length) contains a single open reading frame of 288 amino acids with a DNA-binding domain closely related to that of the human RFLAT-1 protein and a putative transactivator N-terminal domain rich in proline and alanine residues. The mouse Klf13 gene seems to be the homologue of the human RFLAT1 gene. The mouse Klf14 sequence is homologous to a human genomic sequence from chromosome 17 that is believed to code for a protein with three zinc fingers at the end of its C-terminal domain. Using reverse transcription-polymerase chain reaction, we showed ubiquitous expression of Klf13 and Klf14 in adult mice. A third member of this family was also identified in a human EST database; this sequence was found to be identical to KLF11 (TIEG2), recently identified by Cook et al. (1998, J. Biol. Chem. 273: 25929-25936). The corresponding mouse cDNA was isolated and sequenced. The three genes were localized in the human and the rat: chromosomes 15 (human KLF13), 17q21.3-q22 (human KLF14; HGMW-approved symbol SP6), and 2p25 (human KLF11) and chromosomes 1q31-q32 (rat Klf13), 10q31-q32.1 (rat Klf14) (SP6), and 6q16-q21 (rat Klf11).

Amino Acid Sequence↗
PubMed2000

Lipoglycans are putative ligands for the human pulmonary surfactant protein A attachment to mycobacteria. Critical role of the lipids for lectin-carbohydrate recognition.

The human pulmonary surfactant protein A (hSP-A) has been implicated in the early capture and phagocytosis of the pathogenic Mycobacterium tuberculosis by alveolar macrophages. In this report, we examined the interaction of alveolar proteinosis patient hSP-A with Mycobacterium bovis BCG, the vaccinating strain, as a model of pathogenic mycobacteria, and Mycobacterium smegmatis, a nonpathogenic strain. We found that hSP-A binds to the surface of M. bovis BCG, but also to a slightly lesser extent, to M. smegmatis, indicating that hSP-A does not discriminate between virulent and nonpathogenic strains. Among the various glycoconjugates isolated from the mycobacterial envelope, we found that the best ligands are the two major lipoglycans: the mannosylated lipoarabinomannan (ManLAM) and the lipomannan. In contrast, the mannose-capped arabinomannan, structurally close to the ManLAM, as well as the LAMs from the non pathogenic M. smegmatis are poorly recognized by hSP-A. These results clearly show that the presence of both the terminal mannose residues and the phophatidyl-myo-inositol anchor are necessary to achieve the highest binding affinity. Selective removal of either the terminal mannose or the acyl residues esterifying the glycerol moiety of the ManLAM abrogates the interaction with hSP-A, further supporting the notion that the hSP-A recognition of the carbohydrate epitopes of the lipoglycans is dependent of the presence of the fatty acids.

Carbohydrate Conformation↗
PubMed2000

Localization of 54 rat genes, and definition of new synteny groups conserved in the human and the rat.

In order to improve the rat gene map and comparative mapping with the human and the mouse, we determined the chromosome localization of 54 rat genes. Most genes encode transcription factors or other regulatory proteins of cancer relevance. The human homologs of four genes were also assigned to their respective chromosome. These data generated anchor points between the recently established radiation hybrid maps and the genetic and cytogenetic maps. They improve comparative mapping between the rat, the mouse, and the human gene maps, and in particular they disclose four new synteny groups conserved in the rat and the human. These new localizations should also be useful for the identification of genes involved in the control of quantitative traits such as cancer susceptibility or diabetes.

Animals↗
PubMed2000

Digestive lipases: from three-dimensional structure to physiology.

Human gastric lipase (HGL) is a lipolytic enzyme that is secreted by the chief cells located in the fundic part of the stomach. HGL plays an important role in lipid digestion, since it promotes the subsequent hydrolytic action of pancreatic lipase in duodenal lumen. Physiological studies have shown that HGL is able of acting not only in the highly acid stomach environment but also in the duodenum in synergy with human pancreatic lipase (HPL). Recombinant HGL (r-HGL) was expressed in the baculovirus/insect cell system in the form of an active protein with a molecular mass of 45 kDa. The specific activities of r-HGL were found to be similar to that of the native enzyme when tested on various triacylglycerol (TG) substrates. The 3-D structure of r-HGL was the first solved within the mammalian acid lipase family. This globular enzyme (379 residues) shows a new feature, different from the other known lipases structures, which consists of a core domain having the alpha/beta hydrolase fold and a cap domain including a putative 'lid' of 30 residues covering the active site of the lipase (closed conformation). HPL is the major lipolytic enzyme involved in the digestion of dietary TG. HPL is a 50 kDa glycoprotein which is directly secreted as an active enzyme. HPL was the first mammalian lipase to be solved structurally, and it revealed the presence of two structural domains: a large N-terminal domain (residues 1-336) and a smaller C-terminal domain (residues 337-449). The large N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site. A surface loop called the lid domain (C237-C261) covers the active site in the closed conformation of the lipase. The 3-D structure of the lipase-procolipase complex illustrates how the procolipase might anchor the lipase at the interface in the presence of bile salts: procolipase binds to the C-terminal domain of HPL and exposes the hydrophobic tips of its fingers at the opposite site of its lipase-binding domain. These hydrophobic tips help to bring N-terminal domain into close conformation with the interface where the opening of the lid domain probably occurs. As a result of all these conformational changes, the open lid and the extremities of the procolipase form an impressive continuous hydrophobic plateau, extending over more than 50 A. This surface might able to interact strongly with a lipid-water interface. The biochemical, histochemical and clinical studies as well as the 3-D structures obtained will be a great help for a better understanding of the structure-function relationships of digestive lipases.

Amino Acid Sequence↗
PubMed2000

Functional expression in insect cells, one-step purification and characterization of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp).

Phospholipase D (PLD) is an important enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. In this study, large amounts of a recombinant plant PLD alpha were secreted into the culture medium of baculovirus-infected insect cells and purified to homogeneity in the form of a fully active enzyme. The transient production of recombinant PLD alpha yielded a protein (rPLD alpha a, 88 kDa) together with a shorter form (rPLD alpha b, 87 kDa), which accumulated in the medium. N-Terminal amino acid sequencing of the rPLD alpha a and rPLD alpha b showed that rPLD alpha b resulted from proteolytic cleavage at Gly8-Ile9. Immunoblotting showed that both rPLD alpha a and rPLD alpha b are recognized by a polyclonal antibody previously raised against native soybean PLD alpha. One-step calcium-dependent octyl-Sepharose chromatography was used to obtain the two highly purified forms of rPLD alpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry. The N-terminal region of PLD alpha is homologous with the C2 domains which are present in a number of enzymes known to be involved in signal transduction and/or phospholipid metabolism. The truncated rPLD alpha b lacks the first acidic amino acid in its N-terminus, which is probably involved in the calcium binding site. The rPLD alpha b was thus easily eluted from the octyl-Sepharose column by decreasing the calcium concentration of the buffer from 50 to 30 mM, whereas, the rPLD alpha a was eluted after chelating calcium ions with EDTA. The purified rPLD alpha yield reached a level of 10 mg per liter of serum-free culture medium. The availability of baculovirus-derived rPLD alpha constitutes a valuable source of enzyme for future crystallographic studies to determine its three-dimensional structure.

Amino Acid Sequence↗
PubMed1999

Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern.

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.

Amino Acid Sequence↗
PubMed1999

Crystal structure of human gastric lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest.

Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We report here the structure of recombinant human gastric lipase at 3.0-A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain belonging to the alpha/beta hydrolase-fold family and a "cap" domain, which is analogous to that present in serine carboxypeptidases. It possesses a classical catalytic triad (Ser-153, His-353, Asp-324) and an oxyanion hole (NH groups of Gln-154 and Leu-67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under a segment consisting of 30 residues, which can be defined as a lid and belonging to the cap domain. The displacement of the lid is necessary for the substrates to have access to Ser-153. A phosphonate inhibitor was positioned in the active site that clearly suggests the location of the hydrophobic substrate binding site. The lysosomal acid lipase was modeled by homology, and possible explanations for some previously reported mutations leading to the cholesterol ester storage disease are given based on the present model.

Amino Acid Sequence↗
PubMed1999

The cysteine residues of recombinant human gastric lipase.

Recombinant human gastric lipase (rHGL) and three of its cysteine mutants (cysteine 227, 236, and 244 substitued for threonine or serine) were expressed in the baculovirus/insect cell system and purified to homogeneity by performing a two-step procedure. Substituting Ser for Cys 227 and Cys 236 resulted in mutant lipases with a significantly lower level of activity (30% and 22%, respectively) on a short chain triglyceride (tribuyrin) substrate, while the mutation at position 244 only slightly reduced the activity. Using 4, 4'-dithiopyridine (4-PDS) as a sulfhydryl reagent on the above mutants, it was possible to clearly identify the single sulfhydryl residue at position 244 and consequently, the disulfide bridge at position 227-236. No potential disulfide bridges were formed during the protein folding between cysteines 227-244 or between cysteines 236-244, as thought to occur in the case of rabbit gastric lipase (RGL). The present results are consistent with the recently determined 3D-structure of rHGL.

Amino Acid Substitution↗
PubMed1999

Rat Chromosome 2: assignment of the genes encoding cyclin B1, interleukin 6 signal transducer, and proprotein convertase 1 to the Mcs1-containing region and identification of new microsatellite markers.

The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus. We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1. We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA. These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers. A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23.

Animals↗
PubMed1999

Site-directed removal of N-glycosylation sites in human gastric lipase.

Human gastric lipase (HGL) is a highly glycosylated protein, as glycan chains account for about 15% of the molecular mass of the native HGL. Four potential N-glycosylation consensus sites (Asn15, 80, 252 and 308) can be identified from the HGL amino acid sequence. We studied the functional role of the individual N-linked oligosaccharide chains by removing one by one all the N-glycosylation sites, via Ala residue replacement by site-directed mutagenesis of Ser and Thr residues from the consensus sequences Asn-X-Ser/Thr. Mutagenized cDNA constructs were heterologously expressed in the baculovirus/insect cell system. Removal of oligosaccharides either at Asn15, 80 or 252 was found to have no significant influence on the enzymatic activity measured in vitro. However, the absence of glycosylation at Asn308, as well as a total deglycosylation, reduced the specific enzymatic activity of recombinant HGL (r-HGL), measured on short- and long-chain triglycerides, to about 50% of normal values. Furthermore, biosynthesis and secretion of r-HGL markedly dropped when all four potential glycosylation sites were mutated. The kinetics of the interfacial adsorption of r-HGL and the completely deglycosylated r-HGL (four-site mutant) were found to be identical when recording the changes with time of the surface pressure either at the air-water interface or in the presence of an egg phosphatidylcholine (PtdCho) monomolecular film spread at various initial surface pressures. This indicates that both recombinant HGLs are identical, as far as recognition of phospholipid film and adsorption on PtdCho are concerned. The N-glycosylation of HGL may contribute to the enzyme stability in the stomach, as under acidic conditions the degradation by pepsin of the unglycosylated r-HGL is increased.

Animals↗