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P Gros

Publications and source records attributed to P Gros.

At least 91 records · Page 5Linked to original sources
PubMed1999

Physical delineation of a 700-kb region overlapping the Looptail mutation on mouse chromosome 1.

The mouse looptail (Lp) mutation is an established model for neural tube defects with homozygous Lp embryos showing an open neural tube from the caudal midbrain to the tip of the tail. Heterozygous Lp mice are characterized by a "looped-tail" and wobbly head movements. The Lp gene has been mapped to a 0.6-cM interval on mouse chromosome 1 delineated by two clusters of markers, Fcer1gamma/Usf1/D1Mit113/D1Wsu1 on the proximal side and Fcer1alpha/Spna1/D1Mit149 distally. In the present study, we have created a high-resolution physical map of the Lp genetic interval that is based on long-range restriction mapping by PFGE, fluorescence in situ hybridization analysis of interphase nuclei and extended chromatid fibers, and the assembly of a cloned contig. This contig consists of 25 independent and overlapping BAC clones and 3 YAC clones. The combined analysis indicates that the 0.6-cM genetic interval for Lp corresponds to a minimal physical interval of 700 kb that is delineated by D1Mit113 proximally (two crossovers) and Fcer1alpha distally (one crossover). The overall gene order and intergene distances for the region were determined to be D1Mit113-<150 kb-Nhlh1-250 kb-Atp1alpha2-280 kb-Fcer1alpha. Partial sequencing of BAC clones from the contig yielded 42 new STS markers for this region of mouse chromosome 1. Sequence analysis of the BAC clones and assignment of ESTs from the human transcript map to the cloned contig allowed the placement of four new transcription units within this region: Pc326, Kiaa0253, and Pea15 were positioned in the Nhlh1/Atp1alpha2 nonrecombinant interval, while Girk3 was located distal to Atp1alpha2.

Animals↗
PubMed1999

Bactericidal antibody recognition of meningococcal PorA by induced fit. Comparison of liganded and unliganded Fab structures.

MN12H2 is a bactericidal antibody directed against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. Binding of MN12H2 to PorA at the meningococcal surface activates the classical complement pathway resulting in bacterial lysis. We have determined the crystal structure of the unliganded MN12H2 Fab fragment in two different crystal forms and compared it with the structure of the Fab in complex with a P1.16-derived peptide. The unliganded Fabs have elbow bend angles of 155 degrees and 159 degrees, whereas the liganded Fab has a more closed elbow bend of 143 degrees. Substantial differences in quaternary and tertiary structure of the antigen binding site are observed between the unliganded and liganded MN12H2 Fab structures that can be attributed to peptide binding. The variable light and heavy chain interface of the liganded Fab is twisted by a 5 degrees rotation along an axis approximately perpendicular to the plane of the interface. Hypervariable loops H1, H2, and framework loop FR-H3 follow this rotation. The hypervariable loop H3 undergoes conformational changes but remains closely linked to hypervariable loop L1. In contrast with the binding site expansion seen in other Fab-peptide structures, the MN12H2 binding site is narrowed upon peptide binding due to the formation of a "false floor" mediated by arginine residue 101 of the light chain. These results indicate that PorA epitope P1.16 of N. meningitidis is recognized by the complement-activating antibody MN12H2 through induced fit, allowing the formation of a highly complementary immune complex.

Antibodies, Bacterial↗
PubMed1999

The Lps locus: genetic regulation of host responses to bacterial lipopolysaccharide.

Lipopolysaccharide (LPS), an abundant glycolipid of the outer membrane of gram-negative bacteria, is able to provoke a generalized proinflammatory response in the infected host. Genetic regulation of this trait has been localized to the Lps locus on mouse chromosome 4. Several inbred mouse strains, including C3H/HeJ, C57BL/10ScNCr and C57BL/10ScCr, bear mutations at the Lps locus (Lps(d)) that confer hyporesponsiveness to the immunostimulatory properties of LPS and susceptibility to overwhelming gram-negative bacterial infection. The phenotypic expression of Lps(d) is pleiotropic, affecting several cell types crucial to host defense, including the macrophage. By positional cloning, Toll-like receptor 4 (Tlr4), a transmembrane protein with a cytoplasmic domain that bears homology to the Interleukin-1 receptor, has been identified as the gene encoded by Lps. Tlr4 is a member of a novel gene family that participates in host defense against microbial infection in plants, invertebrates and mammals. Discovery of the molecular basis of the Lps mutation represents a significant advance in defining the fundamental mechanisms of cellular activation by LPS.

Animals↗
PubMed1999

Host resistance to infection: genetic control of lipopolysaccharide responsiveness by TOLL-like receptor genes.

Gram-negative bacterial lipopolysaccharide evokes a protective inflammatory response in the normal host. Through genetic analysis of mutant mice, the gene encoding Toll-like receptor 4 (Tlr4) was recently identified as a critical component of this host defense mechanism. Tlr4 is a member of an ancient gene family that regulates antimicrobial host defense in plants, invertebrates and mammals.

Animals↗
PubMed1999

[Short-term dynamics of intertidal microphytobenthos biomass. Mathematical model].

We formulate a deterministic mathematical model to describe the dynamics of the microphytobenthos of intertidal mudflats. It is 'minimal' because it only takes into account the essential processes governing the functioning of the system: the autotrophic production, the active upward and downward migrations of epipelic microalgae, the saturation of the mud surface by a biofilm of diatoms and the global net loss rates of biomass. According to the photic environment of the benthic diatoms inhabiting intertidal mudflats, and to their migration rhythm, the model is composed of two sub-systems of ordinary differential equations; they describe the simultaneous evolution of the biomass 'S' concentrated in the mud surface biofilm--the photic layer--and of the biomass 'F' diluted in the topmost centimetre of the mud--the aphotic layer. Qualitatively, the model solutions agree fairly well with the in situ observed dynamics of the S + F biomass. The study of the mathematical properties of the model, under some simplifying assumptions, shows the convergence of solutions to a stable cyclic equilibrium, whatever the frequencies of the physical synchronizers of the production. The sensitivity analysis reveals the necessity of a better knowledge of the processes of biomass losses, which so far are uncertain, and may further vary in space and time.

Biomass↗
PubMed1999

Consequence of Nramp1 deletion to Mycobacterium tuberculosis infection in mice.

129sv mice functionally deleted of the antimicrobial resistance gene, Nramp1, were found to be as resistant as wild-type mice to infection with the virulent H37Rv strain of Mycobacterium tuberculosis, as determined by monitoring bacterial growth in major organs and recording host survival times. Death of infected mice of both types was associated with extensive infection-induced pathology in the lungs but not in other major organs. These findings are in keeping with the view that Nramp1 is of limited importance in resistance to tuberculosis in mice.

Animals↗
PubMed1999

Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7.

Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes. Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-type Nramp1(G169) cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1(D169) allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defective S. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.

Alleles↗
PubMed1999

Functional complementation of the malvolio mutation in the taste pathway of Drosophila melanogaster by the human natural resistance-associated macrophage protein 1 (Nramp-1).

The malvolio (mvl) gene of Drosophila melanogaster encodes a protein with a high degree of homology to natural resistance-associated macrophage proteins (Nramps). This family of integral membrane proteins, many of which appear to function as cation transporters, is remarkably conserved in several phylogenetically distinct species. In Drosophila melanogaster, the protein Mvl is expressed in macrophages and in differentiated neurons; loss-of-function mutations lead to defects in gustatory behaviour. The human Nramp-1 protein was expressed in Drosophila melanogaster using the hsp70 promoter. Overexpression in normal animals does not lead to any alterations in their behaviour or physiology. In mutants, however, ubiquitous expression of human Nramp-1 can totally rescue the taste defect. This finding that Nramp-1 can complement the taste defect in mvl mutants provides a potent means of exploiting behavioural genetics to dissect the function of Nramp-1 and to identify other molecules involved with this transport system.

Animals↗
PubMed1999

Giant cell tumor of the sternum. Report of a case with a 17q isochromosome.

We report a case of giant cell tumor of the manubrium with radiological evidence of aggressiveness. Few cases of giant cell tumor of the sternum have been published. Extensive surgery offers the best hopes of recovery. Cytogenetic studies evidenced several clonal abnormalities including a 17q isochromosome. The TP53 suppressor gene is located at 17q13.1. Whether loss of one of the TP53 alleles may have contributed to the aggressive behavior of our patient's tumor is discussed.

Bone Neoplasms↗
PubMed1999

[Chondroma of the soft tissues of the hand. Report of a recurrent case].

A twenty six-year-old man presented a mass of the fifth left finger. X Ray and pathological examination after surgical treatment showed an extraskeletal chondroma. One month later, this tumor recurred, leading to a second surgical procedure. At one year follow up there was no evidence of new recurrence. A review of the literature shows that recurrence of this type of tumor is extremely rare. Also, it stresses the importance of a proper diagnosis which allows an appropriate treatment initially and after a recurrence.

Adult↗
PubMed1998

Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain.

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.

Amino Acid Sequence↗
PubMed1998

Residues in P-glycoprotein catalytic sites that react with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is a specific covalent inhibitor of P-glycoprotein ATPase activity (M. K. Al-Shawi, and A. E. Senior, 1993, J. Biol. Chem. 268, 4197-4206). Complete inhibition occurs at a reaction stoichiometry of 1 mol NBD/mol P-glycoprotein, and the reagent has proved valuable in understanding catalytic mechanisms, particularly in relation to catalytic site cooperativity (A. E. Senior, and S. Bhagat, 1998, Biochemistry 37, 831-836). The actual location of reaction in the amino acid sequence has not yet been determined. Using a combined mutagenesis and biochemical approach we establish here that the initial reaction of NBD-Cl is with Cys within the Walker A consensus sequence of the N- or C-terminal nucleotide site (Cys-431 or Cys-1074 of human P-glycoprotein). Reaction with either Cys yields full inhibition. It was further found that inhibition consists of dithiothreitol (DTT)-reversible and DTT-irreversible components. The former predominates at low pH and the latter at higher pH. This demonstrates that, at higher pH, intramolecular transfer of NBD from Cys to Lys occurs, probably to the proximate Walker A Lys (Lys-433 or Lys-1076 of human P-glycoprotein). After transfer of NBD to Lys, P-glycoprotein ATPase remains fully inhibited.

4-Chloro-7-nitrobenzofurazan↗
PubMed1998

Interaction of PC4 with melted DNA inhibits transcription.

PC4 is a nuclear DNA-binding protein that stimulates activator-dependent class II gene transcription in vitro. Recent biochemical and X-ray analyses have revealed a unique structure within the C-terminal domain of PC4 that binds tightly to unpaired double-stranded (ds)DNA. The cellular function of this evolutionarily conserved dimeric DNA-binding fold is unknown. Here we demonstrate that PC4 represses transcription through this motif. Interaction with melted promoters is not required for activator-dependent transcription in vitro. The inhibitory activity is attenuated on bona fide promoters by (i) transcription factor TFIIH and (ii) phosphorylation of PC4. PC4 remains a potent inhibitor of transcription in regions containing unpaired ds DNA, in single-stranded DNA that can fold into two antiparallel strands, and on DNA ends. Our observations are consistent with a novel inhibitory function of PC4.

Amino Acid Sequence↗
PubMed1998

Crystallography & NMR system: A new software suite for macromolecular structure determination.

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

Computer Simulation↗
PubMed1998

Functional characterization of ORCTL2--an organic cation transporter expressed in the renal proximal tubules.

Chromosome 11p15.5 harbors a gene or genes involved in Beckwith-Wiedemann syndrome that confer(s) susceptibility to Wilms' tumor, rhabdomyosarcoma, and hepatoblastoma. We have previously identified a transcript at 11p15.5 which encodes a putative membrane transport protein, designated organic cation transporter-like 2 (ORCTL2), that shares homology with tetracycline resistance proteins and bacterial multidrug resistance proteins. In this report, we have investigated the transport properties of ORCTL2 and show that this protein can confer resistance to chloroquine and quinidine when overexpressed in bacteria. Immunohistochemistry analyses performed with anti-ORCTL2 polyclonal antibodies on human renal sections indicate that ORCTL2 is localized on the apical membrane surface of the proximal tubules. These results suggest that ORCTL2 may play a role in the transport of chloroquine and quinidine related compounds in the kidney.

Animals↗
PubMed1998

Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification.

The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 +/- 0.06 versus pH 6.6 +/- 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton "leak", as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase-containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.

Animals↗