Biomedical subjects

P Gros

Publications and source records attributed to P Gros.

At least 19 recordsLinked to original sources

PubMed2006

Complex genetic control of susceptibility to Mycobacterium bovis (Bacille Calmette-Guérin) infection in wild-derived Mus spretus mice.

Susceptibility to Mycobacterium bovis Bacille Calmette-Guérin (BCG) is genetically controlled by Nramp1 (Slc11a1). Inbred mouse strains harbor either the resistance (Nramp1(G169)) or the susceptibility (Nramp1(D169)) allele at Nramp1. Mus spretus (Nramp1(G169); SPRET/EiJ) is shown to display an intermediate level of BCG replication in the spleen (log(10) colony-forming units (CFU) approximately 5), compared to resistant A/J (log(10)CFU approximately 4.0) and susceptible C57BL/6J (log(10)CFU approximately 6.0) mice. The presence of genetic modifiers of Nramp1-dependent susceptibility to M. bovis (BCG) infection in Mus spretus was analyzed by whole-genome scanning in 175 mice of an informative (C57BL/6J x SPRET/EiJ) x C57BL/6J backcross. Nramp1 showed a major effect (D1Mcg4, P<1e(-4)), but additional single marker effects were identified on chromosomes 4 (D4Mit150) and x (DXMit249) in male mice, and on chromosome 9 (D9Mit77) and 17 (D17Mit81) in female mice. A strong interaction between Nramp1 and the major histocompatibility locus was also noted in female mice. The mapped loci may act as modifiers of Nramp1 action, and constitute novel entry points for the parallel search of loci regulating susceptibility to mycobacterial infections in humans.

Animals↗

PubMed2006

Tissue, cellular and sub-cellular localization of the Vangl2 protein during embryonic development: effect of the Lp mutation.

Loop-tail (Lp) mice show a very severe neural tube defect, craniorachischisis, which is caused by mis-sense mutations in the Vangl2 gene. The membrane protein Vangl2 belongs to a highly conserved group of proteins that regulate planar polarity in certain epithelia, and that are also important for convergent extension movements during gastrulation and neurulation. A specific anti-Vangl2 antiserum was produced and used to examine the tissue, cell type, and sub-cellular localization of Vangl2 during embryogenesis. Vangl2 protein is expressed at high levels in the neural tube and shows a dynamic expression profile during neurulation. After neural tube closure, robust Vangl2 staining is detected in several neural and neurosensory tissues, including cerebral cortex, dorsal root ganglia, olfactory epithelium, retina, mechanosensory hair cells of the cochlea, and optic nerve. Vangl2 is also expressed during organogenesis in a number of tubular epithelia, including the bronchial tree, intestinal crypt/villus axis, and renal tubular segments derived from ureteric bud and from metanephric mesenchyme. Examination of Vangl2 localization in the neural tubes and cochleas of the normal and Lp/Lp embryos shows disruption of normal membrane localization of Vangl2 in independent alleles at Lp (Lp, Lp(m1Jus)) as well as overall decrease in the expression level.

Animals↗

PubMed2005

Supercooled liquid-like solvent in trypsin crystals: implications for crystal annealing and temperature-controlled X-ray radiation damage studies.

The study of temperature-dependent physical changes in flash-cooled macromolecular crystals is pertinent to cryocrystallography and related issues such as crystal annealing, X-ray radiation damage and kinetic crystallography. In this context, the unit-cell volume of flash-cooled trigonal and orthorhombic trypsin crystals has been monitored upon warming from 100 to 200 K and subsequent re-cooling to 100 K. Crystals of both forms were obtained under the same crystallization conditions, yet they differ in solvent content and channel size. An abrupt non-reversible unit-cell volume decrease is observed at 185 K in orthorhombic and at 195 K in trigonal crystals as the temperature is increased; this result is consistent with ultra-viscous solvent leaving the crystals. Concomitant appearance of ice rings in the diffraction patterns suggests that the transported solvent forms crystalline ice. These results demonstrate that solvent in flash-cooled protein crystals is liquid-like near its crystallization temperature, as has been proposed, yet controversially discussed, for the case of pure water. The use of mineral oil prevents the unit-cell volume decrease in trigonal but not in orthorhombic crystals. The observation of liquid-like solvent has implications in the development of annealing protocols and points a way to the rational design of temperature-controlled crystallographic studies that aim either at studying specific radiation damage or at trapping enzymatic intermediate states.

Animals↗

PubMed2005

[Inflammatory pseudotumor of lymph node].

INTRODUCTION: Inflammatory pseudotumor of lymph node is a rare case in the etiology of fever of unknown origin. OBSERVATION: We report the observation of a woman, aged 40, hospitalized with intermittent fever revealing under-diaphragm adenopathy related to inflammatory pseudotumor of lymph node. CONCLUSION: Inflammatory pseudotumor of lymph node is a rare pathology whose nosological definition is unclear. It should probably be considered as belonging to a category different from the inflammatory pseudotumor of other organs. The diagnosis presents itself in case of isolated adenopathy or prolonged fever and is based on an anatomopathology that essentially calls to mind a lymphoma. The evolution of the condition is shown to be favorable : it can lead to a spontaneous remission, or call for a non-steroid anti-inflammatory treatment, or a steroid therapy.

Adult↗

PubMed2005

Genetic control of susceptibility to Candida albicans in susceptible A/J and resistant C57BL/6J mice.

The importance of host factors in determining susceptibility to systemic Candida albicans infections is evident in both humans and mice. We have used a mouse model to study the genetic basis of susceptibility, using the inbred strains A/J and C57BL/6J, which are susceptible and resistant, respectively, based on different parameters of the response to infection. To identify genes responsible for this differential host response, brain and kidney fungal load were measured in 128 [A/J x C57BL/6J] F(2) mice 48 h after infection with 5 x 10(4) C. albicans blastospores. Segregation analysis in this informative population identified complement component 5 (C5/Hc) as the major gene responsible for this differential susceptibility (LOD of 22.7 for kidney, 19.0 for brain), with a naturally occurring mutation that causes C5 deficiency leading to enhanced susceptibility. C5 was also found to control heart fungal load, survival time, and serum TNF-alpha levels during infection. Investigation of the response to C. albicans challenge in a series of AcB/BcA recombinant congenic strains validated the importance of C5 in determining the host response. However, the strains BcA67 and BcA72 showed discordant phenotypes with respect to their C5 status, suggesting additional complexity in the genetic control of the inter-strain difference in susceptibility observed in A/J and C57BL/6J following systemic infection with C. albicans.

Analysis of Variance↗

PubMed2004

Phenotypic expression of pyruvate kinase deficiency and protection against malaria in a mouse model.

The recombinant congenic mouse strains AcB55 and AcB61 are extremely resistant to malaria (Plasmodium chabaudi AS) despite the presence of susceptibility alleles at the known Char1/Char2 resistance loci. Resistance in AcB55 and AcB61 is controlled by a locus on chromosome 3 (Char4) shown to be allelic with or tightly linked to a loss-of-function mutation in pyruvate kinase (Pklr). AcB55 and AcB61 show important splenomegaly prior to infection caused by the expansion of the red pulp, and display histological signs of extramedullary erythropoiesis in the liver. Examination of splenic cell populations by flow cytometry demonstrates elevated numbers of TER119-positive erythroid precursor cells (>30% of total spleen cells), while RNA expression studies show elevated expression of erythrocyte-specific transcripts such as globin, transferrin receptor, and Nramp2/Slc11a2 in the spleen of both strains. Hematological profiling in both strains is consistent with the presence of anemia as evidenced by low total erythrocyte counts, decreased hemoglobin, as well as abnormally high numbers of circulating reticulocytes (15-20%). These results strongly suggest that the mutant Pklr allele (Pklr(269A)) of AcB55/61 strains causes hemolytic anemia compensated by constitutive erythropoiesis, which in turn protects the mice against P. chabaudi infection. The possible molecular basis of the Pklr protective effect is discussed and is under current investigation in these two strains.

Anemia, Hemolytic↗

PubMed2004

Genetic analysis of innate immunity in resistance to Candida albicans.

Systemic candidiasis is a significant cause of nosocomial infections and the mechanisms of defense against Candida albicans in humans remain poorly understood. Studies in animal models have demonstrated the importance of innate immunity in controlling the response to infection. Although Th1 cytokines have been shown to direct the overall outcome of infection, the precise role of the Th1/Th2 response and, more generally, the adaptive immune response as a whole, in systemic candidiasis, appears to apply mainly to the development of resistance to reinfection. A genetic approach to the identification of host factors regulating pathogenesis and susceptibility to C. albicans infection has been used in humans and in mouse models of infection. Mouse mutants bearing experimentally induced mutations in specific genes have provided a systematic tool for directly assessing the role of individual proteins in C. albicans susceptibility. Inbred mouse strains have been valuable in showcasing the spectrum of naturally occurring variations in initial susceptibility to infection, and type of disease developed. Crosses between resistant and susceptible strains have led to the detection of additional gene effects affecting innate immunity. Of particular interest is the major effect of a naturally occurring loss-of-function mutation in the C5 complement component that has become fixed in many inbred strains. These and other studies have shown that both a functional complement pathway and robust inflammatory response are critical for resistance to C. albicans.

Animals↗

PubMed2003

[Liposomal amphotericin B treatment of cutaneous leishmaniasis contracted in Djibouti and resistant to meglumine antimoniate].

Pentavalent antimony (PA) compounds remain the main therapeutic agents of cutaneous leishmaniasis (CL). CL infection resistant to PA is difficult to cure, limited by severe side effects and requiring a long course treatment of parenteral administration of recommended second line drugs. We report a case of CL unresponsive to meglumine antimoniate contracted in Djibouti, successfully treated with a short course treatment of AmBisome. In this case the subject had a recurrent thick crusted erythematous lesion on his left elbow associated with spreading micropapula on arms and thorax. The diagnosis of CL was confirmed by direct examination and genomic amplification by PCR of skin samples, cultures were negative. A short course treatment of parenteral AmBisome (18 mg/kg) has lead to clinical cure with no side effects and no relapse. In our hospital, the high cost of medication was counterbalanced by easiest administration, reduction of hospitalization duration, absence of adverse events and a gain of comfort. For this patient, a short course treatment of AmBisome proved to be a suitable alternative to traditional drugs used in CL resistant to PA.

Adult↗

PubMed2002

Susceptibility to malaria as a complex trait: big pressure from a tiny creature.

Malaria, which is a major infectious disease worldwide, is caused by the Plasmodium parasite, one of the longest-known parasites infecting humans. The malaria situation is complicated by the emergence of drug resistance and the lack of an effective vaccine. Genetic factors play a key role in disease susceptibility, progression and outcome. Interestingly, an increasing large number of polymorphisms associated with resistance and susceptibility in humans have been found in proteins from erythrocytes, the site of Plasmodium replication. Some of these deleterious alleles have been selected by direct genetic pressure from the parasite in endemic areas of malaria. A number of additional gene effects have been mapped both in humans and in mice using population studies and experimental models of malaria, respectively. These recent studies have started to reveal additional aspects of the complex host-parasite interactions.

Animals↗

PubMed2002

Complex genetic control of susceptibility to malaria in mice.

Malaria is a major infectious disease worldwide, with over 1 million deaths in African children every year. The molecular pathways of pathogenesis of the Plasmodium parasite and the host mechanisms of defense against this infection remain poorly understood. Epidemiological studies, together with linkage analyses in endemic areas have clearly pointed at a genetic component of innate susceptibility and severity of disease. In humans, this genetic trait is complex, and has been studied in a mouse experimental model over the past few years. Inbred strains of mice show different degrees of susceptibility to infection with Plasmodium chabaudi, and the genetic component of these inter-strain differences has been studied in standard informative backcross and F2 populations, as well as in recombinant inbred strains and more recently, in recombinant congenic strains. These studies have shown that genetic susceptibility to malaria is also complex in mice, and have led to the mapping of major susceptibility Char (Chabaudi resistance) loci, located on chromosomes 9 (Char1), 8 (Char2), 17 (Char3) and 3 (Char4).

Animals↗

PubMed2002

Acquisition of Mn(II) in addition to Fe(II) is required for full virulence of Salmonella enterica serovar Typhimurium.

The roles of the genes feoB (ABC ferrous iron transporter), mntH (proton-dependent manganese transporter), and sitABCD (putative ABC iron and/or manganese transporter) in Salmonella pathogenicity were investigated by using mutant strains deficient in one, two, or three transporters. Our results indicated that sitABCD encodes an important transporter of Mn(II) and Fe(II) which is required for full virulence in susceptible animals (Nramp1(-/-)) and for replication inside Nramp1(-/-) macrophages in vitro. The mntH sitABCD double mutant (mutant MS) showed minimal Mn(II) uptake and increased sensitivity to H(2)O(2) and to the divalent metal chelator 2,2'-dipyridyl (DP) and was defective for replication in macrophages. In vivo MS appeared to be as virulent as the sitABCD mutant in Nramp1(-/-) animals. The ferrous iron transporter Feo was required for full virulence in 129/Sv Nramp1(-/-) mice, and infection with multiple mutants lacking FeoB was not fatal. The sitABCD feoB mutant (mutant SF) and the mntH sitABCD feoB mutant (mutant MSF) showed minimal Fe(II) uptake and were slightly impaired for replication in susceptible macrophages. MSF showed reduced growth in minimal medium deficient in divalent cations. The role of the mntH gene, which is homologous to mammalian Nramp genes, was also investigated after overexpression in the double mutant MS. MntH preferred Mn(II) over Fe(II) and could suppress MS sensitivity to H(2)O(2) and to DP, and it also improved the intracellular survival in Nramp1(-/-) macrophages. This study indicates that acquisition of Mn(II), in addition to Fe(II), is required for intracellular survival and replication of Salmonella enterica serovar Typhimurium in macrophages in vitro and for virulence in vivo.

Animals↗

PubMed2002

Regulation of NRAMP1 gene expression by 1alpha,25-dihydroxy-vitamin D(3) in HL-60 phagocytes.

The natural resistance-associated macrophage protein 1 (Nramp1) is a proton-dependent transporter of divalent metals. We studied NRAMP1 expression during HL-60 differentiation induced by VD and VD agonists. NRAMP1 and CD14 gene expression differed in kinetics of induction, mRNA levels and stability, and response to VD combined with PMA, whereas a combination of VD and IFN-gamma induced similar up-regulation. NRAMP1 protein expression paralleled the accumulation of mRNA and was localized in the phagosomal membrane after phagocytosis. A promoter construct extending 647 bp upstream of NRAMP1 ATG showed myeloid-specific transcription in transient transfection assays, which was up-regulated by VD in HL-60. In HL-60 clones stably transfected with this construct, transcription was apparently induced through indirect VD genomic effects, and there was accordance between the levels of reporter transcription and endogenous NRAMP1 mRNA in response to VD but not to IFN-gamma. Thus, VD genomic effects stimulate NRAMP1 transcription and protein expression in maturing phagocytes.

5' Flanking Region↗

PubMed2001

Characterization of the iron transporter DMT1 (NRAMP2/DCT1) in red blood cells of normal and anemic mk/mk mice.

Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mk reticulocytes, suggesting a subsequent defect in Fe(++) transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non-iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.

Anemia↗

PubMed2001

Conditional optimization: a new formalism for protein structure refinement.

Conditional optimization allows unlabelled loose-atom refinement to be combined with extensive application of geometrical restraints. It offers an N-particle solution for the assignment of topology to loose atoms, with weighted gradients applied to all possibilities. For a simplified test structure consisting of a polyalanine four-helical bundle, this method shows a large radius of convergence using calculated diffraction data to at least 3.5 A resolution. It is shown that with a new multiple-model protocol to estimate sigma(A) values, this structure can be successfully optimized against 2.0 A resolution diffraction data starting from a random atom distribution. Conditional optimization has potentials for map improvement and automated model building at low or medium resolution limits. Future experiments will have to be performed to explore the possibilities of this method for ab initio phasing of real protein diffraction data.

Crystallization↗

PubMed2001

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site.

Escherichia coli outer membrane protease OmpT has previously been classified as a serine protease with Ser(99) and His(212) as active site residues. The recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed. Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants. Our results support the involvement of a nucleophilic water molecule that is activated by the Asp(210)/His(212) catalytic dyad. Activity is also strongly dependent on Asp(83) and Asp(85). Both may function in binding of the water molecule and/or oxyanion stabilization. The proposed mechanism implies a novel proteolytic catalytic site.

Amino Acid Sequence↗

PubMed2001

Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site.

OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria. Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel beta-barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase-shaped beta-barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His-Asp dyad and an Asp-Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)

Amino Acid Sequence↗

PubMed2001

Identification of a new malaria susceptibility locus (Char4) in recombinant congenic strains of mice.

The genetic component of susceptibility to malaria is complex, both in humans and in the mouse model of infection. Two murine loci on chromosomes 8 (Pchr/Char2) and 9 (Char1) have previously been mapped in F(2) crosses, and play an important role in regulating blood parasitemia and survival to infection with Plasmodium chabaudi. These loci explain only part of the interstrain phenotypic variance, and their penetrance and expressivity vary in different inbred strains. Novel loci regulating response to P. chabaudi infection were investigated by using an alternative strategy based on a newly derived set of AcB/BcA recombinant congenic strains bred from malaria-susceptible A/J (A) and resistant C57BL/6J (B6). One of the AcB strains, AcB55, is shown to be highly resistant to infection despite 83% susceptible A genomic composition, including susceptibility alleles at Char1 and Pchr/Char2. Early onset of parasite clearance in AcB55 is associated with lower peak parasitemia and absence of mortality. Linkage analysis in an informative (AcB55 x A)F(2) population, using peak parasitemia as a quantitative trait, located a new B6-derived resistance locus on chromosome 3 (lod score = 6.57) that we designate Char4. A second, suggestive linkage on chromosome 10 (lod score = 2.53) shows additive effect with Char4 on peak parasitemia. Char4 maps to a small congenic B6 fragment in AcB55 that should facilitate the search for candidate genes. Our findings provide an entry point for parallel association studies in humans between the syntenic 4q21-4q25 region and susceptibility to disease in endemic areas of malaria.

Alleles↗

PubMed2001

Functional expression of multidrug resistance protein 1 in Pichia pastoris.

Overexpression of the multidrug resistance-associated protein (MRP1) causes multidrug resistance in cultured cells. MRP1 transports a large number of glutathione, glucuronide, and sulfate-conjugated organic anions by an ATP-dependent efflux mechanism. Six other MRP proteins exist (MRP2-7), and mutations in some of these genes cause major pathological conditions in humans. A detailed characterization of the structure and mechanism of action of these proteins requires an efficient expression system from which large amounts of active protein can be obtained. We report the expression of a recombinant MRP1 in the methylotrophic yeast Pichia pastoris. The protein is expressed in the membrane fraction of these cells, as a stable and underglycosylated 165 kDa peptide. Expression levels are very high, and 30 times superior to those seen in multidrug-resistant HeLa/MRP1 transfectants. MRP1 expressed in P. pastoris binds 8-azido[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion, which can be competed by a molar excess of ADP and ATP. Under hydrolysis conditions (at 37 degrees C), orthovanadate induces trapping of the 8-azido[alpha-(32)P]nucleotide in MRP1, which can be further modulated by known MRP1 ligands. MRP1 is also labeled by a photoactive analogue of rhodamine 123 (IAARh123) in P. pastoris/MRP1 membranes, and this can be competed by known MRP1 ligands. Finally, MRP1-positive membrane vesicles show ATP-dependent uptake of LTC(4). Thus, MRP1 expressed in P. pastoris is active and shows characteristics of MRP1 expressed in mammalian cells, including drug binding, ligand-modulated formation of the MRP1-MgADP-P(i) intermediate (ATPase activity), and ATP-dependent substrate transport. The successful expression of catalytically active and transport-competent MRP1 in P. pastoris should greatly facilitate the efficient production and isolation of the wild type or inactive mutants of MRP1, or of other MRP proteins for structural and functional characterization.

ATP Binding Cassette Transporter, Subfamily B, Mem↗