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P Gros

Publications and source records attributed to P Gros.

At least 73 records · Page 4Linked to original sources
PubMed2000

Identification of the Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter.

The Escherichia coli mntH (formerly yfeP) gene encodes a putative membrane protein (MntH) highly similar to members of the eukaryotic Nramp family of divalent metal ion transporters. To determine the function of E. coli MntH, a null mutant was created and MntH was overexpressed both in wild-type E. coli and in the metal-dependent mutant hflB1(Ts). At the restrictive temperature 42 degrees C, the mntH null mutation reduces the suppression of hflB1(Ts) thermosensitivity by exogenous divalent metals. Conversely, overexpression of MntH restores growth at 42 degrees C, increases suppression of the ts phenotype by Fe(II) and Ni(II) and renders hflB1(Ts) cells hypersensitive to Mn(II). Transport studies in intact cells show that MntH selectively facilitates uptake of 54Mn(II) and 55Fe(II) in a temperature-, time- and proton-dependent manner. Competition studies in uptake assays and growth inhibition experiments in hflB1(Ts) mutants together indicate that MntH is a divalent metal cation transporter of broad substrate specificity. The functional characteristics of MntH suggest that it corresponds to the previously described manganese transporter of E. coli. This study indicates that proton-dependent divalent metal ion uptake has been preserved in the Nramp family from bacteria to humans.

Base Sequence↗
PubMed2000

Super-infection by Bacillus thuringiensis H34 or 3a3b can lead to death in mice infected with the influenza A virus.

Bacterial super-infections are the main cause of complication and mortality after influenza virus (IAV) infection. Since Bacillus thuringiensis (Bt) is considered non-pathogenic for humans and is widely sprayed in urban areas, the aim of this work was to evaluate the potential pathogenicity of a combined infection Bt-IAV in a mouse model of pneumonia. Bacteria used for super-infections were Bt serotype H34 isolated from human infection and the insecticidal strain 3a3b obtained from a commercial source. Virus strain was A/Scotland/20/74 (H3N2) adapted to BALB/c mice by serial lung passage. Combined infection with 4% of the viral lethal dose 50% (LD(50)) and 10(2) spores of Bt H34 killed 40% of the mice. Mortality rates increased up to 55% and 100% when combined infections were done with respectively 10(4) and 10(7) spores. The insecticidal strain Bt 3a3b was less pathogenic than Bt H34. A dose of 10(4) spores associated with 4% of IAV LD(50) killed 50% of the mice. This inoculum must be compared with the doses usually sprayed in agriculture: 10(11) spores m(-2). Total protection against super-infection was obtained when mice were treated with amantadine. Even if only a few cases of Bt human infection have been reported, these results suggest a possible risk for workers spraying Bt-based biopesticides during flu outbreaks.

Administration, Oral↗
PubMed2000

Functional analysis of a tryptophan-less P-glycoprotein: a tool for tryptophan insertion and fluorescence spectroscopy.

P-glycoprotein (Pgp) functions as an ATP-dependent drug efflux pump to confer multidrug resistance to tumor cells. In the absence of a high-resolution structure for this protein, several important and intriguing aspects of Pgp structure and function remain poorly understood. Fluorescence spectroscopy of endogenous or genetically engineered tryptophan residues represents a potentially powerful method to probe static and dynamic aspects of Pgp at high resolution. We have used site-directed mutagenesis to modify the wild-type (WT) mouse mdr3 Pgp for tryptophan fluorescence spectroscopy by replacement of all 11 tryptophan residues individually with phenylalanine. None of the 11 tryptophans were found to be absolutely essential for Pgp activity, because Chinese hamster ovary cells transfected and overexpressing this mutant Trp-less mdr3 cDNA (mdr3F(1-11)) become multidrug-resistant and can carry out active transport of vinblastine, colchicine, and Calcein-AM. The mdr3F(1-11) mutant has reduced activity compared with WT Mdr3, and shows a unique pattern of drug resistance clearly distinct from WT and, as opposed to the latter, can neither confer FK-506 resistance nor functionally complement ste6 in yeast. Studies with Pgp mutants containing either single or double tryptophan residues or with chimeric molecules constructed between wild-type Pgp and mdr3F(1-11) indicated that no single tryptophan residue was responsible for the reduced activity of the mdr3F(1-11) mutant. Likewise, all but one chimeric Pgp preserved the unique drug resistance profile of the mdr3F(1-11) mutant. Altogether, we show that a Trp-less Pgp is functionally active and can be used as a molecular backbone for insertion of tryptophans in strategic locations to probe various aspects of Pgp function.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2000

[Malignant paraganglioma of the uterus].

We report a malignant uterine paraganglioma in a 41-year-old woman who underwent a hysterectomy for meno-metrorrhagia. It was initially thought to be a leiomyoma in necrobiosis. The clinical outcome was characterized by an early regional recurrence (in the left Fallopian tube). Later, vertebral and lung metastasis occurred, leading to death 22 months after the initial diagnosis. Paragangliomas are uncommon neuroendocrine tumors, related to pheochromocytomas. They are mainly found in the para-aortic and retroperitoneal region, and less commonly in the pelvic area. Location in the uterus is extremely rare: 5 cases were previously reported and only one malignant.

Adult↗
PubMed1999

Crystals of acetylated SecB diffract to 2.3-A resolution.

The molecular chaperone SecB is part of the protein translocation pathway in Escherichia coli. SecB was purified from an overproducing strain and crystallized, resulting in crystals diffracting to 2.3-A resolution. The analysis of electrospray ionization mass spectra of dissolved crystals of SecB indicated that we have crystallized an acetylated form of SecB. Sequence analysis suggests that the protein is fully acetylated at its N-terminus in vivo, indicating that potential deacetylation is artificially introduced by purification methods. The high degree of acetylation that we observed might account for the fact that the crystals obtained as described in this study diffract to higher resolution than those in previously reported trials.

Acetylation↗
PubMed1999

Large scale purification of detergent-soluble P-glycoprotein from Pichia pastoris cells and characterization of nucleotide binding properties of wild-type, Walker A, and Walker B mutant proteins.

P-glycoprotein (Pgp; mouse MDR3) was expressed in Pichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wild-type mouse MDR3 Pgp were studied using 2'-(3')-O-(2,4,6-trinitrophenyl)adenosine tri- and diphosphate. Both analogs were found to bind with K(d) in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.

Amino Acid Sequence↗
PubMed1999

Adhesion mechanism of human beta(2)-glycoprotein I to phospholipids based on its crystal structure.

Human beta(2)-glycoprotein I is a heavily glycosylated five-domain plasma membrane-adhesion protein, which has been implicated in blood coagulation and clearance of apoptotic bodies from the circulation. It is also the key antigen in the autoimmune disease anti-phospholipid syndrome. The crystal structure of beta(2)-glycoprotein I isolated from human plasma reveals an elongated fish-hook-like arrangement of the globular short consensus repeat domains. Half of the C-terminal fifth domain deviates strongly from the standard fold, as observed in domains one to four. This aberrant half forms a specific phospholipid-binding site. A large patch of 14 positively charged residues provides electrostatic interactions with anionic phospholipid headgroups and an exposed membrane-insertion loop yields specificity for lipid layers. The observed spatial arrangement of the five domains suggests a functional partitioning of protein adhesion and membrane adhesion over the N- and C-terminal domains, respectively, separated by glycosylated bridging domains. Coordinates are in the Protein Data Bank (accession No. 1QUB).

Antibodies, Antiphospholipid↗
PubMed1999

Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron.

Genetic studies in animal models of microcytic anemia and biochemical studies of transport have implicated the Nramp2 gene in iron transport. Nramp2 generates two alternatively spliced mRNAs that differ at their 3' untranslated region by the presence or absence of an iron-response element (IRE) and that encode two proteins with distinct carboxy termini. Antisera raised against Nramp2 fusion proteins containing either the carboxy or amino termini of Nramp2 and that can help distinguish between the two Nramp2 protein isoforms (IRE: isoform I; non-IRE: isoform II) were generated. These antibodies were used to identify the cellular and subcellular localization of Nramp2 in normal tissues and to study possible regulation by dietary iron deprivation. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney. Dietary iron starvation results in a dramatic upregulation of the Nramp2 isoform I in the proximal portion of the duodenum only, whereas expression in the rest of the small intestine and in kidney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum, immunostaining studies of tissue sections show that Nramp2 protein expression is abundant under iron deplete condition and limited to the villi and is absent in the crypts. In the villi, staining is limited to the columnar absorptive epithelium of the mucosa (enterocytes), with no expression in mucus-secreting goblet cells or in the lamina propria. Nramp2 expression is strongest in the apical two thirds of the villi and is very intense at the brush border of the apical pole of the enterocytes, whereas the basolateral membrane of these cells is negative for Nramp2. These results strongly suggest that Nramp2 is indeed responsible for transferrin-independent iron uptake in the duodenum. These findings are discussed in the context of overall mechanisms of iron acquisition by the body.

Alternative Splicing↗
PubMed1999

Pax-3 regulates neurogenesis in neural crest-derived precursor cells.

The peripheral nervous system consists of multiple neural lineages derived from the neural crest (NC). Pax-3 is expressed in the NC and when mutated in the splotch mouse (Sp) results in the loss of derivatives from this precursor cell population. We have investigated the role of Pax-3 in regulating the generation of neurons from NC-derived precursor cells in vitro. Pax-3 mRNA in NC cultures is initially expressed in all NC but is subsequently only retained in neurons, suggesting a role in their generation. To determine whether Pax-3 is involved in neuron development, we first examined the generation of sensory-like neurons in NC cultures from Sp mice. Fivefold less sensory-like neurons were generated in NC cultures from Sp homozygous mice as compared to wild-type littermates. The role of Pax-3 in sensory neuron generation was then directly examined in dorsal root ganglia cultures by down-regulating the expression of Pax-3 protein with antisense oligonucleotides. It was found that antisense oligonucleotides inhibited 80-90% of newly generated sensory neurons; however, there was no significant effect on the survival of sensory neurons or the precursor population. These results suggest that Pax-3 has a role in regulating the differentiation of peripheral neurons.

Animals↗
PubMed1999

TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages.

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.

Animals↗
PubMed1999

[Chronic diarrhea due to lymphocytic colitis treated with piroxicam beta cyclodextrin].

BACKGROUND: Lymphocytic colitis is a chronic diarrhea syndrome with no endoscopically detectable anomaly and histological colonic lesions. CASE REPORT: A 52-year-old woman was hospitalized for chronic diarrhea. She experienced 10 to 30 liquid bowel movements per day. Symptoms had begun 8 days after taking piroxicam fl-cyclodextrin. Intraepithelial cell counts on rectum and colon endoscopic biopsies showed more than 20% lymphocytes, giving the diagnosis of lymphocytic colitis. The patient was given 5ASA 3 g/24 h. Symptoms regressed in less than one week. DISCUSSION: Lymphocytic colitis is uncommon. The cause remains unknown but a secondary autoimmune mechanism triggered by drugs has been suggested. This case would implicate nonsteroidal antiinflammatory drugs. Certain authors have demonstrated that they can be involved in the development of collagen colitis.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

[Mesenteric cystic lymphangioma].

BACKGROUND: Cystic lymphangioma is an uncommon congenital malformation usually encountered in children and often discovered fortuitously. CASE REPORT: A 46-year-old man was hospitalized for acute alcoholic hepatitis with edematoascitic decompensation. The abdominal and pelvic CT scan showed homogeneous hepatomegaly and a tumoral process involving the terminal ileal loops without signs of occlusion or node enlargement. Pathology reported benign mesenteric multicystic lymphangioma. DISCUSSION: Cystic lymphangiomas account for 7% of all intra-abdominal cystic formations. The ileal localization predominates with little or no clinical expression. Ultrasonography and CT scan provide complementary information on the relations with other organs. Magnetic resonance imaging is currently the gold standard allowing precise diagnosis of the anatomic relations and identifying intracystic hemorrhage. The pathology examination is required to eliminate possible benign multicystic mesothelioma which may occasionally take on an aggressive form. Complete surgical excision provides cure.

Humans↗
PubMed1999

The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes.

The natural resistance associated macrophage protein (Nramp) gene family is composed of two members in mammals, Nramp1 and Nramp2. Nramp1 is expressed primarily in macrophages and mutations at this locus cause susceptibility to infectious diseases. Nramp2 has a much broader range of tissue expression and mutations at Nramp2 result in iron deficiency, indicating a role for Nramp2 in iron metabolism. To get further insight into the function and mechanism of action of Nramp proteins, we have generated isoform specific anti-Nramp1 and anti-Nramp2 antisera. Immunoblotting experiments indicate that Nramp2 is present in a number of cell types, including hemopoietic precursors, and is coexpressed with Nramp1 in primary macrophages and macrophage cell lines. Nramp2 is expressed as a 90-100-kD integral membrane protein extensively modified by glycosylation (>40% of molecular mass). Subcellular localization studies by immunofluorescence and confocal microscopy indicate distinct and nonoverlapping localization for Nramp1 and Nramp2. Nramp1 is expressed in the lysosomal compartment, whereas Nramp2 is not detectable in the lysosomes but is expressed primarily in recycling endosomes and also, to a lower extent, at the plasma membrane, colocalizing with transferrin. These findings suggest that Nramp2 plays a key role in the metabolism of transferrin-bound iron by transporting free Fe2+ across the endosomal membrane and into the cytoplasm.

Animals↗
PubMed1999

Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4)

Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.

Amino Acid Sequence↗