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P Gros

Publications and source records attributed to P Gros.

At least 109 records · Page 6Linked to original sources
PubMed1998

Mutations in the nucleotide-binding sites of P-glycoprotein that affect substrate specificity modulate substrate-induced adenosine triphosphatase activity.

The amino- and carboxy-terminal nucleotide-binding domains (NBD1 and NBD2) of P-glycoprotein (P-gp) share over 80% sequence identity. Almost all of NBD1 can be exchanged by corresponding NBD2 segments with no significant loss of function, except for a small segment around the Walker B motif. Within this segment, we identified two sets of residues [ERGA --> DKGT (522-525) and T578C] that, when replaced by their NBD2 counterparts, cause dramatic alterations of the substrate specificity of the protein [Beaudet, L., and Gros, P. (1995) J. Biol. Chem. 270, 17159-17170]. We wished to gain insight into the molecular basis of this defect. For this, we overexpressed the wild-type mouse Mdr3 and variants bearing single or double mutations at these positions in the yeast Pichia pastoris. P-gp-specific ATPase activity was measured in yeast plasma membrane preparations after detergent solubilization and reconstitution in Escherichia coli proteoliposomes. P-gp proteoliposomes from P. pastoris showed a strong verapamil- and valinomycin-stimulated ATPase activity, with characteristics (KM, Vmax) similar to those measured in mammalian cells. Mutations did not appear to affect the KM for Mg2+ATP ( approximately 0.4 mM), but maximum velocity (Vmax) of the drug-stimulated ATPase activity was severely affected in a substrate/modulator-specific fashion. Indeed, all mutants showed complete loss of verapamil-induced ATPase, while all retained at least some degree of valinomycin-induced ATPase activity. Photolabeling studies with [125I]iodoarylazidoprazosin, including competition with MDR drugs and modulators, suggested that drug binding was not affected in the mutants. The altered drug resistance profiles of the ERGA --> DKGT(522-525) and T578C mutants in vivo, together with the observed alterations in substrate-induced ATPase activity of these proteins, suggest that the residues involved may form part of a signal pathway between the membrane regions (substrate binding) and the ATP binding sites.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites.

Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Mutational analysis of the P-glycoprotein first intracellular loop and flanking transmembrane domains.

The role of individual intracellular (IC) loops linking transmembrane (TM) domains in P-glycoprotein (P-gp) function remains largely unknown. The high degree of sequence conservation of these regions in the P-gp family and other ABC transporters suggests an important role in a common mechanism of action of these proteins. To gain insight into this problem, we have randomly mutagenized a portion of TM2, the entire IC1 loop, TM3, the entire extracellular loop (EC2), and part of TM4, and analyzed the effect of such mutations on P-gp function. Random mutagenesis was carried out using Taq DNA polymerase and dITP under conditions of low polymerase fidelity, and the mutagenized segments were reintroduced in the full length mdr3 cDNA by homologous recombination in the yeast Saccharomyces cerevisiae strain JPY201. The biological activity of mutant P-gp variants was analyzed in yeast by their ability to confer cellular resistance to the antifungal drug FK506 and the peptide ionophore valinomycin, and by their ability to complement the yeast Ste6 gene and restore mating in a yeast strain bearing a null mutation [Raymond, M., et al. (1992) Science 256, 232-4] at this locus. The analysis of 782 independent yeast transformants allowed the identification of 49 independent mutants bearing single amino acid substitutions in the mutagenized segment resulting in an altered P-gp function. The mutants could be phenotypically classified into two major groups, those that resulted in partial or complete overall loss of function and those that seemed to affect substrate specificity. Several of the mutants affecting overall activity mapped in IC1; in particular we identified a segment of four consecutive mutation sensitive residues (TRLT, positions 169-172) with such a phenotype. On the other hand, we identified a cluster of mutants affecting substrate specificity within the short EC2 segment and in the adjacent portion of the neighboring TM4 domain. Expression and partial purification of a representative subset of these mutants showed that in all but two cases, loss of function was associated with loss of drug-induced ATPase activity of P-gp. Therefore, it appears that TM domains, IC and EC loops, are structurally and functionally tightly coupled in the process of drug stimulatable ATPase characteristic of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Epitope insertion favors a six transmembrane domain model for the carboxy-terminal portion of the multidrug resistance-associated protein.

The overexpression of the multidrug resistance protein, MRP, in mammalian cells is associated with pleiotropic resistance to cytotoxic drugs. MRP is an integral membrane protein which belongs to the family of ATP-binding cassette transporters. Secondary structure predictions combined with biochemical analyses suggest that MRP encodes 11 transmembrane (TM) domains in the amino-terminal half of the protein and four or six transmembrane domains in the carboxy-terminal half of the protein. To gain insight into the membrane topology of the carboxy-terminal half of MRP, small, antigenic hemagglutinin (HA) epitopes (YPYDVPDYAS) were inserted within six predicted hydrophilic subfragments of this region (938, 1001, 1084, 1175, 1222, 1295). These epitope-tagged MRP variants were expressed in HeLa cells to evaluate their ability to confer resistance to the drug etoposide (VP-16). Insertion of the HA epitopes at positions 938, 1001, and 1222 resulted in functional proteins, while epitope insertion at positions 1084, 1175, and 1295 abrogated MRP function. The intracellular versus extracellular location of the HA epitopes present in biologically active MRP variants was then established in intact and permeabilized cells by immunofluorescence using an anti-HA antibody. Epitopes inserted at positions 1001 and 1222 were located on the extracellular side of the plasma membrane, while the epitope inserted at position 938 was located intracellularly. These results are consistent with a six TM rather than a four TM domain model for the membrane portion of the carboxy-terminal half of MRP.

ATP-Binding Cassette Transporters↗
PubMed1998

Mapping of genetic modulators of natural resistance to infection with Salmonella typhimurium in wild-derived mice.

Despite antibiotic therapy and vaccination programs, microbial diseases continue to be the leading cause of morbidity and mortality worldwide. The genetic basis of the host response to infection is complex, and its understanding has been facilitated through the study of mouse models of human infectious diseases. Genetic variation in resistance of mice to infection with Salmonella typhimurium has been recognized for over 50 years and shown to be a multifactorial trait. We have studied the genetic basis of resistance or susceptibility to infection with S. typhimurium in the wild-derived inbred mouse Mus musculus molossinus (MOLF/Ei). MOLF/Ei mice are extremely susceptible to infection with S. typhimurium despite the presence of resistance alleles at Nramp1 and Lps. To identify genes that modulate the expression of natural resistance or susceptibility to infection with S. typhimurium in MOLF/Ei, we have performed a genome-wide study using an F2 intercross between C56BL/6J and MOLF/Ei inbred mice. We have mapped three QTLs that significantly affect survival time following lethal infection with S. typhimurium. The Salmonella-resistant phenotype was linked to Nramp1 on proximal chromosome 1 (maximum lod score of 18.8 at D1Mcg4) and to a newly mapped region on mouse chromosome 11 (maximum lod score of 7.0 at D11Mit5). The third QTL conferred recessive susceptibility and was located on mouse chromosome 1, approximately 25 cM distal to Nramp1 (maximum lod score of 4.8 at D1Mit100).

Animals↗
PubMed1998

Macrophage NRAMP1 and its role in resistance to microbial infections.

The identification and characterization of genetic factors influencing natural susceptibility to infectious diseases in humans and in model organisms, such as the laboratory mouse, can provide new insight into the basic mechanisms of host defense against infections. In the mouse, resistance or susceptibility to infection with intracellular pathogens such as Salmonella, Mycobacterium and Leishmnania is controlled by the Natural resistance associated macrophage protein (Nramp1) gene on chromosome 1, which influences the rate of intracellular replication of these parasites in macrophages. Nramp1 codes for an integral membrane protein, which is expressed exclusively in macrophage/monocytes and polymorphonuclear leukocytes. The protein is localized to the endosomal/lysosomal compartment of the macrophage and is rapidly recruited to the membrane of the particle-containing phagosome upon phagocytosis. Nramp defines a novel family of functionally related membrane proteins including Nramp2, which was recently shown to be the major transferrin-independent uptake system of the intestine in mammals. This observation supports the hypothesis that the phagocyte-specific Nramp1 protein may regulate the intraphagosomal replication of antigenically unrelated bacteria by controlling divalent cation concentrations at that site. Recent genetic studies have found that allelic variants at the human NRAMP1 locus are associated with susceptibility to leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis) and possibly with the onset of rheumatoid arthritis.

Animals↗
PubMed1998

Structural organization and chromosomal localization of the human Na,K-ATPase beta 3 subunit gene and pseudogene.

We have cloned and characterized the Na,K-ATPase beta 3 subunit gene (ATP1B3), and a beta 3 subunit pseudogene (ATP1B3P1), from a human PAC genomic library. The beta 3 subunit gene is > 50 kb in size and is split into 7 exons. The exon/intron organization of the beta 3 subunit gene is identical to that of the Na,K-ATPase beta 3 subunit gene, indicating that these two genes evolved from a common evolutionary ancestor. Comparison of the promoter region of the human and mouse beta 3 subunit gene reveals a high degree of homology within a 300-bp segment located immediately upstream of the translation start site, suggesting that control elements that serve to regulate the cell-specific expression of the beta 3 subunit gene are likely to be located within this conserved region. Dot blot analysis of beta 3 subunit transcripts revealed expression within virtually all human tissues, while in situ hybridization showed expression of beta 3 mRNA in both neurons and glia of rat brain. Fluorescence in situ hybridization with PAC DNA clones localized ATP1B3 to the q22-->23 region of Chromosome (Chr) 3, and the beta 3 pseudogene to the p13-->15 region of Chr 2.

Amino Acid Sequence↗
PubMed1998

cDNA cloning, structural organization, and expression of the sheep NRAMP1 gene.

Mouse resistance to several intracellular pathogens including Mycobacteria, Leishmania, and Salmonella is under the control of the Chromosome (Chr) 1 Natural Resistance Associated Macrophage Protein I gene (Nramp1). This gene could have an economic and health importance for domestic animals and humans as well. Therefore, equivalents of the NRAMP1 gene have been cloned by several research groups in various animal species. To study in sheep the influence of the NRAMP1 gene on the susceptibility to intracellular pathogens induced diseases, we have cloned the sheep NRAMP1 cDNA by screening a splenic cDNA library. The genomic organization of the sheep NRAMP1 gene was then determined by sequencing the exon/intron boundaries. The transcription start points (tsp) from the NRAMP1 mRNA have been located with primer extension experiments. RT-PCR reactions have been used to determine the profile of mRNA expression of this gene.

Amino Acid Sequence↗
PubMed1998

Infection genomics: Nramp1 as a major determinant of natural resistance to intracellular infections.

The scope of the tuberculosis (TB) epidemic in the world today is enormous, with about 30 million active cases. Current research into preventing the spread of TB is focused on development of new drugs to inactivate Mycobacterium tuberculosis, the causative agent of TB, as well as on identifying the critical steps of host defense to infection with Mycobacteria, which might also yield therapeutic targets. Our infection genomics approach toward the latter strategy has been to isolate and characterize a mouse gene, Bcg (Nramp1), which controls natural susceptibility to infection with Mycobacteria, as well as Salmonella and Leishmania. Through comparative genomics, we have identified the homologous human NRAMP1 gene, alleles of which are now being used for tests of linkage with TB and leprosy.

Alleles↗
PubMed1998

[Helminth diagnosis in pathologic anatomy].

Most publications devoted to diagnosis of helminths emphasize characteristic parasitological features. Histological feature are seldom described in detail. The purpose of this study was to propose a diagnostic method suitable for use by histologists who, unlike parasitologists, do not visualize the whole worm but rather pieces randomly scattered over a slide. It is relatively easy to distinguish helminths which have smooth muscle, no respiratory or circulatory system, and no coelom from arthropods which have striated muscle, both respiratory and circulatory systems, and coelom. At the adult stage, roundworms or nemathelminths present an external cuticle that may have patterned markings. The visceral cavity is empty. Adults can dwell either in the intestine in which case they are oviparous (oxyuris, ascaris, Ancylostoma, Strongyloides stercoralis, Trichuris...) or in tissue in which case they are viviparous (filaria). Larva of some species can be found in tissues. Adult tape-worms are devoid of cuticle and have a mesenchymatous visceral cavity. Cestodes which are segmented and have no digestive tract (taenias) are readily distinguishable from trematodes which are not segmented and have a digestive tract (faciolasis) Some cestodes unable thrive in man can cause cyst formation (hydatidosis, sparganosis, cysticercosis, coenurosis). On the basis of symptoms and histological features, it is usually possible to diagnose the genus and even the species of the offending helminth provided that the parasite has not been excessively damaged and that a sufficient number of sections are available.

Animals↗
PubMed1998

[Cutaneous-splanchnic neurofibromatosis].

BACKGROUND: Segmental neurofibromatosis (NF V) is ten times less frequent than Recklinghausen disease. Would the risk of visceral involvement in this uncommon form of neurofibromatosis warrant systematic imaging procedures? CASE REPORT: A 31-year-old man consulted for a voluminous plexiform neurofibroma in the left lumbar area. More ventrally, on the left side, there was also a café au lait spot. There were no Lisch nodules. The chest and abdominopelvic computed tomography and magnetic resonance imaging showed intramuscular tumoral extension, two neurofibromas in the 9th intercostal space and a voluminous 5-cm tumor situated in the left adrenal area. After resection pathology examination of the surgical specimen confirmed the diagnosis of ganglioneuroma. DISCUSSION: In this patient, all the neurofibromas and the café of lait spot developed in the territories of the left T10 and adjacent spinal roots. This was also true for the ganglioneuroma which developed on the deep sympathetic ramus to the adrenal gland which originates essentially from roots T8 to T11. This would place this case in the second subgroup of NF V in Roth's classification. Only six other cases have been reported in the literature. Such deep localizations are very likely to be underestimated, raising the problem of their detection and the correct protocol to follow asymptomatic forms, especially to detect disease progression to malignant degeneration which has a poor prognosis. Patients with a NF V should receive genetic counselling with a search for a family history, other signs of neurofibromatosis and Lisch nodules. In young patients, the risk of deep asymptomatic spread underlines the importance of regional computed tomographic or magnetic resonance explorations.

Adult↗
PubMed1998

[Autoimmune cholangitis successfully treated with corticotherapy. One case].

Autoimmune cholangitis is a rare cause of chronic liver disease which has recently been described and associates the clinical, biological, and histological patterns of primary biliary cirrhosis without serum anti-mitochondrial antibodies. We report a case of this disease in a 67-year-old female. The patient presented with jaundice and marked biological cholestasis associated with pulmonary fibrosis and salivary and lacrymal sicca syndrome. Serum anti-smooth muscle antibodies were found without anti-mitochondrial antibodies. Corticotherapy resulted in rapid improvement of clinical and hepatic abnormalities, as well as of pulmonary lesions. The patient was still healthy 18 months later, with low dose corticotherapy. This report emphasizes the possible effectiveness of corticotherapy in autoimmune cholangitis.

Aged↗
PubMed1998

[Cutaneous vasculitis disclosing cat-scratch disease].

BACKGROUND: We describe a case of cat-scratch disease ingnaugurated by vascular purpura and discuss the role of the causal agent, Bartonella henselae. CASE REPORT: A 49-year-old woman presented vascular purpura without fever. Skin biopsy demonstrated leukocytoclasic vasculitis. She owned a cat and a had a scratch scar on the back of her left hand. A few days later, two inflammatory epitrochlear lymph nodes suppurated. Catscratch disease was confirmed by serology and PRC analysis of pus aspirated from the nodes. The purpura resolved spontaneously in three weeks. Left axillary adenopathy developed and suppurated. In spite of four antibiotics, the nodes subsided only ten months later, leaving scars. DISCUSSION: Two arguments favor Bartonella henselae as the causal agent in this cutaneous vasculitis. The simultaneous onset of the two diseases and the absence of another cause of vasculitis. This patient did not have Bartonella henselae endocarditis which could have explained this vasculitis. Only one case of hypersensitivity vasculitis has been described during cat-scratch disease. The exceptional feature of this association is perhaps the result of the unawareness of moderate or asymptomatic cat-scratch disease. Bartonella henselae was possibility caused hypersensitivity vasculitis.

Bartonella henselae↗
PubMed1997

Binding of steroid modulators to recombinant cytosolic domain from mouse P-glycoprotein in close proximity to the ATP site.

We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J. -C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658]. The present work shows that a longer 371-705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Delta6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 microM. The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching. A similar differential interaction was observed with full-length purified P-glycoprotein. The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations. In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.

ATP Binding Cassette Transporter, Subfamily B, Mem↗