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V David

Publications and source records attributed to V David.

At least 55 records · Page 3Linked to original sources
PubMed1995

The amino-terminal part of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-rich region acts as a stimulator.

The intracellular bacterial pathogen Listeria monocytogenes moves inside the host-cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin-driven bacterial propulsion, we have now generated several L. monocytogenes strains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino-terminal part is critical for F-actin assembly and movement. The internal proline-rich repeats and the carboxy-terminal domains are not essential. However, in vitro motility assays have demonstrated that mutants lacking the proline-rich repeats domain of ActA moved two times slower (6+/-2 micrometers min(-1)) than the wild type (13 +/-3 micrometers min(-1)). In addition, phosphatase treatment of protein extracts of cells infected with the L. monocytogenes strains expressing the ActA variants suggested that phosphorylation may not be essential for ActA activity.

Actins↗
PubMed1994

A comparative study of self-administration of morphine into the amygdala and the ventral tegmental area in mice.

BALB/c mice were unilaterally implanted with a guide-cannula, the tip of which was positioned 1.5 mm above either the amygdala (AMY) or the ventral tegmental area (VTA). On each experimental day, a stainless-steel injection cannula was inserted into these structures in order to compare the self-administration of two doses of morphine (5 ng or 50 ng) in independent groups using a spatial discrimination task in a Y-maze. During the acquisition phase, both AMY and VTA injected mice showed a regular self-administration response at the two doses used. The latency to trigger the injection was short, particularly in the VTA group. Subcutaneous injection of naloxone (4 mg/kg) in trained mice reduced the number of self-administrations to a level near to chance in both groups, which suggests that the drug-seeking behavior observed is effectively dependent on an opiate receptor-mediated mechanism. However the rate of extinction was more rapid in AMY than in VTA injected mice. The 'perseveration' response exhibited by the VTA group during the withdrawal precipitated by naloxone may probably be due to the strong motivational and/or rewarding effect of morphine when injected in this brain structure during acquisition.

Amygdala↗
PubMed1994

Differentiation of intracranial morphine self-administration behavior among five brain regions in mice.

BALB/c mice were unilaterally implanted with a guide cannula, the tip of which was positioned 1.5 mm above either the lateral hypothalamus (LH) the medial hypothalamus (MH), the mesencephalic central gray area (CG), or either the dorsal (DRF) or ventral parts (VRF) of the reticular formation. On each day of the experimental period a stainless steel injection cannula was inserted into these brain structures to compare the self-administration of two doses of morphine (5 ng or 50 ng), using a spatial discrimination task in a Y-maze. At the dose of 5 ng, LH-, MH-, CG-, and VRF-injected mice all showed a regular self-administration response. At the dose of 50 ng, a discrimination between the reinforced arm and the neutral arm of the Y-maze was observed in LH-, MH-, and VRF-injected mice. Animals of the MH group exhibited the highest level of discrimination performance. At this dose, long injection latencies (> 15 min) were recorded in the CG group, which constrained us to reduce the number of daily trials from 10 to 4. In these modified conditions, CG animals clearly self-injected the dose of 50 ng of morphine. Subcutaneous injections of naloxone (4 mg/kg) reduced the number of self-administrations of morphine at each of the four responding structures. Marked signs of physical dependence (escape attempts) were observed in the four groups but with a higher frequency in CG and MH animals. When the injections of naloxone were suspended, a regular self-administration reappeared.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Prognosis of first-trimester twin pregnancies: polychotomous logistic regression analysis.

PURPOSE: To determine which clinical and sonographic characteristics independently affect the prognosis of first-trimester twin pregnancies and to develop formulas for calculating the probabilities of the pregnancy resulting in two, one, or no liveborn infants. MATERIALS AND METHODS: The authors prospectively identified 137 twin pregnancies with two heartbeats at first-trimester sonography and with known pregnancy outcome. Stepwise polychotomous logistic regression analysis was used to identify characteristics that have an independent statistically significant relationship with pregnancy outcome and to develop outcome-prediction formulas. RESULTS: Of 137 patients, 110 (80.3%) had viable twins, 12 (8.8%) had one infant, and 15 (10.9%) had none. Gestational age, chorionicity, and sonographic findings were independent, statistically significant (P < .05) prognostic factors, while maternal age, method of conception, and indication for sonography were not. CONCLUSION: The logistic regression formulas involving the three independent prognostic factors can be used in the first trimester to predict pregnancy outcome.

Adult↗
PubMed1993

Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin).

A cDNA clone encoding the human endoplasmic reticulum (ER) resident protein IP90 was isolated and sequenced. It predicts a transmembrane protein with a large ER luminal region showing sequence similarity to calreticulin and a short cytoplasmic domain containing a COOH-terminal RKPRRE sequence that may be relevant to its retention in the ER. It is 95% homologous to the canine ER membrane phosphorprotein called pp90 or calnexin (Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J. J., II, Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). Previously, in lymphocytes, we have characterized IP90 as a protein associated with partially assembled multichain proteins including the T cell receptor, the membrane immunoglobulin, and the heavy chain of the major histocompatibility complex class I molecules (Hochstenbach, F., David, V., Watkins, S., and Brenner, M. B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4734-4738). Here, we show that within a short metabolic labeling period, IP90 associates transiently with many different newly synthesized proteins. However, in a T cell line that cannot assemble a complete T cell receptor because it lacks the alpha subunit, the unassembled T cell receptor beta chains, which are retained in the ER, remain associated with IP90 throughout a prolonged chase time period. Together, these data offer further evidence suggesting that IP90 may act in assisting protein assembly and/or in the retention within the ER of unassembled protein subunits.

Amino Acid Sequence↗
PubMed1993

Analysis of 160 CF chromosomes: detection of a novel mutation in exon 20.

The cystic fibrosis (CF) gene has been cloned and a major mutation identified (delta F508). This 3-bp deletion has been found in approximately 70% of CF chromosomes. We have used the strategy of denaturing gradient gel electrophoresis followed by direct sequencing of the polymerase chain reaction products, in order to detect other mutations in exons 10, 11 and 20 of the CF transmembrane conductance regulator gene. A new mutation, F1286-S, was found in exon 20. It involves a nucleotide change of T-->C at nucleotide 3989 and changes a phenylalanine into serine at position 1286 of the protein.

Adolescent↗
PubMed1993

Localization of seven new genes around the HLA-A locus.

A yeast artificial chromosome (YAC B30) with a 320 kb insert of genomic DNA which includes the HLA-A gene was used to screen a cDNA library of human duodenal mucosa. Seven cDNA clones were isolated which correspond to seven new non-HLA class I structural genes. These new genes are located within a region that may well contain the gene responsible for hemochromatosis and have therefore been named HCG I-VII (Hemochromatosis Candidate Gene). HCG I, III, V and VI are probably single copy genes, situated at 180, 155, 140 and 230 kb centromeric to HLA-A, respectively. HCG II, IV and VII present several copies: one copy of HCG II, one of HCG IV and one of HCG VII are centromeric to HLA-A (at 30, 70 and 100 kb respectively). Another copy of HCG IV is 20 kb telomeric to HLA-A. Each of the genes localized on the YAC B30 is associated with an CpG/HTF island.

Blotting, Southern↗
PubMed1993

[Molecular genetics of hemochromatosis].

Haemochromatosis is an inherited disorder of iron metabolism characterized by a general iron over loading. Without diagnosis and early treatment, it is a serious and potentially fatal disease by cardiac failure or hepatocellular carcinoma in particular. Gene prevalence was estimated at 0.06 in Brittany, so that haemochromatosis may be the most common genetic disease in this area. The biochemical defect of the disease is unknown; only one fact is well established: the iron absorption through duodenal mucosa is excessive. However we don't know if it is a primary event. The gene is also unknown but in 1975 it was located on the short arm of chromosome 6, closely linked to the HLA class I region, less than 1 cM from HLA-A. None of the genes coding for the known iron proteins could be the haemochromatosis gene because of their chromosomal localization. In order to locate this gene with precision, we have used a reverse genetic approach now called positional cloning. Characterization of new polymorphic markers and linkage disequilibrium analysis, have led us to locate the gene within a 350 kb region around HLA-A. We have then searched for all the structural genes in this region. Seven new genes have been so identified and located with precision. A structural analysis of these genes was undertaken to find an eventual abnormality in patients.

Hemochromatosis↗
PubMed1992

Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly.

In the endoplasmic reticulum (ER), newly synthesized subunits of the T-cell antigen receptor (TCR), membrane-bound immunoglobulin (mIg), and major histocompatibility complex (MHC) class I antigens must fold correctly and assemble completely into multimeric protein complexes prior to transport to the cell surface. Although folding and assembly may occur spontaneously, the concept that molecular chaperones facilitate these events is emerging. Here, an intracellular protein of 90-kDa apparent molecular mass, denoted IP90, was shown to be an ER resident protein that associated with partial complexes of the TCR, mIg, and MHC class I proteins but was absent from fully assembled complexes. We speculate that IP90 might participate in folding and assembly processes of these and other multisubunit protein complexes during their transit through the ER.

Animals↗
PubMed1992

A continuous restriction map from HLA-E to HLA-F. Structural comparison between different HLA-A haplotypes.

The class I region of the human major histocompatibility complex contains genes encoding the classical transplantation antigens (HLA-A, B, and C), at least three new class I genes (HLA-E, F, and G) and many class I pseudogenes (including HLA-H). By pulse field gel electrophoresis and using five rare cutter enzymes, we have constructed a precise and continuous map of 1200 kilobases (kb) around HLA-A. The blots were hybridized with HLA-A, E, and F-specific probes and with new probes derived from yeast artificial chromosomes and cosmids of the class I region. We have compared the genomic organization of the same 1200 kb in three homozygous lymphoblastoid cell lines corresponding to three different HLA haplotypes (A3, A24, and A31). The differences in size observed may have been caused by insertions and deletions and may prove valuable in understanding the evolution of the HLA chromosomal region.

Genes, MHC Class I↗
PubMed1992

Germ line variable regions that match hypermutated sequences in genes encoding murine anti-hapten antibodies.

We asked whether there are germ line immunoglobulin variable (V) segments that match sites of hypermutation in V regions encoding murine antibodies. Murine germ line DNA was probed with a panel of short deoxyoligonucleotides identical in sequence to segments of hypermutated V regions from hybridomas generated in the BALB/c response to the hapten 2-phenyloxazolone (Ox). Germ line sequences that match mutations in both heavy and kappa light chain V regions were identified, and clones of some of these germ line V segments were obtained. Comparison of these clones with hypermutated V regions revealed regions of identity ranging in size from 7 to over 50 nucleotides. In an effort to separate the effects of antigen selection from the mutagenic process, we also searched for matches to a panel of silent mutations in VH regions from germinal center B cells. Fourteen silent mutations occur among a collection of 36 hypermutated VH regions from two separate germinal centers of C57BL/6 mice stimulated with the hapten 4-hydroxy-3-nitrophenyl. Matches to nine of these silent mutations can be found among published sequences of C57BL/6 VH regions of the J558 family. Taken together, these data are consistent with the possibility that a template-dependent mutational process, like gene conversion, may contribute to somatic hypermutation.

Animals↗
PubMed1992

Familial screening for genetic haemochromatosis by means of DNA markers.

Genetic haemochromatosis (HFE) is a frequent and potentially fatal disease. Early phlebotomies may prevent complications. The recessive gene for HFE is unknown but closely linked to the HLA-A locus. No direct test for homozygosity for HFE is currently available, apart from HLA typing within the family of a patient with confirmed HFE. During a reverse genetic approach to identify the gene, we found three anonymous genomic probes (P3, P5, and I.82) derived from previously cloned YACs and physically mapped in the HLA class I region. P3 and P5 probes recognise 3 loci (P3A, P3B, P5) and I.82 one locus about 100 kb from HLA-A. Using five biallelic polymorphisms (I.82/BglII, P3B/EcoRV, P3B/PstI, P5/HindIII, P3A/PstI), we tested 198 HLA typed subjects from the families of 22 haemochromatosis patients. The information from the five polymorphisms was sufficient to identify unequivocally extended restriction haplotypes in all families. The restriction haplotypes cosegregate with the HFE allele and enable identification of genotypically identical sibs in all families studied. The linked DNA markers described in this article avoid the disadvantages of HLA serological typing and can be used in genetic counselling of HFE families.

DNA↗