The CYP2C19*17 variant is not independently associated with clopidogrel response.

BACKGROUND: Cytochrome P450 2C19 (CYP2C19) is the principal enzyme responsible for converting clopidogrel into its active metabolite, and common genetic variants have been identified, most notably CYP2C19*2 and CYP2C19*17, that are believed to alter its activity and expression, respectively. OBJECTIVE: We evaluated whether the consequences of the CYP2C19*2 and CYP2C19*17 variants on clopidogrel response were independent of each other or genetically linked through linkage disequilibrium (LD). PATIENTS/METHODS: We genotyped the CYP2C19*2 and CYP2C19*17 variants in 621 members of the Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study and evaluated the effects of these polymorphisms singly and then jointly, taking into account LD, on clopidogrel prodrug level, clopidogrel active metabolite level, and adenosine 5'-diphosphate (ADP)-stimulated platelet aggregation before and after clopidogrel exposure. RESULTS: The CYP2C19*2 and CYP2C19*17 variants were in LD (|D'| = 1.0; r(2)  = 0.07). In association analyses that did and did not account for the effects of CYP2C19*17, CYP2C19*2 was strongly associated with levels of clopidogrel active metabolite (ß = -5.24, P = 3.0 × 10(-9) and ß = -5.36, P = 3.3 × 10(-14) , respectively) and posttreatment ADP-stimulated platelet aggregation (ß = 7.55, P = 2.9 × 10(-16) and ß = 7.51, P = 7.0 × 10(-15) , respectively). In contrast, CYP2C19*17 was marginally associated with clopidogrel active metabolite levels and ADP-stimulated platelet aggregation before (ß = 1.57, P = 0.04 and ß = -1.98, P = 0.01, respectively) but not after (ß = 0.40, P = 0.59 and ß = -0.13, P = 0.69, respectively) adjustment for the CYP2C19*2 variant. Stratified analyses of CYP2C19*2/CYP2C19*17 genotype combinations revealed that CYP2C19*2, and not CYP2C19*17, was the primary determinant in altering clopidogrel response. CONCLUSIONS: Our results suggest that CYP2C19*17 has a small (if any) effect on clopidogrel-related traits and that the observed effect of this variant is due to LD with the CYP2C19*2 loss-of-function variant.

Phylogenetic analysis of the nucleoprotein gene of measles viruses prevalent in Nantong, Jiangsu Province, China, during 2010.

Measles control in China is monitored in part by surveillance of circulating wild-type viruses. The objective of this study was genetic characterization and phylogenetic analysis of measles strains in the Nantong City region of Jiangsu province, China, during 2010. Sera from suspected cases were tested for IgM antibodies and measles virus isolated by inoculation of transport medium onto Vero/SLAM cells. Isolated strains were phylogenetically analysed according to the nucleotide sequence of the C-terminal region of the nucleoprotein gene amplified by RT-PCR. The results revealed 34 cases confirmed by positive IgM, for an incidence of 0·45/100 000. Six isolates identified were all clustered within genotype H1. The findings reported here support continued endemic transmission of measles virus in China.

Selective expression of murine prostate stem cell antigen in fetal and adult tissues and the transgenic adenocarcinoma of the mouse prostate model of prostate carcinogenesis.

Prostate stem cell antigen (PSCA) is a GPI-anchored membrane protein whose expression is reportedly up-regulated in a majority of human prostate cancers, including advanced stages and metastases. In this study, we investigate the expression pattern of the murine orthologue of PSCA by in situ hybridization in fetal and adult mouse tissues. Murine PSCA is expressed during fetal development in the urogenital sinus, skin, and gastrointestinal tract. The expression in these tissues is restricted to the most superficial cell layer. In the adult mouse, expression is highest in the mucosal lining of the urinary tract. In the normal adult prostate, expression of PSCA is detected exclusively in the secretory epithelium. Examination of PSCA during carcinogenesis of the murine prostate in the transgenic adenocarcinoma of the mouse prostate model showed a markedly increased expression in areas of neoplasia. The transgenic adenocarcinoma of the mouse prostate model may represent a valuable model for the study of PSCA as a potential target for immunotherapy of prostate cancer, despite potential differences in the pattern of expression between mice and humans.

Tyrosine phosphorylation regulates the SH3-mediated binding of the Wiskott-Aldrich syndrome protein to PSTPIP, a cytoskeletal-associated protein.

Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that manifests itself in platelet deficiency and a compromised immune system. Analysis of hematopoietic cells from affected individuals reveals that mutations in the Wiskott-Aldrich syndrome protein (WASP) result in structural and functional abnormalities in the cell cortex, consistent with the suggestion that WASP is involved with regulation of the actin-rich cortical cytoskeleton. Here we report that WASP interacts with a recently described cytoskeletal-associated protein, PSTPIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p. This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP. Co-expression of PSTPIP with WASP in vivo results in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain. Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation. One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo. In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASP in vitro. These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton.

The orphan receptor CRF2-4 is an essential subunit of the interleukin 10 receptor.

The orphan receptor CRF2-4 is a member of the class II cytokine receptor family (CRF2), which includes the interferon receptors, the interleukin (IL) 10 receptor, and tissue factor. CRFB4, the gene encoding CRF2-4, is located within a gene cluster on human chromosome 21 that comprises three interferon receptor subunits. To elucidate the role of CRF2-4, we disrupted the CRFB4 gene in mice by means of homologous recombination. Mice lacking CRF2-4 show no overt abnormalities, grow normally, and are fertile. CRF2-4 deficient cells are normally responsive to type I and type II interferons, but lack responsiveness to IL-10. By approximately 12 wk of age, the majority of mutant mice raised in a conventional facility developed a chronic colitis and splenomegaly. Thus, CRFB4 mutant mice recapitulate the phenotype of IL-10-deficient mice. These findings suggest that CRF2-4 is essential for IL-10-mediated effects and is a subunit of the IL-10 receptor.

Disruption of a single allele of the nerve growth factor gene results in atrophy of basal forebrain cholinergic neurons and memory deficits.

Administration of nerve growth factor (NGF) to aged or lesioned animals has been shown to reverse the atrophy of basal forebrain cholinergic neurons and ameliorate behavioral deficits. To examine the importance of endogenous NGF in the survival of basal forebrain cholinergic cells and in spatial memory, mice bearing a disruption mutation in one allele of the NGF gene were studied. Heterozygous mutant mice (ngf+/-) have reduced levels of NGF mRNA and protein within the hippocampus and were found to display significant deficits in memory acquisition and retention in the Morris water maze. The behavioral deficits observed in NGF-deficient mice were accompanied by both shrinkage and loss of septal cells expressing cholinergic markers and by a decrease in cholinergic innervation of the hippocampus. Infusions of NGF into the lateral ventricle of adult ngf+/- mice abolished the deficits on the water maze task. Prolonged exposure to NGF may be required to induce cognitive effects, because reversal of the acquisition deficit was seen after long (5 weeks) but not short (3 d) infusion. Although NGF administration did not result in any improvement in the number of septal cells labeled for choline acetyltransferase, this treatment did effectively correct the deficits in both size of cholinergic neurons and density of cholinergic innervation of the hippocampus. These findings demonstrate the importance of endogenous NGF for survival and function of basal forebrain cholinergic neurons and reveal that partial depletion of this trophic factor is associated with measurable deficits in learning and memory.

Human trks: molecular cloning, tissue distribution, and expression of extracellular domain immunoadhesins.

Using molecular cloning techniques, human homologs of the known members of the trk family of neurotrophin receptors have been cloned and sequenced. Overall, there is a high degree of similarity between the human sequences and those from other mammals; however, there are differences in splicing patterns. There are two spliced forms of the extracellular domain of trkC in the human, a finding that has not been described in other species. In contrast, fewer spliced forms were detected of the intracellular domains of human trkB and trkC than has been described in other mammals. Northern analysis and in situ hybridization experiments indicate that the human trks are expressed in a similar pattern to that described in other mammals. Expression of the trk extracellular domains as fusion proteins with IgG heavy chain yields soluble molecules that mimic intact trks in their binding specificity and affinity. These soluble chimeras block the biological activity of their cognate neurotrophin(s) in vitro.

Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells.

The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor. Cell cycle analysis shows that stem cells that do not express the receptor have a greater percentage of the population in G0 when compared with the flk-2/flt-3-positive population. Development of agonist antibodies to the receptor shows a proliferative role for the receptor in stem cell populations. Stimulation with an agonist antibody gives rise to an expansion of both myeloid and lymphoid cells and this effect is enhanced by the addition of kit ligand. These studies serve to further illustrate the importance of the flk-2/flt-3 receptor in the regulation of the hematopoietic stem cell.

Suppression of mammalian 5' splice-site defects by U1 small nuclear RNAs from a distance.

One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both.

Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.

Physiological platelet synthesis is thought to require the humoral activities of meg-CSF and thrombopoietin, which respectively promote proliferation and maturation of megakaryocytic cells. A meg-CSF/thrombopoietin-like protein that is present in plasma of irradiated pigs has been purified and cloned. This protein binds to and activates the c-mpl protein, a member of the cytokine receptor superfamily. The isolated Mpl ligand shares homology with erythropoietin and stimulates both megakaryocytopoiesis and thrombopoiesis.

Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons.

Homologous recombination was utilized to generate mice with a deletion in the coding sequence of the nerve growth factor (NGF) gene. Animals homozygous for NGF disruption failed to respond to noxious mechanical stimuli, and histological analysis revealed profound cell loss in both sensory and sympathetic ganglia. Within dorsal root ganglia, effects of the mutation appeared to be restricted to small and medium peptidergic neurons. These observations confirm the critical dependence of sensory and sympathetic neurons on NGF and demonstrate that other neurotrophins are not able to compensate for the loss of NGF action on these cells. Examination of the central nervous system revealed that, in marked contrast with neurons of sensory and sympathetic ganglia, basal forebrain cholinergic neurons differentiate and continue to express phenotypic markers for the life span of the null mutant mice. Thus, differentiation and initial survival of central NGF-responsive neurons can occur in the absence of NGF.