Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 g/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)- containing FcR chain. Conversely, thrombin only activated at high concentrations ( 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2 mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)– containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature. (Circ Res. 2004;94:1598-1605.)

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Diversity in expression of phosphorus (P) responsive genes in Cucumis melo L.

Background: Phosphorus (P) is a major limiting nutrient for plant growth in many soils. Studies in model species have identified genes involved in plant adaptations to low soil P availability. However, little information is available on the genetic bases of these adaptations in vegetable crops. In this respect, sequence data for melon now makes it possible to identify melon orthologues of candidate P responsive genes, and the expression of these genes can be used to explain the diversity in the root system adaptation to low P availability, recently observed in this species. Methodology and Findings: Transcriptional responses to P starvation were studied in nine diverse melon accessions by comparing the expression of eight candidate genes (Cm-PAP10.1, Cm-PAP10.2, Cm-RNS1, Cm-PPCK1, Cm-transferase, Cm-SQD1, Cm-DGD1 and Cm-SPX2) under P replete and P starved conditions. Differences among melon accessions were observed in response to P starvation, including differences in plant morphology, P uptake, P use efficiency (PUE) and gene expression. All studied genes were up regulated under P starvation conditions. Differences in the expression of genes involved in P mobilization and remobilization (Cm-PAP10.1, Cm-PAP10.2 and Cm-RNS1) under P starvation conditions explained part of the differences in P uptake and PUE among melon accessions. The levels of expression of the other studied genes were diverse among melon accessions, but contributed less to the phenotypical response of the accessions. Conclusions: This is the first time that these genes have been described in the context of P starvation responses in melon. There exists significant diversity in gene expression levels and P use efficiency among melon accessions as well as significant correlations between gene expression levels and phenotypical measurements.

Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von Willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αIIbβ3

OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.

CLEC-2 expression is maintained on activated platelets and on platelet microparticles

The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in FcγRIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-gamma) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 x 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

Differential antibody isotype expression to the major Paracoccidioides brasiliensis antigen in juvenile and adult form paracoccidioidomycosis

We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms. Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients' sera. Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients. IgG1 subclass levels, however, were comparable between the two clinical forms. Patients with the juvenile form had higher (P <.001) IgG4, but lower(P =.03) IgG2 levels than patients with the adult form. The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients. In contrast, high levels of the IgG2 isotype, regulated by interferon-gamma, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients. IgG3 was either absent or detected at low levels. These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-l or Th-2) dominant cytokine profile. A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed. Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form. (C) Elsevier, Paris.

Quantification of bovine cytokine gene expression using real-time RT-PCR methodology

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene expression in placentation of farm animals: An overview of gene function during development

Eutherian mammals share a common ancestor that evolved into two main placental types, i.e., hemotrophic (e.g., human and mouse) and histiotrophic (e.g., farm animals), which differ in invasiveness. Pregnancies initiated with assisted reproductive techniques (ART) in farm animals are at increased risk of failure; these losses were associated with placental defects, perhaps due to altered gene expression. Developmentally regulated genes in the placenta seem highly phylogenetically conserved, whereas those expressed later in pregnancy are more species-specific. To elucidate differences between hemotrophic and epitheliochorial placentae, gene expression data were compiled from microarray studies of bovine placental tissues at various stages of pregnancy. Moreover, an in silico subtractive library was constructed based on homology of bovine genes to the database of zebrafish - a nonplacental vertebrate. In addition, the list of placental preferentially expressed genes for the human and mouse were collected using bioinformatics tools (Tissue-specific Gene Expression and Regulation [TiGER] - for humans, and tissue-specific genes database (TiSGeD) - for mice and humans). Humans, mice, and cattle shared 93 genes expressed in their placentae. Most of these were related to immune function (based on analysis of gene ontology). Cattle and women shared expression of 23 genes, mostly related to hormonal activity, whereas mice and women shared 16 genes (primarily sexual differentiation and glycoprotein biology). Because the number of genes expressed by the placentae of both cattle and mice were similar (based on cluster analysis), we concluded that both cattle and mice were suitable models to study the biology of the human placenta. (C) 2011 Elsevier B.V. All rights reserved.

Distribution of exoantigens and a 43-kDa glycoprotein (gp43) in the yeast and mycelial forms of Paracoccidioides brasiliensis

Objective. Paracoccidioides brasiliensis antigens (strain 113) were located at ultrastructural level in both yeast and mycelial forms of the fungus. The reactivity of the sera employed was analysed. Materials and methods. Immunofluorescence and ultrastructural protein A-gold immunolabelling techniques were performed using two polyclonal antisera: one against P. brasiliensis exoantigens and the other against a 43-kDa glycoprotein (gp43). Immunoblotting assays were employed to define reactivity of these antisera with somatic and metabolic antigens of both forms of the fungus. Results. The techniques employed revealed in both yeast and mycelial forms of P. brasiliensis a similar antigenic distribution. The antigens deposits were seen within the cytoplasm, and over the cell wall of the fungus. The anti-exoantigen serum recognized several bands in both forms of the fungus. The anti-gp43 serum reacted strongly with the 43-kDa fraction and weakly with few other fractions. Conclusions. Immunocytochemical techniques suggest a protein synthesis within the cytoplasm followed by excretion through the cell wall. Similar results employing both polyclonal antisera were obtained.

Expressão da glicoproteína-p e da mrp1 em tecidos de cães portadores de leishmaniose visceral

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)