Analysis of the effects of snake venom proteinases on the activity of human plasma C1 esterase inhibitor, alpha 1-antichymotrypsin and alpha 2-antiplasmin

Incubation of C1 esterase inhibitor with Crotalid, Viperid and Colubrid snake venoms resulted in enzymatic inactivation of the inhibitor. Intact inhibitor (104 kDa) was converted into an active intermediate species of 89 kDa and then a further cleavage resulted in formation of an 86-kDa inactive inhibitor. In contrast, C1 esterase inhibitor did not lose activity during incubation with Elapid venoms; however, the intact inhibitor was gradually converted to an active species of 89 kDa during the incubation. Human alpha 1-antichymotrypsin was inactivated by all venoms tested, including those from the Elapid family. The 67-kDa intact inhibitor was converted by the venom proteinases to an inactive 63-kDa form. The results suggest that this acute-phase plasma protein is readily susceptible to inactivation by venom proteinases. Human alpha 2-antiplasmin (68 kDa) was cleaved to form a 61-kDa active intermediate, which then underwent a second cleavage to produce an inactive 53-kDa product. Elapid venoms had no effect on alpha 2-antiplasmin activity and did not cleave this inhibitor. All inhibitors were inactivated with catalytic amounts of venom proteinases. No stable proteinase-proteinase inhibitor complexes were detected, and no random proteolysis of the inhibitors occurred.     

West African and Amerindian ancestry and risk of myocardial infarction and metabolic syndrome in the Central Valley population of Costa Rica

Genetic ancestry and environmental factors may contribute to the ethnic differences in risk of coronary heart disease (CHD), metabolic syndrome (MS) or its individual components. The population of the Central Valley of Costa Rica offers a unique opportunity to assess the role of genetic ancestry in these chronic diseases because it derived from the admixture of a relatively small number of founders of Southern European, Amerindian, and West African origin. We aimed to determine whether genetic ancestry is associated with risk of myocardial infarction (MI), MS and its individual components in the Central Valley of Costa Rica. We genotyped 39 ancestral informative markers in cases (n = 1,998) with a first non-fatal acute MI and population-based controls (n = 1,998) matched for age, sex, and area of residence, to estimate individual ancestry proportions. Odds ratios (ORs) and 95% confidence intervals (95% CI) were estimated using conditional (MI) and unconditional (MS and its components) logistic regression adjusting for relevant confounders. Mean individual ancestry proportions in cases and controls were 57.5 versus 57.8% for the Southern European, 38.4 versus 38.3% for the Amerindian and 4.1 versus 3.8% for the West African ancestry. Compared with Southern European ancestry, each 10% increase in West African ancestry was associated with a 29% increase in MI, OR (95% CI) = 1.29 (1.07, 1.56), and with a 30% increase on the risk of hypertension, OR (95% CI) = 1.30 (1.00, 1.70). Each 10% increase in Amerindian ancestry was associated with a 14% increase on the risk of MS, OR (95% CI) = 1.14 (1.00, 1.30), and 20% increase on the risk of impaired fasting glucose, OR (95% CI) = 1.20 (1.01, 1.42). These results show that the high variability of admixture proportions in the Central Valley population offers a unique opportunity to uncover the genetic basis of ethnic differences on the risk of disease.     

Complete mitochondrial DNA sequences of the frigate tuna Auxis thazard and the bullet tuna Auxis rochei.

The complete mitochondrial DNA sequence of the frigate tuna Auxis thazard and two divergent mitotypes (Mitotype I and Mitotype II) of the bullet tuna Auxis rochei have been determined. The total length of the mitogenomes was 16,506, 16,501 and 16,503 bp, respectively. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs and 22 tRNAs) and organization similar to those observed in most other vertebrates. The major non-coding region (control region) ranged between 843 and 847 bp in length, and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Auxis with respect to other tuna fish. Molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.     

Characterization of eight microsatellite markers in the white sea bream, Diplodus sargus (Teleostei, Sparidae)

The white sea bream, Diplodus sargus (Teleostei, Sparidae), is a species with a high commercial importance in Mediterranean aquaculture. There is currently little information available about the genetic characteristics of cultured populations. In this survey, we have developed eight polymorphic microsatellites for the white sea bream using an enriched genome library protocol. All of them were polymorphic in the 67 individuals tested, 32 of which were wild specimens, and 35 were individuals from a captive F(1) broodstock. These markers can potentially be useful tools for use in population genetic studies.     

Analysis of 17,576 potentially functional SNPs in three case-control studies of myocardial infarction

Myocardial infarction (MI) is a common complex disease with a genetic component. While several single nucleotide polymorphisms (SNPs) have been reported to be associated with risk of MI, they do not fully explain the observed genetic component of MI. We have been investigating the association between MI and SNPs that are located in genes and have the potential to affect gene function or expression. We have previously published studies that tested about 12,000 SNPs for association with risk of MI, early-onset MI, or coronary stenosis. In the current study we tested 17,576 SNPs that could affect gene function or expression. In order to use genotyping resources efficiently, we staged the testing of these SNPs in three case-control studies of MI. In the first study (762 cases, 857 controls) we tested 17,576 SNPs and found 1,949 SNPs that were associated with MI (P<0.05). We tested these 1,949 SNPs in a second study (579 cases and 1159 controls) and found that 24 SNPs were associated with MI (1-sided P<0.05) and had the same risk alleles in the first and second study. Finally, we tested these 24 SNPs in a third study (475 cases and 619 controls) and found that 5 SNPs in 4 genes (ENO1, FXN (2 SNPs), HLA-DPB2, and LPA) were associated with MI in the third study (1-sided P<0.05), and had the same risk alleles in all three studies. The false discovery rate for this group of 5 SNPs was 0.23. Thus, we have identified 5 SNPs that merit further examination for their potential association with MI. One of these SNPs (in LPA), has been previously shown to be associated with risk of cardiovascular disease in other studies.     

Mechanisms of intake induction of a low-nutritious food in sheep (Ovis aries)

Intake induction refers to the phenomenon by which animals increase consumption of a less-valued meal when followed by a highly-preferred food relative to when followed by no food or by the same less-preferred food. In the Training phase of the present experiment, we assessed the induction effect in sheep using a within-subject design where learning could be tested while controlling for digestive state. Results showed that, once intake reached stability, subjects ate more low-nutritious food (oat hay) when followed than when preceded by a preferred food (soybean meal), supporting the learning hypothesis of induction. The objective of the second, Revaluation, phase of the experiment was to explore the associative mechanism of induction, for which we paired gastrointestinal malaise caused by lithium chloride intoxication with consumption of soybean meal or a control food (wheat bran). Despite subjects partially rejecting soybean meal relative to controls after the aversive conditioning protocol, oat hay consumption seemed unaffected by soybean meal devaluation. We conclude that intake induction in sheep may rely on changes in hedonic properties of the low-nutritious food based on its association with post-ingestive feedback from the preferred food (hedonic hypothesis), but not on an explicit anticipation of the latter (signalling hypothesis).     

Isolation from opossum serum of a metalloproteinase inhibitor homologous to human alpha 1B-glycoprotein.

Fractionation of opossum (Didelphis virginiana) serum with (NH4)2SO4, followed by chromatography on DEAE-Sepharose, phenyl-Sepharose, and Mono Q HR 5/5, has resulted in the isolation in homogeneous condition of a metalloproteinase inhibitor designated oprin (opossum proteinase inhibitor). Oprin is a single-chain glycoprotein (26% carbohydrate) with an estimated Mr = 52,000, pI = 3.5, and E(1%/1 cm) = 11. Oprin inhibited snake venom metalloproteinases, but showed no activity on venom serine proteinases or on bacterial metalloproteinases. Incubation of Crotalus atrox alpha-proteinase (EC 3.4.24.1) with oprin, and analysis of the reaction products by chromatography on Mono Q HR 5/5 and by electrophoresis under nondenaturing conditions, indicated formation of an inactive enzyme/inhibitor complex. The complex dissociated during SDS/polyacrylamide gel electrophoresis. An opossum liver cDNA library was immunoscreened, and clones containing cDNA encoding for part of the open reading frame for oprin were isolated. The cDNA inserts contained nucleotide sequences corresponding to two internal amino acid sequences of oprin which had been separately determined by protein sequence analysis. Protein database screening using a 211 amino acid sequence deduced from one of the cDNA inserts showed no significant homology to known proteinase inhibitors. There was, however, a 36% identity with human alpha 1B-glycoprotein, a plasma protein of unknown function related to the immunoglobulin supergene family. In addition, the amino-terminal sequence of oprin showed 46% identity with human alpha 1B-glycoprotein in a 26 amino acid residue overlap.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Scavenger Receptor Class B Type I in Hepatitis C Virus Entry: Kinetics and Molecular Determinants

Scavenger receptor class B type I (SR-BI) is an essential receptor for hepatitis C virus (HCV) and a cell surface high-density-lipoprotein (HDL) receptor. The mechanism of SR-BI-mediated HCV entry, however, is not clearly understood, and the specific protein determinants required for the recognition of the virus envelope are not known. HCV infection is strictly linked to lipoprotein metabolism, and HCV virions may initially interact with SR-BI through associated lipoproteins before subsequent direct interactions of the viral glycoproteins with SR-BI occur. The kinetics of inhibition of cell culture-derived HCV (HCVcc) infection with an anti-SR-BI monoclonal antibody imply that the recognition of SR-BI by HCV is an early event of the infection process. Swapping and single-substitution mutants between mouse and human SR-BI sequences showed reduced binding to the recombinant soluble E2 (sE2) envelope glycoprotein, thus suggesting that the SR-BI interaction with the HCV envelope is likely to involve species-specific protein elements. Most importantly, SR-BI mutants defective for sE2 binding, although retaining wild-type activity for receptor oligomerization and binding to the physiological ligand HDL, were impaired in their ability to fully restore HCVcc infectivity when transduced into an SR-BI-knocked-down Huh-7.5 cell line. These findings suggest a specific and direct role for the identified residues in binding HCV and mediating virus entry. Moreover, the observation that different regions of SR-BI are involved in HCV and HDL binding supports the hypothesis that new therapeutic strategies aimed at interfering with virus/SR-BI recognition are feasible.Hepatitis C virus (HCV) is a global blood-borne pathogen, with 3% of the world''s population chronically infected. Most infections are asymptomatic, yet 60 to 80% become persistent and lead to severe fibrosis and cirrhosis, hepatic failure, or hepatocellular carcinoma (3). Currently available therapies are limited to the administration of pegylated alpha interferon in combination with ribavirin, which are expensive and often unsuccessful, with significant side effects (23, 36). Thus, the development of novel therapeutic approaches against HCV remains a high priority (18, 40, 60). Targeting the early steps of HCV infection may represent one such option, and much effort is being devoted to uncovering the mechanism of viral attachment and entry.The current view is that HCV entry into target cells occurs after attachment to specific cellular receptors via its surface glycoproteins E1 and E2 (27). The molecules to which HCV initially binds might constitute a diverse collection of cellular proteins, carbohydrates, and lipids that concentrate viruses on the cell surface and determine to a large extent which cell types, tissues, and organisms HCV can infect.CD81, claudin 1 (CLDN1), occludin (OCLN), and scavenger receptor class B type I (SR-BI) were previously shown to play essential roles in HCV cell entry (15, 22, 26, 35, 42, 43, 50, 63, 64).Recent reports suggest that CD81 engagement triggers intracellular signaling responses, ultimately leading to actin remodeling and the relocalization of CD81 to tight junctions (TJ) (11). Thus, CD81 may function as a bridge between the initial interaction of the virus with receptors on the basolateral surface of the hepatocyte and the TJ where two of the HCV entry molecules, CLDN1 and OCLN, are located. CD81 acts as a postbinding factor, and the TJ proteins CLDN1 and OCLN seem to be involved in late steps of HCV entry, such as HCV glycoprotein-dependent cell fusion (9, 11, 22). The discovery of TJ proteins as entry factors has added complexity to the model of HCV entry, suggesting parallels with other viruses like coxsackievirus B infection, where an initial interaction of the viral particle with the primary receptor decay-accelerating factor induces the lateral movement of the virus from the luminal surface to TJ, where coxsackievirus B binds coxsackievirus-adenovirus receptor and internalization takes place (17).Much less is known about the specific role of SR-BI in virus entry: neither the specific step of the entry pathway that SR-BI is involved in nor the protein determinants that mediate such processes are known. SR-BI is a lipoprotein receptor of 509 amino acids (aa) with cytoplasmic C- and N-terminal domains separated by a large extracellular domain (1, 13, 14). It is expressed primarily in liver and steroidogenic tissues, where it mediates selective cholesteryl ester uptake from high-density lipoprotein (HDL) and may act as an endocytic receptor (45, 46, 51, 52). SR-BI was originally identified as being a putative receptor for HCV because it binds soluble E2 (sE2) through interactions with E2 hypervariable region 1 (HVR1) (8, 50). RNA interference studies as well as the ability to block both HCV pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) infections with anti SR-BI antibodies have confirmed its involvement in the HCV entry process (7, 8, 15, 26, 33, 63). Intriguingly, lipoproteins were previously shown to modulate HCV infection through SR-BI (12). It was indeed previously demonstrated that two natural ligands of SR-BI, HDL and oxidized low-density lipoprotein, can improve and inhibit HCV entry, respectively (57, 59). Moreover, small-molecule inhibitors of SR-BI-mediated lipid transfer (block of lipid transfer BLT-3 and BLT-4) abrogate the stimulation of HCV infectivity by human serum or HDL, suggesting that the enhancement of viral infection might be dependent on the lipid exchange activity of SR-BI (20, 58).We previously generated high-affinity monoclonal antibodies (MAbs) specific for human SR-BI and showed that they were capable of inhibiting the binding of SR-BI to sE2 and blocking HCVcc infection of human hepatoma cells (15). The HDL-induced enhancement of infection had no impact on the ability of the anti-SR-BI MAbs to block HCV infection, and the antibodies were effective in counteracting HCV infection even in the absence of lipoproteins. These data demonstrated that SR-BI participates in the HCV infection process as an entry receptor by directly interacting with viral glycoproteins. Here we have used one of the anti-SR-BI MAbs to show that SR-BI participates in an early step of HCV infection. By assays of binding of sE2 to SR-BI molecules from different species and to SR-BI mutants, we identified species-specific SR-BI protein residues that are required for sE2 binding. The functional significance of these observations was confirmed by the finding that SR-BI mutants with reduced binding to sE2 were also impaired in their ability to restore the infectivity of an SR-BI-knocked-down Huh-7.5 cell line. Finally, we demonstrated that SR-BI mutants with impaired sE2 binding can still form oligomeric structures and that they can bind the physiological ligand HDL and mediate cholesterol efflux, suggesting that distinct protein determinants are responsible for the interaction with HDL and the HCV particle.