Anticoagulant and antiplatelet agents: their clinical and device application(s) together with usages to engineer surfaces

An essential aspect of the treatment of patients with cardiovascular disease is the use of anticoagulant and antiplatelet agents for the prevention of further ischaemic events and vascular death resulting from thrombosis. Aspirin and heparin have been the standard therapy for the management of such conditions to date. Recently, numerous more potent platelet inhibitors together with anticoagulant agents have been developed and tested in randomized clinical trials. This article reviews the current state of the art of antiplatelet and anticoagulant therapy in light of its potential clinical efficacy. It then focuses on the usages of these agents in order to improve the performance of clinical devices such as balloon catheters, coronary stents, and femoropopliteal bypass grafting and extra corporeal circuits for cardiopulmonary bypass. The article then goes on to look at the usage of these agents more specifically heparin, heparan, hirudin, and coumarin in the development of more biocompatible scaffolds for tissue engineering.     

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.     

QSAR study on phenolic activity: need of positive hydrophobic term (log P) in QSAR

The phenolic activity (log 1/C) of a large series of phenols against L1210 leukaemia cells was modelled using physicochemical parameters other than conventional electronic and steric parameters. Attempts have also been made to examine need or otherwise of the hydrophobic parameter, log P, in such studies. The results have shown that contribution of log P in modelling log 1/C is favourable.     

Regulation of cell motility by tyrosine phosphorylated villin

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.     

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.     

Cytokine polymorphisms and histologic expression in autopsy studies: contribution of TNF-alpha and TGF-beta 1 to the pathogenesis of autoimmune-associated congenital heart block

Although Abs to SSA/Ro-SSB/La are necessary for the development of congenital heart block (CHB), the low frequency suggests that fetal factors are contributory. Because CHB involves a cascade from inflammation to scarring, polymorphisms of the TNF-alpha promoter region and codons 10 and 25 of the TGF-beta gene were evaluated in 88 children (40 CHB, 17 rash, 31 unaffected siblings) and 74 mothers from the Research Registry for Neonatal Lupus (NL). Cytokine expression was assessed in autopsy material from two fetuses with CHB. Significantly increased frequency of the -308A (high-producer) allele of TNF-alpha was observed in all NL groups compared with controls. In contrast, the TGF-beta polymorphism Leu(10) (associated with increased fibrosis) was significantly higher in CHB children (genotypic frequency 60%, allelic frequency 78%) than unaffected offspring (genotypic frequency 29%, p = 0.016; allelic frequency 56%, p = 0.011) and controls, while there were no significant differences between controls and other NL groups. For the TGF-beta polymorphism, Arg(25), there were no significant differences between NL groups and controls. In fetal CHB hearts, protein expression of TGF-beta, but not TNF-alpha, was demonstrated in septal regions, extracellularly in the fibrous matrix, and intracellularly in macrophage infiltrates. Age-matched fetal hearts from voluntary terminations expressed neither cytokine. TNF-alpha may be one of several factors that amplify susceptibility; however, the genetic studies, backed by the histological data, more convincingly link TGF-beta to the pathogenesis of CHB. This profibrosing cytokine and its secretion/activation circuitry may provide a novel direction for evaluating fetal factors in the development of a robust animal model of CHB as well as therapeutic strategies in humans.     

Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences

The antigenic diversity of human immunodeficiency virus type 1 (HIV-1) represents a significant challenge for vaccine design as well as the comprehensive assessment of HIV-1-specific immune responses in infected persons. In this study we assessed the impact of antigen variability on the characterization of HIV-1-specific T-cell responses by using an HIV-1 database to determine the sequence variability at each position in all expressed HIV-1 proteins and a comprehensive data set of CD8 T-cell responses to a reference strain of HIV-1 in infected persons. Gamma interferon Elispot analysis of HIV-1 clade B-specific T-cell responses to 504 overlapping peptides spanning the entire expressed HIV-1 genome derived from 57 infected subjects demonstrated that the average amino acid variability within a peptide (entropy) was inversely correlated to the measured frequency at which the peptide was recognized (P = 6 x 10(-7)). Subsequent studies in six persons to assess T-cell responses against p24 Gag, Tat, and Vpr peptides based on autologous virus sequences demonstrated that 29% (12 of 42) of targeted peptides were only detected with peptides representing the autologous virus strain compared to the HIV-1 clade B consensus sequence. The use of autologous peptides also allowed the detection of significantly stronger HIV-1-specific T-cell responses in the more variable regulatory and accessory HIV-1 proteins Tat and Vpr (P = 0.007). Taken together, these data indicate that accurate assessment of T-cell responses directed against the more variable regulatory and accessory HIV-1 proteins requires reagents based on autologous virus sequences. They also demonstrate that CD8 T-cell responses to the variable HIV-1 proteins are more common than previously reported.     

An umbraviral protein,involved in long-distance RNA movement,binds viral RNA and forms unique,protective ribonucleoprotein complexes

Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [TMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with TMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.