Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264-7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A - studies in platelets, monocytes, and human endothelial cells

During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide plus interferon-gamma-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-alpha-induced monocyte chemoattractant protein production in primary human endothelial cells.

G protein ss gamma subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of G alpha(i)/(o)beta y subunits. Expression of G alpha transducin, an agent which sequesters G protein beta gamma (G beta y) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G beta gamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G beta gamma subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G beta gamma subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G beta gamma subunits functionally mediate inhibition of transmitter release by alpha 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

On the system of rational difference equations x(n) = A+(1)over-bar y(n-p), y(n) = A+(gamma(n-1))over-bar x(n-r)y(n-5)

In this paper, we study the behavior of the positive solutions of the system of two difference equations [GRAPHICS] where p >= 1, r >= 1, s >= 1, A >= 0, and x(1-r), x(2-r),..., x(0), y(1-max) {p.s},..., y(0) are positive real numbers. (c) 2005 Elsevier Inc. All rights reserved.

Asymmetric phase-transfer-catalyzed synthesis of five-membered cyclic gamma-Amino acid precursors

The first example of an intramolecular enantioselective Michael addition of nitronates onto conjugated systems utilizing a chiral phase-transfer catalyst is described. A range of five-membered gamma-nitro esters with up to three stereocentres have been prepared and the relative and absolute configurations proven by chemical and crystallographic methods. The products are rapidly obtained and are precursors to five-membered cyclic gamma-amino acids.

Dengue: desenvolvimento de ferramentas moleculares e caracterização de cepas virais quanto a inibição da sinalização do Interferon do tipo I (IFN-I)

A infecção pelo vírus da dengue é um problema de saúde pública global que põe em risco cerca de 2,5 bilhões de pessoas no mundo, com uma incidência de 50-100 milhões de casos resultando em cerca de 24.000 mortes por ano. Os mecanismos envolvidos na resposta imune inata atuam imediatamente após o contato do hospedeiro com os antígenos virais, levando à secreção de interferon do tipo I (IFN-I), a principal citocina envolvida na resposta antiviral. Entender como o sistema IFN-I é inibido em células infectadas pelo vírus dengue pode fornecer valiosas informações sobre a patogênese da doença. Propomos neste estudo analisar a inibição da via de sinalização do IFN-I por diferentes cepas isoladas no estado de Pernambuco, assim como o desenvolvimento de um vírus recombinante da dengue expressando a proteína Gaussia luciferase, para estudos futuros de replicação e imunopatogênese. A fim de estudar a via de sinalização do IFN-I, foram selecionadas cepas dos quatro sorotipos de dengue para crescimento, concentração e titulação viral. Foi utilizada a linhagem celular BHK-21-ISRE-Luc-Hygro que expressa o gene firefly luciferase fusionado a um promotor induzido pelo IFN-I (ISRE - Interferon Stimulated Response Element). Observamos que todos os sorotipos em estudo foram capazes de inibir, em diferentes proporções, a resposta ou sinalização do IFN-I. Com o intuito de auxiliar as pesquisas em dengue, desenvolvemos um vírus repórter de dengue expressando o gene repórter da Gaussia luciferase. Células transfectadas com o transcrito in vitro de um dos clones resultou em imunofluorescência positiva, porém não houve recuperação de partículas infectivas. Outros clones deverão ser testados para recuperação de vírus recombinante repórter. Juntos, os dados da caracterização das cepas em estudo e a recuperação de partículas infectivas da construção realizada neste trabalho deverão contribuir para as pesquisas em imunopatogênese, replicação viral e desenvolvimento de antivirais contra o dengue

Amyloid peptides incorporating a core sequence from the amyloid beta peptide and gamma amino acids: relating bioactivity to self-assembly

A series of heptapeptides comprising the core sequence Ab(16–20), KLVFF, of the amyloid b peptide coupled with paired N-terminal c-amino acids are investigated in terms of cytotoxicity reduction and binding to the full Ab peptide, both pointing to inhibition of fibrillisation for selected compounds. This is related to the self-assembly capacity of the heptapeptides.

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.

Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: uncoupling of gamma-aminobutyrate synthesis from efflux in a chemically defined medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular gamma-aminobutyrate (GABA(i)). The glutamate is then decarboxylated to GABA(i), a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA(e)) in response to acidification. In addition, high levels of GABA(i) (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA(i) in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA(i). These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.

The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activitor of T cells (LAT) in platelets stimulated by collagen and convulxin.

There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosine-phosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor gamma-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor gamma-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation.

Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain.

We have recently shown that collagen activates platelets through a pathway dependent on the Fc receptor gamma-chain and the tyrosine kinase Syk. We report here that the Fc receptor gamma-chain and the candidate collagen receptor glycoprotein VI (GPVI) co-associate. Furthermore, cross-linking GPVI stimulates a similar pattern of tyrosine phosphorylation to that stimulated by collagen, including tyrosine phosphorylation of Fc receptor gamma-chain. These results support a model where GPVI couples collagen-stimulation of platelets to phosphorylation of the Fc receptor gamma-chain leading to activation of Syk and phospholipase Cgamma2.

The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.

Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus.

Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets.

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor gamma-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor gamma-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor gamma-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin alpha2beta1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcgammaRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor gamma-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin alpha2beta1 and involves phosphorylation of the Fc receptor gamma-chain, its association with Syk and subsequent phosphorylation of phospholipase Cgamma2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking.

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.

Phenotypic and genotypic characterization of avian Escherichia coli O86:K61 isolates possessing a gamma-like intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli 086:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli 086:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type I and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.