A cross-sectional study to quantify the prevalence of avian influenza viruses in poultry at intervention and non-intervention live bird markets in central Vietnam, 2014.

In Vietnam, live bird markets are found in most populated centres, providing the means by which fresh poultry can be purchased by consumers for immediate consumption. Live bird markets are aggregation points for large numbers of poultry, and therefore, it is common for a range of avian influenza viruses to be mixed within live bird markets as a result of different poultry types and species being brought together from different geographical locations. We conducted a cross-sectional study in seven live bird markets in four districts of Thua Thien Hue Province in August and December, 2014. The aims of this study were to (i) document the prevalence of avian influenza in live bird markets (as measured by virus isolation); and (ii) quantify individual bird-, seller- and market-level characteristics that rendered poultry more likely to be positive for avian influenza virus at the time of sale. A questionnaire soliciting details of knowledge, attitude and avian influenza practices was administered to poultry sellers in study markets. At the same time, swabs and faecal samples were collected from individual poultry and submitted for isolation of avian influenza virus. The final data set comprised samples from 1,629 birds from 83 sellers in the seven live bird markets. A total of 113 birds were positive for virus isolation; a prevalence of 6.9 (95% CI 5.8-8.3) avian influenza virus-positive birds per 100 birds submitted for sale. After adjusting for clustering at the market and individual seller levels, none of the explanatory variables solicited in the questionnaire were significantly associated with avian influenza virus isolation positivity. The proportions of variance at the individual market, seller and individual bird levels were 6%, 48% and 46%, respectively. We conclude that the emphasis of avian influenza control efforts in Vietnam should be at the individual seller level as opposed to the market level.

Structural requirements of Syk kinase for Fcgamma receptor-mediated phagocytosis.

The tyrosine kinase Syk plays a critical role in the phagocytic pathway mediated by Fcgamma receptors (FcgammaR). In transfected COS1 cells co-expression of Syk enhances FcgammaR mediated phagocytosis. The other member of the Syk kinase family, the highly homologous tyrosine kinase Zap70, also plays a role in signaling by immunoglobulin gene family receptors, but does not increase the phagocytic efficiency of FcgammaRs. The homologous tandem SH2 and kinase domains of Syk and Zap70 are separated by a nonhomologous region referred to as the unique domain. Zap70's inability to enhance phagocytosis was not due to unique domain tyrosine 292, previously implicated in negative regulation of Zap70 function. We determined the regions of Syk important for its interaction with the phagocytic pathway. An intact kinase domain was required for Syk's effect on phagocytosis. Furthermore, the Syk variant SykB, lacking 23 amino acids in the unique region, signaled for phagocytosis as efficiently as did Syk. We then constructed exchange chimeras between Syk and Zap70 and determined the contributions of the SH2, unique and kinase domains to phagocytic signaling. Our data suggest that the Syk kinase domain, which has high intrinsic kinase activity, is important for facilitating phagocytic signaling by FcgammaRI and FcgammaRIIIA.

Immobilization of alpha-amylase to a composite temperature-sensitive membrane for starch hydrolysis.

A composite membrane was made by casting hydrogel onto a nonwoven polyester support and used for enzyme immobilization. The hydrogel consists of N-isopropylacrylamide, cross-linker N, N'-methylenebis(acrylamide), 2-hydroxyethyl methacrylate, soluble starch, and N-(acryloxy)succinimide (NAS). The composite membrane is temperature-sensitive with a lower critical solution temperature (LCST) around 35 degreesC. It responds to temperature change by swelling below the LCST and shrinking above the LCST, corresponding to opening and closing of the membrane pores. alpha-Amylase was immobilized to the membrane by covalent bonds through reacting with the high reactive ester groups in NAS. The membrane-immobilized enzyme retained 32% of specific activity toward soluble starch when compared with that of free enzyme, and its properties were characterized and compared with those of the free enzyme. The immobilized enzyme was more thermally stable than the free enzyme. Kinetic constants, (Km) and the activation energy of the immobilized enzyme were both larger than those of the free enzyme. Starch hydrolysis with the immobilized enzyme was investigated in two-compartment permeation cells with a composite membrane between the cells. Reaction was carried out by hydrolyzing soluble starch in the donor side and collecting the hydrolyzed products in the receptor side. This reactor could be operated with temperature cycling to enhance the reaction and facilitate separation of products from the substrate. The best operating condition is cycling the temperature between 50 and 20 degrees C every 5 min. The membrane reactor was operated up to eight times for successive starch hydrolysis.

The Syk family of protein tyrosine kinases in T-cell activation and development.

The processes of T-cell development and activation employ similar immature and mature receptors as well as similar signal transduction pathways to achieve different outcomes. Many signaling molecules are shared between the receptor signaling pathways, including two families of cytoplasmic protein tyrosine kinases, the Src family and the Syk family. The two Syk family members expressed in T cells, Syk and ZAP-70, are structurally similar but are expressed at different times during thymic development and during T-cell activation. These two kinases, although they share many physical features, differ in terms of biochemical activity and regulation. We discuss the overlapping and distinct characteristics of Syk and ZAP-70 in T-cell signaling and the potential biological importance of their differences.

Preparation and characterization of alpha-amylase-immobilized thermal-responsive composite hydrogel membranes.

Composite hydrogel membranes of crosslinked poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyet hyl methacrylate) [P(NIPAAm-NAS-HEMA)] with starch, as a macropore forming agent, on nonwoven polyester was prepared. The membranes could swell and de-swell around the characteristic lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAAm). It was demonstrated that the presence of macropores in the membranes could improve the immobilization efficiency as well as lead to a short responding time upon temperature change across the LCST. Immobilized alpha-amylase could retain as high as 33% of the activity of the free enzyme with a loading level of 0.60-0.65 mg/cm2 when the membrane preparation and enzyme immobilization conditions were optimized. The half time (T0.5) for the swelling or de-swelling response of the gel phase within the membranes was less than 2 min, and the 90% time (T0.9) was less than 6 min. The permeability for maltose through the membranes could change as much as 4.9-fold when the temperature was raised above or reduced below the LCST.

Pre-T cell receptor signals are responsible for the down-regulation of Syk protein tyrosine kinase expression.

Thymocyte development proceeds through two critical checkpoints that involve signaling events through two different receptors, the TCR and the pre-TCR. These receptors employ two families of protein tyrosine kinases to propagate their signals, the Src and Syk families. Genetic and biochemical evidence has shown that the Src family kinases are critical for normal T cell maturation. ZAP-70, a Syk family kinase, has similarly been implicated as a critical component in thymocyte development. Although genetic evidence has suggested that Syk is involved during thymocyte development, a definitive study of Syk expression has not been performed. In this paper we report our reanalysis of Syk expression in subpopulations of murine and human thymocytes by intracellular staining and flow cytometry using anti-Syk mAbs. Syk is expressed at increased levels during the stages in which pre-TCR signaling occurs. Furthermore, Syk is down-regulated after the pre-TCR checkpoint has been passed. Syk may play an important role in thymic development during pre-TCR signal transduction. Finally, incomplete down-regulation of Syk expression was noted in human thymocytes, offering a possible explanation for the distinct phenotypes of mice and humans deficient in ZAP-70.

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling.

Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha.

We have identified the palmitoylated cysteine residues of alpha q and alpha s, alpha subunits of two heterotrimeric G proteins. Mutational substitutions of serines for cysteines 9 and 10 in alpha q and cysteine 3 in alpha s profoundly alter behavior of the subunits expressed in HEK293 cells. Neither mutant alpha subunit incorporates palmitate; both mutant proteins are found in the soluble rather than the particulate fraction; mutant alpha q or alpha s cannot couple a co-expressed receptor to stimulation of phospholipase C or adenylylcyclase, respectively; cysteine substitution prevents a mutationally activated alpha q (R183C) from stimulating phospholipase C directly, and reduces but does not abolish the ability of a similarly activated alpha s (R201C) to stimulate cAMP synthesis. Substitution of a myristoylation sequence for the palmitoylation sites leads to labeling of alpha q and alpha s by myristate, rather than by palmitate. Myristoylation restores the abilities of both nonpalmitoylated alpha q and alpha s to attach to membranes and, in the case of alpha q, restores its ability to stimulate phospholipase C, whether triggered by the R183C mutation or by receptor activation. These findings identify palmitoylation as a critical determinant of membrane attachment for alpha q and alpha s and show that this modification is required for normal signaling by these proteins.

Differential requirements for ZAP-70 in TCR signaling and T cell development.

The Syk/ZAP-70 family of protein tyrosine kinases is indispensable for normal lymphoid development. Syk is necessary for the development of B cells and epithelial gammadelta T cells, whereas ZAP-70 is essential for the normal development of T cells and TCR signaling. In this study, we show that although development of the alphabeta lineage was arrested in the thymus, CD3-positive T cells, primarily of the gammadelta lineage, were present in the lymph nodes of mice lacking ZAP-70. Moreover, in the absence of ZAP-70, dendritic epidermal T cells were fewer in number and of abnormal morphology, and intestinal intraepithelial lymphocytes, normally containing a large proportion of gammadelta T cells, were markedly reduced. These data suggest that gammadelta T cells show a variable dependence upon ZAP-70 for their development. Biochemical analyses of thymocytes revealed a lack of basal zeta-chain tyrosine phosphorylation. However, several other substrates were inducibly tyrosine phosphorylated following TCR stimulation. Thus, TCR-mediated signaling in ZAP-70-deficient thymocytes is only partially impaired. These studies suggest that Syk compensates only partially for the loss of ZAP-70, and that there is an absolute requirement of ZAP-70 for alphabeta T cells and epithelial gammadelta T cells, but not for some gammadelta T cells in peripheral lymphoid tissues.