Specificity of the SH2 domains of SHP-1 in the interaction with the immunoreceptor tyrosine-based inhibitory motif-bearing receptor gp49B

Inhibitory receptors on hemopoietic cells critically regulate cellular function. Despite their expression on a variety of cell types, these inhibitory receptors signal through a common mechanism involving tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM), which engages Src homology 2 (SH2) domain-containing cytoplasmic tyrosine or inositol phosphatases. In this study, we have investigated the proximal signal-transduction pathway of an ITIM-bearing receptor, gp49B, a member of a newly described family of murine NK and mast cell receptors. We demonstrate that the tyrosine residues within the ITIMs are phosphorylated and serve for the association and activation of the cytoplasmic tyrosine phosphatase SHP-1. Furthermore, we demonstrate a physiologic association between gp49B and SHP-1 by coimmunoprecipitation studies from NK cells. To address the mechanism of binding between gp49B and SHP-1, binding studies involving glutathione S-transferase SHP-1 mutants were performed. Utilizing the tandem SH2 domains of SHP-1, we show that either SH2 domain can interact with phosphorylated gp49B. Full-length SHP-1, with an inactivated amino SH2 domain, also retained gp49B binding. However, binding to gp49B was disrupted by inactivation of the carboxyl SH2 domain of full-length SHP-1, suggesting that in the presence of the phosphatase domain, the carboxyl SH2 domain is required for the recruitment of phosphorylated gp49B. Thus, gp49B signaling involves SHP-1, and this association is dependent on tyrosine phosphorylation of the gp49B ITIMs, and an intact SHP-1 carboxyl SH2 domain.     

A dynamical investigation of acrylodan-labeled mutant phosphate binding protein

The static and dynamical behavior of a fluorescently labeled mutant of the Escherichia coli periplasmic phosphate binding protein (PBP) was investigated through steady-state and time-resolved fluorescence spectroscopy. As a means of developing a biorecognition element for inorganic phosphate (P(i)), alanine-197 of PBP was replaced with a cysteine. This site was then labeled with an environmentally sensitive fluorophore. The fluorescence emission of the mutant PBP labeled with acrylodan (MPBP-AC) proved to be sensitive to micromolar concentrations of P(i), as indicated by a 50% increase in the steady-state emission intensity. Steady-state results indicated that the labeling protocol was specific for cys-197 only and did not label the wild-type PBP; thus, a site-selective labeling protocol was developed. Time-resolved measurements were used to determine the influence of the dynamics of MPBP-AC on the process of signal transduction. Time-resolved anisotropy measurements revealed that rotational dynamics were best described by a model with two independent motions: the global motion of the protein and the local motion of the acrylodan probe. The rates of both global and local rotational reorientation of MPBP-AC were faster when the protein was P(i)-bound rather than P(i)-free. This was a result of structural changes involving or surrounding both the P(i)-binding site (global changes) and the residues in near proximity to the fluorescent reporter group (local changes). Recovery of the semiangle (theta) indicated that local structural changes in MPBP-AC took place when P(i) was bound to the protein. Acrylodan gained mobility when MPBP-AC bound P(i), as indicated by the fact that theta increased by approximately 5 degrees. In addition, dynamic quenching measurements confirmed that structural changes occurred locally near the cys-197. Acrylodan became more accessible to iodide when MPBP-AC bound P(i), as demonstrated by the 35% increase in the value of the bimolecular quenching constant.     

Termination of translation in eukaryotes: new results and new hypotheses

The viscoelastic properties of the human and canine pharyngeal tissue in tension were evaluated, based on both an experimental protocol-consisting of cyclic load, tensile stress relaxation, and incremental step load tests-and the quasi-linear viscoelastic theory. The reduced stress relaxation function and the elastic response of the pharyngeal tissues were derived from the experimental results specifically obtained from those tissues. The characteristic features of viscoelastic property were obtained for both human and canine pharyngeal tissues by applying the quasi-linear viscoelastic theory and compared with each other. The material properties of the pharyngeal tissue were sought to facilitate the three-dimensional biomechanical model of the pharyngeal function by using the finite element method.     

Genetic evaluation by BLUP in two-breed terminal crossbreeding systems under dominance

To obtain estimates of breeding values by BLUP using Henderson's mixed-model equations, it is necessary to invert the covariance matrix for each random effect in the model. In a model in which the genotypic value is included as a random effect (genotypic model), it is necessary to invert the genotypic covariance matrix. Under additive inheritance, the inverse of the genotypic covariance matrix can be computed efficiently. Under dominance inheritance, however, an efficient method to invert the genotypic covariance matrix has not yet been developed, especially for crossbred populations. Thus, the use of a genotypic model for BLUP is not suitable for genetic evaluation in large, crossbred populations. We present an equivalent model in which the genotypic effect is partitioned into additive and dominance effects. With this equivalent model, methods used for within-breed genetic evaluation by BLUP can be used for a two-breed terminal cross under dominance.     

Why good people do bad things

The very context of organizational decision making tests autonomous moral agency for managers, and the moral price they pay may be great. Misunderstandings, miscommunication, fear and isolation all play a role in why good people do bad things. Self-knowledge, good communication, alternative support systems and management by walking around (MBWA) all help good people (who happen to be managers) avoid doing bad things.     

Mercury-induced autoimmunity in Brown Norway rats: kinetics of changes in RT6+ T lymphocytes correlated with IgG isotypes of circulating autoantibodies to laminin 1

Repeated exposure to mercury causes various autoimmune effects in rats of the Brown Norway (BN) strain. Previous studies from our laboratory have shown that on day 15 of HgCl2 treatment BN rats exhibit a relative decrease in RT6.2+ T cells. At the same time, they produce high levels of autoantibodies to renal antigens and experience a membranous glomerulonephropathy. In contrast, Lewis (LEW) rats are resistant to autoimmunity caused by mercury and do not demonstrate a decrease in RT6+ cells after administration of HgCl2. In the present paper we provide novel information on the correlation between changes in RT6.2+ lymph node T cells and the production of autoantibodies to laminin 1, obtained by detailed kinetic studies of HgCl2-treated BN rats. We have confirmed a decrease in the percentage of RT6.2+ lymphocytes on day 15 of mercury treatment, despite a significant increase in the number of peripheral lymphocytes. No such changes were observed in LEW rats. We have determined that on day 15 the percentage decrease in RT6+ cells is evident in both RT6.2+CD4+ and RT6.2+CD8+ T cell subsets. Kinetic studies demonstrated that significant changes in the percentage of RT6.2+ cells are first observed by day 8 and continue through days 11 and 15. We have also observed a significant percent decrease in CD4+ T lymphocytes as well as an increase in CD4-CD8- cells. The dramatic increase in the percentage of these double negative cells at the level of peripheral lymphoid tissues does not appear to be due to higher thymic output, since there was a decrease in the percentage of TCR+Thy1+ cells, a phenotype that is associated with recent thymic emigrants. Finally, we have demonstrated that 100% of HgCl2-treated BN rats had circulating antibodies that reacted with both mouse and rat laminin 1, i.e. are autoantibodies to laminin 1. These autoantibodies were predominantly of the IgG1 and IgG2a isotype, possibly as the result of a polarized autoimmune response driven by Type 2 cytokines. A kinetic investigation showed that significant levels of IgG1 and IgG2a autoantibodies to laminin 1 were first presentin the circulation by day 11. The inverse correlation between levels of RT6.2+ T lymphocytes and autoantibodies to laminin 1 suggests that mercury may induce autoimmune responses in BN rats by its effects on these immunoregulatory cells.     

Clinical and experimental studies on preventing and treating anaphylactic asthma with Zusanli point immunotherapy

We have studied on preventing and treating anaphylactic asthma with Zusanli (S36) point immunotherapy (ZPIT). Sixty-nine patients were observed. The results showed that the clinical curative effect of ZPIT was not only much higher than that of conventional desensitization therapy, but also the patients' total IgE level was reduced, anti-acarid IgE was lowered, SIgA level was raised, the absolute eosinophilic granulocyte level dropped and pulmonary function recovered. Animal experiment results showed that the ZPIT could more effectively suppress the guinea pigs' anaphylactic asthma allergized by albumin and more obviously resist the guinea pigs' bronchial spasm induced by histamine and acetylcholine than the conventional desensitization therapy and injected normal saline. The immunomodulating action of the ZPIT are elucidated from clinical study and animal experiment in the paper.