Tumor necrosis factor-alpha induces functionally active hyaluronan-adhesive CD44 by activating sialidase through p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated human monocytic cells

Interaction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-alpha and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-alpha-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-alpha. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-alpha-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-alpha production. Blocking TNF-alpha production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-alpha-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-alpha-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells.     

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.     

Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate

During embryonic development, organs arise along the gut tube as a series of buds in a stereotyped anterior-posterior (A-P) pattern. Using chick-quail chimeras and in vitro tissue recombination, we studied the interactions governing the induction and maintenance of endodermal organ identify focusing on the pancreas. Though several permissive signals in pancreatic development have been previously identified, here we provide evidence that lateral plate mesoderm sends instructive signals to the endoderm, signals that induce expression of the pancreatic genes Pdx1, p48, Nkx6.1, glucagon, and insulin. Moreover, this instructive signal directs cells to form ectopic insulin-positive islet-like clusters in endoderm that would otherwise form more rostral organs. Once generated, endocrine cells no longer require interaction with mesoderm, but nonendocrine cells continue to require permissive signals from the mesoderm. Stimulation of activin, BMP, or retinoic acid signaling is sufficient to induce Pdx1 expression in endoderm anterior to the pancreas. Lateral plate mesoderm appears to pattern the endoderm in a posterior-dominant fashion as first noted in the patterning of the neural tube at the same embryonic stage. These findings argue for a central role of the mesoderm in coordinating the A-P pattern of all three primary germ layers.     

Vav1 and Ly-GDI two regulators of Rho GTPases,function cooperatively as signal transducers in T cell antigen receptor-induced pathways

The Rho family GTPases are pivotal for T cell signaling; however, the regulation of these proteins is not fully known. One well studied regulator of Rho GTPases is Vav1; a hematopoietic cell-specific guanine nucleotide exchange factor critical for signaling in T cells, including stimulation of the nuclear factor of activated T cells (NFAT). Surprisingly, Vav1 associates with Ly-GDI, a hematopoietic cell-specific guanine nucleotide dissociation inhibitor of Rac. Here, we studied the functional significance of the interaction between Vav1 and Ly-GDI in T cells. Upon organization of the immunological synapse, both Ly-GDI and Vav1 relocalize to T cell extensions in contact with the antigen-presenting cell. Ly-GDI is phosphorylated on tyrosine residues following T cell receptor stimulation, and it associates with the Src homology 2 region of an adapter protein, Shc. In addition, the interaction between Ly-GDI and Vav1 requires tyrosine phosphorylation. Overexpression of Ly-GDI alone is inhibitory to NFAT stimulation and calcium mobilization. However, when co-expressed with Vav1, Ly-GDI enhances Vav1 induction of NFAT activation, phospholipase Cgamma phosphorylation, and calcium mobilization. Moreover, Ly-GDI does not alter the regulation of these phenomena when coexpressed with oncogenic Vav1. Since oncogenic Vav1 does not bind Ly-GDI, this suggests that the functional cooperativity of Ly-GDI and Vav1 is dependent upon their association. Thus, our data suggest that the interaction of Vav1 and Ly-GDI creates a fine tuning mechanism for the regulation of intracellular signaling pathways leading to NFAT stimulation.     

Direct binding of human immunodeficiency virus type 1 Nef to the major histocompatibility complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking

Nef, an essential pathogenic determinant for human immunodeficiency virus type 1, has multiple functions that include disruption of major histocompatibility complex class I molecules (MHC-I) and CD4 and CD28 cell surface expression. The effects of Nef on MHC-I have been shown to protect infected cells from cytotoxic T-lymphocyte recognition by downmodulation of a subset of MHC-I (HLA-A and -B). The remaining HLA-C and -E molecules prevent recognition by natural killer (NK) cells, which would otherwise lyse cells expressing small amounts of MHC-I. Specific amino acid residues in the MHC-I cytoplasmic tail confer sensitivity to Nef, but their function is unknown. Here we show that purified Nef binds directly to the HLA-A2 cytoplasmic tail in vitro and that Nef forms complexes with MHC-I that can be isolated from human cells. The interaction between Nef and MHC-I appears to be weak, indicating that it may be transient or stabilized by other factors. Supporting the fact that these molecules interact in vivo, we found that Nef colocalizes with HLA-A2 molecules in a perinuclear distribution inside cells. In addition, we demonstrated that Nef fails to bind the HLA-E tail and also fails to bind HLA-A2 tails with deletions of amino acids necessary for MHC-I downmodulation. These data provide an explanation for differential downmodulation of MHC-I allotypes by Nef. In addition, they provide the first direct evidence indicating that Nef functions as an adaptor molecule able to link MHC-I to cellular trafficking proteins.     

Coumarin glucosides from Cruciata taurica

Two new coumarin glycosides (1 and 2) along with two known coumarin glucosides, daphnin (3) and daphnetin glucoside (4) were isolated from the aerial parts of Cruciata taurica. The structures of the new compounds were elucidated by spectral methods and chemical means as 7-O-(6' -acetoxy-beta-D-glucopyranosyl)-8-hydroxycoumarin (1) and 7-O-[6 '-O-(3',4'-dihydroxycinnamoyl)-beta-D-glucopyranosyl]-8-hydroxycoumarin (2). The phylogenetic significance of coumarins in C. taurica was discussed.     

Possible cytoskeletal structures of rainbow trout sperm revealed by electron microscopic observation after detergent extraction

Despite the considerable research interest in fish sperm ultrastructure, little is known about the functions of different sperm cell components. Our electron microscopic study was aimed at identifying possible tissue-specific cytoskeletal structures in spermatozoa of rainbow trout Oncorhynchus mykiss (Teleostei, Salmoniformes, Salmonidae; formerly Salmo gairdneri). Based on the known resistance of the cytoskeleton to nonionic detergents, we compared the ultrastructure of unextracted and Triton-extracted sperm cells. Besides the nucleus, the centrioles and the axoneme, there were also other structures preserved in Triton-treated spermatozoa: the lateral extensions (sidefins) and a thin layer corresponding in position to the membrane-like structure underlying the midpiece plasma membrane in intact cells. Because of their stability, it could be hypothesized that these cytoplasmic components are likely to have cytoskeletal nature. They are possibly analogous to the well known tissue-specific cytoskeletal components of mammalian spermatozoa with periaxonemal and submitochondrial localization.     

Chaperone-assisted expression,purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

Efficient gene delivery into murine ovarian cells by intraovarian injection of plasmid DNA and subsequent in vivo electroporation

We describe the use of direct injection of circular plasmid DNA and subsequent in vivo electroporation (EP) for efficient gene delivery to the ovarian cells, including follicular cells and oocytes of mice. When Trypan blue (TB) was injected into the central portion of an ovary by a glass micropipette, rapid dispersion of TB to each preantral and antral follicle was observed. Injections of lacZ-expressing plasmid DNA and subsequent in vivo EP resulted in transfection of follicles with efficiencies ranging from 8-60%, together with cells in the thecal portion of the ovary. Of the lacZ-positive follicles, some oocytes were also positive for lacZ activity. These findings suggest that a solution introduced inside the ovary is rapidly dispersed to each follicle. With this technique, we expect great progress in genetic engineering in murine ovary.