Activation of prothrombin by ASP, a serine protease released from Aeromonas sobria

The effect of a serine protease (ASP) secreted from Aeromonas sobria on plasma coagulation was investigated. Proteolytically active ASP promoted human plasma coagulation in a dose-dependent manner. Consistent with the preference for a factor Xa-specific oligo-peptide substrate, ASP produced enzymatic activity from human prothrombin but not from factors IX and X. ASP cleaved prothrombin to produce enzymatically active 37 kDa-fragment displaying the same molecular mass as alpha-thrombin. ASP is the first bacterial serine protease that produces alpha-thrombin, through which ASP may contribute to the induction of thrombotic tendency in disseminated intravascular coagulation complicated with sepsis caused by A. sobria infections.     

Prion infection correlates with hypersensitivity of P2X7 nucleotide receptor in a mouse microglial cell line

We recently established mouse microglial cells persistently infected with mouse-adapted scrapie ME7 (ScMG20/ME7) for in vitro study of prion pathogenesis. Here, we found that ScMG20/ME7 cells were hypersensitive to P2X7 receptor agonists, as demonstrated by sustained Ca(2+) influx, membrane pore formation, cell death, and interleukin-1beta release. P2X7 mRNA expression was upregulated in these cells, and also in scrapie-infected mice brains. Treatment with pentosan polysulfate eliminated the infectivity and disease-related forms of prion protein from ScMG20/ME7 cell cultures, however, hypersensitivity of P2X7 receptors remained. These results suggest that prion infections may strongly affect the P2X7 receptor system in mouse microglial cells.     

Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

A novel growth factor in rat spleen which promotes proliferation of hepatocytes in primary culture

We have identified a factor from adult rat spleen which stimulates the proliferation of rat hepatocytes. The activity was found in the spleen soluble matrix fraction (1,300xg supernatant of inter-cellular fraction). No activity was found in the spleen cell homogenate, in the spleen insoluble matrix fraction or rat serum. After 4 days of incubation with the spleen factor, the cell number increased 4-fold higher than that at inoculation. The growth stimulation were observed in both fetal bovine serum supplemented medium and hormonally defined medium which contains insulin, epidermal growth factor, glucagon, growth hormone and prolactin. The level of activity in the spleen soluble matrix was not affected by partial hepatectomy or trypsinization. These data indicate that the spleen factor is different from previously characterized effectors of hepatocyte proliferation. The novel factor has been named spleen derived growth factor (SDGF).     

Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.     

beta -Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport

The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.     

Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction.

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them. The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles. Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B). It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not. Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs). These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana.     

Thyrotropin releasing hormone stimulation of GABA-gated but not basal chloride ion influx in rat cerebellum

Potential interaction between gamma-amino butyric acid (GABA) and thyrotropin-releasing hormone (TRH) in eliciting a variety of central nervous system (CNS)-related biologic activities is well known; however, the mechanism underlying this interaction is not clearly defined. To gain further insight into this interaction, we examined the effects of TRH and two of its central nervous system selective analogs, DN 1417 and TA 0910, on basal and GABA-mediated chloride ion influx into rat cerebellar neurosynaptosomes. The results of these studies show that TRH may facilitate GABA action by augmenting chloride ion influx and hyperpolarization of cerebellar neurons.     

Modulation of Formalin-Evoked Hyperalgesia by Intrathecal N-Type Ca Channel and Protein Kinase C Inhibitor in the Rat

1. -CgTx attenuated formalin-evoked biphasic flinches, while PKC inhibitor (STU) attenuated phase 2 and was reversed by PDBu.2. -CgTx and STU suppressed the increase in CSF-glutamate after formalin injection.3. Morphine completely suppressed both increased flinching and CSF glutamate release.4. Thus, -CgTx (N-type Ca channels) may regulate neurotransmitter release evoked by C fiber activation and the formalin-evoked hyperalgesia may possibly be provoked as a result of PKC activation elicited by both presynaptic neurotransmitter release and activation of NMDA receptors in the spinal neurons.