Intraovarian thrombin and activated protein C signaling system regulates steroidogenesis during the periovulatory period

In addition to its role in blood coagulation, thrombin directly stimulates protease-activated receptors (PAR) or interacts with thrombomodulin (THBD) to activate membrane-bound protein C which stimulates PAR1 and PAR4 receptors to promote downstream pleiotropic effects. Our DNA microarray, RT-PCR, and immunostaining analyses demonstrated ovarian expression of THBD, activated protein C (APC) receptor [endothelial protein C receptor (EPCR)], as well as PAR1 and PAR4 receptors in mice. After treatment of gonadotropin-primed immature mice with an ovulatory dose of human chorionic gonadotropin (hCG) (a LH surrogate), major increases in the expression of THBD, EPCR, PAR1, and PAR4 were detected in granulosa and cumulus cells of preovulatory follicles. Immunoassay analyses demonstrated sustained increases in ovarian prothrombin and APC levels after hCG stimulation. We obtained luteinizing granulosa cells from mice treated sequentially with equine CG and hCG. Treatment of these cells with thrombin or agonists for PAR1 or PAR4 decreased basal and forskolin-induced cAMP biosynthesis and suppressed hCG-stimulated progesterone production. In cultured preovulatory follicles, treatment with hirudin (a thrombin antagonist) and SCH79797 (a PAR1 antagonist) augmented hCG-stimulated progesterone biosynthesis, suggesting a suppressive role of endogenous thrombin in steroidogenesis. Furthermore, intrabursal injection with hirudin or SCH79797 led to ipsilateral increases in ovarian progesterone content. Our findings demonstrated increased ovarian expression of key components of the thrombin-APC-PAR1/4 signaling system after LH/hCG stimulation, and this signaling pathway may allow optimal luteinization of preovulatory follicles. In addition to assessing the role of thrombin and associated genes in progesterone production by the periovulatory ovary, these findings provide a model with which to study molecular mechanisms underlying thrombin-APC-PAR1/4 signaling.     

Functional studies on three novel HCNH2 mutations in Taiwan: identification of distinct mechanisms of channel defect and dissociation between glycosylation defect and assembly defect

Mutations of the KCNH2 with decreased channel activity lead to congenital long QT syndrome (LQTS). We studied the electrophysiological, glycosylation, trafficking and assembly properties of three novel KCNH2 mutations identified in Taiwanese patients with LQTS (p.N633D, p.R744fs, and p.P923fs). When expressed in HEK293T cells, p.N633D and p.R744fs HERG channels displayed no HERG current while p.P923fs HERG channel showed HERG current with significantly lower (34%) current density and faster inactivation kinetics. In Western blot analysis, pR744fs was the only one with glycosylation defect, which was in consistence with the confocal microscopic findings showing that p.R744fs-GFP was the only one with trafficking defect. However, p.R744fs-GFP differed from pR744fs in being fully glycosylated while p.R744fs fusion with GFP at the N-terminus revealed glycosylation defect. To access the assembly capacity of each mutant, co-immunoprecipitation was conducted. Wild type (WT), p.N633D, and p.P923fs HERG protein showed assembly ability while p.R744fs failed to assemble with neither WT nor itself.In conclusion, we identified three novel LQTS-related KCNH2 mutations and each had a distinct mechanism of channel defect. For p.R744fs mutant, adding GFP to the C-terminus rescued the glycosylation defect but the channel was still assembly defective indicating a dissociation between glycosylation and assembly defects.     

Role of taurine on acid secretion in the rat stomach

Background  

Taurine has chemical structure similar to an inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Previous studies on GABA in the stomach suggest GABAergic neuron is involved in acid secretion, but the effects of taurine are poor understood.     

Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology

Dendritic arborization is a critical neuronal differentiation process. Here, we demonstrate that syndecan-2 (Sdc2), a synaptic heparan sulfate proteoglycan that triggers dendritic filopodia and spine formation, regulates dendritic arborization in cultured hippocampal neurons. This process is controlled by sterile α and TIR motif-containing 1 protein (Sarm1), a negative regulator of Toll-like receptor 3 (TLR3) in innate immunity signaling. We show that Sarm1 interacts with and receives signal from Sdc2 and controls dendritic arborization through the MKK4-JNK pathway. In Sarm1 knockdown mice, dendritic arbors of neurons were less complex than those of wild-type littermates. In addition to acting downstream of Sdc2, Sarm1 is expressed earlier than Sdc2, which suggests that it has multiple roles in neuronal morphogenesis. Specifically, it is required for proper initiation and elongation of dendrites, axonal outgrowth, and neuronal polarization. These functions likely involve Sarm1-mediated regulation of microtubule stability, as Sarm1 influenced tubulin acetylation. This study thus reveals the molecular mechanism underlying the action of Sarm1 in neuronal morphogenesis.     

Ischemic preconditioning ameliorates microcirculatory disturbance through downregulation of TNF-alpha production in a rat cremaster muscle model

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IB--NF-B-TNF- (tumor necrosis factor-) pathway in IPC's ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n=8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF- expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF- protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IB- phosphorylation and NF-B (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-B activation and subsequently reduced TNF- expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles.     

Periovulatory changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in hormonally induced immature female rats

Immature female rats were treated with PMSG and human CG to induce ovulation. Sequential treatment with these hormones allowed us to investigate variations in the production of inhibin subunits shortly before ovulation and during the induced luteal phase. Using this model, we found that expression patterns for the alpha-, beta A-, and beta B-subunits were similar to those observed in mature cycling animals: administration of PMSG (to mimic the gonadotropin surge) led to a sharp increase in the expression of all three subunits in large preovulatory follicles whereas injection with human CG (to induce ovulation) caused a decrease in the levels of the respective mRNAs. In contrast to mature females, shortly before ovulation, levels of inhibin alpha-subunit mRNA were low in small antral follicles (approximately 350 microns). In addition, at that time, inhibin beta A- and beta B-subunits mRNAs were present in several large follicles (greater than 500 microns). More than 2 days after ovulation, inhibin beta A- and beta B-subunit mRNAs could not be detected in small antral size follicles (approximately 350 microns) of hormonally induced females. On the other hand, hybridization signals for the inhibin alpha-subunit were observed in some small antral and preantral size follicles, while signals were very low or undetectable in a large number of atretic follicles. Using this synchronized ovulation model, hybridization patterns for inhibin beta A-subunit mRNA was observed in interstitial cells, 8-10 h after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Updates in Gastrointestinal Oncology – insights from the 2008 44th annual meeting of the American Society of Clinical Oncology

Tyrosine Kinase Inhibitors (TKI) have significantly changed the landscape of current cancer therapy. Understanding of mechanisms of aberrant TK signaling and strategies to inhibit TKs in cancer, further promote the development of novel agents. ABT-869, a novel ATP-competitive receptor tyrosine kinase inhibitor is a potent inhibitor of members of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor families. ABT-869 showed potent antiproliferative and apoptotic properties in vitro and in animal cancer xenograft models using tumor cell lines that were "addicted" to signaling of kinases targeted by ABT-869. When given together with chemotherapy or mTOR inhibitors, ABT-869 showed at least additive therapeutic effects. The phase I trial for ABT-869 was recently completed and it demonstrated respectable efficacy in solid tumors including lung and hepatocellular carcinoma with manageable side effects. Tumor cavitation and reduction of contrast enhancement after ABT-869 treatment supported the antiangiogenic activity. The correlative laboratory studies conducted with the trial also highlight potential biomarkers for future patient selection and treatment outcome. Parallel to the clinical development, in vitro studies on ABT-869 resistance phenotype identified novel resistance mechanism that may be applicable to other TKIs. The future therapeutic roles of ABT-869 are currently been tested in phase II trials.     

Posttranslational modification of ras proteins: detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification

Products of ras genes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttraslational modification by fatty acid. In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification of ras proteins prior to the fatty acid acylation. The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel. The fatty acid acylation does not contribute to this mobility shift. This modification is affected by the dprl mutation which has recently been shown to affect the processing of yeast RAS proteins. To further characterize the nature of the modification event, we have cloned DPR1 gene from the DNA of Saccharomyces cerevisiae. The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb. Genes related to the DRP1 appear to be present in a distantly related yeast, Schizosaccharomyces pombe as well as in guinea pig and human cells.