THE ROLE OF SYNTAXIN AND TOMOSYN IN PLATELET SECRETION

Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the identity of the functional syntaxin has been controversial. Previous studies, using anti-syntaxin antibodies in permeabilized platelets, suggested roles for both syntaxin-2 and -4. These conclusions were experimentally tested using platelets from syntaxin knockout mice and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient that lacks syntaxin-11. Platelets from syntaxin-2 and syntaxin-4 single or double knockout mice had no significant secretion defect. However, platelets from the FHL4 patient had a robust defect, though their morphology, activation, and cargo levels appeared normal. Semi-quantitative western blotting showed that syntaxin-11 is the most abundant syntaxin in both human and murine platelets. Co-immunoprecipitation experiments showed that syntaxin-11 forms SNARE complexes with VAMP-8 and SNAP-23. These data conclusively demonstrate that syntaxin-11, but not syntaxin-2, or -4, is required for platelet exocytosis. We also show that a syntaxin binding protein, tomosyn-1, is important for platelet exocytosis and hemostasis. Tomosyn-1 was identified from platelet extracts using affinity chromatography, RT-PCR analysis, and western blotting analysis. Tomosyn-1 was co-immunoprecipitated with syntaxin-11/SNAP-23 from both resting and activated platelet extracts. Platelets from tomosyn-1-/- mice displayed a secretion defect, but their morphology and activation appeared normal. Tomosyn-1-/- mice showed impaired thrombus formation in two different injury models. Given the importance of platelet secretion to hemostasis, it is hoped that the insights gained from these studies in this dissertation will help to identify new and more valuable therapeutic targets to control clot formation

The effects of feeding ration and cheliped autotomy on the growth and expression of ecdysteroid receptor in early juvenile mud crabs, Scylla paramamosain

This study investigated the combined effects of feeding ration and cheliped autotomy on the intermolt duration, molting success, molt increments in size and weight, and ecdysteroid receptor gene (SpEcR) expression of early juvenile mud crab Scylla paramamosain. Newly molted second stage juvenile crabs (C2) were subjected to four feeding conditions; optimal, suboptimal (1/2 optimal), low (1/4 optimal) ration and starvation, and autotomy (intact vs. cheliped autotomy) in a 4 x 2 factorial design until all crabs successfully molted or died. A significant interaction of feeding ration and cheliped autotomy on intermolt duration was identified. With reduced feeding ration, both intact and cheliped autotomized crabs showed increased time and desynchrony of molting, but decreased carapace size and body weight. Importantly, all crabs with different feeding rations even the low ration had high rates of molting success (> 95%), while the crabs subjected to starvation died without molting. When fed optimal ration, the mean intermolt duration of the cheliped autotomized crabs was significantly prolonged, while no such effect was found between autotomized and intact crabs subjected to suboptimal or low feeding ration. The qRT-PCR revealed that the expression of SpEcR showed a general trend of inhibited by reduced feeding ration, which was consistent with observed significantly increased intermolt duration. Interestingly, the transcript level of SpEcR was only significantly affected by cheliped autotomy under the optimal and suboptimal feeding rations but not for the low feeding ration. Together, the results of this study suggest that the S. paramamosain early juveniles have a strong tolerance for fluctuations in food availability. In addition, the availability of food and limb autotomy could significantly affect growth, molting duration and synchrony of the crabs, which appeared to reflect in SpEcR expression level that involved in the regulation of molting and limb regeneration process of the juvenile crabs

The retinoid X receptor from mud crab: new insights into its roles in ovarian development and related signaling pathway

In arthropods, retinoid X receptor (RXR) is a highly conserved nuclear hormone receptor. By forming a heterodimeric complex with the ecdysone receptor (EcR), RXR is known to be vital importance for various physiological processes. However, in comparison to EcR, the RXR signaling pathway and its roles in crustacean reproduction are poorly understood. In the present study, the RXR mRNA was detected in the ovarian follicular cells of mud crab Scylla paramamosain (SpRXR) and during ovarian maturation, its expression level was found to increase significantly. In vitro experiment showed hat both SpRXR and vitellogenin (SpVg) mRNA in the ovarian explants were significantly induced by 20-hydroxyecdysone (20E) but not methyl farnesoate (MF). However, differing from the in vitro experiment, injection of MF in in vivo experiment significantly stimulated the expressions of SpRXR and SpVg in female crabs at early vitellogenic stage, but the ecdysone and insect juvenile hormone (JH) signaling pathway genes were not induced. The results together suggest that both MF and SpRXR play significant roles in regulating the expression of SpVg and ovarian development of S. paramamosain through their own specific signaling pathway rather than sharing with the ecdysone or the insect JH

Cardiac Chemical Exchange Saturation Transfer MR Imaging Tracking of Cell Survival or Rejection in Mouse Models of Cell Therapy

Purpose: To examine whether cardiac chemical exchange saturation transfer (CEST) imaging can be serially and noninvasively used to probe cell survival or rejection after intramyocardial implantation in mice. Materials and Methods: Experiments were compliant with the National Institutes of Health Guidelines on the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. One million C2C12 cells labeled with either europium (Eu) 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A) or saline via the hypotonic swelling technique were implanted into the anterior-lateral left ventricular wall in C57BL/6J (allogeneic model, n = 17) and C3H (syngeneic model, n = 13) mice. Imaging (frequency offsets of ±15 parts per million) was performed 1, 10, and 20 days after implantation, with the asymmetrical magnetization transfer ratio (MTRasym) calculated from image pairs. Histologic examination was performed at the conclusion of imaging. Changes in MTRasym over time and between mice were assessed by using two-way repeated-measures analysis of variance. Results: MTRasym was significantly higher in C3H and C57BL/6J mice in grafts of Eu-HP-DO3A–labeled cells (40.2% ± 5.0 vs 37.8% ± 7.0, respectively) compared with surrounding tissue (−0.67% ± 1.7 vs −1.8% ± 5.3, respectively; P \u3c .001) and saline-labeled grafts (−0.4% ± 6.0 vs −1.2% ± 3.6, respectively; P \u3c .001) at day 1. In C3H mice, MTRasym remained increased (31.3% ± 9.2 on day 10, 28.7% ± 5.2 on day 20; P \u3c .001 vs septum) in areas of in Eu-HP-DO3A–labeled cell grafts. In C57BL/6J mice, corresponding MTRasym values (11.3% ± 8.1 on day 10, 5.1% ± 9.4 on day 20; P \u3c .001 vs day 1) were similar to surrounding myocardium by day 20 (P = .409). Histologic findings confirmed cell rejection in C57BL/6J mice. Estimation of graft area was similar with cardiac CEST imaging and histologic examination (R2 = 0.89). Conclusion: Cardiac CEST imaging can be used to image cell survival and rejection in preclinical models of cell therapy

Intravesical CD74 and CXCR4, Macrophage Migration Inhibitory Factor (MIF) Receptors, Mediate Bladder Pain

BACKGROUND: Activation of intravesical protease activated receptor 4 (PAR4) leads to release of urothelial macrophage migration inhibitory factor (MIF). MIF then binds to urothelial MIF receptors to release urothelial high mobility group box-1 (HMGB1) and elicit bladder hyperalgesia. Since MIF binds to multiple receptors, we investigated the contribution of individual urothelial MIF receptors to PAR4-induced HMGB1 release in vivo and in vitro and bladder pain in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We tested the effect of intravesical pre-treatment with individual MIF or MIF receptor (CD74, CXCR4, CXCR2) antagonists on PAR4-induced HMGB1 release in vivo (female C57/BL6 mice) and in vitro (primary human urothelial cells) and on PAR4-induced bladder hyperalgesia in vivo (mice). In mice, PAR4 induced HMGB1 release and bladder hyperalgesia through activation of intravesical MIF receptors, CD74 and CXCR4. CXCR2 was not involved in these effects. In primary urothelial cells, PAR4-induced HMGB1 release through activation of CD74 receptors. Micturition parameters in mice were not changed by any of the treatments. CONCLUSIONS/SIGNIFICANCE: Urothelial MIF receptors CD74 and CXCR4 mediate bladder pain through release of urothelial HMGB1. This mechanism may set up persistent pain loops in the bladder and warrants further investigation. Urothelial CD74 and CXCR4 may provide novel targets for interrupting bladder pain

Platelet Secretion and Hemostasis Require Syntaxin-binding Protein STXBP5

Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion

Immunorecognition of estrogen and androgen receptors in the brain and thoracic ganglion mass of mud crab,Scylla paramamosain

【英文摘要】 The brain and the thoracic ganglion of a crustacean can synthesize and secrete gonad-stimulating hormone(GSH)which stimulates the maturation of gonad.In the previous experiments,sex steroid hormones(estradiol,testosterone,progesterone,etc.)have been detected from the crustacean.However,the feedback regulation of sex steroid hormones on the brain and the thoracic ganglion of the crustacean has not been reported so far.In the present experiment,monoclonal antibodies were applied to investigate the immuno-reco..

Genetic diversity and differentiation of three populations of Penaeus monodon Fabricus

【英文摘要】 Genetic diversity of two wild Penaeus monodon populations sampled from the coastal waters of Qinglan (Hainan Province of China,HN) and Malaysia (KD),and the F1 generation of a Thailand broodstock population (CP) were examined by vertical polyacrylamide gel electrophoresis. Of 21 loci encoded by ten enzymes,11 were polymorphic. The mean proportions of polymorphic loci of HN,KD and CP were 36.36%,45.45% and 50.00%,with the average heterozygosities of 0.135,0.181 and 0.191,and the effective numbers of alleles ..The National Natural Science Foundation of China under contract No.30471322

Immunocytochemical localization of neuropeptide Y,serotonin,substance P and β-endorphin in optic ganglia and brain of Metapenaeus ensis

【英文摘要】 By using immunocytochemistry method of Strept Avidin-Biotin-Complex, four kinds of antisera raised against rabbits were applied to observe the immunoreactive neurons and neuropils of sero-tonin (5-HT), neuropeptide Y (NPY), substance P (SP) and β-Endorphin (β-Ep) in optic ganglia and brain of Metapenaeus ensis. The results showed that, the 5-HT-immunoreactive cells were located in all the four neuropils of optic ganglia. Immunoreactivity of 5-HT was detected in anterior medial protocerebrum neuropils (AMPN...Supported by the Key Foundation Research Program of Fujian Province (1998-2002

Hepatopancreas cell cultures from mud crab, Scylla paramamosain

Hepatopancreas is an important digestive and endocrine organ in crustacean. However, there are few reports on cell cultures from crabs. Here, the cell cultures of hepatopancreas from Scylla paramamosain was studied in vitro. Both the primary cell culture and subculture were grown in Leibovitz' L-15 medium, M199 medium, or a specially designed medium for S. paramamosain (MSP). The results showed that hepatopancreas cells in vitro grew in compact clusters in 2-3 d. Four types of cells could be identified. They were embryo cells, fibrillar cells, resorptive cells, and blister-like cells, respectively. Some of these cells could be subcultured for three generations. The MSP supported the best survival of these hepatopancreas cells, while M199 medium was the least effective of these three media. Fetal bovine serum and crab muscle extracts as supplements stimulated growth, but the crab hemolymph inhibited cell growth. Taken together, MSP is an appropriate medium for hepatopancreas cell cultures from S. paramamosain and can support cultures through several passages.National Natural Science Foundation of China [40776084]; National High Technology Research and Development Program of China [2006AA10A406