Reciprocal regulation of syndecan-2 and Notch signaling in vascular smooth muscle cells

Vascular cell interactions mediated through cell surface receptors play a critical role in the assembly and maintenance of blood vessels. These signaling interactions transmit important information that alters cell function through changes in protein dynamics and gene expression. Here, we identify syndecan-2 (SDC2) as a gene whose expression is induced in smooth muscle cells upon physical contact with endothelial cells. Syndecan-2 is a heparan sulfate proteoglycan that is known to be important for developmental processes, including angiogenesis. Our results show that endothelial cells induce mRNA expression of syndecan-2 in smooth muscle cells by activating Notch receptor signaling. Both NOTCH2 and NOTCH3 contribute to the increased expression of syndecan-2 and are themselves sufficient to promote its expression independent of endothelial cells. Syndecan family members serve as coreceptors for signaling molecules, and interestingly, our data show that syndecan-2 regulates Notch signaling and physically interacts with NOTCH3. Notch activity is attenuated in smooth muscle cells made deficient in syndecan-2, and this specifically prevents expression of the differentiation marker smooth muscle α-actin. These results show a novel mechanism in which Notch receptors control their own activity by inducing the expression of syndecan-2, which then acts to propagate Notch signaling by direct receptor interaction.     

TRIM50 Protein Regulates Vesicular Trafficking for Acid Secretion in Gastric Parietal Cells

Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.     

Role of soluble epoxide hydrolase phosphatase activity in the metabolism of lysophosphatidic acids

The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of epoxides from arachidonic acid and other unsaturated fatty acids, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, inflammation and pain. While recent findings suggested complementary biological roles for Nterm-phos, its mode of action is not well understood. Herein, we demonstrate that lysophosphatidic acids are excellent substrates for Nterm-phos. We also showed that sEH phosphatase activity represents a significant (20-60%) part of LPA cellular hydrolysis, especially in the cytosol. This possible role of sEH on LPA hydrolysis could explain some of the biology previously associated with the Nterm-phos. These findings also underline possible cellular mechanisms by which both activities of sEH (EH and phosphatase) may have complementary or opposite roles.     

G6PD Genotype and Its Associated Enzymatic Activity in a Chinese Population

Knowledge of the G6PD genotype and its associated enzyme activity is significant for population genetics, diagnosis of disease, and management of patients. We tested 2,872 unrelated subjects from a Hakka population in China for G6PD activity by the WHO standard method and for genotype by DHPLC and DNA sequencing. Among female heterozygotes, 78.5% had relatively normal enzyme activity. The phenotype frequency of G6PD deficiency is 0.028, and the causal allele frequency is 0.060 in females. The accuracy, sensitivity, and specificity of DHPLC are more than 98% for detecting G6PD-deficient hemizygotes, heterozygotes, and homozygotes. Measuring enzyme activity alone is not sufficient for the diagnosis of heterozygotes. A combination of enzyme activity and DNA analysis should be used.     

一种新的抗菌药物靶标蛋白质(3，4-二羟基-2-丁酮-4-磷酸合成酶)的克隆、表达、纯化及酶活性鉴定

[目的]核黄素( vitamin B12,riboflavin)是辅因子黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)和黄素单核苷酸(flavin mononucleotide,FMN)的前体物,对生物体的生物合成至关重要.如果细菌不能够从外界摄取足够的黄素( flavin)就需要自身合成核黄素以维持菌体的生存与增殖.3,4-二羟基-2-丁酮-4-磷酸合成酶(3,4-Dihydroxy-2-butanone-4-phosphate synthase,DHBPs)为核黄素生物合成途径中关键酶之一.在镁离子存在的情况下,DHBPs将5-磷酸核酮糖(ribulose-5 -phosphate,Ru5P)转换成3,4-二羟基-2-丁酮4-磷酸(3,4-dihydroxy-2-Bu-tanone-4-Pho-sphate,DHBP)和甲酸盐(formate),生成的DHBP为核黄素合成的必需原料之一.人类没有合成核黄素的相关途径,因此细菌参与合成核黄素的DHBPs等相关酶就有望成为抗菌药物作用的靶位点.本课题通过对肺炎链球菌的DHBPs进行克隆表达纯化与酶学性质鉴定,为开展其三维结构的解析和抗菌药物设计提供重要的工作基础.[方法]利用PCR技术扩增DHBPs基因,构建重组表达载体pW28-DHBPs.将其转入大肠杆菌(Escherichia coli)BL21( DE3)中表达,用Ni离子亲和层析及离子交换(DEAE)纯化获得有活性的DHBPs后,进行酶学性质鉴定.[结果]酶切和测序证实成功构建了质粒pW28-DHBPs,在E.coli BL21中表达了可溶性DHBPs,纯化后获得了纯度为95％的靶蛋白质,经分子筛分析DHBPs在溶液中以二聚体形式存在.对DHBPs进行酶学性质分析表明,在25℃、pH为7.5和Mg2+存在的情况下,DHBPs具有将5-磷酸核酮糖转换成DHBP和甲酸盐的活性.[结论]第一次成功克隆并在E.coli BL21中表达了一种肺炎链球菌合成核黄素的相关酶—DHBPs,纯化后的重组DHBPs具有较好的5-磷酸核酮糖分解活性,这为解析其三维结构和基于结构进行的新一代抗菌药物设计提供重要的工作基础.     

Analysis of NLRP3 in the development of allergic airway disease in mice

The contribution of NLRP3, a member of the nucleotide-binding domain leucine-rich repeat-containing (NLR) family, to the development of allergic airway disease is currently controversial. In this study, we used multiple allergic asthma models to examine the physiologic role of NLRP3. We found no significant differences in airway eosinophilia, histopathologic condition, mucus production, and airway hyperresponsiveness between wild-type and Nlrp3(-/-) mice in either acute (alum-dependent) or chronic (alum-independent) OVA models. In addition to the OVA model, we did not detect a role for NLRP3 in the development of allergic airway disease induced by either acute or chronic house dust mite Ag exposure. Although we did not observe significant phenotypic differences in any of the models tested, we did note a significant reduction of IL-13 and IL-33 in Nlrp3(-/-) mice compared with wild-type controls in the chronic OVA model without added alum. In all of the allergic airway disease models, the NLRP3 inflammasome-associated cytokines IL-1β and IL-18 in the lung were below the level of detection. In sum, this report surveyed four different allergic asthma models and found a modest and selected role for NLRP3 in the alum-free OVA model. However, this difference did not greatly alter the clinical outcome of the disease. This finding suggests that the role of NLRP3 in allergic asthma must be re-evaluated.     

Complete genomic sequence of goose-origin reovirus from china

We report the full-genome sequence of a goose-origin reovirus (GRV) strain 03G from Zhejiang Province, China. This is the first report of the complete genomic sequence (segments 1 to 10) of GRV. Phylogenetic analyses of the sequence suggest that GRV 03G represents a new species distinct from other established species within the avian reovirus (ARV) group of orthoreoviruses.