Influence of Ca2+ and pH on the folding of the prourotensin II precursor

Proper folding is a crucial step for the trafficking of proteins through the secretory pathway. We hypothesized that the secretory granules of endocrine cells provide optimal folding conditions of prohormone precursors for cleavage. Here, using circular dichroism and in vitro processing on purified prourotensin II (ProUII), we show that the precursor undergoes pH- and Ca(2+)-dependent conformational and stability changes. ProUII has a stable tertiary structure at pH 5.5 in presence of Ca(2+) and is correctly cleaved in these conditions by prohormone convertases. Taken together, our results support the notion that precursors may need to be optimally folded in the lumen of secretory granules for their processing.     

The self-association of two N-terminal interaction domains plays an important role in the tetramerization of TRPC4

Transient receptor potential canonical (TRPC) channels function as cation channels. In a previous study, we identified the molecular determinants involved in promoting TRPC subunit assembly. In the present study, we used size-exclusion chromatography assays to show that the N-terminus of TRPC4 can self-associate and form a tetramer in cellulo. We further showed that the N-terminus of TRPC4 self-associates via the ankyrin repeat domain and the region downstream from the coiled-coil domain. GST pull-down, yeast two-hybrid, and circular dichroism approaches demonstrated that both domains can self-associate. These findings indicated that the self-association of two distinct domains in the N-terminus of TRPC4 is involved in the assembly of the tetrameric channel.     

Effects of caffeic acid phenethyl ester on matrix molecules and angiogenetic and anti-angiogenetic factors in gastric cancer cells cultured on different substrates

Migration, invasion, metastasis and angiogenesis associated with cancer depend on the surrounding microenvironment. Angiogenesis, the growth of new capillaries, is a regulator of cancer growth and a useful target for cancer therapy. We examined matrix protein interactions in a gastric cancer cell culture that was treated with different doses of caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE). We also investigated the relations among the levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), endostatin (ES) and trombospondin-1 (TSP-1). Cytotoxity of CAPE was measured using the 3-(4,5-dmethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We examined the behavior of cells on laminin and collagen I coated surfaces in response to the angiogenic effect of these matrix molecules. We examined the protein alterations of these matrix molecules immunohistochemically and measured the levels of VEGF, MMP-9, ES and TSP-1 using the ELISA test. We showed that application of CAPE to the gastric cancer cell line on tissue culture plastic, laminin and collagen I significantly decreased the VEGF and MMP-9 protein levels. We found that TSP-1 levels were increased significantly in the gastric cancer cells after application of CAPE. The protein levels of gastric cancer cells also were increased significantly when tissue was cultured on laminin and collagen I. Application of CAPE to cells on laminin or collagen I coated surfaces significantly increased all of the proteins except ES. ES levels were increased on the collagen I covered surfaces, but the laminin surface decreased the levels of ES significantly. We demonstrated the beneficial effect of CAPE on a gastric cancer cell line including inhibition of proliferation and induction of some proteins that might be related to decreased angiogenesis.     

粘虫生殖的研究——Ⅱ.补充营养对生殖力的效应

粘虫成虫需补充取食糖类,方能完成飞翔(包括迁飞)、交配、产卵等活动。我们用了18种糖类来测定粘虫取食的范围,并观察糖类对生殖力的效应。试验结果表明:粘虫能取食葡萄糖、果糖、甘露糖、半乳糖、蔗糖、麦芽糖、海藻糖、棉子糖、甘油、甘露醇等,能完成正常生殖活动,产下数百粒卵,或上千粒卵,卵能孵化;但取食水、木糖、山梨糖、乳糖、糊精、淀粉、糖元等后,只能产下几粒卵,卵不能孵化。食量依糖液浓度而稍有变化,产卵前期取食量最高。 文中讨论各种糖的营养效价,并比较了各种糖类对不同昆虫生存、飞翔、生殖等效应的异同。     

CHARACTERISTICS OF BRUCELLAPHAGE.

McDuff, C. R. (University of Wisconsin, Madison), Lois M. Jones, and J. B. Wilson. Characteristics of brucellaphage. J. Bacteriol. 83:324-329. 1962.-Methods of characterizing phage have been applied to a brucellaphage of Russian origin grown on its propagating strain, Brucella abortus R 19. Phage can be propagated by single plaque transfer. Phage titers of about 10(10) particles per ml can be obtained by propagation on a young culture of R 19 in Albimi broth on a shaker at 37 C. After lyophilization, phage retains its activity during storage for at least 20 months at 4 C. Phage is stable in broth at pH values from 6 to 8 for 24 hr at 37 C. Some loss in activity results from heating for 1 hr at 60 C. All activity is lost in the presence of 10% chloroform. It has a slow adsorption rate (K = 3.6 x 10(-11) ml/min), a latent period of 100 min, and a burst size of 121 particles. Electron micrographs indicate that the phage is approximately 65 mmu in diameter, polygonal in shape, with a short tail.     

Mass ivermectin treatment for Onchocerciasis: Lack of evidence for collateral impact on transmission of Wuchereria bancrofti in areas of co-endemicity

There has long been interest in determining if mass ivermectin administration for onchocerciasis has 'unknowingly' interrupted lymphatic filariasis (LF) transmission where the endemicity of the two diseases' overlaps. We studied 11 communities in central Nigeria entomologically for LF by performing mosquito dissections on Anopheline LF vectors. Six of the communities studied were located within an onchocerciasis treatment zone, and five were located outside of that zone. Communities inside the treatment zone had been offered ivermectin treatment for two-five years, with a mean coverage of 81% of the eligible population (range 58–95%). We found 4.9% of mosquitoes were infected with any larval stage of W. bancrofti in the head or thorax in 362 dissections in the untreated villages compared to 4.7% infected in 549 dissections in the ivermectin treated villages (Mantel-Haenszel ChiSquare 0.02, P = 0.9). We concluded that ivermectin annual therapy for onchocerciasis has not interrupted transmission of Wuchereria bancrofti (the causative agent of LF in Nigeria).     

Mutation G827R in matriptase causing autosomal recessive ichthyosis with hypotrichosis yields an inactive protease

Matriptase is a member of the novel family of type II transmembrane serine proteases. It was recently shown that a rare genetic disorder, autosomal recessive ichthyosis with hypotrichosis, is caused by a mutation in the coding region of matriptase. However, the biochemical and functional consequences of the G827R mutation in the catalytic domain of the enzyme have not been reported. Here we expressed the G827R-matriptase mutant in bacterial cells and found that it did not undergo autocatalytic cleavage from its zymogen to its active form as did the wild-type matriptase. Enzymatic activity measurements showed that the G827R mutant was catalytically inactive. When expressed in HEK293 cells, G827R-matriptase remained inactive but was shed as a soluble form, suggesting that another protease cleaved the full-length mature form of matriptase. Molecular modeling based on the crystal structure of matriptase showed that replacing Gly(827) by Arg blocks access to the binding/catalytic cleft of the enzyme thereby preventing autocatalysis of the zymogen form. Our study, thus, provides direct evidence that the G827R mutation in patients with autosomal recessive ichthyosis with hypotrichosis leads to the expression of an inactive protease.