Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers

A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins. Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104. The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers. The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates. Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility. The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C. Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.     

利用DNA分子标志鉴别台湾茶树品种及评估种原之遗传歧异性

为评估台湾茶树种原之遗传歧异性。本研究由100条ISSR引子申筛选出12条可产生多型性条带明显的引子．这些引子共可产生67个的多型性条带。依据每一种原之分子标志数据进行UPGMA法分群分析结果。可将台湾133个茶树种原区分成六大群，包括油茶群、赤芽山茶群、野生茶树群、大叶变种与小叶变种混合群、大叶、小叶及大叶、小叶杂交种混合群及小叶变种群。而主成分向量分析的结果与利用群聚分析得到的亲缘关系树形固结果相符合。台湾茶树种原高比例的遗传歧异度是由台湾的野生茶树所贡献．部分重要栽培种间的相似性仍极高。为了探讨制茶过程封分子级品种鉴定之影响及DNA分子标志应用于咸茶品种鉴定之可行性，本研究分析不同发酵程度的茶类。在制茶过程申封DNA质量之影响。试验结果显示高温杀菁过程严重造成咸茶DNA的降解。利用各种类别咸茶与新鲜茶叶（封照）所抽取之DNA样品进行PCR扩增反应．结果发现分子量小于1,000bp的ISSR DNA条带表现较稳定。     

Treatment with taurine attenuates hepatic apoptosis in NZB/W F1 mice fed with a high-cholesterol diet

Cholesterol-rich diets are known to cause hepatic apoptosis, which has been associated with the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanisms and treatments for hepatic apoptosis in SLE are poorly understood. To clarify the effects of taurine on hepatic apoptosis in SLE, NZB/W F1 mice received control, cholesterol, and cholesterol/taurine diets. Significant reductions of caspase-3 activity, TUNEL-positive cells, and Fas- and mitochondrial- dependent apoptosis were detected in liver from the cholesterol/taurine group as compared to the cholesterol group. Moreover, significant increases of phosphorylated AKT, NF-kappaB (p65), and ERK1/2 proteins were detected in liver from the cholesterol/taurine group as compared to the cholesterol group. In contrast, a significant reduction of phosphorylated p38 protein was observed in the cholesterol/taurine group. These experimental results demonstrated positive effects of taurine against hepatic apoptosis in NZB/W F1 mice fed a high-cholesterol diet and suggested the therapeutic potential of taurine in SLE.     

Rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: application to hydroxyl radical-scavenging ability of aqueous extracts of Food constituents

With the availability of an ultraweak chemiluminescence analyzer, it is possible to monitor the production of a specific oxygen-derived reactive species, such as hydroxyl radical ((*)OH), whenever a suitable chemiluminescent probe is obtainable. Reported herein is the development of a rapid and specific method for detecting (*)OH production using a specific probe, indoxyl-beta-glucuronide (IBG), a low-level chemiluminescence emitter. Using the Fenton reagent as a source of (*)OH, it was shown that IBG could elicit a very strong intensity of chemiluminescence (CL) (16200 +/- 200 photon counts/s). Conversely, IBG was shown to be insensitive to either superoxide radical or hydrogen peroxide with their CL intensities nearly close to the background values (25 +/- 5 and 180 +/- 20 photon counts/s, respectively). Furthermore, it was also shown that this IBG-based CL production could be effectively quenched by the addition of (*)OH scavengers such as sodium salicylate, dimethyl sulfoxide, and penicillamine to the assay system. Taken together, these data indicate that IBG is a specific CL probe suitable for monitoring the production of (*)OH. This system demonstrated inhibitory activities of various aqueous extracts of food constituents on the CL of hydroxyl radicals generated by Fenton's reagents with the order of scavenging efficiencies being Prunus mume > Cordyceps sinensin > Lilium lancifolium > Astragalus membranceus.     

新疆农业高效节水工程资产运行管理模式比较分析

新疆农业高效节水工程项目资产运行管理模式是在新疆节水灌溉工程大规模建设过程中逐渐形成的,在对新疆农业高效节水工程项目资产管理模式归纳总结的基础上,对各种模式优缺点采用定性分析的方法进行了比较,分析了存在的问题,对进一步提高节水工程项目资产管理水平提出了一些建议。     

基于SNP和InDel标记的巴西木薯遗传多样性与群体遗传结构分析

As a typical tropical crop, cassava (Manihot esculenta Crantz) has the characteristics of drought resistance, barren resistance, high biomass and so on. In addition to being used for food and forage, it can also be used for production, processing and starch extraction. Due to highly heterozygous cassava genome, breeding is more difficult. Enriching the genetic diversity of cassava germplasm, comprehensively evaluating its genetic background and traits, and discovering superior alleles that control excellent traits are of great significance for cassava breeding in the future. In order to analyze the genetic diversity, genetic relationship and population structure of cassava germplasm in Brazil, 7946 SNPs and 1997 InDels molecular markers were used. Population structure analysis was performed by ADMIXTURE software, and principal component analysis was performed by GCTA software. Brazilian cassava was divided into nine subgroups, and was roughly consistent with the results of cluster analysis using PHYLIP. Among them, subgroup 1, subgroup 2, subgroup 4, subgroup 6, and subgroup 8 could be clustered together respectively, while the samples of other subgroups could be roughly clustered, and there was a certain cross between the samples. The genetic diversity of cassava germplasm in Brazil (0.274) was higher than the genetic diversity level of cassava germplasm in China and Nigeria. Subgroup 5 of Brazil cassava had a relatively high genetic diversity (0.29). The genetic differentiation of subgroups was low (the genetic differentiation vary from 0.03 to 0.15), but higher than domestic cassava germplasm. The genetic distance between cassava accessions varied from 0.084 to 0.297, with the average of 0.228. The results of this study can provide a basis for subsequent association analysis to identify great alleles and introduction.     

4-Shogaol, an active constituent of dietary ginger, inhibits metastasis of MDA-MB-231 human breast adenocarcinoma cells by decreasing the repression of NF-κB/Snail on RKIP

4-Shogaol is one of the phytoconstituents isolated from dried red ginger, which is commercially available to consumers. Some active constituents from ginger have been found to have anti-inflammatory and antioxidant effects, but studies on 4-shogaol have been relatively rare. This is the first report describing the antimetastasis activities of 4-shogaol and the possible mechanisms. This study determined that 4-shogaol inhibits the migration and invasion of MDA-MB-231 and causes mesenchymal-epithelial transition (MET). In addition, 4-shogaol suppresses the activation of NF-κB and cell migration and invasion induced by TNF-α. Furthermore, 4-shogaol has been shown to inhibit the phosphorylation of IκB and the translocation of NF-κB/Snail in MDA-MB-231. This study shows that RKIP, an inhibitory molecule of IKK, is up-regulated after 4-shogaol treatment and prolongs the inhibitory effects of 4-shogaol. Inhibition of RKIP by shRNA transfection significantly decreases the inhibitory effect of 4-shogaol on the NF-κB/Snail pathway, together with cell migration and invasion, whereas overexpression of Snail suppresses 4-shogaol-mediated metastasis inhibition and E-cadherin upregulation. Finally, the animal model revealed that 4-shogaol effectively inhibits metastasis of MDA-MB-231 in mice. This study demonstrates that 4-shogaol may be a novel anticancer agent for the the treatment of metastasis in breast cancer.     

Effects of whey protein concentrate (WPC) on the distributions of lymphocyte subpopulations in rats with excessive alcohol intake

To investigate the effects of whey protein concentrate (WPC) on antioxidant statuses and the lymphocyte subpopulations in the rats with alcohol intake, the antioxidant statuses in the peripheral blood (PB) and the lymphocyte subpopulations in the PB, spleen, and bone marrow (BM) of the rats fed with WPC (0.334 g/kg) and alcohol (6 g/kg) for 3 months were analyzed. Results showed that the effects of WPC on the glutathione peroxidase and glutathione in the PB, the T and B cells in the spleen, and the B cells in the BM were more apparent in the rats with alcohol intake; however, they are not apparent in the controls. Taken together, our results indicated that the immunity of rats might be enhanced by the increased antioxidant ability after WPC supplementation and the effects of WPC on the lymphocyte subpopulations were mainly in the spleen and BM and not in the PB.     

Green tea polyphenol epigallocatechin-3-gallate protects cells against peroxynitrite-induced cytotoxicity: modulatory effect of cellular G6PD status

Glucose-6-phosphate dehydrogenase (G6PD) plays important roles in the maintenance of cellular redox balance. It was not until recently that the importance of G6PD in regulation of cellular growth and apoptosis emerged. In the present study, we found that G6PD-deficient fibroblasts were more susceptible to peroxynitrite-induced cytotoxicity. Treatment with peroxynitrite generator 3-morpholinosydnonimine (SIN-1) hydrochloride caused apoptosis in human fibroblast in a dose-dependent manner. This was preceded by a decrease in the intracellular level of glutathione (GSH) as well as accumulation of p53. The extent of apoptosis and glutathione depletion were greater in G6PD-deficient fibroblasts than in the normal counterpart. Pretreatment with green tea polyphenol epigallocatechin-3-gallate (EGCG) effectively blocked peroxynitrite-induced glutathione depletion, p53 accumulation, and apoptosis in both normal and G6PD-deficient cells. EGCG, administered to cells alone or as pretreatment, caused activation of Akt. The protective effect was abolished by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin, and LY294002. Our findings suggest that G6PD deficiency enhances the toxicity of peroxynitrite and that EGCG initiates cell survival signaling via the PI3K/akt pathway.     

A cell-based screening identifies compounds from the stem of Momordica charantia that overcome insulin resistance and activate AMP-activated protein kinase

Treatment of insulin resistance is a critical strategy in the prevention and management of type 2 diabetes. The crude extracts from all parts of Momordica charantia L. have been reported by many studies for the effective treatment of diabetes and related complications. However, the exact ingredients responsible for the hypoglycemic effect and the underlying mechanism of their actions have not been well characterized because of the lack of a proper assay and screening system. A new cell-based, nonradioactive, and nonfluorescent screening method was demonstrated in this study to screen for natural products from the stem of M. charantia, aiming to identify hypoglycemic components that can overcome cellular insulin resistance. The results suggest triterpenoids being potential hypoglycemic components of the plant and the mechanism underlying their action involving AMP-activated protein kinase.