Enhanced autophagy plays a cardinal role in mitochondrial dysfunction in type 2 diabetic Goto-Kakizaki (GK) rats: ameliorating effects of (-)-epigallocatechin-3-gallate

Oxidative stress and mitochondrial dysfunction are known to play important roles in type 2 diabetes mellitus (T2DM) and insulin resistance. However, the pathology of T2DM remains complicated; in particular, the mechanisms of mitochondrial dysfunction in skeletal muscle and other insulin-sensitive tissues are as yet unclear. In the present study, we investigated the underlying mechanisms of oxidative stress and mitochondrial dysfunction by focusing on mitochondrial dynamics, including mitochondrial biogenesis and autophagy, in skeletal muscle of a nonobese diabetic animal model--the Goto-Kakizaki (GK) rat. The results showed that GK rats exhibited impaired glucose metabolism, increased oxidative stress and decreased mitochondrial function. These dysfunctions were found to be associated with induction of LC3B, Beclin1 and DRP1 (key molecules mediating the autophagy pathway), while they appeared not to affect the mitochondrial biogenesis pathway. In addition, (-)-epigallocatechin-3-gallate (EGCG) was tested as a potential autophagy-targeting nutrient, and we found that EGCG treatment improved glucose tolerance and glucose homeostasis in GK rats, and reduced oxidative stress and mitochondrial dysfunction in skeletal muscle. Amelioration of excessive muscle autophagy in GK rats through the down-regulation of the ROS-ERK/JNK-p53 pathway leads to improvement of glucose metabolism, reduction of oxidative stress and inhibition of mitochondrial loss and dysfunction. These results suggest (a) that hyperglycemia-associated oxidative stress may induce autophagy through up-regulation of the ROS-ERK/JNK-p53 pathway, which may contribute to mitochondrial loss in soleus muscle of diabetic GK rats, and (b) that EGCG may be a potential autophagy regulator useful in treatment of insulin resistance.     

珠三角典型稻田生态沟渠型人工湿地的非点源污染削减功能

为了评价珠江三角洲地区农业非点源污染控制技术的开发与削减功能,本研究以珠海市斗门区上洲村典型稻田系统为例,提出了一种适应当地实际情况的生态沟渠型人工湿地构建技术,并进行了示范工程建设和防治效果评价。结果表明:该生态沟渠利用农田既有的排灌系统,经过适当改建而成;在满足原有排灌功能的前提下,对稻田排水径流中固体悬浮物(SS)、总磷(TP)、总氮(TN)、化学需氧量(CODCr)、铵态氮(NH4+-N)、生化需氧量(BOD5)的去除效率分别达到71.7%、63.4%、49.9%、26.6%、14.5%和11.6%;对营养型和颗粒态污染物质净化作用效果较好,对有机型污染物净化作用有限;其中对SS和TP的作用最为明显。该技术和工程成本效益良好,既减轻了稻田排水对附近水体的污染负荷,又增加了农田的景观效果。在珠三角及类似的经济发达而人地关系紧张的地区具有一定的推广价值。     

冰灾后南岭五指山森林树种的受损与早期恢复

应用样带调查法,于2009年调查了2008年冰灾后南岭五指山森林优势种及保护树种的受损及恢复情况。在3条长2～7km、宽15m的样带调查区共记录了2832株胸径>2cm的树木,它们隶属于53科96属141种,其中2072株林木受到损伤,总受损率为73%,61%为轻度受损。灾后一年,约84%的个体能通过萌条恢复生长,但16%未出现萌条而死亡或濒临死亡。群落优势类群壳斗科、樟科、木兰科和金缕梅科优势种的受损率和萌条恢复率均较高。共记录了15种国家级保护树种,以广东松(Pinus kwangtungensis)和福建柏(Fokienia hodginsii)最为常见,除三尖杉(Cephalotaxus fortunei)和穗花杉(Amentotaxus argot-aenia)外,其余种类均有不同程度的受损,其中59%死亡或濒临死亡。此外,在43个具3株以下的小频度种中,25%的个体未见恢复而死亡。本研究结果揭示了2008年冰雪灾害对南岭五指山森林林木造成了严重的伤害,大多数树种具有较强的自然恢复能力,能以快速萌条方式进行恢复,但部分濒危珍稀植物和低密度种的死亡可能会改变原有群落的物种组成,进而影响未来群落的演替进程。     

花鲈Wap65-2基因的克隆、理化性质及其表达与哈维氏弧菌感染的相关性

Wap65-2(warm temperature acclimation related 65 kDa protein-2)是鱼类中发现的一种血浆糖蛋白,与细菌感染性免疫应激紧密相关。该研究首次克隆了花鲈Wap65-2基因全长cDNA序列。它由1601个核苷酸组成,包含一个大的开放阅读框,预期编码一个由436个氨基酸组成、相对分子质量为4.87×104的前体蛋白,N端19个残基为信号肽序列。序列和系统进化树分析表明,花鲈Wap65-2与欧洲海鲈进化关系最近,两者氨基酸同源性高达80.7%。健康花鲈Wap65-2基因mRNA主要在肝中表达,心和肌肉中少量表达。qRT-PCR分析揭示,哈维氏弧菌(Vibrio harveyi)感染感染12h后,花鲈肝中Wap65-2基因mRNA表达显著上调,24h时达到峰值,为健康对照的6.89倍。原核表达花鲈Wap65-2并制备抗血清。Western blot分析表明,哈维氏弧菌感染12h后,花鲈血清中Wap65-2含量显著增加,36h时达到最大值,为健康对照的5.33倍。综上所述,花鲈Wap65-2基因的表达与其细菌感染性免疫应激紧密相关。     

Bone marrow-derived mesenchymal stem cells differentiation into tubular epithelial-like cells in vitro

The possibility of differentiating bone marrow‐derived mesenchymal stem cells (BMSCs) into tubular epithelial‐like cells is explored in vitro. Purified BMSCs from Sprague–Dawley rats were obtained by density gradient centrifugation. Third generation BMSCs were divided into six groups and were cultured under different conditions. The expression of alkaline phosphatase and cytokeratin (CK)‐18 protein was detected through staining and immunocytochemistry, respectively, and the expression of E‐cadherin proteins was recorded through immunofluorescence. Some cells in ischemia/reperfusion (I/R), all‐trans retinoic acid (ATRA), epidermal growth factor (EGF) and bone morphogenetic protein‐7 (BMP‐7) groups turned positive, whereas the positive cells in the combined group significantly increased compared with the other groups. Compared with the control group, the positive expression rates of CK‐18 in the I/R, ATRA, EGF, BMP‐7 and the combined group were 11·50% ± 3·84%, 27·40% ± 2·70%, 29·60% ± 4·51%, 26·80% ± 5·00% and 44·00% ± 3·16%, respectively, and CK‐18 mRNA expression in the combined group was obviously higher than that in the other groups (P < 0·01). Immunofluorescence detection showed that E‐cadherin expression was not detectable in the control group, whereas the positive expression rates of E‐cadherin in the I/R, ATRA, EGF, BMP‐7 and the combined group were 6·75% ± 2·13%, 16·40% ± 2·69%, 18·25% ± 3·50%, 16·06% ± 2·00% and 30·26% ± 5·16%, respectively. The addition of ATRA, EGF and BMP‐7 induces BMSCs differentiation into tubular epithelial‐like cells in stimulated acute renal failure microenvironment in vitro. Copyright © 2011 John Wiley & Sons, Ltd.     

AP—PCR结合进化树分析在幽门螺杆菌地域起源特征研究中的价值

目的探讨随机引物PCR（Arbitrary primerPCR,AP—PCR）结合克隆测序及生物信息学分析等方法在研究幽门螺杆菌（Helicobacter pylori,Hpylori）菌株地域起源特征中的价值和意义。方法针对临床分离培养的Hpylori菌株的基因组DNA,采用一组10nt的寡核苷酸引物进行随机PCR扩增,选取相对保守的片段进行回收、克隆及测序,测序的基因序列提交GenBank数据库进行序列相似性的BLAST比对,收集BLAST比对得到的同源性较高不同地域来源螺杆菌的对应序列,用ClustalX软件进行排序,采用Mega4．0软件中的邻位相连法（Neighbor-joining）和最大简约法（Maximum—parsimony）进行进化树分析。结果随机引物扩增及筛选克隆测序得到的基因产物为NADH脱氢酶G和H亚单位的部分编码序列,与27株不同地域来源H．pylori及1株猫科动物来源螺杆菌菌株的同源性均高达90％以上,表明Hpylori中NADH脱氢酶基因序列为保守结构,进化树分析显示：采用AP．PCR方法得到的Hpylori临床菌株的基因序列,显示出东亚菌株来源的遗传特征,与具有东亚菌株特征的美洲秘鲁Sat464和Shi470菌株、韩国的52、51菌株、日本的1757菌株遗传距离较近,与南亚、欧洲菌株距离较远,与非洲的SouthAffica7菌株和猫科动物来源的Sheeba菌株的遗传距离最远。结论不仅某些特殊基因可以反映地域差异,随机定位相对保守的基因片段同样可以反映Hpylori的地域起源特征。AP—PCR、测序等技术方法与进化树分析相结合是探讨Hpylori地域起源特征的一种更为便捷有效的新方法。     

A hybrid gene team model and its application to genome analysis

It is well-known that functionally related genes occur in a physically clustered form, especially operons in bacteria. By leveraging on this fact, there has recently been an interesting problem formulation known as gene team model, which searches for a set of genes that co-occur in a pair of closely related genomes. However, many gene teams, even experimentally verified operons, frequently scatter within other genomes. Thus, the gene team model should be refined to reflect this observation. In this paper, we generalized the gene team model, that looks for gene clusters in a physically clustered form, to multiple genome cases with relaxed constraints. We propose a novel hybrid pattern model that combines the set and the sequential pattern models. Our model searches for gene clusters with and/or without physical proximity constraint. This model is implemented and tested with 97 genomes (120 replicons). The result was analyzed to show the usefulness of our model. We also compared the result from our hybrid model to those from the traditional gene team model. We also show that predicted gene teams can be used for various genome analysis: operon prediction, phylogenetic analysis of organisms, contextual sequence analysis and genome annotation. Our program is fast enough to provide a service on the web at http://platcom.informatics.indiana.edu/platcom/. Users can select any combination of 97 genomes to predict gene teams.     

PA‐MSHA inhibits proliferation and induces apoptosis through the up‐regulation and activation of caspases in the human breast cancer cell lines

To investigate the effects of PA‐MSHA (Pseudomonas aeruginosa‐mannose sensitive hemagglutinin) on inhibiting proliferation of breast cancer cell lines and to explore its mechanisms of action in human breast cancer cells. MCF‐10A, MCF‐7, MDA‐MB‐468, and MDA‐MB‐231HM cells were treated with PA‐MSHA or PA (Heat‐killed P. aeruginosa) at different concentrations and different times. Changes of cell super‐microstructure were observed by transmission electron microscopy. Cell cycle distribution and apoptosis induced by PA‐MSHA were measured by flow cytometry (FCM) with PI staining, ANNEXIN V‐FITC staining and Hoechst33258 staining under fluorescence microscopy. Western blot was used to evaluate the expression level of apoptosis‐related molecules. A time‐dependent and concentration‐dependent cytotoxic effect of PA‐MSHA was observed in MDA‐MB‐468 and MDA‐MB‐231HM cells but not in MCF‐10A or MCF‐7 cells. The advent of PA‐MSHA changed cell morphology, that is to say, increases in autophagosomes, and vacuoles in the cytoplasm could also be observed. FCM with PI staining, ANNEXIN V‐FITC and Hoechst33258 staining showed that the different concentrations of PA‐MSHA could all induce the apoptosis and G₀–G₁ cell cycle arrest of breast cancer cells. Cleaved caspase 3, 8, 9, and Fas protein expression levels were strongly associated with an increase in apoptosis of the breast cancer cells. There was a direct relationship with increased concentrations of PA‐MSHA but not of PA. Completely different from PA, PA‐MSHA may impart antiproliferative effects against breast cancer cells by inducing apoptosis mediated by at least a death receptor‐related cell apoptosis signal pathway, and affecting the cell cycle regulation machinery. J. Cell. Biochem. 108: 195–206, 2009. © 2009 Wiley‐Liss, Inc.