Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel

Coronaviruses contain a small envelope membrane protein with cation-selective ion channel activity mediated by its transmembrane domain (ETM). In a computational study, we proposed that ion channel activity can be explained by either of two similar ETM homopentameric transmembrane α-helical bundles, related by a ∼50° rotation of the helices. Later, we tested this prediction, using site-specific infrared dichroism of a lysine-flanked isotopically labeled ETM peptide from the virus responsible for the severe acute respiratory syndrome, SARS, reconstituted in lipid bilayers. However, the data were consistent with the presence of a kink at the center of the ETM α-helix, and it did not fit completely either computational model. Herein, we have used native ETM, without flanking lysines, and show that the helix orientation is now consistent with one of the predicted models. ETM only produced one oligomeric form, pentamers, in the lipid-mimic detergent dodecylphosphocholine and in perfluorooctanoic acid. We thus report the correct backbone model for the pentameric α-helical bundle of ETM. The disruptive effects caused by terminal lysines probably highlight the conformational flexibility required during ion channel function.     

Insulin resistance and impaired functional vasodilation in obese Zucker rats

Individuals with metabolic syndrome exhibit insulin resistance and an attenuated functional vasodilatory response to exercise. We have shown that impaired functional vasodilation in obese Zucker rats (OZRs) is associated with enhanced thromboxane receptor (TP)-mediated vasoconstriction. We hypothesized that insulin resistance, hyperglycemia/hyperlipidemia, and the resultant ROS are responsible for the increased TP-mediated vasoconstriction in OZRs, resulting in impaired functional vasodilation. Eleven-week-old male lean Zucker rats (LZRs) and OZRs were fed normal rat chow or chow containing rosiglitazone (5 mg.kg(-1).day(-1)) for 2 wk. In another set of experiment, LZRs and OZRs were treated with 2 mM tempol (drinking water) for 7-10 days. After the treatments, spinotrapezius muscles were prepared, and arcade arteriolar diameters were measured following muscle stimulation and arachidonic acid (AA) application (10 muM) in the absence and presence of the TP antagonist SQ-29548 (1 muM). OZRs exhibited higher insulin, glucose, triglyceride, and superoxide levels and increased NADPH oxidase activity compared with LZRs. Functional and AA-induced vasodilations were impaired in OZRs. Rosiglitazone treatment improved insulin, glucose, triglyceride, and superoxide levels as well as NADHP oxidase activity in OZRs. Both rosiglitazone and tempol treatment improved vasodilatory responses in OZRs with no effect in LZRs. SQ-29548 treatment improved vasodilatory responses in nontreated OZRs with no effect in LZRs or treated OZRs. These results suggest that insulin resistance and the resultant increased ROS impair functional dilation in OZRs by increasing TP-mediated vasoconstriction.     

眼镜蛇毒对大鼠脊髓乙酰胆碱酯酶表达的影响

目的 探讨眼镜蛇毒对大鼠腰髓乙酰胆碱酯酶（AchE）表达的影响。方法 将蛇毒注射到大鼠左小腿下部,采用亚铁氰化铜法示AchE,观察大鼠腰髓前角AchE阳性神经元在眼镜蛇毒中毒组、生理盐水组、正常对照组的变化。结果 蛇毒组大鼠AchE阳性神经元比对照组表达增强。结论 眼镜蛇毒对腰髓的AchE阳性神经元表达有上调作用。     

Multistep mass tagging coupled with 2D LC-MS--an approach to increasing the number of identified proteins.

Identification of large numbers of proteins from complex biological samples is a continuing challenge in the area of quantitative proteomics. We introduce here a simple and reliable multistep mass tagging technique using our recently developed solid phase mass tagging reagents. When coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D-LC/nano-ESI-MS), this method allows enhanced protein identification when tested on samples from prokaryotic and eukaryotic sources. The proteome of Escherichia coli D21 grown to either mid-exponential or stationary phase, and the membrane proteome from established breast cancer cell lines BT474 and MCF7 were used as model systems in these experiments. In both experiments, the numbers of total identified proteins are at least twice the numbers identified from a single tagging cycle. The sample complexity can be effectively reduced with corresponding increases in protein identification using the multistep method. The strategy described here represents a potentially powerful technique for large-scale qualitative and quantitative proteome research.     

奶山羊转基因供核细胞的再饥饿对核移植胚胎发育的影响

为提高转基因奶山羊体细胞核移植胚胎早期发育率,将经转染外源基因的山羊胎儿成纤维细胞经饥饿培养(含0.5?S的DMEM)5天后分成两部分:第一部分细胞-80℃或液氮冻存,试验前复苏后直接用作供核细胞(试验组Ⅰ),或复苏后恢复培养(含10% FCS的DMEM)2-5天后再饥饿5天用作供核细胞(试验组Ⅱ);第二部分细胞作传代培养(含10% FCS的DMEM)2天后再饥饿5 天用作供核细胞(试验组Ⅲ)。将上述不同处理的供核细胞进行细胞周期与存活率的检测,并将该供核细胞移入去除遗传物质的山羊MⅡ期卵母细胞的卵周隙内,经电融合、化学激活后,将核移植(NT)胚胎经0.8%琼脂糖包理后移入临时寄母输卵管内,培养6天后回收并观察NT胚胎的早期发育。结果,试验组Ⅱ所用供核细胞中G_0/G_1期细胞所占比例及其存活率分别为95.68%、99.9%,均显著地高于试验组Ⅰ(88.66%、80%);试验组Ⅱ的桑椹及囊胚期NT胚胎的发育率(66.09%)显著地高于试验组Ⅰ(22.00%)与试验组Ⅲ(50.51%)。将以上发育的NT胚胎分别移入同步发情的受体后,35 天作B超妊娠诊断,试验组Ⅱ的受体妊娠率为45.83%,显著地高于试验组Ⅰ(20.00%)与试验组Ⅲ(9.58%)。流式细胞仪分析结果表明,饥饿后的供核细胞经冷冻,复苏后恢复培养2-5天,再经饥饿处理,能显著地提高G_0/G_1期细胞的比例及细胞存活率;应用该细胞所组建的NT胚不仅具有较高的桑椹与囊胚期发育率,而且具有较高的受体妊娠率。     

von Hippel-Lindau病的分子机制研究

VHL病(Von Hippel-Lindau disease)是一种遗传性肿瘤综合征,由VHL抑癌基因突变引起.研究表明,VHL蛋白在体内具有多种功能,VHL基因突变形式和部位的差异所造成的VHL蛋白功能增加、减少或缺失可能是导致肿瘤不同表型的重要原因.     

Soya food intake and risk of endometrial cancer among Chinese women in Shanghai: population based case-control study

Objective To evaluate the association of intake of soya food, a rich source of phytoestrogens, with the risk of endometrial cancer.Design Population based case-control study, with detailed information on usual soya food intake over the past five years collected by face to face interview using a food frequency questionnaire.Setting Urban Shanghai, China.Participants 832 incident cases of endometrial cancer in women aged of 30 to 69 years diagnosed during 1997-2001 and identified from the Shanghai Cancer Registry; 846 control women frequency matched to cases on age and randomly selected from the Shanghai Residential Registry.Main outcome measures Odds ratios for risk of endometrial cancer in women with different intakes of soya foods.Results Regular consumption of soya foods, measured as amount of either soya protein or soya isoflavones, was inversely associated with the risk of endometrial cancer. Compared with women with the lowest quarter of intake, the adjusted odds ratio of endometrial cancer was reduced from 0.93 to 0.85 and 0.67 with increasing quarter of soya protein intake (P for trend 0.01). A similar inverse association was observed for soya isoflavones and soya fibre intake. The inverse association seemed to be more pronounced among women with high body mass index and waist:hip ratio.Conclusion Regular intake of soya foods is associated with a reduced risk of endometrial cancer.     

Effect of metal ions on the activity of the catalytic domain of calcineurin

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa. No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.