N+注入和60Co-γ辐照对柠檬酸发酵菌黑曲霉的诱变效应

首次尝试了应用两种核技术手段(同时)对黑曲霉进行诱变,即将大剂量^60Co-γ辐照的黑曲霉孢于直接进行低能氮离子注入,使处于休眠状态下的黑曲霉抱子同时受至^60Co-γ辐照和N^ 注入的作用。通过溴甲酚绿指示性平板辅助筛选和摇瓶发酵。获得了1株产酸提高18．44％、转化率达1034．5％的M3代菌株CN05,为柠檬酸发酵菌黑曲霉的进一步育种工作提供了诱变参数和高产出发菌株。     

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.     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇(PEG)诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

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。     

南方红豆杉响应不同传粉式样的结实表现

采用开花前雌株短枝套袋隔离传粉、花期人工授粉、风媒传粉 对照 及其传粉障碍等几种传粉式样 ,比较不同传粉 授粉 方式对南方红豆杉 Taxus chinensis var.mairei 结实的影响 ;在风媒传粉条件下 ,研究了南方红豆杉不同种植行向、树冠不同方位、上下内外不同层面的结实表现 .结果表明 :南方红豆杉存在孤雌生殖现象 ,平均单枝结实率为 2 8.7% ,与风媒传粉相比较 ,存在极显著差异 P<0 .0 1 ;人工授粉能显著提高结实率 P<0 .0 5 2 0 .7% ;2～ 3月份开花期 ,江汉平原盛行东北风 ,在风媒传粉条件下 ,东西向种植的南方红豆杉有更多的授粉机会 ,其结实情况极显著优于南北向种植的 P<0 .0 1 ;东西向种植的南方红豆杉树冠果实的空间分布 ,受传粉条件包括风向、冠层方位及距传粉源的距离、冠层内外上下层次等因素的影响 ,方位间的结实量按东南西北方位依次为 :2 30 .375粒、185 .6 2 5粒、12 8.813粒、10 5 .4 38粒 ,呈递减趋势 ,冠外层结实量普通高于冠内层 ,树冠东向下外层结实量最高 4 16 .75粒 ,北向下内层最低 2 2 .0 0粒 .     

Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

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+.     

应用树状DNA杂交(DDH)对生殖道尖锐湿疣中HPV DNA的分型检测

从手术切除的50例生殖道尖锐湿疣新鲜标本中,以及15例正常人血清中,提取基因组DNA,同时用树状DNA杂交(dendrimer DNA hybridizalion,DDH)技术和PCR进行HPV DNA的分型检测.结果50例尖锐湿疣中,以DDH方法检测,感染HPV6型者20例,感染11型者24例,6/11型混合感染者3例,阴性3例,总检测率达94%;以PCR方法检测,HPV6型感染者21例,11型感染者24例,6/11型混合感染者3例,阴性2例,总检测率为96%.15例正常人血清中,以DDH方法检测,HPV感染的假阳性率为0%;以PCR检测,假阳性率为6.67%.还以HPV阳性标本对DDH方法做了敏感度的测定,结果阳性病例DNA检测最低浓度为97.28pg/ml.研究表明,DDH技术具有较高敏感性和高特异性,且成本较低,操作安全简便,可适用于基层中小医院较大样本量筛查.