太平洋牡蛎(Crassostrea gigas)类α-1,2-岩藻糖基转移酶的密码子优化与原核表达

牡蛎消化组织内存在的类A型血型组织抗原是其特异性富集诺如病毒的主要原因,FUT2(Fucosyltransferase 2,α-1,2-岩藻糖基转移酶)是A型血型组织抗原合成的关键酶.本研究在前期克隆了太平洋牡蛎(Crassostrea gigas)类FUT2基因cDNA全长的基础上,根据大肠杆菌密码子偏爱性优化并合成了类FUT2基因,插入原核表达载体pRSET A构建pRSET-mof,将其转化大肠杆菌BL21,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导.经SDS-PAGE分析显示,在37℃、IPTG终浓度为0.8 mmol/L的条件下,诱导4h后出现大小约为46 kDa的特异性目的条带.利用His亲和层析柱纯化及超滤管浓缩目的蛋白,得到单一条带,说明纯化效果良好.Western blot分析显示,目的蛋白与抗6×His标签单克隆抗体、抗人FUT2单克隆抗体均能发生特异性反应,表明优化后的太平洋牡蛎类FUT2基因在大肠杆菌系统中成功表达.本研究结果为今后研究太平洋牡蛎类FUT2基因的功能,进一步探索牡蛎特异性富集诺如病毒的分子机理奠定了基础.     

六个群体翘嘴红鲌肌肉生化组成的比较

比较分析了中国六群体翘嘴红鲌(Culter alburnus)的肌肉生化组成.试验材料分别取自太湖、兴凯湖、梁子湖、浮桥河水库和南湾水库五个野生群体,以及由太湖野生群体经人工驯养、繁殖数代获得的养殖群体.分析结果表明,各群体之间翘嘴红鲌肌肉的水分、灰分和粗蛋白含量差异不显著(P＞0.05).而养殖群体的粗脂肪含量显著高于其它五个野生群体(P＜0.05).南湾水库群体的多不饱和脂肪酸和必需脂肪酸含量均最高,必需脂肪酸含量由高到低依次为南湾水库群体＞浮桥河水库群体＞兴凯湖群体＞梁子湖群体＞养殖群体＞太湖群体.氨基酸总含量由高到低依次为:太湖群体＞兴凯湖群体＞南湾水库群体＞梁子湖群体＞浮桥河水库群体＞养殖群体,而必需氨基酸含量的高低顺序为:太湖群体＞兴凯湖群体＞梁子湖群体＞南湾水库群体＞浮桥河水库群体＞养殖群体.相比之下,太湖群体的必需氨基酸指数(EAAI)最高,其次依次为:南湾水库群体＞梁子湖群体＞兴凯湖群体＞浮桥河水库群体＞养殖群体.各群体的大多数必需氨基酸氨基酸分(AAS)和化学分(CS)均大于1,仅兴凯湖群体的赖氨酸、苯丙氨酸+酪氨酸的AAS低于1,养殖群体的赖氨酸和缬氨酸的AAS和赖氨酸、蛋氨酸+半胱氨酸、苯丙氨酸+酪氨酸和缬氨酸的CS小于1,而成为各自的限制性氨基酸.综合所得的结果,野生群体的肌肉营养品质明显优于养殖群体,而野生群体中太湖、兴凯湖和南湾水库群体的肌肉品质相对较优.     

优质抗寒、抗病酿造葡萄新品种‘北红’

 ‘北红’葡萄新品种由‘玫瑰香’与‘山葡萄’杂交育成。浆果在北京地区9月底成熟。果粒圆形,蓝黑色,果粒质量1.57g,果穗质量160.0 g,浆果可溶性固形物含量23.8 %～ 27.0 %,含酸量0.89～1.26 %。早果性及丰产性强。抗寒、抗病能力特强。酿成的酒深宝石红色,酒体平衡,酒质上等。     

利用抑制消减杂交技术分离辣椒细胞质雄性不育育性恢复相关EST

 以辣椒(Capsicum annuum L. ) 细胞质雄性不育系23A、121A, 和其相应的近等基因恢复系23C、121C为试验材料, 利用抑制消减杂交( SSH) 技术成功构建了CMS恢复基因诱导表达的消减cDNA文库。结合高密度点阵膜杂交差异筛选, 获得了282个阳性克隆。通过测序, 除去重复序列共得到175个Unique ESTs。在GenBank上进行BLAST分析, 55个EST片段未找到对应的同源序列, 可能代表了新基因;120个EST片段找到了对应的同源序列, 包括103个已知功能基因和17个未知功能基因。按照MIPS功能分类法, 将其分为14个功能组, 涉及代谢、胁迫应答、蛋白活性、转录因子、信号转导等多方面的功能。     

Effect of atorvastatin on neointimal hyperplasia of venous autografts in rats

AIM: To investigate the effect of atorvastatin on neointimal hyperplasia of autogenous vein graft in rats. METHODS: The model of autogenous vein graft was prepared by transplanting the external jugular vein into aorta in Wistar rats. The rats were divided into three groups: sham operation group, graft control group and graft experimental group. From three days after transplantation, the rats of autograft experimental group were treated by atorvastatin at a dosage of 5 mg·kg-1·d-1. Four weeks after treatment, venous autografts were removed at autopsy and cut into 4 μm sections. Histopathological examination was carried out to analysis the neointimal hyperplasia of grafted veins. Immunohistochemical staining was conducted to evaluate SMα-actin and PCNA expression of neointimal cells in venous autografts. RESULTS: In venous autograft control and experimental groups, SMα-actin-positive smooth muscle cells were proliferated and accumulated excessively in venous autografts, which resulted in significant neointimal formation and vascular lumen narrowing. Neointima quantitative assay revealed that the neointimal hyperplasia of venous autografts was suppressed obviously in graft experimental group, and its neointimal area and NIA/MA ratio of venous autografts were significantly lower than those in graft control group (P<0.01). Immunohistochemical assay indicated that the PCNA labeling index of neointimal cells was significantly lower in graft experimental group than that in graft control group (P<0.01). CONCLUSION: Atorvastatin significantly inhibits the proliferation of neointimal smooth muscle cells and the development of neointimal hyperplasia of venous autografts in rats. Atorvastatin is a powerful inhibitor of restenosis after vascular reconstructive operation with a potential for therapeutic use.     

Identification of serum differential proteins in colorectal adenoma and early malignantly transformed adenoma by proteomics

AIM: To investigate the associated proteins and sensitive biomarkers for early diagnosis of colorectal adenocarcinoma by comparing the results of differential proteomic analysis between colorectal adenoma and early malignantly transformed adenoma. METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expressions of colorectal adenoma and early malignantly transformed adenoma. Proteins expressed differentially among groups were detected, cut out and analyzed by MALDI-TOF/TOF mass spectrometry. RESULTS: Two-dimensional protein maps of colorectal adenoma and early malignantly transformed adenoma were analyzed with gel-analysis software, an average of 1 672 spots in adenoma, 1 732 in early malignantly transformed adenoma were observed. 28 spots of a 1.5-fold change were found, including 15 proteins down-regulated and 13 up-regulated in early malignantly transformed adenoma, in which 23 proteins were identified by mass spectrometry, the rate of identification was 82.14%. 13 differential proteins were attained, 8 were up-regulated and 5 were down-regulated, which was classified to 6 categories, including protease inhibitor, complement, immunoglobulin, keratoproteins, signal transduction protein and function-unknown proteins. CONCLUSION: The changes of serum proteins in early malignantly transformed adenoma from adenoma can be identified by proteomic technology. Proteins detected in the study may provide new biomarkers correlated with biological behavior of colorectal adenocarcinoma.     

Role of nestin in the maintenance of the structure of podocytes

AIM:To investigate the expression of nestin, a kind of cytoskeletal protein in cultured murine podocytes and the role of nestin in the maintenance of the podocyte structure.METHODS:The immortalized murine podocytes were cultured. The expression of nestin was determined by immunofluorescence. In differentiated podocytes, the expression of nestin was knock-down by RNAi. The effect of nestin knock-down was examined by Western blotting and immunofluorescence. The projections longer than maximal length of the cell body in which the cells transfected with siRNA and control vector that contained nonhomologous oligo were counted, respectively. RESULTS:Nestin siRNA markedly reduced or abolished nestin expression. In cells transfected with nestin siRNA, the percentage of cells with processes was significantly lower than that in cells transfected with control vector (77.0%±6.3% vs 16.0%±4.6%, n=3, P<0.01). CONCLUSION:Nestin may play an important role in maintaining normal function of podocytes.     

13-MTD induces apoptosis of MCF-7 breast cancer cells by activating MAPK pathway and inhibiting Akt signaling

［ABSTRACT］AIM: To study the effect of 13-methyltetradecanoic acid (13-MTD), a saturated branched-chain fatty acid, on apoptotic induction in breast carcinoma cell line MCF-7 and its underlying mechanisms. METHODS: Breast carcinoma cell line MCF-7 and normal breast epithelial cells MaEC were treated with solvent or 13-MTD at concentration of 140 mg/L. Apoptosis was analyzed by flow cytometry. Phosphorylation of JNK, p38, FADD and Akt after treated with 13-MTD were detected by Western blotting. RESULTS: 13-MTD effectively induced apoptosis of breast carcinoma cell line MCF-7 and no influence to normal breast epithelial cells MaEC, which were confirmed by flow cytometry analysis, was observed. The results of Western blotting showed that obvious increase in p38 and JNK phosphorylation. No significant difference of FADD phosphorylation was observed. However, evidently decrease in Akt phosphorylation was found after treated with 13-MTD. CONCLUSION: 13-MTD was a new safe, effective chemotherapeutic drug. Its underlying mechanisms are through activating MAPK pathway and inhibiting Akt pathway to induce the cancer cells apoptosis.     

Effects of salvianolic acid B on neurons during oxygen/glucose deprivation and reintroduction

AIM: To observe the damage induced in the primary cultured rat cortical neurons by oxygen/glucose deprivation and reintroduction, and to investigate the neuroprotective mechanism of salvianolic acid B (SalB). METHODS: Primary cultured rat cortical neurons were randomly divided into the control group, the model group and the SalB group. The cell model was established by oxygen/glucose deprivation for 3 h followed oxygen/glucose reintroduction for 24 h. The cortical neurons viability was determined by MTT assay. The leakage rate of lactate dehydrogenase (LDH) was measured by chromatometry. The mitochondrial membrane potentials (MMP) and the apoptosis rate were quantitatively analyzed by flow cytometry. The cytosolic free calcium was assessed using LSCM. The morphologic changes of neuronal nuclei were observed by Hoechst 33342 fluorescence staining. RESULTS: Compared to the model group, the cortical neurons viability, the survival rate and the fluorescence value of MMP in the SalB group were obviously increased (P<0.05,P<0.01). In addition, in the SalB group, the leakage rate of LDH, the fluorescence intensity of cytosolic free calcium and the apoptosis rate were significantly lower than those in the model group (P<0.01). CONCLUSION: The neuroprotective mechanism of SalB in the oxygen/glucose deprivation and reintroduction neurons would be due to the fact that SalB maintains the MMP and the calcium homeostasis.