人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

将人胱硫醚β-合酶(CBS)基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta (DE3)菌株,构建了高效表达CBS的重组菌E.coli Rosetta (pGEX4T-1-CBS)。重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基。将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1%CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1mmol/L的S-腺苷甲硫氨酸(AdoMet)可使CBS单位酶活提高5.1倍,达到735U/mg。同时构建了表达CBS_1-413(删除了CBS羧基端调控域138个氨基酸残基)的重组菌E.coli Rosetta (pETDuet-1-CBS_1-413),经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965U/mg; 还表达和纯化了胱硫醚β-裂解酶(CBL),并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法。     

大桑菊合剂的提取工艺研究

目的:大桑菊合剂为医院制剂,由桑叶、菊花、薄荷、连翘、鱼腥草等中药经乙醇提取制成,为完善大桑菊合剂的制备工艺,对该制剂的制备工艺进行初步的研究。方法:①实验设计:以加水量、提取时间、提取次数和醇沉浓度为考察因素,各因素均取3水平,采用正交试验L(934)优选制备工艺。②含量测定方法:以大桑菊饮合剂中总黄酮的含量为指标,以芦丁为标准品,利用紫外分光光度计于503nm下测定吸光值,计算样品中总黄酮的含量,以此作为筛选大桑菊合剂最佳制备工艺的重要指标。结果:最佳制备工艺为不浸泡,加8倍水,提取3次,每次1小时。结论:经正交设计优化的大桑菊合剂的中药制备工艺合理、可行。     

CHEK2*1100delC and susceptibility to breast cancer: a collaborative analysis involving 10,860 breast cancer cases and 9,065 controls from 10 studies

Previous studies of families with multiple cases of breast cancer have indicated that a frameshift alteration in the CHEK2 gene, 1100delC, is associated with an elevated frequency of breast cancer in such families, but the risk associated with the variant in other situations is uncertain. To evaluate the breast cancer risk associated with this variant, 10,860 breast cancer cases and 9,065 controls from 10 case-control studies in five countries were genotyped. CHEK2*1100delC was found in 201 cases (1.9%) and 64 controls (0.7%) (estimated odds ratio 2.34; 95% CI 1.72–3.20; P=.0000001). There was some evidence of a higher prevalence of CHEK2*1100delC among cases with a first-degree relative affected with breast cancer (odds ratio 1.44; 95% CI 0.93–2.23; P=.10) and of a trend for a higher breast cancer odds ratio at younger ages at diagnosis (P=.002). These results confirm that CHEK2*1100delC confers an increased risk of breast cancer and that this risk is apparent in women unselected for family history. The results are consistent with the hypothesis that CHEK2*1100delC multiplies the risks associated with susceptibility alleles in other genes to increase the risk of breast cancer.     

利用DGGE评价不同培养基回收番茄根际细菌类群的能力

用营养肉汤、YG、根系分泌物、土壤浸渍液4种培养基从番茄根际分离培养细菌,并结合变性梯度凝胶电泳(DGGE)技术,对4种培养基回收番茄根际细菌种群的能力进行了比较研究。结果表明,不同培养基和培养温度,回收到的细菌种群有一定差异;低营养浓度的YG培养基在较低的培养温度20℃下进行较长时间的培养,比高营养浓度营养肉汤培养基产生更多、更具代表性的细菌;以根系分泌物为基础的培养基从番茄根际回收到的优势菌群最多。该研究初步建立了用DGGE技术对不同培养基回收分离细菌种群能力进行评价的方法。     

In vitro Anti-viral Activity of the Total Alkaloids from Tripterygium hypoglaucum against Herpes Simplex Virus Type 1

Herpes simplex virus type 1 (HSV-1) is a commonly occurring human pathogen worldwide. There is an urgent need to discover and develop new alternative agents for the management of HSV-1 infection. Tripterygium hypoglaucum (level) Hutch (Celastraceae) is a traditional Chinese medicine plant with many pharmacological activities such as anti-inflammation, anti-tumor and antifertility. The usual medicinal part is the roots which contain about a 1% yield of alkaloids. A crude total alkaloids extract was prepared ...     

维生素C和B_1对铅致小鼠睾丸损伤保护作用研究

目的探讨维生素C、B1联合用药拮抗铅对睾丸的毒性作用。方法 1.30只雄性小鼠随机分为2组(15只/组):空白对照组和铅染毒组。染铅组雄性小鼠每日腹腔注射醋酸铅20 mg/kg,空白对照组给予等容量的生理盐水,一日一次,第42日结束以上实验。将小鼠取血,用试剂盒检测血浆中谷胱甘肽的含量、谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)的活性。将睾丸组织经过处理后,在光镜及透射电镜下观察睾丸组织结构变化;2.45只雄性小鼠随机分为3组(15只/组):空白对照组、铅染毒组和维生素联合干预组。染铅组雄性小鼠每日腹腔注射醋酸铅20 mg/kg,维生素干预组在染铅后,即时每组小鼠给予维生素C和维生素B1,一日一次,第42日结束。处死小鼠,取血,用试剂盒检测血浆中谷胱甘肽的含量,谷胱甘肽过氧化物酶(GPx)和谷胱甘肽还原酶(GR)的活性。在光镜以及透射电镜下观察睾丸组织结构变化。结果1.与空白对照组比较,醋酸铅染毒组小鼠血浆中还原型谷胱甘肽(GSH)的水平降低,GSH/GSSG的比例降低,GPX活性升高,GR活性降低;光镜下睾丸组织生精小管变薄,各级生精细胞、支持细胞减少,部分细胞核出现核固缩;3.电镜下染毒组小鼠睾丸组织超微结构变化明显,支持细胞溶酶体增多,线粒体肿胀空泡化;2.与醋酸铅染毒组比较,维生素干预组小鼠血浆中还原型谷胱甘肽(GSH)的水平升高,GSH/GSSG的比例升高,GPx活性降低,GR活性升高;光镜观察结果:与染铅组相比,维生素B1和C联合干预组中破坏的睾丸生精上皮有所恢复;电镜观察结果:与染铅组相比,维生素B1和C联合干预组睾丸组织超微结构基本恢复正常,生精细胞和支持细胞结构正常,细胞器丰富。结果 1.醋酸铅使体内GSH的水平降低,GPX活性升高,GR活性降低,从而诱导氧化应激的发生,导致染铅小鼠睾丸的损伤;2.维生素干预组小鼠血浆中还原型谷胱甘肽(GSH)的水平升高,GSH/GSSG的比例升高,GPx活性降低,GR活性升高,维生素干预组小鼠血浆中氧化型谷胱甘肽还原为还原型谷胱甘肽,从而发挥抗氧化作用,对小鼠睾丸起到保护作用。     

Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.     

Association of N-Terminal Pro-Brain Natriuretic Peptide with Cognitive Function and Depression in Elderly People with Type 2 Diabetes

Background

Type 2 diabetes mellitus is associated with risk of congestive heart failure (CHF), cognitive dysfunction and depression. CHF itself is linked both to poor cognition and depression. The ventricular N-terminal pro-brain natriuretic peptide (NT-proBNP) is a marker of CHF, suggesting potential as a marker for cognitive impairment and/or depression. This was tested in the Edinburgh Type 2 Diabetes Study (ET2DS).

Methodology and Principal Findings

Cross-sectional analysis of 1066 men and women aged 60–75 with type 2 diabetes. Results from seven neuropsychological tests were combined in a standardised general cognitive ability factor, ‘g’. A vocabulary-based test estimated pre-morbid cognitive ability. The Hospital Anxiety and Depression Scale (HADS) assessed possible depression. After adjustment for age and sex, raised plasma NT-proBNP was weakly associated with lower ‘g’ and higher depression scores (ß −0.09, 95% CI −0.13 to −0.03, p = 0.004 and ß 0.08, 95% CI 0.04 to 0.12, p<0.001, respectively). Comparing extreme quintiles of NT-proBNP, subjects in the highest quintile were more likely to have reduced cognitive ability (within the lowest tertile of ‘g’) and ‘possible’ depression (HADS depression ≥8) (OR 1.80; 95% CI: 1.20, 2.70; p = 0.005 and OR 2.18; 95% CI: 1.28, 3.71; p = 0.004, respectively). Associations persisted when pre-morbid ability was adjusted for, but as expected were no longer statistically significant following the adjustment for diabetes-related and vascular co-variates (β −0.02, 95% CI −0.07 to 0.03, p>0.05 for ‘g’; β 0.03, 95% CI −0.02 to 0.07, p>0.05 for depression scores).

Conclusion

Raised plasma NT-proBNP was weakly but statistically significantly associated with poorer cognitive function and depression. The prospective phases of the ET2DS will help determine whether or not NT-proBNP can be considered a risk marker for subsequent cognitive impairment and incident depression and whether it provides additional information over and above traditional risk factors for these conditions.     

血清脂联素与载脂蛋白A5 及2 型糖尿病的关系

目的:检测血清脂联素(APN)水平,分析血清APN浓度与血脂、血清载脂蛋白A5(apoA5)及2型糖尿病的关系。方法:收集2型糖尿病(T2DM)210例,健康体检者112例,采用ELISA法检测血浆脂联素水平,双抗体夹心ELISA法检测血清载脂蛋白A5(apoA5)水平,7600-020E全自动生化分析仪检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)等,放射免疫分析仪检测血胰岛素水平。结果:T2DM患者血清APN浓度明显低于健康对照组,LDL-C、TG及TC均高于对照组(P<0.05)。T2DM患者血清apoA5浓度(200.3±51.2)ng/ml,显著低于健康对照组(229.8±56.5)ng/ml,P<0.05。Pearson相关分析显示经年龄、性别校正后APN水平与LDL-C、TG呈负相关,与HDL-C呈正相关;T2DM组APN与apoA5呈正相关(P<0.05)。结论:T2DM患者血清APN水平显著降低,本研究证实低水平血清APN和apoA5不仅与血脂代谢密切相关,还可作为T2DM患者早期监测的指标,对其预后评价具有积极的意义。     

尿微量蛋白联合血凝在冠心病诊断中的检测价值

目的:探讨尿微量蛋白联合血清纤维蛋白原在冠心病的诊断价值。方法:选取同期在我院治疗的24例稳定型心绞痛的患者,36例诊断为不稳定型心绞痛的患者和30例诊断为急性心肌梗死的患者,并选择同期30例来我院体检健康志愿者为对照组。分析以上4组患者发病时尿微量蛋白及血清纤维蛋白原的变化情况。结果:与对照组比,3个冠心病组的尿微量蛋白及血清纤维蛋白原的含量显著升高(P<0.05),与稳定型心绞痛组比,不稳定型心绞痛组的尿微量蛋白及血清纤维蛋白原的含量显著升高(P<0.05);与不稳定型心绞痛组比,急性心梗的尿微量蛋白及血清纤维蛋白原的含量显著升高(P<0.05)。3组病患的尿微量蛋白及血清纤维蛋白原之间呈正相关关系(r=0.852,P<0.05)。结论:心肌梗死和心绞痛患者尿微量蛋白及血清纤维蛋白原含量较健康成人含量高,提示尿微量蛋白及血清纤维蛋白原的含量有助于对心肌梗死和心绞痛的诊断,对急性心肌梗死的诊断价值较高。