不同细胞破碎方法对无细胞蛋白表达系统细胞抽提物活性的影响

无细胞蛋白表达系统由于能够有效表达膜蛋白等有毒性蛋白,因此近二十年受到了关注,其蛋白表达产率有了显著的提高。细胞抽提物活性的高低是无细胞蛋白表达系统高效运行的关键,若找到简单易行的活性评估方法,将大大降低成本及时间。葡萄糖-6-磷酸脱氢酶 (glucose-6-phosphate dehydrogenase, G-6-PDH)是糖代谢的戊糖磷酸途径中的关键调控酶,该酶可以被用来评估细胞抽提物的活性。以G-6-PDH的活性为指标对无细胞蛋白表达系统中的抽提物活性进行评价,并利用G-6-PDH活性评价体系对机械破碎、高压破碎以及超声破碎三种破碎方法进行了比较,得出了三种破碎方法的最佳破碎条件。机械破碎最佳破碎条件是5 000r/min, 用直径0.1 mm玻璃珠,破碎6次;高压破碎的最佳破碎压力为1 300bar;超声破碎最佳破碎条件是功率强度为总功率的60%,破碎30次。酶活性测定结果显示机械破碎和超声破碎得到的抽提物活性比高压破碎得到的抽提物活性略高。     

Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

一种快速提取细菌总DNA的方法研究

随着分子生物学技术应用于环境微生物研究的深入开展,占自然界微生物物种总数的90%以上的不能人工培养或培养困难的微生物已经可以借助分子生物学技术进行功能基因的开发和利用。而快速得到纯度较高,结构完整的细菌染色体DNA成为这一技术得以实现的前提。本文报道了利用高温处理和SDS的裂解作用相结合而建立的一种快速、简便的提取细菌染色体DNA的方法。经过脉冲电泳实验证明,利用本方法提取得到的几种革兰氏阳性和革兰氏阴性菌株的基因组DNA结构完整,并且无明显降解,无须经过纯化,可以直接进行PCR扩增和酶切等分子生物学操作,将此方法进一步应用于土壤环境DNA的提取方面,同样达到了快速得到大片段、高质量的环境微生物基因组的目的,为研究未培养的环境微生物多样性打下了坚实的基础,同时为环境基因组的提取提供了一个新的途径。     

Novel function of cardiac protein kinase D1 as a dynamic regulator of Ca2+ sensitivity of contraction

Although the function of protein kinase D1 (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 (histone deacetylase 5/7) and CREB, implicating this kinase in the development of dysfunction seen in heart failure. Additional studies have shown that PKD also phosphorylates multiple sarcomeric substrates to regulate myofilament function. Initial studies examined PKD through adenoviral vector expression of wild type PKD, constitutively active PKD (caPKD), or dominant negative PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated reticular pattern, suggesting the importance of PKD in Ca(2+) signaling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca(2+) sensitivity with an unexpected electrical stimulus dependence to this response. This desensitization was accompanied by stimulus-dependent increases in cTnI phosphorylation in control and caPKD cells with a more pronounced effect in the latter. Electrical stimulation also provoked phosphorylation of regulatory site Ser(916) on PKD. The functional importance of this phospho-Ser(916) event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. Dominant negative PKD expression resulted in sensitization of the myofilaments to Ca(2+) and blocked stimulus-dependent increases in cTnI phosphorylation. Taken together, these data reveal that localized PKD may play a role as a dynamic regulator of Ca(2+) sensitivity of contraction in cardiac myocytes.     

Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype

Standard biochemical tests have revealed that hemin and menadione auxotrophic Staphylococcus aureus small-colony variants (SCVs) exhibit multiple phenotypic changes. To provide a more complete analysis of the SCV phenotype, two genetically defined mutants with a stable SCV phenotype were comprehensively tested. These mutants, generated via mutations in menD or hemB that yielded menadione and hemin auxotrophs, were subjected to phenotype microarray (PM) analysis of over 1,500 phenotypes (including utilization of different carbon, nitrogen, phosphate, and sulfur sources; growth stimulation or inhibition by amino acids and other nutrients, osmolytes, and metabolic inhibitors; and susceptibility to antibiotics). Compared to parent strain COL, the hemB mutant was defective in utilization of a variety of carbon sources, including Krebs cycle intermediates and compounds that ultimately generate ATP via electron transport. The phenotype of the menD mutant was similar to that of the hemB mutant, but the defects in carbon metabolism were more pronounced than those seen with the hemB mutant. In both mutant strains, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth. Other phenotypes of SCV mutants, such as hypersensitivity to sodium selenite, sodium tellurite, and sodium nitrite, were also uncovered by the PM analysis. Key results of the PM analysis were confirmed in independent growth studies and by using Etest strips for susceptibility testing. PM technology is a new and efficient technology for assessing cellular phenotypes in S. aureus.     

鸦胆子油乳联合NP 方案治疗晚期非小细胞肺癌的临床疗效观察

目的:观察鸦胆子油乳联合NP方案化疗与单纯NP方案化疗对晚期肺小细胞癌患者的疗效、生活质量等因素的影响。方法:将154例患有晚期非小细胞肺癌患者随机分为两组:治疗组,在NP方案化疗基础上使用鸦胆子油乳治疗;对照组,单纯接受NP方案化疗。对154例患者追踪3个月,观察并比较患者的疗效、体质量、生活质量、毒副反应等方面的差异。结果:在NP方案化疗基础上使用鸦胆子油乳治疗在患者疗效评价方面与单纯NP方案化疗相比不具有统计学差异(P>0.05);在提高患者体质量、改善患者临床症状和生活质量等方面均优于单纯NP方案化疗(P<0.05);在化疗所引发的白细胞、血红蛋白和血小板降低等部分毒副反应发生率方面低于单纯NP方案化疗组(P<0.05)。结论:鸦胆子油乳联合NP方案化疗治疗晚期非小细胞肺癌不仅可以起到化疗增效的作用,而且能够明显提高患者生活质量,有效的改善临床症候,降低化疗引发的毒副反应,这一联合治疗方案可以在临床治疗中广泛推广。     

应用SSR标记分析大豆种质资源的遗传多样性

利用SSR分子标记分析了119个大豆品种的遗传多样性,结果表明：30对SSR引物在119份材料中共检测出159个等位变异,平均每对引物检测到5．30个等位变异;河北省农家品种中平均每对引物检测到5．17个等住变异,育成品种4．87个,省外品种4．93个,表明地方品种的遗传多样性高于育成品种。河北省农家品种、育成品种和省外育成品种依据SSR数据获得的品种间相似系数总体平均值相近,分别为0．698、0．698、0．672,但河北省农家品种较育成品种具有较大的变化幅度。119个品种可被划分为3个类群,在一定程度上能把育成品种和农家品种分开,并反映了一定的品种地域来源。