Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions

Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K(mapp) for UDP-glucose in vivo was 1.4mM (in the presence or absence of glucose-6-P). The in vitro value was 6mM and was reduced by glucose-6-P to 0.8mM. Under both conditions (in vitro and in vivo) V(max) was 0.2 m Units per oocyte in the absence of glucose-6-P. V(max) in vivo was stimulated 2-fold by glucose-6-P, whereas, in vitro, a 10-fold increase was obtained. Glucose-6-P required for 50% activation in vivo was 15 microM and, depending on substrate concentrations, 50-100 microM in vitro. The prevailing enzyme obtained in vitro was the glucose-6-P-dependent form, which may be converted to the independent species by dephosphorylation. This transformation could not be observed in vivo. We suggest that enzyme activation by glucose-6-P in vivo is due to allosteric effects rather than to dephosphorylation of the enzyme. Regulatory mechanisms other than allosteric activation and covalent phosphorylation are discussed.     

石斛总RNA提取方法的研究

采用TRIZOL法、异硫氰酸胍法、Tris-硼酸法和改良的RNA提取方法提取石斛的总RNA,并通过凝胶电泳、紫外分光光度法检测提取的RNA样品的品质。研究结果表明:改良的RNA提取方法提取的RNA具有28S rRNA和18S rRNA两条清晰的条带,且无降解。OD260nm/OD280nm接近2.0,具有较高的纯度。其它三种方法获得的RNA品质较差,有降解和弥散现象。将改良的RNA提取方法提取的RNA逆转录成cDNA,经RAPD扩增,出现清晰的条带,进一步证明改良的RNA提取方法提取的RNA具有很高的纯度,可以满足进一步分子生物学研究的要求。     

A method to extract intact and pure RNA from mycobacteria

We describe a high-yielding, simple, and aerosol-free protocol for the isolation of RNA from mycobacteria that does not require sophisticated instruments. The method yielded 50 μg of RNA from 10⁷ cells, 50 times more than a recently reported method. Our method can extract total RNA from aerobically grown bacteria and from in vitro hypoxia-induced dormant bacilli and mycobacteria residing within infected macrophages.     

一种提取高质量油菜种子和种皮RNA的方法

提取高质量的RNA是从基因表达水平上研究油菜种子和种皮发育的必要条件。现有方法因为油菜种子脂肪、多酚和多糖,难以快速获得完整、高纯度的油菜种子总RNA。本试验针对油菜种子和种皮特点,利用苯酚-氯仿抽提后用无水乙醇沉淀RNA,建立了在油菜种子和种皮中快速提取高质量总RNA的提取方法,电泳分析表明28S rRNA亮度约为18S rRNA的2倍;紫外分光光度计检测A260/A280介于1.8～2.0之间。用该法分离的RNA,已成功用于RT-PCR、Northern blot分析和基因全长的克隆等分子生物学研究。     

Isolation of RNA from apple skin

A method has been developed for the isolation of RNA from apple skin. The method involves an adaptation of the Manning (1991) method and includes a high-salt extraction step and a final purification step through a CsCl cushion. The RNA isolated was of high quality and produced good hybridization signals in northern blot analyses.     

日本结缕草总RNA提取和mRNA分离方法的优化

介绍了分别以日本结缕草根部、叶部、匍匐茎等为材料的总RNA提取的改良步骤,质量和浓度检测及其注意事项,并采用Promega公司的Poly ATract Systems Ⅲ(Z5300)试剂盒进行mRNA分离,尽管这一方法成熟可行,但仍存在洗脱体积较大,常需先沉淀浓缩后才能进行后续试验(如cDNA合成),在制备少量mRNA时回收率较低等问题。据此,对mRNA分离方法作了改良,介绍了mRNA分离的优化步骤,通过增加体系形成了一种更适合于日本结缕草mRNA分离的方法,从而为日本结缕草的分子生物学研究提供基础资料。结果证明所提取的总RNA和mRNA完整,质量较高,并应用到日本结缕草cD-NA文库的构建。     

一种快速、高效的橡胶树胶乳总RNA提取方法

胶乳是橡胶树（Hevea brasiliensis）乳管中特殊的细胞质,主要由橡胶粒子、黄色体、F-W粒子和普通细胞质成分构成,其中橡胶粒子占20％-40％,蛋白含量高达1％-2％。由于高比例橡胶粒子和蛋白质的干扰,目前使用的胶乳RNA提取方法都具有步骤繁琐、胶乳需求量大、操作技巧性强不易掌握等缺点。为快速、高效地获取高质量的胶乳RNA,我们在现有方法的基础上摸索出一套步骤简单、容易操作、快速、高效提取橡胶树胶乳总RNA的简易方法,获得了较好的实验效果。紫外分光光度计、RT-PCR和RACE分析结果表明,使用该方法提取的胶乳RNA质量完全能够满足相应的分子操作,但所需时间仅为目前常用方法的50％,RNA获得率提高了2-3倍,操作难度大大降低。     

In vitro and in vivo chromosomal aberrations induced by megazol

With the re-emergence of Human African Trypanosomiasis (HAT) on the one hand, which are increasingly resistant to current therapies, and the stage-dependent effectiveness or even the prohibitive cost of these therapies on the other hand, megazol, a 5-nitroimidazole thiadiazole highly active against various trypanosomal species, was assessed for its genotoxic potential. Very little information has become available until now. Two batches of megazol were provided by two different suppliers: Far-Manguinhos, a part of the Fiocruz foundation, under the Brazilian Minister of Health, and Delphia, a French company. These two batches, obtained by different synthetic routes, were studied by means of the in vitro micronucleus assay on L5178Y mouse lymphoma cells, in its microscale version. Both batches of magazol displayed a strong genotoxic activity in this screening assay. A second batch from Delphia was then investigated by use of two tests, i.e. the in vitro metaphase analysis with human lymphocytes and the in vivo micronucleus test in rat bone-marrow. Megazol was shown to be a potent inducer of in vitro and in vivo chromosomal aberrations. Although megazol is a potent trypanocidal agent and is orally bio-available, its toxicity dictates that it should not be developed further for the treatment of HAT and Chagas disease. All development work has therefore been discontinued.     

Isolation of High Quality DNA and RNA from Leaves of the Carnivorous Plant Drosera rotundifolia

Drosera rotundifolia belongs to the family of the sundews, a large group of carnivorous plants that carry stalked glands on the upper leaf surface to attract, trap and digest insects for food. Therefore, such plants can live in relatively poor ecosystems. They are frequently used as medicinal herbs and have various other interesting characteristics associated with them. In attempts to evaluate the gene pool of these plants, we experienced that many published protocols for nucleic acid isolation failed to yield DNA and RNA of sufficient quality for analysis. Therefore, we have developed CTAB (hexadecyltrimethylammoniumbromide)-based extraction protocols for the routine isolation of high-quality DNA and RNA from small amounts of in vitro-grown Drosera rotundifolia leaves. The methods developed are simple, fast and effective. The obtained DNA could be analyzed by PCR, restriction endonucleases and DNA gel blotting, and the obtained RNA was of sufficient quality for RT-PCR and RNA gel blotting.     

A Method for Isolation of Total RNA from Fruit Tissues of Banana

We describe a rapid and efficient method for isolation of total RNA from banana fruit tissues. The RNA was extracted with a high ionic strength buffer at room temperature. The proteins, genomic DNA and secondary metabolites in the extract were then removed by precipitation with pre-cooled potassium acetate and repeated phenol/chloroform/isoamyl alcohol extractions. The RNA was recovered by ethanol precipitation. It was relatively free of ribonucleases and was suitable for RT-PCR and northern blot analysis. The procedure can be completed in less than 4 hours.     

何首乌总RNA提取方法的比较及改进

目的：探讨不同提取方法对提取何首乌总RNA的质量影响,寻找适于何首乌成熟叶组织RNA的提取方法。方法：以何首乌成熟叶组织为材料,采用SDS／酸酚法、常规CTAB法及改良的TRIzol试剂法分别进行实验,并对所提取RNA的质量进行验证。结果：采用3种方法都能提取出RNA,但质量差异较大。其中改良的TRIzol试剂法能有效抑制次生物质的影响,提取的RNA产量可达70-110μg／g,纯度高于其他2种方法,D260nm／D280nm值为1．85～1．97。结论：改良的TRIzol试剂法操作简便,提取的RNA完整性和纯度较高,可以满足下一步实验的要求。     

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.     

In vitro and in vivo interactions of aluminum on NTPDase and AChE activities in lymphocytes of rats

Al adjuvants are used in vaccines to increase the immune response. NTPDase and AChE play a pivotal role and act in the regulation of the immune system. The effect of Al exposure in vitro and in vivo on NTPDase and AChE activities in the lymphocytes of rats was determined. In vitro, ATP hydrolysis was decreased by 20.4% and 17.3% and ADP hydrolysis was decreased by 36.5% and 34.8%, in groups D and E, respectively, when compared to the control. AChE activity was increased by 157.3%, 152.5%, 74.7% and 90.8% in groups B, C, D, and E, respectively, when compared to the control. In vivo, ATP hydrolysis was increased by 85% and 86% and ADP hydrolysis was increased by 104.2% and 74%, in Al plus citrate and Al groups, respectively, when compared to the control. AChE activity was increased by 50.7% in Al plus citrate and by 28.6% in Al groups, when compared to the control. Our results show that Al exposure both in vitro and in vivo altered NTPDase and AChE activities in lymphocytes. These results may demonstrate the ability of Al to elicit the immune system, where NTPDase and AChE activities can act as purinergic and cholinergic markers in lymphocytes.     

一种提取小鼠表皮总RNA的新方法

目的:建立一种从小鼠表皮组织提取高质量RNA的方法。方法:用热击法分离小鼠表皮,用TRIzol法提取RNA,用紫外分光光度计测定RNA的产率和纯度,用琼脂糖电泳和RT-PCR检测RNA的质量和完整性。结果:采用新方法提取的小鼠表皮总RNA,其D260nm/D280nm值为1.8～2.0,大于1.5,且RNA产率高于100μg/g;琼脂糖电泳出现5S、18S和28S等3条清晰的rRNA条带,而且28SrRNA条带的亮度约为18S的2倍;用新方法制备的总RNA可成功地用于RT-PCR实验。结论:采用热击法分离表皮并结合TRIzol法可提取到高质量、完整性好的小鼠表皮总RNA,并能用于相关的分子生物学实验。     

一种简便的从石蜡包埋组织中提取RNA的方法

目的:建立一种从石蜡包埋组织提取高质量RNA的简便、经济的方法。方法:运用TRIzol法和改进的AGPC(酸-异硫氰酸胍-苯酚-氯仿)法等2种提取方法,从50例石蜡包埋组织中提取RNA,用紫外分光光度仪测定RNA的产率和纯度;用管家基因β-肌动蛋白做RT-PCR,检测RNA的质量。结果:TRIzol法提取RNA的成功率为96%(48/50),AGPC法为94%(47/50),2种方法所得RNA的吸光度值及得率在统计学上均无明显差异。结论:AGPC法和TRIzol法的提取效率相似,且提取费用只有TRIzol法的一半左右,是一种简便、经济、适合大量提取石蜡包埋组织中的RNA的方法,可用于临床。