肾癌组织中端粒酶活性的测定与分析

端粒酶 (Ｔｅｌｏｍｅｒａｓｅ)是一种核糖核蛋白 ,依赖酶分子中的ＲＮＡ模板 ,通过逆转录合成染色体末端的端粒。近年来研究证实 ,端粒酶与细胞衰老及细胞分裂过程密切相关 ,尤其在恶性肿瘤组织中活性异常高。因此通过测定肿瘤组织、癌旁细胞及脱落细胞等端粒酶活性 ,可用于癌症的临床诊断、疗效观察、愈后及病理机制的研究。国外已经有对肾癌组织的端粒酶活性进行测定但都为定性 ,国内未见报道。本实验采用ＴＲＡＰ -ＥＬＩＳＡ技术对肾癌组织中端粒酶活性进行定量检测 ,现将结果报告如下。1　材料与方法1 1　样本来源 :组织标本均来自白…     

不同碘摄入水平对小鼠甲状腺组织Ⅰ型脱碘酶基因表达及酶活性的影响

甲状腺功能与碘摄入水平有密切的关系,甲状腺功能主要是通过其合成的甲状腺激素来实现的.脱碘反应是调节甲状腺激素生物活性的重要方式,不同碘营养状态下甲状腺组织I型脱碘酶(D1)基因表达及活性的变化是甲状腺功能调节的重要机制.在成功建立碘缺乏与不同程度碘过量的.Babl/c小鼠模型的基础上,采用实时荧光定量PCR法检测甲状腺组织DlmRNA表达水平,同时以125I-rT3,作为底物,结合离子交换层析技术测定甲状腺D1活性,此外,应用竞争结合放射免疫分析方法对甲状腺组织激素进行了检测.结果显示:碘缺乏时,D1 mRNA表达和D1活性均显著升高,甲状腺组织内T3与T4水平均显著降低,但T3/T4明显升高;碘过量可明显抑制D1 mRNA表达,但对D1活性并无显著影响,甲状腺组织内T3/T4有所下降.上述结果提示,甲状腺I型脱碘酶在甲状腺功能调节中具有重要的作用,碘缺乏时D1 mRNA表达及活性的上调可促进T4向T3的转化,以满足机体代谢的需要,而过量碘对Dl基因表达的抑制可防止过多的T3对机体的损伤,具有重要的生理意义.     

Adaptive Proteome Diversification by Nonsynonymous A-to-I RNA Editing in Coleoid Cephalopods

RNA editing by the ADAR enzymes converts selected adenosines into inosines, biological mimics for guanosines. By doing so, it alters protein-coding sequences, resulting in novel protein products that diversify the proteome beyond its genomic blueprint. Recoding is exceptionally abundant in the neural tissues of coleoid cephalopods (octopuses, squids, and cuttlefishes), with an over-representation of nonsynonymous edits suggesting positive selection. However, the extent to which proteome diversification by recoding provides an adaptive advantage is not known. It was recently suggested that the role of evolutionarily conserved edits is to compensate for harmful genomic substitutions, and that there is no added value in having an editable codon as compared with a restoration of the preferred genomic allele. Here, we show that this hypothesis fails to explain the evolutionary dynamics of recoding sites in coleoids. Instead, our results indicate that a large fraction of the shared, strongly recoded, sites in coleoids have been selected for proteome diversification, meaning that the fitness of an editable A is higher than an uneditable A or a genomically encoded G.     

Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease

Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer''s disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing.     

一种提取小型昆虫总RNA的有效方法

实验采用SDS,乙酸钾(KAc)等常规化学试剂从蚜虫等小型昆虫中获得完整且纯度较高的RNA。进一步的实验证明提取的RNA可用于RT-PCR以及cDNA文库的建立等。该方法简捷、相对安全和快速、提取效率较高,适合于中小型昆虫RNA的提取。     

Processing of RNA by cytoplasmic extract

Nuclei are isolated from HeLa cells by a low speed centrifugation procedure. These nuclei can carry out RNA synthesis at 30°. The effect of cytoplasmic extract on the transport and processing of RNA is measured. In the presence of cytoplasmic extract the precursor to 4S RNA is processed to 4S RNA and an RNA species of 7–9S is transported outside the nuclei.Abbreviations HEPES N-2-Hydroxymethylpiperazine-N'-2'-ethanesulfonic acid - DTT dithiothreitol - cDNA complementary DNA     

Induction of rat liver malic enzyme messenger RNA activity by insulin and by fructose

Several human normal and neoplastic cell lines were screened for production of PDGF receptor competing activity. Conditioned medium from two sarcomas and one glioma blocked 125I-PDGF binding to human foreskin fibroblasts in a dose-dependent manner. In each case this effect was abolished when the conditioned medium was pretreated with PDGF-antiserum, indicating that the receptor competing activity was immunologically related to PDGF. Direct evidence for de novo synthesis of a PDGF-like component in the cultures was afforded by 35S-cysteine labeling of the three cell lines, followed by immunoprecipitation with PDGF antiserum. This resulted in the specific precipitation of a 31,000 molecular weight labeled protein, which upon reduction was split into two polypeptides of molecular weights 17,000 and 16,500. The significance of these findings in view of the recently discovered structure homology between PDGF and the transforming gene product of simian sarcoma virus, p28sis, is discussed.