转移核糖核酸发现五十年——tRNA发现者Zamecnik辞世

Crick(1955)预见到细胞内存在一种小分子RNA称为"衔接子",并假定它是一种沟通DNA与蛋白质序列之间的中介体。Zamecnik(1955)发现sRNA(tRNA),Hoagland(1954)发现氨基酸激活酶(氨基酰-tRNA合成酶)。镁离子稳定tRNA的二级结构(1963),由此,可以得到大的寡核苷酸片断,甚至tRNA的半分子。这项技术大大推动了tRNA及其他RNA序列分析工作的迅速进展。Holley(1965)首次测定出酵母tRNAAla全部核苷酸序列,并设计出一种"三叶草"二级结构。Crick(1966)较早地提出"摆动假说",即反密码子5’端的碱基并不与密码子3’端碱基严格配对,允许有一定的摆动。Rich(1974)等用X射线晶体衍射法,阐明了酵母tRNAPhe的高级结构(呈L型)。2000年,英国剑桥科学家,使用在冰箱保存了15年的酵母tRNAPhe晶体,重新测定它的L型结构是正确的,并严格确定其中10个镁离子和一个精胺分子的精确部位。     

信号蛋白中甲硫氨酸残基的可逆性氧化和甲硫氨酸亚砜还原酶

含硫氨基酸甲硫氨酸在体内易被胞内、外活性氧氧化为甲硫氨酸-R,S-亚砜。蛋白质肽链中的甲硫氨酸残基被氧化后,蛋白活性发生显著改变,如钙调素与钙调素结合蛋白亲和力的下降、钙离子/钙调素依赖性蛋白激酶Ⅱ的激活、钾离子通道ShC/B失活动力学的改变。多数生物都存在一个msrA基因和1～3个msrB基因,编码两种序列和结构都明显不同的酶:甲硫氨酸亚砜还原酶A(MsrA)和甲硫氨酸亚砜还原酶B(MsrB),分别还原甲硫氨酸-S-亚砜和甲硫氨酸-R-亚砜。两种酶的催化机制基本相同,其活性中心结构互为镜像。两种还原酶分布于体内不同器官及各种亚细胞结构。对于MsrA活性的研究,已有30年的历史,最初主要集中在低等生物,已发现MsrA对于延缓衰老和神经退行性疾病具有重要作用,也是致病菌的主要毒力因子。最近10年对MsrB也进行了系统研究,并取得了重要进展。人们正在逐渐认识到这些酶在细胞信号蛋白分子活性调节中的重要作用。     

Higher urine desmosine levels are associated with mortality in patients with acute lung injury

Desmosine is a stable breakdown product of elastin that can be reliably measured in urine samples. We tested the hypothesis that higher baseline urine desmosine would be associated with higher mortality in 579 of 861 patients included in the recent Acute Respiratory Distress Syndrome Network trial of lower tidal volume ventilation (1). We also correlated urine desmosine levels with indexes of disease severity. Finally, we assessed whether urine desmosine was lower in patients who received lower tidal volumes. Desmosine was measured by radioimmunoassay in urine samples from days 0, 1, and 3 of the study. The data were expressed as a ratio of urine desmosine to urine creatinine to control for renal dilution. The results show that higher baseline (day 0) urine desmosine-to-creatinine concentration was associated with a higher risk of death on adjusted analysis (odds ratio 1.36, 95% confidence interval 1.02-1.82, P=0.03). Urine desmosine increased in both ventilator groups from day 0 to day 3, but the average rise was higher in the 12-ml/kg predicted body weight group compared with the 6-ml/kg predicted body weight group (P=0.053, repeated-measures model). In conclusion, patients with acute lung injury ventilated with lower tidal volumes have lower urine desmosine levels, a finding that may reflect reduced extracellular matrix breakdown. These results illustrate the value of evaluating urinary biological markers that may have prognostic and pathogenetic significance in acute lung injury.     

Statistical diagnostics emerging from external quality control of real-time PCR

Besides the application of conventional qualitative PCR as a valuable tool to enrich or identify specific sequences of nucleic acids, a new revolutionary technique for quantitative PCR determination has been introduced recently. It is based on real-time detection of PCR products revealed as a homogeneous accumulating signal generated by specific dyes. However, as far as we know, the influence of the variability of this technique on the reliability of the quantitative assay has not been thoroughly investigated. A national program of external quality assurance (EQA) for real-time PCR determination involving 42 Italian laboratories has been developed to assess the analytical performance of real-time PCR procedures. Participants were asked to perform a conventional experiment based on the use of an external reference curve (standard curve) for real-time detection of three cDNA samples with different concentrations of a specific target. In this paper the main analytical features of the standard curve have been investigated in an attempt to produce statistical diagnostics emerging from external quality control. Specific control charts were drawn to help biochemists take technical decisions aimed at improving the performance of their laboratories. Overall, our results indicated a subset of seven laboratories whose performance appeared to be markedly outside the limits for at least one of the standard curve features investigated. Our findings suggest the usefulness of the approach presented here for monitoring the heterogeneity of results produced by different laboratories and for selecting those laboratories that need technical advice on their performance.     

Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF

Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.     

利用DDRT-PCR技术分析神经生长因子低亲和力受体诱导的细胞凋亡过程中基因表达的差异

转染了Ｐ７５ＮＧＦＲ的Ｒ２神经细胞系Ｒ２Ｌ１在去血清的培养时可以诱导细胞凋亡的发生．此凋亡可以被ＲＮＡ合成抑制剂放线菌素Ｄ和蛋白质合成抑制剂环己酰胺所抑制．利用ＤＤＲＴ－ＰＣＲ技术比较了去血清培养的发生凋亡的Ｒ２Ｌ１细胞与有血清培养的不发生凋亡的Ｒ２Ｌ１细胞以及去血清培养的不发生凋亡的Ｒ２Ｐ细胞基因表达的差异．克隆了数个特异或差异表达的短ｃＤＮＡ片段,经Ｎｏｒｔｈｅｒｎ杂交证实其中两个片段ＬＩＡＲＥＳＴ－１和ＬＩＡＲＥＳＴ－２表达量在凋亡细胞中显著高于不发生凋亡的细胞中,ＧｅｎＢａｎｋ检索表明此二片段为新的ｃＤＮＡ序列并给予登录号Ｕ４７３１５和Ｕ４７３１６．另有一个ｃＤＮＡ片段ＬＩＡＲＣＤ－３在凋亡细胞中受到了明显的抑制,经检索为一已知的与前强啡肽原上游调控区结合的ＤＮＡ结合蛋白ｃＤＮＡ编码区的一部分,首次被证实它与Ｐ７５ＮＧＦＲ诱导的神经细胞凋亡调控关联     

The Urinary Proteomic Profile Implicates Key Regulators for Urologic Chronic Pelvic Pain Syndrome (UCPPS): A MAPP Research Network Study

Urologic chronic pelvic pain syndrome (UCPPS) is a condition of unknown etiology characterized by pelvic pain and urinary frequency and/or urgency. As the proximal fluid of this syndrome, urine is an ideal candidate sample matrix for an unbiased study of UCPPS. In this study, a large, discovery-phase, TMT-based quantitative urinary proteomics analysis of 244 participants was performed. The participants included patients with UCPPS (n = 82), healthy controls (HC) (n = 94), and disparate chronic pain diseases, termed positive controls (PC) (n = 68). Using training and testing cohorts, we identified and validated a small and distinct set of proteins that distinguished UCPPS from HC (n = 9) and UCPPS from PC (n = 3). The validated UCPPS: HC proteins were predominantly extracellular matrix/extracellular matrix modifying or immunomodulatory/host defense in nature. Significantly varying proteins in the UCPPS: HC comparison were overrepresented by the members of several dysregulated biological processes including decreased immune cell migration, decreased development of epithelial tissue, and increased bleeding. Comparison with the PC cohort enabled the evaluation of UCPPS-specific upstream regulators, contrasting UCPPS with other conditions that cause chronic pain. Specific to UCPPS were alterations in the predicted signaling of several upstream regulators, including alpha-catenin, interleukin-6, epidermal growth factor, and transforming growth factor beta 1, among others. These findings advance our knowledge of the etiology of UCPPS and inform potential future clinical translation into a diagnostic panel for UCPPS.