The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies

Recent results suggest that cytoplasmic mRNAs can form translationally repressed messenger ribonucleoprotein particles (mRNPs) capable of decapping and degradation, or accumulation into cytoplasmic processing bodies (P-bodies), which can function as sites of mRNA storage. The proteins that function in transitions between the translationally repressed mRNPs that accumulate in P-bodies and mRNPs engaged in translation are largely unknown. Herein, we demonstrate that the yeast translation initiation factor Ded1p can localize to P-bodies. Moreover, depletion of Ded1p leads to defects in P-body formation. Overexpression of Ded1p results in increased size and number of P-bodies and inhibition of growth in a manner partially suppressed by loss of Pat1p, Dhh1p, or Lsm1p. Mutations that inactivate the ATPase activity of Ded1p increase the overexpression growth inhibition of Ded1p and prevent Ded1p from localizing in P-bodies. Combined with earlier work showing Ded1p can have a positive effect on translation, these results suggest that Ded1p is a bifunctional protein that can affect both translation initiation and P-body formation.     

非酒精性脂肪肝大鼠PPARα基因表达及脂代谢和胰岛素水平的变化

目的观察非酒精性脂肪肝（NAFLD）大鼠肝组织中PPARα基因的表达,并用PPARct激动剂进行干预,探讨其与胰岛素抵抗、脂代谢紊乱的关系。方法大鼠随机分为①正常对照组、②高脂模型组、③PPARα激动剂干预组,利用高脂饮食建立大鼠非酒精性脂肪肝模型。12周后,检测大鼠血脂、肝功能、血糖、胰岛素水平及胰岛素抵抗指数;RT-PCR法分析PPARα基因的表达;观察肝脏的形态学改变。结果PPARa激动剂可降低NAFLD大鼠转氨酶、血脂水平及胰岛素抵抗指数,可促进NAFLD大鼠中PPARa基因的表达;肝脏形态学明显改善。结论PPARα激动剂能改善NAFLD大鼠脂质代谢紊乱,有明显的保肝降酶作用,具有适度的胰岛素增敏作用。PPARα及其配体在NAFLD发病机制及治疗中的进一步深入研究,将为临床防治NAFLD提供新的思路。     

SD雌性大鼠性发育早期性器官等脏器和性激素的动态变化

目的探讨正常SD雌性大鼠性成熟前不同日龄段的脏器与促黄体生成素（LH）、促卵泡素（FSH）、雌二醇（E2）等性激素的变化及其关系。方法从生产群中取出60窝密度状态一致的SD大鼠,在不同日龄随机选取雌性大鼠,检测15、25、32、40日龄时大鼠体重、主要脏器指数,子宫、卵巢组织变化和15、25、32、40、60日龄大鼠血清LH、FSH、E2水平。结果记录了SD雌性大鼠性成熟前各脏器指数和卵巢、子宫组织变化,结果显示大鼠卵巢、子宫的增长速度大于体重的增长,而其他脏器增速大都小于体重的增长。本研究还记录了血清LH、FSH、E2水平在不同日龄段的变化规律,表明血清LH、E2浓度在32日龄时出现较为明显升高。结论不同日龄大鼠脏器指数的动态变化提示大鼠性器官在性发育早期得到机体的优先发育。血清LH、E2水平在32日龄时有了明显升高,提示性腺轴功能已经激活。60日龄大鼠血清性激素水平的波动类似于动情周期的规律性变化,推测大鼠在60日龄前即已进入性成熟,这些结果将为大鼠性发育的相关研究提供重要的参考数据。     

Developing human laboratory models of smoking lapse behavior for medication screening

Use of human laboratory analogues of smoking behavior can provide an efficient, cost-effective mechanistic evaluation of a medication signal on smoking behavior, with the result of facilitating translational work in medications development. Although a number of human laboratory models exist to investigate various aspects of smoking behavior and nicotine dependence phenomena, none have yet modeled smoking lapse behavior. The first instance of smoking during a quit attempt (i.e. smoking lapse) is highly predictive of relapse and represents an important target for medications development. Focusing on an abstinence outcome is critical for medication screening as the US Food and Drug Administration approval for cessation medications is contingent on demonstrating effects on smoking abstinence. This paper outlines a three-stage process for the development of a smoking lapse model for the purpose of medication screening. The smoking lapse paradigm models two critical features of lapse behavior: the ability to resist the first cigarette and subsequent ad libitum smoking. Within the context of the model, smokers are first exposed to known precipitants of smoking relapse (e.g. nicotine deprivation, alcohol, stress), and then presented their preferred brand of cigarettes. Their ability to resist smoking is then modeled and once smokers 'give in' and decide to smoke, they participate in a tobacco self-administration session. Ongoing and completed work developing and validating these models for the purpose of medication screening is discussed.     

2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro

Pharmacologic approaches to studying palmitoylation are limited by the lack of specific inhibitors. Recently, screens have revealed five chemical classes of small molecules that inhibit cellular processes associated with palmitoylation (Ducker, C. E., L. K. Griffel, R. A. Smith, S. N. Keller, Y. Zhuang, Z. Xia, J. D. Diller, and C. D. Smith. 2006. Discovery and characterization of inhibitors of human palmitoyl acyltransferases. Mol. Cancer Ther. 5: 1647-1659). Compounds that selectively inhibited palmitoylation of N-myristoylated vs. farnesylated peptides were identified in assays of palmitoyltransferase activity using cell membranes. Palmitoylation is catalyzed by a family of enzymes that share a conserved DHHC (Asp-His-His-Cys) cysteine-rich domain. In this study, we evaluated the ability of these inhibitors to reduce DHHC-mediated palmitoylation using purified enzymes and protein substrates. Human DHHC2 and yeast Pfa3 were assayed with their respective N-myristoylated substrates, Lck and Vac8. Human DHHC9/GCP16 and yeast Erf2/Erf4 were tested using farnesylated Ras proteins. Surprisingly, all four enzymes showed a similar profile of inhibition. Only one of the novel compounds, 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one [Compound V (CV)], and 2-bromopalmitate (2BP) inhibited the palmitoyltransferase activity of all DHHC proteins tested. Hence, the reported potency and selectivity of these compounds were not recapitulated with purified enzymes and their cognate lipidated substrates. Further characterization revealed both compounds blocked DHHC enzyme autoacylation and displayed slow, time-dependent inhibition but differed with respect to reversibility. Inhibition of palmitoyltransferase activity by CV was reversible, whereas 2BP inhibition was irreversible.     

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence-blocking compounds

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.     

Multiple functions of aromatic-carbohydrate interactions in a processive cellulase examined with molecular simulation

Proteins employ aromatic residues for carbohydrate binding in a wide range of biological functions. Glycoside hydrolases, which are ubiquitous in nature, typically exhibit tunnels, clefts, or pockets lined with aromatic residues for processing carbohydrates. Mutation of these aromatic residues often results in significant activity differences on insoluble and soluble substrates. However, the thermodynamic basis and molecular level role of these aromatic residues remain unknown. Here, we calculate the relative ligand binding free energy by mutating tryptophans in the Trichoderma reesei family 6 cellulase (Cel6A) to alanine. Removal of aromatic residues near the catalytic site has little impact on the ligand binding free energy, suggesting that aromatic residues immediately upstream of the active site are not directly involved in binding, but play a role in the glucopyranose ring distortion necessary for catalysis. Removal of aromatic residues at the entrance and exit of the Cel6A tunnel, however, dramatically impacts the binding affinity, suggesting that these residues play a role in chain acquisition and product stabilization, respectively. The roles suggested from differences in binding affinity are confirmed by molecular dynamics and normal mode analysis. Surprisingly, our results illustrate that aromatic-carbohydrate interactions vary dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, these results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering strategies for biomass utilization. Generally, these results suggest that nature employs aromatic-carbohydrate interactions with a wide range of binding affinities for diverse functions.     

Three-dimensional structure of a flavivirus dumbbell RNA reveals molecular details of an RNA regulator of replication

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). The 3′ UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs). DBs regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we solved the x-ray crystal structure of the Donggang virus DB to 2.1Å resolution and used structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.     

Harmonised Australian Reference Intervals for Serum PINP and CTX in Adults

Bone turnover markers (BTMs) are classified as either formation or resorption markers. Their concentrations in blood or urine of adults are considered to reflect the rate of bone remodelling and may be of use in the management of patients with bone disease. Major inter-method differences exist for BTMs, and harmonisation of methods is currently being pursued at an international level. Based on published data, this article describes age- and sex-specific Australian consensus reference intervals for adults for serum procollagen type I amino-terminal propeptide (s-PINP) and serum β-isomerised carboxy-terminal cross-linking telopeptide of type I collagen (s-CTX).