Fast conformational exchange between the sulfur-free and persulfide-bound rhodanese domain of E. coli YgaP

Rhodanese domains are abundant structural modules that catalyze the transfer of a sulfur atom from thiolsulfates to cyanide via formation of a covalent persulfide intermediate that is bound to an essential conserved cysteine residue. In this study, the three-dimensional structure of the rhodanese domain of YgaP from Escherichia coli was determined using solution NMR. A typical rhodanese domain fold was observed, as expected from the high homology with the catalytic domain of other sulfur transferases. The initial sulfur-transfer step and formation of the rhodanese persulfide intermediate were monitored by addition of sodium thiosulfate using two-dimensional ¹H–¹⁵N correlation spectroscopy. Discrete sharp signals were observed upon substrate addition, indicting fast exchange between sulfur-free and persulfide-intermediate forms. Residues exhibiting pronounced chemical shift changes were mapped to the structure, and included both substrate binding and surrounding residues.     

ADP Signaling in Vascular Endothelial Cells: ADP-DEPENDENT ACTIVATION OF THE ENDOTHELIAL ISOFORM OF NITRIC-OXIDE SYNTHASE REQUIRES THE EXPRESSION BUT NOT THE KINASE ACTIVITY OF AMP-ACTIVATED PROTEIN KINASE*

ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser¹¹⁷⁹ and Ser⁶³⁵ and dephosphorylation at Ser¹¹⁶ in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y₁ receptor antagonist MRS 2179 or by knockdown of P2Y₁ using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser¹¹⁷⁹ and Ser⁶³⁵ phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-β (CaMKKβ) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser⁶³⁵ phosphorylation and eNOS activity but did not affect eNOS Ser¹¹⁷⁹ phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKKβ knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKKβ kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKKβ is necessary for ADP signaling to eNOS.     

Rapid detection of noroviruses in fecal samples and shellfish by nucleic acid sequence-based amplification

The purpose of this study was to determine the efficacy of a nucleic acid sequence-based amplification (NASBA) method of detecting noroviruses in artificially and naturally contaminated shellfish. We used 58 fecal samples that tested positive for noroviruses with electron microscopy (EM) to develop an NASBA assay for these viruses. Oligonucleotide primers targeting the polymerase coding region were used to amplify the viral RNA in an isothermal process that resulted in the accumulation of RNA amplicons. These amplicons were detected by hybridization with digoxigenin-labeled oligonucleotide probes that were highly specific for genogroup I (GI) and genogroup II (GII) of noroviruses. The expected band of 327 bp appeared in denaturing agarose gel without any nonspecific band. The specific signal for each amplicon was obtained through Northern blotting in many repeats. All fecal samples of which 46 (79.3%) belonged to GII and 12 (20.6%) belonged to GI were positive for noroviruses by EM and by NASBA. Target RNA concentrations as low as 5 pg/ml were detected in fecal specimens using NASBA. When the assay was applied to artificially contaminated shellfish, the sensitivity to nucleic acid was 100 pg/1.5 g shellfish tissue. The potential use of this assay was also confirmed in naturally contaminated shellfish collected from different ponds in Guangzhou city of China, of which 24 (18.76%) out of 128 samples were positive for noroviruses; of these, 19 (79.6%) belonged to GII and 5 (20.4%) belonged to GI. The NASBA assay provided a more rapid and efficient way of detecting noroviruses in fecal samples and demonstrated its potential for detecting noroviruses in food and environmental samples with high specificity and sensitivity.     

Adenoviral-mediated interleukin-18 expression in mesenchymal stem cells effectively suppresses the growth of glioma in rats

Glioma is the most common primary intracranial malignant tumor. Despite advances in surgical techniques and adjuvant radio- and chemotherapies, the prognosis for patients with glioma remains poor. We have explored the effects of using genetically modified mesenchymal stem cells (MSCs) to treat malignant glioma in rats. Mesenchymal stem cells isolated from Sprague-Dawley rats can directly suppress the growth of C6 cells in vitro. MSCs transplanted intratumorally can also significantly inhibit the growth of glioma and prolong survival in C6 glioma-bearing models. MSCs producing Interleukin-18 infected by adenoviral vector inhibited glioma growth and prolonged the survival of glioma-bearing rats. Transplantation of IL-18 secreting MSCs was associated with enhanced T cell infiltration and long-term anti-tumor immunity. Thus, IL-18 may be an effective adoptive immunotherapy for malignant glioma. When used in conjunction with MSCs as targeting vehicles in vivo, IL-18 may offer a promising new treatment option for malignant glioma.     

大肠杆菌-链霉菌穿梭表达载体pMF的构建及其应用

【目的】构建能定点整合到链霉菌(Streptomyces)染色体上的高效表达载体。【方法】以链霉菌自杀型表达载体pLSB2为基础,通过插入链霉菌噬菌体ΦC31整合酶基因int和attP位点(Phage attachment site),构建了能在大肠杆菌和链霉菌之间进行接合转移并定点整合到链霉菌染色体上的表达载体pMF。将pMF转化大肠杆菌ET12567(pUZ8002),并分别接合转移天蓝色链霉菌(Streptomyces coelicolorM145)、变铅青链霉菌(Streptomyces lividansTK24)和红色糖多孢菌(Saccharopolyspora erythraea2338),挑取接合子进行PCR和Southern杂交检测。将来自刺糖多孢菌S08-4的S-腺苷甲硫氨酸合成酶基因(SAM-s)克隆到载体pMF的启动子下游,接合转移到天蓝色链霉菌中。【结果】表明pMF成功整入链霉菌染色体,并且检测到目的蛋白的表达。【结论】构建的pMF载体可作为外源基因定点整合表达的有效工具,为后续的基因功能研究以及链霉菌的遗传改造奠定了基础。     

海蜇(Rhopilema esculentum)even-skipped基因的克隆及生物信息学分析

Even-skipped(eve)是一个含有同源框结构域的转录调控因子,在体节形成、神经分化、尾芽发育等过程中发挥着重要作用.为探究eve在钵水母变态发育中的作用,本研究以海蜇转录组测序获得的eve基因类似序列为基础,通过RACE技术获得了海蜇eve基因(ReEve)的cDNA全长,并已提交GenBank数据库,登录号...     

Design,synthesis, and biological evaluation of triazole-pyrimidine-methylbenzonitrile derivatives as dual A2A/A2B adenosine receptor antagonists

A series of novel dual A_2A/A_2B AR antagonists based on the triazole-pyrimidine-methylbenzonitrile core were designed and synthesised. The A_2A AR antagonist cAMP functional assay results were encouraging for most target compounds containing quinoline or its open-ring bioisosteres. In addition, compound 7i displayed better inhibitory activity on A_2B AR (IC₅₀ 14.12 nM) and higher potency in IL-2 production than AB928. Moreover, molecular docking studies were carried out to explain the rationality of molecular design and the activity of compound 7i. Further studies on 7f and 7i revealed good liver microsomes stabilities and acceptable in vivo PK profiles. This study provides insight into the future development of dual A_2A/A_2B AR antagonists for cancer immunotherapy.