A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.     

Competitive study of monoclonal antibodies against the HIV-1 Gp41 core structure

Monoclonal antibodies (MAbs) 50.69, 98.6, and T26 bind specifically to the core structure of the human immunodeficiency virus type 1 (HIV-1) envelope transmembrane glycoprotein (gp41). To clarify the specificity of the anti-core structure MAbs, we performed competitive assays using the MAbs to the H9 human T cell line infected with the IIIB strain of HIV-1 (H9/IIIB). Bound MAb 50.69 inhibited MAb 98.6 binding unidirectionally. The reason for the unidirectional cross competition between MAbs 50.69 and 98.6 is not clear, but these results help to define the antigenic structure of gp41 on the surface of infected cells.     

Butyrate-induced GPR41 activation inhibits histone acetylation and cell growth

Butyrate has been recently identified as a natural ligand of the G-protein-coupled receptor 41(GPR41).In addition,it is an inhibitor of histone deacetylase(HDAC).Butyrate treatment results in the hyperacetylation of histones,with resultant multiple biological effects including inhibition of proliferation,induction of cell cycle arrest,and apoptosis,in a variety of cultured mammalian cells.However,it is not clear whether GPR41 is actively involved in the above-mentioned processes.In this study,we generated a stable cell line expressing the hGPR41 receptor in order to investigate the involvement of GPR41 on butyrate-induced biochemical and physiologic processes.We found that GPR41 activation may be a compensatory mechanism to counter the increase in histone H3 acetylation levels induced by butyrate treatment.Moreover,GPR41 had an inhibitory effect on the anti-proliferative,pro-apoptotic effects of butyrate.GPR41 expression induced cell cycle arrest at the G1-stage,while its activation by butyrate can cause more cells to pass the G1 checkpoint.These results indicated that GPR41 was associated with histone acetylation and might be involved in the acetylation-related regulation of cell processes including proliferation,apoptosis,and the cell cycle.     

Upregulation of the let-7 microRNA with precocious development in lin-12/Notch hypermorphic Caenorhabditis elegans mutants

The lin-12/Notch signaling pathway is conserved from worms to humans and is a master regulator of metazoan development. Here, we demonstrate that lin-12/Notch gain-of-function (gf) animals display precocious alae at the L4 larval stage with a significant increase in let-7 expression levels. Furthermore, lin-12(gf) animals display a precocious and higher level of let-7 gfp transgene expression in seam cells at L3 stage. Interestingly, lin-12(gf) mutant rescued the lethal phenotype of let-7 mutants similar to other known heterochronic mutants. We propose that lin-12/Notch signaling pathway functions in late developmental timing, upstream of or in parallel to the let-7 heterochronic pathway. Importantly, the human microRNA let-7a was also upregulated in various human cell lines in response to Notch1 activation, suggesting an evolutionarily conserved cross-talk between let-7 and the canonical lin-12/Notch signaling pathway.     

Kinase-dependent adhesion to fibronectin: regulation by calreticulin

We studied the phosphorylation (activation status) of c-Src and CaMKII in MEFs either wild type for calreticulin, calreticulin-null, or rescued with full-length calreticulin. We found that calreticulin-null cells were poorly spread on the substratum and formed few, if any, focal contacts. Fibronectin expression and deposition were lower in calreticulin-null MEFs compared to calreticulin-expressing cells, which also exhibited increased c-Src and CaMKII phosphorylation (activity). Plating MEFs on preformed fibronectin rescued the poor adhesive phenotype of calreticulin-null cells, and caused a decrease in c-Src Y418 phosphorylation (activity). c-Src inhibition caused the calreticulin-null MEFs to become well spread on the substratum and to make many prominent focal contacts. Calmodulin and CaMKII inhibition caused similar results, along with a notable increase in paxillin phosphorylation (activation). To test if the calcium storage function of calreticulin was responsible for these effects, we manipulated intracellular [Ca(2+)]. Lowering [Ca(2+)](ER) caused an increase in c-Src phosphorylation and a decrease in fibronectin abundance. Conversely, increasing [Ca(2+)] caused opposite effects. These results suggest that calreticulin regulates both the c-Src and calmodulin/CaMKII pathways, enabling cells to be better spread on the substratum by allowing greater fibronectin deposition and increased focal contact formation.     

伪狂犬病病毒宿主关闭蛋白的基因克隆及结构分析

为研究伪狂犬病病毒 (pseudorabiesvires ,PRV)UL4 1基因编码的病毒宿主关闭蛋白 (VHS)的结构与功能 ,通过PCR扩增得到含UL4 1基因完整编码区的 1174bp片段 ,将该片段克隆到表达载体pGEX KG中GST下游 ,在大肠杆菌BL2 1(DE3)中实现了GST VHS融合蛋白的高效表达 .序列分析发现VHS蛋白具有 4个保守区 ,并且第 3个保守区与核酸内切酶结构域FEN 1(1A76 )高度同源 .PROSPECT软件预测的伪狂犬病病毒VHS蛋白三维结构中含有 10个α螺旋和 2 2个β折叠 ,与单纯疱疹病毒Ⅰ型的VHS蛋白三维结构十分相似 .     

Simulation of the N-terminus of HIV-1 glycoprotein 41000 fusion peptide in micelles.

In this paper, the N-terminus of glycoprotein-41, the HIV-1 fusion peptide, was studied by molecular dynamics simulations in an explicit sodium dodecyl sulfate micelle. The simulation provides a detailed picture of the equilibrium structure and peptide stability as it interacts with the micelle. The equilibrium location of the peptide shows the peptide at the surface of the micelle with hydrophobic residues interacting with the micelle's core. At equilibrium, the peptide adopts an alpha-helical structure from residues 5-16 and a type-1 beta-turn from 17-20 with the other residues exhibiting more flexible conformations. The primary hydrophobic interactions with the micelle are from the leucine and phenylalanine residues (Leu-7, Phe-8, Leu-9, Phe-11, Leu-12) while the alanine and glycine residues (Ala-1, Gly-3, Gly-5, Ala-6, Gly-10, Gly-13, Ala-14, Ala-15, Gly-16, Gly-10, Ala-21) interact favorably with water molecules. The results suggest that Phe-8, part of the highly conserved FLG motif of the fusion peptide, plays a key role in the interaction of the peptide with membranes. Our simulations corroborate experimental investigations of the fusion peptide in SDS micelles, providing a high-resolution picture that explains the experimental findings.     

HIV-1 gp41的酵母表面展示及表达优化

以His标签检测蛋白的表达, 利用酿酒酵母表面展示系统, 成功地将HIV-1 gp41片段锚定在酵母表面, 并检测到gp41的活性。以pMD18T-gp41为模板, 通过PCR技术克隆了gp41基因, 将gp41基因通过双酶切连接到载体pICAS-His上,构建了gp41酵母表面展示载体, 并将其转化至酿酒酵母(Saccharomyces cerevisiae)MT8-1中。重组菌经培养, 利用免疫荧光染色方法进行染色, 显微镜观察发现重组酵母细胞表面有绿色荧光, 流式细胞仪结果进一步证实gp41正确折叠展示于酵母细胞表面。采用不同浓度的葡萄糖培养基进行表达优化。当葡萄糖浓度为1%时, 82.46%的酵母细胞表达了gp41抗原; 随着葡萄糖浓度升高, 蛋白表达受到抑制。     

Immunogenic Properties of Peptides Mimicking a HIV-1 gp41 Epitope Recognized by Virus-Neutralizing Antibodies 2F5

Phage display was used to obtain peptides mimicking a HIV-1 gp41 conserved epitope recognized by virus-neutralizing monoclonal antibodies (mAb) 2F5. Rabbits and mice were immunized with the peptides exposed on the surface of filamentous bacteriophages. Antibodies to gp41 were detected in the sera of immunized animals. The virus-neutralizing activity of the sera was examined.