S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.     

犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

人用狂犬病疫苗过敏性反应探讨

采用超滤浓缩技术生产人用狂犬病疫苗后,疫苗效力得到明显提高;胆在使用中,包括过敏反应等在内的不良反应等有所增多且严重程度明显增大。以豚鼠为实验动物模型,探讨人用狂犬病疫苗引起过敏反应的原因。结果显示,地鼠民分是引起过敏的重要物质,过敏的严重程度随制品浓缩倍数的增大而增大。     

混合培养对固氮菌和纤维素分解菌生长及固氮的影响

对筛选的自生固氮菌和纤维素分解菌进行混合培养 ,研究了菌数与菌液含氮量的变化情况 ,并与其单独培养进行了比较。实验证明 :在混合培养条件下 ,两种菌能相互利用、相互促进 ,混合培养液的菌数增加 ,固氮菌的固氮能力提高。这两种菌可混合培养制成混合菌剂。。     

A Proteomics Sample Preparation Method for Mature,Recalcitrant Leaves of Perennial Plants

Sample preparation is key to the success of proteomics studies. In the present study, two sample preparation methods were tested for their suitability on the mature, recalcitrant leaves of six representative perennial plants (grape, plum, pear, peach, orange, and ramie). An improved sample preparation method was obtained: Tris and Triton X-100 were added together instead of CHAPS to the lysis buffer, and a 20% TCA-water solution and 100% precooled acetone were added after the protein extraction for the further purification of protein. This method effectively eliminates nonprotein impurities and obtains a clear two-dimensional gel electrophoresis array. The method facilitates the separation of high-molecular-weight proteins and increases the resolution of low-abundance proteins. This method provides a widely applicable and economically feasible technology for the proteomic study of the mature, recalcitrant leaves of perennial plants.     

Differential pattern of spinal sympathetic outflow in response to stimulation of the caudal medullary raphe

In urethan-anesthetized cats, frequency domain analysis was used to explore the mechanisms of differential responses of inferior cardiac (CN), vertebral (VN), and renal (RN) sympathetic nerves to electrical stimulation of a discrete region of the medullary raphe (0-2 mm caudal to the obex). Raphe stimulation in baroreceptor-denervated cats at frequencies (7-12 Hz) that entrained the 10-Hz rhythm in nerve activity decreased CN and RN activities but increased VN activity. The reductions in CN and RN discharges were associated with decreased low-frequency (相似文献   

Constitutive β-catenin activation in osteoblasts impairs terminal osteoblast differentiation and bone quality

Accumulating evidence suggests that Wnt/β-catenin signaling plays a central role in controlling bone mass. We previously reported that constitutive activation of β-catenin (CA-β-catenin) in osteoblasts potentially has side effects on the bone growth and bone remodeling process, although it could increase bone mass. The present study aimed to observe the effects of osteoblastic CA-β-catenin on bone quality and to investigate possible mechanisms of these effects. It was found that CA-β-catenin mice exhibited lower mineralization levels and disorganized collagen in long bones as confirmed by von Kossa staining and sirius red staining, respectively. Also, bone strength decreased significantly in CA-β-catenin mice. Then the effect of CA-β-catenin on biological functions of osteoblasts were investigated and it was found that the expression levels of osteocalcin, a marker for the late differentiation of osteoblasts, decreased in CA-β-catenin mice, while the expression levels of osterix and alkaline phosphatase, two markers for the early differentiation of osteoblasts, increased in CA-β-catenin mice. Furthermore, higher proliferation rate were revealed in osteoblasts that were isolated from CA-β-catenin mice. The Real-time PCR and western blot examination found that the expression level of c-myc and cyclin D1, two G1 progression-related molecules, increased in osteoblasts that were isolated from the CA-β-catenin mice, and the expression levels of CDK14 and cyclin Y, two mitotic-related molecules that can accelerate cells entering into S and G2/M phases, increased in osteoblasts that were isolated from the CA-β-catenin mice. In summary, osteoblastic CA-β-catenin kept osteoblasts in high proliferative state and impaired the terminal osteoblast differentiation, and this led to changed bone structure and decreased bone strength.     

共培养的树突状细胞与CIK细胞的体外增殖和杀瘤活性研究

本文观察树突状细胞与同源CIK细胞共培养后培养物的表型、增殖活性变化,及DC对CIK细胞细胞毒活性的影响。提取健康供血者的PBMC,常规诱导出DC与CIK细胞,用A549肺腺癌细胞裂解液抗原冲击DC,并和CIK细胞共培养,动态观察DC-CIK培养物增殖活性和表型变化;定量检测细胞培养上清中的IFN-γ和IL-12;并用MTT法检测共培养细胞杀伤A549肺腺癌细胞和BEL-7404肝癌细胞的活性。结果表明DC与CIK细胞共培养,通过彼此的相互作用诱导出比CIK细胞增殖活性和杀伤活性更强的细胞群体。经A549肺腺癌细胞裂解液抗原冲击的DC活化的CIK,对A549肺腺癌细胞的杀伤活性高于单纯CIK细胞,差异显著(p<0.05)。二者对BEL-7404肝癌细胞的杀伤活性无显著差异。实验证明DC与CIK共培养细胞是一种增殖活性和细胞毒活性高于CIK细胞的免疫活性细胞,经肿瘤抗原冲击的DC能明显提高CIK对肿瘤细胞的杀伤活性,具有更广阔的应用前景。