The Association of Receptor of Activated Protein Kinase C 1(RACK1) with Infectious Bursal Disease Virus Viral Protein VP5 and Voltage-dependent Anion Channel 2 (VDAC2) Inhibits Apoptosis and Enhances Viral Replication

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Our previous report indicates that IBDV VP5 induces apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2). However, the underlying molecular mechanism is still unclear. We report here that receptor of activated protein kinase C 1 (RACK1) interacts with both VDAC2 and VP5 and that they could form a complex. We found that overexpression of RACK1 inhibited IBDV-induced apoptosis in DF-1 cells and that knockdown of RACK1 by small interfering RNA induced apoptosis associated with activation of caspases 9 and 3 and suppressed IBDV growth. These results indicate that RACK1 plays an antiapoptotic role during IBDV infection via interaction with VDAC2 and VP5, suggesting that VP5 sequesters RACK1 and VDAC2 in the apoptosis-inducing process.     

高粘发酵体系不同搅拌桨的CFD模拟及发酵验证

搅拌桨是高好氧高黏度微生物发酵实现高效反应必不可少的因素之一,不同搅拌桨组合对发酵过程的影响十分重要。威兰胶是由产碱杆菌在高耗氧高粘度发酵体系下合成的胞外微生物多糖,广泛应用于水泥、石油、油墨、食品等行业中。本研究借助于计算流体力学(Computational fluid dynamics,CFD)的方法,以威兰胶发酵液体系为研究体系,研究了6种不同搅拌桨组合在反应器内流体速率分布、剪切速率、和气含率等参数。将模拟效果较好的3种组合用于威兰胶发酵过程。研究表明MB-4-6搅拌桨组合对改善发酵罐内部的溶氧及流场分布效果最明显,威兰胶产量水平提高了13%。同时在该组合下威兰胶的产品粘度得到有效提高。     

禽网状内皮组织增殖病病毒gp90蛋白单克隆抗体的制备及其识别区分析

为了制备禽网状内皮组织增殖病病毒(REV)gp90蛋白的单克隆抗体,应用His-gp90融合蛋白免疫BALB/c小鼠,取免疫鼠的脾细胞与骨髓瘤细胞(SP2/0)进行融合,经过筛选、3次亚克隆后获得3株稳定分泌抗REV-gp90蛋白的单克隆抗体杂交瘤细胞株,分别命名为3G5-B8、3G5-A10和1G12。经间接ELISA(Enzyme-linked immunosorbent assay)方法检测,单克隆抗体的亲和力解离常数(Kd)分别为6.483×10–10、4.844×10–10和9.330×10–10,3株单抗的亚型分别为Ig G1、Ig G1和Ig G2b。经Western blotting和间接免疫荧光实验检测,3株单抗均能识别REV感染DF-1细胞后产生的gp90蛋白。以Western blotting方法利用单抗检测不同截短的gp90蛋白,初步确定3G5-B8和3G5-A10 2株单抗抗原识别区均位于gp90蛋白第200-245位氨基酸,而1G12株单抗识别区包含第230-235位氨基酸。这些单抗为REV的诊断和致病机理研究奠定了基础。     

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.     

Artificial leaf aids analysis of chlorophyll fluorescence and P700 absorbance in studies involving microalgae

Measuring chlorophyll fluorescence and P700 absorbance has been widely used to study photosynthesis in both terrestrial plants and algae. However, in order to apply these measurement techniques to study microalgae, a concentrated suspension of algae, which is usually prepared by centrifugation, is required. In this study, instead of using centrifugation, we concentrated microalgae on a nitrocellulose membrane using filtration to create an ‘artificial leaf’ before analysis. Overall, we were able to generate values of the appropriate photosynthetic parameters that were comparable to those obtained when chlorophyll fluorescence and P700 absorbance were measured following centrifugation. There were no statistically significant differences (P > 0.05) between the artificial leaf method and the traditional cuvette method for determining chlorophyll fluorescence or P700 absorbance at appropriate chlorophyll concentrations. We were also able to reduce background noise by using a filter membrane as a carrier. Therefore, an artificial leaf has the potential to be a valuable tool for phycologists interested in studying microalgal photosynthesis by enabling them to eliminate tedious centrifugation steps. In addition, fluorometers commonly used for studying the leaves of higher plants will also be suitable for studying microalgae.