eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells

ObjectiveWe investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs).MethodsCortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.ResultsMdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.ConclusionseEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.     

NdhO,a Subunit of NADPH Dehydrogenase,Destabilizes Medium Size Complex of the Enzyme in Synechocystis sp. Strain PCC 6803

Two mutants that grew faster than the wild-type (WT) strain under high light conditions were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in ssl1690 encoding NdhO. Deletion of ndhO increased the activity of NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET), while overexpression decreased the activity. Although deletion and overexpression of ndhO did not have significant effects on the amount of other subunits such as NdhH, NdhI, NdhK, and NdhM in the cells, the amount of these subunits in the medium size NDH-1 (NDH-1M) complex was higher in the ndhO-deletion mutant and much lower in the overexpression strain than in the WT. NdhO strongly interacts with NdhI and NdhK but not with other subunits. NdhI interacts with NdhK and the interaction was blocked by NdhO. The blocking may destabilize the NDH-1M complex and repress the NDH-CET activity. When cells were transferred from growth light to high light, the amounts of NdhI and NdhK increased without significant change in the amount of NdhO, thus decreasing the relative amount of NdhO. This might have decreased the blocking, thereby stabilizing the NDH-1M complex and increasing the NDH-CET activity under high light conditions.     

药物组合物封闭式权利要求的解读与侵权判定 ——由最高人民法院（2012)民提字第10号判决案例谈起

从最高人民法院的一个典型案例出发，探讨药物组合物封闭式权利要求保护范围的解读及其专利侵权判定标准，比较其他国家 的相关规定和判例，并对药物组合物封闭式权利要求的专利授权、确权审查、侵权判定以及申请文件撰写技巧提出见解，以供借鉴和参考。     

Phylogeny and comparative genomic analysis of Pteriomorphia (Mollusca: Bivalvia) based on complete mitochondrial genomes

The subclass Pteriomorphia is a morphologically diverse and economically important group of Mollusca. We retrieved 42 mitochondrial genomes (mtGenomes) of Pteriomorphia and concatenated protein-coding genes, rRNAs and tRNAs to assess phylogenetic relationships and divergence times among the families with maximum likelihood (ML) and Bayesian inference (BI) analyses. Both ML and BI analyses strongly support the same topology except for the position of Atrina pectinata. Our study confirms the monophyly of the families Arcidae, Mytilidae, Pteriidae, Ostreidae and Pectinidae. Within Pteriomorphia, we recovered two clusters, one comprising Mytilidae, Arcidae and Pectinidae, the other consisting of Ostreidae, Pteriidae and Pinnidae, but we did not confirm a basal position for any family. The phylogenetic trees suggest that Ostreidae, Pteriidae and Pinnidae should be grouped as the order Ostreoida. Divergence times of major families are estimated as follows: Arcidae, 315.9 Ma; Pectinidae, 384.4 Ma; Ostreidae, 240.8 Ma; Mytilidae, 390.8 Ma. Comparative analysis indicates a low-level codon usage bias (with an average of 50.29) in mtGenomes of Pteriomorphia. In Mytilidae and Ostreidae, the codon usage bias was under mutation pressure rather than selection. Contrastingly, mutation is not the main factor in defining the codon usage in Pectinidae and Pteriidae. Among Ostreidae, Pectinidae and Mytilidae, Ka/Ks ratios range from 0.00 to 1.22 and most values (89.11%) are less than 0.20, indicating that most genes are under strong negative or purifying selection. The protein-coding gene orders show dramatically different patterns in Pteriomorphia. There is no gene block even consisting of two genes that is shared by five families.     

The protective effect of cycloastragenol on aging mouse circadian rhythmic disorder induced by d-galactose

Aging process in mammals is associated with a decline in amplitude and a long period of circadian behaviors which are regulated by a central circadian regulator in the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. It is unclear whether enhancing clock function can retard aging. Using fibroblasts expressing per2::lucSV and senescent cells, we revealed cycloastragenol (CAG), a natural aglycone derivative from astragaloside IV, as a clock amplitude enhancing small molecule. CAG could activate telomerase to antiaging, but no reports focused on its effects on circadian rhythm disorders in aging mice. Here we analyze the potential effects of CAG on d -galactose-induced aging mice on the circadian behavior and expression of clock genes. For this purpose, CAG (20 mg/kg orally), was administered daily to d -galactose (150 mg/kg, subcutaneous) mice model of aging for 6 weeks. An actogram analysis of free-running activity of these mice showed that CAG significantly enhances the locomotor activity. We further found that CAG increase expressions of per2 and bmal1 genes in liver and kidney of aging mouse. Furthermore, CAG enhanced clock protein BMAL1 and PER2 levels in aging mouse liver and SCN. Our results indicated that the CAG could restore the behavior of circadian rhythm in aging mice induced by d -galactose. These data of present study suggested that CAG could be used as a novel therapeutic strategy for the treatment of age-related circadian rhythm disruption.