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.     

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E  ^R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.     

Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus

Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~ 3.8 Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.     

Mapping QTLs and meta-QTLs for two inflorescence architecture traits in multiple maize populations under different watering environments

Drought significantly affects the architectural development of maize inflorescence, which leads to massive losses in grain yield. However, the genetic mechanism for traits involved in inflorescence architecture in different watering environments, remains poorly understood in maize. In this study, 19 QTLs for tassel primary branch number (TBN) and ear number per plant (EN) were detected in 2 F_2:3 populations under both well-watered and water-stressed environments by single environment mapping with composite interval mapping (CIM); 11/19 QTLs were detected under water-stressed environments. Moreover, 21 QTLs were identified in the 2 F_2:3 populations by joint analysis of all environments with a mixed linear model based on composite interval mapping (MCIM), 11 QTLs were involved in QTL × environment interactions, seven epistatic interactions were identified with additive by additive/dominance effects. Remarkably, 12 stable QTLs (sQTLs) were simultaneously detected by single environment mapping with CIM and joint analysis through MCIM, which were concentrated in ten bins across the chromosomes: 1.05_1.07, 1.08_1.10, 2.01_2.04, 3.01, 4.06, 4.09, 5.06_5.07, 6.05, 7.00, and 7.04 regions. Twenty meta-QTLs (mQTLs) were detected across 19 populations under 51 watering environments using a meta-analysis, and 34 candidate genes were predicted in corresponding mQTLs regions to be involved in the regulation of inflorescence development and drought resistance. Therefore, these results provide valuable information for finding quantitative trait genes and to reveal the genetic mechanisms responsible for TBN and EN under different watering environments. Furthermore, alleles for TBN and EN provide useful targets for marker-assisted selection to generate high-yielding maize varieties.     

Production of Bio-Energy from Pig Manure: A Focus on the Dynamics Change of Four Parameters under Sunlight-Dark Conditions

This study investigated the effect of sunlight-dark conditions on volatile fatty acids (VFAs), total ammonium nitrogen (TAN), total alkalinity (TA) and pH during pig manure (PM) digestion and then the subsequent influence on biogas yield of PM. PM₁ and PM₂ were performed in a transparent reactor and a non-transparent reactor, respectively. Two sets of experiments were conducted with a temperature of 35.0±2.0 °C and a total solid concentration of 8.0% to the digestion material. The dynamic change of the four parameters in response to sunlight-dark conditions resulted in variations of the physiological properties in the digester and affected the cumulative biogas production (CBP). PM₁ obtained higher CBP (15020.0 mL) with a more stable pH and a lower TAN concentration (1414.5 mg/L) compared to PM₂ (2675.0 mL and 1670.0 mg/L, respectively). The direct path coefficients and indirect path coefficients between the four parameters and CBP were also analyzed.     

Effect of Chronic Administration of Low Dose Rapamycin on Development and Immunity in Young Rats

Mammalian target of rapamycin (mTOR) regulates cell growth, cell differentiation and protein synthesis. Rapamycin, an inhibitor of mTOR, has been widely used as an immunosuppressant and anti-cancer drug. Recently, mTOR inhibitors have also been reported to be a potential anti-epileptic drug, which may be effective when used in young patients with genetic epilepsy. Thus, a suitable dose of rapamycin which can maintain the normal function of mTOR and has fewer side effects ideally should be identified. In the present study, we first detected changes in marker proteins of mTOR signaling pathway during development. Then we determined the dose of rapamycin by treating rats of 2 weeks of age with different doses of rapamycin for 3 days and detected its effect on mTOR pathway. Young rats were then treated with a suitable dose of rapamycin for 4 weeks and the effect of rapamycin on mTOR, development and immunity were investigated. We found that the expression of the marker proteins of mTOR pathway was changed during development in brain hippocampus and neocortex. After 3 days of treanent, 0.03 mg/kg rapamycin had no effect on phospho-S6, whereas 0.1, 0.3, 1.0 and 3.0 mg/kg rapamycin inhibited phospho-S6 in a dose-dependent manner. However, only 1.0 mg/kg and 3.0 mg/kg rapamycin inhibited phospho-S6 after 4 weeks treatment of rapamycin. Parallel to this result, rats treated with 0.1 and 0.3 mg/kg rapamycin had no obvious adverse effects, whereas rats treated with 1.0 and 3.0 mg/kg rapamycin showed significant decreases in body, spleen and thymus weight. Additionally, rats treated with 1.0 and 3.0 mg/kg rapamycin exhibited cognitive impairment and anxiety as evident by maze and open field experiments. Furthermore, the content of IL-1β, IL-2, IFN-γ, TNF-α in serum and cerebral cortex were significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. The expression of DCX was also significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. However, rats treated with 1.0 mg/ kg rapamycin exhibited fewer and milder side effects than those treated with 3.0 mg/kg. In summary, all these data suggest that there is not a rapamycin dose that can inhibit mTOR for epilepsy without causing any side effects, but 1 mg /kg may be the optimal dose for young rats for suppressing mTOR with relatively few side effects.