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.     

A Method for Identification and Analysis of Non-Overlapping Myeloid Immunophenotypes in Humans

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN^-CD33⁺HLA-DR^- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies.     

Splenic gene expression profiling in White Leghorn layer inoculated with the Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (SE) is a foodborne pathogen that can threaten human health through contaminated poultry products. Live poultry, chicken eggs and meat are primary sources of human salmonellosis. To understand the genetic resistance of egg‐type chickens in response to SE inoculation, global gene expression in the spleen of 20‐week‐old White Leghorn was measured using the Agilent 4 × 44 K chicken microarray at 7 and 14 days following SE inoculation (dpi). Results showed that there were 1363 genes significantly differentially expressed between inoculated and non‐inoculated groups at 7 dpi (I7/N7), of which 682 were up‐regulated and 681 were down‐regulated genes. By contrast, 688 differentially expressed genes were observed at 14 dpi (I14/N14), of which 371 were up‐regulated genes and 317 were down‐regulated genes. There were 33 and 28 immune‐related genes significantly differentially expressed in the comparisons of I7/N7 and I14/N14 respectively. Functional annotation revealed that several Gene Ontology (GO) terms related to immunity were significantly enriched between the inoculated and non‐inoculated groups at 14 dpi but not at 7 dpi, despite a similar number of immune‐related genes identified between I7/N7 and I14/N14. The immune response to SE inoculation changes with different time points following SE inoculation. The complicated interaction between the immune system and metabolism contributes to the immune responses to SE inoculation of egg‐type chickens at 14 dpi at the onset of lay. GC, TNFSF8, CD86, CD274, BLB1 and BLB2 play important roles in response to SE inoculation. The results from this study will deepen the current understanding of the genetic response of the egg‐type chicken to SE inoculation at the onset of egg laying.     

Hepatitis B Virus Pre-S2 Mutant Induces Aerobic Glycolysis through Mammalian Target of Rapamycin Signal Cascade

Hepatitis B virus (HBV) pre-S2 mutant can induce hepatocellular carcinoma (HCC) via the induction of endoplasmic reticulum stress to activate mammalian target of rapamycin (MTOR) signaling. The association of metabolic syndrome with HBV-related HCC raises the possibility that pre-S2 mutant-induced MTOR activation may drive the development of metabolic disorders to promote tumorigenesis in chronic HBV infection. To address this issue, glucose metabolism and gene expression profiles were analyzed in transgenic mice livers harboring pre-S2 mutant and in an in vitro culture system. The pre-S2 mutant transgenic HCCs showed glycogen depletion. The pre-S2 mutant initiated an MTOR-dependent glycolytic pathway, involving the eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), Yin Yang 1 (YY1), and myelocytomatosis oncogene (MYC) to activate the solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1), contributing to aberrant glucose uptake and lactate production at the advanced stage of pre-S2 mutant transgenic tumorigenesis. Such a glycolysis-associated MTOR signal cascade was validated in human HBV-related HCC tissues and shown to mediate the inhibitory effect of a model of combined resveratrol and silymarin product on tumor growth. Our results provide the mechanism of pre-S2 mutant-induced MTOR activation in the metabolic switch in HBV tumorigenesis. Chemoprevention can be designed along this line to prevent HCC development in high-risk HBV carriers.     

SOME REMARKS ON CHENJIAWO FAUNA──On the First Appearance of Peking Man Fauna

I.CORRELATIONBETWEENL0ESSSECTI0NAND0XYGENIS0TOPESTAGESIntheYuanloessarea,manyscientiststhinkthatthesedimentsarecontinuousandtheclimaticrecordsareperfect.Thisdoesnotappeartobeaccurate.Sedimentationwasdiscontinuousinmanysections.Forexample,manyscientistsarguethatS2corresp0ndstooxygenisot0peStage7(Liu,l985,KuklaandAn,l989;Dingetal.,l990).lnfact,S2includesthreelayersfS2SSl,S2LLlandS2SS2.Dingetal.(l990)reportedthatthereisathickunitofloessbetweenS2SSlandS2SS2intheBaicao…     

Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban

Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in canine cardiac microsomes were found to be stimulated by heparin and various other polyanions. Prior treatment of the microsomes with the ionophores alamethicin or A23187 produced no change in the extent of stimulation of the ATPase activity by heparin yet eliminated net calcium uptake. This finding and a lack of change in the stoichiometric ratio of mol of calcium transported/mol of ATP hydrolyzed (calcium:ATP) suggest that the effect of heparin is on the calcium pump rather than on a parallel calcium efflux pathway. Certain polycationic compounds including poly-L-arginine and histone inhibited both cardiac and fast skeletal muscle microsomal calcium uptake and also produced no change in the stoichiometric ratio of calcium to ATP. Several lines of evidence indicate that the polyanionic compounds tested stimulate calcium uptake by interacting with phospholamban, the putative phosphorylatable regulator of the cardiac sarcoplasmic reticulum calcium pump, whereas polycationic compounds appear to interact with the pump. (i) Heparin stimulated calcium uptake to the same extent as protein kinase A or trypsin, whereas prior phosphorylation or tryptic cleavage of phospholamban from the membrane abolished the stimulatory effect of heparin. (ii) Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in fast skeletal muscle microsomes, which lack phospholamban, were unaffected by heparin. (iii) Purified cardiac (Ca2+ + Mg2+)-ATPase activity was no longer stimulated by heparin yet was still inhibited by polycationic compounds. The heparin-induced stimulation of calcium uptake was dependent on the pH and ionic strength of the heparin-containing preincubation medium, hence electrostatic interactions appear to play a significant role in heparin's stimulatory action. The data are consistent with an inhibitory role of the positively charged cytoplasmic domain of phospholamban with respect to calcium pump activity and the relief of the inhibition upon reduction in phospholamban's positive charge by phosphorylation or binding of polyanions.     

Ginsenoside Rg1 protects H9c2 cells against nutritional stress-induced injury via aldolase /AMPK/PINK1 signalling

Insufficient nutrients supply will greatly affect the function of cardiac myocytes. The adaptive responses of cardiac myocytes to nutritional stress are not fully known. Ginsenoside Rg1 is one of the most pharmacologically active components in Panax Ginseng and possesses protective effects on cardiomyocyte. Here, we investigate the effects of ginsenoside Rg1 on H9c2 cells which were subjected to nutritional stress. Nutritional stress-induced by glucose deprivation strongly induced cell death and this response was inhibited by ginsenoside Rg1. Importantly, glucose deprivation decreased intracellular ATP levels and mitochondrial membrane potential. Ginsenoside Rg1 rescued ATP levels and mitochondrial membrane potential in nutrient-starved cells. For molecular mechanisms, ginsenoside Rg1 increased the expressions of PTEN-induced kinase 1 (PINK1) and p-AMPK in glucose deprivation treated H9c2 cells. Reducing the expression of aldolase in H9c2 cells inhibited ginsenoside Rg1′s actions on PINK1 and p-AMPK. Further, the nutritional stress mice were used to verify the mechanisms obtained in vitro. Ginsenoside Rg1 increased the expressions of aldolase, p-AMPK, and PINK1 in starved mice heart. Taken together, our results reveal that ginsenoside Rg1 limits nutritional stress-induced H9c2 cells injury by regulating the aldolase /AMP-activated protein kinase/PINK1 pathway.     

Morpho-anatomical and physiological responses to waterlogging stress in different barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) genotypes

Waterlogging is one of the major stresses limiting crop production worldwide. The understanding of the mechanisms of plant adaptations to waterlogging stress helps improve plant tolerance to stress. In this study, physiological responses and morpho-anatomical adaptations of seven different barley genotypes were investigated under waterlogging stress. The results showed that the waterlogging-tolerant varieties (TX9425, Yerong, TF58) showed less reduction in plant height, SPAD (soil–plant analyses development analyses) value, tillers, shoot and root biomasses than did the waterlogging-sensitive varieties (Franklin, Naso Nijo, TF57). Under waterlogging stress condition, the tolerant genotypes also showed a much larger number of adventitious roots than did the sensitive genotypes. More intercellular spaces and better integrated chloroplast membrane structures were observed in the leaves of the waterlogging-tolerant cultivars, which is likely due to increased ethylene content, decreased ABA content and less accumulation of O₂^.?. The ability to form new adventitious roots and intercellular spaces in shoots can also be used as selection criteria in breeding barley for waterlogging tolerance.