iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

Deregulation of allosteric response of Lactococcus lactis prolidase and its effects on enzyme activity

The allosteric behaviour of Lactococcus lactis prolidase (Xaa-Pro dipeptidase) of this proline-specific peptidase was investigated where it was hypothesized that intersubunit interactions between a loop structure and three residues near the active site contributed to this behaviour. Seven mutant prolidases were constructed, and it was observed that the loopless mutant and His303 substitution inactivated the enzyme. Ser307 substitution revealed that this residue influenced the substrate binding, as judged from its kinetic constants and substrate specificity; however, this residue did not contribute to allostery of prolidase. R293S mutation resulted in the disappearance of the allosteric behaviour yielding a Hill constant of 0.98 while the wild type had a constant of 1.58. In addition, the R293S mutation suppressed the substrate inhibition that was observed in other mutants and wild type. The K_m value of R293S was 2.9-fold larger and V_max was approximately 50% less as compared to the wild type. The results indicated that Arg293 increased the affinity for substrates while introducing allosteric behaviour and substrate inhibition. Computer modelling suggested that negative charges on the loop structure interacted with Arg293 and Ser307 to maintain these characteristics. It was, therefore, concluded that Arg293, His303, Ser307 and the loop contributed to the enzyme's allosteric characteristics.     

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD⁺ and NADPH/NADP⁺. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].     

Molecular characterization of the Oncidium orchid HDR gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, the last step of the methylerythritol phosphate pathway

Two pathways are used by higher plants for the biosynthesis of isoprenoid precursors: the mevalonate pathway in the cytosol and a 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in the plastids, with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) catalyzing the last step in the MEP pathway. In order to understand the contribution of MEP pathway in isoprenoid biosynthesis of Oncidium orchid, a full-length cDNA corresponding to HDR from the flower tissues of Oncidium Gower Ramsey was cloned. The deduced OncHDR amino acid sequence contains a plastid signal peptide at the N-terminus and four conserved cysteine residues. RT-PCR analysis of HDR in Oncidium flowering plants revealed ubiquitous expression in organs and tissues, with preferential expression in the floral organs. Phylogenetic analysis revealed evolutionary conservation of the encoding HDR protein sequence. The genomic sequence of the HDR in Oncidium is similar to that in Arabidopsis, grape, and rice in structure. Successful complementation by OncHDR of an E. coli hdr ⁻ mutant confirmed its function. Transgenic tobacco carrying the OncHDR promoter-GUS gene fusion showed expression in most tissues, as well as in reproductive organs, as revealed by histochemical staining. Light induced strong GUS expression driven by the OncHDR promoter in transgenic tobacco seedlings. Taken together, our data suggest a role for OncHDR as a light-activated gene.     

Seasonal variation of mixing depth and its influence on phytoplankton dynamics in the Zeya reservoir, China

In reservoirs or lakes, mixing depth affects growth and loss rates of phytoplankton populations. Based on 1-year data from the Zeya reservoir, China, we scaled the mixing depth throughout a whole year by utilizing cluster analysis, and then investigated its influence on phytoplankton dynamics and other physical and chemical parameters. Over the whole year, all physical and chemical parameters except TN and temperature had significant correlations with mixing depth, indicating that mixing depth is one of the important driving factors influencing water environment. According to mixing depth, a year can be divided into three different periods, including the thermally stratified period, isothermally mixed period, and transition period between them. When considering the former two different periods separately, mixing depth had no correlation with the phytoplankton biovolume. However, over the whole year a significant correlation was observed, which indicated that the influence of mixing depth on phytoplankton growth in the Zeya reservoir still followed Diehl’s theory. Furthermore, according to the steady-state assumption, a unimodal curve (mixing depth—phytoplankton biovolume) with a significant peak appearing at a mixing depth of 2 m was observed, closely agreeing with Diehl’ prediction.     

Propofol Reduces Inflammatory Reaction and Ischemic Brain Damage in Cerebral Ischemia in Rats

Our previous studies demonstrated that inflammatory reaction and neuronal apoptosis are the most important pathological mechanisms in ischemia-induced brain damage. Propofol has been shown to attenuate ischemic brain damage via inhibiting neuronal apoptosis. The present study was performed to evaluate the effect of propofol on brain damage and inflammatory reaction in rats of focal cerebral ischemia. Sprague–Dawley rats underwent permanent middle cerebral artery occlusion, then received treatment with propofol (10 or 50 mg/kg) or vehicle after 2 h of ischemia. Neurological deficit scores, cerebral infarct size and morphological characteristic were measured 24 h after cerebral ischemia. The enzymatic activity of myeloperoxidase (MPO) was assessed 24 h after cerebral ischemia. Nuclear factor-kappa B (NF-κB) p65 expression in ischemic rat brain was detected by western blot. Cyclooxygenase-2 (COX-2) expression in ischemic rat brain was determined by immunohistochemistry. ELISA was performed to detect the serum concentration of tumor necrosis factor-α (TNF-α). Neurological deficit scores, cerebral infarct size and MPO activity were significantly reduced by propofol administration. Furthermore, expression of NF-κB, COX-2 and TNF-α were attenuated by propofol administration. Our results demonstrated that propofol (10 and 50 mg/kg) reduces inflammatory reaction and brain damage in focal cerebral ischemia in rats. Propofol exerts neuroprotection against ischemic brain damage, which might be associated with the attenuation of inflammatory reaction and the inhibition of inflammatory genes.     

High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B,Type I,PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI''s C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.     

DDPH对低氧内皮细胞条件培养液诱导肺动脉平滑肌细胞增殖及α-SM-actin表达的影响

目的探讨1-(2,6-二甲基苯氧基)-2-(3,4-二甲氧基苯乙氨基)丙烷盐酸盐(DDPH)抑制低氧内皮细胞条件培养液(HECCM)诱导肺动脉平滑肌细胞增殖及对α-SM-actin表达的影响.方法利用低氧内皮细胞条件培养液建立猪肺动脉平滑肌细胞(PASMC)的增殖模型;以四甲基偶氮唑盐(MTT)比色法、α平滑肌肌动蛋白(α-SM-actin)为指标,采用免疫细胞化学染色法观察低氧内皮细胞条件培养液对肺动脉平滑肌细胞增殖的影响以及DDPH对低氧内皮细胞条件培养液促肺动脉平滑肌细胞增殖后的逆转效应.结果低氧内皮细胞条件培养液显著促进肺动脉平滑肌细胞增殖,低氧内皮细胞条件培养液促肺动脉平滑肌细胞增殖后,肺动脉平滑肌细胞的表型发生转化,由收缩表型转化为合成表型,肺动脉平滑肌细胞胞浆内的α-SM-actin含量下降;DDPH能显著抑制低氧内皮细胞条件培养液对肺动脉平滑肌细胞的增殖作用,并使肺动脉平滑肌细胞的表型发生逆转,即由合成表型逆转为具有执行正常收缩功能的收缩表型,肺动脉平滑肌细胞胞浆内的α-SM-actin含量回升.结论提示DDPH能显著抑制低氧内皮细胞条件培养液促肺动脉平滑肌细胞的增殖作用,其作用机制可能是通过肺动脉平滑肌细胞的表型发生逆转来实现的.     

Progesterone inhibits the estrogen-induced phosphoinositide 3-kinase-->AKT-->GSK-3beta-->cyclin D1-->pRB pathway to block uterine epithelial cell proliferation

The mammalian cell cycle is regulated by the cyclin/cyclin-dependent kinase (CDK) phosphorylation of the retinoblastoma (pRB) family of proteins. Cyclin D1 with its CDK4/6 partners initiates the cell cycle and acts as the link between extracellular signals and the cell cycle machinery. Estradiol-17beta (E2) stimulates uterine epithelial cell proliferation, a process that is completely inhibited by pretreatment with progesterone (P4). Previously, we identified cyclin D1 localization as a key point of regulation in these cells with E2 causing its nuclear accumulation and P4 retaining it in the cytoplasm with the resultant inhibition of pRB phosphorylation. Here we show that E2 stimulates phosphoinositide 3-kinase to activate phosphokinase B/AKT to effect an inhibitory phosphorylation of glycogen synthase kinase (GSK-3beta). This pathway is suppressed by P4. Inhibition of the GSK-3beta activity in P4-treated uteri by the specific inhibitor, LiCl, reversed the nuclear accumulation of cyclin D1 and in doing so, caused pRB phosphorylation and the induction of downstream genes, proliferating cell nuclear antigen and Ki67. Conversely, inhibition of phosphoinositide 3 kinase by LY294002 or Wortmanin reversed the E2-induced GSK-3beta Ser9 inhibitory phosphorylation and blocked nuclear accumulation of cyclin D1. These data show the reciprocal actions of E2 and P4 on the phosphoinositide 3-kinase through to the GSK-3beta pathway that in turn regulates cyclin D1 localization and cell cycle progression. These data reveal a novel signaling pathway that links E2 and P4 action to growth factor-mediated signaling in the uterus.     

Characterization of HSCD5, a novel human stearoyl-CoA desaturase unique to primates

Stearoyl-CoA desaturase (SCD) is an integral membrane protein of the endoplasmic reticulum (ER) that catalyzes the formation of monounsaturated fatty acids from saturated fatty acids. Recent studies suggest that SCD is a key regulator of energy metabolism and has implications in dislipidemia and obesity. Four SCD isoforms (SCD1-4) have been identified in mouse. In human, only one SCD isoform has been characterized so far. Here we report that the previously reported human ACOD4 gene encodes a distinct stearoyl-CoA desaturase, hSCD5. GenBank database mining revealed orthologues of hSCD5 in the primates, but not in the rodents. In transiently transfected 293 cells, hSCD5 co-localized with calnexin on ER membrane. Microsome fractions prepared from hSCD1 and hSCD5 transfected cells displayed similar delta 9 desaturase activity. Quantitative real-time RT-PCR analysis suggested that hSCD5 was abundantly expressed in adult brain and pancreas. These data suggested that hSCD5 plays a role distinct from that of hSCD1 during development and in normal physiological conditions.