Selective restriction of Nef-defective human immunodeficiency virus type 1 by a proteasome-dependent mechanism

The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) infectivity by facilitating an early postentry step in the virus life cycle. We report here that the addition of MG132 or lactacystin, each a specific inhibitor of cellular proteasome activity, preferentially enhances cellular permissiveness to infection by Nef-defective versus wild-type HIV-1. Pseudotyping by the glycoprotein of vesicular stomatitis virus rendered Nef-defective HIV-1 particles minimally responsive to the enhancing effects of proteasome inhibitors. These results suggest that Nef enhances the infectivity of HIV-1 particles by reducing their susceptibility to proteasomal degradation in target cells.     

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.     

Cellular environment is important in controlling V-ATPase dissociation and its dependence on activity

One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation into its component V(1) and V(0) domains, which in yeast occurs in response to glucose depletion. V-ATPase complexes containing the Vph1p isoform of subunit a (VCC) are targeted to the vacuole, and Stv1p-containing complexes (SCC) are targeted to the Golgi. Overexpression of Stv1p results in mistargeting of SCC to the vacuole. We have investigated the role of the a subunit isoform and cellular environment in controlling dissociation using vacuolar protein sorting (vps) mutants that accumulate proteins in either the prevacuolar compartment (PVC) (vps27Delta) or a post-Golgi compartment (PGC) (vps21Delta). Dissociation of both VCC and SCC depends upon cellular environment, with dissociation most complete in the vacuole and least complete in the PVC. The dependence of dissociation on V-ATPase activity was also investigated using both concanamycin and inactivating mutations. Concanamycin partly blocks dissociation of both VCC and SCC in all three compartments, with inhibition generally greater for SCC than VCC. The R735Q mutant of Vph1p results in loss of both ATPase and proton transport, whereas the R735K mutant lacks proton transport but has 10% of wild type ATPase activity. For VCC in the vacuole, dissociation is completely blocked for the R735Q but not the R735K mutant. Significant dissociation of VCC is observed for both mutants in the PVC and PGC, indicating that V-ATPase activity is not absolutely required for dissociation. Similar results were obtained for SCC, although dissociation of SCC is again generally more sensitive to activity than VCC. These results suggest that the cellular environment is important both in controlling in vivo dissociation of the V-ATPase and the dependence of this process on catalytic activity. Moreover, catalytic activity is not absolutely required for V-ATPase dissociation.     

Redox control of G(1)/S cell cycle regulators during nitric oxide-mediated cell cycle arrest

Redox regulation of cell cycle progression during nitric oxide (NO) mediated cytostasis is not well-understood. In this study, we investigated the role of the intracellular antioxidant glutathione (GSH) in regulating specific signaling events that are associated with NO-mediated cell cycle arrest. Manipulation of intracellular GSH content through pharmacological inhibition of glutamate-cysteine ligase (GCL) indicated that GSH depletion potentiated nitrosative stress, DNA damage, phosphorylation of the tumor suppressor p53 (Ser-18) and upregulation of p21(cip1/waf1) upon NO stimulation. However, we found that neither overexpression of a dominant negative p53 nor pharmacological inhibition of p53 with cyclic pifithrin-alpha (cPFT-alpha) was sufficient to reverse NO-mediated cell cycle arrest or hypophosphorylation of retinoblastoma protein (Rb). We found that the decrease in cyclin D1 levels induced by NO was GSH-sensitive implying that the redox regulation of NO-mediated cytostasis was a multifaceted process and that both p53/p21(cip1/waf1) and p53 independent cyclin D1 pathways were involved. Together, our results demonstrate that GSH serves as an important component of cellular protective mechanisms against NO-derived nitrosative stress to regulate DNA damage checkpoint.     

Prostaglandin E(2) protects human lung fibroblasts from cigarette smoke extract-induced apoptosis via EP(2) receptor activation

Prostaglandin E(2) (PGE(2)) has been shown to have a strong cytoprotective effect, inhibiting apoptosis. In the present study, we evaluated whether PGE(2) has a protective effect on cigarette smoke extract (CSE)-induced apoptosis in human lung fibroblasts. Apoptosis was assessed by various methods, including DNA content analysis. CSE (15%-20%) led to apoptosis and induced imbalance in favor of pro- over anti-apoptotic protein expression and activated caspases. PGE(2) blocked CSE-induced apoptosis and modulated the balance of pro- and anti-apoptotic proteins and decreased the activation of caspases. This anti-apoptotic effect was mediated via EP(2) receptor activation as the EP(2) agonist butaprost mimicked PGE(2) activity and siRNA for the EP(2) receptor blocked it. An adenylyl cyclase inhibitor was found to abolish the PGE(2)-mediated cytoprotective effect. Correspondingly, c-AMP analogs blocked CSE-induced apoptosis. Consistently, the protein kinase A (PKA) inhibitor KT-5720 abolished PGE(2)-mediated protection. PGE(2) and butaprost phosphorylated Bad and KT-5720 blocked phosphorylation. These results suggest that PGE(2) inhibits CSE-induced apoptosis via EP(2) receptor activation and activation of PKA, which leads to an alteration in the balance between pro- and anti-apoptotic factors. Through such a mechanism, PGE(2) may alter survival of cells in the smoke-exposed lungs, thus affecting the pathogenesis of cigarette smoke-induced disease.     

Osterix is a key target for mechanical signals in human thoracic ligament flavum cells

Mechanical stress is considered to be an important factor in the progression of thoracic ossification of the ligament flavum (TOLF). To elucidate the mechanism underlying mechanical stress-induced TOLF, we investigated the effect of stretching on cultured flavum ligament cells derived from TOLF and non-TOLF patients. We found that the mRNA expression of alkaline phosphatase (ALP), osteocalcin, Runx2, and osterix, but not that of Dlx5 and Msx2, was significantly increased by stretching in TOLF cells. In addition, the effect seems to be finely tuned by stretching-triggered activation of distinct mitogen-activated protein kinase cascades. Specifically, a p38 specific inhibitor, SB203580, significantly inhibited stretching-induced osterix expression as well as ALP activity, whereas a specific inhibitor of ERK1/2, U0126, prevented stretching-induced Runx2 expression. We showed that overexpression of osterix resulted in a significant increase of ALP activity in TOLF cells, and osterix-specific RNAi completely abrogated the stretching-induced ALP activity, indicating that osterix plays a key role in stretching-stimulated osteogenic effect in TOLF cells. These results suggest that mechanical stress plays important roles in the progression of TOLF through induction of osteogenic differentiation of TOLF cells, and our findings support that osterix functions as a molecular link between mechanostressing and osteogenic differentiation.     

Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.     

Potential involvement of the cyclooxygenase-2 pathway in hepatocellular carcinoma-associated angiogenesis

Angiogenesis plays a crucial role in tumor development and growth. The present study was carried out to investigate the potential involvement of the cyclooxygenase-2 (Cox-2) pathway in the regulation of angiogenesis in hepatocellular carcinoma (HCC). We inhibited Cox-2 expression in HCC cell line HuH-7 by selective Cox-2 inhibitor (SC-58635) or Cox-2 siRNA. Conditioned media (CMs) from HuH-7 cells were used in angiogenic assays in vitro and in vivo. Compared with CMs from untreated and negative siRNA treated HuH-7 cells, CMs from SC-58635 and Cox-2 siRNA treated HuH-7 dramatically suppressed the proliferation, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) in vitro and neovascularization in vivo. These inhibitory effects could be partially reversed by the addition of exogenous PGE2 to CMs. Furthermore, Cox-2 inhibition by SC-58635 resulted in PGE2 reduction accompanied by the down-regulation of four PGE2 receptor (EP receptor) subtypes. Treatment with SC-58635 led to the down-expression of proangiogenic factors such as VEGF, HGF, FGF2, ANGPT1 and ANGPT2 in HCC. An approximately 78% reduction of VEGF level has been found in the CM from SC-58635 treated HuH-7. Our results suggest an involvement of Cox-2 in the control of HCC-associated angiogenesis. PGE2 as a vital angiogenic factor may act directly on endothelial cells to promote HuH-7-stimulated angiogenic process. Moreover, Cox-2/PGE2/EP/VEGF pathway possibly also contributes to tumor angiogenesis in HCC. This study provides the rationale for clinical studies of Cox-2 inhibitors on the treatment or chemoprevention of HCC.     

Epigallocatechin-3-gallate对原代大鼠脂肪细胞脂肪分解及HSL、TGH转录表达的影响

体内能量代谢是维持机体正常生理活动的基础,而脂肪细胞的脂解是能量代谢的核心反应之一,调控脂肪酸从TG库释放,后由血清白蛋白转运至体内各个组织以满足能量需要。如果脂解作用出现障碍,就会影响机体能量的平衡进而引发肥胖和胰岛素抵抗等疾病。表没食子儿茶素没食子酸酯(Epig     

The myocardial response to adrenomedullin involves increased cAMP generation as well as augmented Akt phosphorylation

In this work we aimed to observe (1) the changes in adrenomedullin (AM) and its receptor system - calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) - in myocardial ischemic injury and (2) the response of injuried myocardia to AM and the phosphorylation of Akt to illustrate the protective mechanism of AM in ischemic myocardia. Male SD rats were subcutaneously injected with isoproterenol (ISO) to induce myocardial ischemia. The mRNA levels of AM, CRLR, RAMP1, RAMP2 and RAMP3 were determined by RT-PCR. Protein levels of Akt, phosphor-Akt, CRLR, RAMP1, RAMP2 and RAMP3 were assayed by Western blot. Results showed that, compared with that of the controls, ISO-treated rats showed lower cardiac function and myocardial injury. The mRNA relative amount of AM, CRLR, RAMP1, RAMP2 and RAMP3 in the myocardia of ISO-treated rats was increased. The elevated mRNA levels of CRLR, RAMP1, RAMP2 and RAMP3 were positively correlated with AM content in injured myocardia. The protein levels of CRLR, RAMP1, RAMP2 and RAMP3 in injured myocardia were increased compared with that of control myocardia. AM-stimulated cAMP generation in myocardia was elevated in the ISO group, and was antagonized by AM(22-52) and CGRP(8-37). Western blot analyses revealed that AM significantly enhanced Akt phosphorylation in injured myocardia, which was blocked by pretreatment with AM(22-52) or CGRP(8-37). Ischemia-injured myocardia hyper-expressed AM and its receptors - CRLR, RAMP1, RAMP2 and RAMP3 - and the response of ischemic myocardia to AM was potentiated, and the level of Akt phosphorylation was also increased, which suggests that changes in cardiac AM/AM receptor might play an important role in the pathogenesis of myocardial ischemic injury.