Upregulated YB-1 protein promotes glioblastoma growth through a YB-1/CCT4/mLST8/mTOR pathway

Y-box–binding protein 1 (YB-1) is a multifunctional RNA binding protein involved in virtually every step of RNA metabolism. However, the functions and mechanisms of YB-1 in one of the most aggressive cancers, glioblastoma, are not well understood. In this study, we found that YB-1 protein was markedly overexpressed in glioblastoma and acted as a critical activator of both mTORC1 and mTORC2 signaling. Mechanistically, YB-1 bound the 5′UTR of CCT4 mRNA to promote the translation of CCT4, a component of the CCT chaperone complex, that in turn activated the mTOR signaling pathway by promoting mLST8 folding. In addition, YB-1 autoregulated its own translation by binding to its 5′UTR, leading to sustained activation of mTOR signaling. In patients with glioblastoma, high protein expression of YB-1 correlated with increased expression of CCT4 and mLST8 and activated mTOR signaling. Importantly, the administration of RNA decoys specifically targeting YB-1 in a mouse xenograft model resulted in slower tumor growth and better survival. Taken together, these findings uncover a disrupted proteostasis pathway involving a YB-1/CCT4/mLST8/mTOR axis in promoting glioblastoma growth, suggesting that YB-1 is a potential therapeutic target for the treatment of glioblastoma.     

Centrally mediated cardiovascular actions of dynorphin A(1-8) on rat hippocampal formation.

The effects of dynorphin A(1-8) (DA1-8) microinjected into various areas of the hippocampal formation (HF) on the mean blood pressure (MBP) and heart rate (HR) were investigated in the alpha-chloralose-anesthetized rat. Intra-HF injection of DA1-8 dose-dependently (0.5-50 nmol) reduced MBP and HR. Depressor and bradycardic responses also occurred following microinjection of the excitatory amino acid l-glutamate (1 M, 0.2-0.4 microliter) into the DA1-8-sensitive sites. By contrast, administration of 4% lidocaine (0.2-0.4 microliter) into the same HF sites failed to affect MBP and HR. Pretreatment of the HF with the kappa opioid receptor antagonist nor-binaltorphimine at a dose of 2 to 4 nmol, which itself had no significant influence on basal MBP and HR, almost totally abolished the depressor and bradycardic responses induced by HF injection of DA1-8. DA1-8 at a dose of 10 nmol produced no significant alterations in the frequency of respiration and blood PaO2 and PaCO2 and artificial ventilation did not change the cardiovascular responses of DA1-8. Atropine given i.v. almost totally eliminated the bradycardia and partially prevented the hypotensive responses to intra-HF DA1-8. The data indicate that exogenous administration of DA1-8 into the HF is capable of producing substantial inhibition of peripheral cardiovascular function. Because lidocaine was without effect, the hypotension and bradycardia most likely resulted from an augmentation of an excitatory process rather than from direct inhibition of hippocampal neurons around the injection sites. The effects appear to involve activation of kappa opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression

BACKGROUND: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored co-receptor. OBJECTIVES/METHODS: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. RESULTS AND CONCLUSIONS: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.     

Notch1/Hes1信号通路与糖尿病心肌缺血再灌注关系研究进展

Notch1信号通路是一种进化上高度保守的信号通路,在哺乳动物的胚胎期和成年期调节细胞的稳态和组织、器官的分化和发育。Hes1是Notch1信号通路下游关键靶基因,其作为碱性螺旋-环-螺旋蛋白转录抑制因子,在许多器官发育过程中起重要作用。糖尿病并发心肌梗死后及时恢复血流可能造成心肌缺血再灌注损伤,是围术期防治的重点。Notch1/Hes1信号通路的异常与糖尿病心肌缺血再灌注损伤的发生、发展密切相关。本文就Notch1/Hes1信号通路与糖尿病心肌缺血再灌注损伤关系的研究进展作一综述。     

Involvement of Rho-kinase pathway for angiotensin II-induced plasminogen activator inhibitor-1 gene expression and cardiovascular remodeling in hypertensive rats

Angiotensin II (Ang II) is a potent stimulator of plasminogen activator inhibitor-1 (PAI-1) expression, which is an important regulator of pathogenesis of atherosclerosis. Rho-kinase, a downstream target protein of small GTP-binding protein Rho, plays a key role for various cellular functions. We evaluated the cardioprotective effects of a specific Rho-kinase inhibitor, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), and an Ang II type 1 receptor antagonist, candesartan, on PAI-1 gene expression and cardiovascular remodeling in Ang II-induced hypertensive rats. Rats given Ang II alone (200 ng.kg(-1).min(-1)) were compared with rats also receiving Ang II plus Y-27632 or Ang II plus candesartan. Ang II-induced PAI-1 mRNA up-regulation in the left ventricle was inhibited by Y-27632 and candesartan. In addition, increased RhoA protein, Rho-kinase, and c-fos gene expression, and myosin light chain phosphorylation were suppressed by Y-27632 and candesartan. In contrast, Y-27632 had no effect on Ang II-stimulated phospho-p42/p44 extracellular signal-regulated kinases (ERK) and phospho-p70S6 kinase activities, which are reported to be involved in Ang II-induced protein synthesis. Moreover, activated Ang II-induced phosphorylation of ERK and p70S6 kinase were blocked by candesartan. Y-27632 or candesartan administration resulted in significant improvements in the wall-to-lumen ratio, perivascular fibrosis, and myocardial fibrosis. These results suggested that differential activation of Rho-kinase and ERK pathways may play a critical role in Ang II-induce PAI-1 gene expression, and up-regulation of Rho-kinase plays a key role in the pathogenesis of Ang II-induced hypertensive rats. Thus, inhibition of the Rho-kinase pathway may be at least a useful therapeutic strategy for treating cardiovascular remodeling.     

变异链球菌表面蛋白SpaP P区真核表达质粒的构建及表达

背景:变异链球菌是龋病的主要致龋菌,针对介导变异链球菌非蔗糖依赖性黏附的重要毒力因子SpaP的基因疫苗在理论上能对抗变异链球菌黏附于牙面和进一步破坏牙体硬组织.目的:构建变异链球菌表面蛋白P区(SpaP/P)真核表达质粒pVAX1-spapiP,并观察其在哺乳动物细胞COS.7中的表达.方法:通过基因重组技术,构建真核表达质粒pVAX1-spap/P,并经酶切分析、测序分析鉴定正确后,采用脂质体转染法,将其转染至COS-7细胞中,然后经免疫组织化学SABC法检测其在细胞中的表达.结果与结论:真核表达质粒pVAX1-spap/P经EcoR Ⅰ和Xba Ⅰ双酶切分析,证实携带1.2 kb的目的基因spapiP片段,经测序分析,目的基因正向插入到预先设计的载体位点处.pVAX1-spap/P转染的细胞胞质呈褐色,pVAX1空载体质粒转染的细胞胞质中无着色.证实实验成功构建真核表达质粒pVAX1-spap/P,所携带的基因序列正确,能够在真核细胞COS-7中正确表达目的蛋白.     

Mithramycin A induces apoptosis by regulating the mTOR/Mcl-1/tBid pathway in androgen-independent prostate cancer cells

Mithramycin A (Mith) is an aureolic acid-type polyketide produced by various soil bacteria of the genus Streptomyces. Mith inhibits myeloid cell leukemia-1 (Mcl-1) to induce apoptosis in prostate cancer, but the molecular mechanism underlying this process has not been fully elucidated. The aim of this study was therefore to investigate the detailed molecular mechanism related to Mith-induced apoptosis in prostate cancer cells. Mith decreased the phosphorylation of mammalian target of rapamycin (mTOR) in both cell lines overexpressing phospho-mTOR compared to RWPE-1 human normal prostate epithelial cells. Mith significantly induced truncated Bid (tBid) and siRNA-mediated knock-down of Mcl-1 increased tBid protein levels. Moreover, Mith also inhibited the phosphorylation of mTOR on serine 2448 and Mcl-1, and increased tBid protein in prostate tumors in athymic nude mice bearing DU145 cells as xenografts. Thus, Mith acts as an effective tumor growth inhibitor in prostate cancer cells through the mTOR/Mcl-1/tBid signaling pathway.     

SIRT1与INS/IGF-1通路的关系及对NIT-1细胞胰岛素分泌的影响

目的探讨哺乳动物沉默信息调节因子沉默信息调节因子1（SIRT1）与胰岛素/胰岛素样生长因子（INS/IGF-1）通路的关系,观察SIRT1的表达是否可通过INS/IGF-1通路并影响胰岛素瘤细胞（NIT-1细胞）的胰岛素分泌,为2型糖尿病发病机制提供新的实验依据。方法利用SIRT1激活剂白藜芦醇（RE）和抑制剂尼克酰胺（NIC）分别对NIT-1细胞进行刺激,应用RT-PCR免疫细胞化学方法检测INS/IGF-1通路主要因子PI3K、AKT、FOXO1的mRNA及蛋白表达水平;放射免疫、免疫细胞化学、RT-PCR检测NIT-1细胞胰岛素分泌及表达量的变化,细胞计数法检测细胞倍增时间的变化。结果SIRT1激活剂和抑制剂分别使SIRT1在mRNA水平上出现高、低表达的同时影响了NIT-1细胞的胰岛素分泌量,并影响了INS/IGF-1通路主要因子PI3K、AKT的表达,但对FOXO1的影响,SIRT1的高表达并没有使其产生变化,提示SIRT1影响NIT-1胰岛素分泌通路的复杂性。结论SIRT1可以通过调控INS/IGF-1通路,进而影响NIT-1细胞胰岛素分泌。     

Discovery of common human genetic variants of GTP cyclohydrolase 1 (GCH1) governing nitric oxide, autonomic activity, and cardiovascular risk

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants' contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3'-untranslated region (3'-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3'-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension.     

E/NS1 modifications of dengue 2 virus after serial passages in mammalian and/or mosquito cells

OBJECTIVE: Dengue viruses are routinely maintained in nature by transmission cycles involving the passage of virus between humans and AEDES mosquitoes. The number of dengue virus lineages has been increasing over time. The aim of this study was to identify the genetic diversity of dengue 2 virus serially transferred in mammalian and/or mosquito cells. METHODS: The E/NS1 gene of dengue 2 virus variants derived from serial passages in Vero or C6/36 cells, or alternately in both cell systems, was amplified and sequenced in order to observe gene modification after serial passages. RESULTS: Three nucleotides (two in E and one in NS1) or two amino acids (one each in E and NS1) changed in the virus that was continuously cultured in Vero cells for 20 passages, whereas four nucleotides (two each in E and NS1) or three amino acids (one in E and two in NS1) changed in the virus cultured for 30 passages. The genome of dengue 2 virus remained stable even when the virus was serially transferred in C6/36 cells for 30 generations. However, there was one amino acid substitution (E46 I-->V) resulting from a single nucleotide change in the E region of dengue 2 virus alternately transferred in C6/36 and Vero cells for either 20 or 30 passages. In addition, dengue 2 virus obtained from serially cultured Vero cells usually replicated better when it reinfected Vero cells, reflecting its high adaptation fitness to the host cell. CONCLUSIONS: It is concluded that genetic changes of dengue 2 virus are constrained in Vero (mammalian) cells, resulting in a variety of genome-related quasispecies populations. Some populations of the virus are subsequently selected by and genetically (at least in the E/NS1 portion of the viral genome) maintained in C6/36 (mosquito) cells during replicative competition.     

利多卡因对肺癌A549细胞增殖、侵袭及AMPK/mTOR/4EBP1信号通路的影响

目的探讨利多卡因对肺癌A549细胞增殖、侵袭及单磷酸腺苷活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)/真核细胞翻译起始因子4E结合蛋白1(4EBP1)信号通路的影响。方法设肺癌A549细胞组,5-氟尿嘧啶组(50μmol/L),利多卡因低剂量组(100μmol/L)、高剂量组(200μmol/L),以上各组每组设6个平行孔,培养72 h。试验结束后,采用细胞计数试剂盒8(CCK-8)法测定细胞增殖水平,transwell小室测定细胞侵袭水平,实时荧光定量PCR及蛋白免疫印迹法测定细胞AMPK、mTOR、4EBP1 mRNA和蛋白,以及磷酸化mTOR(p-mTOR)、磷酸化4EBP1(p-4EBP1)蛋白表达水平。结果与肺癌A549细胞组比较,5-氟尿嘧啶组、利多卡因低剂量组、利多卡因高剂量组吸光度值、存活率及mTOR、4EBP1 mRNA和蛋白、p-mTOR蛋白、p-4EBP1蛋白表达水平降低,穿膜数减少(P<0.05),AMPK mRNA和蛋白表达水平升高(P<0.05);与5-氟尿嘧啶组比较,利多卡因低、高剂量组吸光度值、存活率及mTOR、4EBP1 mRNA和蛋白、p-mTOR蛋白、p-4EBP1蛋白表达水平升高,穿膜数增加(P<0.05),AMPK mRNA和蛋白表达水平降低(P<0.05);与利多卡因低剂量组比较,利多卡因高剂量组吸光度值、存活率及mTOR、4EBP1 mRNA和蛋白、p-mTOR蛋白、p-4EBP1蛋白表达水平降低,穿膜数减少(P<0.05),AMPK mRNA和蛋白表达水平升高(P<0.05)。结论利多卡因对肺癌A549细胞增殖、侵袭具有明显抑制作用,其机制可能与利多卡因通过激活AMPK表达进而抑制mTOR/4EBP1信号通路的激活有关。     

亚洲人群白细胞介素8-251基因多态性与胃癌易感性的Meta分析

背景:炎性因素在胃癌发生过程中起重要作用,白细胞介素8在许多肿瘤组织均有明显表达.目的:定量分析白细胞介素8-251基因位点多态性与亚洲人群胃癌易感性的关系.方法:检索Medline,Embase和CNKI数据库,共纳入9篇以亚种人群为研究对象的病例对照研究.采用Stata 10.0统计软件对其结果进行定量分析,同时依据研究对象来源进行分层分析,综合评价白细胞介素8-251基因位点多态性与胃癌发生的关系.结果与结论:白细胞介素8-251 TT基因位点及A基因位点在(AA+AT)在非中国亚洲人群的合并OR值及95%CI分别为[0.83,0.71～0.96]和[1.13,1.00～1.28],具有显著性差异.白细胞介素8-251 AA基因位点及T基因位点(AT+TT)在亚洲人群的合并OR值及95%CI分别为[1.15,0.91～1.45]和[0.99,0.90～1.07],均无显著性差异.白细胞介素8-251 TT基因位点和A基因位点在(AA+AT)在中国人群的合并OR值及95%CI分别为[1 02,0.85～1.22]和[1.00,0.87～1.15],均无显著性差异.因此,白细胞介素8-251基因多态性与非中国亚洲人群胃癌易感性有关.携带白细胞介素8-251 A基因位点的非中国亚洲人群可能是胃癌高危人群,而携带TT等位基因的非中国亚洲人群可能是胃癌的低危人群.     

A SALL4/MLL/HOXA9 pathway in murine and human myeloid leukemogenesis

The embryonic self-renewal factor SALL4 has been implicated in the development of human acute myeloid leukemia (AML). Transgenic mice expressing the human SALL4B allele develop AML, which indicates that this molecule contributes to leukemia development and maintenance. However, the underlying mechanism of SALL4-dependent AML progression is unknown. Using SALL4B transgenic mice, we observed that HoxA9 was significantly upregulated in SALL4B leukemic cells compared with wild-type controls. Downregulation of HoxA9 in SALL4B leukemic cells led to decreased replating capacity in vitro and delayed AML development in recipient mice. In primary human AML cells, downregulation of SALL4 led to decreased HOXA9 expression and enhanced apoptosis. We found that SALL4 bound a specific region of the HOXA9 promoter in leukemic cells. SALL4 overexpression led to enhanced binding of histone activation markers at the HOXA9 promoter region, as well as increased HOXA9 expression in these cells. Furthermore, we observed that SALL4 interacted with mixed-lineage leukemia (MLL) and co-occupied the HOXA9 promoter region with MLL in AML leukemic cells, which suggests that a SALL4/MLL pathway may control HOXA9 expression. In summary, our findings revealed a molecular mechanism for SALL4 function in leukemogenesis and suggest that targeting of the SALL4/MLL/HOXA9 pathway would be an innovative approach in treating AML.     

Direct blockade of inflammatory hypernociception by peripheral A1 adenosine receptors: Involvement of the NO/cGMP/PKG/KATP signaling pathway

Through activation of the A1 adenosine receptors (A1Rs) at both the central and peripheral level, adenosine produces antinociception in a wide range of tests. However, the mechanisms involved in the peripheral effect are still not fully understood. Therefore, the mechanisms by which peripheral activation of A1Rs reduces inflammatory hypernociception (a decrease in the nociceptive threshold) were addressed in the present study. Immunofluorescence of rat dorsal root ganglion revealed significant expression of A1Rs in primary sensory neurons associated with nociceptive pathways. Functionally, peripheral activation of A1Rs reduced inflammatory hypernociception because intraplantar (i.pl.) administration of an A1R antagonist (DPCPX) enhanced carrageenan-induced hypernociception. On the other hand, local (paw) administration of CPA (a selective A1R agonist) reversed mechanical hypernociception induced by carrageenan or by the directly acting hypernociceptive mediator prostaglandin E₂ (PGE₂). Down-regulation of A1Rs expression in primary nociceptive neurons by intrathecal treatment with antisense oligodeoxinucleotides significantly reduced peripheral antinociceptive action of CPA. Direct blockade of PGE₂ inflammatory hypernociception by the activation of A1Rs depends on the nitric oxide/cGMP/Protein Kinase G/KATP signaling pathway because the peripheral antinociceptive effect of CPA was prevented by pretreatment with inhibitors of neuronal nitric oxide synthase (N-propyl-l-arginine), guanylyl cyclase (ODQ), and Protein Kinase G (KT5823) as well as with a KATP blocker (glibenclamide). However, this effect of CPA was not reduced by naloxone, excluding the participation of endogenous opioids. These results suggest that the peripheral activation of A1R plays a role in the regulation of inflammatory hypernociception by a mechanism that involves the NO/cGMP/PKG/KATP intracellular signaling pathway.