中华心血管病杂志2005年度继续教育园地思考多选题答案

第1期急性冠状动脉综合征的概念演化与治疗策略更新:1.A、B、C、D;2.A、D;3.A、B。第2期低分子肝素与血栓栓塞性疾病:1.A、B、C;2.B、C;3.D;4·A、C、D。第3期神经内分泌拮抗剂在慢性心力衰竭治疗中的常见问题:1.B;2.D;3.A;4.D。第4期心率与心血管病危险性:1.B;2.A;3.C;4.D。第5期神经内分泌拮抗治疗慢性心力衰竭的几个热点问题:1.A;2.C;3.E。第6期肾上腺髓质素与心血管病研究现状:1.B;2.C;3.D;4.D。第7期对中国2004年、欧洲2003年高血压防治指南和JNC7血压分类的比较及评价:1.B;2.C;3.B。第8期心肾综合征研究进展:1.B;2.A…     

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2012年第二届亚太地区幽门螺杆菌会议

会议主题:科学向临床医学的转化:亚太地区新千年幽门螺杆菌感染特别工作组:早期胃癌的内镜诊断和治疗;H.pylori的微生物学、免疫学和分子遗传学会议热点:H.pylori与胃癌;H.pylori的分子流行病学;H.pylori的最佳治疗方法;H.pylori感染中具有争议的问题:胃食管反流病,消化不良,NSAID肠病以及胃外疾病。时间:2012年1月13～15日     

慢性肾衰血液透析患者血浆儿茶酚胺水平及其心率变异性分析

目的 :观察心透患者血浆儿茶酚胺水平与心率变异性 ,并探讨两者的相关性。方法 :测定慢性肾衰血透患者血浆儿茶酚胺水平及记录 2 4h动态心电图 ,分析其心率变异性。结果 :慢性肾衰血液透析患者血浆多巴胺和去甲肾上腺素水平较对照组明显升高 （分别为 P <0 .0 1和 P≤ 0 .0 5 ） ;心率变异性时域指标 SDNN,SDANN ,SDNNindex及 r MSSD均较对照组明显降低 （P<0 .0 1） ;血浆多巴胺水平与 HRV呈明显的负相关 （SDNN:r=- 0 .45 ,P≤ 0 .0 1;SDANN:r=- 0 .43,P≤ 0 .0 1;SDNN index:r=- 0 .5 1,P<0 .0 1;r MSSD:r=- 0 .49,P<0 .0 1） ,血浆去甲肾上腺素水平亦呈类似的相关性。结论 :尿毒症心脏交感神经病变以血浆儿茶酚胺水平升高和心率变异性降低为特点 ,提示交感神经作用代偿性增强     

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中华心血管病杂志2004年度继续教育园地思考多选题答案

第 1期心血管超声医学的研究进展: 1.D; 2.C; 3.B; 4 A。β-受体阻滞剂在心肌梗死中的应用: 1.D; 2.D; 3.A、B、C。第 2期贯彻循证医学的原则作好动脉粥样硬化的预: 1.D; 2.B、D; 3.C; 4.B、C。高血压病肾脏损害的诊断与防治: 1.A、B、C、D; 2.A、B、D; 3.D。第 3期易损斑块及易损患者的新定义及危险分层: 1.A、B、C、D; 2.A、B、C; 3.D; 4.A、B、C、D。β受体阻滞剂治疗慢性心力衰竭临床试验的启示: 1 C;2 B; 3 D; 4 A。第 4期冠心病整体防治中他汀类药物的重要地位: 1.A、B、C、D; 2.A; 3.C。高血压与心力衰…     

Levels of plasma des-γ-carboxy protein C and prothrombin in patients with liver diseases

AIM: To study the plasma des-γ-carboxy protein C activity, antigen and prothrombin levels in patients with liver diseases and their clinical significance. METHODS: Plasma protein C activity (PC:C) was detected by chromogenic assay and antigen (PC:Ag) and des-γ-carboxy protein C (DCPC) were detected by ELISA. Total prothrombin and unabsorbed prothrombin in plasma were detected by ecarin chromogenic assay. RESULTS: Compared with the control, the levels of PC:C and PC:Ag in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC) were lower (PCC: 104.65&#177;23.0%,62.50&#177;24.89%, 56.75&#177;20.14%, PC:Ag: 5.31&#177;1.63 μg/mL, 2.28&#177;1.15 μg/mL, 2.43&#177;0.79 μg/mL, P&lt;0.05). The levels of PC:Ag in patients with acute viral hepatitis (AVH) also was lower (2.98&#177;0.91 μg/mL, P&lt;0.01), but PC:C was close to the control (93.76&#177;30.49%, P&gt;0.05). The levels of DCPC in patients with HCC were remarkably higher (0.69&#177;0.29 μg/mL,1.18&#177;0.63 μg/mL, 0.45&#177;0.21 μg/mL, P&lt;0.05) and its averagewas up to 50% of total PC:Ag. But those of DCPC in patients with AVH were not significantly different from the control. The levels of total prothrombin were lower in patients with LC, but higher in patients with HCC. The levels of unabsorbed prothrombin were predominantly higher than those of other groups. CONCLUSION: PC:C and PC:Ag in patients with liverdiseases (except PC:C in AVH) were lower. The total prothrombin was lower in patients with LC. The higher level of unabsorbed prothrombin may be used as a scanning marker for HCC. DCPC may be used as a complementary marker in the diagnosis of HCC.     

中华人民共和国国家标准(GB/T714-2005)《文后参考文献著录规范》

<正>l专著类①著录格式主要责任者.题名:其他题名信息[文献类型标志].其他责任者.版本项.出版地:出版者,出版年:引文页码[引用日期].获取和访问途径.②示例余敏.出版集团研究[M]北京:中国书籍出版社,2001:179-193.     

ERK2 but not ERK1 plays a key role in hepatocyte replication: an RNAi-mediated ERK2 knockdown approach in wild-type and ERK1 null hepatocytes

The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 have been implicated in various physiological events, and specific targeting of these MAPKs could affect cell proliferation in many cell types. First, to evaluate the potential specific roles of these two MAPKs, we analyzed the mitogenic response in regenerating liver after partial hepatectomy (PH) and in primary culture of hepatocytes isolated from ERK1-deficient mice. We show that ERK1 knockout and wild-type (wt) cells replicate with the same kinetics after PH in liver, in vivo, and in primary cultures of hepatocytes, in vitro. Indeed, Cyclin D1 and Cdk1 appear to be expressed concomitantly in knockout and wt cells, highlighting that hepatocytes progress in the cell cycle independently of the presence of ERK1. Second, we specifically abolished ERK2 expression by RNA interference in mouse and rat hepatocytes. We investigated whether small interfering RNA (siRNA) targeting ERK2 could specifically inhibit its expression and interfere with the process of replication. In ERK1-deficient hepatocytes, silencing ERK2 expression by RNA interference and ERK2 activation by U0126 clearly demonstrate that DNA replication is regulated by an ERK2-dependent mechanism. Furthermore, in rat wt hepatocytes, whereas ERK2 targeting inhibits late G(1) and S phase progression, ERK1 silencing is devoid of any effect on cell proliferation, indicating that ERK1 cannot rescue ERK2 deficiency. CONCLUSION: Our results emphasize the importance of the MAPK cascade in hepatocyte replication and allow us to conclude that ERK2 is the key form involved in this regulation, in vivo and in vitro.     

Both ERK1 and ERK2 Are Required for Enterovirus 71 (EV71) Efficient Replication

It has been demonstrated that MEK1, one of the two MEK isoforms in Raf-MEK-ERK1/2 pathway, is essential for successful EV71 propagation. However, the distinct function of ERK1 and ERK2 isoforms, the downstream kinases of MEKs, remains unclear in EV71 replication. In this study, specific ERK siRNAs and selective inhibitor U0126 were applied. Silencing specific ERK did not significantly impact on the EV71-caused biphasic activation of the other ERK isoform, suggesting the EV71-induced activations of ERK1 and ERK2 were non-discriminative and independent to one another. Knockdown of either ERK1 or ERK2 markedly impaired progeny EV71 propagation (both by more than 90%), progeny viral RNA amplification (either by about 30% to 40%) and protein synthesis (both by around 70%), indicating both ERK1 and ERK2 were critical and not interchangeable to EV71 propagation. Moreover, suppression of EV71 replication by inhibiting both early and late phases of ERK1/2 activation showed no significant difference from that of only blocking the late phase, supporting the late phase activation was more importantly responsible for EV71 life cycle. Taken together, this study for the first time identified both ERK1 and ERK2 were required for EV71 efficient replication and further verified the important role of MEK1-ERK1/2 in EV71 replication.     

Phosphorylation releases constraints to domain motion in ERK2

Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR ¹³C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with k_ex ≈ 300 s⁻¹ (k_AB ≈ 240 s⁻¹/k_BA ≈ 60 s⁻¹) and p_A/p_B ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N- and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of k_ex ≈ 500 s⁻¹ (k_AB ≈ 15 s⁻¹/k_BA ≈ 485 s⁻¹) and p_A/p_B ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.The MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling and a model for protein kinase activation mechanisms (1). ERK2 can be activated by MAP kinase kinases 1 and 2 (MKK1 and 2) through dual phosphorylation of Thr and Tyr residues located at the activation loop (Thr183 and Tyr185, numbered in rat ERK2) (1, 2). Phosphorylation at both sites is required for kinase activation, resulting in increased phosphoryl transfer rate and enhanced affinity for ATP and substrate (3).Conformational changes accompanying the activation of ERK2 have been documented by X-ray structures of the inactive, unphosphorylated (0P-ERK2) and the active, dual-phosphorylated (2P-ERK2) forms (4, 5). Phosphorylation rearranges the activation loop, leading to new ion-pair interactions between phospho-Thr and phospho-Tyr residues and basic residues in the N- and C-terminal domains of the kinase core structure. This leads to a repositioning of active site residues surrounding the catalytic base, enabling recognition of the Ser/Thr-Pro sequence motif at phosphorylation sites and exposing a recognition site for interactions with docking sequences in substrates and scaffolds (6).Less is known about how changes in internal motions contribute to kinase activation. Previous studies using hydrogen-exchange mass spectrometry (HX-MS) and electron paramagnetic resonance spectroscopy (7–9) led to a model where conformational mobility at the hinge linking the N- and C-terminal domains is increased by phosphorylation, therefore releasing constraints needed for activation. Such a model differs from other types of autoinhibitory mechanisms in protein kinases, which involve interactions with domains outside the kinase core (10, 11). However, how hinge flexibility regulates ERK2 is unknown.NMR relaxation dispersion methods enable protein dynamics to be monitored by measuring exchange between conformational states (12). In particular, Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experiments report on motions on slow (100–2,000 s⁻¹) timescales (13), which are often important for enzymatic function (13–16). In the CPMG experiment, exchange between different conformational states is probed with varying times between “refocusing” pulses. Conformational exchange leads to imperfect refocusing, thus decreasing the intensity of the NMR signal. Increasing the pulse frequency allows less chance for conformational exchange, and therefore increased NMR signal intensity. For a given pulse frequency, analysis of the signal intensity yields the effective relaxation rate for the resonance, R_2,eff. This is typically plotted as a relaxation dispersion curve, which can be fit to a two-state conformational exchange process (e.g., A ⇌ B interconversion). Fitting extracts the populations and the exchange rates between states, thus reflecting the thermodynamics and kinetics of the system (17, 18).Here we performed CPMG relaxation dispersion experiments at multiple field strengths to compare the dynamic properties of [¹³C]methyl-labeled ERK2 in its phosphorylated and unphosphorylated states. The results demonstrate that phosphorylation causes a significant change in exchange dynamics throughout the kinase core, consistent with a global domain motion. Increasing hinge mobility by introducing mutations at the hinge also promotes domain motion within the core but with differing kinetics and populations. Taken together, the results show that large changes in dynamics accompany ERK2 phosphorylation, which are influenced by conformational mobility at the hinge. We propose that the activation of ERK2 involves removing inhibitory constraints to domain motion, which are conferred by the internal architecture of the kinase.     

ERK2 enters the nucleus by a carrier-independent mechanism

In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay. GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.     

Elevated activation of ERK1 and ERK2 accompany enhanced liver injury following alcohol binge in chronically ethanol-fed rats

Background: Binge drinking after chronic ethanol consumption is one of the important factors contributing to the progression of steatosis to steatohepatitis. The molecular mechanisms of this effect remain poorly understood. We have therefore examined in rats the effect of single and repeat ethanol binge superimposed on chronic ethanol intake on liver injury, activation of mitogen‐activated protein kinases (MAPKs), and gene expression. Methods: Rats were chronically treated with ethanol in liquid diet for 4 weeks followed by single ethanol binge (5 gm/kg body weight) or 3 similar repeated doses of ethanol. Serum alcohol and alanine amino transferase (ALT) levels were determined by enzymatic methods. Steatosis was assessed by histology and hepatic triglycerides. Activation of MAPK, 90S ribosomal kinase (RSK), and caspase 3 were evaluated by Western blot. Levels of mRNA for tumor necrosis factor alpha (TNFα), early growth response‐1 (egr‐1), and plasminogen activator inhibitor‐1 (PAI‐1) were measured by real‐time qRT‐PCR. Results: Chronic ethanol treatment resulted in mild steatosis and necrosis, whereas chronic ethanol followed by binge group exhibited marked steatosis and significant increase in necrosis. Chronic binge group also showed significant increase (compared with chronic ethanol alone) in the phosphorylation of extracellular regulated kinase 1 (ERK1), ERK2, and RSK. Phosphorylation of c‐Jun N‐terminal kinase (JNK) and p38 MAPK did not increase by the binge. Ethanol binge, after chronic ethanol intake, caused increase in mRNA for egr‐1 and PAI‐1, but not TNFα. Conclusions: Chronic ethanol exposure increases the susceptibility of rat liver to increased injury by 1 or 3 repeat binge. Among other alterations, the activated levels of ERK1, and more so ERK2, were remarkably amplified by binge suggesting a role of these isotypes in the binge amplification of the injury. In contrast, p38 MAPK and JNK1/2 activities were not amplified. These binge‐induced changes were also reflected in the increases in the RNA levels for egr‐1 and PAI‐1. This study offers chronic followed by repeat binge as a model for the study of progression of liver injury by ethanol and highlights the involvement of ERK1 and ERK2 isotypes in the amplification of liver injury by binge ethanol.     

ERK1 and ERK2, two microtubule-associated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ.

Tyrosine hydroxylase (TH) is phosphorylated at four sites in situ and in vivo, and the protein kinases that phosphorylate three of these sites (Ser8,Ser19,Ser40) have been identified. In intact cells, the phosphorylation of the fourth site (Ser31) is increased in response to phorbol esters or nerve growth factor (NGF). Here, we show that Ser31 is phosphorylated by ERK1 and ERK2, two myelin basic protein and microtubule-associated protein kinases. Extracts of NGF- or bradykinin-treated PC12 rat pheochromocytoma cells were fractionated on Mono Q columns. Protein kinase activity toward Ser31 in TH was present in two peaks corresponding to myelin basic protein kinase activities previously identified as ERK1 and ERK2. Phosphorylation of purified TH in vitro by both kinases was selective for Ser31 up to at least 0.6 mol of phosphate per mol of TH subunit. Treatment of intact PC12 cells with bradykinin or NGF increased both the phosphorylation of TH-Ser31 in situ and the catalytic activity of ERKs (measured subsequently in vitro with myelin basic protein as substrate). Pretreatment of the cells with genistein (a protein-tyrosine kinase inhibitor) decreased the bradykinin- but not the NGF-induced changes in both TH-Ser31 phosphorylation and ERK activity. Genistein also inhibited the increases in Ser31 phosphorylation produced by phorbol dibutyrate, muscarine, and Ba2+. The data indicate that ERK activity is responsible for phosphorylating TH at Ser31 in intact cells and suggest that TH-Ser31 phosphorylation may be regulated by multiple signaling pathways that converge at or prior to the activation of the ERKs.     

Expression of phosphorylated ERK1/2 and homeodomain protein CDX2 in cholangiocarcinoma

Purpose The extracellular signal-regulated kinase (ERK) 1/2 pathway plays important roles in the regulation of cell proliferation, differentiation and cell survival. The caudal-related homeobox protein CDX2 is essential for the development of the intestine, and is related to gastric and gallbladder cancers with the intestinal phenotype. However, the roles of ERK1/2 phosphorylation (pERK1/2) and CDX2 in cholangiocarcinogenesis remain unknown.Methods We investigated the expression of pERK1/2, CDX2 and MUC2 in Thai cholangiocarcinoma (CCA) specimens by means of immunohistochemical staining, and compared the expression of these proteins with clinicopathological factors.Results The pERK1/2 protein was expressed in 29 of 59 (49.2%) CCA cases. Interestingly, in tubular-type CCA, the frequency of pERK1/2 expression was associated with a higher grade of differentiation (P = 0.001). CDX2 expression was observed in 22 of the 59 (37.3%) CCA cases, showed a relationship with MUC2 expression (P = 0.001), and was much higher in papillary-type than tubular-type CCA (P = 0.002).Conclusion These results imply that pERK1/2 may be important for the differentiation of tubular-type CCA, while CDX2 is related to the intestinal phenotype of papillary-type CCA.     

NEK2及ERK2在乳腺癌中的表达及意义

目的通过检测乳腺原位癌及浸润性癌组织中NIMA相关蛋白激酶NEK2和细胞外信号调节激酶ERK2的表达,探讨其在乳腺癌变过程中发挥的作用。方法采用免疫组织化学ElivisionTMplus两步法,研究86例乳腺浸润性癌、10例乳腺原位癌、20例乳腺组织的表达情况。结果 NEK2在3组中的表达情况分别为87.2%、50.0%、10.0%。ERK2在3组中的表达情况分别为96.7%、80.0%、10.0%。NEK2与ERK2的表达呈正相关。NEK2蛋白和ERK2蛋白的表达均与患者发生肿瘤的淋巴结转移、病理学分级及临床分期呈明显正相关,而与患者的年龄,肿物大小,月经情况,组织分型无关。结论 ERK2相关信号传导通路和NEK2共同作用参与了乳腺癌的形成过程。