PHLPP1对结直肠癌细胞生长抑制及细胞周期阻滞的作用

目的探讨PHLPP1对结直肠癌细胞生长和细胞周期的影响。方法以荧光定量PCR和Western印迹方法检测结直肠癌细胞HT29、Lovo、SW480和人正常结肠上皮细胞FHC中PHLPP1表达水平。在结直肠癌细胞中转染pcDNA3.1-PHLPP1,筛选稳定转染的细胞株用荧光定量PCR和Western印迹方法测定细胞中PHLPP1水平。MTT测定结直肠癌细胞增殖情况,PI单染法测定结直肠癌细胞周期变化,Annexin V-FITC/PI双染法检测结直肠癌细胞凋亡情况,Western印迹检测细胞周期蛋白(Cyclin)D1、细胞周期依赖性蛋白激酶(Cdk)4和凋亡蛋白活化型Caspase-3、活化型Caspase-9(Cleaved Caspase-9)表达水平。结果 PHLPP1表达水平由高到低依次为:FHCSW480HT29Lovo,选用Lovo细胞做后续实验。pcDNA3.1-PHLPP1转染可以上调结直肠癌细胞中PHLPP1表达水平。高表达PHLPP1的结直肠癌细胞的增殖能力降低,细胞G0/G1期比例升高,细胞中CyclinD1、Cdk4蛋白水平降低,细胞凋亡率升高,细胞中活化型Caspase-3、活化型Caspase-9蛋白水平也升高。结论 PHLPP1在结直肠癌细胞中表达下调,上调其表达可以将结直肠癌细胞周期阻滞在G0/G1期,诱导结直肠癌细胞凋亡,降低结直肠癌细胞增殖能力。     

Effects of IGF-1 and oxLDL on expression of phosphatase PHLPP1 in vascular smooth muscular cells

Objective To investigate the effects of insulin-like growth factor-1 （IGF-1） and oxidized low density lipoprotein （oxLDL） on expression ofphosphatase PHLPP 1 in vascular smooth muscle cells （VSMCs）. Methods Rabbit aortic VSMCs were cultured. VSMCs proliferation ability was determined by measuring cell number and mitochondrial dehydrogenase （MD） activity with MTT assay. Western blot was used to detect the protein expression ofphosphatase PHLPP1. Results IGF-1 （100ug/L） increased cell number and MD activity to 3.02 and 3.59 times of that in control group, oxLDL（501xg/ml） elevated the above two parameters to 2.03 and 2.91 times respectively. Western blot showed that IGF-1 and oxLDL inhibited the expression of PHLPPI to 39.27% and 40.26% of the control group （P〈0.01 ）. Conclusion IGF- 1 and oxLDL may enhance the proliferation of VSMCs by decreasing the expression ofphosphatase PHLPP 1.     

老年血液透析患者动静脉内瘘功能不良危险因素

目的 分析老年血液透析患者发生动静脉内瘘(AVF)功能不良的危险因素。方法 选取104例老年慢性肾衰竭患者,均在医院接收血液透析治疗,于透析治疗6个月时,行超声检查评估患者AVF功能,将发生AVF功能不良患者纳入发生组,未发生AVF功能不良患者纳入未发生组。询问并记录两组一般资料,分析老年血液透析患者发生AVF功能不良的危险因素。结果 稳定透析6个月时,发生AVF功能不良占26.92%,未发生AVF功能不良占73.08%;发生组AVF狭窄占比多于未发生组,差异有统计学意义(P<0.05);发生组全血糖化血红蛋白(GHB)、总胆固醇(TC)水平高于未发生组,血清D-二聚体(D-D)、血钙(Ca)、血磷(P)水平高于未发生组,差异有统计学意义(P<0.001)。经Logistic回归分析显示,AVF狭窄、全血GBH、TC、血清D-D、Ca、P过表达是老年血液透析患者发生AVF功能不良的危险因素(P<0.05)。结论 AVF狭窄、全血GBH、TC、血清D-D、Ca、P过表达均能够增加老年血液透析患者发生AVF功能不良的发生风险。     

冠状动脉介入诊疗中血管迷走神经反射21例

目的探讨血管迷走神经反射的发生特点及预防和治疗措施。方法总结941例经股动脉逆行冠状动脉造影术后患者的临床资料,分析其中21例血管迷走神经反射的发生特点。结果共发生血管迷走神经反射21例,其中冠状动脉造影术3例,经皮冠状动脉介入治疗18例。拔管压迫止血时发生16例,拔管后30min内发生3例,2h内发生2例。结论经股动脉穿刺留置鞘管在拔除动脉鞘后,患者可能会发生拔管后迷走神经反射并发症,发生迅速且后果严重,确诊后应立即行相应的处理。     

5000例12导联同步动态心电图的分析

目的研究12导联同步动态心电图（DCG）检测缺血性ST段和心律失常发生率。方法回顾性分析5000饲资料，分析冠心痛心肌缺血、无症状性心肌缺血（SMI）和心律失常的发生率。结果1506例冠心病组中，912例发生缺血性ST段下降，234例发生一过性损伤型ST段抬高。716例SMI组，发生缺血性ST段下降的655例，发生一过性损伤型ST段抬高的61例。1466例以心悸为主诉组，发生缺血性ST段下降859例、发生一过性损伤型ST段抬高26例、发生各种心律失常的共1091例。1312例健康体检组，发生缺血性舛段下降556倒、发生损伤型ST段抬高15倒、发生心律失常973例。结论DCG可检测一过性心律失常和心肌缺血的发生，可对缺血性ST段改变进行定位诊断。     

成年哺乳动物海马齿状回神经发生的研究进展

神经干细胞（NSCs）在一定条件下可增殖分化成神经元和神经胶质细胞，参与神经功能的修复过程，称之为神经发生。传统观念认为，神经发生主要存在于胚胎期。近年研究发现，成年哺乳动物的中枢神经系统也存在神经发生，生理状态下神经发生较少，而病理状态下神经发生较多。这为临床上神经损伤的修复提供了理论依据。现将成年哺乳动物海马齿状回神经发生的研究进展综述如下。     

首次心房颤动后冠状动脉缺血事件的发生率及其相关因素

目的探讨发生首次心房颤动(房颤AF)后冠状动脉缺血事件的发生率及其相关因素。方法回顾分析543例患者首次发生房颤后的完整资料并随访2006年。观察冠状动脉缺血事件发生情况,及其伴随疾病并与无发生冠状动脉缺血事件患者进行比较。结果首次发生AF后随访4.0±1.2年后,有80/543例(14.7%),发生冠状动脉缺血事件,相关疾病有高血压、糖尿病、阻塞性肺病、高血脂等。结论以往无冠心病的中老年患者首次房颤后发生冠状动脉缺血事件较高,房颤可作为新发生冠状动脉缺血性心脏病一种危险因素。     

冠心病患者肥胖时程的影响因素

肥胖，特别是腹型肥胖是冠心病的独立危险因素。有关肥胖与冠心病发生的时间关系即肥胖发生后多长时间发生冠心病鲜有报道。本研究对冠心病患者进行了腹围的测量和调查，记录其发生肥胖的最早时间，对肥胖与冠心病发生的时程关系进行了研究。     

神经发生、血管内皮生长因子与血管性认知损害

神经发生是神经前体细胞自我增殖和分化产生新神经元的动态过程。研究证实,海马神经发生可改善认知功能,并且血管内皮生长因子(vascular endothelial growth factor, VEGF)在神经发生中发挥着重要的调控作用。文章就 VEGF 促进神经发生的机制以及神经发生改善血管性认知损害的作用进行了综述。     

老年慢性阻塞性肺疾病急性加重期通气治疗患者谵妄发生情况及其影响因素

目的 分析老年慢性阻塞性肺疾病急性加重期(AECOPD)通气治疗患者谵妄发生情况及其影响因素。方法 选择182例老年AECOPD患者,统计患者住院治疗期间谵妄发生情况并分组(发生组与未发生组),调查患者基线资料,分析老年AECOPD通气治疗患者谵妄发生的相关因素。结果 住院期间,182例患者中66例(36.26%)发生谵妄;发生组机械通气时间较未发生组长,血清脑源性神经营养因子(BDNF)、白蛋白(ALB)水平较未发生组低,使用咪达唑仑镇静占比较未发生组高,差异有统计学意义(P<0.05);经Logistic回归分析结果显示,机械通气时间较短是老年AECOPD通气治疗患者谵妄发生的保护因素(OR<1,P<0.05),血清BDNF、ALB低表达是老年AECOPD通气治疗患者谵妄发生的危险因素(OR>1,P<0.05)。结论 老年AECOPD通气治疗患者谵妄发生率较高,机械通气时间较长、血清BDNF、ALB低表达均为患者发生谵妄的影响因素。     

PHLiPPing the switch on Akt and protein kinase C signaling.

The Ser/Thr-specific phosphatase PHLPP [pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase] provides 'the brakes' for Akt and protein kinase C (PKC) signaling. The two isoforms of this recently discovered family, PHLPP1 and PHLPP2, control the amplitude and duration of signaling of Akt and PKC by catalyzing the dephosphorylation of the hydrophobic phosphorylation motif, a C-terminal phosphorylation switch that controls these kinases. Aberrant regulation of either kinase accompanies many diseases, notably diabetes and cancer. By specifically dephosphorylating the hydrophobic motif, PHLPP controls the degree of agonist-evoked signaling by Akt and the cellular levels of PKC. This review focuses on the function of PHLPP1 and PHLPP2 in modulating signaling by Akt and PKC.     

USP1 regulates AKT phosphorylation by modulating the stability of PHLPP1 in lung cancer cells

Background

Hyperactivation of phosphatidylinositol 3-kinase/Akt signaling is commonly associated with human tumors including lung cancers. PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), which terminates Akt signaling by directly dephosphorylating and inactivating Akt, has been identified as a tumor suppressor. The protein level of PHLPP1 is regulated by E3 ligase beta-TRCP, however, the deubiquitinase for PHLPP1 is still not known.

Methods

The mRNA levels of USP1 and PHLPP1 in lung cancer cells and tissues were determined by real-time PCR. The half-life of PHLPP1 was detected by CHX assay. The interaction between USP1 and PHLPP1 was examined by immunoprecipitation and GST pull-down assay.

Results

Both USP1 and PHLPP1 are low expressed in lung cancer cells and tissues and silencing of USP1 by RNA interference significantly decreased the half-life of PHLPP1, which in turn amplified Akt1 phosphorylation. Our data identified a novel USP1-PHLPP1-Akt signaling axis, and decreased USP1 level in lung cancer cells may play an important role in lung cancer progress.     

Protein phosphatase PHLPP1 controls the light-induced resetting of the circadian clock

The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) differentially attenuates Akt, PKC, and ERK1/2 signaling, thereby controlling the duration and amplitude of responses evoked by these kinases. PHLPP1 is expressed in the mammalian central clock, the suprachiasmatic nucleus, where it oscillates in a circadian fashion. To explore the role of PHLPP1 in vivo, we have generated mice with a targeted deletion of the PHLPP1 gene. Here we show that PHLPP1-null mice, although displaying normal circadian rhythmicity, have a drastically impaired capacity to stabilize the circadian period after light-induced resetting, producing a large phase shift after light resetting. Our findings reveal that PHLPP1 exerts a previously unappreciated role in circadian control, governing the consolidation of circadian periodicity after resetting.     

Isoform-specific defects of insulin stimulation of Akt/protein kinase B (PKB) in skeletal muscle cells from type 2 diabetic patients

Aims/hypothesis The serine/threonine kinase Akt/protein kinase B (PKB) is required for the metabolic actions of insulin. Controversial data have been reported regarding Akt defective activation in the muscle of type 2 diabetic patients. Because three Akt isoforms exist, each having a distinct physiological role, we investigated the contribution of isoform-specific defects to insulin signalling in human muscle. Methods The phosphorylation pattern and kinase activity of each Akt isoform were compared in primary myotubes from healthy control participants and type 2 diabetic patients. Phosphorylation of Ser⁴⁷³ and of Thr³⁰⁸ in each isoform was determined after immunoprecipitation in myotubes treated or not with insulin. Results Muscle cells from diabetic patients displayed defective insulin action and a drastic reduction of insulin-stimulated activity of all Akt isoforms. This was associated with specific defects of their phosphorylation pattern in response to insulin, with impaired Akt2- (and to a lower extent Akt3-) Ser⁴⁷³ phosphorylation, and with altered Akt1-Thr³⁰⁸ phosphorylation. These defects were not due to faulty phosphoinositide-dependent protein kinase 1 (PDK1) production or activation. Rather, we found higher levels of the Akt2-Ser⁴⁷³-specific protein phosphatase PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) in muscle from diabetic patients, which may contribute to the alteration of Akt2-Ser⁴⁷³ phosphorylation. Conclusions/interpretation These results suggest that several mechanisms affecting Akt isoforms, including deregulated production of PHLPP1, could underlie the alterations of skeletal muscle insulin signalling in type 2 diabetes. Taking into account the recently described isoform-specific metabolic functions of Akt, our results provide mechanistic insight that may contribute to the defective regulation of glucose and lipid metabolisms in the muscle of diabetic patients.     

蛋白激酶B调节骨桥蛋白在肝癌HepG2细胞中的表达

目的 转移相关基因骨桥蛋白(OPN)在肝癌中的表达方式、途径尚不清楚，检测OPN在HepG2细胞中转染蛋白激酶B(Akt)前后的表达，旨在探讨磷脂酰肌醇3激酶信号途径中的关键基因Akt与OPN表达的关系。方法 用脂质体介导的基因转染法将含有Akt基因的质粒转染HepG2细胞，并用Western blot鉴定；OPN的表达用Northern blot和Western blot方法检测。结果 Akt基因成功转染HepG2细胞，Western blot能检测到HepG2细胞中外源表达的Akt基因；Northern blot和Western blot检测发现，Akt在核酸和蛋白水平调节OPN的表达；在无血清培养条件下，OPN在HepG2中结构性表达量很少或无表达,转染活性型Akt后OPN表达升高；在有血清培养条件下,HepG2细胞转染缺陷型Akt基因后OPN表达下降。结论 Akt调节转移相关基因OPN在肝癌细胞中的表达，提示可通过使Akt基因失活来阻断OPN产生，从而抑制肝癌转移。     

Ischemia-reperfusion injury and cardioprotection: investigating PTEN,the phosphatase that negatively regulates PI3K,using a congenital model of PTEN haploinsufficiency

Activation of the PI3K/Akt pathway protects the heart from ischemia-reperfusion injury (IRI). The phosphatase PTEN is the main negative regulator of this pathway. We hypothesized that reduced PTEN levels could protect against IRI. Isolated perfused mouse hearts from PTEN^+/− and their littermates PTEN^+/+ (WT), were subjected to 35 min global ischemia and 30 min reperfusion, with and without 2, 4 or 6 cycles ischemic preconditioning (IPC). The end point was infarct size, expressed as a percentage of the myocardium at risk (I/R%). PTEN and Akt levels were determined using Western blot analysis. Unexpectedly, there were no significant differences in infarction between PTEN^+/− and WT (42.1 ± 5.0% Vs. 45.6 ± 3.3%). However, the preconditioning threshold was significantly reduced in the PTEN^+/− Vs. WT, with 4 cycles of IPC being sufficient to reduce I/R%, compared to 6 cycles in the WT (4 cycles IPC: 29.8. ± 3.69% in PTEN^+/− Vs. 45.5. ± 5.08% in WT, P < 0.01). In addition, the ratio between the phospho/total Akt (Ser473 and Thr308) was slightly but significantly increased in the PTEN^+/− indicating an upregulation of PI3K/Akt pathway. Interestingly, the levels of the other phosphatases that may negatively regulate the PI3K/Akt pathway (PP2A, SHIP2 and PHLPP) were not significantly different between littermates and PTEN^+/−. In conclusion, PTEN haploinsufficiency alone does not induce cardioprotection in this model; however, it reduces the threshold of protection induced by IPC. Returned for 1. Revision: 29 November 2007 1. Revision received: 5 May 2008 Returned for 2. Revision: 2 June 2008 2. Revision received: 5 June 2008     

Tumor necrosis factor-{alpha} decreases Akt protein levels in 3T3-L1 adipocytes via the caspase-dependent ubiquitination of Akt

TNF-alpha is a mediator of insulin resistance in sepsis, obesity, and type 2 diabetes and is known to impair insulin signaling in adipocytes. Akt (protein kinase B) is a crucial signaling mediator for insulin. In the present study we examined the posttranslational mechanisms by which short-term (<6-h) exposure of 3T3-L1 adipocytes to TNF-alpha decreases Akt levels. TNF-alpha treatment both increased the ubiquitination of Akt and decreased its protein level. The decrease in protein was associated with the presence of an (immunoreactive) Akt fragment after TNF-alpha treatment, indicative of Akt cleavage. The broad-spectrum caspase inhibitor t-butoxycarbonyl-Asp(O-Me)-fluoromethyl ketone markedly suppressed these effects of TNF-alpha. The caspase-6 inhibitor Z-Val-Glu(OMe)-Ile-Asp(OMe)-CH(2)F potently suppressed Akt ubiquitination, degradation, and fragment formation, whereas the proteasome inhibitor Z-Leu-Leu-Leu-CHO modestly attenuated the decline in Akt levels. Exposure to TNF-alpha also enhanced the association of Akt with an E3 ligase activity. Adipocytes preexposed to TNF-alpha for 5 h and then stimulated with insulin for 30 min exhibited decreased levels of Akt, phosphorylated Akt, as well as phosphorylated Mdm2, which is a known direct substrate of Akt, and glucose uptake. Caspase inhibition attenuated these inhibitory effects of TNF-alpha. Collectively, our results suggest that TNF-alpha induces the caspase-dependent degradation of Akt via the cleavage and ubiquitination of Akt, which results in its degradation through the 26S proteasome. Furthermore, the caspase- and proteasome-mediated degradation of Akt due to TNF-alpha exposure leads to impaired Akt-dependent insulin signaling in adipocytes. These findings expand the mechanism by which TNF-alpha impairs insulin signaling.