Inhibitory effect of sulfonylureas on protein phosphatase activity in rat pancreatic islets

We have measured protein phosphatase (PP) activity in crude homogenates as well as in the total 105,000×g supernatant and precipitate fractions from normal rat pancreatic islets. On the basis of the inhibition produced by either 1 nM or 1 μM okadaic acid, both PP₁ and PP₂A activity were present in crude islet homogenates in equivalent proportions (53% and 47%, respectively); PP₁ was the main activity present in the precipitate, whereas in the supernatant it was PP₂A. Tolbutamide, glybenclamide and glyclazide significantly decreased PP activity in islet homogenates in a dose-dependent manner, with a K _i0.5 value that in the case of glybenclamide correlated with its K _d for binding site, its EC₅₀ on K_ATP channel, and its EC₅₀ on insulin release. These data indicate that PPs play a role in the control of insulin secretion and suggest a further possible target for sulfonylureas within their overall action as insulin secretagogues. Received: 19 September 1996 / Accepted in revised form: 20 December 1996     

p38 MAP-kinase in cultured adult rat ventricular cardiomyocytes: expression and involvement in hypertrophic signalling

Both alpha-adrenoceptor- and beta-adrenoceptor-stimulation lead to hypertrophic growth of the myocardium. But only beta-adrenoceptor-stimulation requires the pre-cultivation of cells with active TGF-beta. In order to define signalling molecules that are specifically involved in beta-adrenoceptor-dependent hypertrophy, changes in expression and hypertrophic responsiveness during pre-cultivation with TGF-beta were investigated. Isolated adult ventricular cardiomyocytes from rats were either cultured in 20% (v/v) foetal calf serum (FCS) to activate autocrine released TGF-beta or used without pre-treatment. Protein synthesis was analysed by (14)C-phenylalanine incorporation. Expression of signalling molecules was determined by immunoblotting. During cultivation of cardiomyocytes with active TGF-beta only the expression of p38 MAP-kinase increased. Subsequent stimulation of beta-adrenoceptors induced protein synthesis in a p38 MAP-kinase-dependent way. However, stimulation of beta-adrenoceptors activated p38 MAP-kinase irrespective of pre-treatment with TGF-beta. In the absence of this cytokine, hyperosmolarity or reconstitution of mechanical activity increased protein synthesis via p38 MAP-kinase activation in freshly isolated cells. In conclusion, activation of p38 MAP-kinase is a newly identified necessary signalling step required for beta-adrenoceptor induced hypertrophic growth. Like activation of adenyl cyclase, activation of p38 MAP-kinase is up-stream of the TGF-beta-induced coupling to the regulation of protein synthesis. Reconstitution of mechanical activity mimics the co-activation required and induced by TGF-beta.     

蛋白酪氨酸磷酸酶1B与细胞信号转导

蛋白酪氨酸磷酸酶-1B(PTP-1B)通过对细胞内不同蛋白底物脱磷酸化参与不同的生理反应,在胰岛素、瘦素等多条细胞信号通路中发挥作用.因其参与多种生理、病理过程,近年来PTP-1B抑制剂的提取和研制成为相关疾病防治的新方向.     

高脂饮食诱导的肥胖大鼠胰腺蛋白酪氨酸磷酸酶1B表达增高

目的观察高脂饮食诱导的肥胖大鼠胰腺中蛋白酪氨酸磷酸酶1B（PTP-1B）的表达。方法选取SD大鼠20只，随机分为正常对照组10只，给予常规饲料；肥胖模型组10只，给予高脂饲料，共喂养12周。测定大鼠空腹胰岛素、血糖、甘油三酯、总胆固醇，附睾脂肪垫重量；观察肝脏形态学改变；进行葡萄糖耐量试验和胰岛素释放试验；用免疫组化和蛋白印迹法检测大鼠胰腺组织中PTP-1B蛋白的表达。用免疫沉淀法检测胰岛素受体（IR）和胰岛素受体底物1（IRS-1）磷酸化程度。结果（1）肥胖组胰岛素敏感指数显著低于对照组（0．36±0．18 vs 0．91±0．28，P〈0．05）；（2）肥胖组大鼠葡萄糖耐量受损，葡萄糖刺激的胰岛素Ⅰ相分泌反应受损；（3）肥胖组大鼠胰腺中PTP-1B蛋白与对照组相比，含量增加86％（P〈0．01）。肥胖组胰腺中胰岛素诱导的IR和IRS-1磷酸化程度都降低。结论高脂饮食诱导的肥胖大鼠胰腺中PTP-1B蛋白表达量升高，从而使胰岛素诱导的IR和IRS-1磷酸化程度降低，可能是肥胖状态下引发胰岛产生胰岛素抵抗的机制之一。     

蛋白酪氨酸磷酸酶抑制剂对VacA空泡活性的影响

目的　幽门螺杆菌 (Hp)的空泡毒素 (VacA)受体属于与细胞信号转导密切相关的受体蛋白酪氨酸磷酸酶家族 (RPTP) ,但VacA导致空泡形成的具体机制尚不清楚。研究干扰信号转导后空泡形成的变化 ,可为临床治疗有毒Hp菌株感染提供新思路。 方法　采用Hp液体培养上清浓缩液作为粗制VacA毒素 ,将蛋白酪氨酸磷酸酶抑制剂 (PTPI)及蛋白酪氨酸激酶抑制剂 (PTKI)木黄酮倍比稀释后 ,与VacA共同作用于胃癌细胞 ,观察VacA空泡毒性的变化。结果　PTPI浓度达到 2 .7μmol/L时 ,即产生对VacA空泡活性显著的抑制作用 (A550 =0 .46± 0 .0 6比 0 .5 9± 0 .0 4,P <0 .0 5 ) ,至 2 1.5 μmol/L时抑制作用可达 10 0 % (A550 =0 .0 9± 0 .0 2 )。PTPI浓度高于 2 1.5 μmol/L时可见部分细胞死亡。将PTPI作用于已产生空泡的细胞 ,采用能引起空泡抑制作用的 4个浓度梯度 2 1.5、10 .8、5 .4及 2 .7μmol/L ,2 4h后PTPI组与空白对照相比 ,A550 无显著差异。 16～ 5 0 0 μmol/L浓度木黄酮均未对空泡活性产生明显的影响。结论　PTPI对VacA空泡活性具有显著的抑制作用 ,间接证实VacA受体的属性为受体RPTP ,VacA毒素的作用可能与干扰细胞内的信号转导过程有关     

Rheb activates protein synthesis and growth in adult rat ventricular cardiomyocytes

The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of protein synthesis, growth and proliferation in mammalian cells, is implicated in the development of cardiac hypertrophy. Ras homolog enriched in brain (Rheb) positively regulates mTORC1. We have studied whether Rheb is sufficient to activate mTOR signaling and promote protein synthesis and cardiac hypertrophy in adult rat ventricular cardiomyocytes (ARVC). Rheb was overexpressed via an adenoviral vector in isolated ARVC. Overexpression of Rheb in ARVC activated mTORC1 signaling, several components of the translational machinery and stimulated protein synthesis. Our direct visualization approach to determine ARVC size revealed that overexpression of Rheb also induced cell growth and indeed did so to similar extent to the hypertrophic agent, phenylephrine (PE). Despite potent activation of mTORC1 signaling, overexpression of Rheb did not induce expression of the cardiac hypertrophic marker mRNAs for brain natriuretic peptide and atrial natriuretic factor, while PE treatment did markedly increase their expression. All the effects of Rheb were blocked by rapamycin, confirming their dependence on mTORC1 signaling. Our findings reveal that Rheb itself can activate both protein synthesis and cell growth in ARVC and demonstrate the key role played by mTORC1 in the growth of cardiomyocytes.     

The proteasome and its role in nuclear protein maintenance

The cellular proteome is in a dynamic state of synthesis and degradation. Degradation of extracellular proteins is mainly mediated non-specifically by the lysosomes or due to released proteases, while the proteolysis of intracellular including nuclear proteins is catalyzed by the ubiquitin-proteasome pathway. Furthermore, the proteasomal system is largely responsible for the removal of unfolded and oxidatively damaged proteins. Taking into account the role of ubiquitin and proteasome system in protein metabolism, studies of its spatial organization within the cell are of great importance. For the understanding of cellular, including nuclear, protein maintenance the distribution of the proteasomes in both the nucleus and the cytosol and their response upon oxidative stress is of great interest. Although, the functional diversity of the cells is ensured by the three dimensional organization of the nucleus, nuclear proteins are also prone to oxidation and have to be removed from the cellular environment by the nuclear proteasome. Interestingly, nuclear proteins are partly degraded within the nucleus, whereas some are exported from the nucleus to the cytosol. Proteasomes are transported unidirectionally from the cytoplasm to the nucleus with a possible countervail during mitosis. This review is focused largely on the specifics of cellular proteasome distribution and on nuclear protein maintenance under physiological and oxidative stress conditions.     

Increased 19 kDa protein phosphorylation and protein kinase C activity in pressure-overload cardiac hypertrophy

Summary The aim of the study was to determine the role of protein kinase C (PKC) in protein phosphorylation in hypertrophied C. myocytes, particularly the phosphorylation of the 19 kDa protein which corresponds to myosin light chains. In myocardial hypertrophy the PKC activity in the cytosolic fraction of tissue homogenate was increased up to 253 % of control hearts, and in membrane fraction up to 140 % of the control value. Phorbol ester (TPA), the specific activator of protein kinase C, stimulated phosphorylation of the 19 kDa protein obtained from isolated myocytes to 181 ± 9 % of control value in normal and to 248 ± 66 % in hypertrophic myocytes. Taken together, these data suggest that protein kinase C might be involved in the increased phosphorylation of cardiac myosin light chain protein in myocardial hypertrophy.     

代谢综合征患者内脏脂肪组织蛋白酪氨酸磷酸酶1B表达增多

目的研究蛋白酪氨酸磷酸酶1B(PTP-1B)在代谢综合征(MS)发病中的作用。方法51例患者分为对照(NC)组、2型糖尿病(T2DM)组和MS组,测定FPG、FIns,Western印迹法测定内脏脂肪组织PTP-1B表达水平。结果与NC组相比,T2DM组和MS组FPG、Ln(HOMA-IR)和PTP-1B表达水平明显升高。与T2DM组相比,MS组腰围和PTP-1B显著升高。校正年龄后PTP-1B与FPG、Ln(HOMA-IR)、腰围(WC)和SBP均呈正相关。结论内脏脂肪组织PTP-1B表达升高与肥胖、糖代谢紊乱等MS危险因素明显相关,可见内脏脂肪组织PTP-1B表达异常与MS发生发展关系密切。     

蛋白酪氨酸磷酸酶抗体对成人隐匿性自身免疫性糖尿病的诊断价值

目的探讨蛋白酪氨酸磷酸酶抗体（IA-2A）对成人隐匿性自身免疫性糖尿病（LADA）的诊断价值。方法采用放射配体法检测2027例初诊2型糖尿病（T2DM）患者的IA．2A和谷氨酸脱羧酶抗体（GAD-Ab）,分析IA-2A在初诊T2DM患者中的分布及其与临床特征的关系。结果初诊T2DM患者中IA-2A阳性检出率低于GAD-Ab阳性检出率（2．2％7）S10．6％,P〈0．01）。联合GAD-Ab和IA-2A检测,可使LADA阳性检出率达11．5％。IAm2A在低体重、低c肽水平患者中阳性检出率高;随着起病年龄增大,IA-2A阳性检出率下降（P〈0．05）。与T2DM组比较,单独IA-2A阳性患者使用胰岛素治疗的比例高（P〈0．05）。高滴度IA-2A阳性LADA患者具有起病年龄小（P〈0．01）、体重轻、高血压比例低、空腹C肽低、合并GAD-Ab阳性和使用胰岛素比例高（P〈0．05）等特点。结论IA-2A检测对LA—DA具有辅助诊断价值,与GAnAb联合检测可提高LADA诊断的敏感性。     

Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes

Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility and was shown previously to activate Akt through an undefined mechanism. Here we report Pak1 as a potential PDK2 that is essential for Akt activity in cardiomyocytes. Both Pak1 and Akt can be activated by multiple hypertrophic stimuli or growth factors in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. Pak1 overexpression induces Akt phosphorylation at both Ser473 and Thr308 in cardiomyocytes. Conversely, silencing or inactivating Pak1 gene diminishes Akt phosphorylation in vitro and in vivo. Purified Pak1 can directly phosphorylate Akt only at Ser473, suggesting that Pak1 may be a relevant PDK2 responsible for AKT Ser473 phosphorylation in cardiomyocytes. In addition, Pak1 protects cardiomyocytes from cell death, which is blocked by Akt inhibition. Our results connect two important regulators of cellular physiological functions and provide a potential mechanism for Pak1 signaling in cardiomyocytes.     

Mitochondrial phosphoglycerate mutase 5 uses alternate catalytic activity as a protein serine/threonine phosphatase to activate ASK1

Phosphoglycerate mutase (PGAM) is an enzyme of intermediary metabolism that converts 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. Here, we discovered PGAM5 that is anchored in the mitochondrial membrane lacks PGAM activity and instead associates with the MAP kinase kinase kinase ASK1 and acts as a specific protein Ser/Thr phosphatase that activates ASK1 by dephosphorylation of inhibitory sites. Mutation of an active site His-105 in PGAM5 abolished phosphatase activity with ASK1 and phospho-Thr peptides as substrates. The Drosophila and Caenorhabditis elegans orthologs of PGAM5 also exhibit specific Ser/Thr phosphatase activity and activate the corresponding Drosophila and C. elegans ASK1 kinases. PGAM5 is unrelated to the other known Ser/Thr phosphatases of the PPP, MPP, and FCP families, and our results suggest that this member of the PGAM family has crossed over from small molecules to protein substrates and been adapted to serve as a specialized activator of ASK1.     

A baculovirus-encoded protein tyrosine phosphatase gene induces enhanced locomotory activity in a lepidopteran host

Enhanced locomotory activity (ELA), such as wandering, is a normal behavior that occurs at the end of the larval stage in lepidopteran (butterflies and moths) insects. Baculovirus infection can also induce ELA in lepidopteran larvae. The belief is that the virus induces this behavior to increase its transmission [Goulson, D. (1997) Oecologia 109, 219-228]. Here we show that a baculovirus-encoded protein tyrosine phosphatase (PTP) gene (ptp) induces ELA that is activated by light. ELA was induced in silkworm Bombyx mori infected with the baculovirus B. mori nucleopolyhedrovirus (BmNPV) beginning at approximately 3.75 days postinfection (p.i.) and continued until 4.75 days p.i. The intensity of the ELA was dramatically reduced immediately before death at 5.25 days p.i. Light activated the intensity of the ELA by approximately 3-fold, and larvae with ELA showed positive phototropism. ELA was not induced in larvae of B. mori infected with a BmNPV ptp knockout mutant (BmPTPD). However, when a silkworm-derived ptp gene (Bmptp-h) was inserted into BmPTPD, ELA was partially recovered. Bmptp-h was identified from silkworms at 2 days after the start of the natural wandering stage. The deduced amino acid sequence of Bmptp-h showed 48.2% identify (80.7% similarity) to the deduced amino acid sequence of BmNPV ptp. On the basis of the high homology and larval stage at which Bmptp-h was isolated, we postulate that the modern baculovirus may have acquired its ptp gene from an ancestral host and that this gene was selectively maintained because it increases virus transmission.     

PTP-1B基因突变的PCR-RFLP与肥胖的相关性

采用多聚酶联反应-限制性片段长度多态性方法，对110例单纯肥胖者、110例肥胖合并2型糖尿病者、120例正常对照者PTPlB基因ISV5＋3666、303位编码子进行酶切研究。结果显示PTPlB基因的P303P突变可能与肥胖发生相关，ISV5＋3666delT突变与肥胖发生不相关。     

Change of nuclear protein phosphatase activity in the liver of old rats

Nuclear protein phosphatase (NPP-ase) activity was measured in non-histone protein (NHP) preparations obtained from liver nuclei of young and old rats with exogenous substrates labelled with ³²P. NPP-ase activity can be measured in a rather broad range of pH between 5.5 and 7.5 and it shows several characteristic differences between young (4–6 months) and old (24–28 months) animals: (a) under the effect of NPP-ase a limited dephosphorylation of nuclear phosphoprotein (NPP) substrate ensues reaching a 25 per cent liberation maximum of incorporated ³²P in the case of NHP preparations from young animals; the same liberation maximum is over 45 per cent in the case of old animals, (b) an age-related change of NPP-ase activity is assumed to occur in old rats in comparison with young ones. The NPP-ase activity measurable for its homologous—NPP—substrate increases in old animals while there is a concomitant decrease of casein phosphatase activity. The possible implication of the phosphorylation of chromosomal non-histone proteins in the cellular aging is discussed.     

2型猪链球菌低分子量酪氨酸磷酸酶的制备及酶学研究

目的 表达纯化2型猪链球菌(Streptococcus suis 2,SS2)低分子量酪氨酸磷酸酶(Low molecular weight protein tyrosine phosphatase,LMW-PTP),测定LMW-PTP磷酸酶活性并鉴定其酶活性位点,为后续的功能鉴定奠定基础。方法 分别构建野生型LMW-PTP基因表达质粒pET28a:LMW-PTP、突变型LMW-PTP基因表达质粒pET28a:LMW-PTPC33A和pET28a:LMW-PTPR39A,重组质粒转化E.coil BL21(DE3),筛选阳性转化子,通过IPTG诱导表达后经SDS-PAGE鉴定表达产物。镍柱亲和层析纯化得到重组蛋白经Western blot鉴定,野生型LMW-PTP免疫新西兰兔制备兔多克隆抗体。以对硝基苯酚二钠六水(PNPP-Na)为底物检测重组蛋白的磷酸酶活性。结果 成功构建野生型LMW-PTP基因表达质粒pET28a:LMW-PTP、突变型LMW-PTP基因表达质粒pET28a:LMW-PTPC33A、pET28a:LMW-PTPR39A,在大肠杆菌中野生型和突变型LMW-PTP均以包涵体的形式存在,相对分子量为23 kDa,包涵体经镍柱纯化及透析复性获得纯度较高的野生型和突变型LMW-PTP。制备得到的抗LMW-PTP的兔多克隆抗体的效价为1∶102 400,且重组蛋白均能与His-Tag单抗和兔多抗血清发生反应。对野生型蛋白进行磷酸酶活性检测显示其具有酶活性,酶活为2.1 nmol/(min·μg),突变型LMW-PTPC33A、LMW-PTPR39A无磷酸酶活性。结论 成功表达了具有磷酸酶活性的LMW-PTP,并鉴定出Cys³³和Arg³⁹是LMW-PTP的活性位点。为后续寻找LMW-PTP在2型猪链球菌致病过程中毒力相关的靶蛋白奠定了基础。     

蛋白酪氨酸磷酸酶2自身抗体微量平板放射结合检测法的建立与临床应用

目的建立蛋白酪氨酸磷酸酶2自身抗体（IA-2A）的微量平板放射结合检测法（RBA）,并初步评价其临床应用价值。方法经纯化的^35S-IA-2抗原与血清在96孔V形底平板中缓慢振荡孵育24h,而后转入已包被蛋白A的Millipore平板中,洗涤后采用多功能液体闪烁发光仪计数。检测162例1型糖尿病（T1DM）患者、210例新诊2型糖尿病（T2DM）患者和224名健康者血清中IA-2A浓度,初步评价其临床应用效果。结果（1）该法检测IA-2A批内CV4．1％～10．0％,批间CV5．7％～12．8％。（2）国际第4次糖尿病自身抗体检测标准化评估回报结果显示,其灵敏度为72％,特异度为98％。与放射配体法（RLA）结果判定一致率为96．5％,检测值呈显著正相关（r=0．962,P〈0．01）。（3）IA-2A在T1DM患者中阳性率为22．8％,明显高于健康者的0．89％（P〈0．01）;在T2DM患者中阳性率为2．4％,与健康者相比差异无统计学意义。（4）该法检测指血IA-2A与RLA法检测静脉血结果判定一致率为100％。结论微量平板RBA法灵敏度、特异度、重复性好,能适用于末梢血IA-2A检测,具有较好的临床应用价值。     

Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII

Frequency-dependent acceleration of relaxation (FDAR) is an important intrinsic mechanism that allows for diastolic filling of the ventricle at higher heart rates, yet its molecular mechanism is still not understood. Previous studies showed that FDAR is dependent on functional sarcoplasmic reticulum (SR) and can be abolished by phosphatase or by Ca/CaM kinase (CaMKII) inhibition. Additionally, CaMKII activity/autophosphorylation has been shown to be frequency-dependent. Thus, we tested the hypothesis that CaMKII phosphorylation of SR Ca(2+)-handling proteins (Phospholamban (PLB), Ca(2+) release channel (RyR)) mediates FDAR. Here we show that FDAR occurs abruptly in fluo-4 loaded isolated rat ventricular myocytes when frequency is raised from 0.1 to 2 Hz. The effect is essentially complete within four beats (2 s) with the tau of [Ca(2+)](i) decline decreasing by 42+/-3%. While there is a detectable increase in PLB Thr-17 and RyR Ser-2814 phosphorylation, the increase is quantitatively small (PLB<5%, RyR approximately 8%) and the time-course is clearly delayed with regard to FDAR. The low substrate phosphorylation indicates that pacing of myocytes only mildly activates CaMKII and consistent with this CaMKIIdelta autophosphorylation did not increase with pacing alone. However, in the presence of phosphatase 1 inhibition pacing triggered a net-increase in autophosphorylated CaMKII and also greatly enhanced PLB and RyR phosphorylation. We conclude that FDAR does not rely on phosphorylation of PLB or RyR. Even though CaMKII does become activated when myocytes are paced, phosphatases immediately antagonize CaMKII action, limit substrate phosphorylation and also prevent sustained CaMKII autophosphorylation (thereby suppressing global CaMKII effects).     

增殖细胞核抗原和p53在反流性食管炎黏膜的表达及其意义

目的　探讨增殖细胞核抗原 (PCNA)和p5 3在反流性食管炎 (RE)及治疗干预后内镜下恢复正常的食管上皮的表达及其意义。方法　 2 13例RE及 2 6例治疗后内镜下恢复正常的食管上皮活检标本 ,组织病理学HE染色 ,并采用卵白素 生物素 过氧化物酶复合物法测定PCNA和p5 3的表达。结果　随着食管黏膜从正常到RE和非典型增生 ,PCNA阳性细胞数分别为 (192± 12 0 ) /mm2 、(32 6± 2 0 0 ) /mm2 、(5 96± 2 0 8) /mm2 ,各组之间差异有显著性 (P <0 0 1) ;p5 3阳性表达率逐渐升高 ,分别为5 9%、34 4 %、5 7 6 %,各组之间差异有显著性 (P <0 0 1)。 2 6例RE病人经干预治疗后内镜下食管黏膜形态恢复正常 ,其食管黏膜的PCNA阳性细胞数为 (30 6± 2 14) /mm2 ,p5 3阳性率为 2 6 9%,均较干预前的 (4 83± 2 18) /mm2 和 6 1 5 %明显降低 (P <0 0 1) ;但仍然高于正常对照组 (P <0 0 1)。结论　随食管反流性病变的加重 ,PCNA及p5 3蛋白的表达增加 ;RE患者经干预治疗后内镜下恢复正常者 ,其食管黏膜在分子水平尚未达到正常 ,故仍需维持治疗和内镜随访。