HeLa细胞中激酶NUAK1调控Tuberiun的磷酸化

NUAK1是LKB1的下游激酶之一,可被LKB1磷酸化而激活,但对其在LKB1相关信号通路中的功能仍缺乏了解.本研究发现在HeLa细胞中重建LKB1表达后NUAK1与tuberin蛋白免疫共沉淀,提示在野生型LKB1存在时NUAK1与tuberin可能存在直接相互作用.进一步的激酶活性测定和in vivo蛋白磷酸化实验表明:在HeLa细胞中葡萄糖匾乏条件下,野生型LKB1可显著激活NUAK1的激酶活性;而被激活的NUAK1可明显提高tuberin 的磷酸化水平,使用NUAK1 siRNA pool干扰NUAK1的表达则几乎将tuberin的磷酸化水平降低为零.上述结果表明NUAK1可能介导了LKB1对tuberin磷酸化的调节,进而下调mTOR通路,抑制蛋白质合成与细胞生长增殖.     

牛磺酸抗心肌成纤维细胞增殖作用的实验研究

目的研究牛磺酸(Taurine,Tau)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导新生大乳鼠心肌成纤维细胞(myofibroblasts,myo Fbs)增殖的抑制作用,并探讨其作用机制.方法用AngⅡ诱导新生大乳鼠myo Fbs增殖,建立心肌纤维化(myofibrosis,MF)模型.采用四甲基偶氮唑盐(MTT)法检测细胞增殖;羟脯氨酸试剂盒检测胶原含量;免疫细胞化学染色检测p-PKCα的膜转位及表达;Western blot检测p-PKCα和p-ERK1/2蛋白表达及含量.结果 Tau(30,60,120)mmol/L可明显抑制AngⅡ诱导的Myo Fbs增殖及胶原合成,同AngⅡ组比较差异具有统计学意义(P0.05或P0.01),并且呈现出剂量依赖性.应用免疫细胞染色Western blot并且结合图像分析,结果表明:Tau可抑制p-PKCα膜转位和表达,与AngⅡ组相比差异具有统计学意义(P0.01).结论 Tau可能通过减少p-PKCα的表达及膜转位,从而抑制MyoFbs增殖和胶原含量的增加,抑制MF,逆转心肌重塑.     

心肌酶谱与白细胞对AMI疗效联合监测初探

心肌酶谱检测现已成为急性心肌梗塞（ＡＭＩ）诊断及疗效监测的主要手段之一,在临床中发现,ＡＭＩ患者其末梢血中白细胞也异常增高,且与心肌酶如（ＣＫ－ＭＢ、ＣＫ、ＨＢＤＨ、ＧＯＴ等）有良好的正相关性,二者联合监测对于判定ＡＭＩ疗效具有一定实际作用,现报告如下：１材料与方法１．１病例资料急性心肌梗塞患者共８例,均为本院住院患者,其中男性４例,女性４例,年龄３０～６６岁,平均年龄为５５岁。１．２方法监测仪器为雅培ＣＣＸ全自动生化分析仪。心肌酶测定试剂购于上海长征公司,病人血常规测定采用末梢血,经Ｆ－８２０…     

Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase—2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion,outgrowth,differentiation and expression of serine proteinases by the blastocyst,so it is regarded as a vital factor in blastocyst implantation.Although the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated,the roles of the signaling molecules in the extracellular matrix-integrin signal transduction pathway in blastocyst implantation are unknown.This limits the understanding of blastocyst implantation and ECM-integrin signal transduction pathway.In the present study,in vitro blastocyst culture and indirect immunocytochemistry,matrix metalloproteinases(MMPs) zymography and antisense oligodeoxynucleotide(ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal transduction pathways,focal adhesion kinase(FAK),in mouse blastocysts and its influence on mouse blastocyst adhesion,outgrowth and MMP-2.The results showed that mouse blastocysts expressed FAK.FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth.Fibronectin triggered pro-MMP-2 and 64kD MMP-2 activities.The antisense ODN to FAK attnuated pro-MMP-2 and 64kD MMP-2 activites which decreased abruptly and tended to disappear with increasting concentrations of the antisense ODN.Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN.Up to 20μg/mL of the antisense ODN concentration,the adhesion and out-growth rates were decreased in a dose-dependent manner.The results indicated that FAK influenced mouse blastocyst adhesion,outgrowth and MMP-2 activity by intracellular signal transduction.In other words,FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.     

PDGF-BB活化PSCs的分子机制研究

血小板生长因子(PDGF)是诱导胰星状细胞(PSCs)活化的重要细胞因子，其分子机制尚不清楚。用PDGF-BB诱导体外培养的PSCs，旨在探讨PDGF信号转导通路在胰纤维化形成的分子发生机制中的可能作用。PSCs与不同刺量PDGF-BB(10ng／mL，25ng／mL和50ng／mL)共培养，24h后收集细胞，采用Western-blotting检测，PSC中磷酸化细胞外信号调节激酶-1(pERK1)蛋白水平和RT-PCR检测Colal(Ⅰ)mRNA的表达。结果表明，不同刺量PDGF-BB作用于PSCs后，各组pERK1蛋白表达比空白对照组有不同程度上调；Colal(Ⅰ)mRNA在PSCs中表达也明显上调，且在一定范围内与PDGF-BB刺量呈正相关。     

鼠脑己糖激酶的纯化及铝离子对该酶的影响

应用蔗糖溶液提取,葡萄糖－６－磷酸溶解,ＤＥＡＥ－纤维素离子交换柱层析,可从鼠脑中纯化出己糖激酶（ＨＫ）,纯化的ＨＫ在ＰＡＧＥ中,呈现单一的蛋白质染色带,经ＳＤＳ－ＰＡＧＥ测得其分子量为９７７２３。该ＨＫ受Ａｌ＾３＋的抑制,Ａｌ＾３＋对该ＨＫ的抑制作用表现为所谓的“负协同作用”现象,即在低底物（ＡＴＰ）浓度下表现为非竞争性抑制,在高底物浓度下表现为竞争性竞争性抑制。柠檬酸钠、ＥＤＴＡ和邻苯二酚可拮     

Reverse micelles extraction of nattokinase： From model system to real system

Nattokinase (also named subtilisin NAT) was pri- marily isolated from a traditional fermented food in Japan, ‘‘Natto’’, by Sumi et al.[1]. Since then more and more reports of pharmacological researches on natto- kinase demonstrated that nattokinase showed many advantages, such as longer half life, greater fibrinolytic activity than plasmin, availability for both oral admini- stration and intravenous instillation, both preventive and therapeutic measure against thrombosis, less risk of he…     

辐照灭菌用于纳豆激酶制剂的实验研究

对60钴γ-射线辐照前后的纳豆激酶(NK)制剂有效成分、酶活及活菌数等技术指标进行了实验研究,结果表明,不同剂量的60钴γ-射线辐照对纳豆激酶制剂中的总皂甙含量和纳豆激酶酶活影响不大,30 kGy时总皂甙含量仍可达到原始含量的105%～125%,NK酶活达到原始酶活的90%以上;纳豆芽孢杆菌、大肠菌群和金黄色葡萄球菌等活菌数对60钴γ-射线辐照十分敏感,5 kGy的60钴γ-射线辐照处理后纳豆芽孢杆菌、大肠菌群和金黄色葡萄球菌数量下降99%以上.     

北京棒杆菌天冬氨酸激酶突变体A380H的酶学性质

同源序列比对和蛋白质结构分析表明,A380为天冬氨酸激酶(aspartate kinase,AK)的绝对保守位点,对该位点进行定点突变、分离纯化和性质表征.结果表明:与野生型(WT)相比,突变体A380H的V_(max)提高4.28倍;突变体A380H的最适温度由28℃提高至35℃,最适pH值仍为7.5,半衰期由4.5h缩短至3.5h;底物抑制剂对WT和突变体均有抑制作用,苏氨酸和赖氨酸呈协同抑制作用,苏氨酸单独存在时对A380H具有激活或抑制减弱作用;Mg~(2+),Ni ~(2+)对WT有激活作用,1,5mmol/L的Cu~(2+)对A380H有激活作用;与WT相比,甲醇和异丙醇对A380H的抑制作用增强,正丁醇和乙腈对A380H的抑制作用减弱且表现出激活作用.     

基于关键酶表达的发酵调控与分子改造策略对E.coli产丁二酸的影响

考察E.coli JM001及其重组菌株E.coli JM002生物发酵生产丁二酸的性能。E.coli JM001在两阶段发酵产丁二酸过程中通过在有氧培养阶段添加乙酸钠,即可提高丁二酸的生产能力,厌氧阶段的丁二酸收率可达84%,但会有较多的副产物乙酸和丙酮酸积累。以E.coli JM001为出发菌株,敲除其磷酸烯醇式丙酮酸羧化酶(PPC)并导入来源于枯草芽孢杆菌的磷酸烯醇式丙酮酸羧化激酶(PCK)基因,构建了重组菌株E.coli JM002,该重组菌株在两阶段发酵的有氧培养过程中不需添加乙酸钠,转厌氧后菌株也具有转化葡萄糖合成丁二酸的能力,丁二酸收率可达86%,副产物积累很少。