胎衣不下奶牛母体胎盘黏着斑激酶异常表达分析及验证

目的通过对黏着斑激酶(focal adhesion kinase,FAK)在胎衣不下(retained fetal membranes,RFM)奶牛胎盘组织中差异表达的检测及验证,分析FAK在RFM中的作用。方法选取无其他疾病影响的胎衣自然排出组和RFM组奶牛,提取两组奶牛胎盘组织中的总蛋白和总RNA,利用Western blot及荧光定量PCR法对其FAK蛋白及mRNA水平进行检测。结果与胎衣自然排出组相比,RFM组奶牛母体胎盘组织中FAK蛋白及mRNA水平明显降低(P0.05)。结论发生RFM的奶牛母体胎盘中FAK表达下调,提示FAK可能参与该疾病的发生与发展。     

胎衣不下奶牛母体胎盘中血管内皮生长因子A异常表达的分析

目的分析胎衣不下(retained fetal membrane,RFM)奶牛母体胎盘组织中血管内皮生长因子A(vascular endothelial growth factor-A,VEGFA)的表达情况。方法提取RFM及胎衣正常排出奶牛母体胎盘组织中的总蛋白,采用荧光定量PCR及Western blot法检测两组奶牛母体胎盘组织中VEGFA的mRNA转录及蛋白表达水平。结果与胎衣正常排出组相比,RFM组奶牛母体胎盘组织中VEGFA的mRNA转录及蛋白表达水平均显著降低,差异均有统计学意义(P 0. 01)。结论发生RFM的奶牛母体胎盘组织中VEGFA表达减少,提示VEGFA可能参与该病的发生发展。     

胎衣不下奶牛母体胎盘组织基质相互作用分子1异常表达分析

目的分析胎衣不下(retained placenta,RP)奶牛母体胎盘组织中基质相互作用分子1(stromal interaction molecule 1,STIM1)的表达变化,探讨STIM1与奶牛胎衣不下的相关性。方法选取年龄、胎次、体重、泌乳量均相近且身体状况良好的的胎衣不下和胎衣正常排出奶牛,分别为R组和N组,收集两组奶牛母体胎盘组织,提取总RNA和蛋白,利用Q-PCR及Western blot法检测STIM1基因转录及蛋白表达水平。结果 R组STIM1基因转录及蛋白表达水平均下调,与N组相比差异有统计学意义(P0.05)。结论下调表达的STIM1可能通过降低胞内Ca2+浓度影响奶牛RP的发生。     

丝裂原活化蛋白激酶信号转导通路在脑缺血再灌注损伤中的作用及机制

目的探讨丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号转导通路在脑缺血再灌注损伤(cerebral ischemia/reperfusion,I/R)中的作用及机制。方法将大鼠随机分为6组:未使用p38MAPK抑制剂SB203580的假手术组(Sham组)、使用抑制剂的假手术组(Sham/SB组)、未使用抑制剂再灌注24 h组(I/R 24 h组)、未使用抑制剂再灌注48 h组(I/R 48 h组)、使用抑制剂再灌注24 h组(SB 24 h组)和使用抑制剂再灌注48 h组(SB 48 h组)。采用大脑中动脉线栓法(middle cerebral artery occlusion,MCAO)复制大鼠局灶性脑缺血再灌注模型,使用抑制剂的各组于造模前30 min经腹腔注射SB203580(5 mg/kg,溶于5 mg/ml DMSO)。采用免疫荧光双标法检测大鼠大脑中动脉(middle cerebral artery,MCA)和脑微血管(microvascular,Mic.V)周围IgG的渗出;Western blot和RT-PCR法检测大鼠脑组织中p38、一氧化氮合酶(inducible nitric oxide synthase,iNOS)、基质金属蛋白酶-9(matrix metalloproteinase,MMP-9)、胶原Ⅳ型(collagenⅣ)蛋白和基因表达的变化。结果随着再灌注时间的延长,大鼠脑血管内IgG渗出量增加(P<0.01);经p38MAPK抑制剂预处理后,IgG的渗出程度有所减轻。脑缺血再灌注后,大鼠脑组织中p38、iNOS、MMP-9蛋白的表达水平和基因mRNA的转录水平均明显升高(P<0.05),经p38MAPK抑制剂预处理后,能显著抑制p38、iNos、MMP-9蛋白的表达水平和基因mRNA的转录水平(P<0.05);collagenⅣ蛋白的表达水平和基因mRNA的转录水平随着缺血时间的延长而逐渐下降(P<0.05),经p38MAPK抑制剂预处理后,其下降程度有所减轻(P<0.05)。结论 MAPK通路中的关键蛋白p38、iNOS、MMP-9在脑缺血时表达显著增加,其过度表达直接导致血脑屏障的破坏,抑制其表达,从而遏制对血脑屏障的破坏,有望为I/R的治疗提供新的途径。     

胎衣不下奶牛胎盘组织差异表达蛋白的筛选及鉴定

目的筛选胎衣不下奶牛胎盘组织中的差异表达蛋白,并对其鉴定,为研究奶牛胎衣不下的发生机理和治疗提供依据。方法收集胎衣不下奶牛及胎衣正常排出奶牛胎盘组织各5份,应用双向凝胶电泳(two-dimensional polyacrylamidegel electrophoresis,2D-PAGE)技术对胎盘组织中蛋白进行分离,银染显色后获得差异表达蛋白点,并采用基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)技术对其进行鉴定。结果与胎衣正常排出组相比,胎衣不下组奶牛胎牛胎盘中差异表达蛋白点共240个,其中表达上调蛋白点212个,表达下调蛋白点28个;母体胎盘中差异表达蛋白点共214个,其中表达上调蛋白点134个,表达下调蛋白点80个,共鉴定出5种差异表达蛋白。与胎衣正常排出组相比,胎衣不下组奶牛胎牛胎盘中牛血清白蛋白(bovine serum albumin,BSA)、α-烯醇化酶(Alpha enolase)表达下调,谷胱甘肽转移酶(glutathione transferases,GST)表达上调;母体胎盘中膜联蛋白V(Annexin V)、Alpha enolase和膜联蛋白A2(Annexin A2)表达均上调。结论发现的5种差异表达蛋白可能与奶牛胎衣不下的发生发展相关。     

奶牛胎衣不下与胎儿胎盘中iNOS、eNOS及MMP-2表达的相关性

目的分析奶牛胎衣不下(Retained fetal membranes,RFM)与胎儿胎盘中诱导型一氧化氮合酶(Induction nitric oxid esynthase,iNOS)、内皮型一氧化氮合酶(Endothelial nitric oxide synthase,eNOS)和基质金属蛋白酶-2(Matrix metallopeptidase-2,MMP-2)表达的相关性,探讨RFM发生的分子机制。方法 RT-PCR扩增、克隆产后胎衣正常脱落(No retention of fetal membranes,NRFM)和RFM奶牛胎儿胎盘中iNOS、eNOS和MMP-2基因,以牛β-actin基因为内参,采用荧光定量PCR法检测RFM和NRFM奶牛胎儿胎盘组织中iNOS、eNOS和MMP-2基因表达的差异。结果克隆的iNOS、eNOS和MMP-2基因片段与GenBank中登录的核苷酸序列同源性均达100%。RFM组iNOS基因mRNA的转录水平明显高于NRFM组(P<0.05),eNOS和MMP-2基因mRNA的转录水平则明显低于NRFM组(P<0.05)。结论 iNOS、eNOS和MMP-2基因mRNA的转录水平与RFM的发生密切相关。     

bta-mir-423-5p在胎衣不下奶牛母体胎盘中的差异表达分析及验证

目的分析及验证bta-mir-423-5p在胎衣不下奶牛母体胎盘中的差异表达。方法选取各种因素无明显差异,且排除其他疾病影响的胎衣正常排出奶牛(对照组)和胎衣不下奶牛(试验组)各3头,对照组在奶牛产后胎衣排出后,立即采集母体胎盘组织约150 mg,试验组在奶牛产后12 h,收集母体胎盘组织约150 mg。采用Solexa高通量测序技术对两组母体胎盘组织间bta-mir-423-5p的差异表达进行分析;利用Real-time Q-PCR法进行验证。结果试验组奶牛母体胎盘中bta-mir-423-5p的表达量明显低于对照组(P0.01)。结论 bta-mir-423-5p在胎衣不下奶牛母体胎盘中异常表达,表明其可能参与胎衣不下的发生,为奶牛胎衣不下发病机理的研究提供了实验依据。     

NIRF蛋白对妊娠早期绒毛滋养细胞增殖、凋亡的影响及其作用机制

目的观察NIRF蛋白对妊娠早期绒毛滋养细胞增殖、凋亡的影响及其作用机制。方法取正常妊娠妇女人工流产绒毛组织(8~10周),按常规分离方法获得滋养细胞,进行原代培养。通过上/下调NIRF在滋养细胞中的表达后,采用qRT-PCR法检测p53基因mRNA的转录水平;Western blot法检测p53蛋白的表达水平;MTT法检测滋养细胞的增殖能力;Annexin V/PI双染色法检测细胞凋亡情况。结果与未转染组比较,NIRF上/下调后,p53基因m RNA转录水平差异无统计学意义(P0.05);NIRF上调,p53蛋白表达水平明显下降(P0.05),早期绒毛滋养细胞增殖能力显著增强(P0.05),凋亡率差异无统计学意义(P0.05);NIRF下调,p53蛋白的表达水平明显升高(P0.05),早期绒毛滋养细胞增殖能力明显减弱(P0.05),凋亡率明显上升(P0.05)。结论 NIRF在蛋白水平上抑制了p53的表达,促进了滋养细胞的增殖。     

硫氧还蛋白相互作用蛋白上调对胰岛β细胞衰老的影响及其作用机制

目的探讨硫氧还蛋白相互作用蛋白(thioredoxin interacting protein,TXNIP)上调对胰岛β细胞衰老的影响及其可能的作用机制。方法采用慢病毒构建TXNIP过表达(Ad-TXNIP-GFP)及沉默(TXNIP-ShRNA)稳转INS-1胰岛β细胞株,Western blot检测衰老相关基因p16、p21、Rb、p38 MAPK磷酸化及p53蛋白表达水平。分别采用p38MAPK抑制剂SB203580、p53抑制剂Pifithrin-β预处理细胞后,Western blot检测上述蛋白的表达水平。结果 TXNIP过表达可促进p21、p16、Rb、p53蛋白表达、p38 MAPK的磷酸化(P 0. 01)及INS-1细胞衰老,TXNIP沉默可抑制p21、p16、Rb、p53蛋白表达、p38 MAPK的磷酸化(P 0. 05)及INS-1细胞衰老;p38 MAPK和p53抑制剂均可抑制p21、Rb蛋白的表达(P 0. 05),减轻细胞衰老;p38 MAPK抑制剂可抑制p53蛋白表达(P 0.05),而p53抑制剂并不影响p38 MAPK的磷酸化及p16蛋白的表达(P 0. 05)。结论 TXNIP可促进p38 MAPK磷酸化以及后续p16、p53蛋白表达上调,进而增加p21、Rb蛋白表达,从而引起INS-1细胞衰老。     

Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg²⁺ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg²⁺. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg²⁺. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg²⁺ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF. It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg²⁺. This work was presented at the Twenty-Third Southeastern Regional Lipid Conference, held October 26–28, in Cashiers, North Carolina.     

A multiplexed protein kinase assay

We report a novel protein kinase assay designed for high-throughput detection of one or many kinases in a complex mixture. A solution-phase phosphorylation reaction is performed on 900 different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation, phosphoserine, phosphothreonine, and phosphotyrosine are chemically labeled, and the substrates are hybridized to a microarray with oligonucleotides complementary to the tags to read out the phosphorylation state of each peptide. Because protein kinases act on more than one peptide sequence, each kinase can be characterized by a unique signature of phosphorylation activity on multiple substrates. Using this method, we determined signatures for 26 purified kinases and demonstrated that enzyme mixtures can be screened for activity and selectivity of inhibition.     

Electrochemical investigations of sarcoma-related protein kinase inhibition

An electrochemical biosensor was developed for the determination of sarcoma (Src)-related protein kinase-catalyzed phosphorylation reactions in the presence of adenosine 5′-γ-ferrocenoyl triphosphate (Fc-ATP). The sensing platform is based on a highly specific amino acid sequence Glu-Gly-Ile-Tyr-Asp-Val-Pro (EGIYDVP), to which a Fc-PO₂ moiety can be transferred from Fc-ATP by the action of the Src kinase. The enzyme kinetics and kinase inhibition were investigated by square wave voltammetry (SWV). The kinetic parameters K_m and V_max were determined for Src protein kinase with respect to Fc-ATP co-substrate and were found to be 200 μM and 115 μA cm⁻² min, for phosphorylation of the EGIYDVP peptide substrate. Furthermore, the Src-catalyzed phosphorylation of Tyr was investigated in the presence of the small molecule inhibitors PP1, PP2, SU6656, and roscovitine. PP3 does not inhibit Src activity and was used as a control. The percent inhibition at half concentration, IC₅₀, values were determined for all inhibitors under the study and were estimated to be in the 5–30 nM range. The electrochemical study suggests that the increase in inhibition efficiency was in the order PP3 < SU6656 < roscovitine < PP2 < PP1.     

Derivatives of Di-O-octanoylglycerol and mono-O-octylglycerol as modulators of protein kinase C and diacylglycerol kinase activities

Twelve analogs of 1,2-di-O-octanoylglycerol modified at C-3 and three quaternaryN-alkyl-ammonium derivatives of glycerol were synthesized. The compounds were testedin vitro as potential modulators of the calcium activated, phospholipid dependent protein kinase C (PKC) and diacylglycerol (DAG) kinase activities in order to understand the molecular interactions of these enzymes with their natural activators, inhibitors, or substrates. PKC activity was assayed by measuring histone H₁ phosphorylation, and the compounds synthesized were tested either in the presence (inhibitors) or in the absence (activators) of 1,2-di-O-octanoyglycerol analogs with the phosphatidylserine/Ca²⁺ mixture. DAG kinase activity was measured by the incorporation of phosphate into 1,2-di-O-oleoyl-sn-glycerol in the presence of the various analogs synthesized. In regard to PKC activity, the assays revealed that 1,2-di-O-octanoylglycerol analogs are inactive when modified at C-3 with groups which do not permit hydrogen bonding. Under our conditions, di-O-octanoylthioglycerol, which has been reported as inactive, was able to activate PKC in the presence of phosphatidylserine. It has been shown to give a synergistic activation with diacylglycerol and had no affinity for the phorbol ester receptor binding site, suggesting thatO-octanoylthioglycerol interacts with the enzyme at a different site from the phorbol ester receptor binding site. PKC and DAG kinase activities are inhibited byN-alkyl-ammonium compounds (IC₅₀ 24 μM) only when either two 8-carbon alkyl or acyl chains are present at the 1- and 2-positions of the glycerol backbone. The fact that these compounds have a strong effect on the binding of [³H]phorbol 12,13-dibutyrate to protein kinase C, and also inhibit DAG kinase, may suggest binding to the DAG site of the regulatory domain of PKC.     

Functional classification of protein kinase binding sites using Cavbase

Increasingly, drug-discovery processes focus on complete gene families. Tools for analyzing similarities and differences across protein families are important for the understanding of key functional features of proteins. Herein we present a method for classifying protein families on the basis of the properties of their active sites. We have developed Cavbase, a method for describing and comparing protein binding pockets, and show its application to the functional classification of the binding pockets of the protein family of protein kinases. A diverse set of kinase cavities is mutually compared and analyzed in terms of recurring functional recognition patterns in the active sites. We are able to propose a relevant classification based on the binding motifs in the active sites. The obtained classification provides a novel perspective on functional properties across protein space. The classification of the MAP and the c-Abl kinases is analyzed in detail, showing a clear separation of the respective kinase subfamilies. Remarkable cross-relations among protein kinases are detected, in contrast to sequence-based classifications, which are not able to detect these relations. Furthermore, our classification is able to highlight features important in the optimization of protein kinase inhibitors. Using small-molecule inhibition data we could rationalize cross-reactivities between unrelated kinases which become apparent in the structural comparison of their binding sites. This procedure helps in the identification of other possible kinase targets that behave similarly in "binding pocket space" to the kinase under consideration.     

喹啉类蛋白酪氨酸激酶抑制剂的研究进展

蛋白酪氨酸激酶的过度表达与多种肿瘤的发生、发展及转移相关,抑制酪氨酸激酶活性可有效抑制肿瘤。目前已有11个小分子蛋白酪氨酸激酶抑制剂作为抗肿瘤药物上市,1个由美国FDA批准接受预登记的药物,超过100个候选药物处于临床试验阶段。其中喹唑啉结构的酪氨酸激酶抑制剂已成为临床上最常见的抗肿瘤化疗药物类别之一。综述了近年来与喹唑啉结构相似的具有喹啉骨架的酪氨酸激酶抑制剂研究进展。     

Mechanism-based fluorescent reporter for protein kinase A detection

A novel mechanism-based fluorescent reporter was designed for the detection of protein kinase A (PKA), which is known to mediate a variety of cellular responses in most eukaryotic cells. The probe consists of a specific binding peptide sequence, LRRRRFAFC, conjugated with 2'-thioethyl-5-(or -6)-carboxyfluoresceinamide (FAMS; 2) and 5-(or 6-)carboxytetramethylrhodamine (TAMRA) at the cysteine and leucine residues, respectively. In the absence of PKA, the two fluorophores associate by hydrophobic interactions, forming an intramolecular ground-state dimer; this results in fluorescein quenching (>93 %). Upon PKA addition, the reporter reacts with the sulfhydryl functionality at Cys199 through a disulfide-exchange mechanism. FAMS is subsequently released, resulting in significant fluorescence amplification. The remaining peptide sequence, which acts as an inhibitor, is attached covalently to the enzyme. Our results suggest that this type of sensors could have far-reaching applications in the molecular sensing of enzymes.     

NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase

Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.     

Synthetic caged DAG-lactones for photochemically controlled activation of protein kinase C

Switching on kinases: Synthetic caged DAG-lactones have been developed and showed decreases of two orders of magnitude, relative to the corresponding parent compounds, in their binding affinities towards PKC. The caged compounds had no effect on the translocation of PKC until after photoactivation. This approach is a potentially powerful tool for probing the PKC signaling cascade.