Downregulation of cold-inducible RNA-binding protein activates mitogen-activated protein kinases and impairs spermatoRenic function in mouse testes

Cold-inducible RNA-binding protein （CIRP） is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purpose of this study was to investigate the functions of CIRP in the testes. We employed RNAi technique to knock down the expression of CIRP in the testes, and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown. Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling （TUNEL） assay, and mitogen-activated protein kinase （MAPK） signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis. We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes. Compared to controls, the mean seminiferous tubule diameter （MSTD） and the thickness of the germ cell layers decreased following siRNA treatment, whereas the percentage of apoptotic seminiferous tubules increased. The p44/p42, p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP. In conclusion, we discovered that downregulation of CIRP resulted in increased germ cell apoptosis, possibly viathe activation of the p44/p42, p38 and SAPK/JNK MAPK pathways.

Overinhibition of Mitogen-Activated Protein Kinase Inducing Tau Hyperphosphorylation

To reveal the relationship between mitogen-acti-vated protein kinase (MAPK)and tau phosphorylation, we used different concentration of PD98059, an inhibitor of MEK (MAPKkinase), to treat mice neuroblastma (N2a) cell line for 6 h. It showed that theactivity of MAPKdecreased in a dose-dependent manner. But Western blot and immunofluo-rescence revealed that justwhen the cells were treated with 16 mu mol/L PD98059, tau was hyperphosphorylated at Ser396/ 404 andSerl99/202 sites. We obtained the conclusion that overinhibited MAPK induced tauhyperphosphorylation at Ser396/404 and Serl99/202 sites.

下调富含脯氨酸蛋白11表达对食管癌耐药细胞EC9706/DDP耐药性的影响及其机制

目的:探讨下调富含脯氨酸蛋白11(PRR11)表达对食管癌耐药细胞耐药性的影响,阐明其相关机制。方法:采用顺铂(DDP)浓度递增间断刺激人食管癌EC9706细胞建立DDP耐药细胞株EC9706/DDP,MTT法检测EC9706/DDP细胞药敏性,实时荧光定量PCR(RT-qPCR)法和Western blotting法检测EC9706/DDP细胞及其亲本EC9706细胞中PRR11 mRNA和蛋白表达水平。将EC9706/DDP细胞分为对照组、sh-NC组(转染sh-NC)、sh-PRR11组(转染sh-PRR11)、sh-NC+DDP组(转染sh-NC后用4 mg·L^(-1)DDP处理)和sh-PRR11+DDP组(转染sh-PRR11后用4 mg·L^(-1)DDP处理),RT-qPCR法检测各组细胞中PRR11 mRNA表达水平,Western blotting法检测各组细胞中PRR11、磷脂酰肌醇3-激酶(PI3K)p110α、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、P-糖蛋白(P-gp)和多药耐药相关蛋白1(MRP1)蛋白表达水平,流式细胞术检测各组细胞凋亡率。结果:成功获得DDP耐药细胞株EC9706/DDP,耐药指数为7.23±0.86。与EC9706细胞比较,EC9706/DDP细胞中PRR11 mRNA和蛋白表达水平升高(P<0.05)。分别与对照组和sh-NC组比较,sh-PRR11组细胞中PRR11 mRNA和蛋白表达水平降低(P<0.05),细胞的DDP半数抑制浓度(IC50)降低(P<0.05)。与sh-NC组比较,sh-NC+DDP组和sh-PRR11组细胞中PI3K p110α、p-AKT、P-gp和MRP1蛋白表达水平降低(P<0.05),细胞凋亡率升高(P<0.05);分别与sh-NC+DDP组和sh-PRR11组比较,sh-PRR11+DDP组细胞中PI3Kp110α、p-AKT、P-gp和MRP1蛋白表达水平降低(P<0.05),细胞凋亡率升高(P<0.05)。结论:下调EC9706/DDP耐药细胞中PRR11基因的表达,可抑制耐药相关蛋白的表达,逆转对DDP耐药,并诱导细胞凋亡,其作用机制可能与抑制PI3K/AKT信号通路激活有关。

Metformin promotes anti-tumor immunity in STK11 mutant NSCLC through AXIN1-dependent upregulation of multiple nucleotide metabolites

Background:Metformin has pleiotropic effects beyond glucose reduction,including tumor inhibition and immune regulation.It enhanced the anti-tumor effects of programmed cell death protein 1(PD-1)inhibitors in serine/threonine kinase 11(STK11)mutant non-small cell lung cancer(NSCLC)through an axis inhibition protein 1(AXIN1)-dependent manner.However,the alterations of tumor metabolism and metabolites upon metformin administration remain unclear.Methods:We performed untargeted metabolomics using liquid chromatography(LC)-mass spectrometry(MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis.Results:According to the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database,most metabolites were annotated into metabolism,including nucleotide metabolism.Next,the differentially expressed metabolites in H460(refers to H460 cells),H460_met(refers to metformin-treated H460 cells),and H460_KO_met(refers to metformin-treated Axin1-/-H460 cells)were distributed into six clusters based on expression patterns.The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis.We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated.Furthermore,these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes(STING)pathway independently of AXIN1.Conclusion:Relying on AXIN1,metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC,indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.

Old Drug for New Use： Searching for MEK1 （Mitogen- Activated Protein Kinase Kinase 1） Inhibitor by the Computer Aided Drug Design

An old drug with a new use can significantly reduce the cost and time for new drug research and development. MAPK （Mitogen-activated protein kinase） plays a very important key role in signal transduction pathways of cell proliferation and differentiation. According to the statistics, there are about 30% persons who suffered from cancers related to the MAPK signal transduction pathways. Therefore, many researchers are focused on blocking these pathways in cancers therapies. Ras/Raf/MEK/ERK, however, is one of very important pathways among MAPK message transduction pathways. More and more information about MEK protein inhibitors are unveiled in several recent years. In the present study, the authors utilized MEK inhibitors which were already published and their activities were available to construct 2D-QSAR model by using CADD （multiple linear regression）. Then, the authors searched certified FDA drugs （Drugs@FDA 6184 drugs） making preliminary screening. The secondary screening on 3D structures were followed by using Docking, Scoring and Pharmacophore analysis to find out most suitable MEK inhibitors to become a fundamental database in drug discovery. The results are shown the ALogP, number of aromatic rings, number of hydrogen bond acceptors and number of hydrogen bond donors are all in positive correlation. According to the equation from 2D-QSAR model, the results conform to the previous description.

Gαq/11介导的细胞信号转导在两肾一夹肾性高血压大鼠主动脉中的变化

实验以两肾一夹肾性高血压大鼠为模型 ,研究主动脉Gαq/ 11介导的细胞信号转导的变化及其意义。制备两肾一夹肾性高血压大鼠模型 ,分别于术后第 1、2、4和 8周测定动脉血压 ,用免疫印迹法检测主动脉组织中Gαq/ 11亚单位和细胞外信号调节激酶 1/ 2 (ERK1/ 2 )含量 ,测定磷脂酶C (phospholipaseC ,PLC)活性。结果显示高血压组大鼠于术后第 2周血压开始升高 ;主动脉Gαq/ 11和ERK1/ 2含量于术后第 1周增加 (分别为 5 7 5 3%和40 16 % ) ,并维持在高水平 (P <0 0 1) ;术后主动脉PLC活性亦增加 (P <0 0 5 )。实验表明 ,肾素依赖性高血压能引起大鼠主动脉Gαq/ 11介导的细胞信号转导系统激活 ,并参与高血压的发生和发展。

家族性进行性色素过度沉着症家系丝氨酸/苏氨酸蛋白激酶11基因的克隆和序列鉴定

目的进行家族性进行性色素过度沉着症(FPH)家系中患者丝氨酸/苏氨酸蛋白激酶11(STK11)基因的克隆和序列鉴定,以期检测FPH发生是否由于STK11突变所致。方法从FPH患者永生化的B淋巴细胞中提取总RNA,反转录聚合酶链式反应(RT-PCR)获取cDNA序列,加入特异性引物经PCR扩增STK11 cDNA双链,再经过HindⅢ、EcoRI双酶切PCR产物及质粒载体pcDNA3.0,酶切产物经回收、连接,产物转化到大肠杆菌DH5α,选取阳性克隆菌落进行PCR鉴定、酶切鉴定和测序分析。结果成功构建重组质粒pcDNA3.0-STK11,其测序结果与美国国立生物技术信息中心查询的正常序列结果一致。结论该FPH家系发病并非由于STK11突变所致,在初步定位FPH致病基因的19p13.1-pter区段存在尚未发现的致病基因。

非小细胞肺癌组织中丝苏氨酸激酶11与细胞死亡调解子抗体表达检测的临床意义

目的探讨非小细胞肺癌组织中丝苏氨酸激酶11（STK11）、细胞死亡调解子抗体（BIM）表达检测的临床意义。方法选取2014年3月至2015年3月河北省胸科医院接收的非小细胞肺癌患者65例，收集切除后的非小细胞肺癌组织标本及癌旁正常组织。采用免疫组织化学技术对标本中STK11、BIM的表达进行研究。结果非小细胞肺癌组织中的STK11阳性表达率低于旁癌组织[63．08％（41／65）比92．31％（60／65）]（x^2=16．022，P〈0．05），非小细胞肺癌组织中的BIM的阳性表达率高于旁癌组织[61．54％（40／65）比21．54％（14／65）]（x^=21．413，P〈0．05）。淋巴结转移患者非小细胞肺癌组织中STK11阳性表达率显著低于未转移淋巴结患者[41．38％（12／29）比80．56％（29／36）]（P〈0．05）；Ⅰ～Ⅱ期患者非小细胞肺癌组织中STK11的阳性表达率显著高于Ⅲ～Ⅳ期[72．55％（37／51）比28．57％（4／14）]（P〈0．05）；高分化患者的非小细胞肺癌组织中STK11阳性表达率显著高于中分化、低分化[89．47％（17／19）比51．61％（16／31）、53．33％（8／15）]（P〈0．05）。腺癌患者非小细胞肺癌组织中STK11阳性率显著高于鳞癌[78．13％（25／32）比48．48％（16／33）]（P〈0．05）。Ⅰ～Ⅱ期的BIM阳性表达率显著高于Ⅲ～Ⅳ期[70．59％（36／51）比28．27％（4／14）]（P〈0．05）；高分化患者非小细胞肺癌组织阳性表达率显著高于中分化、低分化[89．47％（17／19）比45．16％（14／31）、60．oo％（9／15）]（P〈0．05）。腺癌BIM阳性率高于鳞癌[75．oo％（24／32）比48．48％（16／33）]（P〈O．05）。结论STK11、BIM表达水平可作为判定非小细胞肺癌转移及预后的生物学指标。

基于数据挖掘分析STK11基因在前列腺癌中的表达及意义

目的通过研究丝苏氨酸激酶11(STK11)在前列腺癌(PCa)组织中的表达,阐明其对PCa预后的意义。方法运用LinkedOmics、GEO和GEPIA大数据分析STK11表达与PCa临床病理学参数和前列腺癌恶性进程的相关性及对预后的影响。再运用The Human Protein Atlas中的免疫组化结果分析STK11在不同等级PCa组织中的表达情况。结果LinkedOmics、GEO和GEPIA数据库的分析结果表明在PCa中,STK11的表达与T分期、N分期、淋巴结转移和前列腺癌恶性进程呈显著正相关性,且高表达STK11的前列腺癌其总生存率和无病生存率显著低于低表达STK11的前列腺癌(P<0.05)。免疫组化的结果表明:低等级PCa组织中的STK11表达显著低于高等级PCa组织中的表达,且主要定位于癌细胞的胞质和胞膜。结论STK11是一种重要的不良预后指标,可以作为预测前列腺癌患者转移发生、判断预后的有效生物标志物。

白细胞介素11预防大鼠肠道缺血性损伤的机制探讨

目的探讨重组人白细胞介素 1 1 (recombinanthumaninterleukin 1 1 ,rhIL 1 1 )预处理对大鼠肠道缺血再灌注损伤保护作用的分子机制。方法夹闭大鼠肠系膜上动脉 1h后松夹建立缺血再灌注模型。将 56只雄性Wistar大鼠随机分为正常对照组(A)、单纯缺血再灌注组 (B)、生理盐水对照组 (C)和rhIL 1 1预处理组 (D)。C组和D组分别于夹闭前 2天给予生理盐水 (0 .2 5ml/只/d)或rhIL 1 1 (60 0 μg/kg/d)。于再灌注 6h和 2 4h时分别处死各组大鼠 ,以凋亡细胞原位末端标记技术 (TdT mediatedd UTPnickendlabeling,TUNEL)检测组织中细胞凋亡情况 ,用免疫组织化学法检测抗凋亡因子bcl 2及蛋白激酶caspase 3和caspase 8的表达水平。同时以苏木素 伊红 (hematoxylin eosin,HE)染色观察形态学病理改变。 结果HE及TUNEL检测显示 ,rhIL 1 1预处理组大鼠肠屏障功能的损伤程度较B、C组显著减轻 ,凋亡细胞数量减少 ,caspase 3和caspase 8的表达水平也低于B、C组 ,而bcl 2的表达量增加。结论rhIL 1 1预处理对大鼠缺血再灌注损伤的保护作用可能与抑制caspase 3和caspase 8的表达 ,激活bcl 2的表达有关。

Mitogen-activated protein kinase signal pathways play an important role in right ventricular hypertrophy of tetralogy of Fallot

Background Tetralogy of Fallot （TOF） is the most common malformation of children with an incidence of approximately 10% of congenital heart disease patients. There can be a wide spectrum to the severity of the anatomic defects, which include ventricular septal defect, aortic override, right ventricular outflow tract obstruction, and right ventricular hypertrophy. We examined the relationship between right ventricular hypertrophy in patients with TOF and the gene expression of factors in the mitogen-activated protein kinase （MAPK） signal pathway. Methods To gain insight into the characteristic gene（s） involved in molecular mechanisms of right ventricular hypertrophy in TOF, differential mRNA and micro RNA expression profiles were assessed using expression-based micro array technology on right ventricular biopsies from young TOF patients who underwent primary correction and on normal heart tissue. We then analyzed the gene expression of the MAPK signal pathway using reverse transcription-polymerase chain reaction （RT-PCR） in normals and TOF patients. Results Using the micro RNA chip V3.0 and human whole genome oligonucleotide microarray VI.0 to detect the gene expression, we found 1068 genes showing altered expression of at least two-fold in TOF patients compared to the normal hearts, and 47 micro RNAs that showed a significant difference of at least two-fold in TOF patients. We then analyzed these mRNAs and micro RNAs by target gene predicting software Microcosm Targets version 5.0, and determined those mRNA highly relevant to the right ventricular hypertrophy by RT-PCR method. There were obvious differences in the gene expression of factors in the MAPK signal pathway when using RT-PCR, which was consistent to the results of the cDNA microarray.Conclusion The upregulation of genes in the MAPK signal pathway may be the key events that contribute to right ventricular hypertrophy and stunted angiogenesis in patients with TOF.

A cotton mitogen-activated protein kinase (GhMPK6) is involved in ABA-induced CAT1 expression and H_2O_2 production

The mitogen-activated protein kinase （MAPK） cascade is one of the a pivotal role in the regulation of stress and developmental signals in plants. major and evolutionally conserved signaling pathways and plays Here, we identified one gene, GhMPK6, encoding an MAPK protein in cotton. GFP fluorescence assay demonstrated that GhMAPK6 is a cytoplasm localized protein. Quantitative RT-PCR analysis revealed that mRNA accumulation of GhMPK6 was significantly promoted by abscisic acid （ABA）. Overexpression of GhMPK6 gene in the T-DNA insertion mutant atmkkl （SALK_015914） conferred a wild-type phenotype to the transgenic plants in response to ABA. Under ABA treatment, cotyledon greening/expansion in GhMPK6 transgenic lines and wild type was significantly inhibited, whereas the atmkkl mutant showed a relatively high cotyledon greening/expansion ratio. Furthermore, CAT1 expression and H2O2 levels in leaves of GhMPK6 transgenic lines and wild type were remarkably higher than those of atmkkl mutant with ABA treatment. Collectively, our results suggested that GhMPK6 may play an important role in ABA-induced CAT1 expression and H2O2 production.

Neuroprotective Effects of Raloxifene on Aβ_(25-35)-induced Damages in PC12 Cells via Mitogen-activated Protein Kinase Signaling Pathway

Objective To investigate the neuroprotective effects and the mechanism of this protection of raloxifene （RLX）, a selective estrogen receptor modulator.Methods MTT assay and flow cytometry with annexin V-FITC/PI staining were performed to evaluate the neuroprotective effects of RLX on Aβ25-35-induced toxicity. The potential mechanisms were studied by Western blotting in cultured rat pheochromocytoma cells （PC12 cells）.Results RLX（1 000 nmol/L）, in combination with Aβ25-35 （30 llmol/L）, increased the cell viability （P 〈0.001）, and reduced the number of apoptotic cells （P 〈0.05）. RLX attenuated Aβ25-35-induced loss of △ψm （P 〈0.01）. The changing of △ψm was similar to the variation of apoptosis. PD98059 （inhibitor of ERK1/2） inhibited the effects of RLX on cell viability and phosphorylation of cleaved caspase-9. No significant difference of cell viability or phosphorylation of cleaved caspase-9 had been found when PC12 cells were incubated with SB203580 （inhibitor of p38MAPK） or SP600125 （inhibitor of JNK）. Afl25.35 induced a time-dependent phosphorylation of p38MAPK and JNK. In PC12 cells treated solely with RLX, ERK1/2 was activated （P〈0.01）. In PC12 cells treated with Aβ25-35 and RLX, Aβ2545-induced phosphorylation of p38MAPK and JNK were inhibited （P〈0.01 and P〈0.001, respectively）.Conclusion RLX inhibited Af125.35-induced cell apoptosis by activating the ERK1/2 pathway in PC12 cells. RLX also attenuated Aβ25-35-induced activation of p38MAPK and JNK. The mitochondria pathway Was involved in this inhibitory effect.

Arabidopsis RNA polymerase Ⅱ C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity

Mitogen-activated protein kinase(MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1(C-terminal domain phosphatase-like 1)as a negative regulator of microbe-associated molecular pattern(MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide fg22. Furthermore, fg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with fg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefy luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.

水稻MAPK基因Os MPK4的克隆鉴定、蛋白表达和转基因载体构建

克隆鉴定了一个水稻促分裂原活化蛋白激酶（mitogen-activated protein kinases, MAPK）基因OsMPK4．OsMPK4基因cDNA全长1483bp，包含一个1131bp的开放阅读框，编码一个由376个氨基酸组成的蛋白，预测分子量为42．8kD．OsMPK4基因位于水稻第10号染色体上，由6个外显子和5个内含子组成．OsMPK4蛋白具有MAPK的11个保守结构域及磷酸化位点TEY模体．系统进化树分析表明，OsMPK4属于B组MAPK成员，与已知水稻MAPK蛋白有56％～74％的一致性．原核表达了OsMPK4基因，纯化获得重组OsMPK4融合蛋白，并构建OsMPK4的转基因双元载体，用于OsMPK4的生化功能及其生物学功能研究．

线粒体融合蛋白2及Ras-MAPK信号通路与乳腺癌的研究进展

线粒体融合蛋白2(Mfn2)是一种高度保守的跨膜GTP酶,由新鉴定的抑癌基因Mfn2编码,它的抗肿瘤作用跟丝裂原活化蛋白激酶(MAPK)信号通路有关。MAPK是Ras信号通路下游的一个主要支路,在细胞恶变和肿瘤浸润、转移过程中起着重要作用。本文对Mfn2的分子结构、调控机制及其在乳腺癌中与Ras-MAPK信号通路关系的最新研究进展作一综述。

Down-regulation of HIV-1 Infection by Inhibition of the MAPK Signaling Pathway

The human immunodeficiency virus type 1 （HIV-1） can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase （MAPK） signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase （ERK） pathway inhibitor, PD98059, the Jun N-terminal kinase （JNK） pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1NL4-3 luciferase reporter virus and HIV-1NL4-3 virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-INL4-3 luciferase reporter virus inhibition activity but no HIV-1NL4-3 virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.

Genome-wide evolution of MAPKs family and their expression in response to bacterial infection in seahorse Hippocampus erectus

Seahorses have evolved many unique biological traits,including a male brood pouch,the absence of caudal and pelvic fins,and the lack of spleen and gut-associated lymphatic tissue.The mitogenactivated protein kinases(MAPKs)are known to be involved in various important biological processes including growth,differentiation,immunity,and stress responses.Therefore,we hypothesized that the adaptive evolution and expression of the MAPK gene family in seahorse may differ from those of other teleost species.We identified positive selection sites in the erk2,erk5,jnk1,and p38αMAPK genes of the lined seahorse Hippocampus erectus and tiger-tailed seahorse Hippocampus comes.A novel expression profile of MAPK cascade genes was found in seahorse larvae during the first day after birth based on the RNA-seq data of H.erectus,which refl ected vital signs of immune response to its parental immune system.The expression patterns of the four positively selected MAPK genes were analyzed following the bacterial challenge of Vibrio fortis,revealing their upregulation pattern in brood pouch and other immune tissues.This study enriched our knowledge of the evolution of the H.erectus MAPK subfamilies,and could help better understanding the functional role of MAPKs in teleosts.

Transthyretin—A Key Gene Involved in Regulating Learning and Memory in Brain, and Providing Neuroprotection in Alzheimer Disease via Neuronal Synthesis of Transthyretin Protein

Transthyretin (TTR), a carrier protein present in the liver and choroid plexus of the brain, has been shown to be responsible for binding thyroid hormone thyroxin (T4) and retinol in plasma and cerebrospinal fluid (CSF). TTR aids in sequestering of beta-amyloid peptides Aβ deposition, and protects the brain from trauma, ischemic stroke and Alzheimer disease (AD). Accordingly, hippocampal gene expression of TTR plays a significant role in learning and memory as well as in simulation of spatial memory tasks. TTR via interacting with transcription factor CREB regulates this process and decreased expression leads to memory deficits. By different signaling pathways, like MAPK, AKT, and ERK via Src, TTR provides tropical support through megalin receptor by promoting neurite outgrowth and protecting the neurons from traumatic brain injury. TTR is also responsible for the transient rise in intracellular Ca2+ via NMDA receptor, playing a dominant role under excitotoxic conditions. In this review, we tried to shed light on how TTR is involved in maintaining normal cognitive processes, its role in learning and memory, under memory deficit conditions;by which mechanisms it promotes neurite outgrowth;and how it protects the brain from Alzheimer disease (AD).

Serine659 in ClC-2-Target Site for Phosphorylation by MAPK

In order to further investigate the role of ClC-2（ClC=chloride-ion channel） played in the regulation of cell proliferation and differentiation, the capablity of ClC-2 phosphorylation catalyzed by mitogen-activated protein ki-nase（MAPK） was studied. A mutation of 659Ser to Ala（S659A） of the rabbit ClC-2 cDNA in the consensus sequence of MAPK phosphorylation was introduced by overlap extension polymerase chain reaction（PCR）. Recombinant vectors pGEX-4T-1/ClC-2-2CT and pGEX-4T-1/ClC-2CT（S659A） were constructed. They were transformed to E. coli BL21, expressed by isopropy-β-D-thiogalactoside（IPTG） induction, the recombinant proteins were subjected to purification by glutathione sepharose 4B affinity chromatography. In vitro phosphorylation of the fusion proteins catalyzed by MAPK was performed. The results show that fusion protein GST/ClC-2CT（wild type） can be phosphorylated by MAPK, and this phosphorylation can be restrained by the inhibitor p42/44MAPK, PD98095; while the phosphorylation level of fusion protein GST/ClC-2CT（S659A）（mutant） was significantly reduced. Therefore, ClC-2 can be phosphorylated by MAPK and the target site of the phosphorylation is most likely the 659Ser residue.