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1.
AIM:To analyze the subcellular localization of Arnt2 in rat cerebellar granule neurons (CGNs). METHODS:Based on the amino acids sequence of Arnt2 (LOCUS：NP_036913), the subcellular localization of Arnt2 in eukaryotic cells and the nuclear export signals (NES) of Arnt2 were predicted in CBS bioinformatics database. The subcellular localization of Arnt2 in rat cerebellar granule neurons was detected by the method of laser scanning confocal microscopy (LSM) analysis. RESULTS:It was predicted that Arnt2 located in nuclei of eukaryotic cells with the most probability, while located in cytoplasmic mitochondria with a slight possibility. A nuclear export signal was found in Arnt2 amino acids sequence, it was identified to be the leucine of No.143 that located in N-terminal of Arnt2 amino acids sequence. Finally, the result of LSM analysis shows nuclear localization of Arnt2 in rat CGNs. CONCLUSION:Arnt2 is located in nuclei of normal rat CGNs, it suggests that Arnt2 has the tendency to translocate into mitochondria after induced by some of inducible factors, for both the possibility of mitochondria localization and NES exist in Arnt2 amino acids sequence.  相似文献   

2.
AIM：To analyze the changes of Arnt2 subcellular localization in the process of cerebellar granule neurons (CGNs) apoptosis induced by low concentration of potassium.METHODS：Western blotting was used to detect the variation of Arnt2 proteins in nuclei extracts of CGNs in the process of apoptosis induced by low concentration of potassium.Laser scanning confocal microscopy (LSM) was used to detect the changes of Arnt2 subcellular localization.RESULTS：In nuclei extracts of CGNs,when treated with low concentration of potassium,Arnt2 protein was up-regulated obviously at 30 min and peaked at 1 h,returned to the level of normal control at 6 h.LSM analysis result showed that Arnt2,which located in normal CGNs nuclei,was translocated into cytoplasm after induced by low concentration of potassium gradually.At the late stage of apoptosis,Arnt2 proteins located in cytoplasm and overlapped with HSP60 completely,which was regarded as a protein of mitochondria localization.CONCLUSIONS：Arnt2 protein translocates from nuclei into cytoplasm (probably into mitochondria) during CGNs apoptosis evoked by low concentration of potassium;suggesting that Arnt2 is involved in apoptosis probably by transmitting apoptotic or survival signals evoked by potassium directly.  相似文献   

3.
AIM: To observe the expression of neuronal Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) involved in neuronal apoptosis evoked by low K+ and to investigate the relationship between ARNT2 and neuronal apoptosis. METHODS: After neuron apoptosis model was established, the changes of mRNA and protein of ARNT2 during apoptosis were investigated by RT-PCR and Western blotting, respectively. Immunofluorescence was analyzed by confocal microscopy to probe the subcellular localization of ARNT2. RESULTS: Induced by low K+, the expression of ARNT2 mRNA was up-regulated obviously at the point of 30 min, and peaks at the point of 1 h. This up-regulated expression lasted for 12 h, and the variation of ARNT2 protein was similar to that of mRNA. The results of immunofluorescence analyzed by confocal microscopy showed that the localization of ARNT2 protein was in the nucleus. CONCLUSION: ARNT2 locate in nuclei of normal cerebellar granule neurons of rat. During the process of apoptosis evoked by low K+, both mRNA and protein of ARNT2 are overexpressed.  相似文献   

4.
AIM: To screen and identify the differentially expressed genes in lymphocytes of patients with unstable angina in order to find the molecular mechanism of unstable angina . METHODS: Suppression subtractive hybridizations (SSH) and dot blot hybridizations were performed to screen the relatively differentially expressed genes in lymphocyte RNA between the patients with unstable angina pectoris and stable angina pectoris. The obtained expressed sequence tags (ESTs) were used as probes to perform Reverse Northern blot with forward and reverse suppression products. And the positive ESTs were performed RNA slot hybridization with unstable and stable angina group. The obtained ESTs were sequenced and analyzed using BLAST (nr) at NCBI. RESULTS: Three up-regulated ESTs in the unstable angina group, and one down-regulated EST in the stable angina group were obtained. All of them are sequences of known genes. CONCLUSION: All these ESTs may be associated with the unstablization of plaque of coronary artery in patients with unstable angina.  相似文献   

5.
利用SSH分离TDZ诱导金钗石斛成花相关基因   总被引：1，自引：0，他引：1  
闻真珍  张娥  刘运权  刘伟 《园艺学报》2013,40(8):1591-1599
 以金钗石斛（Dendrobium nobile Lindl.）腋芽为材料,利用抑制消减杂交（SSH）技术构建了TDZ处理后金钗石斛腋芽的正向和反向SSH文库。通过PCR和反向Northern杂交验证后,得到的正向文库的314个阳性克隆和反向文库的59个克隆测序后经过聚类,最终得到39条受TDZ正向调控的EST序列,以及21条受其反向调控的EST序列。对其中某些差异基因进行半定量RT-PCR,结果显示这些基因的表达受TDZ的影响。利用Blastn和Blastx进行比对分析,其中20个没有同源序列,属于未知功能基因。其余序列的同源基因编码的产物参与包括光合作用、合成代谢、转录调控等多种生物学过程。在反向文库中分离到1个MADS-box基因的同源序列和1个Sos同源基因,而正向文库中存在多个反转录转座子,这些结果表明TDZ可以影响开花过程中的某些转录因子和信号基因的表达,进而促进金钗石斛开花。  相似文献   

6.
紫外线照射对梁平柚果皮基因表达的影响   总被引：1，自引：0，他引：1  
 采用SSH技术以紫外照射的梁平柚[Citrus grandis（L.）Osbeck]果皮作为实验方（tester）,以未被照射的正常果皮作为驱动方（driver）,构建了一个梁平柚果皮紫外诱导基因的正向差减文库。经菌液PCR检测后随机挑取200个阳性克隆测序,获得168条表达序列标签（ESTs）。比对这168条ESTs,发现有分属于57个基因的114条ESTs与已知基因高度同源,54条ESTs同源性较低或没有同源性。功能分析发现,这些ESTs主要参与抗逆防御、生长发育、细胞凋亡、转录与翻译、细胞分化、信号传导、能量代谢、糖类及氨基酸代谢以及次生代谢等。  相似文献   

7.
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8.
AIM: To assess the effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells (VSMCs). METHODS: Wistar rats were ovariectomized and subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). 2 weeks after implantation, the expression of caveolin-1 gene in endothelium-denuded aortic tissue was examined by RT-PCR. Furthermore, Northern blotting was used to analyze the mRNA expression of caveolin-1 in cultured rat VSMCs. RESULTS: RT-PCR showed that expression of caveolin-1 gene was significantly higher in OVX+E group than that in OVX+V group. Northern blot analysis showed that the mRNA expression of caveolin-1 was higher in VSMCs pretreated with 17β-estradiol (17β-E2) than that in VSMCs without 17β-E2 pretreatment. CONCLUSION: Estrogen up-regulates the gene expression of caveolin-1 in the vascular wall, partially indicating the cardiovascular effect of estrogen.  相似文献   

9.
ZOU Fei  XIAO Jun  CAI Shao-xi  JIN Yue 《园艺学报》2005,21(10):2050-2053
AIM: To reveal the molecular mechanisms of heat adaptation by study the change of gene expression in rat liver when the rats exposed to hyperthermia stress and heat adaptation. METHODS: Differentially expressed genes in rat liver in association with heat adaptation were identified using the suppression subtractive hybridization (SSH) technique to prepare subtracted cDNA libraries in heat adaptation. The PCR-amplified cDNA fragments generated by SSH were then ligated into the plasmid pGEM-T (Promega). PCR-select differential screening technique was used to filtrate differentially expressed genes. Each of these differentially expressed genes was sequenced and was compared to known sequences in the GenBank database. RESULTS: eight up-regulating expressed genes segments were isolated from the up-regulating differentially expressed library, three of which were known gene such as p53 and five genes were new expressed sequence tags. CONCLUSION: Up-regulating expressed genes such as p53 maybe participate in the signal transduction pathway of heat adaptation.  相似文献   

10.
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11.
AIM: To clone the full-length of 75A EST. METHODS: After the extraction of total RNA from primary cultured rat cerebellar granule cell of 7DIV in the medium containing 25 nmol/L KCl,T4 DNA ligase-me-RESULTS: The first round of 5'RACE produce a 2.5 kb band, and 75A EST was identified to be partial sequence of Neuron-derived orphan receptor (Nor1) gene. After two more rounds RACE, we firstly cloned the full-length of Nor-1 cDNA. diated 5’RACE was used to retrieve 5’unknown sequence of 75A EST, and the first round 5’RACE PCR product was subcloned into pGEM-T easy vector for sequence and homogeneous analysis.CONCLUSION: T4 DNA ligase mediated 5'RACE is an efficient method to retrieve information about the 5' termini of mRNAs, and lay a foundation for further study which role Nor1 play in the cerebellar granule cell's differentiation or survive.  相似文献   

12.
AIM: Centrifuge training can improve forward acceleration (+Gz) endurance. This study was to analyzed the gene expression of rat heart affected by centrifuge, and to research the molecular mechanism of improving+Gz endurance by centrifuge training. METHODS: Differential expressed genes between high+Gz endurance (+16Gz) rats, of test group after trained12 d and control were screened using suppression subtractive hybridization (SSH) and dot blot hybridization. The obtained expressed sequence tags (ESTs)were used as probes to perform RNA slot hybridization with heart total RNA isolated from each gruop of centrifuge training and high+Gz endurance and low+Gz endurance (+12Gz) rats, respectively. The positive ESTs were sequenced and analyzed using BLAST(nr) at NCBI.RESULTS: Three down-regulated ESTs were obtained from heart samples, all of them are new, and their expression levels were decreasing during centrifuge training. CONCLUSION: Centrifuge training can significantly affect the special gene expressions of rat heart, and the expression changes of these genes may be ralated to the mechainism that+Gz endurance can be improved by centrifuge training.  相似文献   

13.
 为分析柑橘果实贮藏过程中枯水相关特异基因的表达情况,以纽荷尔脐橙（Citrus Sinensis Osbeck）采摘后贮藏至略见枯水果实的果肉为测验方（Tester）,以刚采摘果实的果肉为驱动方（Driver）,采用抑制性差减杂交（Suppression Subtractive Hybridization,SSH）技术成功构建了贮藏果实差减cDNA文库,共获得656个阳性克隆。随机挑取克隆进行菌液PCR分析,插入片段长度主要集中在100 ~ 1 000 bp。成功对213个克隆进行测序,获得有效序列143条,经序列分析,共得到53条uniESTs序列。在NCBI基因库中对这53个uniESTs进行BLAST分析,比对结果参照MIPS的分类标准,按功能分为14类,其中参与代谢、能量及蛋白合成的ESTs最多,分别占到了全部ESTs的17%、11%和13%。  相似文献   

14.
AIM: To explore the possible mechanism of tert-butyl hydroperoxide (t-BHP)-induced apoptosis in rat cortical neurons. METHODS: Primary cultured rat cortical neurons were performed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cell apoptosis and mitochondrial transmembrane potential (ΔΨm) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer. Bcl-2 and Bax protein, cytosolic cytochrome c, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were detected by Western blotting. RESULTS: After exposure of cortical neurons to tBHP (25-400 μmol/L), the cell viability was reduced. ΔΨm and cellular GSH content were also decreased significantly. The level of Bcl-2 protein was reduced and Bax was elevated. Meanwhile, tBHP exposure resulted in cytochrome c release, caspase-3 and PARP proteolysis, DNA fragmentation and eventually neuron apoptosis. CONCLUSION: Mitochondrial damage may mediate tBHP- induced apoptosis in cortical neurons.  相似文献   

15.
AIM: To obtain differentially expressed cDNA fragments in the cerebellum of rats and screen unknown expressed sequence tag (EST). METHODS:Suppression subtractive hybridization (SSH) was carried out, in which the cDNA fragments of cerebellum were taken as "tester" and correspondingly that of cerebrum and brain stem as "driver". The homogeneous sequences between the tester and driver were excluded and the rare sequences in cerebellum were enriched by SSH. The differentially expressed cDNA fragments were further cloned for the construction of subtracted cDNA libraries and sequencing. RESULTS: 32 clones were selected and 34 cDNA fragments were sequenced. 8 of 32 were proved to be true positive with reverse Northern assay, 13 of 34 fragments were identified to be new cDNA fragment and given the gene sequence numbers by GenBank (AW288461-AW288474).CONCLUSION:SSH is very useful method for screening differentially expressed genes.Our data may be helpful to understand the molecular mechanism of brain function.  相似文献   

16.
AIM:To investigate the influence of ovariectomy and estrogen replacement treatment on profile of gene expression in myocardium by cDNA microarray,and to characterize the targeting genes of estrogen.METHODS:cDNA microarray containing 1 400 rat cDNAs was used to study the genes differentially expressed in myocardium between sham (Ⅰ),ovariectomy (Ⅱ,OVX) and estrogen replacement treatment (Ⅲ,OVX+E2) group.Then down-regulated genes in myocardium of OVX rats were further confirmed by RT-PCR.RESULTS:177 genes were differentially expressed in myocardium between sham and OVX rats,with 91 genes up-regulated and 86 genes down-regulated in OVX rats.164 genes were differentially expressed in myocardium between OVX and OVX+E2 rats,with 113 genes up-regulated and 54 genes down-regulated in OVX rats.There were 54 genes differentially expressed in OVX compared to sham and OVX+E2.They are involved in membrane channels and transporters (18),cell receptors (9),intracellular transducers/effectors/modulator (7) and metabolism (6).Most of the genes (45) were down-regulated in OVX rats and up-regulated in OVX+E2 rats.RT-PCR test confirmed the results of cDNA microarray.CONCLUSIONS:Long-term estrogen replacement may influence the expression of genes involved in membrane channels and transporters,cell receptors,intracellular transducers/effectors/ modulator and metabolism.Long-term estrogen replacement has some beneficial effects on ionic concentration and cardiac function which partially comes from the results of influence of expression on Na+,K+-ATPase and Na+/H+ exchanger.Estrogen has an inhibitory effect on the expression of dopamine receptor,which partially clarify the myocardial protection of estrogen.  相似文献   

17.
郭亚鹤  赵祯  张映  连勇  陈钰辉  刘富中 《园艺学报》2017,44(10):1914-1925
利用实时荧光定量PCR技术,研究茄子单性结实SSH-c DNA文库中的96条EST序列在单性结实自交系和非单性结实自交系果实发育过程中的表达模式。分析表明,在低温条件下,相对表达量有显著差异的EST序列有31条,总体上调表达的EST序列有17条,总体下调表达的EST序列有14条。通过NCBI对EST序列进行Blastx比对,得到与其同源性高的序列信息。其中5条EST与抵御低温相关,6条EST与植物激素代谢相关,多条EST与植物代谢过程中的蛋白质和碳水化合物合成相关,1条EST无比对结果,可能为新基因。差异表达序列可作为研究茄子单性结实和耐低温的候选基因。  相似文献   

18.
AIM: To study the effect and mechanism of humanin (HN) on inhibition of excitatory neurotoxicity induced by N-methyl-D-asparate(NMDA). METHODS: Cortical neurons from newborn SD rat were primarily cultured. The purity of the cells was assessed by immunofluorescence technique. The neurons were randomly divided into normal group, NMDA group, NG-nitro-L-arginine methyl ester(L-NAME) group and HN group. The activity of the neurons was detected by MTT assay. Nitric oxide (NO) concentration was detected by NO detection kit, and the neuronal apoptosis was examined by Hoechst staining. The expression of p-p38 MAPK in the neurons was determined by Western blotting. RESULTS: Immunofluorescence test showed that 90% cells were neuron-specific enolase(NSE)-positive staining cells. The activity of the neurons in L-NAME group and HN group was lower than that in normal group, but higher than that in NMDA group. NO concentration, the numbers of apoptotic cells, and the expression of p-p38 MAPK in L-NAME group and HN group were higher than those in normal group, but lower than those in NMDA group. Compared with HN group, the activity of the neurons decreased, the numbers of apoptotic cells and the expression of p-p38 MAPK increased in L-NAME group. CONCLUSION: HN inhibits the apoptosis of neurons partly by inhibiting the NO toxicity, thus protecting the neurons.  相似文献   

19.
AIM: To observe the role of cyclooxygenase-2 (COX-2) in injury induced by hypoxia and reoxygenation in cultured rat cortical neurons and protective effects of COX-2 specific inhibitor NS398.METHODS: Primary rat cortical neuronal cells were cultured. Experiments were divided into control group, hypoxia/reoxygenation group and hypoxia/reoxygenation with COX-2 inhibitor group. Cell viability was measured by MTT assay. COX-2 protein expression was examined by Western blotting. Apoptosis was measured by DNA agarose electrophoresis.RESULTS: The expression levels of COX-2 increased significantly after neurons were treated with hypoxia and reoxygenation, compared with control group and hypoxia/reoxygenation with COX-2 inhibitor group (P＜0.05). COX-2 specific inhibitor NS398 protected neurons from death (P＜0.05 and P＜0.01), DNA fragmentation analysis showed DNA fragmentation was inhibited significantly by NS398.CONCLUSION: COX-2 is involved in the pathogenesis of neuron apoptosis induced by hypoxia/reoxygenation. COX-2 specific inhibitor significantly protects cortical neurons against hypoxia/reoxygenation injury and inhibits apoptosis induced by hypoxia.  相似文献   

20.
大花蕙兰授粉后子房cDNA差异表达片段序列分析   总被引：1，自引：0，他引：1  
 应用mRNA差异显示(DDRT-PCR) 方法比较分析了大花蕙兰(Cymbidium hybridium ) 授粉后2 d与未授粉子房的基因表达差异。结果分离到了7个在授粉后子房中特异表达的cDNA片段, 分别命名为CDD-313, CDD-272, CDD-265, CDD-243, CDD-193, CDD-218,CDD-470。在GenBank中进行同源性比较发现前4个没有同源序列与之匹配; 后3个片段推导的氨基酸序列分别与ABC型transporter, GTPase和40S核糖体蛋白质S3 (RPS3) 有高度同源性。反向Northern杂交进一步证实它们存在, 并在授粉子房中高度表达。其中, 最为有意义的是CDD-470, 其推定的氨基酸与参与细胞生长分化的植物源多功能蛋白RPS3高度同源性, 进而推测其与RPS3有相似的功能, 可能与兰花子房发育有关, 这为解释花发育分子机制提供了新资料。  相似文献   

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