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11.
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以光敏生物素标记的R3 cDNA克隆为模型,采用正交设计的方法,对影响核酸杂交反应的主要因素:杂交反应温度,反应时间,探针浓度及甲酰胺浓度作了综合性分析。当探针浓度为800μg/L,甲酰胺浓度为10mol/L,50℃杂交反应6h,可获得最高灵敏度(10pg)。 相似文献
13.
目的:构建吗啡依赖模型大鼠脑cDNA文库.方法:建立大鼠吗啡躯体依赖模型后,取鼠脑进行总RNA的抽提、mRNA的纯化,以mRNA为模板合成第一链cDNA后用LD-PCR方法合成双链cDNA,用限制性内切酶SfiⅠA、B分别双酶切双链cDNA与pTRG载体,连接后的cDNA文库转化XL1-Blue MRF′ Kan 超级感受态并收菌,完成cDNA文库构建.结果与结论:PCR结果显示,cDNA片段连接效率>90%,cDNA插入片段在1 kb左右.吗啡依赖大鼠脑cDNA文库总容量为3.05×106,满足下一步细菌双杂交文库筛选要求. 相似文献
14.
Pharmacologic preconditioning effects: Prostaglandin E1 induces heat-shock proteins immediately after ischemia/reperfusion of the mouse liver 总被引:1,自引:0,他引:1
Ken-ichi Matsuo M.D. Shinji Togo M.D. Ph.D. Hitoshi Sekido M.D. Ph.D. Tomoyuki Morita M.D. Ph.D. Masako Kamiyama Ph.D. Daisuke Morioka M.D. Ph.D. Toru Kubota M.D. Ph.D. Yasuhiko Miura M.D. Ph.D. Kuniya Tanaka M.D. Ph.D. Takashi Ishikawa M.D. Ph.D. Yasushi Ichikawa M.D. Ph.D. Itaru Endo M.D. Ph.D. Hitoshi Goto M.D. Ph.D. Hiroyuki Nitanda M.D. Ph.D. Yasushi Okazaki M.D. Ph.D. Yoshihide Hayashizaki M.D. Ph.D. Hiroshi Shimada M.D. Ph.D. 《Journal of gastrointestinal surgery》2005,9(6):758-768
Prostaglandin E1 (PGE1) has several potential therapeutic effects, including cytoprotection, vasodilation, and inhibition of platelet aggregation. This study investigates the protective action of PGE1 against hepatic ischemia/reperfusion injury in vivo using a complementary DNA microarray. PGE1 or saline was continuously administered intravenously to mice in which the left lobe of the liver was made ischemic for 30 minutes and then reperfused. Livers were harvested 0, 10, and 30 minutes postreperfusion. Messenger RNA was extracted, and the samples were labeled with two different fluorescent dyes and hybridized to the RIKEN set of 18,816 full-length enriched mouse complementary DNA microarrays. Serum alanine aminotransferase and aspartate aminotransferase levels at 180 minutes postreperfusion were significantly lower in the PGE1-treated group than in the saline-treated group. The cDNA microarray analysis revealed that the genes encoding heat-shock protein (HSP) 70, glucose-regulated protein 78, HSP86, and glutathione S-transferase were upregulated at the end of the ischemic period (0 minutes postreperfusion) in the PGE1 group. Our results suggested that PGE1 induces HSPs immediately after ischemia reperfusion. HSPs might therefore play an important role in the protective effects of PGE1 against ischemia/reperfusion injury of the liver. 相似文献
15.
神经节细胞胶质瘤恶变及其差异表达基因分析 总被引:7,自引:3,他引:4
目的探讨神经节细胞胶质瘤恶性进展的分子变化,为进一步研究分子病因奠定基础。方法取同一患者不断恶化的3次手术标本,行cDNA微阵列检测,分析在不同阶段持续存在的差异表达基因。结果初发(WHO II级)、复发(WHO III级)和再发(WHO IV级)的标本与正常脑组织相比,差异表达基因共19条,其中下调者16条,上调者3条。在下调的基因中,功能明确者有抑制RAF1/MEK/ERKs激酶通路激活的磷酸酰乙醇胺结合蛋白,抑制肿瘤血管增生、侵袭,调控细胞凋亡的碳酰还原酶;抑制c-myc基因表达的肺库否样因子;参与人DNA切除修复的多聚酶ε。结论神经节细胞胶质瘤恶变过程中持续存在的分子变化,以抑制肿瘤增殖基因表达下调为主,其中RAF激酶抑制蛋白、DNA多聚酶ε和碳酰还原酶是参与细胞信号传导和DNA损伤修复等具重要功能的基因,值得进一步研究。 相似文献
16.
Akira Horiuchi Robin A Felder† 《Clinical and experimental pharmacology & physiology》1996,23(S3):150-154
1. In orefer to develop a simple, efficient system for the highlvel expression of dopamine recetors in eukaryotic cells, we have studied the effects of n-butyrate on the expression of rat D1A dopamine receptor cDNA in mouse fibroblast LTK- cells as compared with those of n-butyrate on endogenous D1 receptor levles in opossum kidney cells.
2. In the transfected LTK- cell mebranes with pRc/CMVD1A receptor cDNA, a selective D1 dopamine antagonist, [3 H]-SCH 23390, exhibited a Kd of 0.9 ± 0.1 nmol/L and a Bmax of 0.35 ± 0.05 pmol/mg protien (n = 5).
3. Addtion of n-butrate (2–10 mmol/L) to the culture medium for 48h dose-dependently increased the D1A receptor level up to 1.5 ± 0.3 pmol/mg protien (n = 7), although the Kd values were not affected. The increase in receptor level was accompanined by an elevation of selective D1 agoinist-induced adenyly cyclase activity.
4. In contrast, n-butyrate treatment (2–10 mmol/L) did not affect either endogenous D1 receptor levels or fendoldopmainduced adenylyl cyclase activity in possum kidney cells.
5. These results sugges n-butyrate is a sueful tool for obtaining high-level expression of D1A dopamine receptor cDNA in mouse fibroblast LTK- cells. 相似文献
2. In the transfected LTK- cell mebranes with pRc/CMVD
3. Addtion of n-butrate (2–10 mmol/L) to the culture medium for 48h dose-dependently increased the D
4. In contrast, n-butyrate treatment (2–10 mmol/L) did not affect either endogenous D
5. These results sugges n-butyrate is a sueful tool for obtaining high-level expression of D
17.
AstudyonimprovementofexpressionofhumanG-CSFcDNAtransferredwithretroviraldouble-copyvectorGuoBaoyu(郭葆玉);ZhangSuying(张淑英);XuHui... 相似文献
18.
高密度cDNA微阵列技术检测乳腺癌差异表达基因 总被引:1,自引:1,他引:0
目的 描绘乳腺癌的基因差异表达谱,以寻找乳腺癌新的肿瘤相关候选基因和分子标志。方法 用含14000个cDNA克隆的表达微阵列检测乳腺癌组织、乳腺癌旁组织及乳腺非癌组织的mRNA表达,并分析其表达的差异。结果 乳腺癌组织与乳腺非癌组织比较有80个差异表达基因,乳腺癌组织与乳腺癌旁组织比较有57个差异表达基因,乳腺癌旁组织与乳腺非癌组织比较有29个差异表达基因,在乳腺癌组织与乳腺非癌组织和乳腺癌旁组织比较中有14个差异表达基因是一致的。结论 用微阵列技术对乳腺癌的肿瘤相关基因和分子标志作初步探索,为了解中国女性乳腺癌发生发展的分子机制、发展新的诊断和治疗手段提供了信息。 相似文献
19.
Proteolytic Processing Mechanisms in the Biosynthesis of Neuroendocrine Peptides: The Subtilisin-like Proprotein Convertases 总被引:1,自引:0,他引:1
Yves Rouill Stephen J. Duguay Kaare Lund Machi Furuta Qiuming Gong Gregory Lipkind Anthony A. Oliva Jr. Shu Jin Chan Donald F. Steiner 《Frontiers in neuroendocrinology》1995,16(4)
The recent discovery of a novel family of precursor processing endoproteases has greatly accelerated progress in understanding the complex mechanisms underlying the maturation of prohormones, neuropeptides, and many other precursor-derived proteins. At least six members of this family have been found thus far in mammalian species, several having alternatively spliced isoforms, and related enzymes have been identified in many invertebrates, including molluscs, insects, nematodes, and coelenterates. The proprotein convertases are all dependent on calcium for activity and all possess highly conserved subtilisin-like domains with the characteristic catalytic triad of this serine protease (ordered Asp, His, and Ser along the polypeptide chain). Two members of this family, PC2(SPC2) and PC1/PC3(SPC3), appear to play a preeminent role in neuroendocrine precursor processing. Both convertases are expressed only in the brain and in the extended neuroendocrine system, while another important family member—furin/PACE (SPC1)—is expressed more ubiquitously, in almost all tissues, and at high levels in liver. SPC2 and SPC3 exhibit acidic pH optima and other properties which enhance their activity in the acidic, calcium-enriched environment of the dense-core secretory granules of the regulated pathway in neuroendocrine cells, while furin has a neutral pH optimum and is localized predominantly to the trans Golgi network where it is retained by a C-terminal transmembrane domain. Furin processes a wide variety of precursors in the constitutive pathway, such as those of growth factors, receptors, coagulation factors, and viral glycoproteins. Recent findings on the processing of proopiomelanocortin, proinsulin, proglucagon, and several other neuroendocrine precursors by SPC2 and SPC3 are discussed, along with information on the structure, properties, evolution, developmental expression, and regulation or the convertases. An inherited defect in the fat/fat mouse which affects the processing of proinsulin, and probably also many other prohormones, due to a point mutation in carboxypeptidase E has recently been identified and has begun to provide new insights into the functional integration of the individual processing steps. 相似文献
20.
S. Iwamoto E. Kajii T. Omi T. Kamesaki Y. Akifuji S. Ikemoto 《European journal of haematology》1993,50(5):286-291
Abstract: A patient who represented acute hemolytic crisis was studied. Analysis of the erythrocyte membrane proteins by SDS-PAGE revealed a deficiency of band 4.2. In the family, the sister of the patient who had been clinically normal was also shown to be deficient in band 4.2. Binding studies showed that the propositus' membranes were able to bind normal band 4.2 protein as much as control. It was suggested that the binding sites for the protein were prepared on the membrane. We analyzed the band 4.2 cDNA of the propositus and detected a mutation that changes a codon for alanine to one for threonine at residue 142. Band 4.2 exon III of genomic DNA which included the mutation site was amplified and sequenced directly in the family members, and it was revealed that only the homozygotes of the mutation allele manifested band 4.2 deficiency and the parents, who were heterozygotes, showed normal amounts of band 4.2. Recently, the same mutation was reported as Protein 4.2NIPPON in another 4 cases (Bouhassira et al. Blood 1992: 79: 1846–1854). This study supports the hypothesis that this mutation is the pathogenetic cause of band 4.2 deficiency and not a polymorphism. 相似文献