检索结果-内蒙古大学图书馆

Magnetic intracellular labeling of mammalian cells by combining (FDA-approved) superparamagnetic iron oxide MR contrast agents and commonly used transfection agents

引用

ACADEMIC RADIOLOGY 2002年第Sup2期9卷 S484-S487页

作者： Frank, JA Zywicke, H Jordan, EK Mitchell, J Lewis, BK Miller, B Bryant, LH Bulte, JWM NIH Expt Neuroimaging Sect Lab Diagnost Radiol Res Bethesda MD 20892 USA

来源：评论

学校读者我要写书评

暂无评论

Mapping the agonist binding site of the nicotinic acetylcholine receptor - Orientation requirements for activation by covalent agonist

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第17期275卷 12651-12660页

作者： Sullivan, DA Cohen, JB Harvard Univ Sch Med Dept Neurobiol Boston MA 02115 USA

To characterize the structural requirements for ligand orientation compatible with activation of the Torpedo nicotinic acetylcholine receptor (nAChR), we used Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to tether primary or quaternary amines at defined positions within the agonist binding site of nAChRs containing mutant alpha- or gamma-subunits expressed in Xenopus oocytes, 4-(N-Maleimido)benzyltrimethylammonium and 2-aminoethylmethanethiosulfonate acted as irreversible antagonists when tethered at alpha Y93C, alpha Y198C, or gamma E57C, as well as at alpha N94C (2-aminoethylmethanethiosulfonate only), [2-(Trimethylammonium)ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine to binding site Cys, also acted as an irreversible antagonist when tethered at alpha Y93C, alpha N94C, or gamma E57C. However, MTSET modification of alpha Y198C resulted in prolonged activation of the nAChR not reversible by washing but inhibitable by subsequent exposure to non competitive antagonists. Modification of alpha Y198C (or any of the other positions tested) by [(trimethylammonium)methyl]methanethiosulfonate resulted only in irreversible inhibition, while modification of alpha Y198C by [3-(trimethylammonium)propyl]methanethiosulfonate resulted in irreversible activation of nAChR, but at lower efficacy than by MTSET, Thus changing the length of the tethering arm by less than 1 Angstrom in either direction markedly effects the ability of the covalent trimethylammonium to activate the nAChR, and agonist activation depends on a very selective orientation of the quaternary ammonium within the agonist binding site.

关键词：乙酰胆碱/代谢亲和力标记物/药理学烷化作用结合部位半胱氨酸/化学剂量效应关系药物甲磺酸乙酯/类似物和衍生物甲磺酸乙酯/药理学指示剂和试剂/药理学动力学甲磺酸盐类/药理学模型化学诱变烟碱激动剂/化学膜片钳术蛋白质结构四级季胺类化合物/代谢季胺类化合物/药理学受体烟碱/化学时间因素转录遗传爪蟾属/胚胎学动物

来源：评论

学校读者我要写书评

暂无评论

Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第33期275卷 25585-25594页

作者： Katsumi, A Tuley, EA Bodó, I Sadler, JE Washington Univ Sch Med Howard Hughes Med Inst Dept Med St Louis MO 63110 USA Washington Univ Sch Med Dept Biochem & Mol Biophys St Louis MO 63110 USA

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines, Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), CyS2010 and Cys(2048). BY partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (1-4), Cys(1987)-Cys(2041) (2-5), Cys(1991)-Cys(2045) (3-6), and Cys(1976)- Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.

关键词：丙氨酸/化学烷化作用氨基酸序列 COS细胞色谱法高压液相计算机模拟溴化氰/药理学胱氨酸/化学二聚作用二硫化物类二硫苏糖醇/药理学乙基马来酰亚胺/药理学糖基化指示剂和试剂/药理学质谱分析法分子序列数据诱变定点磷化氢类/药理学质粒/代谢蛋白质结构三级吡啶类/药理学重组蛋白质类/化学序列分析蛋白质序列同源性氨基酸巯基试剂/药理学嗜热菌蛋白酶/药理学时间因素 vonWillebrand因子/化学 vonWillebrand因子/遗传学动物人类

来源：评论

学校读者我要写书评

暂无评论

Analysis of N-linked oligosaccharides released from glycoproteins separated by two-dimensional gel electrophoresis

引用

ANALYTICAL BIOCHEMISTRY 2000年第1期284卷 49-59页

作者： Charlwood, J Skehel, JM Camilleri, P SmithKline Beecham Pharmaceut Harlow CM19 5AW Essex England

Protocols have been developed for the characterization of carbohydrate covalently attached (N-linked) to an asparagine residue in glycoproteins, after separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Mixtures of proteins teach at a level from 0.5 to 50 mu g) were resolved in the first dimension according to their isoelectric points (pI, followed by separation in the orthogonal axis on the basis of their molecular weights. Glycans were released directly from excised gel spots after digestion with PNGase F, with or without prior treatment with trypsin. In a third method, glycoproteins were electroblotted onto poly(vinylidene difluoride) before glycans were released by PNGase F. For all these procedures profiles of the neutral and sialic acid-containing oligosaccharide mixtures were obtained after derivatization with 3-acetamido-6-aminoacridine, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and/or high-performance liquid chromatography. Potential applications to proteomics are discussed. (C) 2000 Academic Press.

关键词：色谱法高压液相色谱法液相电泳凝胶双向/方法电泳聚丙烯酰氨凝胶糖蛋白类/分析糖蛋白类/血液糖蛋白类/代谢氢离子浓度指示剂和试剂/药理学寡糖类/分析寡糖类/血液多糖类/代谢乙烯类聚合物/药理学原黄素/类似物和衍生物原黄素/药理学品红类染料/药理学光谱法质量基质辅助激光解吸电离甲胎蛋白类/化学动物小鼠

来源：评论

学校读者我要写书评

暂无评论

Intracellular sodium modulates mitochondrial calcium signaling in vascular endothelial cells

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第45期275卷 35402-35407页

作者： Sedova, M Blatter, LA Loyola Univ Stritch Sch Med Dept Physiol Maywood IL 60153 USA

We have investigated the role of extramitochondrial Na+ for the regulation of mitochondrial Ca2+ concentration ([Ca2+](m)) in permeabilized single vascular endothelial cells. [Ca2+], was measured by loading the cells with the membrane-permeant Ca2+ indicator fluo-3/AM: and subsequent removal of cytoplasmic fluo-3 by surface membrane permeabilization with digitonin. An elevation of extramitochondrial Ca2+ resulted in a dose-dependent increase in the rate of Ca2+ accumulation into mitochondria (k(0.5) = 3 muM) via the mitochondrial Ca2+ uniporter. In the presence of 10 mM extramitochondrial Na+ ([Na+](em)), repetitive application of brief pulses of high Ca2' (2-10 muM) to simulate cytoplasmic [Ca2+] oscillations caused transient increases of [Ca2+], characterized by a fast rising phase that was followed by a slow decay. Removal of extramitochondrial Naf or inhibition of mitochondrial Na+/Ca2+ exchange with clonazepam blocked mitochondrial Ca2+ efflux and resulted in a net accumulation of Ca2+ by the mitochondria. Half-maximal activation of mitochondrial Na+/Ca2+ exchange occurred at [Na+](em) = 4.4 mM, which is well within the physiological range of cytoplasmic [Na+]. This study provides evidence that Ca2+ efflux from the mitochondria in vascular endothelial cells occurs solely via Na+/Ca2+ exchange and emphasizes the important role of intracellular Nat for mitochondrial Ca2+ regulation.

关键词：苯胺化合物/药理学钙/代谢钙/药代动力学细胞系细胞膜/代谢氯硝西泮/药理学细胞质/代谢洋地黄皂甙/药理学剂量效应关系药物内皮血管/代谢荧光染料/药理学指示剂和试剂/药理学动力学显微镜检查共焦线粒体/代谢信号传导钠/药理学钠/生理学光谱法荧光呫吨类/药理学动物牛

来源：评论

学校读者我要写书评

暂无评论

FlaA1, a new bifunctional UDP-GlcNAc C₆ dehydratase C₄ reductase from Helicobacter pylori

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第45期275卷 34873-34880页

作者： Creuzenet, C Schur, MJ Lin, JJ Wakarchuk, WW Lam, JS Univ Guelph Dept Microbiol Guelph ON N1G 2W1 Canada Natl Res Council Canada Ottawa ON K1A 0R6 Canada

FlaA1 is a small soluble protein of unknown function in Helicobacter pylori, It has homologues that are essential for the virulence of numerous medically relevant bacteria. FlaA1 was overexpressed as a histidine-tagged protein and purified to homogeneity by nickel chelation and cation exchange chromatography, Spectrophotometric assays, capillary electrophoresis, and mass spectrometry analyses showed that FlaA1 is a novel bifunctional C-6 dehydratase/C-4 reductase specific for UDP-GlcNAc. It converts UDP-GlcNAc into a UDP-4-keto-6-methyl-GlcNAc intermediate, which is stereospecifically reduced into UDP-QuiNAc. Substrate conversions as high as 80% were obtained at equilibrium. The K-m and V-max for UDP-GlcNAc were 159 muM and 65 pmol/min, respectively. No exogenous cofactor was required to obtain full activity of FlaA1, Additional NADH was only used with poor efficiency for the reduction step. The biochemical characterization of FlaA1 is important for the elucidation of biosynthetic pathways that lead to the formation of 2,6-deoxysugars in medically relevant bacteria. It establishes unambiguously the first step of the pathway and provides the means of preparing the substrate UDP-QuiNAc, which is necessary for the study of downstream enzymes.

关键词：细菌蛋白质类苯甲醛类/药理学印迹法蛋白质碳水化合物脱氢酶类/化学色谱法凝胶色谱法离子交换克隆分子剂量效应关系药物电泳毛细管遗传互补测验葡萄糖/代谢螺杆菌幽门/酶学组氨酸/代谢氢裂合酶类/化学氢裂合酶类/遗传学氢裂合酶类/代谢指示剂和试剂/药理学动力学质谱分析法模型化学诱变镍/代谢氧化还原酶类/化学氧化还原酶类/遗传学氧化还原酶类/代谢蛋白质结合分光光度法底物特异性时间因素

来源：评论

学校读者我要写书评

暂无评论

The functional unit of the renal type IIa Na⁺/P_i cotransporter is a monomer

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第34期275卷 26113-26120页

作者： Köhler, K Forster, IC Lambert, G Biber, J Murer, H Univ Zurich Inst Physiol CH-8057 Zurich Switzerland

The composition of the functional unit of the rat renal type IIa Na+/P-i cotransporter (NaPi-IIa) was investigated by using two approaches based on the differential sensitivities of the wild type (WT) and mutant S460C proteins to 2-aminoethyImethanethiosulfonate hydrobromide (MTSEA), a charged cysteine modifier. Transport activity of S460C is completely blocked after incubation in MTSEA, whereas that of the WT remains unaffected. First, Xenopus laevis oocytes were coinjected with cRNAs coding for the WT and S460C in different proportions, and the transport inhibition after MTSEA incubation was assayed by electrophysiology. The relationship between MTSEA inhibition and proportion of cRNA was consistent with that for a functional monomer, Second, concatameric proteins were constructed that either comprised two WT proteins (WT-WT), two S460C mutants (S460C-S460C), or one of each (WT-S460C), Western blots of oocytes injected with fusion protein cRNA showed bands at similar to 200 kDa, whereas a main band at similar to 90 kDa was obtained for the WT cRNA alone. The kinetic properties of concatamers were the same as for the single proteins. Transport activity of the WT-WT concatamer was unaffected by MTSEA incubation, fully inhibited for S460C-S460C, but 50% inhibited for WT-S460C, This behavior was also consistent with NaPi-IIa being a functional monomer.

关键词：载体蛋白质类/拮抗剂和抑制剂载体蛋白质类/遗传学甲磺酸乙酯/类似物和衍生物甲磺酸乙酯/药理学指示剂和试剂/药理学肾/代谢分子量卵母细胞/代谢钠-磷协同转运蛋白质类钠-磷协同转运蛋白质类 Ⅱa型构效关系协同转运子爪蟾光滑动物大鼠

来源：评论

学校读者我要写书评

暂无评论

Activation of DNA-dependent protein kinase by single-stranded DNA ends

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第3期275卷 1541-1550页

作者： Hammarsten, O DeFazio, LG Chu, G Stanford Univ Sch Med Dept Med Stanford CA 94305 USA Stanford Univ Sch Med Dept Biochem Stanford CA 94305 USA

DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination, The kinase is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKCS. To define the DNA structure required for kinase activation, we synthesized a series of DNA molecules and tested their interactions with purified DNA-PKCS. The addition of unpaired single strands to blunt DNA ends increased binding and activation of the kinase. When single-stranded loops were added to the DNA ends, binding was preserved, but kinase activation was severely reduced. Obstruction of DNA ends by streptavidin reduced both binding and activation of the kinase, Significantly, short single-stranded oligonucleotides of 3-10 bases were capable of activating DNA-PKCS. Taken together, these data indicate that kinase activation involves a specific interaction with free single-stranded DNA ends. The structure of DNA-PKCS, contains an open channel large enough for double-stranded DNA and an adjacent enclosed cavity with the dimensions of single-stranded DNA. The data presented here support a model in which duplex DNA binds to the open channel, and a single-stranded DNA end is inserted into the enclosed cavity to activate the kinase.

关键词：抗原核结合竞争性/药物作用晶体学 DNA/代谢 DNA解旋酶类 DNA 单链/代谢 DNA活化蛋白激酶 DNA结合蛋白质类/代谢酶激活指示剂和试剂/药理学动力学核蛋白质类/代谢蛋白质构象蛋白质丝氨酸苏氨酸激酶/代谢抗生蛋白链菌素/药理学

来源：评论

学校读者我要写书评

暂无评论

Introduction of C3 exoenzyme into cultured endothelium by lipofectamine

引用

ANALYTICAL BIOCHEMISTRY 2000年第2期285卷 260-264页

作者： Borbiev, T Nurmukhambetova, S Liu, F Verin, AD Garcia, JGN Johns Hopkins Univ Sch Med Johns Hopkins Asthma & Allergy Ctr Div Pulm & Crit Care Med Baltimore MD 21224 USA

来源：评论

学校读者我要写书评

暂无评论

A calcium-activated cation current by an alternatively spliced form of Trp3 in the heart

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第50期275卷 39055-39060页

作者： Ohki, G Miyoshi, T Murata, M Ishibashi, K Imai, M Suzuki, M Jichi Med Sch Dept Pharmacol Minami Kawachi Tochigi 3290498 Japan

To investigate a cDNA encoding cation current, we isolated an alternatively spliced form of a rat Trp3, designated Trp3sv, Trp3sv encodes 736 amino acids with a unique N terminus and six transmembrane segments. Expression of the cRNA in Xenopus oocytes was successfully performed. The cation selective current appeared after the addition of ionomycin or induced by prolonged depolarization but not by hyperpolarization. This induction was not observed by a treatment with thapsigargin, phorbol ester, or ATP, Na+, K+, tetraethylammonium, and divalent cations were permeable, while N-methyl-glucamine and chloride were nominally impermeable ions, The currents were not inhibited by flufenamate ruthenium red but nonspecifically by 2 mM Gd3+. Northern as well as Western blot suggested lower levels of the expression observed in some organs, while reverse transcriptase-polymerase chain reaction suggested that it widely spread among various organs. Therefore, we may conclude that N-terminal spliced valiant of Trp3, Trp3sv, encodes a calcium-activated cation channel in various organs.

关键词：腺苷三磷酸/药理学选择性剪接氨基酸序列氨基酸类/化学印迹法 RNA 印迹法蛋白质脑/代谢钙/代谢钙通道/遗传学钙通道/代谢阳离子氯化物/药理学克隆分子 DNA 互补/代谢酶抑制剂/药理学钆/药理学指示剂和试剂/药理学离子霉素/药理学离子载体/药理学葡甲胺/药理学分子序列数据心肌/代谢卵母细胞/代谢膜片钳术佛波醇酯类/药理学钾/药理学逆转录聚合酶链反应钌红/药理学序列同源性氨基酸钠/药理学 TRPC阳离子通道四乙基铵/药理学毒胡萝卜内酯/药理学组织分布爪蟾属动物大鼠

来源：评论

学校读者我要写书评

暂无评论

建议与咨询留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

时间限定

文献类型

馆藏选择

核心期刊

语言

文献类型

帮助

文字说明：

检索规则说明：

检索范例：

分类表

所选分类

限定检索结果

文献类型

馆藏范围

日期分布

学科分类号

主题

机构

作者

语言

请选择保存的检索档案：

请选择收藏分类：

通借通还

建议与咨询 留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

时间限定

文献类型

馆藏选择

核心期刊

语言

文献类型

帮助

文字说明：

检索规则说明：

检索范例：

分类表

所选分类

限定检索结果

文献类型

馆藏范围

日期分布

学科分类号

主题

机构

作者

语言

请选择保存的检索档案： 新增检索档案 确定 取消

请选择收藏分类： 新增自定义分类 确定 取消

通借通还

建议与咨询留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

请选择保存的检索档案：

请选择收藏分类：