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Crystallographic titration of cubic insulin crystals:: pH affects GluB13 switching and sulfate binding

ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2003年第4期59卷 670-676页

作者： Diao, JS Florida State Univ Inst Mol Biophys Tallahassee FL 32306 USA

Structures of porcine insulin crystals soaked in 1 M sodium sulfate at pH 5.00, 5.53, 5.80, 6.00, 6.16, 6.26, 6.35, 6.50, 6.98 and 9.00 have been determined at between 1.7 and 1.9 Angstrom resolution. GluB13 exhibits a single conformation at pHless than or equal to5.80, two conformations between pH 6.00 and 6.98 and a single conformation at pH 9.00. Between pH 6.00 and 6.98, the conformation of GluB13 switches from one rotamer to another rotamer. Between pH 6.16 and 6.26, PheB1 undergoes a significant conformational change. By pH 9.00 many residues have undergone relatively large shifts and HisB10 exhibits a double conformation. As a result of the pH increase, the occupancy of the sulfate ion decreases from a maximum of 1.00 at pH 5.00 to a minimum of 0.46 at pH 6.50. Comparison of the structures, the observed and calculated structure factors and map correlation coefficients indicate that the porcine insulin structure changes gradually as a function of pH.

关键词：结晶谷氨酸/化学氢离子浓度胰岛素/化学模型分子蛋白质结合蛋白质构象硫酸盐类/化学硫酸盐类/代谢猪 X线衍射/统计学和数值数据动物

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Structural and functional changes in heparan sulfate proteoglycan expression associated with the myofibroblastic phenotype

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AMERICAN JOURNAL OF PATHOLOGY 2003年第3期162卷 977-989页

作者： Thomas, G Clayton, A Thomas, J Davies, M Steadman, R Univ Wales Coll Med Inst Nephrol Cardiff CF14 4XN S Glam Wales

The principal cells implicated as the source of the extracellular matrix in areas of progressive fibrosis are fibroblasts with the phenotypic appearance of myofibroblasts. This report describes differences in heparan sulfate proteoglycan expression between myofibroblasts and normal fibroblasts, associated with impaired responses to fibroblast growth factor-2 (FGF-2). Although both cell types responded to platelet-derived growth factor, myofibroblasts, unlike fibroblasts, did not proliferate to FGF-2. A response was acquired, however, when myofibroblasts were incubated with FGF-2 in the presence of heparan sulfate (HS) and heparin. Selective digestion with pronase, NaOH/NaBH4, heparinase I, or low pH nitrous acid showed that each HS-glycosaminoglycan region comprised a pronase-resistant peptide separating two HS chains. The HS-glycosaminoglycan chains from myofibroblasts were larger (K-av, 0.32;molecular weight, 50 kd) than those from fibroblasts (K-av, 0.4;molecular weight, 33 kd), although their disaccharide composition was identical. The chains from myofibroblasts, however, contained three, compared to two, heparinase 1-resistant sequences separated by larger contiguous areas of low sulfation. Furthermore, although there was no difference in FGF-2-binding affinity between the two cell types I the chains secreted by myofibroblasts had twice the binding capacity of those from fibroblasts. Thus, it is likely that the difference in response to FGF-2 is because of a difference in FGF-2 sequestration and receptor interaction with FGF-2-HS complexes. A comparative investigation into HS fine structure is being undertaken to examine these findings in more detail.

关键词：细胞分裂细胞系二糖类/化学二糖类/分离和提纯成纤维细胞生长因子2/药理学成纤维细胞/药物作用成纤维细胞/生理学流式细胞术生长物质/药理学类肝素硫酸蛋白聚糖类/生物合成类肝素硫酸蛋白聚糖类/化学类肝素硫酸蛋白聚糖类/分离和提纯动力学肌骨骼/药物作用肌骨骼/生理学血小板源性生长因子/药理学放射性同位素稀释技术受体成纤维细胞生长因子/生理学硫酸盐类/代谢硫放射性同位素动物大鼠

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Short-term exposure of cartilage to blood results in chondrocyte apoptosis

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AMERICAN JOURNAL OF PATHOLOGY 2003年第3期162卷 943-951页

作者： Hooiveld, M Roosendaal, G Wenting, M van den Berg, M Bijlsma, J Lafeber, F Univ Med Ctr Utrecht Dept Rheumatol & Clin Immunol NL-3508 GA Utrecht Netherlands Univ Med Ctr Utrecht Van Creveldklin Natl Hemophilia Ctr NL-3508 GA Utrecht Netherlands

Studies have shown that joint bleeding leads to cartilage degradation independent of concurrent synovitis. We hypothesized that the blood-induced cartilage damage is because of increased chondrocyte apoptosis after short-term exposure of whole blood or isolated mononuclear cells plus red blood cells to cartilage. Human cartilage tissue samples were co-cultured for 4 days with whole blood (50% v/v) or with mononuclear cells plus red blood cells (50% v/v equivalents). Cartilage matrix proteoglycan synthesis ((SO42-)-S-35 incorporation) was determined after 4 days as well as at day 16 (after a 12-day recovery period in the absence of any additions). To test the involvement of apoptosis a specific caspase-3 inhibitor (acDEVDcho, 0 to 500 mumol/L) as well as a pan-caspase inhibitor (zVADfmk, 0 to 500 mumol/L) were added. Chondrocyte apoptosis was evaluated by immunohistochemical staining of single-strand DNA and by terminal dUTP nick-end labeling. Cartilage co-cultured with whole blood as well as mononuclear cells plus red blood cells induced a long-term inhibition of proteoglycan synthesis (74% and 78% inhibition on day 16, respectively). Immunohistochemistry showed a threefold increase in apoptotic chondrocytes in cultures with 50% whole blood as well as with mononuclear cells plus red blood cells. Both the specific caspase-3 inhibitor and the pan-caspase inhibitor partially restored proteoglycan synthesis in the cartilage after blood exposure. This effect was accompanied by a decrease in the number of apoptotic chondrocytes. These data suggest that a single joint hemorrhage (a 4-day exposure of cartilage to 50% v/v blood) results in induction of chondrocyte apoptosis, responsible for the observed inability of the chondrocytes to restore the proteoglycan synthesis during recovery from a short-term exposure to blood. This reduced restoration could eventually lead to cartilage degeneration and ultimately joint destruction.

关键词：细胞凋亡/生理学尸体解剖软骨关节/细胞学软骨关节/病理学软骨关节/生理学软骨细胞/细胞学软骨细胞/药物作用软骨细胞/病理学软骨细胞/生理学共同培养技术半胱氨酸蛋白酶抑制剂/药理学肱骨白细胞介素1/药理学动力学蛋白聚糖类/拮抗剂和抑制剂蛋白聚糖类/生物合成硫酸盐类/代谢硫放射性同位素肿瘤坏死因子α/药理学人类

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Inhibition of microbial H₂S production in an oil reservoir model column by nitrate injection

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2002年第3期58卷 400-408页

作者： Myhr, S Lillebo, BLP Sunde, E Beeder, J Torsvik, T Univ Bergen Dept Microbiol N-5020 Bergen Norway STATOIL N-4035 Stavanger Norway Norsk Hydro AS Res Ctr N-3900 Porsgrunn Norway

The effect of nitrate addition on microbial H2S production in a seawater-flooded oil reservoir model column with crude oil as carbon and energy source was investigated. Injection of 0.5 mM nitrate for 2.5-3.5 months led to complete elimination of H2S (initially 0.45-0.67 mM). The major decline in H2S level coincided with the first complete nitrate consumption and production of nitrite. When nitrate was excluded, H2S production resumed after approximately 2.5 months and reached previous levels after approximately 5 months. Using a fluorescent antibody technique, three populations each of sulfate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB) were monitored. SRB dominated the anoxic zone prior to nitrate addition, comprising 64-93% of the total bacterial population. The monitored NRB constituted less than 6% and no increase was observed during nitrate addition (indicating that other, unidentified, NRB populations were present). After 1-3 months without significant H2S production (3.5-5.5 months with nitrate), the SRB population collapsed, the fraction being reduced to 9-25%. The dominant SRB strain in the column, which constituted on average 94% of the monitored SRB population, was partly/completely inhibited by 56/75 muM nitrite in batch culture tests. Similar nitrite concentrations (50-150 muM) were detected in the column when the H2S level declined, indicating that nitrite inhibition was the main cause of H2S elimination. The results from this study indicate that nitrate/nitrite can be used to prevent detrimental SRB activity in oil reservoirs.

关键词：醇类/分析细菌/生长和发育细菌/代谢脂肪酸类/分析燃料油/微生物学硫化氢/代谢工业用油/微生物学注射硝酸盐类/投药和剂量硝酸盐类/药理学人口密度硫酸盐类/代谢硫还原细菌/药物作用硫还原细菌/生长和发育硫还原细菌/代谢

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The structure of human liver fructose-1,6-bisphosphate aldolase

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2001年第11期57卷 1526-1533页

作者： Dalby, AR Tolan, DR Littlechild, JA Univ Exeter Sch Biol & Chem Sci Exeter EX4 4QD Devon England Boston Univ Dept Biol Boston MA 02215 USA

The X-ray crystallographic structure of the human liver isozyme of fructose-1,6-bisphosphate aldolase has been determined by molecular replacement using a tetramer of the human muscle isozyme as a search model. The liver aldolase (B isozyme) crystallized in space group C2, with unit-cell parameters a = 291.1, b = 489.8, c = 103.4 Angstrom, alpha = 90, beta = 103.6, gamma = 90 degrees. These large unit-cell parameters result from the presence of 18 subunits in the asymmetric unit: four catalytic tetramers and a dimer from a fifth tetramer positioned on the twofold crystallographic axis. This structure provides further insight into the factors affecting isozyme specificity. It reveals small differences in secondary structure that occur in regions previously determined to be isozyme specific. Two of these regions are at the solvent-exposed enzyme surface away from the active site of the enzyme. The most significant changes are in the flexible C-terminal region of the enzyme, where there is an insertion of an extra alpha -helix. Point mutations of the human liver aldolase are responsible for the disease hereditary fructose intolerance. Sequence information is projected onto the new crystal structure in order to indicate how these mutations bring about reduced enzyme activity and affect structural stability.

关键词：氨基酸序列结合部位结晶结晶学 X线果糖不耐受果糖-二磷酸醛缩酶/化学果糖-二磷酸醛缩酶/遗传学同工酶类/化学同工酶类/代谢肝/酶学模型分子分子序列数据突变柔韧性蛋白质构象序列同源性氨基酸底物特异性硫酸盐类/代谢人类

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Microbial sulfate reduction with acetate: process performance and composition of the bacterial communities in the reactor at different salinity levels

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2001年第6期55卷 787-793页

作者： Muthumbi, W Boon, N Boterdaele, R De Vreese, I Top, EM Verstraete, W State Univ Ghent Fac Agr & Appl Biol Sci Lab Microbial Ecol & Technol B-9000 Ghent Belgium

Microbial sulfate reduction with acetate as carbon source and electron donor was investigated at salinity levels between 0.53 and 1.48%. The experiment was carried out in a 2.3-l upflow anaerobic sludge blanket reactor inoculated with granular methanogenic sludge. A pH of 8.3, a temperature of 32 +/-1 degreesC and a chemical oxygen demand (COD)/SO42--S ratio of 2 were maintained in the reactor throughout the experiment. Sulfate reduction and the composition of the dominant bacterial communities in the reactor were monitored. The results showed that a maximal conversion rate for SO42--S of 14 g l(-1) day(-1) and a conversion efficiency of more than 90% were obtained at a salinity level of 1.26-1.39%. A further increase in the salinity level led to reactor instability. Denaturant gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from total bacterial DNA extracted from the inoculum and reactor sludge showed that salinity level had an impact on the composition of the bacterial communities in the reactor. However, no clear relationship was found between reactor performance and the composition of the dominant bacterial communities in the reactor.

关键词：乙酸盐类/代谢厌氧生活细菌/分类细菌/遗传学细菌/生长和发育细菌/代谢生物反应器梭菌属/分类梭菌属/遗传学梭菌属/生长和发育梭菌属/代谢 DNA 细菌/分析 DNA 细菌/遗传学 DNA 核糖体/分析 DNA 核糖体/遗传学电泳聚丙烯酰氨凝胶氢离子浓度分子序列数据氧化还原假单胞菌属/分类假单胞菌属/遗传学假单胞菌属/生长和发育假单胞菌属/代谢 RNA 核糖体 16S/遗传学氯化钠/代谢硫酸盐类/代谢温度

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Characterization of determinants for the specificity of Arabidopsis thioredoxins h in yeast complementation

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第41期275卷 31641-31647页

作者： Bréhélin, C Mouaheb, N Verdoucq, L Lancelin, JM Meyer, Y Univ Perpignan UMR CNRS 5096 Lab Genome & Dev Plantes F-66860 Perpignan France Univ Lyon 1 Lab Resonance Magnet Nucl Biomol CNRS F-69622 Villeurbanne France Ecole Super Chim Phys & Elect F-69622 Villeurbanne France

The disruption of the two thioredoxin genes in Saccharomyces cerevisiae leads to a complex phenotype, including the inability to use methionine sulfoxide as sulfur source, modified cell cycle parameters, reduced H2O2 tolerance, and inability to use sulfate as sulfur source. Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements only some aspects of the phenotype, depending on the expressed thioredoxin: AtTRX2 or AtTRX3 induce methionine sulfoxide assimilation and restore a normal cell cycle. In addition AtTRX2 also confers growth on sulfate but no H2O2 tolerance, In contrast, AtTRX3 does not confer growth on sulfate but induces H2O2 tolerance. We have constructed hybrid proteins between these two thioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information needed for H2O2 tolerance is located in the N-terminal part of AtTRX3. In addition, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate. All these data suggest that the multiple Arabidopsis thioredoxins h originate from a totipotent ancestor with all the determinants necessary for interaction with the different thioredoxin target proteins. After duplications each member evolved by losing or masking some of the determinants.

关键词：氨基酸序列氨基酸取代/遗传学拟南芥属/酶学拟南芥属/遗传学结合部位印迹法蛋白质细胞周期进化分子遗传互补测验过氧化氢/药理学甲硫氨酸/类似物和衍生物甲硫氨酸/代谢模型分子分子序列数据突变氧化还原表型蛋白质构象重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/代谢酵母菌酿酒/细胞学酵母菌酿酒/药物作用酵母菌酿酒/酶学酵母菌酿酒/遗传学序列比对底物特异性硫酸盐类/代谢

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Characterization of a sulfur-regulated oxygenative alkylsulfatase from Pseudomonas putida S-313

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第41期275卷 31661-31667页

作者： Kahnert, A Kertesz, MA Univ Manchester Sch Biol Sci Manchester M13 9PT Lancs England ETH Zentrum Swiss Fed Inst Technol Inst Microbiol CH-8092 Zurich Switzerland

The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid identity). The AtsK protein catalyzed the alpha-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C-4 to C-12, required oxygen and Fe2+ for activity and released succinate, sulfate, and the corresponding aldehyde as products. Enzyme activity was optimal at pH 7 and was strongly stimulated by ascorbate. Unlike most other characterized alpha-ketoglutarate-dependent dioxygenases, AtsK accepted a range of alpha-keto acids as co-substrates, including alpha-ketoglutarate (K-m 140 mu M), alpha-ketoadipate, alpha-ketovalerate, and alpha-ketooctanoate. The measured K-m values for hexyl sulfate and SDS were 40 and 34 mu M, respectively. The apparent M-r of the purified enzyme of 121,000 was consistent with a homotetrameric structure, which is unusual for this enzyme superfamily, members of which are usually monomeric or dimeric, The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytic alkylsulfatase and a novel member of the alpha-ketoglutarate-dependent dioxygenase family.

关键词：醛类/代谢链烷磺酸盐类/代谢氨基酸序列抗坏血酸/药理学催化作用/药物作用大肠杆菌/遗传学氢离子浓度铁/代谢酮戊二酸类/代谢动力学分子序列数据分子量氧/代谢氧合酶类/化学氧合酶类/遗传学氧合酶类/分离和提纯氧合酶类/代谢蛋白质结构四级假单胞菌恶臭/酶学假单胞菌恶臭/代谢重组蛋白质类/化学重组蛋白质类/代谢序列比对序列分析蛋白质底物特异性琥珀酸盐/代谢硫酸酯酶类/化学硫酸酯酶类/遗传学硫酸酯酶类/分离和提纯硫酸酯酶类/代谢硫酸盐类/代谢

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Characterization of proexosite I on prothrombin

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第22期275卷 16428-16434页

作者： Anderson, PJ Nesset, A Dharmawardana, KR Bock, PE Vanderbilt Univ Sch Med Dept Pathol Nashville TN 37232 USA

Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin, Quantitative affinity chromatography and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand, hirudin(54-65) ([5F]Hir(54-65) (SO3-), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir(54-65)(SO3-) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human prothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 mu M and 25 +/- 2 nM, respectively, demonstrating a 130-fold increase in affinity for the active proteinase. The bovine proteins similarly showed a 150-fold higher affinity of [5F]Hir(54-65)(SO3-) for thrombin compared with prothrombin, despite a 2-5-fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr(63)-sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir(54-65)(SO3-) to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a similar to 100-fold increase in affinity when thrombin is formed.

关键词：结合部位结合竞争性催化作用色谱法亲和酶激活凝血酶原/代谢硫酸盐类/代谢凝血酶/代谢动物牛人类

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Oncostatin M and transforming growth factor-β1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第24期275卷 18061-18069页

作者： Cichy, J Puré, E Wistar Inst Anat & Biol Philadelphia PA 19104 USA Jagiellonian Univ Inst Mol Biol PL-31120 Krakow Poland Ludwig Inst Canc Res New York NY 10158 USA

CD44, a receptor for hyaluronan (HA), has been implicated in tumor growth and metastasis. Most CD44-positive cells fail to exhibit constitutive HA receptor function but CD44-mediated HA binding on hematopoetic cells can be induced by antibody cross-linking of the receptor and by physiologic stimuli, including cytokines. me now demonstrate that oncostatin M (OSM) and transforming growth factor-beta 1, cytokines known to regulate the growth of tumor cells, stimulate HA binding in lung epithelial-derived tumor cells. In lung epithelial-derived tumor cells, cytokine-induced binding resulted from post-translational modification of the receptor. OSM-induced HA binding was associated with a reduction in N-linked carbohydrate content of CD44. In addition, OSM induced Hcl binding via a novel mechanism requiring sulfation of chondroitin sulfate chains linked to CD44. The mechanism underlying transforming growth factor-beta 1 induced HA binding was distinct from the effects of OSM. The data presented indicate that modulation of the glycosylation and sulfation of CD44 by cytokines provides mechanisms for regulating cell adhesion during tumor growth and metastasis.

关键词：抗原 CD44/代谢癌/代谢电泳聚丙烯酰氨凝胶酶联免疫吸附测定流式细胞术糖基化透明质酸/代谢肺肿瘤/代谢抑瘤素M 肽类/代谢蛋白质加工转译后硫酸盐类/代谢转化生长因子β/代谢肿瘤细胞培养的人类

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