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排序方式: 共有286条查询结果,搜索用时 31 毫秒
71.
72.
Johansson E Bjornberg O Nyman PO Larsen S 《The Journal of biological chemistry》2003,278(30):27916-27922
The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-A resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted beta-barrel surrounded by two beta-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residue-long DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser118, Arg122, Thr130, and Glu145. These water molecules are potential nucleophile candidates in the deamination reaction. 相似文献
73.
By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the alpha- and beta-phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [3H]dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km >100 micro m). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 micro m) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism. 相似文献
74.
The dut gene, which encodes Escherichia coli deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), has been recloned to increase overexpression of the enzyme and to enable simplification of the purification protocol into a one-step procedure. The gene was cloned into the vector pET-3a and expressed in E. coli BL21(DE3) pLysS under the control of a bacteriophage T7 promotor. Induction results in production of dUTPase corresponding to 60% of the extracted protein. Phosphocellulose chromatography at low pH was utilised for one-step purification, resulting in a homogenous preparation of the recombinant protein with a highly specific activity. The yield of purified enzyme is 500 mg per litre of bacterial culture, a significant increase compared to previously employed methods. 相似文献
75.
Jennifer L. Nyman Frank Caccavo Al B. Cunningham Robin Gerlach 《Bioremediation Journal》2002,6(1):39-55
Ferrous iron [Fe(II)] reductively transforms heavy metals in contaminated groundwater, and the bacterial reduction of indigenous ferric iron [Fe(III)] to Fe(II) has been proposed as a means of establishing redox reactive barriers in the subsurface. The reduction of Fe(III) to Fe(II) can be accomplished by stimulation of indigenous dissimilatory metal-reducing bacteria (DMRB) or injection of DMRB into the subsurface. The microbially produced Fe(II) can chemically react with contaminants such as Cr(VI) to form insoluble Cr(III) precipitates. The DMRB Shewanella algae BrY reduced surface-associated Fe(III) to Fe(II), which in batch and column experiments chemically reduced highly soluble Cr(VI) to insoluble Cr(III). Once the chemical Cr(VI) reduction capacity of the Fe(II)/Fe(III) couple in the experimental systems was exhausted, the addition of S. algae BrY allowed for the repeated reduction of Fe(III) to Fe(II), which again reduced Cr(VI) to Cr(III). The research presented herein indicates that a biological process using DMRB allows the establishment of a biogeochemical cycle that facilitates chromium precipitation. Such a system could provide a means for establishing and maintaining remedial redox reactive zones in Fe(III)-bearing subsurface environments. 相似文献
76.
Sutinen J Kannisto K Korsheninnikova E Fisher RM Ehrenborg E Nyman T Virkamäki A Funahashi T Matsuzawa Y Vidal H Hamsten A Yki-Järvinen H 《American journal of physiology. Endocrinology and metabolism》2004,286(6):E941-E949
Highly active antiretroviral therapy (HAART) has improved the prognosis of human immunodeficiency virus (HIV)-infected patients but is associated with severe adverse events, such as lipodystrophy and insulin resistance. Rosiglitazone did not increase subcutaneous fat in patients with HAART-associated lipodystrophy (HAL) in a randomized, double-blind, placebo-controlled trial, although it attenuated insulin resistance and decreased liver fat content. The aim of this study was to examine effects of rosiglitazone on gene expression in subcutaneous adipose tissue in 30 patients with HAL. The mRNA concentrations in subcutaneous adipose tissue were measured using real-time PCR. Twenty-four-week treatment with rosiglitazone (8 mg/day) compared with placebo significantly increased the expression of adiponectin, peroxisome proliferator-activated receptor-gamma (PPARgamma), and PPARgamma coactivator 1 and decreased IL-6 expression. Expression of other genes involved in lipogenesis, fatty acid metabolism, or glucose transport, such as acyl-CoA synthase, adipocyte lipid-binding protein, CD45, fatty acid transport protein-1 and -4, GLUT1, GLUT4, keratinocyte lipid-binding protein, lipoprotein lipase, PPARdelta, and sterol regulatory element-binding protein-1c, remained unchanged. Rosiglitazone also significantly increased serum adiponectin concentration. The change in serum adiponectin concentration was inversely correlated with the change in fasting serum insulin concentration and liver fat content. In conclusion, rosiglitazone induced significant changes in gene expression in subcutaneous adipose tissue and ameliorated insulin resistance in patients with HAL. Increased expression of adiponectin might have mediated most of the favorable insulin-sensitizing effects of rosiglitazone in these patients. 相似文献
77.
Transient kinetics of the equine infectious anemia virus deoxyuridine 5'-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one-step mechanism for substrate binding. A C-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C-terminus is not a prerequisite for substrate binding, but indispensable for catalysis. 相似文献
78.
The multisubunit enzyme V-ATPase harbours isoforms of individual subunits. a3 is one of four 116 kDa subunit a isoforms, and it is crucial for bone resorption. We used small interfering RNA (siRNA) molecules to knock down a3 in rat osteoclast cultures. Labeled siRNA-molecules entered osteoclasts via endocytosis and knocked down the a3 mRNA. Bone resorption was decreased in siRNA-treated samples due to decreased acidification and osteoclast inactivation. Expression of a1 did not respond to decreased a3 levels, suggesting that a1 does not compensate for a3 in osteoclast cultures. Subunit a3 is thus an interesting target for novel nucleic acid therapy. 相似文献
79.
80.
Heidi Marjonen Alejandra Sierra Anna Nyman Vladimir Rogojin Olli Gr?hn Anni-Maija Linden Sampsa Hautaniemi Nina Kaminen-Ahola 《PloS one》2015,10(5)
The adverse effects of alcohol consumption during pregnancy are known, but the molecular events that lead to the phenotypic characteristics are unclear. To unravel the molecular mechanisms, we have used a mouse model of gestational ethanol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first 8 days of gestation (GD 0.5-8.5). Early neurulation takes place by the end of this period, which is equivalent to the developmental stage early in the fourth week post-fertilization in human. During this exposure period, dynamic epigenetic reprogramming takes place and the embryo is vulnerable to the effects of environmental factors. Thus, we hypothesize that early ethanol exposure disrupts the epigenetic reprogramming of the embryo, which leads to alterations in gene regulation and life-long changes in brain structure and function. Genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and three miRNAs in ethanol-exposed, adolescent offspring at postnatal day (P) 28. We confirmed this result by using two other tissues, where three candidate genes are known to express actively. Interestingly, we found a similar trend of upregulated gene expression in bone marrow and main olfactory epithelium. In addition, we observed altered DNA methylation in the CpG islands upstream of the candidate genes in the hippocampus. Our MRI study revealed asymmetry of brain structures in ethanol-exposed adult offspring (P60): we detected ethanol-induced enlargement of the left hippocampus and decreased volume of the left olfactory bulb. Our study indicates that ethanol exposure in early gestation can cause changes in DNA methylation, gene expression, and brain structure of offspring. Furthermore, the results support our hypothesis of early epigenetic origin of alcohol-induced disorders: changes in gene regulation may have already taken place in embryonic stem cells and therefore can be seen in different tissue types later in life. 相似文献