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Sample records for nadh-cytochrome b5 reductase

  1. Reductive detoxification of arylhydroxylamine carcinogens by human NADH cytochrome b5 reductase and cytochrome b5.

    PubMed

    Kurian, Joseph R; Chin, Nathaniel A; Longlais, Brett J; Hayes, Kristie L; Trepanier, Lauren A

    2006-10-01

    Heterocyclic and aromatic amine carcinogens are thought to lead to tumor initiation via the formation of DNA adducts, and bioactivation to arylhydroxylamine metabolites is necessary for reactivity with DNA. Carcinogenic arylhydroxylamine metabolites are cleared by a microsomal, NADH-dependent, oxygen-insensitive reduction pathway in humans, which may be a source of interindividual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. On the basis of our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxification of arylhydroxylamine carcinogens was catalyzed by NADH cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). We found that reduction of the carcinogenic hydroxylamines of the aromatic amine 4-aminobiphenyl (4-ABP; found in cigarette smoke) and the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP; found in grilled meats) was indeed catalyzed by a purified system containing only human b5R and cyt b5. Specific activities were 56-346-fold higher in the purified system as compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (K(m) values) in both systems. The stoichiometry for b5R and cyt b5 that yielded the highest activity in the purified system was also similar to that found in native HLM ( approximately 1:8 to 1:10). Polyclonal antisera to either b5R or cyt b5 significantly inhibited N-hydroxy-4-aminobiphenyl (NHOH-4-ABP) reduction by 95 and 89%, respectively, and immunoreactive cyt b5 protein content in individual HLM was significantly correlated with individual reduction of both NHOH-4-ABP and N-hydroxy-PhIP (NHOH-PhIP). Finally, titration of HLM into the purified b5R/cyt b5 system did not enhance the efficiency of reduction activity. We conclude that b5R and cyt b5 are together solely capable of the reduction of

  2. Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction

    PubMed Central

    Sacco, James C.; Trepanier, Lauren A.

    2010-01-01

    Objectives NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Methods Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semi-quantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Results Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06–1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (p < 0.0001, r = 0.42; p = 0.01, r = 0.23, respectively), and expression of both proteins correlated with one another (p < 0.0001; r = 0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency. Conclusion These studies indicate that while novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low frequency cSNPs only appear to minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction. PMID:19997042

  3. Defining the Role of the NADH-Cytochrome-b5 Reductase 3 in the Mitochondrial Amidoxime Reducing Component Enzyme System.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Bork, Bettina; Bittner, Florian; Mendel, Ralf; Clement, Bernd

    2016-10-01

    The importance of the mitochondrial amidoxime reducing component (mARC)-containing enzyme system in N-reductive metabolism has been studied extensively. It catalyzes the reduction of various N-hydroxylated compounds and therefore acts as the counterpart of cytochrome P450- and flavin-containing monooxygenase-catalyzed oxidations at nitrogen centers. This enzyme system was found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism. The synergy of three components (mARC, cytochrome b5, and the appropriate reductase) is crucial to exert the N-reductive catalytic effect. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein cytochrome b5 in N-reductive metabolism. To date, the corresponding reductase involved in N-reductive metabolism has yet to be defined because previous investigations have presented ambiguous results. Using small interfering RNA-mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in vitro, we provide evidence that NADH-cytochrome-b5 reductase 3 is the principal reductase involved in the mARC enzyme system and is an essential component of N-reductive metabolism in human cells. In addition, only minimal levels of cytochrome-b5 reductase 3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase.

  4. A novel L218P mutation in NADH-cytochrome b5 reductase associated with type I recessive congenital methemoglobinemia.

    PubMed

    Arikoglu, Tugba; Yarali, Nese; Kara, Abdurrahman; Bay, Ali; Bozkaya, Ikbal O; Tunc, Bahattin; Percy, Melanie J

    2009-01-01

    The presence of central cyanosis that is unrelated to cardiopulmonary causes alerts clinicians to a possible diagnosis of methemoglobinemia. Congenital methemoglobinemia due to deficiency of nicotinamide-adenine dinucleotide (NADH)-cytochrome b5 reductase (cb(5)r) is an autosomal recessive disorder characterized by life long cyanosis. Here we report a six-year old boy who presented with central cyanosis and upon examination revealed a methemoglobin level of 19.0%. Sequencing the CYB5R3 gene identified a homozygous T-->C transition at base c.653, which changed codon 218 from leucine to proline (L218P) in cb(5)r protein. Treatment with ascorbic acid relieved the cyanosis and returned methemoglobin levels to normal. PMID:19579085

  5. Methemoglobin reduction by NADH-cytochrome b(5) reductase in Propsilocerus akamusi larvae.

    PubMed

    Maeda, Shintaro; Kobori, Hiroki; Tanigawa, Minoru; Sato, Katsuya; Yubisui, Toshitsugu; Hori, Hiroshi; Nagata, Yoko

    2015-07-01

    For oxygen respiration, a methemoglobin (metHb) reduction system is needed in the cell because metHb cannot bind oxygen. We examined the insect Propsilocerus akamusi larvae to elucidate the metHb reduction system in an organism that inhabits an oxygen-deficient environment. NADH-dependent reduction of metHb in coelomic fluid suggested the coexistence of cytochrome b5 reductase (b5R) and cytochrome b5 with hemoglobin in the fluid and that these proteins were involved in physiological metHb reduction in the larvae. The presence of b5R was revealed by purifying b5R to homogeneity from the midge larvae. Using purified components, we showed that larval metHb was reduced via the NADH-b5R (FAD)-cytochrome b5-metHb pathway, a finding consistent with that in aerobic vertebrates, specifically humans and rabbits, and b5R function between mammal and insect was conserved. b5R was identified as a monomeric FAD-containing enzyme; it had a molecular mass of 33.2 kDa in gel-filtration chromatography and approximately 37 kDa in SDS-PAGE analysis. The enzyme's optimal pH and temperature were 6.4 and 25 °C, respectively. The apparent Km and Vmax values were 345 μM and 160 μmol min(-1) mg(-1), respectively, for ferricyanide and 328 μM and 500 μmol min(-1) mg(-1), respectively, for 2,6-dichlorophenolindophenol. The enzyme reaction was inhibited by benzoate, p-hydroxymercuribenzoate, iodoacetamide, and iodoacetate, and was not inhibited by metal ions or EDTA. PMID:25829149

  6. NADH-cytochrome b5 reductase in a Turkish family with recessive congenital methaemoglobinaemia type I.

    PubMed

    Percy, M J; Aslan, D

    2008-10-01

    The development of cyanosis at birth, the so-called blue baby syndrome, alerts paediatricians to the presence of congenital heart disease. In rare cases where the arterial blood gas analysis is normal the cyanosis is a consequence of methaemoglobinaemia. There are three distinct origins of methaemoglobinaemia; the presence of a haemoglobin variant, environmental toxicity and deficiency of cytochrome b5 reductase (cb(5)r). Two children born to two sets of first-degree related parents were cyanotic from birth. Differential diagnosis eliminated cardiac and pulmonary abnormalities. Measurement of methaemoglobin levels confirmed recessive congenital methaemoglobinaemia (RCM) and treatment with ascorbic acid was commenced. In the absence of neurological defects, type I disease was diagnosed. Sequence analysis of CYB5R3 revealed two different missense mutations (one which is novel, Ile85Ser) in the two families. Neither of the mutations was located in the FAD or the NADH binding sites of cb(5)r, thus supporting a diagnosis of type I disease. PMID:18820099

  7. Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene.

    PubMed

    Kugler, W; Pekrun, A; Laspe, P; Erdlenbruch, B; Lakomek, M

    2001-04-01

    Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535 G-->A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757 G-->A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379 A-->G transition (M127V). The second allele carried a sequence difference at the invariant 3' splice-acceptor dinucleotide of intron 4 (IVS4-2A-->G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)(+) RNA was analyzed by RT-PCR and sequencing. The results show that RNA is made from the allele harboring the 3'-splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001. PMID:11295830

  8. A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase

    PubMed Central

    1992-01-01

    Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr approximately 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylation consensus are replaced by a reticulocyte-specific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid- specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms. PMID:1577871

  9. Identification of three new mutations in the NADH-cytochrome b5 reductase gene responsible for recessive congenital methemoglobinemia type II

    SciTech Connect

    Mota-Vieira, L.; Kaplan, J.C.; Kahn, A.; Leroux, A.

    1994-09-01

    Recessive congenital methemoglobinemia (RCM; McKusick N{degrees}25800) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases: in type I form, cyanosis is the only symptom and the enzyme is only defective in red blood cells; in type II form, cyanosis is associated with severe mental retardation and neurological impairment and the enzyme defect is systemic. We have identified three new molecular defects in two unrelated patients with type II RCM. A homozygous C{r_arrow}T transition in codon 218 (Arg) was detected in the cDNA of one patient, resulting in a premature stop codon (TGA) in exon 8. Restriction enzyme analysis of genomic DNA confirmed the homozygosity of the propositus and heterozygosity for an identical defect in both parents. The second patient was found to be a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation (T{r_arrow}C) with substitution of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele showed a 3 bp deletion of nucleotides 815-817 of the cDNA. The CTG ATG sequence at position 814-819 in exon 9 coding for Leu-271 and Met-272 was replaced by the CTG triplet, with conservation of the Leu-271 and loss of the Met-272. To our knowledge, these are the first examples of a homozygous nonsense mutation and of a compound heterozygous mutation detected in the cytb5r gene. This finding supports the diversity of genetic defects in the cytb5r gene leading to the severe form of the disease.

  10. NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene

    PubMed Central

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by 32P-postlabeling was characterized as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP. PMID:27404282

  11. NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene.

    PubMed

    Stiborová, Marie; Indra, Radek; Moserová, Michaela; Frei, Eva; Schmeiser, Heinz H; Kopka, Klaus; Philips, David H; Arlt, Volker M

    2016-08-15

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by (32)P-postlabeling was characterized as 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP. PMID:27404282

  12. NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer.

    PubMed

    Lund, Rikke R; Leth-Larsen, Rikke; Caterino, Tina Di; Terp, Mikkel G; Nissen, Jeanette; Lænkholm, Anne-Vibeke; Jensen, Ole N; Ditzel, Henrik J

    2015-11-01

    Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFβ and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are

  13. The induction of microsomal NADPH:cytochrome P450 and NADH:cytochrome b(5) reductases by long-term salt treatment of cotton (Gossypium hirsutum L.) and bean (Phaseolus vulgaris L.) plants.

    PubMed

    Brankova, Liliana; Ivanov, Sergei; Alexieva, Vera

    2007-09-01

    We studied the effect of salinity on the activity of microsomal NADPH:cytochrome P450 reductase (CPR, EC 1.6.2.4) and NADH:ferricytochrome b(5) oxidoreductase (B5R, EC 1.6.2.2) in two dicotyledonous plant species differing in their sensitivity to salt, cotton (Gossypium hirsutum L. cv Ogosta) and common bean (Phaseolus vulgaris L. cv Dobrujanski 7). A significant inhibition of fresh weight of salt-treated bean plants was observed, while cotton was affected to a much lesser degree. NaCl application resulted in a significant increase in the activity of both reductases, but was more pronounced in salt-tolerant cotton. We suppose that alterations in B5R and CPR activities may be targeted to the maintenance of membrane lipids. Most probably, plants use both enzymes (B5R and CPR) and their respective electron donors (NADH and NADPH) to reduce cytochrome b(5), which can donate reducing equivalents to a series of lipid-modification reactions such as desaturation and hydroxylation.

  14. Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b/sub 5/ reductase

    SciTech Connect

    Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

    1987-06-01

    A cDNA coding for human liver NADH-cytochrome b/sub 5/ reductase was cloned from a human liver cDNA library constructed in phage lambdagt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b/sub 5/ reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb/sub 5/R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb/sub 5/R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

  15. Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver.

    PubMed

    Arinç, E; Cakir, D

    1999-02-01

    Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme

  16. Recessive congenital methaemoglobinaemia: cytochrome b(5) reductase deficiency.

    PubMed

    Percy, Melanie J; Lappin, Terry R

    2008-05-01

    Some 60 years ago, Quentin Gibson reported the first hereditary disorder involving an enzyme when he deduced that familial methaemoglobinaemia was caused by an enzymatic lesion associated with the glycolysis pathway in red blood cells. This disorder, now known as recessive congenital methaemoglobinaemia (RCM), is caused by NADH-cytochrome b5 reductase (cb(5)r) deficiency. Two distinct clinical forms, types I and II, have been recognized, both characterized by cyanosis from birth. In type II, the cyanosis is accompanied by neurological impairment and reduced life expectancy. Cytochrome b(5) reductase is composed of one FAD and one NADH binding domain linked by a hinge region. It is encoded by the CYB5R3 (previously known as DIA1) gene and more than 40 mutations have been described, some of which are common to both types of RCM. Mutations associated with type II tend to cause incorrect splicing, disruption of the active site or truncation of the protein. At present the description of the sequence variants of cb(5)r in the literature is confusing, due to the use of two conventions which differ by one codon position. Herein we propose a new system for nomenclature of cb(5)r based on recommendations of the Human Genome Variation Society. The development of a heterologous expression system has allowed the impact of naturally occurring variants of cb(5)r to be assessed and has provided insight into the function of cb(5)r. PMID:18318771

  17. Congenital Recessive Methemoglobinemia Revealed in Adulthood: Description of a New Mutation in Cytochrome b5 Reductase Gene.

    PubMed

    Forestier, Alexandra; Pissard, Serge; Cretet, Justine; Mambie, Adeline; Pascal, Laurent; Cliquennois, Manuel; Cambier, Nathalie; Rose, Christian

    2015-01-01

    Methemoglobinemia can be acquired (oxidizing drugs or chemicals products) or inherited either by mutations affecting globin chains [M hemoglobins (M Hbs)] or by defects in the enzymatic system involved in the reduction of spontaneous Hb oxidation: nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. It is encoded by the CYB5R3 gene: there are two phenotypes of autosomal recessive congenital methemoglobinemia, in type II CYB5R deficiency is generalized and affects all cells, leading to an early onset, whereas in type I, the enzyme deficiency is restricted to erythrocytes, usually discovered in infancy but not exclusively. We report a new case of methemoglobinemia discovered in a patient from Bahrain who exhibited an unknown dyspnea at the age of 37 years without trigger events or oxidizing products. We discovered a new mutation in the CYB5R3 gene: exon 9, codon 266 (delGAG) (GLU) (CYB5R3: c.726_729delGAG) in the homozygous state. Appearance of methemoglobinemia in an adult usually suggests an acquired cause but our case illustrated that it could also reveal a type I mutation of cytochrome b5 reductase. PMID:26291966

  18. Study of the Individual Cytochrome b5 and Cytochrome b5 Reductase Domains of Ncb5or Reveals a Unique Heme Pocket and a Possible Role of the CS Domain*

    PubMed Central

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R.; Battaile, Kevin P.; Lovell, Scott; Benson, David R.; Zhu, Hao

    2010-01-01

    NADH cytochrome b5 oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b5 (b5), CHORD-SGT1 (CS), and cytochrome b5 reductase (b5R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b5 and b5R domains (Ncb5or-b5 and Ncb5or-b5R, respectively) and compared them with human microsomal b5 (Cyb5A) and b5R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b5 reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His89 and His112, consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b5 family shown to have such a heme environment. Like other b5 family members, Ncb5or-b5 has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b5 differs from Cyb5A with respect to location of the second heme ligand (His112) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b5R to Ncb5or-b5 is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b5 and b5R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b5R domains suggest that the CS domain facilitates docking of the b5 and b5R domains. Trp114 is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b5R domain to the b5 domain. PMID:20630863

  19. Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.

    PubMed

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R; Battaile, Kevin P; Lovell, Scott; Benson, David R; Zhu, Hao

    2010-09-24

    NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain. PMID:20630863

  20. Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability*

    PubMed Central

    Rahaman, Md. Mizanur; Reinders, Fabio G.; Koes, David; Nguyen, Anh T.; Mutchler, Stephanie M.; Sparacino-Watkins, Courtney; Alvarez, Roger A.; Miller, Megan P.; Cheng, Dongmei; Chen, Bill B.; Jackson, Edwin K.; Camacho, Carlos J.; Straub, Adam C.

    2015-01-01

    NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC50 = ∼275 μm), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC50 = 10.81 μm) and ZINC39395747 (IC50 = 9.14 μm), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development. PMID:26001785

  1. Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability.

    PubMed

    Rahaman, Md Mizanur; Reinders, Fabio G; Koes, David; Nguyen, Anh T; Mutchler, Stephanie M; Sparacino-Watkins, Courtney; Alvarez, Roger A; Miller, Megan P; Cheng, Dongmei; Chen, Bill B; Jackson, Edwin K; Camacho, Carlos J; Straub, Adam C

    2015-07-01

    NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC50 = ∼275 μM), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC50 = 10.81 μM) and ZINC39395747 (IC50 = 9.14 μM), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development. PMID:26001785

  2. Structure of Physarum polycephalum cytochrome b 5 reductase at 1.56 Å resolution

    PubMed Central

    Kim, Sangwoo; Suga, Michihiro; Ogasahara, Kyoko; Ikegami, Terumi; Minami, Yoshiko; Yubisui, Toshitsugu; Tsukihara, Tomitake

    2007-01-01

    Physarum polycephalum cytochrome b 5 reductase catalyzes the reduction of cytochrome b 5 by NADH. The structure of P. polycephalum cytochrome b 5 reductase was determined at a resolution of 1.56 Å. The molecular structure was compared with that of human cytochrome b 5 reductase, which had previously been determined at 1.75 Å resolution [Bando et al. (2004 ▶), Acta Cryst. D60, 1929–1934]. The high-resolution structure revealed conformational differences between the two enzymes in the adenosine moiety of the FAD, the lid region and the linker region. The structural properties of both proteins were inspected in terms of hydrogen bonding, ion pairs, accessible surface area and cavity volume. The differences in these structural properties between the two proteins were consistent with estimates of their thermostabilities obtained from differential scanning calorimetry data. PMID:17401193

  3. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    PubMed

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans. PMID:26806391

  4. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    PubMed

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans.

  5. DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours.

    PubMed Central

    Marín, A.; López de Cerain, A.; Hamilton, E.; Lewis, A. D.; Martinez-Peñuela, J. M.; Idoate, M. A.; Bello, J.

    1997-01-01

    The level of expression of enzymes that can activate or detoxify bioreductive agents within tumours has emerged as an important feature in the development of these anti-tumour compounds. The levels of two such reductase enzymes have been determined in 19 human non-small-cell lung tumours and 20 human breast tumours, together with the corresponding normal tissue. DT-diaphorase (DTD) enzyme levels (both expression and activity) were determined in these samples. Cytochrome b5 reductase (Cytb5R) activity was also assessed. With the exception of six patients, the levels of DTD activity were below 45 nmol min(-1) mg(-1) in the normal tissues assayed. DTD tumour activity was extremely variable, distinguishing two different groups of patients, one with DTD activity above 79 nmol min(-1) mg(-1) and the other with levels that were in the same range as found for the normal tissues. In 53% of the lung tumour samples, DTD activity was increased with respect to the normal tissue by a factor of 2.4-90.3 (range 79-965 nmol min[-1] mg[-1]). In 70% of the breast tumour samples, DTD activity was over 80 nmol min(-1) mg(-1) (range 83-267 nmol min[-1] mg[-1]). DTD expression measured by Western blot correlated well with the enzyme activity measured in both tumour and normal tissues. The levels of the other reductase enzyme, Cytb5R, were not as variable as those for DTD, being in the same range in both tumour and normal tissue or slightly higher in the normal tissues. The heterogeneous nature of DTD activity and expression reinforces the need to measure enzyme levels in individual patients before therapy with DTD-activated bioreductive drugs. Images Figure 1 Figure 2 PMID:9328153

  6. Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous

    PubMed Central

    Gutiérrez, María Soledad; Rojas, María Cecilia; Sepúlveda, Dionisia; Baeza, Marcelo; Cifuentes, Víctor; Alcaíno, Jennifer

    2015-01-01

    The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous. PMID:26466337

  7. Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria*

    PubMed Central

    Neve, Etienne P. A.; Nordling, Åsa; Andersson, Tommy B.; Hellman, Ulf; Diczfalusy, Ulf; Johansson, Inger; Ingelman-Sundberg, Magnus

    2012-01-01

    Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b5 reductase (CYB5R), cytochrome b5 (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b5 type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b5 type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase). PMID:22203676

  8. Amidoxime reductase system containing cytochrome b5 type B (CYB5B) and MOSC2 is of importance for lipid synthesis in adipocyte mitochondria.

    PubMed

    Neve, Etienne P A; Nordling, Asa; Andersson, Tommy B; Hellman, Ulf; Diczfalusy, Ulf; Johansson, Inger; Ingelman-Sundberg, Magnus

    2012-02-24

    Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b(5) reductase (CYB5R), cytochrome b(5) (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b(5) type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b(5) type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase). PMID:22203676

  9. Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water.

    PubMed Central

    Gupta, S. K.; Gupta, R. C.; Seth, A. K.; Gupta, A. B.; Bassin, J. K.; Gupta, A.

    1999-01-01

    An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents. PMID:10534899

  10. Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis

    PubMed Central

    Kratzer, Regina; Leitgeb, Stefan; Wilson, David K.; Nidetzky, Bernd

    2005-01-01

    Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo–keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/Km) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 Å (1 Å=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8–9 kJ/mol. PMID:16336198

  11. B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy.

    PubMed

    Shao, Fang-Yuan; Du, Zhi-Yun; Ma, Dong-Lei; Chen, Wen-Bo; Fu, Wu-Yu; Ruan, Bi-Bo; Rui, Wen; Zhang, Jia-Xuan; Wang, Sheng; Wong, Nai Sum; Xiao, Hao; Li, Man-Mei; Liu, Xiao; Liu, Qiu-Ying; Zhou, Xiao-Dong; Yan, Hai-Zhao; Wang, Yi-Fei; Chen, Chang-Yan; Liu, Zhong; Chen, Hong-Yuan

    2015-10-13

    The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy. PMID:26439985

  12. B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy.

    PubMed

    Shao, Fang-Yuan; Du, Zhi-Yun; Ma, Dong-Lei; Chen, Wen-Bo; Fu, Wu-Yu; Ruan, Bi-Bo; Rui, Wen; Zhang, Jia-Xuan; Wang, Sheng; Wong, Nai Sum; Xiao, Hao; Li, Man-Mei; Liu, Xiao; Liu, Qiu-Ying; Zhou, Xiao-Dong; Yan, Hai-Zhao; Wang, Yi-Fei; Chen, Chang-Yan; Liu, Zhong; Chen, Hong-Yuan

    2015-10-13

    The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy.

  13. Reduction of amphetamine hydroxylamine and other aliphatic hydroxylamines by benzamidoxime reductase and human liver microsomes.

    PubMed

    Clement, B; Behrens, D; Möller, W; Cashman, J R

    2000-10-01

    For the reduction of N-hydroxylated derivatives of strongly basic functional groups, such as amidines, guanidines, and aminohydrazones, an oxygen-insensitive liver microsomal system, the benzamidoxime reductase, has been described. To reconstitute the complete activity of the benzamidoxime reductase, the system required cytochrome b(5), NADH-cytochrome b(5)-reductase, and the benzamidoxime reductase, a cytochrome P450 enzyme, which has been purified to homogeneity from pig liver. It was not known if this enzyme system was also capable of reducing aliphatic hydroxylamines. The N-hydroxylation of aliphatic amines is a well-known metabolic process. It was of interest to study the possibility of benzamidoxime reductase reducing N-hydroxylated metabolites of aliphatic amines back to the parent compound. Overall, N-hydroxylation and reduction would constitute a futile metabolic cycle. As examples of medicinally relevant compounds, the hydroxylamines of methamphetamine, amphetamine, and N-methylamine as model compounds were investigated. Formation of methamphetamine and amphetamine was analyzed by newly developed HPLC methods. All three hydroxylamines were easily reduced by benzamidoxime reductase to their parent amines with reduction rates of 220.6 nmol min(-1) (mg of protein)(-1) for methamphetamine, 5.25 nmol min(-1) (mg of protein)(-1) for amphetamine, and 153 nmol min(-1) (mg of protein)(-1) for N-methylhydroxylamine. Administration of synthetic hydroxylamines of amphetamine and methamphetamine to primary rat neuronal cultures produced frank cell toxicity. Compared with amphetamine or the oxime of amphetamine, the hydroxylamines were significantly more toxic to primary neuronal cells. The benzamidoxime reductase is therefore involved in the detoxication of these reactive hydroxylamines.

  14. Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5.

    PubMed

    Yamazaki, H; Johnson, W W; Ueng, Y F; Shimada, T; Guengerich, F P

    1996-11-01

    Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450. PMID:8910324

  15. Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains.

    PubMed

    Crawford, N M; Smith, M; Bellissimo, D; Davis, R W

    1988-07-01

    The sequence of nitrate reductase (EC 1.6.6.1) mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase. PMID:3393528

  16. The N-Reductive System Composed of Mitochondrial Amidoxime Reducing Component (mARC), Cytochrome b5 (CYB5B) and Cytochrome b5 Reductase (CYB5R) Is Regulated by Fasting and High Fat Diet in Mice

    PubMed Central

    Jakobs, Heyka H.; Mikula, Michal; Havemeyer, Antje; Strzalkowska, Adriana; Borowa-Chmielak, Monika; Dzwonek, Artur; Gajewska, Marta; Hennig, Ewa E.; Ostrowski, Jerzy; Clement, Bernd

    2014-01-01

    The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism. PMID:25144769

  17. The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells*

    PubMed Central

    Plitzko, Birte; Ott, Gudrun; Reichmann, Debora; Henderson, Colin J.; Wolf, C. Roland; Mendel, Ralf; Bittner, Florian; Clement, Bernd; Havemeyer, Antje

    2013-01-01

    The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system. PMID:23703616

  18. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus Zymoseptoria tritici reveals novel functionalities implicated in virulence

    PubMed Central

    Derbyshire, Mark C.; Michaelson, Louise; Parker, Josie; Kelly, Steven; Thacker, Urvashi; Powers, Stephen J.; Bailey, Andy; Hammond-Kosack, Kim; Courbot, Mikael; Rudd, Jason

    2015-01-01

    Septoria tritici blotch (STB) caused by the Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. Z. tritici is currently a major target for agricultural fungicides, especially in temperate regions where it is most prevalent. Many fungicides target electron transfer enzymes because these are often important for cell function. Therefore characterisation of genes encoding such enzymes may be important for the development of novel disease intervention strategies. Microsomal cytochrome b5 reductases (CBRs) are an important family of electron transfer proteins which in eukaryotes are involved in the biosynthesis of fatty acids and complex lipids including sphingolipids and sterols. Unlike the model yeast Saccharomyces cerevisiae which possesses only one microsomal CBR, the fully sequenced genome of Z. tritici bears three possible microsomal CBRs. RNA sequencing analysis revealed that ZtCBR1 is the most highly expressed of these genes under all in vitro and in planta conditions tested, therefore ΔZtCBR1 mutant strains were generated through targeted gene disruption. These strains exhibited delayed disease symptoms on wheat leaves and severely limited asexual sporulation. ΔZtCBR1 strains also exhibited aberrant spore morphology and hyphal growth in vitro. These defects coincided with alterations in fatty acid, sphingolipid and sterol biosynthesis observed through GC–MS and HPLC analyses. Data is presented which suggests that Z. tritici may use ZtCBR1 as an additional electron donor for key steps in ergosterol biosynthesis, one of which is targeted by azole fungicides. Our study reports the first functional characterisation of CBR gene family members in a plant pathogenic filamentous fungus. This also represents the first direct observation of CBR functional ablation impacting upon fungal sterol biosynthesis. PMID:26074495

  19. Synthesis and degradation of nitrate reductase during the cell cycle of Chlorella sorokiniana

    NASA Technical Reports Server (NTRS)

    Velasco, P. J.; Tischner, R.; Huffaker, R. C.; Whitaker, J. R.

    1989-01-01

    Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [35S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.

  20. Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant.

    PubMed

    Kratzer, Regina; Kavanagh, Kathryn L; Wilson, David K; Nidetzky, Bernd

    2004-05-01

    Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data

  1. A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk).

    PubMed

    Paquette, Suzanne M; Jensen, Kenneth; Bak, Søren

    2009-12-01

    Gene and genome duplication is a key driving force in evolution of plant diversity. This has resulted in a number of large multi-gene families. Two of the largest multi-gene families in plants are the cytochromes P450 (P450s) and family 1 glycosyltransferases (UGTs). These two families are key players in evolution, especially of plant secondary metabolism, and in adaption to abiotic and biotic stress. In the model plant Arabidopsis thaliana there are 246 and 112 cytochromes P450 and UGTs, respectively. The Arabidopsis P450, cytochromes b(5), NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases website (http://www.P450.kvl.dk) is a sequence repository of manually curated sequences, multiple sequence alignments, phylogenetic trees, sequence motif logos, 3D structures, intron-exon maps, and customized BLAST datasets.

  2. Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2*

    PubMed Central

    Sparacino-Watkins, Courtney E.; Tejero, Jesús; Sun, Bin; Gauthier, Marc C.; Thomas, John; Ragireddy, Venkata; Merchant, Bonnie A.; Wang, Jun; Azarov, Ivan; Basu, Partha; Gladwin, Mark T.

    2014-01-01

    Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production. PMID:24500710

  3. Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP +

    SciTech Connect

    Leitgeb, Stefan; Petschacher, Barbara; Wilson, David K.; Nidetzky, Bernd

    2005-01-11

    Aldo-keto reductases of family 2 employ single site replacement Lys → Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3 Å, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.

  4. Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains

    SciTech Connect

    Crawford, N.M.; Smith, M.; Bellissimo, D.; Davis, R.W. )

    1988-07-01

    The sequence of nitrate reductase mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a {lambda}gt10 cDNA library of Arabidopsis leaf poly(A){sup +} RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being >80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b{sub 5} superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b{sub 5} reductase.

  5. 32 CFR 242b.5 - Voting.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 2 2011-07-01 2011-07-01 false Voting. 242b.5 Section 242b.5 National Defense... SCIENCES § 242b.5 Voting. (a) The concurrence of a majority of the Regents present at a meeting shall be... notation voting, by voting on material circulated to Regents individually or serially, or by polling...

  6. 45 CFR 73b.5 - Hearings.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Hearings. 73b.5 Section 73b.5 Public Welfare... § 73b.5 Hearings. (a) Hearings shall be stenographically recorded and transcribed and the testimony of witnesses shall be taken under oath or affirmation. Hearings will be closed unless an open hearing...

  7. 45 CFR 73b.5 - Hearings.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Hearings. 73b.5 Section 73b.5 Public Welfare... § 73b.5 Hearings. (a) Hearings shall be stenographically recorded and transcribed and the testimony of witnesses shall be taken under oath or affirmation. Hearings will be closed unless an open hearing...

  8. 12 CFR 261b.5 - Exemptions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 false Exemptions. 261b.5 Section 261b.5 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM RULES REGARDING PUBLIC OBSERVATION OF MEETINGS § 261b.5 Exemptions. (a) Except in a case where the agency...

  9. 7 CFR 15b.5 - Assurances required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Assurances required. 15b.5 Section 15b.5 Agriculture... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE General Provisions § 15b.5 Assurances required. (a) Assurances. An applicant for Federal financial assistance to which this part applies shall submit...

  10. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  11. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  12. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  13. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  14. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  15. 15 CFR 8b.5 - Assurances required.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 15 Commerce and Foreign Trade 1 2011-01-01 2011-01-01 false Assurances required. 8b.5 Section 8b.5... Assurances required. (a) Assurances. An applicant for Federal financial assistance to which this part applies shall submit an assurance, on a form specified by the Secretary, that the program or activity will...

  16. 15 CFR 8b.5 - Assurances required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Assurances required. 8b.5 Section 8b.5... Assurances required. (a) Assurances. An applicant for Federal financial assistance to which this part applies shall submit an assurance, on a form specified by the Secretary, that the program or activity will...

  17. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  18. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  19. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  20. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  1. 18 CFR 3b.5 - Legal guardians.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Legal guardians. 3b.5 Section 3b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES COLLECTION, MAINTENANCE, USE, AND DISSEMINATION OF RECORDS OF IDENTIFIABLE...

  2. 32 CFR 242b.5 - Voting.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SCIENCES § 242b.5 Voting. (a) The concurrence of a majority of the Regents present at a meeting shall be necessary for the transaction of business. (b) Unless a written ballot is required by a Regent, no actions taken by the Board need be by written ballot. (c) The Chairman of the Board and of each Committee...

  3. Biochemical responses in mussels Perna perna exposed to diesel B5.

    PubMed

    Nogueira, Lílian; Garcia, Danielly; Trevisan, Rafael; Sanches, Ana Letícia Madeira; da Silva Acosta, Daiane; Dafre, Alcir Luiz; Oliveira, Thiago Yukio Kikuchi; de Almeida, Eduardo Alves

    2015-09-01

    In Brazil B5 blend (5% biodiesel and 95% diesel oil) has been adopted as mandatory fuel since 2010 for automotive vehicles. Since little is known about the effects of B5 exposure can promote on antioxidant system of marine biota this study aimed to assess if B5 can generate modifications in antioxidant parameters of mussels Perna perna. To address this question mussels were exposed to two concentrations of B5 (0.01 mL L(-1) and 0.1 mL L(-1)) for 6h, 12h, 48 h and 168 h. Then the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) were evaluated in gills and digestive gland as well as the contents of glutathione (GSH) and lipid peroxidation by measuring the malondialdehyde concentration (MDA). In the gills, GST activity decreased after 48 h and GR after 12h of exposure to B5. In digestive glands, the activities of SOD, GPx and GR were changed due to treatments. GSH concentration increased in digestive gland after 6h and 12h and in gills after 48 h for B5 0.1 mL L(-1) and after 168 h in the digestive gland for B5 0.01 mL L(-1) treatment. No lipid peroxidation was detected. The integrated biomarker response index (IBR) evidenced a B5 effect in the digestive gland after 168 h of exposure. Regarding the experimental conditions and species used in this study, long-term exposure to B5 is apparently more likely to affect the parameters tested in P. perna mussels.

  4. Technical description of Stack 296-B-5

    SciTech Connect

    Ridge, T.M.

    1994-11-15

    Of particular concern to facilities on the Hanford site is Title 40, Code of Federal Regulations, Chapter 40, Part 61, Subpart H, ``National emission Standards for Emissions of Radionuclides Other Than Radon From Department of Energy Facilities.`` Assessments of facility stacks and potential radionuclide emissions determined whether these stacks would be subject to the sampling and monitoring requirements of 40 CFR 61, Subpart H. Stack 296-B-5 exhausts 221-BB building which houses tanks containing B Plant steam condensate and B Plant process condensate from the operation of the low-level waste concentrator. The assessment of potential radionuclide emissions from the 296-B-5 stack resulted in an effective dose equivalent to the maximally exposed individual of less than 0.1 millirem per year. Therefore, the stack is not subject to the sampling and monitoring requirements of 40 CFR 61, Subpart H. However, the sampling and monitoring system must be in compliance with the Environmental Compliance Manual, WHC-CM-7-5. Currently, 296-B-5 is sampled continuously with a record sampler and continuous air monitor (CAM).

  5. Towards engineering increased pantothenate (vitamin B(5)) levels in plants.

    PubMed

    Chakauya, Ereck; Coxon, Katy M; Wei, Ma; Macdonald, Mary V; Barsby, Tina; Abell, Chris; Smith, Alison G

    2008-11-01

    Pantothenate (vitamin B(5)) is the precursor of the 4'-phosphopantetheine moiety of coenzyme A and acyl-carrier protein. It is made by plants and microorganisms de novo, but is a dietary requirement for animals. The pantothenate biosynthetic pathway is well-established in bacteria, comprising four enzymic reactions catalysed by ketopantoate hydroxymethyltransferase (KPHMT), L: -aspartate-alpha-decarboxylase (ADC), pantothenate synthetase (PS) and ketopantoate reductase (KPR) encoded by panB, panD, panC and panE genes, respectively. In higher plants, the genes encoding the first (KPHMT) and last (PS) enzymes have been identified and characterised in several plant species. Commercially, pantothenate is chemically synthesised and used in vitamin supplements, feed additives and cosmetics. Biotransformation is an attractive alternative production system that would circumvent the expensive procedures of separating racemic intermediates. We explored the possibility of manipulating pantothenate biosynthesis in plants. Transgenic oilseed rape (Brassica napus) lines were generated in which the E. coli KPHMT and PS genes were expressed under a strong constitutive CaMV35SS promoter. No significant change of pantothenate levels in PS transgenic lines was observed. In contrast plants expressing KPHMT had elevated pantothenate levels in leaves, flowers siliques and seed in the range of 1.5-2.5 fold increase compared to the wild type plant. Seeds contained the highest vitamin content, indicating that they might be the ideal target for production purposes.

  6. Bilayer structure and physical dynamics of the cytochrome b5 dimyristoylphosphatidylcholine interaction.

    PubMed Central

    Chester, D W; Skita, V; Young, H S; Mavromoustakos, T; Strittmatter, P

    1992-01-01

    Cytochrome b5 is a microsomal membrane protein which provides reducing potential to delta 5-, delta 6-, and delta 9-fatty acid desaturases through its interaction with cytochrome b5 reductase. Low angle x-ray diffraction has been used to determine the structure of an asymmetrically reconstituted cytochrome b5:DMPC model membrane system. Differential scanning calorimetry and fluorescence anisotropy studies were performed to examine the bilayer physical dynamics of this reconstituted system. These latter studies allow us to constrain structural models to those which are consistent with physical dynamics data. Additionally, because the nonpolar peptide secondary structure remains unclear, we tested the sensitivity of our model to different nonpolar peptide domain configurations. In this modeling approach, the nonpolar peptide moiety was arranged in the membrane to meet such chemically determined criteria as protease susceptibility of carboxyl- and amino-termini, tyrosine availability for pH titration and tryptophan 109 location, et cetera. In these studies, we have obtained a reconstituted cytochrome b5:DMPC bilayer structure at approximately 6.3 A resolution and conclude that the nonpolar peptide does not penetrate beyond the bilayer midplane. Structural correlations with calorimetry, fluorescence anisotropy and acyl chain packing data suggest that asymmetric cytochrome b5 incorporation into the bilayer increases acyl chain order. Additionally, we suggest that the heme peptide:bilayer interaction facilitates a discreet heme peptide orientation which would be dependent upon phospholipid headgroup composition. Images FIGURE 1 FIGURE 2 FIGURE 7 PMID:1600082

  7. Neoplastic lesions of the human liver in relation to the activity of the cytochrome P-450 dependent monooxygenase system.

    PubMed

    Plewka, D; Plewka, A; Nowaczyk, G; Kamiński, M; Rutkowski, T; Ludyga, T; Ziaja, K

    2000-01-01

    We studied the activity of Mixed function oxidase (MFO) in human livers affected by cancer. We determined the content of cytochrome P-450 and b5, as well as the activity of their corresponding reductases, according to generally accepted methods. Liver fragments corresponding with a) healthy tissue, b) tissue at the cancer border and, c) cancerous tissue were collected during surgery from patients with liver cancer. We noted that the developing liver cancer decreased the level of cytochrome P-450, even by a magnitude order. The activity of its corresponding reductase was higher in cancerous than in healthy tissues. Cytochrome b5 behaved in an analogous manner, although the decrease in its content was less significant. NADH-cytochrome b5 reductase activity changes were insignificant.

  8. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 17 Commodity and Securities Exchanges 3 2013-04-01 2013-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  9. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2014 CFR

    2011-04-01

    ... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  10. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 17 Commodity and Securities Exchanges 3 2012-04-01 2012-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  11. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2014 CFR

    2000-04-01

    ... 17 Commodity and Securities Exchanges 3 2000-04-01 2000-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a) Each application for a stay of a trustee's duty...

  12. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2011 CFR

    2005-04-01

    ... 17 Commodity and Securities Exchanges 3 2005-04-01 2005-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  13. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  14. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 3 2011-10-01 2011-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of...

  15. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 3 2014-10-01 2014-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of...

  16. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 3 2012-10-01 2012-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of...

  17. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 3 2013-10-01 2013-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation (Continued) PIPELINE AND HAZARDOUS MATERIALS SAFETY... Containers, and Linings § 178.33b-5 Material. (a) The receptacles must be constructed of...

  18. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 9 2014-07-01 2014-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  19. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 9 2011-07-01 2011-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  20. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 9 2013-07-01 2013-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  1. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 9 2012-07-01 2012-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  2. 29 CFR 2530.200b-5 - Seasonal industries. [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Seasonal industries. 2530.200b-5 Section 2530.200b-5 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR... Provisions § 2530.200b-5 Seasonal industries....

  3. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 17 Commodity and Securities Exchanges 4 2014-04-01 2014-04-01 false Content. 260.10b-5 Section 260.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rule Under Section 310 § 260.10b-5 Content. (a)...

  4. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false How will NIH evaluate applications? 52b.5 Section 52b.5 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating...

  5. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false How will NIH evaluate applications? 52b.5 Section 52b.5 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating...

  6. The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities.

    PubMed

    Billen, M J; Squires, E J

    2009-01-01

    Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5alpha-androst-16-en-3-one (5alpha-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-beta synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17alpha-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-beta synthase as well as the 17alpha-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17alpha-hydroxylase (p<0.013), a transient increase in C17,20 lyase, and an increase in andien-beta synthase activity (p<0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17alpha-hydroxylase, but did not affect the andien-beta synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-beta synthase activity of CYP17A1. PMID:19101629

  7. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

  8. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false How will NIH evaluate applications? 52b.5 Section... INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating and... other pertinent factors, the following: (1) The priority score assigned to the application by an...

  9. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false How will NIH evaluate applications? 52b.5 Section... INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating and... other pertinent factors, the following: (1) The priority score assigned to the application by an...

  10. 42 CFR 52b.5 - How will NIH evaluate applications?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false How will NIH evaluate applications? 52b.5 Section... INSTITUTES OF HEALTH CONSTRUCTION GRANTS § 52b.5 How will NIH evaluate applications? (a) In evaluating and... other pertinent factors, the following: (1) The priority score assigned to the application by an...

  11. 26 CFR 48.4161(b)-5 - Effective date.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... taxes imposed by section 4161(b) are effective with respect to sales made on and after January 1, 1975. ... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Effective date. 48.4161(b)-5 Section 48.4161(b)-5 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED)...

  12. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 8 Aliens and Nationality 1 2012-01-01 2012-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  13. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 8 Aliens and Nationality 1 2011-01-01 2011-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  14. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 8 Aliens and Nationality 1 2014-01-01 2014-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  15. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 8 Aliens and Nationality 1 2013-01-01 2013-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  16. 8 CFR 343b.5 - Verification of naturalization.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Verification of naturalization. 343b.5... CERTIFICATE OF NATURALIZATION FOR RECOGNITION BY A FOREIGN STATE § 343b.5 Verification of naturalization. The application shall not be granted without first obtaining verification of the applicant's naturalization....

  17. 49 CFR 178.33b-5 - Material.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Material. 178.33b-5 Section 178.33b-5 Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS SPECIFICATIONS FOR...

  18. Plant polyphenols as electron donors for erythrocyte plasma membrane redox system: validation through in silico approach

    PubMed Central

    2012-01-01

    Background The plasma membrane redox system (PMRS) has extensively been studied in erythrocytes. The PMRS plays an important role in maintaining plasma redox balance and provides a protective mechanism against oxidative stress. Earlier it was proposed that only NADH or NADPH provided reducing equivalents to PMRS; however, now it is acknowledged that some polyphenols also have the ability to donate reducing equivalents to PMRS. Methods Two different docking simulation softwares, Molegro Virtual Docker and Glide were used to study the interaction of certain plant polyphenols viz. quercetin, epigallocatechin gallate, catechin epicatechin and resveratrol with human erythroyte NADH-cytochrome b5 reductase, which is a component of PMRS and together with the identification of minimum pharmacophoric feature using Pharmagist. Results The derived common minimum pharmacophoric features show the presence of minimum bioactive component in all the selected polyphenols. Our results confirm wet lab findings which show that these polyphenols have the ability to interact and donate protons to the Human NADH-cytochrome b5 reductase. Conclusion With the help of these comparative results of docking simulation and pharmacophoric features, novel potent molecules can be designed with higher efficacy for activation of the PMRS system. PMID:22475026

  19. Li2AlB5O10.

    PubMed

    He, M; Li, H; Chen, X; Xu, Y; Xu, T

    2001-09-01

    A new compound, dilithium aluminium pentaborate, Li(2)AlB(5)O(10), has been synthesized by solid-state reaction and its structure determined by single-crystal X-ray diffraction. This compound is composed of [B(5)O(10)](5-) groups linked by AlO(4) tetrahedra. The [B(5)O(10)](5-) group consists of two hexagonal B-O rings perpendicular to each other connected by tetracoordinated boron. All the B-O rings in this structure can be divided into two groups, with one group approximately parallel and the other perpendicular to the c axis. PMID:11588352

  20. Bioinformatic identification of cytochrome b5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b₅ in parasitic adaptation.

    PubMed

    Takamiya, Shinzaburo; Hashimoto, Muneaki; Mita, Toshihiro; Yokota, Takehiro; Nakajima, Yoshitaka; Yamakura, Fumiyuki; Sugio, Shigetoshi; Fujimura, Tsutomu; Ueno, Takashi; Yamasaki, Hiroshi

    2016-04-01

    We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b5, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b5 and the canonical cytochrome b5 proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b5-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b5 in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b5, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b5 precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b5 and highlight the crucial function of this A. suum adult-specific secretory cytochrome b5 in parasitic adaptation.

  1. Selective and compartmentalized myelin expression of HspB5.

    PubMed

    Quraishe, S; Wyttenbach, A; Matinyarare, N; Perry, V H; Fern, R; O'Connor, V

    2016-03-01

    In the present study, we reveal myelin-specific expression and targeting of mRNA and biochemical pools of HspB5 in the mouse CNS. Our observations are based on in situ hybridization, electron microscopy and co-localization with 2',3'-Cyclic-Nucleotide 3'-Phosphodiesterase (CNPase), reinforcing this myelin-selective expression. HspB5 mRNA might be targeted to these structures based on its presence in discrete clusters resembling RNA granules and the presence of a putative RNA transport signal. Further, sub-cellular fractionation of myelin membranes reveals a distinct sub-compartment-specific association and detergent solubility of HspB5. This is akin to other abundant myelin proteins and is consistent with HspB5's association with cytoskeletal/membrane assemblies. Oligodendrocytes have a pivotal role in supporting axonal function via generating and segregating the ensheathing myelin. This specialization places extreme structural and metabolic demands on this glial cell type. Our observations place HspB5 in oligodendrocytes which may require selective and specific chaperone capabilities to maintain normal function and neuronal support.

  2. Structural stability of W2B5 under high pressure

    NASA Astrophysics Data System (ADS)

    Kumar, N. R. Sanjay; Shekar, N. V. Chandra; Sahu, P. Ch.

    2015-05-01

    High-pressure structural stability studies have been carried out on tungsten boride W2B5 up to maximum pressure of 36 GPa using a Mao-Bell diamond-anvil cell at beamline BR-12 of the ELETTRA synchrotron facility (λ = 0.68881 Å). The hexagonal phase (S.G:P63/mmc) of W2B5 is stable up to the maximum pressure studied. The bulk modulus is estimated to be ~347 GPa using the Birch-Murnaghan equation of state. The variation of lattice parameters and bond lengths B-B and W-B have been studied and the c-axis is seen to be marginally more compressible than the a-axis.

  3. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  4. [Population frequency and age of c.806C > T mutation in CYB5R3 gene as cause of recessive congenital methemoglobinemia in Yakutia].

    PubMed

    Galeeva, N M; Voevoda, M I; Spiridonova, M G; Stepanov, V A; Poliakov, A V

    2013-04-01

    Type-1recessive congenital methemoglobinemia (RCM) is a rare autosomal disease characterized by a deficiency of the soluble form of nicotineamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) and clinically manifests as cyanosis of skin and mucous membranes. In the Russian Federation, type-I RCM is widely disturbed in Yakutia due to the local founder effect. The molecular genetics cause of type-I RCM in Yakutia is mutation c.806C > T in the CYB5R3 gene. In this work we used 13 polymorphic markers, which flanking the CYB5R3 gene to establish the founder haplotype. The age of the mutation was estimated as about 285 +/- 135 years. In this work, we have evaluated the frequency of the c.806 C > T mutation in Yakutia, which averaged 55 : 1000 Yakuts. The calculated frequency of disease was 1: 1250 Yakuts. PMID:23866629

  5. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  6. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  7. Methemoglobin reduction mediated by D-amino acid dehydrogenase in Propsilocerus akamusi (Tokunaga) larvae.

    PubMed

    Kobori, Hiroki; Tanigawa, Minoru; Maeda, Shintaro; Hori, Hiroshi; Yubisui, Toshitsugu; Nagata, Yoko

    2015-06-01

    A methemoglobin (metHb) reduction system is required for aerobic respiration. In humans, Fe(III)-heme-bearing metHb (the oxidized form of hemoglobin), which cannot bind oxygen, is converted to Fe(II)-heme-bearing oxyhemoglobin (oxyHb, the reduced form), which can bind oxygen, in a system comprising NADH, NADH-cytochrome b5 reductase, and cytochrome b5. However, the mechanism of metHb reduction in organisms that inhabit oxygen-deficient environments is unknown. In the coelomic fluid of the larvae of Propsilocerus akamusi, which inhabit a microaerobic environment, we found that metHb was reduced by D-alanine. We purified an FAD-containing enzyme, D-amino acid dehydrogenase (DAD), and component V hemoglobin from the larvae. Using the purified components and spectrophotometric analyses, we showed a novel function of DAD: DAD-mediation of P. akamusi component V metHb reduction with using D-alanine as an electron donor. P. akamusi larvae possess this D-alanine-DAD metHb reduction system in addition to a previously discovered NADH-NADH-cytochrome b5 reductase system. This is the first report of the presence of DAD in a multicellular organism. The molecular mass of DAD was estimated to be 45 kDa. The optimal pH and temperature of the enzyme were 7.4 and 20 °C, respectively, and the optimal substrate was D-alanine. The enzyme activity was inhibited by benzoate and sulfhydryl-binding reagents. PMID:25896287

  8. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... gene provides instructions for making an enzyme called steroid 5-alpha reductase 2. This enzyme is involved ... external genitalia. Mutations in the SRD5A2 gene prevent steroid 5-alpha reductase 2 from effectively converting testosterone ...

  9. Relative Activities and Characteristics of Some Oxidative Respiratory Enzymes from Conidia of Verticillium albo-atrum

    PubMed Central

    Throneberry, G. O.

    1967-01-01

    Conidia of Verticillium albo-atrum Reinke and Berthold, collected from shake cultures grown in Czapek broth, were sonified for 4 or 8 minutes or ground frozen in a mortar to obtain cell-free homogenates. These were assayed for certain enzymes associated with respiratory pathways. Malic dehydrogenase was the most active, glucose-6-P and NADH dehydrogenase were less active, NADH-cytochrome c reductase, NADPH dehydrogenase, and cytochrome oxidase were low in activity, and succinic dehydrogenase and succinic cytochrome c reductase were very low to negligible in activity. No NADH oxidase activity was detected. With the exception of NADH-cytochrome c reductase and possibly succinic dehydrogenase and cytochrome c reductase, there was no evident increase in specific activity of the enzymes during germination. Some NADH-cytochrome c reductase and a small amount of succinic-dehydrogenase and cytochrome c reductase were associated with the particulate fraction from 105,000 × g centrifugation. The other enzymes, including cytochrome oxidase, almost completely remained in the supernatant fraction. Menadione and vitamin K-S(II) markedly stimulated NADH-cytochrome c reductase activity in the supernatant fraction but had much less effect on NADPH-cytochrome c reductase in this fraction or on either of these enzyme systems in the particulate fraction. Electron transport inhibitors affected particulate NADH- and NADPH-cytochrome c reductase activity but had no effect on these in the supernatant fraction. PMID:16656681

  10. Giardia intestinalis Incorporates Heme into Cytosolic Cytochrome b5

    PubMed Central

    Pyrih, Jan; Harant, Karel; Martincová, Eva; Sutak, Robert; Lesuisse, Emmanuel; Hrdý, Ivan

    2014-01-01

    The anaerobic intestinal pathogen Giardia intestinalis does not possess enzymes for heme synthesis, and it also lacks the typical set of hemoproteins that are involved in mitochondrial respiration and cellular oxygen stress management. Nevertheless, G. intestinalis may require heme for the function of particular hemoproteins, such as cytochrome b5 (cytb5). We have analyzed the sequences of eukaryotic cytb5 proteins and identified three distinct cytb5 groups: group I, which consists of C-tail membrane-anchored cytb5 proteins; group II, which includes soluble cytb5 proteins; and group III, which comprises the fungal cytb5 proteins. The majority of eukaryotes possess both group I and II cytb5 proteins, whereas three Giardia paralogs belong to group II. We have identified a fourth Giardia cytb5 paralog (gCYTb5-IV) that is rather divergent and possesses an unusual 134-residue N-terminal extension. Recombinant Giardia cytb5 proteins, including gCYTb5-IV, were expressed in Escherichia coli and exhibited characteristic UV-visible spectra that corresponded to heme-loaded cytb5 proteins. The expression of the recombinant gCYTb5-IV in G. intestinalis resulted in the increased import of extracellular heme and its incorporation into the protein, whereas this effect was not observed when gCYTb5-IV containing a mutated heme-binding site was expressed. The electrons for Giardia cytb5 proteins may be provided by the NADPH-dependent Tah18-like oxidoreductase GiOR-1. Therefore, GiOR-1 and cytb5 may constitute a novel redox system in G. intestinalis. To our knowledge, G. intestinalis is the first anaerobic eukaryote in which the presence of heme has been directly demonstrated. PMID:24297440

  11. [Novel large deletion c.22-1320_633+1224del in the CYB5R3 gene from patients with hereditary methemoglobinemia].

    PubMed

    Galeeva, N M; Nenasheva, S A; Kleĭmenova, I S; Poliakov, A V

    2012-11-01

    Hereditary types I and II methemoglobinemia is a rare autosomal recessive disease due to a deficiency of either soluble or soluble and membrane-bound forms of the enzyme NADH-cytochrome b5 reductase. The molecular genetic bases of both types of the disease consist in changes in the CYB5R3 gene. In this study, the novel and, to date, only large deletion in this gene is described, discovered in two unrelated families with types I and II methemoglobinemia. The common founder haplotype on the chromosomes carrying this mutation was identified. A universal approach for searching for the deletion boundaries was developed, and the c.22-1320_633+1224del deletion breakpoints were determined. In addition, a system for identifying the deletion in heterozygous and homozygous states was designed. PMID:23297489

  12. Study of the Individual Cytochrome b[subscript 5] and Cytochrome b[subscript 5] Reductase Domains of Ncb5or Reveals a Unique Heme Pocket and a Possible Role of the CS Domain

    SciTech Connect

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R.; Battaile, Kevin P.; Lovell, Scott; Benson, David R.; Zhu, Hao

    2010-09-22

    NADH cytochrome b{sub 5} oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b{sub 5} (b{sub 5}), CHORD-SGT1 (CS), and cytochrome b{sub 5} reductase (b{sub 5}R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b{sub 5} and b{sub 5}R domains (Ncb5or-b{sub 5} and Ncb5or-b{sub 5}R, respectively) and compared them with human microsomal b{sub 5} (Cyb5A) and b{sub 5}R (Cyb5R3). A 1.25 {angstrom} x-ray crystal structure of Ncb5or-b{sub 5} reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His{sup 89} and His{sup 112}, consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b{sub 5} family shown to have such a heme environment. Like other b{sub 5} family members, Ncb5or-b{sub 5} has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b{sub 5} differs from Cyb5A with respect to location of the second heme ligand (His{sup 112}) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b{sub 5}R to Ncb5or-b{sub 5} is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b{sub 5} and b{sub 5}R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b{sub 5}R domains suggest that the CS domain facilitates docking of the b{sub 5} and b{sub 5}R domains. Trp{sup 114} is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b{sub 5}R domain to the b{sub 5} domain.

  13. 22 CFR 9b.5 - Temporary Department of State press building passes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Temporary Department of State press building passes. 9b.5 Section 9b.5 Foreign Relations DEPARTMENT OF STATE GENERAL REGULATIONS GOVERNING DEPARTMENT OF STATE PRESS BUILDING PASSES § 9b.5 Temporary Department of State press building passes. A...

  14. 17 CFR 240.12b-5 - Determination of affiliates of banks.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... banks. 240.12b-5 Section 240.12b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... Securities Exchange Act of 1934 General § 240.12b-5 Determination of affiliates of banks. In determining whether a person is an “affiliate” or “parent” of a bank or whether a bank is a “subsidiary” or...

  15. 26 CFR 301.7701(b)-5 - Coordination with section 877.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Coordination with section 877. 301.7701(b)-5 Section 301.7701(b)-5 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) PROCEDURE AND ADMINISTRATION PROCEDURE AND ADMINISTRATION Definitions § 301.7701(b)-5 Coordination...

  16. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  17. Ribonucleotide Reductase-- a Radical Enzyme

    NASA Astrophysics Data System (ADS)

    Reichard, Peter; Ehrenberg, Anders

    1983-08-01

    Ribonucleotide reductases catalyze the enzymatic formation of deoxyribonucleotides, an obligatory step in DNA synthesis. The native form of the enzyme from Escherichia coli or from mammalian sources contains as part of its polypeptide structure a free tyrosyl radical, stabilized by an iron center. The radical participates in all probability in the catalytic process during the substitution of the hydroxyl group at C-2 of ribose by a hydrogen atom. A second, inactive form of the E. coli reductase lacks the tyrosyl radical. Extracts from E. coli contain activities that interconvert the two forms. The tyrosyl radical is introduced in the presence of oxygen, while anaerobiosis favors its removal, suggesting a regulatory role in DNA synthesis for oxygen.

  18. Nitrate reductase from Rhodopseudomonas sphaeroides.

    PubMed Central

    Kerber, N L; Cardenas, J

    1982-01-01

    The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed. PMID:6978883

  19. Effects of charged amino-acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

    PubMed Central

    Qian, Chengmin; Yao, Yong; Ye, Keqiong; Wang, Jinfeng; Tang, Wenxia; Wang, Yunhua; Wang, Wenhu; Lu, Junxia; Xie, Yi; Huang, Zhongxian

    2001-01-01

    The solution structure of oxidized bovine microsomal cytochrome b5 mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045±0.009 nm and 0.088±0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b5. The binding of different surface mutants of cytochrome b5 with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b5 surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b5 complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b5, do occur in the association between cytochrome b5 and cytochrome c. PMID:11714912

  20. Polymer phase partition in the purification of cytochrome P-450 and cytochrome b5 from the yeast Brettanomyces anomalus.

    PubMed

    Kärenlampi, S O; Nikkilä, H; Hynninen, P H

    1986-02-01

    About 0.5% of the total cellular protein in the yeast Brettanomyces anomalus is membrane-bound cytochrome P-450, when this yeast is grown in the presence of 5% glucose as the main carbon and energy source. A partial purification of cytochrome P-450 by phase partition is described. Breakdown of yeast cell walls with microbial enzyme preparations led to extensive losses of this hemoprotein. Instead, by a carefully controlled mechanical breakage as much as 50% of the total cellular cytochrome P-450 could be recovered. During the solubilization of cytochrome P-450 from the cell homogenate with Triton X-100, the protective agents dithiothreitol, EDTA, and butylated hydroxytoluene prevented major losses of the hemoprotein. Applying a three-phase partition system (polyethylene glycol-Ficoll-dextran) to the solubilized whole cell homogenate in the presence of 1 M sodium chloride, followed by a precipitation of the top "oily layer" with 25% polyethylene glycol, a 25- to 60-fold enrichment of cytochrome P-450 was obtained. This corresponds to a specific content of 0.8-2.2 nmol of cytochrome P-450 per milligram of protein. Cytochrome b5 enriched (41%) to the PEG-Ficoll interphase, and NADPH-cytochrome c reductase and "cytochromes P-420" to the Ficoll and dextran phases. The polymer phase partition system thus serves as an excellent initial purification step of cytochrome P-450 without a need for the preparation of the microsomal fraction. Another advantage of the method is that it allows the simultaneous partial purification of cytochrome b5. PMID:3828082

  1. Evidence that biliverdin-IX beta reductase and flavin reductase are identical.

    PubMed Central

    Shalloe, F; Elliott, G; Ennis, O; Mantle, T J

    1996-01-01

    A search of the database shows that human biliverdin-IX beta reductase and flavin reductase are identical. We have isolated flavin reductase from bovine erythrocytes and show that the activity co-elutes with biliverdin-IX beta reductase. Preparations of the enzyme that are electrophoretically homogeneous exhibit both flavin reductase and biliverdin-IX beta reductase activities; however, they are not capable of catalysing the reduction of biliverdin-IX alpha. Although there is little obvious sequence identity between biliverdin-IX alpha reductase (BVR-A) and biliverdin-IX beta reductase (BVR-B), they do show weak immunological cross-reactivity. Both enzymes bind to 2',5'-ADP-Sepharose. PMID:8687377

  2. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  3. Changes in electron transport pathways in endoplasmic reticulum of rapeseed in response to cold.

    PubMed

    de Virville, Jacques Davy; Cochet, Françoise; Tasseva, Guergana; Moreau, François; Zachowski, Alain

    2008-10-01

    We studied changes induced by cold on electron transfer pathways (linked to NADH or NADPH oxidation) in endoplasmic reticulum of rapeseed hypocotyls (Brassica napus L.) from a freezing-sensitive variety (ISL) and freezing-tolerant variety (Tradition). Plantlets were grown at 22 degrees C then submitted to a cold shock of 13 or 35 days at 4 degrees C. We measured the content in NADH, NADPH, NAD and NADP of the hypocotyls and the redox power was estimated by the reduced versus oxidized nucleotide ratio. The contents in cytochromes b (5) and P-450, electron acceptors of NADH and NADPH respectively, were determined by differential spectrophotometry. Finally, activity of both NADH-cytochrome b (5) reductase (E.C.1.6.2.2) and NADPH cytochrome P-450 reductase (E.C.1.6.2.4) was determined by reduction of exogenous cytochrome c. Results show that during cold shock, along with an increase of linolenic acid content, there was a general activation of the NADPH pathway which was observed more quickly in Tradition plantlets than in ISL ones. Due to transfer of electrons that can occur between NADPH reductase and cytochrome b (5), this could favor fatty acid desaturation in Tradition, explaining why linolenic acid accumulation was more pronounced in this variety. Besides, more cytochrome P-450 accumulated in ISL that could compete for electrons needed by the FAD3 desaturase, resulting in a relative slower enrichment in 18:3 fatty acid in these plantlets.

  4. The effect of acute stress and opioid antagonist on the activity of NADPH-P450 reductase in rat Leydig cells.

    PubMed

    Kostić, T; Andrić, S; Marić, D; Kovacević, R

    1998-07-01

    Previous studies indicate that acute immobilization stress (IMO; 2 h) impaired testicular steroidogenesis primarily at the testicular level decreasing the activity of certain steroidogenic enzymes. In the present study unstressed rats as well as IMO rats (2 h) were treated by intratesticular injection of naltrexone methobromide (NMB; peripheral opioid receptor antagonist; 36 microg/testis) or vehicle at the beginning of and at 1 h of the IMO period. In IMO rats the activity of P450c17 was significantly reduced as well as the activity of NADPH-P450 reductase (which catalyzes the transfer of electrons from NADPH to cytochrome P450), while the activity of NADH-b5 reductase was not affected. Present data confirmed previous results that acute IMO reduced testicular P450c17 activity and implicate that decreased activity of NADPH-P450 reductase could be responsible for the inhibition of P450c17 under IMO conditions, while NADH-b5 reductase is probably not involved. NMB treatment antagonized the inhibitory effect of IMO on P450c17 and NADPH-P450 reductase activities. Such results put forward the implication that endogenous opioid peptides are involved in mediating the inhibitory effect of IMO on testicular steroidogenesis, and allow the speculation that NADPH-P450 reductase could be a possible site of such an inhibition. PMID:9712411

  5. 296-B-5 Stack monitoring and sampling system annual system assessment report

    SciTech Connect

    Ridge, T.M.

    1995-02-01

    The B Plant Administration Manual requires an annual system assessment to evaluate and report the present condition of the sampling and monitoring system associated with Stack 296-B-5 at B Plant. The sampling and monitoring system associated with stack 296-B-5 is functional and performing satisfactorily. This document is an annual assessment report of the systems associated with the 296-B-5 stack.

  6. Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii 1

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

    In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID:16668656

  7. Individual variability in the detoxification of carcinogenic arylhydroxylamines in human breast.

    PubMed

    Rhoads, Keelia; Sacco, James C; Drescher, Nicholas; Wong, Amos; Trepanier, Lauren A

    2011-06-01

    Cytochrome b(5) (b5) and NADH cytochrome b(5) reductase (b5R) detoxify reactive hydroxylamine (NHOH) metabolites of known arylamine and heterocyclic amine mammary carcinogens. The aim of this study was to determine whether NHOH reduction for the prototypic arylamine 4-aminobiphenyl (4-ABP) was present in human breast and to determine whether variability in activity was associated with single nucleotide polymorphisms (SNPs) in the coding, promoter, and 3'untranslated region (UTR) regions of the genes encoding b5 (CYB5A) and b5R (CYB5R3). 4-ABP-NHOH reduction was readily detected in pooled human breast microsomes, with a K(m) (280μM) similar to that found with recombinant b5 and b5R, and a V(max) of 1.12 ± 0.19 nmol/min/mg protein 4-ABP-NHOH reduction varied 75-fold across 70 individual breast samples and correlated significantly with both b5 (80-fold variability) and b5R (14-fold) immunoreactive protein. In addition, wide variability in b5 protein expression was significantly associated with variability in CYB5A transcript levels, with a trend toward the same association between b5R and CYB5R3. Although a sample with a novel coding SNP in CYB5A, His22Arg, was found with low reduction and b5 expression, no other SNPs in either gene were associated with outlier activity or protein expression. We conclude that b5 and b5R catalyze the reduction of 4-ABP-NHOH in breast tissue, with very low activity, protein, and messenger RNA expression in some samples, which cannot be attributed to promoter, coding, or 3'UTR SNPs. Further studies are underway to characterize the transcriptional regulation of CYB5A and CYB5R3 and begin to understand the mechanisms of individual variability in this detoxification pathway. PMID:21447608

  8. Individual Variability in the Detoxification of Carcinogenic Arylhydroxylamines in Human Breast

    PubMed Central

    Sacco, James C.; Drescher, Nicholas; Wong, Amos; Trepanier, Lauren A.

    2011-01-01

    Cytochrome b5 (b5) and NADH cytochrome b5 reductase (b5R) detoxify reactive hydroxylamine (NHOH) metabolites of known arylamine and heterocyclic amine mammary carcinogens. The aim of this study was to determine whether NHOH reduction for the prototypic arylamine 4-aminobiphenyl (4-ABP) was present in human breast and to determine whether variability in activity was associated with single nucleotide polymorphisms (SNPs) in the coding, promoter, and 3′untranslated region (UTR) regions of the genes encoding b5 (CYB5A) and b5R (CYB5R3). 4-ABP-NHOH reduction was readily detected in pooled human breast microsomes, with a Km (280μM) similar to that found with recombinant b5 and b5R, and a Vmax of 1.12 ± 0.19 nmol/min/mg protein 4-ABP-NHOH reduction varied 75-fold across 70 individual breast samples and correlated significantly with both b5 (80-fold variability) and b5R (14-fold) immunoreactive protein. In addition, wide variability in b5 protein expression was significantly associated with variability in CYB5A transcript levels, with a trend toward the same association between b5R and CYB5R3. Although a sample with a novel coding SNP in CYB5A, His22Arg, was found with low reduction and b5 expression, no other SNPs in either gene were associated with outlier activity or protein expression. We conclude that b5 and b5R catalyze the reduction of 4-ABP-NHOH in breast tissue, with very low activity, protein, and messenger RNA expression in some samples, which cannot be attributed to promoter, coding, or 3′UTR SNPs. Further studies are underway to characterize the transcriptional regulation of CYB5A and CYB5R3 and begin to understand the mechanisms of individual variability in this detoxification pathway. PMID:21447608

  9. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    PubMed Central

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  10. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells.

    PubMed

    Tamazato Longhi, Mariana; Magalhães, Magna; Reina, Jeffrey; Morais Freitas, Vanessa; Cella, Nathalie

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  11. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2011-01-01

    VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell. PMID:22028781

  12. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus LBB.B5

    PubMed Central

    Hajo, Karima; Lenoci, Leonardo; Bron, Peter A.; Dijkstra, Annereinou; Alkema, Wynand; Wels, Michiel; Siezen, Roland J.; Minkova, Svetlana; van Hijum, Sacha A. F. T.

    2016-01-01

    Lactobacillus delbrueckii subsp. bulgaricus LBB.B5 originates from homemade Bulgarian yogurt and was selected for its ability to form a strong association with Streptococcus thermophilus. The genome sequence will facilitate elucidating the genetic background behind the contribution of LBB.B5 to the taste and aroma of yogurt and its exceptional protocooperation with S. thermophilus. PMID:27795256

  13. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  14. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  15. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  16. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  17. 17 CFR 240.16b-5 - Bona fide gifts and inheritance.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... inheritance. 240.16b-5 Section 240.16b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... gifts and inheritance. Both the acquisition and the disposition of equity securities shall be exempt... securities by will or the laws of descent and distribution....

  18. 26 CFR 1.410(b)-5 - Average benefit percentage test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 5 2010-04-01 2010-04-01 false Average benefit percentage test. 1.410(b)-5...) INCOME TAX (CONTINUED) INCOME TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.410(b)-5 Average benefit percentage test. (a) General rule. A plan satisfies the average benefit percentage test of...

  19. Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity.

    PubMed

    Goosen, Pierre; Storbeck, Karl-Heinz; Swart, Amanda C; Conradie, Riaan; Swart, Pieter

    2011-11-01

    During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ(5) steroid intermediates greatly influences steroidogenic output. Cytochrome-b(5) (Cyt-b(5)), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b(5) on 3βHSD activity was investigated. In COS-1 cells, Cyt-b(5) was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b(5) to have no influence on the K(m) values while significantly increasing the V(max) values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b(5) or anti-Cyt-b(5) IgG. The results presented in this study indicate that Cyt-b(5) augments 3βHSD activity and represents the first documentation of such augmentation in any species. PMID:21930205

  20. 40 CFR Table B-5 to Subpart B of... - Symbols and Abbreviations

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 6 2013-07-01 2013-07-01 false Symbols and Abbreviations B Table B-5 to Subpart B of Part 53 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Performance Characteristics of Automated Methods for SO2, CO, O3, and NO2 Pt. 53, Subpt. B, Table B-5 Table...

  1. A spectroscopic study of uranyl-cytochrome b5/cytochrome c interactions

    NASA Astrophysics Data System (ADS)

    Sun, Mei-Hui; Liu, Shuang-Quan; Du, Ke-Jie; Nie, Chang-Ming; Lin, Ying-Wu

    2014-01-01

    Uranium is harmful to human health due to its radiation damage and the ability of uranyl ion (UO22+) to interact with various proteins and disturb their biological functions. Cytochrome b5 (cyt b5) is a highly negatively charged heme protein and plays a key role in mediating cytochrome c (cyt c) signaling in apoptosis by forming a dynamic cyt b5-cyt c complex. In previous molecular modeling study in combination with UV-Vis studies, we found that UO22+ is capable of binding to cyt b5 at surface residues, Glu37 and Glu43. In this study, we further investigated the structural consequences of cyt b5 and cyt c, as well as cyt b5-cyt c complex, upon uranyl binding, by fluorescence spectroscopic and circular dichroism techniques. Moreover, we proposed a uranyl binding site for cyt c at surface residues, Glu66 and Glu69, by performing a molecular modeling study. It was shown that uranyl binds to cyt b5 (KD = 10 μM), cyt c (KD = 87 μM), and cyt b5-cyt c complex (KD = 30 μM) with a different affinity, which slightly alters the protein conformation and disturbs the interaction of cyt b5-cyt c complex. Additionally, we investigated the functional consequences of uranyl binding to the protein surface, which decreases the inherent peroxidase activity of cyt c. The information of uranyl-cyt b5/cyt c interactions gained in this study likely provides a clue for the mechanism of uranyl toxicity.

  2. Biliverdin reductase isozymes in metabolism.

    PubMed

    O'Brien, Luke; Hosick, Peter A; John, Kezia; Stec, David E; Hinds, Terry D

    2015-04-01

    The biliverdin reductase (BVR) isozymes BVRA and BVRB are cell surface membrane receptors with pleiotropic functions. This review compares, for the first time, the structural and functional differences between the isozymes. They reduce biliverdin, a byproduct of heme catabolism, to bilirubin, display kinase activity, and BVRA, but not BVRB, can act as a transcription factor. The binding motifs present in the BVR isozymes allow a wide range of interactions with components of metabolically important signaling pathways such as the insulin receptor kinase cascades, protein kinases (PKs), and inflammatory mediators. In addition, serum bilirubin levels have been negatively associated with abdominal obesity and hypertriglyceridemia. We discuss the roles of the BVR isozymes in metabolism and their potential as therapeutic targets. PMID:25726384

  3. An electrogenic nitric oxide reductase.

    PubMed

    Al-Attar, Sinan; de Vries, Simon

    2015-07-22

    Nitric oxide reductases (Nors) are members of the heme-copper oxidase superfamily that reduce nitric oxide (NO) to nitrous oxide (N₂O). In contrast to the proton-pumping cytochrome oxidases, Nors studied so far have neither been implicated in proton pumping nor have they been experimentally established as electrogenic. The copper-A-dependent Nor from Bacillus azotoformans uses cytochrome c₅₅₁ as electron donor but lacks menaquinol activity, in contrast to our earlier report (Suharti et al., 2001). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by Cu(A)Nor reconstituted in liposomes involves transmembrane cycling of the PES radical. We show that Cu(A)Nor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa₃, highlighting that bacilli using Cu(A)Nor can exploit NO reduction for increased cellular ATP production compared to organisms using cNor. PMID:26149211

  4. Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

    PubMed

    Chen, Xi'en; Zhang, Yalin

    2015-03-10

    NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides.

  5. Regulation of human adipogenesis by miR125b-5p.

    PubMed

    Rockstroh, Denise; Löffler, Dennis; Kiess, Wieland; Landgraf, Kathrin; Körner, Antje

    2016-01-01

    MicroRNAs (miRNAs) are non-coding RNAs that regulate target gene expression at the post-transcriptional level and are supposed to be implicated in the control of adipogenesis. We aimed to identify miRNAs which are involved in the regulation of human adipogenesis and searched for their molecular targets. Applying microarray-analysis we identified miR125b-5p as upregulated during human adipocyte differentiation, although its role during adipogenesis is unknown. We identified and characterized the matrix metalloproteinase 11 (MMP11) as a direct target of miR125b-5p by showing that miR125b-5p overexpression significantly reduces MMP11 luciferase activity and mutation of any single binding site was sufficient to abolish the miR125b-5p mediated inhibition of luciferase activity. MMP11 overexpression decreased fat accumulation, indicating that MMP11 acts as an anti-adipogenic regulator. In contrast, overexpression of miR125b-5p itself reduced adipogenesis. In summary, we identified miR125b-5p as upregulated during human adipogenesis indicating that miR125b-5p may serve as a regulator of human adipocyte differentiation. We further show that miR125b-5p downregulates the anti-adipogenic MMP11, but directly inhibits adipogenesis itself. Taken together, these data implicate that miR125b-5p can affect human adipogenesis via MMP11 and probably additional targets. PMID:27617174

  6. Regulation of human adipogenesis by miR125b-5p

    PubMed Central

    Rockstroh, Denise; Löffler, Dennis; Kiess, Wieland; Landgraf, Kathrin; Körner, Antje

    2016-01-01

    ABSTRACT MicroRNAs (miRNAs) are non-coding RNAs that regulate target gene expression at the post-transcriptional level and are supposed to be implicated in the control of adipogenesis. We aimed to identify miRNAs which are involved in the regulation of human adipogenesis and searched for their molecular targets. Applying microarray-analysis we identified miR125b-5p as upregulated during human adipocyte differentiation, although its role during adipogenesis is unknown. We identified and characterized the matrix metalloproteinase 11 (MMP11) as a direct target of miR125b-5p by showing that miR125b-5p overexpression significantly reduces MMP11 luciferase activity and mutation of any single binding site was sufficient to abolish the miR125b-5p mediated inhibition of luciferase activity. MMP11 overexpression decreased fat accumulation, indicating that MMP11 acts as an anti-adipogenic regulator. In contrast, overexpression of miR125b-5p itself reduced adipogenesis. In summary, we identified miR125b-5p as upregulated during human adipogenesis indicating that miR125b-5p may serve as a regulator of human adipocyte differentiation. We further show that miR125b-5p downregulates the anti-adipogenic MMP11, but directly inhibits adipogenesis itself. Taken together, these data implicate that miR125b-5p can affect human adipogenesis via MMP11 and probably additional targets. PMID:27617174

  7. 5 alpha-reductase deficiency without hypospadias.

    PubMed Central

    Ng, W K; Taylor, N F; Hughes, I A; Taylor, J; Ransley, P G; Grant, D B

    1990-01-01

    A boy aged 4 with penoscrotal hypospadias and his brother aged 12 with micropenis had typical changes of homozygous 5 alpha-reductase deficiency. After three injections of chorionic gonadotrophin there was a trivial rise in plasma dihydrotestosterone with a normal increase in plasma testosterone. Urine steroid chromatography showed abnormally high 5 beta: 5 alpha ratios and 5 alpha-reductase activity was appreciably reduced in genital skin fibroblasts. The results indicate that 5 alpha-reductase deficiency is not invariably associated with genital ambiguity. PMID:2248513

  8. Genetics Home Reference: sepiapterin reductase deficiency

    MedlinePlus

    ... reductase enzyme. This enzyme is involved in the production of a molecule called tetrahydrobiopterin (also known as ... is responsible for the last step in the production of tetrahydrobiopterin. Tetrahydrobiopterin helps process several building blocks ...

  9. Synthesis of NaB5C bulk ceramics by reaction sintering

    NASA Astrophysics Data System (ADS)

    Morito, Haruhiko; Anzai, Jun; Kimura, Takuma; Yamane, Hisanori

    2015-09-01

    Bulk ceramics of NaB5C were prepared by heating compact bodies of amorphous boron (B) and carbon black (C) powders with Na at 1073 K. The obtained bulk ceramics retained the rectangular shape of their original compacts. The obtained samples had a density of 80.1 ± 0.6% of the theoretical density of NaB5C. NaB5C bulk ceramics were also prepared by heating compacts comprised of B and C powders and Na. The addition of Na to the starting compact bodies increased the relative bulk density to 83.5 ± 0.4%. A fracture bending strength of 195 MPa was measured for the NaB5C bulk sample prepared from the compact of Na, B, and C.

  10. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Grant, M. A.; Hochstein, L. I.

    1984-01-01

    Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

  11. Mutagenesis of the palmitoylation site in vaccinia virus envelope glycoprotein B5.

    PubMed

    Lorenzo, María M; Sánchez-Puig, Juana M; Blasco, Rafael

    2012-04-01

    The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein-protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

  12. Thioredoxin Reductase and its Inhibitors

    PubMed Central

    Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea

    2014-01-01

    Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642

  13. Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells

    PubMed Central

    Sondhi, Varun; Owen, Bryn M.; Liu, Jiayan; Chomic, Robert; Kliewer, Steven A.; Hughes, Beverly A.; Arlt, Wiebke; Mangelsdorf, David J.

    2016-01-01

    Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5flox/flox:Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings. PMID:26974035

  14. The aldo-keto reductase superfamily homepage.

    PubMed

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  15. Modeling and signal analysis of semiconducting B(5)C neutron detectors

    NASA Astrophysics Data System (ADS)

    Harken, Andrew D.

    Neutron detectors are needed for a myriad of applications ranging from military uses to power generation monitors to medical radiation therapy. Recently, a class of semiconducting boron carbide (B5C)/silicon heterojunction diodes were demonstrated to detect thermal neutrons.[1] The B5C-based devices have advantageous features of requiring low operating voltage, low power, are robust and extremely thin while maintaining detection efficiency. A simple model was developed for the analysis of the neutron capture output spectrum from the detectors, which allowed the comparison of several differing styles of planar geometry detectors. The model was also utilized to obtain the functional dependence of the device efficiencies, capture product spectral features, and the capture product energy deposition on capture layer thickness. An all-B5C device construction was determined by the model to be the most efficient form of a B5C-based detector, which reaches nearly 100% detection efficiency with a low probability of false positives. This model showed agreement with output from a full-physics simulation package, GEANT4, and experimental neutron detection spectra from a B5C/Si device. The signals generated in a B5C/Si heterojunction diode during neutron and alpha particle detection experiments were analyzed through fitting of the output current pulses and through capture output spectra. The output current pulse analysis confirmed charge generation and collection from both materials in the diode and demonstrated the suitability of the B5C material for use in an all-semiconducting B5C neutron detector. The experimental output spectra were analyzed and determined to be lower in detected capture product energy than expected, but retained the spectral features that allowed analysis of the detection results. The development of the model and the results from the particle detection experiments show great promise for the future development of B5C neutron detectors. [1]B. W. Robertson, S

  16. MicroRNA-125b-5p mimic inhibits acute liver failure.

    PubMed

    Yang, Dakai; Yuan, Qinggong; Balakrishnan, Asha; Bantel, Heike; Klusmann, Jan-Henning; Manns, Michael P; Ott, Michael; Cantz, Tobias; Sharma, Amar Deep

    2016-01-01

    The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF. PMID:27336362

  17. The role of cytochrome b5 structural domains in interaction with cytochromes P450.

    PubMed

    Sergeev, G V; Gilep, A A; Usanov, S A

    2014-05-01

    To understand the role of the structural elements of cytochrome b5 in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b5 - microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b5 is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b5 to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b5 are mainly determined by the structure of the heme-binding domain.

  18. MicroRNA-125b-5p mimic inhibits acute liver failure

    PubMed Central

    Yang, Dakai; Yuan, Qinggong; Balakrishnan, Asha; Bantel, Heike; Klusmann, Jan-Henning; Manns, Michael P.; Ott, Michael; Cantz, Tobias; Sharma, Amar Deep

    2016-01-01

    The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF. PMID:27336362

  19. Structural and mechanistic insights on nitrate reductases.

    PubMed

    Coelho, Catarina; Romão, Maria João

    2015-12-01

    Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data. PMID:26362109

  20. Respiratory arsenate reductase as a bidirectional enzyme

    USGS Publications Warehouse

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  1. Respiratory arsenate reductase as a bidirectional enzyme

    SciTech Connect

    Richey, Christine; Chovanec, Peter; Hoeft, Shelley E.; Oremland, Ronald S.; Basu, Partha; Stolz, John F.

    2009-05-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  2. 26 CFR 20.2056(b)-5 - Marital deduction; life estate with power of appointment in surviving spouse.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... appointment in surviving spouse. 20.2056(b)-5 Section 20.2056(b)-5 Internal Revenue INTERNAL REVENUE SERVICE... surviving spouse. (a) In general. Section 2056(b)(5) provides that if an interest in property passes from the decedent to his surviving spouse (whether or not in trust) and the spouse is entitled for life...

  3. Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases.

    PubMed

    Gang, D R; Kasahara, H; Xia, Z Q; Vander Mijnsbrugge, K; Bauw, G; Boerjan, W; Van Montagu, M; Davin, L B; Lewis, N G

    1999-03-12

    Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

  4. 26 CFR 1.50B-5 - Limitations with respect to certain persons.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... INCOME TAXES Rules for Computing Credit for Expenses of Work Incentive Programs § 1.50B-5 Limitations... percent of $30,000). If an organization to which section 593 applies is a member of a controlled group (as... estate investment trust is a member of a controlled group (as defined in section 50A (a)(5)), the...

  5. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

    Streeter, J.G.; DeVine, P.J.

    1983-08-01

    Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase. 18 references

  6. Mitigating role of baicalein on lysosomal enzymes and xenobiotic metabolizing enzyme status during lung carcinogenesis of Swiss albino mice induced by benzo(a)pyrene.

    PubMed

    Naveenkumar, Chandrashekar; Raghunandakumar, Subramanian; Asokkumar, Selvamani; Binuclara, John; Rajan, Balan; Premkumar, Thandavamoorthy; Devaki, Thiruvengadam

    2014-06-01

    The lungs mainly serve as a primary site for xenobiotic metabolism and constitute an important defense mechanism against inhalation of carcinogens. Our current study aimed to evaluate the chemotherapeutic efficacy of baicalein (BE) in Swiss albino mice exposed to tobacco-specific carcinogen benzo(a)pyrene [B(a)P] for its ability to mitigate pulmonary carcinogenesis. Here, we report that altered activities/levels of lysosomal enzymes (cathepsin-D, cathepsin-B, acid phosphatase, β-D-galactosidase, β-D-glucuronidase, and β-D-N-acetyl glucosaminidase), phase I biotransformation enzymes (cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, and NADH-cytochrome b5 reductase), and phase II enzymes (glutathione S-transferase, UDP-glucuronyl transferase, and DT-diaphorase) were observed in the B(a)P-induced mice. Treatment with BE significantly restored back the activities/levels of lysosomal enzymes, phase I and phase II biotransformation enzymes. Moreover, assessment of lysosomal abnormalities by transmission electron microscopic examination revealed that BE treatment effectively counteract B(a)P-induced oxidative damages. Protein expression levels studied by immunohistochemistry, immunofluorescence, and immunoblot analysis of CYP1A1 revealed that BE treatment effectively negate B(a)P-induced upregulated expression of CYP1A1. Further analysis of scanning electron microscopic studies in lung was carried out to substantiate the anticarcinogenic effect of BE. The overall data suggest that BE treatment significantly inhibits lysosomal and microsomal dysfunction, thus revealing its potent anticarcinogenic effect.

  7. Electron-transport pathway of the NADH-dependent haem oxygenase system of rat liver microsomal fraction induced by cobalt chloride.

    PubMed Central

    Hino, Y; Minakami, S

    1979-01-01

    The hepatic microsomal haem oxygenase activity of rats treated with CoCl2 was studied kinetically by measuring biliverdin, the immediate product of the reaction. Biliverdin was extracted with diethyl ether/ethanol mixture, and was determined by the difference between A690 and A800. The apparent Km value for NADPH (at 50 microM-haematin) was about 0.2 microM when an NADPH-generating system was used, whereas that for NADH was about 630 microM. Essentially the same Vmax. values were obtained for both the NADH- and NADPH-dependent haem oxygenase reactions. No synergism was observed with NADH and NADPH. The NADH-dependent reaction was competitively inhibited by NADP+, with a Ki of about 10 microM. The inhibitoin of the NADH-dependent reaction by the antibody against rat liver microsomal NADPH-cytochrome c reductase was essentially complete, with a pattern similar to that of the NADPH-dependent reaction. The immunochemical experiment and the comparison of the kinetic values with the reported data on isolated NADH-cytochrome b5 reductase and NADPH--cytochrome c reductase indicated the involvement of the latter enzyme in NADH-dependent haem oxygenation by microsomal fraction in situ. PMID:36076

  8. Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin.

    PubMed Central

    Correll, C. C.; Ludwig, M. L.; Bruns, C. M.; Karplus, P. A.

    1993-01-01

    The structure of phthalate dioxygenase reductase (PDR), a monomeric iron-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP+ reductase (FNR) and ferredoxin, the proteins that reduce NADP+ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe-2S] are very similar in the two systems. Alignment of the X-ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel beta-barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753-3759). Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP+ arises primarily from substitutions in FNR that favor interactions with the 2' phosphate of NADP+. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN. The midpoint potentials for reduction of the flavin and [2Fe-2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant

  9. Promiscuity and diversity in 3-ketosteroid reductases

    PubMed Central

    Penning, Trevor M.; Chen, Mo; Jin, Yi

    2014-01-01

    Many steroid hormones contain a Δ4-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1–AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1–AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’. PMID:25500069

  10. Promiscuity and diversity in 3-ketosteroid reductases.

    PubMed

    Penning, Trevor M; Chen, Mo; Jin, Yi

    2015-07-01

    Many steroid hormones contain a Δ(4)-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1-AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1-AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'.

  11. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  12. Synthesis of Nitrate Reductase in Chlorella

    PubMed Central

    Funkhouser, Edward A.; Shen, Teh-Chien; Ackermann, Renate

    1980-01-01

    Synthesis of nitrate reductase (EC 1.6.6.1) in Chlorella vulgaris was studied under inducing conditions, i.e. with cells grown on ammonia and then transferred to nitrate medium. Cycloheximide (but not chloramphenicol) completely inhibited synthesis of the enzyme, but only if it was added at the start (i.e. at the time of nitrate addition) of the induction period. Cycloheximide inhibition became less effective as induction by nitrate proceeded. Enzyme from small quantities of culture (1 to 3 milliliters of packed cells) was purified to homogeneity with the aid of blue dextran-Sepharose chromatography. Incorporation of radioactivity from labeled arginine into nitrate reductase was measured in the presence and absence of cycloheximide. Conditions were found under which the inhibitor completely blocked the incorporation of labeled amino acid, but only slightly decreased the increase in nitrate reductase activity. The results indicate that synthesis of nitrate reductase from amino acids proceeds by way of a protein precursor which is inactive enzymically. PMID:16661310

  13. The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

    PubMed Central

    Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

    2012-01-01

    Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

  14. Autoxidation of soluble trypsin-cleaved microsomal ferrocytochrome b5 and formation of superoxide radicals.

    PubMed

    Berman, M C; Adnams, C M; Ivanetich, K M; Kench, J E

    1976-07-01

    The rate and mechanism of autoxidation of soluble ferrocytochrome b5, prepared from liver microsomal suspensions, appear to reflect an intrinsic property of membrane-bound cytochrome b5. The first-order rate constant for autoxidation of trypsin-cleaved ferrocytochrome b5, prepared by reduction with dithionite, was 2.00 X 10(-3) +/- 0.19 X 10(-3) S-1 (mean +/- S.E.M., n =8) when measured at 30 degrees C in 10 mM-phosphate buffer, pH 7.4. At 37 degrees C in aerated 10 mM-phosphate buffer (pH 7.4)/0.15 M-KCl, the rate constant was 5.6 X 10(-3) S-1. The autoxidation reaction was faster at lower pH values and at high ionic strengths. Unlike ferromyoglobin, the autoxidation reaction of which is maximal at low O2 concentrations, autoxidation of ferrocytochrome b5 showed a simple O2-dependence with an apparent Km for O2 of 2.28 X 10(-4) M (approx. 20kPa or 150mmHg)9 During autoxidation, 0.25 mol of O2 was consumed per mol of cytochrome oxidized. Cyanide, nucleophilic anions, EDTA and catalase each had little or no effect on autoxidation rates. Adrenaline significantly enhanced autoxidation rates, causing a tenfold increase at 0.6 mM. Ferrocytochrome b5 reduced an excess of cytochrome c in a biphasic manner. An initial rapid phase, independent of O2 concentration, was unaffected by superoxide dismutase. A subsequent slower phase, which continued for up to 60 min, was retarded at low O2 concentrations and inhibited by 65% by superoxide dismutase at a concentration of 3 mug/ml. It is concluded that autoxidation is responsible for a significant proportion of electron flow between cytochrome b5 and O2 in liver endoplasmic membranes, this reaction being capable of generating superoxide anions. A biological role for the reaction is discussed. PMID:183743

  15. Control of dihydrofolate reductase messenger ribonucleic acid production

    SciTech Connect

    Leys, E.J.; Kellems, R.E.

    1981-11-01

    The authors used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.

  16. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae.

    PubMed

    Chang, Qing; Griest, Terry A; Harter, Theresa M; Petrash, J Mark

    2007-03-01

    We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678

  17. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae*

    PubMed Central

    Chang, Qing; Griest, Terry A.; Harter, Theresa M.; Petrash, J. Mark

    2007-01-01

    SUMMARY We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678

  18. Augmentation of CFTR maturation by S-nitrosoglutathione reductase.

    PubMed

    Zaman, Khalequz; Sawczak, Victoria; Zaidi, Atiya; Butler, Maya; Bennett, Deric; Getsy, Paulina; Zeinomar, Maryam; Greenberg, Zivi; Forbes, Michael; Rehman, Shagufta; Jyothikumar, Vinod; DeRonde, Kim; Sattar, Abdus; Smith, Laura; Corey, Deborah; Straub, Adam; Sun, Fei; Palmer, Lisa; Periasamy, Ammasi; Randell, Scott; Kelley, Thomas J; Lewis, Stephen J; Gaston, Benjamin

    2016-02-01

    S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o(-)) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o(-) cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

  19. Cummins Engine Company B5.9 Propane Engine Development, Certification, and Demonstration Project

    SciTech Connect

    The ADEPT Group, Inc.

    1998-12-18

    The objective of this project was to successfuly develop and certify an LPG-dedicated medium-duty original equipment manufacturer (OEM) engine that could be put into production. The engine was launched into production in 1994, and more than 800 B5.9G engines are now in service in the United States and abroad. This engine is now offered by more than 30 bus and truck OEMs.

  20. The standards process: Technical committee X3B5 digital magnetic tape

    NASA Technical Reports Server (NTRS)

    Cheatham, Sam

    1993-01-01

    The definition of X3B5, where it fits in the national and international standards development process, and how it interfaces and influences the world community of standards developers are provided. Details concerning the focus of the committee, how it operates, and what the group sees as the future trends in the area of interchange standards utilizing the multifaceted, ubiquitous magnetic tape are presented.

  1. O(3P) attack on boranes. II. B5H9

    NASA Astrophysics Data System (ADS)

    Cheng, H.-Z.; Bauer, S. H.

    1991-06-01

    When B5H9 is injected into a stream of He that is carrying O(3P) atoms (approximately 100/1), at a total pressure of 5-15 Torr, a blue-green flame develops. The major chemiluminescent species is BO(A 2Π). While its translational and rotational temperatures are ≊350 K, the vibrational temperature in the A state is high, ≊3800 K. From among the many products of this reaction, the OH radical can be most easily quantitated by measuring the intensity of its laser-induced fluorescence. The central streamline from a flow-tube reactor was extracted into an evacuated plenum via a pinhole. The time-intensity profile was calibrated using C2H6 for the fuel. Check runs were made with B2H6. A multistep mechanism was developed for B5H9+O(3P) that simulates the shape as well as the magnitude of the OH concentration over a reactor residence time 0.5-10 ms. Less than a dozen crucial reactions were identified by means of an extended sensitivity analysis. Breakdown schemes for the oxidation of B2H6 and B5H9 have been developed.

  2. Separation of microsomal cytochrome b5 via phase separation in a mixed solution of Triton X-114 and charged dextran.

    PubMed

    Tani, H; Ooura, T; Kamidate, T; Kamataki, T; Watanabe, H

    1998-04-24

    The successful introduction of a charged dextran into the Triton X-114 phase separation system for the selective extraction of cytochrome b5 (cyt. b5) in liver microsomes is described. In the absence of charged dextran, 55% of total microsomal proteins and 84% of cyt. b5 were extracted into the surfactant-rich phase. In the presence of anionic dextran sulfate, the extractability of total microsomal proteins was greatly reduced while that of cyt. b5 was increased. After triplicate extraction, cyt. b5 was purified more than 10-fold from microsomes with a recovery of 91% in the surfactant-rich phase. In view of its operational simplicity, this method provides a good means for the partial purification of cyt. b5 prior to chromatographic separations.

  3. miR-487b-5p Regulates Temozolomide Resistance of Lung Cancer Cells Through LAMP2-Medicated Autophagy.

    PubMed

    Bao, Liang; Lv, Lei; Feng, Jinping; Chen, Yuyu; Wang, Xinhua; Han, Shuguang; Zhao, Hongqing

    2016-08-01

    Temozolomide (TMZ) is a standard agent used in the treatment of various types of cancers, including lung carcinoma, but TMZ resistance is common and accounts for many treatment failures. We investigated miRNA-487b-5p (miR-487b-5p) was highly expressed in A549 and H1299 cells which acquired TMZ resistance. Suppression of miR-487b-5p had overt effects on cellular proliferation and migration in the presence of TMZ. On the other hand, knockdown of miR-487b-5p resulted in increased survival and moderate tumor growth in vivo. In addition, the decreased cellular proliferation following miR-487b-5p suppression was linked to enhanced autophagy, evident by drastically increased levels of LC3-II, BECLIN1, and LAMP2 when miR-487b-5p was knocked down. Further analysis revealed that LAMP2 might be the target gene of miR-487b-5p. In conclusion, our study suggested that miR-487b-5p may be a potential biomarker of acquired TMZ resistance in lung cancer cells, and miR-487b-5p inhibition can be further explored as a chemotherapy target in the treatment of TMZ-resistant lung carcinoma.

  4. Structure of aldose reductase from Giardia lamblia

    PubMed Central

    Ferrell, M.; Abendroth, J.; Zhang, Y.; Sankaran, B.; Edwards, T. E.; Staker, B. L.; Van Voorhis, W. C.; Stewart, L. J.; Myler, P. J.

    2011-01-01

    Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP+-binding site located within the eight β-strands of the interior. PMID:21904059

  5. Steroid 5α-reductase 2 deficiency.

    PubMed

    Mendonca, Berenice B; Batista, Rafael Loch; Domenice, Sorahia; Costa, Elaine M F; Arnhold, Ivo J P; Russell, David W; Wilson, Jean D

    2016-10-01

    Dihydrotestosterone is a potent androgen metabolite formed from testosterone by action of 5α-reductase isoenzymes. Mutations in the type 2 isoenzyme cause a disorder of 46,XY sex development, termed 5α-reductase type 2 deficiency and that was described forty years ago. Many mutations in the encoding gene have been reported in different ethnic groups. In affected 46,XY individuals, female external genitalia are common, but Mullerian ducts regress, and the internal urogenital tract is male. Most affected males are raised as females, but virilization occurs at puberty, and male social sex develops thereafter with high frequency. Fertility can be achieved in some affected males with assisted reproduction techniques, and adults with male social sex report a more satisfactory sex life and quality of life as compared to affected individuals with female social sex. PMID:27224879

  6. Discovery of pinoresinol reductase genes in sphingomonads.

    PubMed

    Fukuhara, Y; Kamimura, N; Nakajima, M; Hishiyama, S; Hara, H; Kasai, D; Tsuji, Y; Narita-Yamada, S; Nakamura, S; Katano, Y; Fujita, N; Katayama, Y; Fukuda, M; Kajita, S; Masai, E

    2013-01-10

    Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

  7. Influence of P ion on Sr2B5O9Cl:Eu for TL dosimetry

    NASA Astrophysics Data System (ADS)

    Oza, Abha H.; Dhoble, N. S.; Dhoble, S. J.

    2015-02-01

    This paper investigates luminescence properties of Sr2B5O9Cl:Eu phosphor prepared by modified solid state diffusion. The influence of Phosphorous ion as codopant is also explained in detail. The structural confirmation of the sample was done using the XRD technique. SEM revealed the microcrystalline nature of the prepared phosphor. The characteristic Eu2+ emission at 437 nm and 423 nm was observed for Sr2B5O9Cl:Eu and Sr2B5O9Cl:P,Eu, respectively under 338 nm excitation. Samples in powder form were irradiated with different doses under γ-ray irradiation with 60Co source and the TL glow curves for both Sr2B5O9Cl:Eu and Sr2B5O9Cl:P,Eu samples were studied. In case of Sr2B5O9Cl:Eu phosphor, single glow curve nature centered on 260 °C with a shoulder peak around 144 °C was observed. However; Sr2B5O9Cl:P,Eu have shown slight different and broad glow curve nature. The TL sensitivity in both the cases was compared with CaSO4:Dy phosphor. Sr2B5O9Cl:Eu sample have shown 1.17 times less sensitivity than CaSO4:Dy and for Sr2B5O9Cl:P,Eu it was found to be equal to CaSO4:Dy and Sr2B5O9Cl:P,Eu is 1.21 times more sensitive than Sr2B5O9Cl:Eu. Other TL properties like dose response, fading and reusability were studied for both the samples. The trapping parameters for both the samples were calculated using computerized glow curve deconvolution and reported in this paper.

  8. Biochemical Characterization of a Haloalkane Dehalogenase DadB from Alcanivorax dieselolei B-5

    PubMed Central

    Li, Anzhang; Shao, Zongze

    2014-01-01

    Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest kcat/Km value toward 1,3-dibromopropane (Km = 0.82 mM, kcat/Km = 16.43 mM−1·s−1). DadB aggregates fast in the buffers with pH≤7.0, while keeps stable in monomer form when pH≥7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications. PMID:24586552

  9. Determination of the topography of cytochrome b5 in lipid vesicles by fluorescence quenching.

    PubMed

    Markello, T; Zlotnick, A; Everett, J; Tennyson, J; Holloway, P W

    1985-06-01

    Cytochrome b5, a protein isolated from the endoplasmic reticulum by detergent extraction, interacts spontaneously with small unilamellar phosphatidylcholine vesicles. When the vesicles are made from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), the tryptophan fluorescence of the cytochrome is enhanced, and when they are made from 1-palmitoyl-2-(dibromostearoyl) phosphatidylcholine (BRPC), the fluorescence is quenched. A series of BRPC were synthesized with bromine atoms at the 6,7, 9,10, 11,12 or 15,16 positions. The vesicles synthesized from each of these lipids were similar in size to those made from POPC. The relative fluorescence intensities of the cytochrome b5 in POPC and 6,7-, 9,10-, 11,12- and 15,16- BRPC were 100, 19.4, 29.4, 37.1, and 54.0, respectively. These data suggest that the exposed tryptophan(s) is (are) at a depth of 0.7 nm below the surface of the vesicle. Bromine is a collisional quencher; hence, these data may indicate the relative position of the lipid annulus around the protein rather than the depth of the protein below the average vesicle surface. Cytochrome b5 contains three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three are fluorescent with an average quantum yield, when in POPC vesicles, of 0.21. Fluorescence lifetime measurements by the demodulation technique indicated heterogeneity of fluorescence lifetimes in all vesicles. The lifetimes in the BRPC vesicles ranged from 2.0 to 2.4 ns compared to a value of 3.3 ns in POPC.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli.

    PubMed

    Hatakeyama, Mayumi; Kitaoka, Takuya; Ichinose, Hirofumi

    2016-07-01

    Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b5 (Cyt-b5) was further coexpressed with CPR, indicating that Cyt-b5 supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b5 but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an "alternative" electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways.

  11. IAU Resolution 2009 B5 - Commission 50 Draft Action Plan - Presentation and Discussion

    NASA Astrophysics Data System (ADS)

    Green, R. F.

    2015-03-01

    IAU Resolution 2009 B5 calls on IAU members to protect the public's right to an unpolluted night sky as well as the astronomical quality of the sky around major research observatories. The multi-pronged approach of Commission 50 includes working with the lighting industry for appropriate products from the solid state revolution, arming astronomers with training and materials for presentation, selective endorsement of key protection issues, cooperation with several other IAU commissions for education and outreach, and provision of clear quantitative priorities for outdoor lighting standards.

  12. Dynamic apparent transition resistance data in spot welding of aluminized 22MnB5.

    PubMed

    Kaars, Jonny; Mayr, Peter; Koppe, Kurt

    2016-09-01

    In-situ resistance measurements of aluminized 22MnB5 steel using a current ramp of 500 A/ms at welding force levels from 2 kN to 8 kN were conducted to obtain data on the dynamic resistance behaviour in spot welding of the material for varying mechanical and electrical loads. The data has been successfully used to calibrate a numerical transition resistance model (KMK-model, Kaars et al., 2016 [1]) in Kaars et al. (2016) [2].

  13. Dynamic apparent transition resistance data in spot welding of aluminized 22MnB5.

    PubMed

    Kaars, Jonny; Mayr, Peter; Koppe, Kurt

    2016-09-01

    In-situ resistance measurements of aluminized 22MnB5 steel using a current ramp of 500 A/ms at welding force levels from 2 kN to 8 kN were conducted to obtain data on the dynamic resistance behaviour in spot welding of the material for varying mechanical and electrical loads. The data has been successfully used to calibrate a numerical transition resistance model (KMK-model, Kaars et al., 2016 [1]) in Kaars et al. (2016) [2]. PMID:27547795

  14. Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

    DOEpatents

    Craft, David L.; Madduri, Krishna M.; Loper, John C.

    2003-01-01

    A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

  15. A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase.

    PubMed

    Wei, Yifeng; Li, Bin; Prakash, Divya; Ferry, James G; Elliott, Sean J; Stubbe, JoAnne

    2015-12-01

    Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism.

  16. Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

    PubMed Central

    Ma, Yan; Ludden, Paul W.

    2001-01-01

    Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding. PMID:11114923

  17. Quorum-sensing-directed protein expression in Serratia proteamaculans B5a.

    PubMed

    Christensen, Allan B; Riedel, Kathrin; Eberl, Leo; Flodgaard, Lars R; Molin, Søren; Gram, Lone; Givskov, Michael

    2003-02-01

    N-Acyl-L-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities, thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore, chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality.

  18. Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions.

    PubMed

    Doceul, Virginie; Hollinshead, Michael; Breiman, Adrien; Laval, Kathlyn; Smith, Geoffrey L

    2012-09-01

    Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.

  19. Ab initio study on structural, electronic properties, and hardness of re-doped W2B5

    NASA Astrophysics Data System (ADS)

    Feng, ShiQuan; Li, XiaoDong; Su, Lei; Li, HaiNing; Yang, Hongyan; Cheng, Xinlu

    2016-11-01

    Using first principle calculations method, we calculated the structural stability, electronic properties and hardness for three Re-doped hexagonal W2B5 compounds. The electronic properties were carried out to discuss the effect of doped Re on the conductivity, chemical bonding components and orbital hybridization of W2B5. What is more, the hardness of these three doped crystals was calculated by a semiempirical method considering the role of metallic components. A Re-doped W2B5 compound with a greater hardness value than that of pure W2B5 was obtained. And the effect of doping on the hardness of hexagonal W2B5was discussed.

  20. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    SciTech Connect

    Iyanagi, Takashi . E-mail: iyanagi@spring8.or.jp

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.

  1. Radical scavengers as ribonucleotide reductase inhibitors.

    PubMed

    Basu, Arijit; Sinha, Barij Nayan

    2012-01-01

    This paper compiled all the previous reports on radical scavengers, an interesting class of ribonucleotide reductase inhibitors. We have highlighted three key research areas: chemical classification of radical scavengers, structural and functional aspects of the radical site, and progress in drug designing for radical scavengers. Under the chemical classification section, we have recorded the discovery of hydroxyurea followed by discussions on hydroxamic acids, amidoximes, hydroxyguanidines, and phenolic compounds. In the next section, we have compiled the structural information for the radical site obtained from different crystallographic and theoretical studies. Finally, we have included the reported ligand based and structure based drug-designing studies.

  2. [Degradation of Steroidal Hormones by Salt-tolerant Altererythrobacter Strain MH-B5: Degradation Characteristics, Metabolites and Its Immobilization].

    PubMed

    Ma, Cong; Qin, Dan; Sun, Qian; Yu, Chang-ping

    2015-12-01

    A steroidal hormone degrading bacterium, named MH-B5, was isolated from saline lagoon. Several steroidal hormones including estrone, 17-β-estradiol, estriol and testosterone could be utilized as sole carbon source by strain MH-B5. Analysis of 16S rRNA gene sequence showed that it belonged to genus Altererythrobacter. Growth of strain MH-B5 was observed at salinities of 0-7% and at pH of 5.5-9.0. The main degradation metabolites of testosterone by strain MH-B5 were identified as 4-androstenedione, 1-dehydrotestosterone and androstadienedione using ultra performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight mass spectrometry (Q-TOF MS). Mono-carrier and multi-carrier cell immobilization techniques were successfully applied to this steroid-degrading bacterium. Batch estrogen degradation study showed that immobilized strain MH-B5 could effectively degrade 2 mg · L⁻¹ of 17β-estradiol and its metabolite estrone. The results mentioned above demonstrated the capability of MH-B5 to degrade both estrogens and testosterone and the potential application of using immobilized MH-B5 cells in bioremediation of steroidal hormone pollution in saline environment. PMID:27012005

  3. Coxsackievirus B5 induced apoptosis of HeLa cells: Effects on p53 and SUMO

    SciTech Connect

    Gomes, Rogerio; Guerra-Sa, Renata; Arruda, Eurico

    2010-01-20

    Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system.

  4. Studies on NADH (NADPH)-cytochrome c reductase (FMN-containing) from yeast. Isolation and physicochemical properties of the enzyme from top-fermenting ale yeast.

    PubMed

    Johnson, M S; Kuby, S A

    1985-10-01

    Only three major NADPH-nitrotetrazolium blue (NTB) reductases may be detected in a unique top-ale yeast (Saccharomyces cerevisiae, Narragansett strain), which appears to be of a near anaerobic type with the absence of cytochromes c and a/a3 and the presence of cytochromes P-450 and b5. Two of these three major NADPH-NTB reductases possessed NADH-NTB reductase activity; the third was specific for NADPH and was isolated in this laboratory (Tryon, E., Cress, M. C., Hamada, M., and Kuby, S. A. (1979) Arch. Biochem. Biophys. 197, 104-118) vis. NADPH-cytochrome c reductase (FAD-containing). A description of the isolation procedure is provided for one of these two NADH(NADPH)-NTB reductases, viz. NADH(NADPH)-cytochrome c reductase (FMN-containing), which accounts for about one-half of the total cyanide-insensitive menadione-activated respiration of this yeast. This NADH(NADPH)-cytochrome c reductase has been isolated from an extract of an acetone powder of the top-fermenting ale yeast, with an apparent purification of more than 67-fold and a final specific activity of 0.41 and 0.31 mumol/min/mg for NADH- and NADPH-dependent reduction, respectively. The isolated enzyme proved to be homogeneous by electrophoresis on cellulose acetate and on polyacrylamide gels. It had a pI of 5.25 (at gamma/2 = 0.05) and a molecular size under nondenaturing conditions (as determined by chromatography on Sephadex G-100 and Sephacryl S-200) of 70,000 daltons. On denaturation, the enzyme dissociated into two similar, if not identical, subunits which possessed a molecular weight of 34,000 by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis and a weight average molecular weight of 35,000 by sedimentation equilibrium in the presence of 4.0 M guanidinium chloride. The absorbance spectrum of NADH(NADPH)-cytochrome c reductase (FMN-containing) showed three maxima at 464, 383, and 278 nm, with extinction coefficients of 9.88, 9.98, and 64.6 mM-1 cm-1, respectively. The reductase, as

  5. The mammalian molybdenum enzymes of mARC.

    PubMed

    Ott, Gudrun; Havemeyer, Antje; Clement, Bernd

    2015-03-01

    The "mitochondrial amidoxime reducing component" (mARC) is the most recently discovered molybdenum-containing enzyme in mammals. All mammalian genomes studied to date contain two mARC genes: MARC1 and MARC2. The proteins encoded by these genes are mARC-1 and mARC-2 and represent the simplest form of eukaryotic molybdenum enzymes, only binding the molybdenum cofactor. In the presence of NADH, mARC proteins exert N-reductive activity together with the two electron transport proteins cytochrome b5 type B and NADH cytochrome b5 reductase. This enzyme system is capable of reducing a great variety of N-hydroxylated substrates. It plays a decisive role in the activation of prodrugs containing an amidoxime structure, and in detoxification pathways, e.g., of N-hydroxylated purine and pyrimidine bases. It belongs to a group of drug metabolism enzymes, in particular as a counterpart of P450 formed N-oxygenated metabolites. Its physiological relevance, on the other hand, is largely unknown. The aim of this article is to summarize our current knowledge of these proteins with a special focus on the mammalian enzymes and their N-reductive activity.

  6. Evaluation of Polymorphisms in the Sulfonamide Detoxification Genes CYB5A and CYB5R3 in Dogs with Sulfonamide Hypersensitivity

    PubMed Central

    Funk-Keenan, J.; Sacco, J.; (Amos) Wong, Y. Y.; Rasmussen, S.; Motsinger-Reif, A.; Trepanier, L. A.

    2015-01-01

    Background Delayed hypersensitivity (HS) reactions to potentiated sulfonamide antimicrobials occur in both dogs and humans, and involve an intermediate hydroxylamine metabolite that is detoxified by cytochrome b5 and NADH cytochrome b5 reductase. Hypothesis/Objectives We hypothesized that polymorphisms in the genes (CYB5A and CYB5R3) encoding these 2 enzymes would be associated with risk of sulfonamide HS in dogs. Animals A total of 18 dogs with delayed HS to potentiated sulfonamide antimicrobials and 16 dogs that tolerated (TOL) a therapeutic course of these drugs without adverse effect. Methods CYB5A and CYB5R3 were sequenced from canine liver, and the promoter, exons, and 3′ untranslated regions of both genes were resequenced from genomic DNA obtained from all dogs. Results Multiple polymorphisms were found in both genes. When controlled for multiple comparisons, the 729GG variant in CYB5R3 was significantly overrepresented in dogs with sulfonamide HS (78% of dogs), compared to TOL dogs (31%; P = .003). Conclusions and Clinical Importance The CYB5R3 729GG variant may contribute to the risk of sulfonamide HS in dogs. Functional characterization of this polymorphism, as well as genotyping in a larger number of HS and TOL dogs, is warranted. PMID:22816446

  7. B5r gene based sequence analysis of Indian buffalopox virus isolates in relation to other orthopoxviruses.

    PubMed

    Singh, R K; Balamurugan, V; Hosamani, M; DE, U K; Chandra, B M; Krishnappa M P, G

    2007-01-01

    We determined complete nucleotide sequence of B5R gene homologue of Vaccinia virus (VACV) in five Buffalopox virus (BPXV) isolates of Indian origin. The obtained sequences were compared with themselves and with corresponding sequences of the other orthopoxviruses. Sequence analysis revealed 99.799.8% and 99.499.7% identities among the BPXV isolates for B5R gene at the nucleotide and amino acid levels, respectively. Sequence identities of B5R gene between BPXV and VACV isolates (98.199.7%) or other orthopoxviruses (95.699.2%) showed highly conserved nature of this protein and a closer relationship of BPXV isolates to VACV than to other orthopoxviruses.

  8. Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum

    PubMed Central

    Li, Xiaochun; Roberti, Rita; Blobel, Günter

    2014-01-01

    Sterols are essential biological molecules in the majority of life forms. Sterol reductases1 including Delta-14 sterol reductase (C14SR), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon-carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B Receptor2 (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Delta-14 sterol reductase (maSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z, a homolog of human C14SR, LBR, and DHCR7, with the cofactor NADPH. The enzyme contains 10 transmembrane segments (TM). Its catalytic domain comprises the C-terminal half (containing TM6-10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5β-reductase structure3 suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves maSR1 can reduce the double bond of a cholesterol biosynthetic intermediate demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases. PMID:25307054

  9. Biliverdin reductase: a target for cancer therapy?

    PubMed Central

    Gibbs, Peter E. M.; Miralem, Tihomir; Maines, Mahin D.

    2015-01-01

    Biliverdin reductase (BVR) is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s) of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1, and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ, and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation. PMID:26089799

  10. Ageing of glutathione reductase in the lens.

    PubMed

    Zhang, W Z; Augusteyn, R C

    1994-07-01

    The distribution of glutathione reductase activity in concentric layers from the lens has been determined as a function of age for 16 species. Primate lenses have almost ten times the level of glutathione reductase found in other species. Comparison with the activity of hexokinase revealed that this is not due to a higher overall rate of metabolism in these lenses. By contrast, the higher activity found in bird and fish lenses reflects a higher metabolic activity in these tissues. In all species, a gradient of activity was observed with the highest specific activity in the outermost cortical fibres, decreasing to virtually no activity in the inner parts of the tissue. No alterations were found in this gradient with increasing age, other than an increase in the amount of nuclear tissue essentially devoid of activity. The maximum activity in the outer cortical fibres was the same, regardless of the age of the lens. The time taken, in different species, for the specific activity to decrease by half, was estimated from the rate of protein accumulation. This time was found to vary from a few days to several years, indicating that the decrease in activity is not due to ageing but rather, it is related to the maturation of fibre cells. These observations are discussed in terms of current concepts of lens ageing and cataract formation. PMID:7835401

  11. Major neutralizing sites on vaccinia virus glycoprotein B5 are exposed differently on variola virus ortholog B6.

    PubMed

    Aldaz-Carroll, Lydia; Xiao, Yuhong; Whitbeck, J Charles; de Leon, Manuel Ponce; Lou, Huan; Kim, Mikyung; Yu, Jessica; Reinherz, Ellis L; Isaacs, Stuart N; Eisenberg, Roselyn J; Cohen, Gary H

    2007-08-01

    Immunization against smallpox (variola virus) with Dryvax, a live vaccinia virus (VV), was effective, but now safety is a major concern. To overcome this issue, subunit vaccines composed of VV envelope proteins from both forms of infectious virions, including the extracellular enveloped virion (EV) protein B5, are being developed. However, since B5 has 23 amino acid differences compared with its B6 variola virus homologue, B6 might be a better choice for such a strategy. Therefore, we compared the properties of both proteins using a panel of monoclonal antibodies (MAbs) to B5 that we had previously characterized and grouped according to structural and functional properties. The B6 gene was obtained from the Centers for Disease Control and Prevention, and the ectodomain was cloned and expressed in baculovirus as previously done with B5, allowing us to compare the antigenic properties of the proteins. Polyclonal antibodies to B5 or B6 cross-reacted with the heterologous protein, and 16 of 26 anti-B5 MAbs cross-reacted with B6. Importantly, 10 anti-B5 MAbs did not cross-react with B6. Of these, three have important anti-VV biologic properties, including their ability to neutralize EV infectivity and block comet formation. Here, we found that one of these three MAbs protected mice from a lethal VV challenge by passive immunization. Thus, epitopes that are present on B5 but not on B6 would generate an antibody response that would not recognize B6. Assuming that B6 contains similar variola virus-specific epitopes, our data suggest that a subunit vaccine using the variola virus homologues might exhibit improved protective efficacy against smallpox.

  12. Purification and characterization of two forms of cytochrome b5 from an arachidonic acid-producing fungus, Mortierella hygrophila.

    PubMed

    Kouzaki, N; Kawashima, H; Chung, M C; Shimizu, S

    1995-06-01

    Two forms of cytochrome b5 have been purified from the microsomes of an arachidonic acid-producing fungus, Mortierella hygrophila IFO 5941, after detergent solubilization. They have monomeric molecular masses of about 16 kDa and 19 kDa. Their absorption spectra are similar to those of mammalian cytochrome b5s. Their amino acid compositions show some similarity to those of mammalian cytochrome b5s, but the contents of some amino acids (glycine, alanine, aspartic acid + asparagine, glutamic acid + glutamine, arginine, proline, histidine, leucine and lysine) are unique to the cytochrome b5s of M. hygrophila. Some of their internal peptide sequences also show close homology with those of some mammals (approx. 65 to 67%), while some others show no or little homology. The addition of various acyl-CoAs to NADH-reduced microsomes caused an abrupt shiftdown of the steady state reduction level of cytochrome b5. This indicates the increased utilization of electrons for the desaturation process and may suggest that the cytochrome b5s of this fungus actually take part in its microsomal desaturation system for polyunsaturated fatty acid biosynthesis as electron carriers. PMID:7786894

  13. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  14. The aldo-keto reductases (AKRs): Overview.

    PubMed

    Penning, Trevor M

    2015-06-01

    The aldo-keto reductase (AKR) protein superfamily contains >190 members that fall into 16 families and are found in all phyla. These enzymes reduce carbonyl substrates such as: sugar aldehydes; keto-steroids, keto-prostaglandins, retinals, quinones, and lipid peroxidation by-products. Exceptions include the reduction of steroid double bonds catalyzed by AKR1D enzymes (5β-reductases); and the oxidation of proximate carcinogen trans-dihydrodiol polycyclic aromatic hydrocarbons; while the β-subunits of potassium gated ion channels (AKR6 family) control Kv channel opening. AKRs are usually 37kDa monomers, have an (α/β)8-barrel motif, display large loops at the back of the barrel which govern substrate specificity, and have a conserved cofactor binding domain. AKRs catalyze an ordered bi bi kinetic mechanism in which NAD(P)H cofactor binds first and leaves last. In enzymes that favor NADPH, the rate of release of NADP(+) is governed by a slow isomerization step which places an upper limit on kcat. AKRs retain a conserved catalytic tetrad consisting of Tyr55, Asp50, Lys84, and His117 (AKR1C9 numbering). There is conservation of the catalytic mechanism with short-chain dehydrogenases/reductases (SDRs) even though they show different protein folds. There are 15 human AKRs of these AKR1B1, AKR1C1-1C3, AKR1D1, and AKR1B10 have been implicated in diabetic complications, steroid hormone dependent malignancies, bile acid deficiency and defects in retinoic acid signaling, respectively. Inhibitor programs exist world-wide to target each of these enzymes to treat the aforementioned disorders. Inherited mutations in AKR1C and AKR1D1 enzymes are implicated in defects in the development of male genitalia and bile acid deficiency, respectively, and occur in evolutionarily conserved amino acids. The human AKRs have a large number of nsSNPs and splice variants, but in many instances functional genomics is lacking. AKRs and their variants are now poised to be interrogated using

  15. Anisotropic and Mechanical Behavior of 22MnB5 in Hot Stamping Operations

    SciTech Connect

    Turetta, A.; Ghiotti, A.; Bruschi, S.

    2007-04-07

    The hot stamping of quenchable High Strength Steels offers the possibility of weight reduction in structural components maintaining the safety requirements together with enhanced accuracy and formability of sheets. The proper design of this technology requires a deep understanding of material behavior during the entire process chain, in terms of microstructural evolution and mechanical properties at elevated temperatures, in order to perform reliable FE simulations and obtain the desired characteristic on final parts. In particular, the analysis of technical-scientific literature shows that accurate data on material rheological behavior are difficult to find; while the lack of knowledge about anisotropic behavior at elevated temperatures is even more evident. To overcome these difficulties, a new experimental set-up was developed to reproduce the thermo-mechanical conditions of the industrial process and evaluate the influence of temperature and strain rate on 22MnB5 flow curves through uniaxial tensile tests; an optical strain measurement system was utilized to evaluate the effective strain after necking. From the same data, plastic anisotropy evolution was determined by means of a specially developed procedure. The influence of different cooling rates was taken into account and the rheological properties were correlated with microstructural changes occurring during deformation, previously evaluated through a dilatometric analysis performed in the same range of temperatures.

  16. Hot Hydroforming of 22MnB5 Tube by Resistance Heating

    NASA Astrophysics Data System (ADS)

    Chu, G. N.; Lin, Y. L.; Ding, M. Q.

    2016-07-01

    To promote the application of high strength steels in automobile bodies, the practicability of hot hydroforming of tube by resistance heating is illustrated. From the results of experiments conducted to measure temperature distributions of the tube during the forming process, a method to improve temperature uniformity has been proposed and achieved. Validity was evaluated by examining the effects of hot gas forming on the microstructure and hardness. Results indicate an obvious temperature difference along the axial direction for two cross-sectional shapes: the temperature in the middle zone of the tube is higher than that at its ends. Both thermal convection and cross-sectional shape have only a limited effect on the temperature distribution. The main reason for non-uniform temperature distribution is the heat transition between the electrodes and the tube ends. The temperature difference decreased as the heating rate increased. In contrast, the temperature distribution was even along the circumferential direction for both cross-sectional shapes. Adjusting the contact resistance is a useful method of reducing the temperature difference. In this study, the temperature difference was successfully decreased to 20°C, while reaching a maximum temperature of 750°C, which is adequate for both forming and quenching. A rectangular component was formed to validate the practicality and efficiency of tube hot hydroforming by resistance heating. The hardness and microstructure met the requirements of 22MnB5, which demonstrates both the forming efficiency and quantity advantages of hot gas hydroforming by resistance heating.

  17. Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites.

    PubMed

    Iribarne, Federico; Paulino, Margot; Aguilera, Sara; Murphy, Miguel; Tapia, Orlando

    2002-05-01

    A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.

  18. Transcripts of anthocyanidin reductase and leucoanthocyanidin reductase and measurement of catechin and epicatechin in tartary buckwheat.

    PubMed

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, Yeji; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

  19. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    PubMed Central

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, YeJi; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

  20. Transcripts of anthocyanidin reductase and leucoanthocyanidin reductase and measurement of catechin and epicatechin in tartary buckwheat.

    PubMed

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, Yeji; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

  1. Ribonucleotide reductases: essential enzymes for bacterial life

    PubMed Central

    Torrents, Eduard

    2014-01-01

    Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria. PMID:24809024

  2. Ribonucleotide reductases: essential enzymes for bacterial life.

    PubMed

    Torrents, Eduard

    2014-01-01

    Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria. PMID:24809024

  3. Ribonucleotide reductases: essential enzymes for bacterial life.

    PubMed

    Torrents, Eduard

    2014-01-01

    Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria.

  4. Monodehydroascorbate reductase mediates TNT toxicity in plants.

    PubMed

    Johnston, Emily J; Rylott, Elizabeth L; Beynon, Emily; Lorenz, Astrid; Chechik, Victor; Bruce, Neil C

    2015-09-01

    The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Due to the scale of affected areas, one of the most cost-effective and environmentally friendly means of removing explosives pollution could be the use of plants. However, mechanisms of TNT phytotoxicity have been elusive. Here, we reveal that phytotoxicity is caused by reduction of TNT in the mitochondria, forming a nitro radical that reacts with atmospheric oxygen, generating reactive superoxide. The reaction is catalyzed by monodehydroascorbate reductase 6 (MDHAR6), with Arabidopsis deficient in MDHAR6 displaying enhanced TNT tolerance. This discovery will contribute toward the remediation of contaminated sites. Moreover, in an environment of increasing herbicide resistance, with a shortage in new herbicide classes, our findings reveal MDHAR6 as a valuable plant-specific target.

  5. The cytochrome bd respiratory oxygen reductases

    PubMed Central

    Borisov, Vitaliy B.; Gennis, Robert B.; Hemp, James; Verkhovsky, Michael I.

    2011-01-01

    Summary Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. PMID:21756872

  6. The cytochrome bd respiratory oxygen reductases.

    PubMed

    Borisov, Vitaliy B; Gennis, Robert B; Hemp, James; Verkhovsky, Michael I

    2011-11-01

    Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated.

  7. Vir Proteins Stabilize VirB5 and Mediate Its Association with the T Pilus of Agrobacterium tumefaciens

    PubMed Central

    Schmidt-Eisenlohr, Heike; Domke, Natalie; Angerer, Christina; Wanner, Gerhard; Zambryski, Patricia C.; Baron, Christian

    1999-01-01

    Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from Agrobacterium tumefaciens, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C. I. Kado, J. Bacteriol. 180:2711–2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction. In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation. VirB5 may therefore be directly involved in pilus assembly, possibly as minor component. In contrast, secreted VirB1* showed no association with the T pilus. In-frame deletions in genes virB1, virB2, virB5, and virB6 blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation. PMID:10601205

  8. Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens.

    PubMed

    Schmidt-Eisenlohr, H; Domke, N; Angerer, C; Wanner, G; Zambryski, P C; Baron, C

    1999-12-01

    Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from Agrobacterium tumefaciens, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C. I. Kado, J. Bacteriol. 180:2711-2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction. In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation. VirB5 may therefore be directly involved in pilus assembly, possibly as minor component. In contrast, secreted VirB1* showed no association with the T pilus. In-frame deletions in genes virB1, virB2, virB5, and virB6 blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation. PMID:10601205

  9. Nitrate Reductase-Deficient Mutants in Barley 1

    PubMed Central

    Somers, David A.; Kuo, Tsung-Min; Kleinhofs, Andris; Warner, Robert L.

    1983-01-01

    Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross-reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate. Images Fig. 1 Fig. 3 PMID:16662774

  10. OBSERVATION OF KINK INSTABILITY DURING SMALL B5.0 SOLAR FLARE ON 2007 JUNE 4

    SciTech Connect

    Srivastava, A. K.; Kumar, Pankaj; Zaqarashvili, T. V.; Khodachenko, M. L. E-mail: pkumar@aries.res.i E-mail: maxim.khodachenko@oeaw.ac.a

    2010-05-20

    Using multi-wavelength observations of SOHO/MDI, SOT-Hinode/blue-continuum (4504 A), G band (4305 A), Ca II H (3968 A), and TRACE 171 A, we present the observational signature of a highly twisted magnetic loop in AR 10960 during the period 04:43 UT-04:52 UT on 2007 June 4. SOT-Hinode/blue-continuum (4504 A) observations show that penumbral filaments of positive polarity sunspot have counterclockwise twist, which may be caused by the clockwise rotation of the spot umbrae. The coronal loop, whose one footpoint is anchored in this sunspot, shows strong right-handed twist in chromospheric SOT-Hinode/Ca II H (3968 A) and coronal TRACE 171 A images. The length and the radius of the loop are L {approx} 80 Mm and a {approx} 4.0 Mm, respectively. The distance between neighboring turns of magnetic field lines (i.e., pitch) is estimated as {approx}10 Mm. The total twist angle, {Phi} {approx} 12{pi} (estimated for the homogeneous distribution of the twist along the loop), is much larger than the Kruskal-Shafranov instability criterion. We detected clear double structure of the loop top during 04:47 UT-04:51 UT on TRACE 171 A images, which is consistent with simulated kink instability in curved coronal loops. We suggest that the kink instability of this twisted magnetic loop triggered a B5.0 class solar flare, which occurred between 04:40 UT and 04:51 UT in this active region.

  11. Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase.

    PubMed

    Quinn, G B; Trimboli, A J; Prosser, I M; Barber, M J

    1996-03-01

    was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.

  12. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

  13. Substrate induction of nitrate reductase in barley aleurone layers.

    PubMed

    Ferrari, T E; Varner, J E

    1969-01-01

    Nitrate induces the formation of nitrate reductase activity in barley (Hordeum vulgare L. cv. Himalaya) aleurone layers. Previous work has demonstrated de novo synthesis of alpha-amylase by gibberellic acid in the same tissue. The increase in nitrate reductase activity is inhibited by cycloheximide and 6-methylpurine, but not by actinomycin D. Nitrate does not induce alpha-amylase synthesis, and it has no effect on the gibberellic acid-induced synthesis of alpha-amylase. Also, there is little or no direct effect of gibberellic acid (during the first 6 hr of induction) or of abscisic acid on the nitrate-induced formation of nitrate reductase. Gibberellic acid does interfere with nitrate reductase activity during long-term experiments (greater than 6 hr). However, the time course of this inhibition suggests that the inhibition may be a secondary one. Barley aleurone layers therefore provide a convenient tissue for the study of both substrate- and hormone-induced enzyme formation.

  14. HD 35502: a hierarchical triple system with a magnetic B5IVpe primary

    NASA Astrophysics Data System (ADS)

    Sikora, J.; Wade, G. A.; Bohlender, D. A.; Shultz, M.; Adelman, S. J.; Alecian, E.; Hanes, D.; Monin, D.; Neiner, C.; MiMeS Collaboration; BinaMIcS Collaboration

    2016-08-01

    We present our analysis of HD 35502 based on high- and medium-resolution spectropolarimetric observations. Our results indicate that the magnetic B5IVsnp star is the primary component of a spectroscopic triple system and that it has an effective temperature of 18.4 ± 0.6 kK, a mass of 5.7 ± 0.6 M⊙, and a polar radius of 3.0^{+1.1}_{-0.5} R_{odot }. The two secondary components are found to be essentially identical A-type stars for which we derive effective temperatures (8.9 ± 0.3 kK), masses (2.1 ± 0.2 M⊙), and radii (2.1 ± 0.4 R⊙). We infer a hierarchical orbital configuration for the system in which the secondary components form a tight binary with an orbital period of 5.668 66(6) d that orbits the primary component with a period of over 40 yr. Least-Squares Deconvolution profiles reveal Zeeman signatures in Stokes V indicative of a longitudinal magnetic field produced by the B star ranging from approximately -4 to 0 kG with a median uncertainty of 0.4 kG. These measurements, along with the line variability produced by strong emission in Hα, are used to derive a rotational period of 0.853 807(3) d. We find that the measured v sin i = 75 ± 5 km s-1 of the B star then implies an inclination angle of the star's rotation axis to the line of sight of 24^{+6}_{-10}{}^circ. Assuming the Oblique Rotator Model, we derive the magnetic field strength of the B star's dipolar component (14^{+9}_{-3} kG) and its obliquity (63± 13deg). Furthermore, we demonstrate that the calculated Alfvén radius (41^{+17}_{-6}R_ast) and Kepler radius (2.1^{+0.4}_{-0.7}R_ast) place HD 35502's central B star well within the regime of centrifugal magnetosphere-hosting stars.

  15. Synthesis of Substituted 2,3,5,6-tetraarylbenzo(1,2-b:5,4-b')difurans

    NASA Technical Reports Server (NTRS)

    Abdul-Aziz, Mahmoud; Auping, Judith V.; Meador, Michael A.

    1995-01-01

    A series of substituted 2,3,5,6-tetraarylbenzo(l,2-b:5,4-b')difurans 1 was synthesized. This synthesis is based upon the photocyclization of 2,5-dibenzoylresorcinol dibenzyl ethers to the corresponding tetrahydrobenzo(1,2-b:5,4-b')difurans. Treatment of the photoproducts with methanesulfonyl chloride in pyridine afforded 1 in overall yields ranging from 30-72%. A number of these compounds have high fluorescence quantum yields (of phi(sub f) = 0.76-0.90), and their fluorescence spectra exhibit large solvatochromic shifts. These compounds may be suitable for use as fluorescent probes.

  16. Comparative anatomy of the aldo-keto reductase superfamily.

    PubMed Central

    Jez, J M; Bennett, M J; Schlegel, B P; Lewis, M; Penning, T M

    1997-01-01

    The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short

  17. [Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

    PubMed

    Gnedenko, O V; Ivanov, A S; Iablokov, E O; Usanov, S A; Mukha, D V; Sergeev, G V; Kuzikov, A V; Moskaleva, N E; Bulko, T V; Shumiantseva, V V; Archakov, A I

    2014-01-01

    Molecular interactions between proteins redox partners (cytochromes P450 3A4, 3A5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 - -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 to testosterone.

  18. [Protein-protein interactions of cytochromes P450 3A4 and 3A5 with their intermediate redox partners cytochromes b5].

    PubMed

    Gnedenko, O V; Ivanov, A S; Yablokov, E O; Usanov, S A; Mukha, D V; Sergeev, G V; Kuzikov, A V; Bulko, T V; Moskaleva, N E; Shumyantseva, V V; Archakov, A I

    2015-01-01

    Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

  19. Regulation of the Neurospora crassa assimilatory nitrate reductase.

    PubMed Central

    Ketchum, P A; Zeeb, D D; Owens, M S

    1977-01-01

    Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source. PMID:19423

  20. An overview on 5alpha-reductase inhibitors.

    PubMed

    Aggarwal, Saurabh; Thareja, Suresh; Verma, Abhilasha; Bhardwaj, Tilak Raj; Kumar, Manoj

    2010-02-01

    Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associated with benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy, urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of 5alpha-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues and hence suppression of androgen action by 5alpha-reductase inhibitors is a logical treatment for BPH as they inhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the first steroidal 5alpha-reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases the prostatic DHT level by 70-90% and reduces the prostatic size. Dutasteride (27) another related analogue has been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5alpha-reductase type I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A number of classes of non-steroidal inhibitors of 5alpha-reductase have also been synthesized generally by removing one or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). In this review all categories of inhibitors of 5alpha-reductase have been covered. PMID:19879888

  1. Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome

    PubMed Central

    Bondareva, Alla A.; Capecchi, Mario R.; Iverson, Sonya V.; Li, Yan; Lopez, Nathan I.; Lucas, Olivier; Merrill, Gary F.; Prigge, Justin R.; Siders, Ashley M.; Wakamiya, Maki; Wallin, Stephanie L.; Schmidt, Edward E.

    2007-01-01

    Thioredoxin reductases (Txnrd)1 maintain intracellular redox homeostasis in most organisms. Metazoans Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1−/− cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd−/− and txnrd+/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells. PMID:17697936

  2. Aldose Reductase, Oxidative Stress, and Diabetic Mellitus

    PubMed Central

    Tang, Wai Ho; Martin, Kathleen A.; Hwa, John

    2012-01-01

    Diabetes mellitus (DM) is a complex metabolic disorder arising from lack of insulin production or insulin resistance (Diagnosis and classification of diabetes mellitus, 2007). DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR; ALR2; EC 1.1.1.21), a key enzyme in the polyol pathway, catalyzes nicotinamide adenosine dinucleotide phosphate-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues of DM including the heart, vasculature, neurons, eyes, and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis) and myocardium (heart failure) leading to severe morbidity and mortality (reviewed in Heather and Clarke, 2011). In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis, and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications. PMID:22582044

  3. Aldose reductase, oxidative stress, and diabetic mellitus.

    PubMed

    Tang, Wai Ho; Martin, Kathleen A; Hwa, John

    2012-01-01

    Diabetes mellitus (DM) is a complex metabolic disorder arising from lack of insulin production or insulin resistance (Diagnosis and classification of diabetes mellitus, 2007). DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR; ALR2; EC 1.1.1.21), a key enzyme in the polyol pathway, catalyzes nicotinamide adenosine dinucleotide phosphate-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues of DM including the heart, vasculature, neurons, eyes, and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis) and myocardium (heart failure) leading to severe morbidity and mortality (reviewed in Heather and Clarke, 2011). In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis, and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications. PMID:22582044

  4. Aldose reductase mediates retinal microglia activation.

    PubMed

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy. PMID:27033597

  5. Aldose reductase mediates retinal microglia activation.

    PubMed

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy.

  6. 17 CFR 240.19b-5 - Temporary exemption from the filing requirements of Section 19(b) of the Act.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... requirements: (1) Form PILOT. The self-regulatory organization: (i) Files Part I of Form PILOT, 17 CFR 249.821... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Temporary exemption from the... of Exchange Members § 240.19b-5 Temporary exemption from the filing requirements of Section 19(b)...

  7. Sperm membrane proteome in wild Japanese macaque (Macaca fuscata) and Sika deer (Cervus nippon).

    PubMed

    Kawase, Osamu; Cao, Shinuo; Xuan, Xuenan

    2015-01-01

    Whereas recent advances in proteome-related techniques have accumulated a lot of information about sperm proteins in model animals, the information in non-model wildlife species is absolutely deficient, although this knowledge would be valuable to regulate wildlife overabundance. To characterize the repertoires of sperm membrane proteins in Japanese overpopulated wildlife, our study focuses on the following two species: Macaca fuscata and Cervus nippon. We enriched sperm membrane proteins by the phase partitioning with Triton X-114, and then separated them by two-dimensional electrophoresis, and, finally, they were comprehensively identified by peptide mass fingerprinting. Sperm membrane proteins were successfully enriched. They included some proteins with unknown function and fertility-related proteins that work in sperm development, motility, capacitation, transport, protection, acrosome reaction, and fertilization. Additionally, beta-defensin 126 and epithelial chloride channel were strongly detected in M. fuscata but not in C. nippon, whereas lactadherin and NADH-cytochrome b5 reductase 1 were strongly detected in C. nippon alone. This study is an initiative case showing that the sperm of wildlife conserve major fertility-related proteins, but express some proteins in a species-specific manner. In the development of a practical method for fertility control, this aspect may be taken into consideration.

  8. Recessive congenital methemoglobinemia caused by a rare mechanism: maternal uniparental heterodisomy with segmental isodisomy of a chromosome 22.

    PubMed

    Huang, Yu-Hsiu; Tai, Chang-Long; Lu, Yung-Hsiu; Wu, Tina Jui-Ting; Chen, Hong-Duo; Niu, Dau-Ming

    2012-08-15

    Recessive congenital methemoglobinemia (RCM) is a very rare disorder caused by NADH-cytochrome b5 reductase (cb5r) deficiency. Two distinct clinical forms, types I and II, caused by cb5r deficiency have been recognized. In type I, the enzyme deficiency is restricted only to erythrocytes with cyanosis being the only major symptom. In contrast, in type II, the enzyme deficiency is generalized to all tissues and associated with neurological impairment, mental and growth retardation and reduced life expectancy, in addition to cyanosis. Recently, we conducted a study on an 11-year-old boy with cb5r deficiency type I. The mutational analysis of the CYB5R3 gene revealed that the boy is homozygous for L72P mutation. Surprisingly, his mother is heterozygous for this L72P mutant, but not his father. Thirteen microsatellite markers of chromosome 22 were selected to analyze the origins of the patient's chromosome 22. The result showed that both of the chromosome 22(s) of this patient came from the maternal side (uniparental heterodisomy of chromosome 22 with segmental isodisomy). This is the first case report of a patient with cb5r deficiency type I resulting from uniparental disomy and also discloses an alternate mechanism whereby this enzymatic disorder can be derived from a single parent. PMID:22658170

  9. Sperm membrane proteome in wild Japanese macaque (Macaca fuscata) and Sika deer (Cervus nippon).

    PubMed

    Kawase, Osamu; Cao, Shinuo; Xuan, Xuenan

    2015-01-01

    Whereas recent advances in proteome-related techniques have accumulated a lot of information about sperm proteins in model animals, the information in non-model wildlife species is absolutely deficient, although this knowledge would be valuable to regulate wildlife overabundance. To characterize the repertoires of sperm membrane proteins in Japanese overpopulated wildlife, our study focuses on the following two species: Macaca fuscata and Cervus nippon. We enriched sperm membrane proteins by the phase partitioning with Triton X-114, and then separated them by two-dimensional electrophoresis, and, finally, they were comprehensively identified by peptide mass fingerprinting. Sperm membrane proteins were successfully enriched. They included some proteins with unknown function and fertility-related proteins that work in sperm development, motility, capacitation, transport, protection, acrosome reaction, and fertilization. Additionally, beta-defensin 126 and epithelial chloride channel were strongly detected in M. fuscata but not in C. nippon, whereas lactadherin and NADH-cytochrome b5 reductase 1 were strongly detected in C. nippon alone. This study is an initiative case showing that the sperm of wildlife conserve major fertility-related proteins, but express some proteins in a species-specific manner. In the development of a practical method for fertility control, this aspect may be taken into consideration. PMID:25277530

  10. Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II.

    PubMed

    Cingolani, Francesca; Casasampere, Mireia; Sanllehí, Pol; Casas, Josefina; Bujons, Jordi; Fabrias, Gemma

    2014-08-01

    Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites.

  11. Role of 5 alpha-reductase in health and disease.

    PubMed

    Randall, V A

    1994-04-01

    The mechanism of androgen action varies in different tissues, but in the majority of androgen target tissues either testosterone or 5 alpha-dihydrotestosterone (DHT) binds to a specific androgen receptor to form a complex that can regulate gene expression. Testosterone is metabolized to DHT by the enzyme 5 alpha-reductase. The autosomal recessive genetic disorder of 5 alpha-reductase deficiency has clearly shown that the requirement for DHT formation varies with different tissues. In this syndrome genetic males contain normal male internal structures including testes, but exhibit ambiguous or female external genitalia at birth; at puberty they undergo partial virilization which includes development of a male gender identity even if brought up as females. Their development suggests that testosterone itself is able to stimulate psychosexual behaviour, development of the embryonic wolffian duct, muscle development, voice deepening, spermatogenesis, and axillary and pubic hair growth; DHT seems to be essential for prostate development and growth, the development of the external genitalia and male patterns of facial and body hair growth or male-pattern baldness. How different hormones operate to regulate genes via the same receptor is currently unknown, but appears to involve cell-specific factors. The 5-alpha-reductase enzyme has proved difficult to isolate biochemically, but recently at least two human isoenzymes have been identified using molecular biological methods. All the various 5 alpha-reductase-deficient kindreds have been shown to have mutations in 5 alpha-reductase 2, the predominant form in the prostate. The biological role of 5 alpha-reductase 1 has not yet been ascertained, but at present it cannot be ruled out that some of the actions ascribed to testosterone are indeed in cells producing DHT via this enzyme. The activity of 5 alpha-reductase is also implicated in benign prostatic hypertrophy, hirsutism and possibly male-pattern baldness; recent evidence

  12. Deorphanization and characterization of the ectopically expressed olfactory receptor OR51B5 in myelogenous leukemia cells

    PubMed Central

    Manteniotis, S; Wojcik, S; Göthert, J R; Dürig, J; Dührsen, U; Gisselmann, G; Hatt, H

    2016-01-01

    The ectopic expression of olfactory receptors (ORs) in the human body has been of major interest in the past decade. Several studies have reported the expression of ORs not only in healthy tissues such as heart, sperm or skin cells, but also in cancerous tissues of the liver, prostate or intestine. In the present study, we detected the expression of OR51B5 in the chronic myelogenous leukemia (CML) cell line K562 and in white blood cell samples of clinically diagnosed acute myelogenous leukemia (AML) patients by reverse transcription-PCR and immunocytochemical staining. The known OR51B5 ligand isononyl alcohol increased the levels of intracellular Ca2+ in both AML patient blood cells and K562 cells. With calcium imaging experiments, we characterized in greater detail the OR51B5-mediated signaling pathway. Here, we observed an involvement of adenylate cyclase and the downstream L-type and T-type calcium channels. In addition, the activation of OR51B5 leads to an inhibition of cell proliferation in K562 cells. In western blot experiments, we found that incubation with isononyl alcohol led to a reduction in p38-MAPK (mitogen-activated protein kinase) phosphorylation that might be responsible for the decreased cell proliferation. In the present study, we characterized the OR51B5-mediated signaling pathway downstream of the activation with isononyl alcohol, which leads to reduced proliferation and therefore provide a novel pharmacological target for CML and AML, the latter of which remains difficult to treat. PMID:27551504

  13. Protonated Dipeptide Losses from b 5 and b 4 Ions of Side Chain Hydroxyl Group Containing Pentapeptides

    NASA Astrophysics Data System (ADS)

    Atik, A. Emin; Yalcin, Talat

    2013-10-01

    In this study, C-terminal protonated dipeptide eliminations were reported for both b 5 and b 4 ions of side chain hydroxyl group (-OH) containing pentapeptides. The study utilized the model C-terminal amidated pentapeptides having sequences of XGGFL and AXVYI, where X represents serine (S), threonine (T), glutamic acid (E), aspartic acid (D), or tyrosine (Y) residue. Upon low-energy collision-induced dissociation (CID) of XGGFL (where X = S, T, E, D, and Y) model peptide series, the ions at m/z 279 and 223 were observed as common fragments in all b 5 and b 4 ion (except b 4 ion of YGGFL) mass spectra, respectively. By contrast, peptides, namely SMeGGFL-NH2 and EOMeGGFL-NH2, did not show either the ion at m/z 279 or the ion at m/z 223. It is shown that the side chain hydroxyl group is required for the possible mechanism to take place that furnishes the protonated dipeptide loss from b 5 and b 4 ions. In addition, the ions at m/z 295 and 281 were detected as common fragments in all b 5 and b 4 ion (except b 4 ion of AYVYI) mass spectra, respectively, for AXVYI model peptide series. The MS4 experiments exhibited that the fragment ions at m/z 279, 223, 295, and 281 entirely reflect the same fragmentation behavior of [M + H]+ ion generated from commercial dipeptides FL-OH, GF-OH, YI-OH, and VY-OH. These novel eliminations reported here for b 5 and b 4 ions can be useful in assigning the correct and reliable peptide sequences for high-throughput proteomic studies.

  14. Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase.

    PubMed

    Lancaster, C Roy D

    2003-11-27

    The three-dimensional structure of Wolinella succinogenes quinol:fumarate reductase (QFR), a dihaem-containing member of the superfamily of succinate:quinone oxidoreductases (SQOR), has been determined at 2.2 A resolution by X-ray crystallography [Lancaster et al., Nature 402 (1999) 377-385]. The structure and mechanism of W. succinogenes QFR and their relevance to the SQOR superfamily have recently been reviewed [Lancaster, Adv. Protein Chem. 63 (2003) 131-149]. Here, a comparison is presented of W. succinogenes QFR to the recently determined structure of the mono-haem containing succinate:quinone reductase from Escherichia coli [Yankovskaya et al., Science 299 (2003) 700-704]. In spite of differences in polypeptide and haem composition, the overall topology of the membrane anchors and their relative orientation to the conserved hydrophilic subunits is strikingly similar. A major difference is the lack of any evidence for a 'proximal' quinone site, close to the hydrophilic subunits, in W. succinogenes QFR.

  15. Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa

    PubMed Central

    Lu, Wanxiang; Yang, Li; Karim, Abdul; Luo, Keming

    2013-01-01

    Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (−)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus. PMID:23741362

  16. Equine 5α-reductase activity and expression in epididymis.

    PubMed

    Corbin, C J; Legacki, E L; Ball, B A; Scoggin, K E; Stanley, S D; Conley, A J

    2016-10-01

    The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies. PMID:27466384

  17. Equine 5α-reductase activity and expression in epididymis.

    PubMed

    Corbin, C J; Legacki, E L; Ball, B A; Scoggin, K E; Stanley, S D; Conley, A J

    2016-10-01

    The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies.

  18. DNA damage induction of ribonucleotide reductase.

    PubMed

    Elledge, S J; Davis, R W

    1989-11-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous. PMID:2513480

  19. Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis.

    PubMed

    Tchobanov, Iavor; Gal, Laurent; Guilloux-Benatier, Michèle; Remize, Fabienne; Nardi, Tiziana; Guzzo, Jean; Serpaggi, Virginie; Alexandre, Hervé

    2008-07-01

    Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. The reasons are the production of ethylphenols that lead to an unpleasant taint described as 'phenolic odour'. Despite its economic importance, Brettanomyces has remained poorly studied at the metabolic level. The origin of the ethylphenol results from the conversion of vinylphenols in ethylphenol by Brettanomyces hydroxycinnamate decarboxylase. However, no information is available on the vinylphenol reductase responsible for the conversion of vinylphenols in ethylphenols. In this study, a vinylphenol reductase was partially purified from Brettanomyces bruxellensis that was active towards 4-vinylguaiacol and 4-vinylphenol only among the substrates tested. First, a vinylphenol reductase activity assay was designed that allowed us to show that the enzyme was NADH dependent. The vinylphenol reductase was purified 152-fold with a recovery yield of 1.77%. The apparent K(m) and V(max) values for the hydrolysis of 4-vinylguaiacol were, respectively, 0.14 mM and 1900 U mg(-1). The optimal pH and temperature for vinylphenol reductase were pH 5-6 and 30 degrees C, respectively. The molecular weight of the enzyme was 26 kDa. Trypsic digest of the protein was performed and the peptides were sequenced, which allowed us to identify in Brettanomyces genome an ORF coding for a 210 amino acid protein.

  20. Restoring virulence to mutants lacking subunits of multiprotein machines: functional complementation of a Brucella virB5 mutant

    PubMed Central

    Sprynski, Nicolas; Felix, Christine; O’Callaghan, David; Vergunst, Annette C.

    2012-01-01

    Complementation for virulence of a non-polar virB5 mutant in Brucella suis 1330 was not possible using a pBBR-based plasmid but was with low copy vector pGL10. Presence of the pBBR-based replicon in wildtype B. suis had a dominant negative effect, leading to complete attenuation in J774 macrophages. This was due to pleiotropic effects on VirB protein expression due to multiple copies of the virB promoter region and over expression of VirB5. Functional complementation of mutants in individual components of multiprotein complexes such as bacterial secretion systems, are often problematic; this study highlights the importance of using a low copy vector. PMID:23650582

  1. Evolution of mechanical properties of boron/manganese 22MnB5 steel under magnetic pulse influences

    NASA Astrophysics Data System (ADS)

    Falaleev, A. P.; Meshkov, V. V.; Vetrogon, A. A.; Shymchenko, A. V.

    2016-02-01

    The boron/manganese 22MnB5 steel can be noted as the widely used material for creation of details, which must withstand high amount of load and impact influences. The complexity and high labor input of restoration of boron steel parts leads to growing interest in the new forming technologies such as magnetic pulse forming. There is the investigation of the evolution of mechanical properties of 22MnB5 steel during the restoration by means of magnetic pulse influence and induction heating. The heating of 22MnB5 blanks to the temperature above 9000C was examined. The forming processes at various temperatures (800, 900 and 9500C) were performed during the experiments. The test measurements allowed to obtain the relationships between the strain and the operation parameters such as induced current, pulse discharge time and the operation temperature. Based on these results the assumption about usage of these parameters for control of deformation process was made. Taking into account the load distribution and the plasticity evolution during the heating process, the computer simulation was performed in order to obtain more clear strain distribution through the processed area. The measurement of hardness and the comparison with the properties evolution during hot stamping processes confirmed the obtained results.

  2. Crystal structure of red chlorophyll catabolite reductase: enlargement of the ferredoxin-dependent bilin reductase family.

    PubMed

    Sugishima, Masakazu; Kitamori, Yuka; Noguchi, Masato; Kohchi, Takayuki; Fukuyama, Keiichi

    2009-06-01

    The key steps in the degradation pathway of chlorophylls are the ring-opening reaction catalyzed by pheophorbide a oxygenase and sequential reduction by red chlorophyll catabolite reductase (RCCR). During these steps, chlorophyll catabolites lose their color and phototoxicity. RCCR catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite. RCCR appears to be evolutionarily related to the ferredoxin-dependent bilin reductase (FDBR) family, which synthesizes a variety of phytobilin pigments, on the basis of sequence similarity, ferredoxin dependency, and the common tetrapyrrole skeleton of their substrates. The evidence, however, is not robust; the identity between RCCR and FDBR HY2 from Arabidopsis thaliana is only 15%, and the oligomeric states of these enzymes are different. Here, we report the crystal structure of A. thaliana RCCR at 2.4 A resolution. RCCR forms a homodimer, in which each subunit folds in an alpha/beta/alpha sandwich. The tertiary structure of RCCR is similar to those of FDBRs, strongly supporting that these enzymes evolved from a common ancestor. The two subunits are related by noncrystallographic 2-fold symmetry in which the alpha-helices near the edge of the beta-sheet unique in RCCR participate in intersubunit interaction. The putative RCC-binding site, which was derived by superimposing RCCR onto biliverdin-bound forms of FDBRs, forms an open pocket surrounded by conserved residues among RCCRs. Glu154 and Asp291 of A. thaliana RCCR, which stand opposite each other in the pocket, likely are involved in substrate binding and/or catalysis.

  3. 4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase.

    PubMed

    Lambooy, J P; Koffman, B M

    1985-01-01

    Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.

  4. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase.

    PubMed

    Leavitt, William D; Bradley, Alexander S; Santos, André A; Pereira, Inês A C; Johnston, David T

    2015-01-01

    The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major ((34)S/(32)S) and minor ((33)S/(32)S, (36)S/(32)S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in (34)S/(32)S (hereafter, [Formula: see text]) to be 15.3 ± 2‰, 2σ. The accompanying minor isotope effect in (33)S, described as [Formula: see text], is calculated to be 0.5150 ± 0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3-0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in (34)εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of [Formula: see text] is similar to the median value of experimental observations compiled from all known published work, where (34)ε r-p = 16.1‰ (r-p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments ([Formula: see text] 17.3 ± 1.5‰, 2σ) and in modern marine sediments ([Formula: see text] 17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the

  5. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase

    PubMed Central

    Leavitt, William D.; Bradley, Alexander S.; Santos, André A.; Pereira, Inês A. C.; Johnston, David T.

    2015-01-01

    The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major (34S/32S) and minor (33S/32S, 36S/32S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in 34S/32S (hereafter, 34εDsrAB) to be 15.3 ± 2‰, 2σ. The accompanying minor isotope effect in 33S, described as 33λDsrAB, is calculated to be 0.5150 ± 0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3–0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in 34εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of 34εDsrAB is similar to the median value of experimental observations compiled from all known published work, where 34εr−p = 16.1‰ (r–p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments (34εSO4−H2S =  17.3 ± 1.5‰, 2σ) and in modern marine sediments (34εSO4−H2S =  17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in

  6. Selective enhancement of xenobiotic metabolizing systems of rat lungs by prolonged exposure to ozone

    SciTech Connect

    Takahashi, Y.; Miura, T.

    1987-04-01

    Male Wistar rats were exposed to 0.2 and 0.4 ppm O/sub 3/ for 4, 8, and 12 weeks and to 0.1 and 0.2 ppm O/sub 3/ for 4 weeks to examine the effects of prolonged exposure to O/sub 3/ on the xenobiotic metabolizing systems in the lung. Exposures to 0.2 and 0.4 ppm O/sub 3/ caused a significant increase in the NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content in a dose-dependent manner during 4-12 weeks, whereas NADH-cytochrome b5 reductase was not altered. After 12 weeks of exposure to 0.4 ppm O/sub 3/, NADPH-cytochrome P-450 reductase and cytochrome P-450 reached a maximum showing 138 and 221% those of the control levels, respectively. At 0.1 ppm O/sub 3/, the cytochrome P-450 content still increased significantly to 152% that of the control on the fourth week. In parallel to the increment of cytochrome P-450 benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase increased significantly during 4-12 weeks of exposures to 0.2 and 0.4 ppm O/sub 3/. After a 4-week exposure to 0.1 and 0.2 ppm O/sub 3/, benzphetamine N-demethylase increased to the largest degree among the enzymes metabolizing four kinds of xenobiotics examined. 7-Ethoxycoumarin O-deethylase also increased significantly but of smaller magnitude, whereas coumarin hydroxylase was not affected. These results show that prolonged exposures to 0.1-0.4 ppm O/sub 3/ persistently enhance pulmonary cytochrome P-450 systems. It is also suggested that some isozyme(s) of pulmonary cytochrome P-450, especially that catalyzing benzphetamine N-demethylation, are preferentially increased by exposure to low levels of O/sub 3/.

  7. The toxic effect of cypermethrin, amitraz and combinations of cypermethrin-amitraz in rats.

    PubMed

    Kanbur, Murat; Siliğ, Yavuz; Eraslan, Gökhan; Karabacak, Mürsel; Soyer Sarıca, Zeynep; Şahin, Serap

    2016-03-01

    In this study, the effects of cypermethrin (CYP), amitraz (AMT) and combined cypermethrin-amitraz (CYP-AMT) on some serum biochemical, oxidative stress and drug-metabolising parameters were investigated in male Wistar albino rats. CYP, AMT and combined CYP-AMT were administered at doses of 80 mg kg(-1) bw(-1) of CYP and 170 mg kg(-1) bw(-1) of AMT for 1 day (single dose), and at doses of 12 mg kg(-1) bw(-1) of CYP and 25 mg kg(-1) bw(-1) of AMT for 40 days by oral gavage. Oxidative stress (malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glucose-6-phosphate dehydrogenase (G6PD)), serum biochemical (glucose, triglyceride, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood urea nitrogen (BUN), creatinine, asparatate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total protein, albumin) in blood/tissues (liver, kidney, brain, spleen and testis) and hepatic drug-metabolising (cytochrome P450 2E1 (CYP2E1), NADH-cytochrome b5 reductase (CYPb5), NADPH-cytochrome c reductase/NADPH cytocrome P450 reductase (CYTC), glutathione S-transferase (GST), glutathione (GSH)) parameters were measured in liver samples taken on days 1 and 40. In result, it was determined that CYP, AMT and their combinations led to significant changes in the parameters investigated, and it was ascertained that long-term exposure to insecticides and the administration of insecticide combinations produced greater toxic effects in comparison with the administration of insecticides alone.

  8. Some physical and immunological properties of ox kidney biliverdin reductase.

    PubMed Central

    Rigney, E M; Phillips, O; Mantle, T J

    1988-01-01

    The liver, kidney and spleen of the mouse and rat and the kidney and spleen of the ox express a monomeric form of biliverdin reductase (Mr 34,000), which in the case of the ox kidney enzyme exists in two forms (pI 5.4 and 5.2) that are probably charge isomers. The livers of the mouse and rats express, in addition, a protein (Mr 46,000) that cross-reacts with antibodies raised against the ox kidney enzyme and may be related to form 2 described by Frydman, Tomaro, Awruch & Frydman [(1983) Biochim. Biophys. Acta 759, 257-263]. Higher-Mr forms appear to exist in the guinea pig and hamster. The ox kidney enzyme has three thiol groups, of which two are accessible to 5,5'-dithiobis-(2-nitrobenzoate) in the native enzyme. Immunocytochemical analysis reveals that biliverdin reductase is localized in proximal tubules of the inner cortex of the rat kidney. Biliverdin reductase antiserum also stains proximal tubules in human and ox kidney. The staining of podocytes in glomeruli of ox kidney with antiserum to aldose reductase is particularly prominent. The localization of biliverdin reductase in the inner cortical zone of rat kidney is similar to that described for glutathione S-transferase YfYf, and it is suggested that one function of this 'intracellular binding protein' may be to maintain a low free concentration of biliverdin to allow biliverdin reductase to operate efficiently. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3060109

  9. miR-146b-5p functions as a tumor suppressor by targeting TRAF6 and predicts the prognosis of human gliomas

    PubMed Central

    Sun, Cuiyun; Yu, Lin; Li, Yanyan; Shi, Cuijuan; Zhou, Xuexia; Bian, Xiuwu; Ping, Yifang; Wen, Yanjun; Zhao, Shujun; Xu, Hui; Ren, Linlin; An, Tongling; Wang, Qian; Yu, Shizhu

    2015-01-01

    Down-regulation of miR-146b-5p contributes to tumorigenesis in several human cancers. However, the relevance of miR-146b-5p to prognosis, proliferation and apoptosis in gliomas remains unknown. In the present study, we demonstrated that miR-146b-5p expression was inversely correlated with grades and Ki-67 index in 147 human glioma specimens, but positively correlated with patients’ survival. Furthermore, two distinct subgroups of patients with grade I-IV gliomas with different prognoses were identified according to miR-146b-5p expression in our specimens. Cox regression showed that miR-146b-5p was an independent predictor for patients’ survival. Overexpression of miR-146b-5p dramatically suppressed glioma cell proliferation and induced apoptosis. Mechanistically, we validated TRAF6 as a direct functional target of miR-146b-5p and found that miR-146b-5p overexpression significantly decreased phosphorylated TAK1 and IκBα, the pivotal downstream effectors of TRAF6. Moreover, TRAF6 expression was positively correlated with glioma grades and Ki-67 index but inversely correlated with miR-146b-5p expression and predicted poor prognosis of glioma patients. In glioblastoma cell lines, silencing of TRAF6 could mimic the anti-tumor effect of miR-146b-5p. Our findings identify miR-146b-5p as a tumor suppressor and novel prognostic biomarker of gliomas, and suggest miR-146b-5p and TRAF6 as potential therapeutic candidates for malignant gliomas. PMID:26320176

  10. Role of cytochrome B5 in modulating peroxide-supported cyp3a4 activity: evidence for a conformational transition and cytochrome P450 heterogeneity.

    PubMed

    Kumar, Santosh; Davydov, Dmitri R; Halpert, James R

    2005-08-01

    The role of cytochrome b(5) (b(5)) in the alpha-naphthoflavone (alpha-NF)-mediated inhibition of H(2)O(2)-supported 7-benzyloxyquinoline (7-BQ) debenzylation by heterologously expressed and purified cytochrome P450 3A4 (CYP3A4) was studied. Although alpha-NF showed negligible effect in an NADPH-dependent reconstituted system, inhibition of 7-BQ oxidation was observed in the H(2)O(2) system. Analysis of the effect of various constituents of a standard reconstituted system on H(2)O(2)-supported activity showed that b(5) alone resulted in a 2.5-fold increase in the k(cat) value and reversed the inhibitory effect of alpha-NF. In addition, titration with b(5) suggested that only 65% of the CYP3A4 participated in the interaction with b(5), consistent with cytochrome P450 (P450) heterogeneity. Study of the influence of b(5) on the kinetics of H(2)O(2)-dependent destruction of the P450 heme moiety suggested two distinct conformers of CYP3A4 with different sensitivity to heme loss. In the absence of b(5), 66% of the wild-type enzyme was bleached in the fast phase, whereas the addition of b(5) decreased the fraction of the fast phase to 16%. Finally, to locate amino acid residues that might influence b(5) action, several active site mutants were tested. Substitution of Ser-119, Ile-301, Ala-305, Ile-369, or Ala-370 with the larger Phe or Trp decreased or even abolished the activation by b(5). Ser-119 is in the B'-C loop, a predicted b(5)-P450 interaction site, and Ile-301 and Ala-305 are closest to the heme. In conclusion, the interaction of b(5) with P450 apparently leads to a conformational transition, which results in redistribution of the CYP3A4 pool. PMID:15870379

  11. Twist1-related miR-26b-5p suppresses epithelial-mesenchymal transition, migration and invasion by targeting SMAD1 in hepatocellular carcinoma

    PubMed Central

    Zhao, Xiulan; Zhao, Nan; Sun, Ran; Zhu, Dongwang; Zhang, Yanhui; Li, Yanlei; Gu, Qiang; Dong, Xueyi; Wang, Meili; An, Jindan

    2016-01-01

    Twist1 is well known to induce epithelial-mesenchymal transition (EMT) and promote tumor metastasis. MicroRNAs (miRNAs) are involved in the EMT process and are associated with metastasis in hepatocellular carcinoma (HCC). In the present study, microRNA-26b-5p (miR-26b-5p) expression was consistently and significantly downregulated in HepG2-Twist1 HCC cell lines compared with HepG2-vector cell lines using microarrays (the HepG2-Twist1 cell line can stably express Twist1). miR-26b- 5p downregulation was directly mediated by Twist1 through binding to the promoter region of miR-26b-5p in HepG2-Twist1 cells by ChIP-seq technology. Both gain- and loss-of-function studies showed that miR-26b-5p dramatically suppressed EMT and the invasion ability of HCC cells in vitro. Using mouse models, tumors derived from miR- 26b-5p-overexpressed HCC cells exhibited a significant reduction in tumorigenicity compared with the control group. Subsequent investigation revealed that miR-26b-5p directly inhibited SMAD family member 1 (SMAD1) expression. miR-26b-5p repressed BMP4/Smad1 signaling following SMAD1 inhibition. Overexpression of SMAD1 reversed the function of miR-26b-5p. In human HCC tissues and mouse xenograft tumors, miR-26b-5p levels were inversely correlated with SMAD1 expression as well as metastasis. Conclusion: miR-26b-5p suppresses Twist1-induced EMT, invasion, and metastasis of HCC cells by targeting SMAD1 and BMP4/Smad1 signaling. This suggests a promising application for miR-26b-5p in anti-HCC therapy. PMID:27027434

  12. The inhibitory activity of aldose reductase in vitro by constituents of Garcinia mangostana Linn.

    PubMed

    Fatmawati, Sri; Ersam, Taslim; Shimizu, Kuniyoshi

    2015-01-15

    We investigated aldose reductase inhibition of Garcinia mangostana Linn. from Indonesia. Dichloromethane extract of the root bark of this tree was found to demonstrate an IC50 value of 11.98 µg/ml for human aldose reductase in vitro. From the dichloromethane fraction, prenylated xanthones were isolated as potent human aldose reductase inhibitors. We discovered 3-isomangostin to be most potent against aldose reductase, with an IC50 of 3.48 µM.

  13. Pentaborate polyanion in the crystal structure of ulexite, NaCaB5O6(OH)6*5H2O

    USGS Publications Warehouse

    Clark, Joan R.; Appleman, Daniel E.

    1964-01-01

    Triclinic ulexite crystals contain isolated borate polyanions [B5O6(OH)6]3- related to the well known pentaborate polyanion [B5O6(OH)4]- by addition of two hydroxyl groups to two opposite B-O triangles. The isolated ulexite polyanions form the [B5O7(OH)4]n3n- chains previously found in crystals of the related mineral probertite, NaCaB5O7(OH)4·3H2O.

  14. Improved technique for fluorescence in situ hybridisation analysis of isolated nuclei from archival, B5 or formalin fixed, paraffin wax embedded tissue.

    PubMed

    Schurter, M J; LeBrun, D P; Harrison, K J

    2002-04-01

    Fluorescence in situ hybridisation (FISH) is an effective method to detect chromosomal alterations in a variety of tissue types, including archived paraffin wax embedded specimens fixed in B5 or formalin. However, precipitating fixatives such as B5 have been known to produce unsatisfactory results in comparison with formalin when used for FISH. This study describes an effective nuclear isolation and FISH procedure for B5 and formalin fixed tissue, optimising the nuclear isolation step and nuclei pretreatments using tonsil and mantle cell lymphoma specimens. The protocol presented can be used to isolate nuclei and perform FISH on B5 or formalin fixed, paraffin wax embedded samples from a variety of tissue types.

  15. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  16. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  17. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  18. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  19. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  20. Domain evolution and functional diversification of sulfite reductases.

    PubMed

    Dhillon, Ashita; Goswami, Sulip; Riley, Monica; Teske, Andreas; Sogin, Mitchell

    2005-02-01

    Sulfite reductases are key enzymes of assimilatory and dissimilatory sulfur metabolism, which occur in diverse bacterial and archaeal lineages. They share a highly conserved domain "C-X5-C-n-C-X3-C" for binding siroheme and iron-sulfur clusters that facilitate electron transfer to the substrate. For each sulfite reductase cluster, the siroheme-binding domain is positioned slightly differently at the N-terminus of dsrA and dsrB, while in the assimilatory proteins the siroheme domain is located at the C-terminus. Our sequence and phylogenetic analysis of the siroheme-binding domain shows that sulfite reductase sequences diverged from a common ancestor into four separate clusters (aSir, alSir, dsr, and asrC) that are biochemically distinct; each serves a different assimilatory or dissimilatory role in sulfur metabolism. The phylogenetic distribution and functional grouping in sulfite reductase clusters (dsrA and dsrB vs. aSiR, asrC, and alSir) suggest that their functional diversification during evolution may have preceded the bacterial/archaeal divergence.

  1. The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase

    ERIC Educational Resources Information Center

    Splittgerber, A. G.; And Others

    1975-01-01

    Describes an undergraduate biochemistry experiment which involves the preparation and kinetics of an oxidation-reduction enzyme system, methemoglobin reductase. A crude enzyme extract is prepared and assayed spectrophotometrically. The enzyme system obeys Michaelis-Menton kinetics with respect to both substrate and the NADH cofactor. (MLH)

  2. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

    NASA Technical Reports Server (NTRS)

    Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.

    1987-01-01

    Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

  3. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    NASA Astrophysics Data System (ADS)

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-09-01

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

  4. Characterization of mitochondrial thioredoxin reductase from C. elegans

    SciTech Connect

    Lacey, Brian M.; Hondal, Robert J. . E-mail: Robert.Hondal@uvm.edu

    2006-08-04

    Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a k {sub cat} of 610 min{sup -1} and a K {sub m} of 610 {mu}M using E. coli thioredoxin as substrate. The reported k {sub cat} is 25% of the k {sub cat} of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.

  5. A detoxifying oxygen reductase in the anaerobic protozoan Entamoeba histolytica.

    PubMed

    Vicente, João B; Tran, Vy; Pinto, Liliana; Teixeira, Miguel; Singh, Upinder

    2012-09-01

    We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ~5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.

  6. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    PubMed Central

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-01-01

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5–8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5–8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5–8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction. PMID:26412036

  7. Domain Evolution and Functional Diversification of Sulfite Reductases

    NASA Astrophysics Data System (ADS)

    Dhillon, Ashita; Goswami, Sulip; Riley, Monica; Teske, Andreas; Sogin, Mitchell

    2005-02-01

    Sulfite reductases are key enzymes of assimilatory and dissimilatory sulfur metabolism, which occur in diverse bacterial and archaeal lineages. They share a highly conserved domain "C-X5-C-n-C-X3-C" for binding siroheme and iron-sulfur clusters that facilitate electron transfer to the substrate. For each sulfite reductase cluster, the siroheme-binding domain is positioned slightly differently at the N-terminus of dsrA and dsrB, while in the assimilatory proteins the siroheme domain is located at the C-terminus. Our sequence and phylogenetic analysis of the siroheme-binding domain shows that sulfite reductase sequences diverged from a common ancestor into four separate clusters (aSir, alSir, dsr, and asrC) that are biochemically distinct; each serves a different assimilatory or dissimilatory role in sulfur metabolism. The phylogenetic distribution and functional grouping in sulfite reductase clusters (dsrA and dsrB vs. aSiR, asrC, and alSir) suggest that their functional diversification during evolution may have preceded the bacterial/archaeal divergence.

  8. Molecular genetics of steroid 5 alpha-reductase 2 deficiency.

    PubMed Central

    Thigpen, A E; Davis, D L; Milatovich, A; Mendonca, B B; Imperato-McGinley, J; Griffin, J E; Francke, U; Wilson, J D; Russell, D W

    1992-01-01

    Two isozymes of steroid 5 alpha-reductase encoded by separate loci catalyze the conversion of testosterone to dihydrotestosterone. Inherited defects in the type 2 isozyme lead to male pseudohermaphroditism in which affected males have a normal internal urogenital tract but external genitalia resembling those of a female. The 5 alpha-reductase type 2 gene (gene symbol SRD5A2) was cloned and shown to contain five exons and four introns. The gene was localized to chromosome 2 band p23 by somatic cell hybrid mapping and chromosomal in situ hybridization. Molecular analysis of the SRD5A2 gene resulted in the identification of 18 mutations in 11 homozygotes, 6 compound heterozygotes, and 4 inferred compound heterozygotes from 23 families with 5 alpha-reductase deficiency. 6 apparent recurrent mutations were detected in 19 different ethnic backgrounds. In two patients, the catalytic efficiency of the mutant enzymes correlated with the severity of the disease. The high proportion of compound heterozygotes suggests that the carrier frequency of mutations in the 5 alpha-reductase type 2 gene may be higher than previously thought. Images PMID:1522235

  9. 5. cap alpha. -reductase activity in rat adipose tissue

    SciTech Connect

    Zyirek, M.; Flood, C.; Longcope, C.

    1987-11-01

    We measured the 5 ..cap alpha..-reductase activity in isolated cell preparations of rat adipose tissue using the formation of (/sup 3/H) dihydrotestosterone from (/sup 3/H) testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5..cap alpha..-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10/sup -8/ M), when added to the medium, caused a 90% decrease in 5..cap alpha..-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5..cap alpha..-reductase activity in each tissue studied.

  10. ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES

    EPA Science Inventory

    ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES.

    S. Lin1, L. M. Del Razo1, M. Styblo1, C. Wang2, W. R. Cullen2, and D.J. Thomas3. 1Univ. North Carolina, Chapel Hill, NC; 2Univ. British Columbia, Vancouver, BC, Canada; 3National Health and En...

  11. Evolving the [Myoglobin, Cytochrome b5] Complex from Dynamic Toward Simple Docking: Charging the Electron-Transfer Reactive Patch

    PubMed Central

    Trana, Ethan N.; Nocek, Judith M.; Knutson, Amanda K.; Hoffman, Brian M.

    2012-01-01

    We describe photo-initiated electron transfer (ET) from a suite of Zn-substituted myoglobin (1Mb) variants to cytochrome b5 (b5). An electrostatic interface redesign strategy has led to the introduction of positive charges in the vicinity of the heme edge through D/E → K charge-reversal mutation combinations at `hotspot' residues (D44, D60, E85), augmented by the elimination of negative charges from Mb or b5 by neutralization of heme propionates. These variations create an unprecedentedly large range in the product of the ET partners' total charges: −5 < −qMbqb5 < 40. The binding affinity (Ka) increases a thousand-fold as −qMbqb5 increases through this range, and exhibits a surprisingly simple, exponential dependence on −qMbqb5. This is explained in terms of electrostatic interactions between a `charged reactive patch' (crp) on each partner's surface, defined as a compact region around the heme edge that (i) contains the total protein charge of each variant, and (ii) encompasses a major fraction of the `reactive region' (Rr) comprising surface atoms with large matrix elements for electron tunneling to the heme. As −qMbqb5 increases, the complex undergoes a transition from fast to slow exchange dynamics on the triplet ET timescale, with a correlated progression in the rate constants for intracomplex (ket) and bimolecular (k2) ET. This progression is analyzed by integrating the crp and Rr descriptions of ET into the textbook steady-state treatment of reversible binding between partners that undergo intracomplex ET, and found to encompass the full range of behaviors predicted by the model. The generality of this approach is demonstrated by applying it to the extensive body of data for the ET complex between the photosynthetic reaction center and cytochrome c2. Deviations from this model also are discussed. PMID:23067206

  12. Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation

    PubMed Central

    Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla, María N.; Luque, Francisco; Corpas, Francisco J.; Barroso, Juan B.

    2015-01-01

    The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement

  13. Coxsackie B5 infection in an adult with fever, truncal rash, diarrhea and splenomegaly with highly elevated ferritin levels.

    PubMed

    Valestra, Paul K; Fornos, Scarlet Herrarte; Gian, John; Cunha, Burke A

    2016-01-01

    Coxsackie viruses are enteroviruses most common in children. Coxsackie B viral infections often present with biphasic fever, headache, pharyngitis, nausea/vomiting, diarrhea and a maculopapular rash that spares the palms and soles. These clinical features may be present in other viral infections. We present a case of a hospitalized adult with rash and fever with highly elevated ferritin levels later found to be due to Coxsackie B5. We believe this is the first case of Coxsackie B infection with otherwise unexplained highly elevated ferritin levels. PMID:27617209

  14. Coxsackieviruses B1, B3, and B5 use decay accelerating factor as a receptor for cell attachment.

    PubMed Central

    Shafren, D R; Bates, R C; Agrez, M V; Herd, R L; Burns, G F; Barry, R D

    1995-01-01

    Receptor binding and subsequent cell-mediated internalization or disassembly are the initial steps in virus replication. Cell surface molecules that participate in this process are the primary determinants of virus tissue tropism. Monoclonal antibody blockade, immunoprecipitation, and DNA transfection were used to identify decay accelerating factor as a major cell attachment receptor for coxsackieviruses B1, B3, and B5. However, expression of human decay acceleration factor on the surface of nonpermissive murine fibroblasts led only to virus attachment without subsequent replication, and it was concluded that an additional cellular cofactor(s) is required to facilitate cell entry and subsequent replication. PMID:7538177

  15. Measurement of nitrous oxide reductase activity in aquatic sediments

    USGS Publications Warehouse

    Miller, L.G.; Oremland, R.S.; Paulsen, S.

    1986-01-01

    Denitrification in aquatic sediments was measured by an N2O reductase assay. Sediments consumed small added quantities of N2O over short periods (a few hours). In experiments with sediment slurries, N2O reductase activity was inhibited by O2, C2H2, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 μM) did not influence activity, and moderate levels (about 150 μM) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N2O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater (Km = 2.17 μM), estuarine (Km = 14.5 μM), and alkaline-saline (Km = 501 μM) environments. An in situ assay was devised in which a solution of N2O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N2O per m2 per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N2O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N2O per m2 per h made with the acetylene block assay.

  16. 3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

    PubMed

    Sheldon, P S; Kekwick, R G; Smith, C G; Sidebottom, C; Slabas, A R

    1992-04-01

    3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

  17. Identification and characterization of 2-naphthoyl-coenzyme A reductase, the prototype of a novel class of dearomatizing reductases.

    PubMed

    Eberlein, Christian; Estelmann, Sebastian; Seifert, Jana; von Bergen, Martin; Müller, Michael; Meckenstock, Rainer U; Boll, Matthias

    2013-06-01

    The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl-coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl-CoA by ATP-dependent or -independent benzoyl-CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2-naphthoyl-CoA reductase was purified from extracts of the naphthalene-degrading, sulphidogenic enrichment culture N47. The oxygen-tolerant enzyme dearomatized the non-activated ring of 2-naphthoyl-CoA by a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin-containing 'old yellow enzyme' family. NCR contained FAD, FMN, and an iron-sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2-naphthoyl-CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl-CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl-CoA reductases.

  18. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases

    NASA Technical Reports Server (NTRS)

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L.; Youn, Buhyun; Lawrence, Paulraj K.; Gang, David R.; Halls, Steven C.; Park, HaJeung; Hilsenbeck, Jacqueline L.; Davin, Laurence B.; Lewis, Norman G.; Kang, ChulHee

    2003-01-01

    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

  19. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases.

    PubMed

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L; Youn, Buhyun; Lawrence, Paulraj K; Gang, David R; Halls, Steven C; Park, HaJeung; Hilsenbeck, Jacqueline L; Davin, Laurence B; Lewis, Norman G; Kang, ChulHee

    2003-12-12

    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

  20. (+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia. Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.

    PubMed

    Dinkova-Kostova, A T; Gang, D R; Davin, L B; Bedgar, D L; Chu, A; Lewis, N G

    1996-11-15

    Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

  1. Characterization of a mouse model of allergy to a major occupational latex glove allergen Hev b 5.

    PubMed

    Hardy, Charles L; Kenins, Linda; Drew, Alexander C; Rolland, Jennifer M; O'Hehir, Robyn E

    2003-05-15

    Allergen-specific immunotherapy is a clinically proven effective treatment for many allergic diseases, including asthma; however, it is not currently available for latex allergy because of the high risk of anaphylaxis. There is, therefore, a crucial need for an animal model of latex allergy in which to develop effective immunotherapy. Previous mouse models of latex allergy either did not characterize the allergic pulmonary immune response or used crude latex extracts, making it difficult to quantify the contribution of individual proteins and limiting their usefulness for developing specific immunotherapy. We immunized mice with recombinant Hev b 5, a defined major latex allergen, or latex glove protein extract, representing the range of occupationally encountered processed latex allergens. The immune response was compared with that seen in ovalbumin-immunized mice. Immunization with Hev b 5 or glove extract elicits hallmarks of allergic pulmonary Th2-type immune responses, comparable to those for ovalbumin, including (1) serum antigen-specific IgE, (2) an eosinophilic inflammatory infiltrate in the lung, (3) increased interleukin-5 in lung bronchoalveolar lavage fluid, and (4) mucus hypersecretion by epithelial cells in the lung airways. This mouse model will aid the development of potentially curative treatments for latex-sensitized individuals, including those with occupational asthma.

  2. Synthesis and thermoluminescence characterizations of Sr2B5O9Cl:Dy3+ phosphor for TL dosimetry.

    PubMed

    Oza, Abha H; Dhoble, N S; Park, K; Dhoble, S J

    2015-09-01

    The photoluminescence (PL) and thermoluminescence (TL) displayed by Dy-activated strontium haloborate (Sr2 B5 O9 Cl) were studied. A modified solid-state reaction was employed for the preparation of the phosphor. Photoluminescence spectra showed blue (484 nm) and yellow (575 nm) emissions due to incorporation of Dy(3+) into host matrix. The Dy-doped (0.5 mol%) Sr2 B5 O9 Cl was studied after exposure to γ-irradiation and revealed a prominent glow curve at 261°C with a small hump around 143°C indicating that two types of traps were generated. The glow peak at the higher temperature side (261°C) was more stable than the lower temperature glow peak. The TL intensity was 1.17 times less than that of the standard CaSO4 :Dy thermoluminescence dosimetry (TLD) phosphor, the phosphor showed a linear dose-response curve for different γ-ray irradiation doses (0.002-1.25 Gy) and fading of 5-7% was observed for higher temperature peaks upon storage. Trapping parameters and their estimated error values have been calculated by Chen's peak shape method and by the initial rise method. Values of activation energies estimated by both these techniques were comparable. The slight difference in activation energy values calculated by Chen's peak shape method indicated the formation of two kinds of traps Furthermore, slight differences in frequency values are due to various escaping and retrapping probabilities.

  3. Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium Colwellia psychrerythraea.

    PubMed

    Ji, Mikyoung; Barnwell, Callie V; Grunden, Amy M

    2015-07-01

    Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems. PMID:26101017

  4. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    DOEpatents

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

    2003-10-21

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  5. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    DOEpatents

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  6. The X-ray crystal structure of APR-B, an atypical adenosine 5'-phosphosulfate reductase from Physcomitrella patens.

    PubMed

    Stevenson, Clare E M; Hughes, Richard K; McManus, Michael T; Lawson, David M; Kopriva, Stanislav

    2013-11-15

    Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where adenosine 5'-phosphosulfate (APS) reductases possess a cluster and 3'-phosphoadenosine 5'-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.

  7. Terpenoid Metabolism in Plastids 1

    PubMed Central

    Camara, Bilal; Bardat, Françoise; Dogbo, Odette; Brangeon, Judy; Monéger, René

    1983-01-01

    A technique for the isolation and the purification of Capsicum annum L. var. Yolo Wonder chromoplasts is described. The degree of purity of the isolated chromoplasts is greatly improved by the absence of MgCl2 in the extraction medium and in the gradient purification system, as shown by electron micrographs and the near absence of antimycin-insensitive NADH:cytochrome c reductase activity and succinate:cytochrome c reductase activity. Furthermore, phosphatidylserine was absent and the phosphatidylethanolamine content was reduced by a factor of 5 in these preparations, which were active in the synthesis of galactolipids, prenylquinones, and carotenoids. Images Fig. 1 PMID:16663194

  8. Implementation of IAU Resolution 2009 B5, "in Defence of the night sky and the right to starlight"

    NASA Astrophysics Data System (ADS)

    Green, Richard F.; Walker, Constance Elaine

    2015-08-01

    IAU Resolution 2009 B5 calls on IAU members to protect the public`s right to an unpolluted night sky as well as the astronomical quality of the sky around major research observatories. The approach of Commission 50 - astronomical site protection - includes working with the lighting industry for appropriate products from rapidly evolving solid state technology, arming astronomers with training and materials for presentation, selective endorsement of key protection issues, cooperation with other IAU commissions for education and outreach with particular current attention to the International Year of Light, and provision of clear quantitative priorities for outdoor lighting standards. In 2012, these priorities were defined as full cut-off shielding, spectral management to minimize output shortward of 500 nm, and zone- and time-appropriate lighting levels. Revisiting the specifics of these priorities will be a topic for current discussion.

  9. VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems?

    PubMed

    Backert, Steffen; Fronzes, Remi; Waksman, Gabriel

    2008-09-01

    Many type-IV secretion systems (T4SSs) of plant and human pathogens assemble a pilus used to inject virulence molecules (effectors) into host target cells. The T4SS of Agrobacterium tumefaciens consists of VirB1-VirB11 and VirD4 proteins. Whether targeting of T4SSs to the host requires a T4SS-adhesin that specifically engages host receptors for delivery of effectors has, until recently, remained unclear. Recent data of Agrobacterium and Helicobacter indicate that two classes of T4SS components, VirB2 and VirB5, might function as adhesins that mediate host-cell targeting through binding to specific host receptors. Here, we discuss this important issue and recent progress in the field. PMID:18706815

  10. Magnetic properties of Nd 55- xCo xFe 30Al 10B 5 cast rods

    NASA Astrophysics Data System (ADS)

    Dan, N. H.; Phuc, N. X.; Hong, L. V.; Kong, H. Z.; Ding, J.

    2003-04-01

    Nd 55- xCo xFe 30Al 10B 5 ( x=0,5,10,15 and 20) rods with a thickness of 1 mm were prepared by the arc-melting copper mold suction-casting method. At appropriate compositions, the coercivity, remanence and Curie temperature of the alloys simultaneously rise considerably. Coercivities larger than 1120 kA/m have been observed at room temperature for rods with x-values between 10 and 20. The remanent magnetization rises from ∼13 to ∼24 A m 2/kg and the Curie temperature increases from ∼450 to ∼625 K when the Co-concentration, x, in the alloys increases from 0 to 20. The magnetic behavior of the alloys has been investigated at low temperatures.

  11. DFT study on the isomerization and tautomerism in vitamins B3 (niacin), B5 (pantothenic acid) and B7 (biotin)

    NASA Astrophysics Data System (ADS)

    Valadbeigi, Younes; Farrokhpour, Hossein; Tabrizchi, Mahmoud

    2014-05-01

    Isomerization and tautomerism of the three water soluble vitamins including B3, B5 and B7 were studied applying density functional theory using B3LYP method in gas and aqueous phases. Activation energies (Ea), Gibbs free energies of activation (ΔG#), and imaginary frequencies of the transition state structures were calculated for all the isomerization and tautomerism reactions. Activation energies of the neutral → zwitterion (amine-enamine) tautomerism in vitamin B3 were 310-360 kJ/mol where these values for the keto-enol tautomerism were 100-130 kJ/mol. It was found that water molecule catalyzes the tautomerism and decreases the activation energies about 90-160 kJ/mol.

  12. Inductive Preheating in Laser Beam Welding of Multimaterial Joints of 22MnB5 and AA6016

    NASA Astrophysics Data System (ADS)

    Kügler, H.; Vollertsen, F.

    Inductive preheating is well known as possibility to heat ferromagnetic materials. In brazing preheating causes an improvement of wetting quality, e.g. smaller wetting angles and longer wetting lengths. In this paper inductive preheating is used to support a laser beam hybrid joining process. Aluminum AA6016 is molten in order to wet the surface of AlSi- coated steel 22MnB5. Investigations on the influence of preheating on wetting characteristics and intermetallic phase seam formation were carried out. Strength values up to 230 MPa have been measured in tensile shear tests. Fraction zone occurs in the aluminum base material indicating uncritical thickness of the intermetallic phase seam at the interface.

  13. Conditional anterograde tracing reveals distinct targeting of individual serotonin cell groups (B5-B9) to the forebrain and brainstem.

    PubMed

    Muzerelle, Aude; Scotto-Lomassese, Sophie; Bernard, Jean François; Soiza-Reilly, Mariano; Gaspar, Patricia

    2016-01-01

    Serotoninergic innervation of the central nervous system is provided by hindbrain raphe nuclei (B1-B9). The extent to which each raphe subdivision has distinct topographic organization of their projections is still unclear. We provide a comprehensive description of the main targets of the rostral serotonin (5-HT) raphe subgroups (B5-B9) in the mouse brain. Adeno-associated viruses that conditionally express GFP under the control of the 5-HT transporter promoter were used to label small groups of 5-HT neurons in the dorsal (B7d), ventral (B7v), lateral (B7l), and caudal (B6) subcomponents of the dorsal raphe (DR) nucleus as well as in the rostral and caudal parts of the median raphe (MR) nucleus (B8 and B5, respectively), and in the supralemniscal (B9) cell group. We illustrate the distinctive and largely non-overlapping projection areas of these cell groups: for instance, DR (B7) projects to basal parts of the forebrain, such as the amygdala, whereas MR (B8) is the main 5-HT source to the hippocampus, septum, and mesopontine tegmental nuclei. Distinct subsets of B7 have preferential brain targets: B7v is the main source of 5-HT for the cortex and amygdala while B7d innervates the hypothalamus. We reveal for the first time the target areas of the B9 cell group, demonstrating projections to the caudate, prefrontal cortex, substantia nigra, locus coeruleus and to the raphe cell groups. The broad topographic organization of the different raphe subnuclei is likely to underlie the different functional roles in which 5-HT has been implicated in the brain. The present mapping study could serve as the basis for genetically driven specific targeting of the different subcomponents of the mouse raphe system.

  14. The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10

    USGS Publications Warehouse

    Afkar, E.; Lisak, J.; Saltikov, C.; Basu, P.; Oremland, R.S.; Stolz, J.F.

    2003-01-01

    The respiratory arsenate reductase from the Gram-positive, haloalkaliphile, Bacillus selenitireducens strain MLS10 was purified and characterized. It is a membrane bound heterodimer (150 kDa) composed of two subunits ArrA (110 kDa) and ArrB (34 kDa), with an apparent Km for arsenate of 34 ??M and Vmax of 2.5 ??mol min-1 mg-1. Optimal activity occurred at pH 9.5 and 150 g l-1 of NaCl. Metal analysis (inductively coupled plasma mass spectrometry) of the holoenzyme and sequence analysis of the catalytic subunit (ArrA; the gene for which was cloned and sequenced) indicate it is a member of the DMSO reductase family of molybdoproteins. ?? 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

  15. Involvement of nitrate reductase in auxin-induced NO synthesis

    PubMed Central

    Erdei, L

    2008-01-01

    It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.1 Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (NG-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation. PMID:19704423

  16. Involvement of nitrate reductase in auxin-induced NO synthesis.

    PubMed

    Kolbert, Zsuzsanna; Erdei, L

    2008-11-01

    It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.1 Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N(G)-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation. PMID:19704423

  17. MiR-181b-5p Downregulates NOVA1 to Suppress Proliferation, Migration and Invasion and Promote Apoptosis in Astrocytoma

    PubMed Central

    Shao, Naiyuan; Wang, Rong; Xue, Lian; Wang, Suinuan; Xia, Xiwei; Yang, Yilin

    2014-01-01

    MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR-181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR-181b-5p bound directly to the 3'-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma. PMID:25299073

  18. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

  19. Using chemical approaches to study selenoproteins - focus on thioredoxin reductases

    PubMed Central

    Hondal, Robert J.

    2009-01-01

    The study of selenocysteine-containing proteins is difficult due to the problems associated with the heterologous production of these proteins. These problems are due to the intricate recoding mechanism used by cells to translate the UGA codon as a sense codon for selenocysteine. The process is further complicated by the fact that eukaryotes and prokaryotes have different UGA recoding machineries. This review focuses on chemical approaches to produce selenoproteins and study the mechanism of selenoenzymes. The use of intein-mediated peptide ligation is discussed with respect to the production of the mammalian selenoenzymes thioredoxin reductase and selenoprotein R, also known as methionine sulfoxide reductase B1. New methods for removing protecting groups from selenocysteine post-synthesis and methods for selenosulfide/diselenide formation are also reviewed. Chemical approaches have also been used to study the enzymatic mechanism of thioredoxin reductase. The approach divides the enzyme into two modules, a large protein module lacking selenocysteine and a small, synthetic selenocysteine-containing peptide. Study of this semisynthetic enzyme has revealed three distinct enzymatic pathways that depend on the properties of the substrate. The enzyme utilizes a macromolecular mechanism for protein substrates, a second mechanism for small molecule substrates and a third pathway for selenium-containing substrates such as selenocystine. PMID:19406205

  20. Cloning and sequence of the human adrenodoxin reductase gene.

    PubMed Central

    Lin, D; Shi, Y F; Miller, W L

    1990-01-01

    Adrenodoxin reductase (ferrodoxin:NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. We cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G + C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of "housekeeping" genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon. Images PMID:2236061

  1. Structural and functional diversity of ferredoxin-NADP(+) reductases.

    PubMed

    Aliverti, Alessandro; Pandini, Vittorio; Pennati, Andrea; de Rosa, Matteo; Zanetti, Giuliana

    2008-06-15

    Although all ferredoxin-NADP(+) reductases (FNRs) catalyze the same reaction, i.e. the transfer of reducing equivalents between NADP(H) and ferredoxin, they belong to two unrelated families of proteins: the plant-type and the glutathione reductase-type of FNRs. Aim of this review is to provide a general classification scheme for these enzymes, to be used as a framework for the comparison of their properties. Furthermore, we report on some recent findings, which significantly increased the understanding of the structure-function relationships of FNRs, i.e. the ability of adrenodoxin reductase and its homologs to catalyze the oxidation of NADP(+) to its 4-oxo derivative, and the properties of plant-type FNRs from non-photosynthetic organisms. Plant-type FNRs from bacteria and Apicomplexan parasites provide examples of novel ways of FAD- and NADP(H)-binding. The recent characterization of an FNR from Plasmodium falciparum brings these enzymes into the field of drug design.

  2. Modulating hemoglobin nitrite reductase activity through allostery: a mathematical model.

    PubMed

    Rong, Zimei; Alayash, Abdu I; Wilson, Michael T; Cooper, Chris E

    2013-11-30

    The production of nitric oxide by hemoglobin (Hb) has been proposed to play a major role in the control of blood flow. Because of the allosteric nature of hemoglobin, the nitrite reductase activity is a complex function of oxygen partial pressure PO2. We have previous developed a model to obtain the micro rate constants for nitrite reduction by R state (kR) and T state (kT) hemoglobin in terms of the experimental maximal macro rate constant kNmax and the corresponding oxygen concentration PO2max. However, because of the intrinsic difficulty in obtaining accurate macro rate constant kN, from available experiments, we have developed an alternative method to determine the micro reaction rate constants (kR and kT) by fitting the simulated macro reaction rate curve (kN versus PO2) to the experimental data. We then use our model to analyze the effect of pH (Bohr Effect) and blood ageing on the nitrite reductase activity, showing that the fall of bisphosphoglycerate (BPG) during red cell storage leads to increase NO production. Our model can have useful predictive and explanatory power. For example, the previously described enhanced nitrite reductase activity of ovine fetal Hb, in comparison to the adult protein, may be understood in terms of a weaker interaction with BPG and an increase in the value of kT from 0.0087M(-1)s(-1) to 0.083M(-1)s(-1).

  3. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions.

  4. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A. Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

  5. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

  6. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

  7. Aldose reductase inhibitory activity of compounds from Zea mays L.

    PubMed

    Kim, Tae Hyeon; Kim, Jin Kyu; Kang, Young-Hee; Lee, Jae-Yong; Kang, Il Jun; Lim, Soon Sung

    2013-01-01

    Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1-7) and 5 anthocyanins (compound 8-12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC(50), 4.78 μ M). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

  8. Two fatty acyl reductases involved in moth pheromone biosynthesis.

    PubMed

    Antony, Binu; Ding, Bao-Jian; Moto, Ken'Ichi; Aldosari, Saleh A; Aldawood, Abdulrahman S

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective 'single pgFARs' produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a 'single reductase' can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  9. Cloning and Sequence Analysis of Two Pseudomonas Flavoprotein Xenobiotic Reductases

    PubMed Central

    Blehert, David S.; Fox, Brian G.; Chambliss, Glenn H.

    1999-01-01

    The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The P. putida gene, xenA, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The P. fluorescens gene, xenB, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyclohexen-1-one. Heterologous expression of xenA and xenB was demonstrated in Escherichia coli DH5α. The transcription initiation sites of both xenA and xenB were identified by primer extension analysis. BLAST analyses conducted with the P. putida xenA and the P. fluorescens xenB sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems. PMID:10515912

  10. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  11. In Vitro Formation of Nitrate Reductase Using Extracts of the Nitrate Reductase Mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum

    PubMed Central

    Ketchum, Paul A.; Sevilla, Cynthia L.

    1973-01-01

    In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)–nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH–nitrate reductase, FADH2–nitrate reductase and reduced methyl viologen (MVH)–nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10−4 M Na2MoO4 and 10−2 M NaNO3 to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase. PMID:4270447

  12. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    NASA Astrophysics Data System (ADS)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.

  13. The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Kunze, Thomas; Clement, Bernd

    2015-04-17

    N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal. PMID:25713076

  14. Homologous electron transport components fail to increase fatty acid hydroxylation in transgenic Arabidopsis thaliana.

    PubMed

    Wayne, Laura L; Browse, John

    2013-01-01

    Ricinoleic acid, a hydroxylated fatty acid (HFA) present in castor ( Ricinus communis) seeds, is an important industrial commodity used in products ranging from inks and paints to polymers and fuels. However, due to the deadly toxin ricin and allergens also present in castor, it would be advantageous to produce ricinoleic acid in a different agricultural crop. Unfortunately, repeated efforts at heterologous expression of the castor fatty acid hydroxylase (RcFAH12) in the model plant Arabidopsis thaliana have produced only 17-19% HFA in the seed triacylglycerols (TAG), whereas castor seeds accumulate up to 90% ricinoleic acid in the endosperm TAG. RcFAH12 requires an electron supply from NADH:cytochrome b5 reductase (CBR1) and cytochrome b5 (Cb5) to synthesize ricinoleic acid. Previously, our laboratory found a mutation in the Arabidopsis CBR1 gene, cbr1-1, that caused an 85% decrease in HFA levels in the RcFAH12 Arabidopsis line. These results raise the possibility that electron supply to the heterologous RcFAH12 may limit the production of HFA. Therefore, we hypothesized that by heterologously expressing RcCb5, the reductant supply to RcFAH12 would be improved and lead to increased HFA accumulation in Arabidopsis seeds. Contrary to this proposal, heterologous expression of the top three RcCb5 candidates did not increase HFA accumulation. Furthermore, coexpression of RcCBR1 and RcCb5 in RcFAH12 Arabidopsis also did not increase in HFA levels compared to the parental lines. These results demonstrate that the Arabidopsis electron transfer system is supplying sufficient reductant to RcFAH12 and that there must be other bottlenecks limiting the accumulation of HFA.

  15. The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Kunze, Thomas; Clement, Bernd

    2015-04-17

    N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.

  16. The Pivotal Role of the Mitochondrial Amidoxime Reducing Component 2 in Protecting Human Cells against Apoptotic Effects of the Base Analog N6-Hydroxylaminopurine*

    PubMed Central

    Plitzko, Birte; Havemeyer, Antje; Kunze, Thomas; Clement, Bernd

    2015-01-01

    N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal. PMID:25713076

  17. Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

    PubMed Central

    Simonov, Alexandr N.; Holien, Jessica K.; Yeung, Joyee Chun In; Nguyen, Ann D.; Corbin, C. Jo; Zheng, Jie; Kuznetsov, Vladimir L.; Auchus, Richard J.; Conley, Alan J.; Bond, Alan M.; Parker, Michael W.; Rodgers, Raymond J.; Martin, Lisandra L.

    2015-01-01

    Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. PMID:26587646

  18. The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

    PubMed Central

    Vienozinskis, J; Butkus, A; Cenas, N; Kulys, J

    1990-01-01

    The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed. PMID:2375745

  19. Selenate reductase activity in Escherichia coli requires Isc iron-sulfur cluster biosynthesis genes.

    PubMed

    Yee, Nathan; Choi, Jessica; Porter, Abigail W; Carey, Sean; Rauschenbach, Ines; Harel, Arye

    2014-12-01

    The selenate reductase in Escherichia coli is a multi-subunit enzyme predicted to bind Fe-S clusters. In this study, we examined the iron-sulfur cluster biosynthesis genes that are required for selenate reductase activity. Mutants devoid of either the iscU or hscB gene in the Isc iron-sulfur cluster biosynthesis pathway lost the ability to reduce selenate. Genetic complementation by the wild-type sequences restored selenate reductase activity. The results indicate the Isc biosynthetic system plays a key role in selenate reductase Fe-S cofactor assembly and is essential for enzyme activity.

  20. Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis[W

    PubMed Central

    Meguro, Miki; Ito, Hisashi; Takabayashi, Atsushi; Tanaka, Ryouichi; Tanaka, Ayumi

    2011-01-01

    The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation. PMID:21934147

  1. Role of 5-hydroxytryptamine 1B (5-HT1B) receptors in the regulation of ethanol intake in rodents

    PubMed Central

    Sari, Youssef

    2012-01-01

    Evidence indicates that the serotonergic system is important in mediating dependence on and craving for alcohol. Among serotonin receptors, 5-hydroxytryptamine 1B (5-HT1B) receptors have been associated with drug abuse including alcohol. In this review, the neurocircuitry involving 5-HT1B receptors in central reward brain regions related to alcohol intake are discussed in detail. Emphasis has been placed on the pharmacological manipulations of 5-HT1B receptor-mediated alcohol intake. Furthermore, 5-HT1B auto- and hetero-receptors regulate alcohol intake through the regulatory mechanism involving release of 5-HT, gamma-aminobutyric acid (GABA), dopamine, and glutamate is evaluated. Thus, interactions between 5-HT1B receptors and these neurotransmitter systems are suggested to modulate alcohol-drinking behavior. This review on the role of 5-HT1B receptors in neurotransmitter release and consequent alcohol intake provides important information about the potential therapeutic role of 5-HT1B receptors for the treatment of alcohol dependence. PMID:23118018

  2. Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

    SciTech Connect

    Nordlund, Henri R. . E-mail: henri.nordlund@uta.fi; Laitinen, Olli H.; Uotila, Sanna T.H.; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S.

    2005-10-14

    The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

  3. miR-146b-5p mediates p16-dependent repression of IL-6 and suppresses paracrine procarcinogenic effects of breast stromal fibroblasts

    PubMed Central

    Al-Ansari, Mysoon M.; Aboussekhra, Abdelilah

    2015-01-01

    Increasing evidence support the critical roles of active stromal fibroblasts in breast cancer development and spread. However, the mediators and the mechanisms of regulation are still not well defined. We have shown here that the tumor suppressor p16INK4A protein inhibits the pro-carcinogenic effects of breast stromal fibroblasts through repressing the expression/secretion of IL-6. Indeed, p16INK4A suppresses IL-6 at the mRNA and protein levels. This effect is mediated trough miR-146b-5p, which inhibits IL-6 expression through a specific sequence at the IL-6 3′UTR. In addition, we present clear evidence that miR-146b-5p inhibition is sufficient to transactivate breast stromal fibroblasts, which promote epithelial-to-mesenchymal-transition in breast cancer cells in a paracrine manner. By contrast, ectopic expression of miR-146b-5p in active fibroblasts abrogated their pro-carcinogenic effects. The physiological importance of miR-146b-5p inhibition was revealed by showing that the levels of pre-miR-146b-5p as well as its mature form are reduced in cancer-associated fibroblasts as compared with their normal adjacent counterparts from cancer-free tissues isolated from the same patients. Interestingly, treatment of active breast stromal fibroblasts with curcumin increased the level of the p16INK4A coding CDKN2A mRNA and miR-146b-5p and suppressed IL-6, which confirms the repressive effect of these two tumor suppressor molecules on IL-6, and shows the possible “normalization” of cancer-related active fibroblasts. These results show that miR-146b-5p has non-cell-autonomous tumor suppressor function through inhibition of IL-6, suggesting that targeting this microRNA in breast stromal fibroblasts could be of great therapeutic value. PMID:26338965

  4. Methylenetetrahydrofolate Reductase C677T: Hypoplastic Left Heart and Thrombosis.

    PubMed

    Spronk, Kimberly J; Olivero, Anthony D; Haw, Marcus P; Vettukattil, Joseph J

    2015-10-01

    The incidence of congenital heart defects is higher in infants with mutation of methylenetetrahydrofolate reductase (MTHFR) gene. The MTHFR C677T gene decreases the bioavailability of folate and increases plasma homocysteine, a risk factor for thrombosis. There have been no reported cases in the literature on the clinical implications of this procoagulable state in the setting of cyanotic heart disease, which itself has prothrombotic predisposition. Two patients with hypoplastic left heart syndrome developed postoperative thrombotic complications, both were homozygous for MTHFR C677T. We present these cases and highlight the implications of MTHFR mutation in the management of complex congenital heart disease. PMID:26467879

  5. Terpenoids from Diplophyllum taxifolium with quinone reductase-inducing activity.

    PubMed

    Wang, Xiao; Zhang, Jiao-Zhen; Zhou, Jin-Chuan; Shen, Tao; Lou, Hong-Xiang

    2016-03-01

    Two new ent-prenylaromadendrane-type diterpenoids, diplotaxifols A (1) and B (2), a new ent-eudesmol, ent-eudesma-4(15),11(13)-dien-6α,12-diol (3), eight new eudesmanolides enantiomers (4-11) of the corresponding compounds from higher plants along with four known ent-eudesmanolides (12-15) were isolated from the 95% EtOH extract of Chinese liverwort Diplophyllum taxifolium. Their structures were elucidated on the basis of MS, NMR and IR spectral data, and confirmed by single-crystal X-ray diffraction analysis. The quinone reductase-inducing activity of the compounds was evaluated. PMID:26656409

  6. Differential Light Induction of Nitrate Reductases in Greening and Photobleached Soybean Seedlings 1

    PubMed Central

    Kakefuda, Genichi; Duke, Stanley H.; Duke, Stephen O.

    1983-01-01

    Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO3−, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO3− and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO3− and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO3−. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO3−, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO3− requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO3− requirement for induction were quite different. Km values for NO3− were identical for both nitrate reductases. PMID:16663185

  7. Electrostatic potentials and electrostatic interaction energies of rat cytochrome b5 and a simulated anion-exchange adsorbent surface.

    PubMed Central

    Roush, D J; Gill, D S; Willson, R C

    1994-01-01

    Electrostatic potentials were determined for the soluble tryptic core of rat cytochrome b5 (using a structure derived from homology modeling) and a simulated anion-exchange surface through application of the linearized finite-difference Poisson-Boltzmann equation with the simulation code UHBD. Objectives of this work included determination of the contributions of the various charged groups on the protein surface to electrostatic interactions with a simulated anion-exchange surface as a function of orientation, separation distance, and ionic strength, as well as examining the potential existence of a preferred contact orientation. Electrostatic interaction free energies for the complex of the model protein and the simulated surface were computed using the electrostatics section of UHBD employing a 110(3) grid. An initial coarse grid spacing of 2.0 A was required to obtain correct boundary conditions. The boundary conditions of the coarse grid were used in subsequent focusing steps until the electrostatic interaction free energies were relatively independent of grid spacing (at approximately 0.5 A). Explicit error analyses were performed to determine the effects of grid spacing and other model assumptions on the electrostatic interaction free energies. The computational results reveal the presence of a preferred interaction orientation; the interaction energy between these two entities, of opposite net charge, is repulsive over a range of orientations. The electrostatic interaction free energies appear to be the summation of multiple fractional interactions between the protein and the anion-exchange surface. The simulation results are compared with those of ion-exchange adsorption experiments with site-directed mutants of the recombinant protein. Comparisons of the results from the computational and experimental studies should lead to a better understanding of electrostatic interactions of proteins and charged surfaces. Images FIGURE 4 FIGURE 5 FIGURE 6 PMID:8061185

  8. Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone.

    PubMed

    Jonsson, Nina; Sävneby, Anna; Gullberg, Maria; Evertsson, Kim; Klingel, Karin; Lindberg, A Michael

    2015-06-01

    Recombination is an important feature in the evolution of the Enterovirus genus. Phylogenetic studies of enteroviruses have revealed that the capsid genomic region (P1) is type specific, while the parts of the genome coding for the non-structural proteins (P2-P3) are species specific. Hence, the genome may be regarded as consisting of two modules that evolve independently. In this study, it was investigated whether the non-structural coding part of the genome in one type could support replication of a virus with a P1 region from another type of the same species. A cassette vector (pCas) containing a full-length cDNA copy of coxsackievirus B5 (CVB5) was used as a replicative backbone. The P1 region of pCas was replaced with the corresponding part from coxsackievirus B3 Nancy (CVB3N), coxsackievirus B6 Schmitt (CVB6S), and echovirus 7 Wallace (E7W), all members of the Enterovirus B species. The replication efficiency after transfection with clone-derived in vitro transcribed RNA was studied and compared with that of pCas. All the recombinant viruses replicated with similar efficiencies and showed threshold cycle (Ct) values, tissue culture infectivity dose 50 %, and plaque-forming unit titers comparable to viruses generated from the pCas construct. In addition to this, a clone without the P1 region was also constructed, and Western Blot and immunofluorescence staining analysis showed that the viral genome could be translated and replicated despite the lack of the structural protein-coding region. To conclude, the replicative backbone of the CVB5 cassette vector supports replication of intraspecies constructs with P1 regions derived from other members of the Enterovirus B species. In addition to this, the replicative backbone can be both translated and replicated without the presence of a P1 region.

  9. Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus.

    PubMed

    Chen, Zhaochun; Earl, Patricia; Americo, Jeffrey; Damon, Inger; Smith, Scott K; Zhou, Yi-Hua; Yu, Fujuan; Sebrell, Andrew; Emerson, Suzanne; Cohen, Gary; Eisenberg, Roselyn J; Svitel, Juraj; Schuck, Peter; Satterfield, William; Moss, Bernard; Purcell, Robert

    2006-02-01

    Chimpanzee Fabs against the B5 envelope glycoprotein of vaccinia virus were isolated and converted into complete mAbs with human gamma 1 heavy chain constant regions. The two mAbs (8AH8AL and 8AH7AL) displayed high binding affinities to B5 (Kd of 0.2 and 0.7 nM). The mAb 8AH8AL inhibited the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro, protected mice from subsequent intranasal challenge with virulent vaccinia virus, protected mice when administered 2 days after challenge, and provided significantly greater protection than that afforded by a previously isolated rat anti-B5 mAb (19C2) or by vaccinia immune globulin. The mAb bound to a conformational epitope between amino acids 20 and 130 of B5. These chimpanzee/human anti-B5 mAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox.

  10. Cytochrome b5 null mouse: a new model for studying inherited skin disorders and the role of unsaturated fatty acids in normal homeostasis.

    PubMed

    Finn, Robert D; McLaughlin, Lesley A; Hughes, Catherine; Song, Chengli; Henderson, Colin J; Roland Wolf, C

    2011-06-01

    Microsomal cytochrome b (5) is a ubiquitous, 15.2 kDa haemoprotein implicated in a number of cellular processes such as fatty acid desaturation, drug metabolism, steroid hormone biosynthesis and methaemoglobin reduction. As a consequence of these functions this protein has been considered essential for life. Most of the ascribed functions of cytochrome b (5), however, stem from in vitro studies and for this reason we have carried out a germline deletion of this enzyme. We have unexpectedly found that cytochrome b (5) null mice were viable and fertile, with pups being born at expected Mendelian ratios. However, a number of intriguing phenotypes were identified, including altered drug metabolism, methaemoglobinemia and disrupted steroid hormone homeostasis. In addition to these previously identified roles for this protein, cytochrome b (5) null mice displayed skin defects closely resembling those observed in autosomal recessive congenital ichthyosis and retardation of neonatal development, indicating that this protein, possibly as a consequence of its role in the de novo biosynthesis of unsaturated fatty acids, plays a central role in skin development and neonatal nutrition. Results from fatty acid profile analysis of several tissues suggest that cytochrome b (5) plays a role controlling saturated/unsaturated homeostasis. These data demonstrate that regional concentrations of unsaturated fatty acids are controlled by endogenous metabolic pathways and not by diet alone.

  11. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

    PubMed

    Milhim, Mohammed; Gerber, Adrian; Neunzig, Jens; Hannemann, Frank; Bernhardt, Rita

    2016-08-10

    Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

  12. Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

    PubMed Central

    French, C E; Nicklin, S; Bruce, N C

    1996-01-01

    Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one. PMID:8932320

  13. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  14. Regulation of a ribonucleoside reductase during the early generative phase in Acetabularia.

    PubMed

    de Groot, E J; Schweiger, H G

    1985-02-01

    The activity of a ribonucleoside reductase was estimated during the life cycle of Acetabularia. During the early generative phase the enzyme activity was dramatically increased. Regulation of the ribonucleoside reductase was observed even in the absence of the nucleus. The increase in activity was inhibited by chloramphenicol but not by cycloheximide. These results indicate that the enzyme is translated on 70 S ribosomes.

  15. Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

    NASA Technical Reports Server (NTRS)

    Warner, R. L.; Huffaker, R. C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

  16. SORGOdb: Superoxide Reductase Gene Ontology curated DataBase

    PubMed Central

    2011-01-01

    Background Superoxide reductases (SOR) catalyse the reduction of superoxide anions to hydrogen peroxide and are involved in the oxidative stress defences of anaerobic and facultative anaerobic organisms. Genes encoding SOR were discovered recently and suffer from annotation problems. These genes, named sor, are short and the transfer of annotations from previously characterized neelaredoxin, desulfoferrodoxin, superoxide reductase and rubredoxin oxidase has been heterogeneous. Consequently, many sor remain anonymous or mis-annotated. Description SORGOdb is an exhaustive database of SOR that proposes a new classification based on domain architecture. SORGOdb supplies a simple user-friendly web-based database for retrieving and exploring relevant information about the proposed SOR families. The database can be queried using an organism name, a locus tag or phylogenetic criteria, and also offers sequence similarity searches using BlastP. Genes encoding SOR have been re-annotated in all available genome sequences (prokaryotic and eukaryotic (complete and in draft) genomes, updated in May 2010). Conclusions SORGOdb contains 325 non-redundant and curated SOR, from 274 organisms. It proposes a new classification of SOR into seven different classes and allows biologists to explore and analyze sor in order to establish correlations between the class of SOR and organism phenotypes. SORGOdb is freely available at http://sorgo.genouest.org/index.php. PMID:21575179

  17. Retrospective approach to methylenetetrahydrofolate reductase mutations in children.

    PubMed

    Özer, Işıl; Özçetin, Mustafa; Karaer, Hatice; Kurt, Semiha G; Şahin, Şemsettin

    2011-07-01

    Methylenetetrahydrofolate reductase reduces methyltetrahydrofolate, a cosubstrate in the remethylation of homocysteine, from methylenetetrahydrofolate. Congenital defects, hematologic tumors, and intrauterine growth retardation can occur during childhood. This study evaluated clinical and laboratory treatment approaches in children diagnosed with methylenetetrahydrofolate reductase mutations. Our group included 23 boys and 14 girls, aged 103.4 ± 70.8 months S.D. Clinical findings of patients and homocysteine, vitamin B12, folate, hemogram, electroencephalography, cranial magnetic resonance imaging, and echocardiography data were evaluated in terms of treatment approach. Our patients' findings included vitamin B12 at 400.4 ± 224.6 pg/mL S.D. (normal range, 300-700 pg/mL), folate at 10.1 ± 4.5 ng/mL S.D. (normal range, 1.8-9 ng/mL), and homocysteine at 8.4 ± 4.7 μmol/L S.D. (normal range, 5.5-17 μmol/L). Eighty-eight percent of patients demonstrated clinical findings. In comparisons involving categorical variables between groups, χ(2) tests were used. No relationship was evident between mutation type, laboratory data, and clinical severity. All mothers who had MTHFR mutations and had babies with sacral dimples had taken folate supplements during pregnancy. To avoid the risk of neural tube defects, pregnant women with a MTHFR mutation may require higher than normally recommended doses of folic acid supplementation for optimum health.

  18. Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

    PubMed

    Davenport, Susie; Le Lay, Pascaline; Sanchez-Tamburrrino, Juan Pablo

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants. PMID:26447683

  19. Properties of seleno-methionine substituted assimilatory nitrate reductase

    SciTech Connect

    Solomonson, L.P.; Barber, M.J. )

    1991-03-11

    Assimilatory NADH:nitrate reductase contains FAD, heme and Mo-pterin arranged in an NADH{yields}FAD{yields}heme{yields}Mo-Pterin{yields}NO{sub 3} electron transfer sequence. A functional Mo-pterin center is essential for all nitrate-reducing activities. To assess the possible functional role of Met, a Se-Met substituted NR was obtained by addition of Se-Met to ammonia-grown Chlorella cells prior to induction of NR activity. Increase in NADH:dehydrogenase partial activities and nitrate reductase protein proceeded normally following induction but little or no nitrate-reducing activity was expressed. This effect was observed with as little as 10{sup {minus}5} Se-Met and was prevented by a 10-fold excess of Met. A less pronounced effect was observed with 10{sup {minus}4}M Se-Cys. The purified Se-Met substituted enzyme exhibited the same apparent physical size, spectral properties and NADH dehydrogenase activities as control NR but was devoid of nitrate-reducing activities. These results suggest that one or more Met residues are essential for the catalytic function of the molybdo-pterin center of assimilatory NR.

  20. Life with too much polyprenol: polyprenol reductase deficiency.

    PubMed

    Gründahl, J E H; Guan, Z; Rust, S; Reunert, J; Müller, B; Du Chesne, I; Zerres, K; Rudnik-Schöneborn, S; Ortiz-Brüchle, N; Häusler, M G; Siedlecka, J; Swiezewska, E; Raetz, C R H; Marquardt, T

    2012-04-01

    Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation. Although an early homozygous stop-codon (c.57G>A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis.

  1. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

    Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  2. A mutant of barley lacking NADH-hydroxypyruvate reductase

    SciTech Connect

    Blackwell, R.; Lea, P. )

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used to show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.

  3. Evolution Alters the Enzymatic Reaction Coordinate of Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    How evolution has affected enzyme function is a topic of great interest in the field of biophysical chemistry. Evolutionary changes from Escherichia coli dihydrofolate reductase (ecDHFR) to human dihydrofolate reductase (hsDHFR) have resulted in increased catalytic efficiency and an altered dynamic landscape in the human enzyme. Here, we show that a subpicosecond protein motion is dynamically coupled to hydride transfer catalyzed by hsDHFR but not ecDHFR. This motion propagates through residues that correspond to mutational events along the evolutionary path from ecDHFR to hsDHFR. We observe an increase in the variability of the transition states, reactive conformations, and times of barrier crossing in the human system. In the hsDHFR active site, we detect structural changes that have enabled the coupling of fast protein dynamics to the reaction coordinate. These results indicate a shift in the DHFR family to a form of catalysis that incorporates rapid protein dynamics and a concomitant shift to a more flexible path through reactive phase space. PMID:25369552

  4. Stereospecificity of (+)-pinoresinol and (+)-lariciresinol reductases from Forsythia intermedia.

    PubMed

    Chu, A; Dinkova, A; Davin, L B; Bedgar, D L; Lewis, N G

    1993-12-25

    Pinoresinol/lariciresinol reductase catalyzes the first known example of a highly unusual benzylic ether reduction in plants; its mechanism of hydride transfer is described. The enzyme was found in Forsythia intermedia and catalyzes the presumed regulatory branch-points in the pathway leading to benzylaryltetrahydrofuran, dibenzylbutane, dibenzylbutyrolactone, and aryltetrahydronaphthalene lignans. Using [7,7'-2H2]-pinoresinol and [7,7'-2H3]lariciresinol as substrates, the hydride transfers of the highly unusual reductase were demonstrated to be completely stereospecific (> 99%). The incoming hydrides were found to take up the pro-R position at C-7' (and/or C-7) in lariciresinol and secoisolariciresinol, thereby eliminating the possibility of random hydride delivery to a planar quinone methide intermediate. As might be expected, the mode of hydride abstraction from NADPH was also stereospecific: using [4R-3H] and [4S-3H]NADPH, it was found that only the 4 pro-R hydrogen was abstracted for enzymatic hydride transfer.

  5. Synthesis and metabolism of inhibitors of ribonucleotide reductase

    SciTech Connect

    Smith, F.T.

    1985-01-01

    In an effort to prepare more effective inhibitors of ribo-nucleotide reductase a series of 2-substituted-4,6-dihydroxypyrimidines was prepared via the appropriately substituted benzamidine. None of the compounds exhibited in vivo activity against L1210 leukemia. No further testing was performed. In order to investigate the metabolism of 3,4-dihydroxybenzohydroxamic acid, a known inhibitor of ribonucleotide reductase, radiolabeled 3,4-dihydroxybenzohydroxamic acid was synthesized by a modification of the procedure of Pichat and Tostain. /sup 14/C-3,4-Dihydroxybenzoic acid was converted to the methyl ester and subsequently reacted with hydroxylamine to give the hydroxamic acid. /sup 14/C-3,4-Dihydroxybenzohydroxamic acid was given i.p. to Sprague-Dawley rats. Excretion occurred mainly (72%) via the urine. HPLC coupled with GC/MS analyses showed that the compound was excreted mainly unchanged. The compound was metabolized to 3,4-dihydroxybenzamide, 4-methoxy-3-hydroxybenzohydroxamic acid, and 4-hydroxy-3-methoxybenzohydroxamic acid. HPLC analysis also showed the lack of formation of any glucuronide or sulfate conjugates through either the hydroxamic acid or catechol functionalities.

  6. Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase

    SciTech Connect

    Slabaugh, M.B.; Mathews, C.K.

    1986-11-01

    Repeated passage of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1000-fold. Analyses of viral protein synthesis by using (/sup 35/S)methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated (/sup 3/H)thymidine into DNA earlier and a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolated readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses.

  7. Metallic Borides, La2Re3B7 and La3Re2B5, Featuring Extensive Boron-Boron Bonding.

    PubMed

    Bugaris, Daniel E; Malliakas, Christos D; Chung, Duck Young; Kanatzidis, Mercouri G

    2016-02-15

    La2Re3B7 and La3Re2B5 have been synthesized in single-crystalline form from a molten La/Ni eutectic at 1000 °C in the first example of the flux crystal growth of ternary rare-earth rhenium borides. Both compounds crystallize in their own orthorhombic structure types, with La2Re3B7 (space group Pcca) having lattice parameters a = 7.657(2) Å, b = 6.755(1) Å, and c = 11.617(2) Å, and La3Re2B5 (space group Pmma) having lattice parameters a = 10.809(2) Å, b = 5.287(1) Å, and c = 5.747(1) Å. The compounds possess three-dimensional framework structures that are built up from rhenium boride polyhedra and boron-boron bonding. La3Re2B5 features fairly common B2 dumbbells, whereas La2Re3B7 has unique one-dimensional subunits composed of alternating triangular B3 and trans-B4 zigzag chain fragments. Also observed in La3Re2B5 is an unusual coordination of B by an octahedron of La atoms. Electronic band structure calculations predict that La2Re3B7 is a semimetal, which is observed in the electrical resistivity data as measured on single crystals, with behavior obeying the Bloch-Grüneisen model and a room-temperature resistivity ρ300 K of ∼375 μΩ cm. The electronic band structure calculations also suggest that La3Re2B5 is a regular metal. PMID:26812202

  8. Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

    PubMed

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M; Toogood, Helen S; Scrutton, Nigel S

    2016-08-01

    Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. PMID:27411040

  9. Prostates, pates, and pimples. The potential medical uses of steroid 5 alpha-reductase inhibitors.

    PubMed

    Tenover, J S

    1991-12-01

    The steroid 5 alpha-reductase enzyme is responsible for the formation of DHT from testosterone. DHT has been the major androgen implicated in the pathogenesis of benign prostatic hyperplasia, male pattern baldness, acne, and idiopathic female hirsutism. Although specific inhibitors of 5 alpha-reductase are not yet generally available for human use, it is expected that they will become available within the next several years. Based on biochemical, histologic, and anatomic information from animals given 5 alpha-reductase inhibitors, preliminary data on their use in humans, and knowledge gained from men with the inherited 5 alpha-reductase deficiency, it is expected that these 5 alpha-reductase inhibitors may have a major role in the medical management of benign prostatic hyperplasia. In addition, it is possible that these compounds will hold promise for the prevention of male pattern baldness and for the treatment of resistant acne and idiopathic hirsutism. PMID:1723383

  10. 20(S)-Ginsenoside Rh2 as aldose reductase inhibitor from Panax ginseng.

    PubMed

    Fatmawati, Sri; Ersam, Taslim; Yu, Hongshan; Zhang, Chunzhi; Jin, Fengxie; Shimizu, Kuniyoshi

    2014-09-15

    The root of Panax ginseng C. A. Meyer (Araliaceae) is a well-known herbal medicine in East Asia. The major bioactive metabolites in this root are commonly identified as ginsenosides. A series of ginsenosides were determined for in vitro human recombinant aldose reductase. This Letter aims to clarify the structural requirement for aldose reductase inhibition. We discovered that only ginsenoside 20(S)-Rh2 showed potent against aldose reductase, with an IC50 of 147.3 μM. These results implied that the stereochemistry of the hydroxyl group at C-20 may play an important role in aldose reductase inhibition. An understanding of these requirements is considered necessary in order to develop a new type of aldose reductase inhibitor. Furthermore, P. ginseng might be an important herbal medicine in preventing diabetic complications.

  11. Natural variation in arsenate tolerance identifies an arsenate reductase in Arabidopsis thaliana.

    PubMed

    Sánchez-Bermejo, Eduardo; Castrillo, Gabriel; del Llano, Bárbara; Navarro, Cristina; Zarco-Fernández, Sonia; Martinez-Herrera, Dannys Jorge; Leo-del Puerto, Yolanda; Muñoz, Riansares; Cámara, Carmen; Paz-Ares, Javier; Alonso-Blanco, Carlos; Leyva, Antonio

    2014-01-01

    The enormous amount of environmental arsenic was a major factor in determining the biochemistry of incipient life forms early in the Earth's history. The most abundant chemical form in the reducing atmosphere was arsenite, which forced organisms to evolve strategies to manage this chemical species. Following the great oxygenation event, arsenite oxidized to arsenate and the action of arsenate reductases became a central survival requirement. The identity of a biologically relevant arsenate reductase in plants nonetheless continues to be debated. Here we identify a quantitative trait locus that encodes a novel arsenate reductase critical for arsenic tolerance in plants. Functional analyses indicate that several non-additive polymorphisms affect protein structure and account for the natural variation in arsenate reductase activity in Arabidopsis thaliana accessions. This study shows that arsenate reductases are an essential component for natural plant variation in As(V) tolerance. PMID:25099865

  12. Aldose reductase catalysis and crystallography. Insights from recent advances in enzyme structure and function.

    PubMed

    Petrash, J M; Tarle, I; Wilson, D K; Quiocho, F A

    1994-08-01

    Enhanced metabolism of glucose via the polyol pathway may play an important role in the pathogenesis of diabetic retinopathy, neuropathy, and nephropathy. Aldose reductase catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step in the polyol pathway. Interruption of the polyol pathway by inhibition of aldose reductase holds considerable promise as a therapeutic measure to prevent or delay the onset and severity of these late complications of diabetes. Dramatic advances in our understanding of the molecular biology, enzymology, and three-dimensional structure of aldose reductase have occurred in recent years, providing new and challenging insights into the enzyme's catalytic mechanism. Recent developments in structure determination of aldose reductase and the implications for evaluation and development of aldose reductase inhibitors are summarized. PMID:8039602

  13. The flavin inhibitor diphenyleneiodonium renders Trichomonas vaginalis resistant to metronidazole, inhibits thioredoxin reductase and flavin reductase, and shuts off hydrogenosomal enzymatic pathways.

    PubMed

    Leitsch, David; Kolarich, Daniel; Duchêne, Michael

    2010-05-01

    Infections with the microaerophilic protozoan parasite Trichomonas vaginalis are commonly treated with metronidazole, a 5-nitroimidazole drug. Metronidazole is selectively toxic to microaerophiles and anaerobes because reduction at the drug's nitro group, which is a precondition for toxicity, occurs only quantitatively in these organisms. In our previous work we identified the flavin enzyme thioredoxin reductase as an electron donor to 5-nitroimidazole drugs in T. vaginalis and observed that highly metronidazole-resistant cell lines lack thioredoxin reductase and flavin reductase activities. In this study we added the flavin inhibitor diphenyleneiodonium (DPI) to T. vaginalis cultures in order to test our hypothesis that metronidazole reduction is catalyzed by flavin enzymes, e.g. thioredoxin reductase, and intracellular free flavins. Indeed, within hours, DPI rendered T. vaginalis insensitive to metronidazole concentrations as high as 1mM and prevented the formation of metronidazole adducts with proteins. Thioredoxin reductase activity was absent from DPI-treated cells and flavin reductase activity was sharply decreased. In addition, DPI-treated cells also upregulated the expression of antioxidant enzymes, i.e. thioredoxin peroxidases and superoxide dismutases, and displayed a fundamentally altered metabolism caused by inactivation of pyruvate:ferredoxin oxidoreductase (PFOR) and concomitant upregulation of lactate dehydrogenase (LDH) activity. Thus, the disruption of the cellular flavin metabolism by DPI mediated metabolic steps which are similar to that of cells with metronidazole resistance induced in vitro. Finally, we present direct evidence that the increased expression of antioxidant enzymes is dispensable for acquiring resistance to metronidazole. PMID:20093143

  14. Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint.

    PubMed

    Turner, Glenn W; Davis, Edward M; Croteau, Rodney B

    2012-06-01

    Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined. PMID:22170164

  15. Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

    PubMed Central

    Cohen, G; Yanko, M; Mislovati, M; Argaman, A; Schreiber, R; Av-Gay, Y; Aharonowitz, Y

    1993-01-01

    The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated. Images PMID:8349555

  16. Peach MYB7 activates transcription of the proanthocyanidin pathway gene encoding leucoanthocyanidin reductase, but not anthocyanidin reductase

    PubMed Central

    Zhou, Hui; Lin-Wang, Kui; Liao, Liao; Gu, Chao; Lu, Ziqi; Allan, Andrew C.; Han, Yuepeng

    2015-01-01

    Proanthocyanidins (PAs) are a group of natural phenolic compounds that have a great effect on both flavor and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs) via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase and anthocyanidin reductase. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5) via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants. PMID:26579158

  17. Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems.

    PubMed

    Ondarza, Raúl N; Hurtado, Gerardo; Tamayo, Elsa; Iturbe, Angélica; Hernández, Eva

    2006-11-01

    This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.

  18. Polymorphisms in the methylene tetrahydrofolate reductase and methionine synthase reductase genes and their correlation with unexplained recurrent spontaneous abortion susceptibility.

    PubMed

    Zhu, L

    2015-01-01

    We aimed to explore the correlation between unexplained recurrent spontaneous abortion and polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes. A case control study was conducted in 118 patients with unexplained recurrent spontaneous abortion (abortion group) and 174 healthy women (control group). The genetic material was extracted from the oral mucosal epithelial cells obtained from all subjects. The samples were subjected to fluorescence quantitative PCR to detect the single nucleotide polymorphisms (SNPs) in the MTHFR (C677T and A1298C) and MTRR (A66G) gene loci. The distribution frequency (18/118, 15.3%) of the MTHFR 677TT genotype was significantly higher in the abortion group (χ2 = 11.006, P = 0.004) than in the control group (2/174, 1.1%); on the other hand, the distribution frequency of the MTHFR A1298C genotype did not significantly differ between the abortion and control groups (χ(2) = 0.441, P = 0.507). The distribution frequency of the MTRR A66G genotype was also significantly higher in the abortion group (14/118, 11.9%; χ(2) = 10.503, P = 0.005) than in the control group (8/174, 4.6%). The MTHFR C677T and MTRR A66G polymorphisms are significantly correlated with the occurrence of spontaneous abortion.

  19. Structural Insight into Methyl-Coenzyme M Reductase Chemistry Using Coenzyme B Analogues

    SciTech Connect

    Cedervall, Peder E.; Dey, Mishtu; Pearson, Arwen R.; Ragsdale, Stephen W.; Wilmot, Carrie M.

    2010-09-07

    Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis: the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme and contains a highly reduced nickel-tetrapyrrole, coenzyme F430. Methyl-SCoM must enter the active site prior to CoBSH, as species derived from methyl-SCoM are always observed bound to the F430 nickel in the deepest part of the 30 {angstrom} long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 {angstrom} predominantly hydrophobic part of the channel close to F430, with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 {angstrom}. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues: pentanoyl, hexanoyl, octanoyl, and nonanoyl derivatives of CoBSH (CoB5SH, CoB6SH, CoB8SH, and CoB9SH, respectively). The data presented here reveal that the shorter CoB5SH mercaptoalkanoyl chain overlays with that of CoBSH but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB6SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB6SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB8SH and CoB9SH analogues can be accommodated in the active site through exclusion of this water. These analogues

  20. Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines

    PubMed Central

    Dluzen, Douglas F.; Sutliff, Aimee K.; Chen, Gang; Watson, Christy J. W.; Ishmael, Faoud T.

    2016-01-01

    The UDP-glucuronosyltransferase (UGT) 2B enzymes are important in the detoxification of a variety of endogenous and exogenous compounds, including many hormones, drugs, and carcinogens. Identifying novel mechanisms governing their expression is important in understanding patient-specific response to drugs and cancer risk factors. In silico prediction algorithm programs were used to screen for microRNAs (miRNAs) as potential regulators of UGT2B enzymes, with miR-216b-5p identified as a potential candidate. Luciferase data suggested the presence of a functional miR-216b-5p binding motif within the 3′ untranslated regions of UGTs 2B7, 2B4, and 2B10. Overexpression of miR-216b-5p mimics significantly repressed UGT2B7 (P < 0.001) and UGT2B10 (P = 0.0018) mRNA levels in HuH-7 cells and UGT2B4 (P < 0.001) and UGT2B10 (P = 0.018) mRNA in Hep3B cells. UGT2B7 protein levels were repressed in both HuH-7 and Hep3B cells in the presence of increasing miR-216b-5p concentrations, corresponding with significant (P < 0.001 and P = 0.011, respectively) decreases in glucuronidation activity against the UGT2B7-specific substrate epirubicin. Inhibition of endogenous miR-216b-5p levels significantly increased UGT2B7 mRNA levels in HuH-7 (P = 0.021) and Hep3B (P = 0.0068) cells, and increased epirubicin glucuronidation by 85% (P = 0.057) and 50% (P = 0.012) for HuH-7 and Hep3B cells, respectively. UGT2B4 activity against codeine and UGT2B10 activity against nicotine were significantly decreased in both HuH-7 and Hep3B cells (P < 0.001 and P = 0.0048, and P = 0.017 and P = 0.043, respectively) after overexpression of miR-216b-5p mimic. This is the first evidence that miRNAs regulate UGT 2B7, 2B4, and 2B10 expression, and that miR-216b-5p regulation of UGT2B proteins may be important in regulating the metabolism of UGT2B substrates. PMID:27474751

  1. Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines.

    PubMed

    Dluzen, Douglas F; Sutliff, Aimee K; Chen, Gang; Watson, Christy J W; Ishmael, Faoud T; Lazarus, Philip

    2016-10-01

    The UDP-glucuronosyltransferase (UGT) 2B enzymes are important in the detoxification of a variety of endogenous and exogenous compounds, including many hormones, drugs, and carcinogens. Identifying novel mechanisms governing their expression is important in understanding patient-specific response to drugs and cancer risk factors. In silico prediction algorithm programs were used to screen for microRNAs (miRNAs) as potential regulators of UGT2B enzymes, with miR-216b-5p identified as a potential candidate. Luciferase data suggested the presence of a functional miR-216b-5p binding motif within the 3' untranslated regions of UGTs 2B7, 2B4, and 2B10. Overexpression of miR-216b-5p mimics significantly repressed UGT2B7 (P < 0.001) and UGT2B10 (P = 0.0018) mRNA levels in HuH-7 cells and UGT2B4 (P < 0.001) and UGT2B10 (P = 0.018) mRNA in Hep3B cells. UGT2B7 protein levels were repressed in both HuH-7 and Hep3B cells in the presence of increasing miR-216b-5p concentrations, corresponding with significant (P < 0.001 and P = 0.011, respectively) decreases in glucuronidation activity against the UGT2B7-specific substrate epirubicin. Inhibition of endogenous miR-216b-5p levels significantly increased UGT2B7 mRNA levels in HuH-7 (P = 0.021) and Hep3B (P = 0.0068) cells, and increased epirubicin glucuronidation by 85% (P = 0.057) and 50% (P = 0.012) for HuH-7 and Hep3B cells, respectively. UGT2B4 activity against codeine and UGT2B10 activity against nicotine were significantly decreased in both HuH-7 and Hep3B cells (P < 0.001 and P = 0.0048, and P = 0.017 and P = 0.043, respectively) after overexpression of miR-216b-5p mimic. This is the first evidence that miRNAs regulate UGT 2B7, 2B4, and 2B10 expression, and that miR-216b-5p regulation of UGT2B proteins may be important in regulating the metabolism of UGT2B substrates. PMID:27474751

  2. Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines.

    PubMed

    Dluzen, Douglas F; Sutliff, Aimee K; Chen, Gang; Watson, Christy J W; Ishmael, Faoud T; Lazarus, Philip

    2016-10-01

    The UDP-glucuronosyltransferase (UGT) 2B enzymes are important in the detoxification of a variety of endogenous and exogenous compounds, including many hormones, drugs, and carcinogens. Identifying novel mechanisms governing their expression is important in understanding patient-specific response to drugs and cancer risk factors. In silico prediction algorithm programs were used to screen for microRNAs (miRNAs) as potential regulators of UGT2B enzymes, with miR-216b-5p identified as a potential candidate. Luciferase data suggested the presence of a functional miR-216b-5p binding motif within the 3' untranslated regions of UGTs 2B7, 2B4, and 2B10. Overexpression of miR-216b-5p mimics significantly repressed UGT2B7 (P < 0.001) and UGT2B10 (P = 0.0018) mRNA levels in HuH-7 cells and UGT2B4 (P < 0.001) and UGT2B10 (P = 0.018) mRNA in Hep3B cells. UGT2B7 protein levels were repressed in both HuH-7 and Hep3B cells in the presence of increasing miR-216b-5p concentrations, corresponding with significant (P < 0.001 and P = 0.011, respectively) decreases in glucuronidation activity against the UGT2B7-specific substrate epirubicin. Inhibition of endogenous miR-216b-5p levels significantly increased UGT2B7 mRNA levels in HuH-7 (P = 0.021) and Hep3B (P = 0.0068) cells, and increased epirubicin glucuronidation by 85% (P = 0.057) and 50% (P = 0.012) for HuH-7 and Hep3B cells, respectively. UGT2B4 activity against codeine and UGT2B10 activity against nicotine were significantly decreased in both HuH-7 and Hep3B cells (P < 0.001 and P = 0.0048, and P = 0.017 and P = 0.043, respectively) after overexpression of miR-216b-5p mimic. This is the first evidence that miRNAs regulate UGT 2B7, 2B4, and 2B10 expression, and that miR-216b-5p regulation of UGT2B proteins may be important in regulating the metabolism of UGT2B substrates.

  3. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    PubMed

    Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

    2006-05-19

    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

  4. Biofilm modifies expression of ribonucleotide reductase genes in Escherichia coli.

    PubMed

    Cendra, Maria del Mar; Juárez, Antonio; Torrents, Eduard

    2012-01-01

    Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed. PMID:23050019

  5. A calibration curve for immobilized dihydrofolate reductase activity assay.

    PubMed

    Singh, Priyanka; Morris, Holly; Tivanski, Alexei V; Kohen, Amnon

    2015-09-01

    An assay was developed for measuring the active-site concentration, activity, and thereby the catalytic turnover rate (k cat) of an immobilized dihydrofolate reductase model system (Singh et al., (2015), Anal. Biochem). This data article contains a calibration plot for the developed assay. In the calibration plot rate is plotted as a function of DHFR concentration and shows linear relationship. The concentration of immobilized enzyme was varied by using 5 different size mica chips. The dsDNA concentration was the same for all chips, assuming that the surface area of the mica chip dictates the resulting amount of bound enzyme (i.e. larger sized chip would have more bound DHFR). The activity and concentration of each chip was measured.

  6. Structure of a bacterial homologue of vitamin K epoxide reductase

    SciTech Connect

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A.

    2010-03-19

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

  7. 5-Alpha-Reductase Inhibitors and Combination Therapy.

    PubMed

    Füllhase, Claudius; Schneider, Marc P

    2016-08-01

    By inhibiting the conversion from testosterone to dihydrotestosterone 5-Alpha reductase inhibitors (5ARIs) are able to hinder prostatic growth, shrink prostate volumes, and improve BPH-related LUTS. 5ARIs are particularly beneficial for patients with larger prostates (>30-40ml). Generally the side effects of 5ARI treatment are mild, and according to the FORTA classification 5ARIs are suitable for frail elderly. 5ARI / alpha-blocker (AB) combination therapy showed the best symptomatic outcome and risk reduction for clinical progression. Combining Phosphodieseterase type 5 inhbibitors (PDE5Is) with 5ARIs counteracts the negative androgenic sexual side effects of 5ARIs, and simultaneously combines their synergistic effects on LUTS. PMID:27476125

  8. Metabolism of the aldose reductase inhibitor ALO1567 in man.

    PubMed Central

    Park, Y H; Hudson, J E; Barker, R C; York, B M; Brazzell, R K

    1991-01-01

    1. The metabolism of the aldose reductase inhibitor, ALO1567, was studied in man. The major biotransformation pathway was aromatic hydroxylation followed by glucuronide conjugation. 2. Hydroxylation occurred at several positions on the fluorene ring. The major metabolite was identified as the 7-hydroxy analogue of ALO1567 and three minor metabolites were characterized as positional isomers of the 7-hydroxy metabolite. 3. Oxidative defluorination and metabolism on the hydantoin ring were also indicated as minor pathways. 4. The capacity of normal subjects to oxidize ALO1567 was indicated by the urinary ratio of the parent drug to the 7-hydroxy metabolite after daily oral administration of 100 mg and 200 mg of ALO1567. Most subjects having higher ALO1567 plasma concentrations showed higher ratios. PMID:1931471

  9. B-factor Analysis and Conformational Rearrangement of Aldose Reductase.

    PubMed

    Balendiran, Ganesaratnam K; Pandian, J Rajendran; Drake, Evin; Vinayak, Anubhav; Verma, Malkhey; Cascio, Duilio

    2014-01-01

    The NADPH-dependent reduction of glucose reaction that is catalyzed by Aldose Reductase (AR) follows a sequential ordered kinetic mechanism in which the co-factor NADPH binds to the enzyme prior to the aldehyde substrate. The kinetic/structural experiments have found a conformational change involving a hinge-like movement of a surface loop (residues 213-224) which is anticipated to take place upon the binding of the diphosphate moiety of NADPH. The reorientation of this loop, expected to permit the release of NADP(+), represents the rate-limiting step of the catalytic mechanism. This study reveals: 1) The Translation/Libration/Screw (TLS) analysis of absolute B-factors of apo AR crystal structures indicates that the 212-224 loop might move as a rigid group. 2) Residues that make the flexible loop slide in the AR binary and ternary complexes. 3) The normalized B-factors separate this segment into three different clusters with fewer residues.

  10. Thioredoxin reductase 1 suppresses adipocyte differentiation and insulin responsiveness

    PubMed Central

    Peng, Xiaoxiao; Giménez-Cassina, Alfredo; Petrus, Paul; Conrad, Marcus; Rydén, Mikael; Arnér, Elias S. J.

    2016-01-01

    Recently thioredoxin reductase 1 (TrxR1), encoded by Txnrd1, was suggested to modulate glucose and lipid metabolism in mice. Here we discovered that TrxR1 suppresses insulin responsiveness, anabolic metabolism and adipocyte differentiation. Immortalized mouse embryonic fibroblasts (MEFs) lacking Txnrd1 (Txnrd1−/−) displayed increased metabolic flux, glycogen storage, lipogenesis and adipogenesis. This phenotype coincided with upregulated PPARγ expression, promotion of mitotic clonal expansion and downregulation of p27 and p53. Enhanced Akt activation also contributed to augmented adipogenesis and insulin sensitivity. Knockdown of TXNRD1 transcripts accelerated adipocyte differentiation also in human primary preadipocytes. Furthermore, TXNRD1 transcript levels in subcutaneous adipose tissue from 56 women were inversely associated with insulin sensitivity in vivo and lipogenesis in their isolated adipocytes. These results suggest that TrxR1 suppresses anabolic metabolism and adipogenesis by inhibition of intracellular signaling pathways downstream of insulin stimulation. PMID:27346647

  11. Increased neurotoxicity of arsenic in methylenetetrahydrofolate reductase deficiency.

    PubMed

    Brouwer, O F; Onkenhout, W; Edelbroek, P M; de Kom, J F; de Wolff, F A; Peters, A C

    1992-01-01

    A 16-year-old girl from Surinam presented with mental deterioration and severe paraparesis with areflexia and bilateral Babinski signs. Laboratory examination showed a hyperhomocysteinemia that was caused by 5,10-methylene-tetrahydrofolate reductase (MTHFR) deficiency. In addition, urine samples contained large amounts of arsenic. An open bag with the pesticide copper acetate arsenite was found to be the source of exposure. In remethylation defects such as MTHFR deficiency, the concentration of methyldonors is severely reduced. As arsenic is detoxified by methylation, we suggest that the MTHFR deficiency in this girl might explain the fact that of all family members exposed to arsenic, only she developed severe clinical signs and symptoms of arsenic poisoning.

  12. Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd)

    SciTech Connect

    Zahedi Avval, Farnaz; Berndt, Carsten; Pramanik, Aladdin; Holmgren, Arne

    2009-02-13

    Motexafin gadolinium (MGd) is an expanded porphyrin anticancer agent which selectively targets tumor cells and works as a radiation enhancer, with promising results in clinical trials. Its mechanism of action is oxidation of intracellular reducing molecules and acting as a direct inhibitor of mammalian ribonucleotide reductase (RNR). This paper focuses on the mechanism of inhibition of RNR by MGd. Our experimental data present at least two pathways for inhibition of RNR; one precluding subunits oligomerization and the other direct inhibition of the large catalytic subunit of the enzyme. Co-localization of MGd and RNR in the cytoplasm particularly in the S-phase may account for its inhibitory properties. These data can elucidate an important effect of MGd on the cancer cells with overproduction of RNR and its efficacy as an anticancer agent and not only as a general radiosensitizer.

  13. Lymphocyte development requires S-nitrosoglutathione reductase1

    PubMed Central

    Yang, Zhiyong; Wang, Zhi-En; Doulias, Paschalis-Thomas; Wei, Wei; Ischiropoulos, Harry; Locksley, Richard M.; Liu, Limin

    2011-01-01

    Nitric oxide is critical to immunity, but its role in the development of the immune system is unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), a protein key to the control of protein S-nitrosylation, is important for the development of lymphocytes. Genetic deletion of GSNOR in mice results in significant decrease in both T and B lymphocytes in the periphery. In thymus, GSNOR deficiency causes excessive protein S-nitrosylation, increases apoptosis, and reduces the number of CD4 single-positive thymocytes. Lymphopenia andincrease in S-nitrosylation and apoptosis in GSNOR-deficient mice are largely abolished by genetic deletion of inducible nitric oxide synthase (iNOS). Furthermore, the protection of lymphocyte development by GSNOR is apparently intrinsic to hematopoietic cells. Thus GSNOR, likely through regulation of S-nitrosylation and apoptosis, physiologically plays aprotective role in the development of the immune system. PMID:20980633

  14. Diuretic and natriuretic effects of sorbinil, an aldose reductase inhibitor.

    PubMed

    Springate, J E; Feld, L G; Van Liew, J B; Fildes, R D; Acara, M A

    1991-04-01

    The renal effects of sorbinil, an aldose reductase inhibitor that interferes with the conversion of glucose to sorbitol, were studied in rats and rabbits before and after fluid deprivation. The intracellular osmolar solute, sorbitol, is found in increasing concentrations from cortex to medulla in the kidney and may be involved in the urinary concentrating mechanism. Oral administration of sorbinil in the rabbit resulted in significant increases in urine flow rate and sodium excretion with a tendency toward decreased urine osmolality and increased potassium excretion both before and after water deprivation. When fluid intake was controlled in the rat study, significant increases in urine flow rate and sodium and potassium excretion and a significant decrease in urine osmolality occurred only in response to fluid deprivation. Thus, sorbinil has diuretic and natriuretic properties and may prevent the normal concentration of urine in the antidiuretic animal.

  15. Purification and characterization of rat lens pyrroline-5-carboxylate reductase.

    PubMed

    Shiono, T; Kador, P F; Kinoshita, J J

    1986-03-19

    delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) has been purified from rat lens and biochemically characterized. Purification steps included ammonium sulfate fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22 degrees C) in 20 mM sodium phosphate/potassium phosphate buffer (pH 7.5) containing 10% glycerol, 7 mM mercaptoethanol and 0.5 mM EDTA. The enzyme, purified to apparent homogeneity, displayed a molecular weight of 240 000 by gel chromatography and 30 000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme is composed of eight subunits. The purified enzyme displays a pH optimum between 6.5 and 7.1 and is inhibited by heavy metal ions and p-chloromercuribenzoate. Kinetic studies indicated Km values of 0.62 mM and 0.051 mM for DL-pyrroline-5-carboxylate as substrate when NADH and NADPH respectively were employed as cofactors. The Km values for the cofactors NADH and NADPH with DL-pyrroline-5-carboxylate as substrate were 0.37 mM and 0.006 mM, respectively. With L-pyrroline-5-carboxylate as substrate, Km values of 0.21 mM and 0.022 mM were obtained for NADH and NADPH, respectively. Enzyme activity is potentially inhibited by NADP+ and ATP, suggesting that delta 1-pyrroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.

  16. The modulation of carbonyl reductase 1 by polyphenols.

    PubMed

    Boušová, Iva; Skálová, Lenka; Souček, Pavel; Matoušková, Petra

    2015-01-01

    Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1.

  17. Functional genomic studies of aldo-keto reductases.

    PubMed

    Petrash, J M; Murthy, B S; Young, M; Morris, K; Rikimaru, L; Griest, T A; Harter, T

    2001-01-30

    Aldose reductase (AR) is considered a potential mediator of diabetic complications and is a drug target for inhibitors of diabetic retinopathy and neuropathy in clinical trials. However, the physiological role of this enzyme still has not been established. Since effective inhibition of diabetic complications will require early intervention, it is important to delineate whether AR fulfills a physiological role that cannot be compensated by an alternate aldo-keto reductase. Functional genomics provides a variety of powerful new tools to probe the physiological roles of individual genes, especially those comprising gene families. Several eucaryotic genomes have been sequenced and annotated, including yeast, nematode and fly. To probe the function of AR, we have chosen to utilize the budding yeast Saccharomyces cerevisiae as a potential model system. Unlike Caenorhabditis elegans and D. melanogaster, yeast provides a more desirable system for our studies because its genome is manipulated more readily and is able to sustain multiple gene deletions in the presence of either drug or auxotrophic selectable markers. Using BLAST searches against the human AR gene sequence, we identified six genes in the complete S. cerevisiae genome with strong homology to AR. In all cases, amino acids thought to play important catalytic roles in human AR are conserved in the yeast AR-like genes. All six yeast AR-like open reading frames (ORFs) have been cloned into plasmid expression vectors. Substrate and AR inhibitor specificities have been surveyed on four of the enzyme forms to identify, which are the most functionally similar to human AR. Our data reveal that two of the enzymes (YDR368Wp and YHR104Wp) are notable for their similarity to human AR in terms of activity with aldoses and substituted aromatic aldehydes. Ongoing studies are aimed at characterizing the phenotypes of yeast strains containing single and multiple knockouts of the AR-like genes. PMID:11306085

  18. Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.

    PubMed

    Perez-Abraham, Romy; Sanchez, Karla Garabiles; Alfonso, Melany; Gubler, Ueli; Siekierka, John J; Goodey, Nina M

    2016-12-01

    Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR. PMID:27544923

  19. Finasteride: the first 5 alpha-reductase inhibitor.

    PubMed

    Sudduth, S L; Koronkowski, M J

    1993-01-01

    Finasteride is a synthetic 4-azasteroid that is a specific competitive inhibitor of 5 alpha-reductase, an intracellular enzyme that converts testosterone to dihydrotestosterone (DHT). It has no binding affinity for androgen receptor sites and itself possesses no androgenic, antiandrogenic, or other steroid hormone-related properties. It is well absorbed after oral administration, with absolute bioavailability in humans of 63% (range 34-108%). The mean time to maximum concentration is 1-2 hours, and it is approximately 90% plasma protein bound. The elimination half-life averages 6-8 hours. The agent is metabolized to a series of five metabolites, of which two are active and possess less than 20% of the 5 alpha-reductase activity of finasteride. Little is known about potential drug interactions, although they appear to be minimal and not clinically relevant. The drug is indicated for the treatment of symptomatic benign prostatic hyperplasia. Its efficacy in regression of prostate gland enlargement is rapid and predictable, although correlation with subsequent improvement in urinary flow and symptoms is highly variable. Dosages of 0.5-100 mg/day regress prostate enlargement; the recommended dosage is 5 mg once/day. Finasteride may hold promise for other DHT-mediated disorders such as acne, facial hirsutism, frontal lobe alopecia, and prostate cancer, but its use in these conditions remains investigational. The frequency of adverse drug events is low, with the most common side effects being impotence, decreased libido, and decreased volume of ejaculate. No reports of intentional overdose have been reported, and dosages of up to 80 mg/day for 3 months have been taken without adverse effect. PMID:7689728

  20. [Decolorization of azo dyes using quinone reductase and quinoid compounds].

    PubMed

    Zhou, Mi; Liu, Guang-Fei; Zhou, Ji-Ti; Jin, Ruo-Fei; Chen, Ming-Xiang; Wang, Yan-Qing

    2009-06-15

    Using quinoid redox mediator and bacterial cellular quinone reductase, we investigated the decolorization ability of gene-engineered strain Escherichia coli YB and the effects of methylhydroquinone (MHQ) pretreatement on decolorization performance of E. coli JM109 and anaerobic sludge. The results indicate that lawsone is an effective accelerator for azo dye decolorization by E. coli YB overexpressing cellular quinone reductase AZR. In the presence of 0.2 mmol x L(-1) lawsone, 75% Amaranth (1 mmol x L(-1)) can be decolorized in 2 h. E. coli YB can also decolorize high concentration of azo dye in the presence of lawsone. Around 50% Amaranth (5 mmol x L(-1)) is decolorized in 8 h. Compared to lawsone, menadione is a less effective mediator. E. coli YB takes 12 h to reach 70% decolorization in the presence of 2.5 mmol x L(-1) menadione. Repeated decolorization studies showed that E. coli YB had stable decolorizing ability in the presence of lawsone. Four rounds of repeated decolorization can be completed in 12 h. Lawsone can also accelerate the decolorization of azo dyes with complex structures such as Acid Scarlet GR and Reactive Brilliant Red K-2BP. With the optimal LQ concentrations, 70% Acid Scarlet GR and Reactive Brilliant Red K-2BP are decolorized in 9 h and 30 h,respectively. Decolorization performances of E. coli JM109 and anaerobic sludge pretreated with MHQ are improved. After MHQ pretreatment,in the presence of lawsone, 80% Amaranth (1 mmol x L(-1)) can be decolorized in 5 h by E. coli JM109, while more than 75% Amaranth can be removed in 11 h by sludge.

  1. Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

    PubMed Central

    Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

    2014-01-01

    Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown. PMID:25002540

  2. Characterization of the nitric oxide reductase from Thermus thermophilus.

    PubMed

    Schurig-Briccio, Lici A; Venkatakrishnan, Padmaja; Hemp, James; Bricio, Carlos; Berenguer, José; Gennis, Robert B

    2013-07-30

    Nitrous oxide (N2O) is a powerful greenhouse gas implicated in climate change. The dominant source of atmospheric N2O is incomplete biological dentrification, and the enzymes responsible for the release of N2O are NO reductases. It was recently reported that ambient emissions of N2O from the Great Boiling Spring in the United States Great Basin are high, and attributed to incomplete denitrification by Thermus thermophilus and related bacterial species [Hedlund BP, et al. (2011) Geobiology 9(6)471-480]. In the present work, we have isolated and characterized the NO reductase (NOR) from T. thermophilus. The enzyme is a member of the cNOR family of enzymes and belongs to a phylogenetic clade that is distinct from previously examined cNORs. Like other characterized cNORs, the T. thermophilus cNOR consists of two subunits, NorB and NorC, and contains a one heme c, one Ca(2+), a low-spin heme b, and an active site consisting of a high-spin heme b and FeB. The roles of conserved residues within the cNOR family were investigated by site-directed mutagenesis. The most important and unexpected result is that the glutamic acid ligand to FeB is not essential for function. The E211A mutant retains 68% of wild-type activity. Mutagenesis data and the pattern of conserved residues suggest that there is probably not a single pathway for proton delivery from the periplasm to the active site that is shared by all cNORs, and that there may be multiple pathways within the T. thermophilus cNOR. PMID:23858452

  3. Characterization of the nitric oxide reductase from Thermus thermophilus

    PubMed Central

    Schurig-Briccio, Lici A.; Venkatakrishnan, Padmaja; Hemp, James; Bricio, Carlos; Berenguer, José; Gennis, Robert B.

    2013-01-01

    Nitrous oxide (N2O) is a powerful greenhouse gas implicated in climate change. The dominant source of atmospheric N2O is incomplete biological dentrification, and the enzymes responsible for the release of N2O are NO reductases. It was recently reported that ambient emissions of N2O from the Great Boiling Spring in the United States Great Basin are high, and attributed to incomplete denitrification by Thermus thermophilus and related bacterial species [Hedlund BP, et al. (2011) Geobiology 9(6)471–480]. In the present work, we have isolated and characterized the NO reductase (NOR) from T. thermophilus. The enzyme is a member of the cNOR family of enzymes and belongs to a phylogenetic clade that is distinct from previously examined cNORs. Like other characterized cNORs, the T. thermophilus cNOR consists of two subunits, NorB and NorC, and contains a one heme c, one Ca2+, a low-spin heme b, and an active site consisting of a high-spin heme b and FeB. The roles of conserved residues within the cNOR family were investigated by site-directed mutagenesis. The most important and unexpected result is that the glutamic acid ligand to FeB is not essential for function. The E211A mutant retains 68% of wild-type activity. Mutagenesis data and the pattern of conserved residues suggest that there is probably not a single pathway for proton delivery from the periplasm to the active site that is shared by all cNORs, and that there may be multiple pathways within the T. thermophilus cNOR. PMID:23858452

  4. Ascorbate free radical reductase mRNA levels are induced by wounding.

    PubMed Central

    Grantz, A A; Brummell, D A; Bennett, A B

    1995-01-01

    A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization. PMID:7784511

  5. Glyphosate inhibition of ferric reductase activity in iron deficient sunflower roots.

    PubMed

    Ozturk, Levent; Yazici, Atilla; Eker, Selim; Gokmen, Ozgur; Römheld, Volker; Cakmak, Ismail

    2008-01-01

    Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mm glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown under Fe deficiency conditions. Application of 1.89 mm glyphosate resulted in almost 50% inhibition of ferric reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower rates of glyphosate (e.g. 0.32 mm and 0.95 mm), ferric reductase was inhibited. Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots were not decreased by the glyphosate applications. To our knowledge, this is the first study reporting the effects of glyphosate on ferric reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase could not be identified. Impaired ferric reductase could be a major reason for the increasingly observed Fe deficiency in cropping systems associated with widespread glyphosate usage.

  6. Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

    PubMed Central

    Snape, J R; Walkley, N A; Morby, A P; Nicklin, S; White, G F

    1997-01-01

    Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively. PMID:9401040

  7. Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India.

    PubMed

    Warang, P P; Kedar, P S; Shanmukaiah, C; Ghosh, K; Colah, R B

    2015-01-01

    We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH-cytochrome b5 reductase (NADH-CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice-site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three-dimensional (3D) structure of human erythrocyte NADH-CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype-phenotype correlation in NADH-CYB5R deficiency. PMID:24266649

  8. Molecular basis of two novel mutations found in type I methemoglobinemia.

    PubMed

    Lorenzo, Felipe R; Phillips, John D; Nussenzveig, Roberto; Lingam, Bindu; Koul, Parvaiz A; Schrier, Stanley L; Prchal, Josef T

    2011-04-15

    Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiency is an autosomal recessive disorder that occurs sporadically worldwide, although endemic clusters of this disorder have been identified in certain ethnic groups. It is present as two distinct phenotypes, type I and type II. Type I methemoglobinemia is characterized by CYB5R3 enzyme deficiency restricted to erythrocytes and is associated with benign cyanosis. The less frequent type II methemoglobinemia is associated with generalized CYB5R3 deficiency affecting all cells and is lethal in early infancy. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients from three distinct ethnic backgrounds, Asian Indian, Mexican and Greek. The CYB5R3 gene of three probands with type I methemoglobinemia and their relatives were sequenced revealing several putative causative mutations; in one subject multiple mutations were present. Two novel mutations, S54R and F157C, were identified and the previously described A179T, V253M mutations were also identified. All these point mutations mapped to the NADH binding domain and or the FAD binding domain. Each has the potential to sterically hinder cofactor binding causing instability of the CYB5R3 protein. Wild-type CYB5R3, as well as two of these novel mutations, S54R and F157C, was amplified, cloned, and purified recombinant peptide obtained. Kinetic and thermodynamic studies of these proteins show that the above mutations lead to decreased thermal stability. PMID:21349748

  9. The use of 5-alpha reductase inhibitors for the prevention of prostate cancer.

    PubMed

    Yu, Eun-mi; El-Ayass, Walid; Aragon-Ching, Jeanny B

    2010-07-01

    The use of 5-alpha-reductase inhibitors has been studied not only in benign prostatic hyperplasia, but as a chemopreventive strategy in prostate cancer. Both finasteride and dutasteride, 5 alpha-reductase inhibitors (5ARI), have been shown to decrease the risk of prostate cancer. The results of the REDUCE trial using the dual alpha-reductase isoenzyme inhibitor dutasteride, has recently been published by Andriole et al. in the New England Journal of Medicine. Certain considerations regarding its use and applicability to men with high risk of developing prostate cancer are herein discussed. PMID:20574153

  10. The 5 alpha-reductase inhibitory components from heartwood of Artocarpus incisus: structure-activity investigations.

    PubMed

    Shimizu, K; Fukuda, M; Kondo, R; Sakai, K

    2000-02-01

    The methanol extract of heartwood of Artocarpus incisus showed potent 5 alpha-reductase inhibitory activity. We investigated the 5 alpha-reductase inhibitory effects of nine compounds isolated from A. incisus. Chlorophorin (IC50 = 37 microM) and artocarpin (IC50 = 85 microM) showed more potent inhibitory effects than did alpha-linolenic acid, which is known as a naturally occurring potent inhibitor. Structure-activity investigations suggested that the presence of an isoprene substituent (prenyl and geranyl) would enhance 5 alpha-reductase inhibitory effects.

  11. An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a subfamily of aldo-keto reductases.

    PubMed

    Ellis, E M; Judah, D J; Neal, G E; Hayes, J D

    1993-11-01

    Protection of liver against the toxic and carcinogenic effects of aflatoxin B1 (AFB1) can be achieved through the induction of detoxification enzymes by chemoprotectors such as the phenolic antioxidant ethoxyquin. We have cloned and sequenced a cDNA encoding an aldehyde reductase (AFB1-AR), which is expressed in rat liver in response to dietary ethoxyquin. Expression of the cDNA in Escherichia coli and purification of the recombinant enzyme reveals that the protein exhibits aldehyde reductase activity and is capable of converting the protein-binding dialdehyde form of AFB1-dihydrodiol to the nonbinding dialcohol metabolite. We show that the mRNA encoding this enzyme is markedly elevated in the liver of rats fed an ethoxyquin-containing diet, correlating with acquisition of resistance to AFB1. AFB1-AR represents the only carcinogen-metabolizing aldehyde reductase identified to date that is induced by a chemoprotector. Alignment of the amino acid sequence of AFB1-AR with other known and putative aldehyde reductases shows that it defines a subfamily within the aldo-keto reductase superfamily. PMID:8234296

  12. Age-Specific Peculiarities of Modulation of Blood Aldo-Keto Reductase Isoenzyme Spectrum.

    PubMed

    Davydov, V V

    2015-12-01

    The aldo-keto reductase spectrum of the blood was studied at different stages of ontogeny to elucidate the role of reduction pathway in utilization of the carbonyl products of free radical oxidation in modulation of organism sensitivity to the damaging effect of stress during ontogeny. The studies revealed the age-specific changes in aldo-keto reductase spectrum in the blood. An analogy of the aldo-keto reductase spectrum structure in animals of early maturity and in old rats was found. The appearance of age specificity of the aldo-keto reductase spectrum in the blood creates metabolic prerequisites for changes in the efficiency of utilization of carbonyl products of free radical oxidation via their reductive transformation.

  13. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line

    SciTech Connect

    Kaufman, R.J.; Schimke, R.T.

    1981-12-01

    During stepwise increases in the methotrexate concentration in culture medium, the authors selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). The authors studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

  14. Participation of NADPH-cytochrome C reductase in thyroid hormone biosynthesis.

    PubMed

    Yamamoto, K; DeGroot, L J

    1975-04-01

    Purified rat liver NADPH-cytochrome c reductase supports iodination of tyrosine in a system including NADPH, cytochrome c and thyroid perioxidase. Catalase inhibits the iodination of tyrosine, while superoxide dismutase has no effect. Antibody developed in the rabbit against purified rat liver NADPH-cytochrome c reductase inhibits both reduction of cytochrome c and tyrosine iodination supported by the enzyme. The antibody forms a single precipitation line with thyroid extract, and inhibits NADPH cytochrome c reductase activity of the thyroid. The antibody partially inhibits iodination in a thyroid mitochondrial-microsomal fraction, but does not inhibit NADH-dependent iodination. The immunochemical studies indicate the participation of NADPH-cytochrome c reductase in thyroidal H2O generation, and the independent existence of NADPH-dependent and NADH-dependent H2O2 generation mechanisms in the thyroid. PMID:235416

  15. Localization and Regulation of Synthesis of Nitrate Reductase in Escherichia coli

    PubMed Central

    Showe, Michael K.; DeMoss, J. A.

    1968-01-01

    The nitrate reductase of Escherichia coli K-12 was localized in a particulate fraction of the cell and it sedimented as if it were bound to a large substructure that is subject to fragmentation during cell disruption procedures. Soluble enzyme, exhibiting a homogenous profile in sucrose gradients, was released from this fraction by an alkaline-heat treatment. Less than 1.5% of total active nitrate reductase apparently occurred in this soluble form during the course of formation of the particulate enzyme. Enzyme synthesis was repressed by aeration in the presence or absence of nitrate. Under anaerobic conditions, nitrate reductase was synthesized at a rate that could be increased 20-fold by the addition of nitrate. When enzyme synthesis was initiated by induction with nitrate or anaerobiosis, biphasic kinetics were obtained. We interpreted the results as evidence for the existence of a redox-sensitive repressor which mediates nitrate reductase regulation. PMID:4869216

  16. Effect of Polygonum hydropiper sulfated flavonoids on lens aldose reductase and related enzymes.

    PubMed

    Haraguchi, H; Ohmi, I; Sakai, S; Fukuda, A; Toihara, Y; Fujimoto, T; Okamura, N; Yagi, A

    1996-04-01

    The sulfated flavonoids in Polygonum hydropiper showed potent inhibiton against lens aldose reductase. Among these flavonoids isorhamnetin 3,7-disulfate (5) was most potent. Kinetic analysis showed that 5 exhibited noncompetitive inhibition against both dl-glyceraldehyde and NADPH.

  17. Synthesis, crystal structure and NLO property of a nonmetal pentaborate [C 6H 13N 2][B 5O 6(OH) 4

    NASA Astrophysics Data System (ADS)

    Liu, Huan-Xin; Liang, Yun-Xiao; Jiang, Xiao

    2008-12-01

    A nonmetal pentaborate [C 6H 13N 2][B 5O 6(OH) 4] ( 1) has been synthesized by 1,4-diazabicyclo[2.2.2] octane (DABCO) and boric acid, and characterized by single-crystal X-ray diffraction, FTIR, elemental analysis, and thermogravimetric analysis. Compound 1 crystallizes in the monoclinic system with space group Cc (no. 9), a=10.205(2) Å, b=14.143(3) Å, c=11.003(2) Å, β=113.97(3)°, V=1451.1(5) Å 3, Z=4. The anionic units, [B 5O 6(OH) 4] -, are interlinked via hydrogen bonding to form a three-dimensional (3D) supramolecular network containing large channels, in which the protonated [C 6H 13N 2] + cations are located. Second-harmonic generation (SHG) measurements on the powder samples reveal that 1 exhibits SHG efficiency approximately 0.9 times that of potassium dihydrogen phosphate (KDP).

  18. Identification of a cytochrome b5-fusion desaturase responsible for the synthesis of triunsaturated sphingolipid long chain bases in the marine diatom Thalassiosira pseudonana.

    PubMed

    Michaelson, Louise V; Markham, Jonathan E; Zäeuner, Simone; Matsumoto, Midori; Chen, Ming; Cahoon, Edgar B; Napier, Johnathan A

    2013-06-01

    Triunsaturated sphingolipid long chain bases (LCBs) have previously been reported in some specialised tissues of marine invertebrates. We report the presence of similar LCBs in the marine diatom Thalassiosira pseudonana and identify the cytochrome b5-fusion desaturase responsible for the introduction of the third double bond at the Δ10 position in d18:3Δ4,8,10. This study extends the catalytic repertoire of the cytochrome b5 fusion desaturase family, also indicating the presence of orthologues in other marine invertebrates. The function of these polyunsaturated sphingolipid LCBs is currently unknown but it was previously suggested that they play an essential role in primitive animals. The identification of the desaturase responsible for their synthesis paves the way for further studies.

  19. A human-human hybridoma producing cytotoxic antibody to HLA-B15, cross-reacting with B17, B5, B35 and B18.

    PubMed

    Hansen, T; Kolstad, A; Thorsby, E; Hannestad, K

    1987-05-01

    Mononuclear blood cells from a multiparous woman were transformed with Epstein Barr virus, and a cell line (Tr2D8) producing anti-HLA antibody was obtained. This cell line was immortalized by hybridization to the human fusion partners KR4 and KR12. While the EBV line died after 7 months, the hybridomas have remained stable for 13 months. The EBV line supernatant (40 micrograms IgM/ml) lysed peripheral blood mononuclear cells (PBMC) bearing B15, B17, B5 and B35. Consistent lysis of B18 bearing cells was only observed with lymphoblastoid cell lines. The supernatant from the Tr2D8 (EBV line X KR4) hybridoma (2.7 micrograms IgM/ml) only lysed B15 bearing PBMC. At a concentration of 13.5 micrograms IgM/ml, the hybridoma antibody lysed lymphoblastoid cell lines bearing B15, B17, B5, B35 and B18.

  20. Synthesis and crystal structure analysis of Li2NaBP2O8 and LiNa2B5P2O14

    NASA Astrophysics Data System (ADS)

    Hasegawa, Toru; Yamane, Hisanori

    2015-05-01

    Single crystals of Li2NaBP2O8 and LiNa2B5P2O14 were prepared at 873-883 K. The XRD reflections of Li2NaBP2O8 single crystal were indexed with triclinic unit-cell parameters of a=5.4344(3) Å, b=7.3793(4) Å, c=7.9840(4) Å, α=103.243(3)°, β=109.270(4)° and γ=87.391(2)°; (space group P 1 bar (No. 2)). Li2NaBP2O8 consists of one-dimensional 1∞[BP2O8]3- chains of BO4 and PO4 tetrahedra in the direction of the c axis, and Li and Na atoms located around the chains. The XRD reflections of the LiNa2B5P2O14 single crystal were indexed with monoclinic unit-cell parameters of a=8.208(3) Å, b=9.151(3) Å, c=8.349(3) Å and β=115.709(7)°; (space group P21/m (No. 11)). In the crystal structure of LiNa2B5P2O14, BO3 trigonal planer and BO4 and PO4 tetrahedra share O atoms and form two-dimensional sheets of 2∞[B5P2O14]3-. One Li and two Na atoms are situated at a large triangular space in the sheets.

  1. Fabrication and characterization of vitamin B5 loaded poly (l-lactide-co-caprolactone)/silk fiber aligned electrospun nanofibers for schwann cell proliferation.

    PubMed

    Bhutto, M Aqeel; Wu, Tong; Sun, Binbin; Ei-Hamshary, Hany; Al-Deyab, Salem S; Mo, Xiumei

    2016-08-01

    Bioengineering strategies for peripheral nerve regeneration have been focusing on the development of alternative treatments for nerve repair. In present study we have blended the Vitamin B5 (50mg) with 8% P(LLA-CL) and P(LLA-CL)/SF solutions and produced aligned electrospun nanofiber mashes and characterized the material for its physiochemical and mechanical characteristics. The vitamin loaded composites nanofibers showed tensile strength of 8.73±1.38 and 8.4±1.37 in P(LLA-CL)/Vt and P(LLA-CL)/SF/Vt nanofibers mashes, respectively. By the addition of vitamin B5 the P(LLA-CL) nanofibers become hydrophilic and the contact angle decreased from 96° to 0° in 6min of duration. The effect of vitamin B5 on Schwann cells proliferation and viability were analyzed by using MTT assay and the number of cells cultured on vitamin loaded nanofiber mashes was significantly higher than the without vitamin loaded nanofiber samples after 5th day (p<0.05) whereas, P (LLA-CL)/SF/Vt exhibit the consistently highest cell numbers after 7th days culture as compare to P (LLA-CL)/Vt. The in vitro vitamin release behavior was observed in PBS solution and released vitamin was calculated by revers phase HPLC method. The sustain release behavior of vitamin B5 were noted higher in P(LLA-CL)/Vt (80%) nanofibers as compared to P (LLA-CL)/SF/Vt (62%) nanofibers after 24h. The present work provided a basis for further studies of this novel aligned nanofibrous material in nerve tissue repair or regeneration.

  2. Fabrication and characterization of vitamin B5 loaded poly (l-lactide-co-caprolactone)/silk fiber aligned electrospun nanofibers for schwann cell proliferation.

    PubMed

    Bhutto, M Aqeel; Wu, Tong; Sun, Binbin; Ei-Hamshary, Hany; Al-Deyab, Salem S; Mo, Xiumei

    2016-08-01

    Bioengineering strategies for peripheral nerve regeneration have been focusing on the development of alternative treatments for nerve repair. In present study we have blended the Vitamin B5 (50mg) with 8% P(LLA-CL) and P(LLA-CL)/SF solutions and produced aligned electrospun nanofiber mashes and characterized the material for its physiochemical and mechanical characteristics. The vitamin loaded composites nanofibers showed tensile strength of 8.73±1.38 and 8.4±1.37 in P(LLA-CL)/Vt and P(LLA-CL)/SF/Vt nanofibers mashes, respectively. By the addition of vitamin B5 the P(LLA-CL) nanofibers become hydrophilic and the contact angle decreased from 96° to 0° in 6min of duration. The effect of vitamin B5 on Schwann cells proliferation and viability were analyzed by using MTT assay and the number of cells cultured on vitamin loaded nanofiber mashes was significantly higher than the without vitamin loaded nanofiber samples after 5th day (p<0.05) whereas, P (LLA-CL)/SF/Vt exhibit the consistently highest cell numbers after 7th days culture as compare to P (LLA-CL)/Vt. The in vitro vitamin release behavior was observed in PBS solution and released vitamin was calculated by revers phase HPLC method. The sustain release behavior of vitamin B5 were noted higher in P(LLA-CL)/Vt (80%) nanofibers as compared to P (LLA-CL)/SF/Vt (62%) nanofibers after 24h. The present work provided a basis for further studies of this novel aligned nanofibrous material in nerve tissue repair or regeneration. PMID:27085042

  3. Experimental investigation of the effect of the material damage induced in sheet metal forming process on the service performance of 22MnB5 steel

    NASA Astrophysics Data System (ADS)

    Zhuang, Weimin; Xie, Dongxuan; Chen, Yanhong

    2016-06-01

    The use of ultra-high strength steels through sheet metal forming process offers a practical solution to the lightweight design of vehicles. However, sheet metal forming process not only produces desirable changes in material properties but also causes material damage that may adversely influence the service performance of the material formed. Thus, an investigation is conducted to experimentally quantify such influence for a commonly used steel (the 22MnB5 steel) based on the hot and cold forming processes. For each process, a number of samples are used to conduct a uniaxial tensile test to simulate the forming process. After that, some of the samples are trimmed into a standard shape and then uniaxially extended until fracture to simulate the service stage. Finally, a microstructure test is conducted to analyze the microdefects of the remaining samples. Based on the results of the first two tests, the effect of material damage on the service performance of 22MnB5 steel is analyzed. It is found that the material damages of both the hot and cold forming processes cause reductions in the service performance, such as the failure strain, the ultimate stress, the capacity of energy absorption and the ratio of residual strain. The reductions are generally lower and non-linear in the former process but higher and linear in the latter process. Additionally, it is found from the microstructure analysis that the difference in the reductions of the service performance of 22MnB5 by the two forming processes is driven by the difference in the micro damage mechanisms of the two processes. The findings of this research provide a useful reference in terms of the selection of sheet metal forming processes and the determination of forming parameters for 22MnB5.

  4. Membrane-Anchored Cytochrome P450 1A2-Cytochrome b5 Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices.

    PubMed

    Jeřábek, Petr; Florián, Jan; Martínek, Václav

    2016-04-18

    Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein-protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2-cyt b5 complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2-cyt b5 complexes, these simulations yielded three stable binding modes (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mAI is the best candidate for the catalytically relevant full-length complex. PMID:26918755

  5. Experimental investigation of the effect of the material damage induced in sheet metal forming process on the service performance of 22MnB5 steel

    NASA Astrophysics Data System (ADS)

    Zhuang, Weimin; Xie, Dongxuan; Chen, Yanhong

    2016-07-01

    The use of ultra-high strength steels through sheet metal forming process offers a practical solution to the lightweight design of vehicles. However, sheet metal forming process not only produces desirable changes in material properties but also causes material damage that may adversely influence the service performance of the material formed. Thus, an investigation is conducted to experimentally quantify such influence for a commonly used steel (the 22MnB5 steel) based on the hot and cold forming processes. For each process, a number of samples are used to conduct a uniaxial tensile test to simulate the forming process. After that, some of the samples are trimmed into a standard shape and then uniaxially extended until fracture to simulate the service stage. Finally, a microstructure test is conducted to analyze the microdefects of the remaining samples. Based on the results of the first two tests, the effect of material damage on the service performance of 22MnB5 steel is analyzed. It is found that the material damages of both the hot and cold forming processes cause reductions in the service performance, such as the failure strain, the ultimate stress, the capacity of energy absorption and the ratio of residual strain. The reductions are generally lower and non-linear in the former process but higher and linear in the latter process. Additionally, it is found from the microstructure analysis that the difference in the reductions of the service performance of 22MnB5 by the two forming processes is driven by the difference in the micro damage mechanisms of the two processes. The findings of this research provide a useful reference in terms of the selection of sheet metal forming processes and the determination of forming parameters for 22MnB5.

  6. Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars1[W][OA

    PubMed Central

    Fan, Ren-Chun; Peng, Chang-Cao; Xu, Yan-Hong; Wang, Xiao-Fang; Li, Yan; Shang, Yi; Du, Shu-Yuan; Zhao, Rui; Zhang, Xiao-Yan; Zhang, Ling-Yun; Zhang, Da-Peng

    2009-01-01

    Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability. PMID:19502355

  7. Studies on WF-3681, a novel aldose reductase inhibitor. I. Taxonomy, fermentation, isolation and characterization.

    PubMed

    Nishikawa, M; Tsurumi, Y; Namiki, T; Yoshida, K; Okuhara, M

    1987-10-01

    WF-3681 was isolated from a cultured filtrate of Chaetomella raphigera as a novel inhibitor of aldose reductase. It was extracted with ethyl acetate and then purified with silica gel chromatography. Its molecular formula was determined to be C13H12O5 by elemental analysis and high resolution electron impact mass spectrometry. IC50 of WF-3681 was 2.5 X 10(-7) M for partially purified aldose reductase of rabbit lens. PMID:3119547

  8. Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6'-deoxychalcone.

    PubMed

    Welle, R; Grisebach, H

    1989-07-01

    Chromatofocusing on Mono P proved to be an efficient purification procedure for the NADPH-dependent reductase from soybean (Glycine max L.) cell cultures which acts together with chalcone synthase in the biosynthesis of 2',4',4-trihydroxychalcone (6'-deoxychalcone). By isoelectric focusing the pI of reductase was determined to be 6.3. Addition of pure soybean reductase to cell-free extracts from stimulated cell cultures of parsley and bean (Phaseolus vulgaris) and from young flowers of Dahlia variabilis caused in each case synthesis of 6'-deoxychalcone. When 4-coumaroyl-CoA was replaced by caffeoyl-CoA in the reductase assay, formation of 2',4',3,4-tetrahydrochalcone (butein) was observed. A polyclonal antireductase antiserum was raised in rabbits and proved to be specific in Ouchterlony diffusion experiments, Western blots and immunotitration. The reductase antiserum showed no cross-reactivity with soybean chalcone synthase (CHS). A biotin/[125I]streptavidin system provided a quantitative Western blot for the reductase. Changes in the activities, amounts of protein, and mRNA activities of reductase and CHS were determined after challenge of soybean cell cultures by elicitor (from Phytophthora megasperma f.sp. glycinea or yeast). For both enzymes a pronounced and parallel increase in activity and amounts of protein was observed after elicitor addition with a maximum at about 16 h after challenge. Parallel increases in mRNA activities occurred earlier. The results indicate a parallel induction of de novo synthesis of reductase and CHS which coact in synthesis of 6'-deoxychalcone. PMID:2500065

  9. Isolation of Assimilatory- and Dissimilatory-Type Sulfite Reductases from Desulfovibrio vulgaris

    PubMed Central

    Lee, Jin-Po; LeGall, Jean; Peck, Harry D.

    1973-01-01

    Bisulfite reductase (desulfoviridin) and an assimilatory sulfite reductase have been purified from extracts of Desulfovibrio vulgaris. The bisulfite reductase has absorption maxima at 628, 580, 408, 390, and 279 nm, and a molecular weight of 226,000 by sedimentation equilibrium, and was judged to be free of other proteins by disk electrophoresis and ultracentrifugation. On gels, purified bisulfite reductase exhibited two green bands which coincided with activity and protein. The enzyme appears to be a tetramer but was shown to have two different types of subunits having molecular weights of 42,000 and 50,000. The chromophore did not form an alkaline ferrohemochromogen, was not reduced with dithionite or borohydride, and did not form a spectrally visible complex with CO. The assimilatory sulfite reductase has absorption maxima at 590, 545, 405 and 275 nm and a molecular weight of 26,800, and appears to consist of a single polypeptide chain as it is not dissociated into subunits by sodium dodecyl sulfate. By disk electrophoresis, purified sulfite reductase exhibited a single greenish-brown band which coincided with activity and protein. The sole product of the reduction was sulfide, and the chromophore was reduced by borohydride in the presence of sulfite. Carbon monoxide reacted with the reduced chromophore but it did not form a typical pyridine ferrohemochromogen. Thiosulfate, trithionate, and tetrathionate were not reduced by either enzyme preparation. In the presence of 8 M urea, the spectrum of bisulfite reductase resembles that of the sulfite reductase, thus suggesting a chemical relationship between the two chromophores. Images PMID:4725615

  10. Characterization of Streptococcus pneumoniae enoyl-(acyl-carrier protein) reductase (FabK).

    PubMed

    Marrakchi, Hedia; Dewolf, Walter E; Quinn, Chad; West, Joshua; Polizzi, Brian J; So, Chi Y; Holmes, David J; Reed, Shannon L; Heath, Richard J; Payne, David J; Rock, Charles O; Wallis, Nicola G

    2003-03-15

    The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to the Bacillus / Lactobacillus / Streptococcus group that includes Staphylococcus aureus and Bacillus subtilis. These two organisms also contain FabK-related genes, suggesting that they may also express a FabK-like enoyl-ACP reductase. However, the genes did not complement a fabI (Ts) mutant and the purified flavoproteins were unable to reduce enoyl-ACP in vitro and did not exhibit NAD(P)H oxidase activity, indicating they were not enoyl-ACP reductases. The restricted occurrence of the FabK enoyl-ACP reductase may be related to the role of substrate-independent NADH oxidation in oxygen-dependent anaerobic energy metabolism.

  11. Measurement of nitrite reductase in leaf tissue of Vigna mungo : A new method.

    PubMed

    Srivastava, R C; Bose, B; Mukerji, D; Mathur, S N; Srivastava, H S

    1979-12-01

    The enzyme nitrite reductase (EC 1.6.6.4) is generally assayed in terms of disappearance of nitrite from the assay medium. We describe a technique which allowed estimation of the enzyme level in leaf tissues of Vigna mungo (L). Hepper in terms of the release of the product (NH3) of the enzyme reaction. The technique is offered as an alternative, possibly more convenient method for assay of nitrite reductase in plant tissue in vivo.

  12. Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi.

    PubMed

    Christmas, P B; Turrens, J F

    2000-02-15

    A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.

  13. Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.

    PubMed Central

    Cramm, R; Siddiqui, R A; Friedrich, B

    1997-01-01

    Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification. PMID:9352929

  14. Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity

    PubMed Central

    2012-01-01

    Background Increasing global demand and reliance on petroleum-derived chemicals will necessitate alternative sources for chemical feedstocks. Currently, 99% of chemical feedstocks are derived from petroleum and natural gas. Renewable methods for producing important chemical feedstocks largely remain unaddressed. Synthetic biology enables the renewable production of various chemicals from microorganisms by constructing unique metabolic pathways. Here, we engineer Escherichia coli for the production of isobutyraldehyde, which can be readily converted to various hydrocarbons currently derived from petroleum such as isobutyric acid, acetal, oxime and imine using existing chemical catalysis. Isobutyraldehyde can be readily stripped from cultures during production, which reduces toxic effects of isobutyraldehyde. Results We adopted the isobutanol pathway previously constructed in E. coli, neglecting the last step in the pathway where isobutyraldehyde is converted to isobutanol. However, this strain still overwhelmingly produced isobutanol (1.5 g/L/OD600 (isobutanol) vs 0.14 g/L/OD600 (isobutyraldehyde)). Next, we deleted yqhD which encodes a broad-substrate range aldehyde reductase known to be active toward isobutyraldehyde. This strain produced isobutanol and isobutyraldehyde at a near 1:1 ratio, indicating further native isobutyraldehyde reductase (IBR) activity in E. coli. To further eliminate isobutanol formation, we set out to identify and remove the remaining IBRs from the E. coli genome. We identified 7 annotated genes coding for IBRs that could be active toward isobutyraldehyde: adhP, eutG, yiaY, yjgB, betA, fucO, eutE. Individual deletions of the genes yielded only marginal improvements. Therefore, we sequentially deleted all seven of the genes and assessed production. The combined deletions greatly increased isobutyraldehyde production (1.5 g/L/OD600) and decreased isobutanol production (0.4 g/L/OD600). By assessing production by overexpression of each

  15. No evidence for a role of Ile587Val polymorphism of EIF2B5 gene in multiple sclerosis in Kashmir Valley of India.

    PubMed

    Zahoor, Insha; Asimi, Ravouf; Haq, Ehtishamul

    2015-12-15

    Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the nervous system with a profound genetic element. It is already known that alterations in Eukaryotic Translation Initiation Factor 2B (EIF2B) gene encoding the five subunits of eIF2B complex cause Vanishing White Matter (VWM) disease of the brain and emerging evidences have advocated certain resemblances between MS and VWM in terms of clinical and epidemiological characteristics, thus validating the association study between EIF2B and MS. Moreover, a recent study has implicated EIF2B5 Ile587Val (rs843358) polymorphism as a susceptibility factor for MS. In order to investigate the association of EIF2B5 Ile587Val polymorphism with MS susceptibility in Kashmir region in India, we screened EIF2B5 Exon 13 in 30 MS patients and 65 controls (a total of 95 participants). During the present course of study, we could not find statistically significant difference in the frequency of Ile587Val between MS patients and controls, thus indicating that such alteration does not appear to influence MS development in Kashmiri population. Our results provide evidence against a major role for Ile587Val polymorphism in MS susceptibility.

  16. Synthesis, characterization and theoretical studies of nonlinear optical crystal Sr2B5O9(OH)·H2O.

    PubMed

    Zhang, Fangyuan; Zhang, Fangfang; Qun, Jing; Pan, Shilie; Yang, Zhihua; Jia, Dianzeng

    2015-04-28

    Strontium borate Sr2B5O9(OH)·H2O (space group C2, No. 5) has been synthesized in high yields using a facile hydrothermal method. The UV-Vis-NIR diffuse reflectance spectrum shows that it has a wide transparency range extending from UV to NIR with the short-wavelength cut off edge below 190 nm. Second-harmonic generation (SHG) has been measured with a 1064 nm laser using the Kurtz and Perry technique, which shows that Sr2B5O9(OH)·H2O is phase matchable and the powder SHG effect is approximately 3 times that of KDP. It also has a high thermal stability up to 500 °C which has been identified by TG, DSC and variable-temperature PXRD. These properties make it possible for application as a UV nonlinear optical (NLO) material. Based on the electronic band structure, the optical refractive indices, birefringence, and SHG coefficients of Sr2B5O9(OH)·H2O are calculated, which are consistent with experiments. In addition, the electronic structure, SHG-weighted electron density and real-space atom-cutting analyses are performed to elucidate the origin of its NLO properties. PMID:25803617

  17. Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

    PubMed

    Sayanova, O; Shewry, P R; Napier, J A

    1999-10-01

    Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

  18. Histidine-41 of the Cytochrome b5 Domain of the Borage Δ6 Fatty Acid Desaturase Is Essential for Enzyme Activity1

    PubMed Central

    Sayanova, Olga; Shewry, Peter R.; Napier, Johnathan A.

    1999-01-01

    Unlike most other plant microsomal desaturases, the Δ6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Δ6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Δ6-fatty acid desaturase resulted in the synthesis and accumulation of Δ6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Δ6-desaturase in transgenic plants failed to produce Δ6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Δ6-fatty acid desaturase is essential for enzymatic activity. PMID:10517856

  19. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    PubMed Central

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  20. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network.

    PubMed

    Couto, Narciso; Wood, Jennifer; Barber, Jill

    2016-06-01

    In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders.

  1. Smith-Lemli-Opitz syndrome is caused by mutations in the 7-dehydrocholesterol reductase gene.

    PubMed Central

    Waterham, H R; Wijburg, F A; Hennekam, R C; Vreken, P; Poll-The, B T; Dorland, L; Duran, M; Jira, P E; Smeitink, J A; Wevers, R A; Wanders, R J

    1998-01-01

    Smith-Lemli-Opitz syndrome is a frequently occurring autosomal recessive developmental disorder characterized by facial dysmorphisms, mental retardation, and multiple congenital anomalies. Biochemically, the disorder is caused by deficient activity of 7-dehydrocholesterol reductase, which catalyzes the final step in the cholesterol-biosynthesis pathway-that is, the reduction of the Delta7 double bond of 7-dehydrocholesterol to produce cholesterol. We identified a partial transcript coding for human 7-dehydrocholesterol reductase by searching the database of expressed sequence tags with the amino acid sequence for the Arabidopsis thaliana sterol Delta7-reductase and isolated the remaining 5' sequence by the "rapid amplification of cDNA ends" method, or 5'-RACE. The cDNA has an open reading frame of 1,425 bp coding for a polypeptide of 475 amino acids with a calculated molecular weight of 54.5 kD. Heterologous expression of the cDNA in the yeast Saccharomyces cerevisiae confirmed that it codes for 7-dehydrocholesterol reductase. Chromosomal mapping experiments localized the gene to chromosome 11q13. Sequence analysis of fibroblast 7-dehydrocholesterol reductase cDNA from three patients with Smith-Lemli-Opitz syndrome revealed distinct mutations, including a 134-bp insertion and three different point mutations, each of which was heterozygous in cDNA from the respective parents. Our data demonstrate that Smith-Lemli-Opitz syndrome is caused by mutations in the gene coding for 7-dehydrocholesterol reductase. PMID:9683613

  2. Relative adrenal insufficiency in mice deficient in 5α-reductase 1

    PubMed Central

    Livingstone, Dawn E W; Di Rollo, Emma M; Yang, Chenjing; Codrington, Lucy E; Mathews, John A; Kara, Madina; Hughes, Katherine A; Kenyon, Christopher J; Walker, Brian R; Andrew, Ruth

    2014-01-01

    Patients with critical illness or hepatic failure exhibit impaired cortisol responses to ACTH, a phenomenon known as ‘relative adrenal insufficiency’. A putative mechanism is that elevated bile acids inhibit inactivation of cortisol in liver by 5α-reductases type 1 and type 2 and 5β-reductase, resulting in compensatory downregulation of the hypothalamic–pituitary–adrenal axis and adrenocortical atrophy. To test the hypothesis that impaired glucocorticoid clearance can cause relative adrenal insufficiency, we investigated the consequences of 5α-reductase type 1 deficiency in mice. In adrenalectomised male mice with targeted disruption of 5α-reductase type 1, clearance of corticosterone was lower after acute or chronic (eightfold, P<0.05) administration, compared with WT control mice. In intact 5α-reductase-deficient male mice, although resting plasma corticosterone levels were maintained, corticosterone responses were impaired after ACTH administration (26% lower, P<0.05), handling stress (2.5-fold lower, P<0.05) and restraint stress (43% lower, P<0.05) compared with WT mice. mRNA levels of Nr3c1 (glucocorticoid receptor), Crh and Avp in pituitary or hypothalamus were altered, consistent with enhanced negative feedback. These findings confirm that impaired peripheral clearance of glucocorticoids can cause ‘relative adrenal insufficiency’ in mice, an observation with important implications for patients with critical illness or hepatic failure, and for patients receiving 5α-reductase inhibitors for prostatic disease. PMID:24872577

  3. Contribution of reductase activity to quinone toxicity in three kinds of hepatic cells.

    PubMed

    Ishihara, Yasuhiro; Tsuji, Kaori; Ishii, Satomi; Kashiwagi, Kyoko; Shimamoto, Norio

    2012-01-01

    Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle, and the arylation of intracellular nucleophiles. The redox cycle is catalyzed by intracellular reductases, and therefore the toxicity of redox cycling quinone is considered to be closely associated with the reductase activity. This study examined the relationship between quinone toxicity and the intracellular reductase activity using 3 kinds of hepatic cells; rat primary hepatocytes, HepG2 and H4IIE. The intracellular reductase activity was; primary hepatocyte >HepG2>H4IIE. The three kinds of cells showed almost the same vulnerability to an arylating quinone, 1,4-naphthoquinone (NQ). However, the susceptibility to a redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was; primary hepatocyte>HepG2>H4IIE. In addition, the cytotoxicity elicited by DMNQ was significantly attenuated in HepG2 cells and almost completely suppressed in primary hepatocytes by diphenyleneiodonium chloride, a reductase inhibitor. These data suggest that cells with a high reductase activity are susceptible to redox cycling quinones. This study provides essential evidence to assess the toxicity of quinone-based drugs during their developmental processes.

  4. X-ray structure of trypanothione reductase from Crithidia fasciculata at 2. 4- angstrom resolution

    SciTech Connect

    Kuriyan, J.; Xiangpeng Kong; Krishna, T.S.R.; Murgolo, N.J.; Field, H.; Cerami, A.; Henderson, G.B. ); Sweet, R.M. )

    1991-10-01

    Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidien conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. The authors report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from {minus}2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4{degree} in the domains that form in site, with relative shifts of as much as 2-3 {angstrom} in residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

  5. Purification and characterization of (+)dihydroflavonol (3-hydroxyflavanone) 4-reductase from flowers of Dahlia variabilis.

    PubMed

    Fischer, D; Stich, K; Britsch, L; Grisebach, H

    1988-07-01

    Individual flowers from inflorescences of Dahlia variabilis (cv Scarlet Star) in young developmental stages contained relatively high activity of (+)-dihydroflavonol (DHF) 4-reductase. The DHF reductase was purified from such flowers to apparent homogeneity by a five-step procedure. This included affinity adsorption on Blue Sepharose and elution of the enzyme with NADP+. By gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis it was shown that DHF reductase contains only one polypeptide chain with a Mr of about 41,000. The reductase required NADPH as cofactor and catalyzed transfer of the pro-S hydrogen of NADPH to the substrate. Flavanones and dihydroflavonols (3-hydroxyflavanones) were substrates for DHF reductase with pH optima of about 6.0 for flavanones and of about 6.8 for dihydroflavonols. Flavanones were reduced to the corresponding flavan-4-ols and (+)-dihydroflavonols to flavan-3,4-cis-diols. Apparent Michaelis constants determined for (2S)-naringenin, (2S)-eriodicytol, (+)-dihydrokaempferol, (+)-dihydroquercetin, and NADPH were, respectively, 2.3, 2, 10, 15, and 42 microM. V/Km values were higher for dihydroflavonols than for flavanones. Conversion of dihydromyricetin to leucodelphinidin was also catalyzed by the enzyme at a low rate, whereas flavones and flavonols were not accepted as substrates. DHF reductase was not inhibited by metal chelators. PMID:3293532

  6. "Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease.

    PubMed Central

    Henderson, G B; Ulrich, P; Fairlamb, A H; Rosenberg, I; Pereira, M; Sela, M; Cerami, A

    1988-01-01

    The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase. PMID:3135548

  7. Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.

    PubMed Central

    Menting, J G; Cornish, E; Scopes, R K

    1994-01-01

    NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4). PMID:7991686

  8. A novel L-xylulose reductase essential for L-arabinose catabolism in Trichoderma reesei.

    PubMed

    Metz, Benjamin; Mojzita, Dominik; Herold, Silvia; Kubicek, Christian P; Richard, Peter; Seiboth, Bernhard

    2013-04-01

    L-Xylulose reductases belong to the superfamily of short chain dehydrogenases and reductases (SDRs) and catalyze the NAD(P)H-dependent reduction of L-xylulose to xylitol in L-arabinose and glucuronic acid catabolism. Here we report the identification of a novel L-xylulose reductase LXR3 in the fungus Trichoderma reesei by a bioinformatic approach in combination with a functional analysis. LXR3, a 31 kDa protein, catalyzes the reduction of L-xylulose to xylitol via NADPH and is also able to convert D-xylulose, D-ribulose, L-sorbose, and D-fructose to their corresponding polyols. Transcription of lxr3 is specifically induced by L-arabinose and L-arabitol. Deletion of lxr3 affects growth on L-arabinose and L-arabitol and reduces total NADPH-dependent LXR activity in cell free extracts. A phylogenetic analysis of known L-xylulose reductases shows that LXR3 is phylogenetically different from the Aspergillus niger L-xylulose reductase LxrA and, moreover, that all identified true L-xylulose reductases belong to different clades within the superfamily of SDRs. This indicates that the enzymes responsible for the reduction of L-xylulose in L-arabinose and glucuronic acid catabolic pathways have evolved independently and that even the fungal LXRs of the L-arabinose catabolic pathway have evolved in different clades of the superfamily of SDRs.

  9. Physiological roles for two periplasmic nitrate reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025).

    PubMed

    Hartsock, Angela; Shapleigh, James P

    2011-12-01

    The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth. PMID:21949073

  10. Molecular cloning, expression and catalytic activity of a human AKR7 member of the aldo-keto reductase superfamily: evidence that the major 2-carboxybenzaldehyde reductase from human liver is a homologue of rat aflatoxin B1-aldehyde reductase.

    PubMed Central

    Ireland, L S; Harrison, D J; Neal, G E; Hayes, J D

    1998-01-01

    The masking of charged amino or carboxy groups by N-phthalidylation and O-phthalidylation has been used to improve the absorption of many drugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxybenzaldehyde (2-CBA) and the pharmaceutically active compound; in humans, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by reduction of the aldehyde group. In the present work, the enzyme responsible for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B1-aldehyde reductase (rAFAR). This novel human aldo-keto reductase (AKR) has been cloned from a liver cDNA library, and together with the rat protein, establishes the AKR7 family of the AKR superfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in extrahepatic tissues. The deduced human and rat protein sequences share 78% identity and 87% similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which distinguish them from the prototypic AKR1 family of AKRs, the catalytic- and NADPH-binding residues appear to be conserved in both families. Certain of the predicted structural features of the AKR7 family members are shared with the AKR6 beta-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B1-8,9-dihydrodiol, hAFAR shows high affinity for the gamma-aminobutyric acid metabolite succinic semialdehyde (SSA) which is structurally related to 2-CBA, suggesting that hAFAR could function as both a SSA reductase and a 2-CBA reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA reductase activity in human liver co-purifies with hAFAR protein. PMID:9576847

  11. Regulation of cytochrome b5 gene transcription by Sp3, GATA-6, and steroidogenic factor 1 in human adrenal NCI-H295A cells.

    PubMed

    Huang, Ningwu; Dardis, Andrea; Miller, Walter L

    2005-08-01

    Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances. PMID:15831526

  12. Constitutive nitrate reductase expression and inhibition in winged bean

    SciTech Connect

    Wu, Shenchuan; Harper, J.E. )

    1990-05-01

    It was found that NO{sub 3}{sup {minus}} had no effect on winged bean nitrate reductase activity (NRA). Similar NRA was expressed in plants grown on NO{sub 3}{sup {minus}}, urea, NH{sub 4}{sup +}, and nil N. This indicated that the primary NR expressed in winged bean was constitutive, rather than substrate-inducible. Maximum NRA in winged bean was obtained in the light. KClO{sub 3} was capable of inhibiting NRA of leaves if added to the root growth medium or to the NR assay medium, indicating possible competition with NO{sub 3}{sup {minus}} at the reduction site. While it has previously been shown that either cycloheximide alone, or both cycloheximide and chloramphenicol impair the synthesis of NR protein, our data unexpectedly demonstrated that cycloheximide had little effect on NRA, whereas chloramphenicol greatly inhibited the expression of NRA in winged bean. One interpretation is that chloroplasts may influence the activity and/or synthesis of constitutive NR proteins.

  13. Optical observation of correlated motions in dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2015-03-01

    Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

  14. Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase.

    PubMed

    Smutná, Tamara; Gonçalves, Vera L; Saraiva, Lígia M; Tachezy, Jan; Teixeira, Miguel; Hrdy, Ivan

    2009-01-01

    Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen. PMID:19011120

  15. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  16. Structural Basis for Activation of Class Ib Ribonucleotide Reductase

    SciTech Connect

    Boal, Amie K.; Cotruvo, Jr., Joseph A.; Stubbe, JoAnne; Rosenzweig, Amy C.

    2010-12-03

    The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn{sub 2}{sup III}-tyrosyl radical (Y{sm_bullet}) or a Fe{sub 2}{sup III}-Y{sm_bullet} cofactor in the NrdF subunit. Whereas Fe{sub 2}{sup III}-Y{sm_bullet} can self-assemble from Fe{sub 2}{sup II}-NrdF and O{sub 2}, activation of Mn{sub 2}{sup II}-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O{sub 2}. The crystal structures reported here of E. coli Mn{sub 2}{sup II}-NrdF and Fe{sub 2}{sup II}-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of Mn{sub 2}{sup II}-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF Mn{sub 2}{sup II} active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of Mn{sub 2}{sup III}-Y{sm_bullet} cofactor assembly.

  17. Loop interactions during catalysis by dihydrofolate reductase from Moritella profunda.

    PubMed

    Behiry, Enas M; Evans, Rhiannon M; Guo, Jiannan; Loveridge, E Joel; Allemann, Rudolf K

    2014-07-29

    Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the well-studied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR. PMID:25014120

  18. Chromate reductase activity in Streptomyces sp. MC1.

    PubMed

    Polti, Marta A; Amoroso, María J; Abate, Carlos M

    2010-02-01

    Biological transformation of Cr(VI) to Cr(III) by enzymatic reduction may provide a less costly and more environmentally friendly approach to remediation. In a previous report a Cr(VI) resistant actinomycete strain, Streptomyces sp. MC1, was able to reduce Cr(VI) present in a synthetic medium, soil extract and soil samples. This is the first time optimal conditions such as pH, temperature, growth phase and electron donor have been elucidated in vitro for Cr(VI) reduction by a streptomycete. Chromate reductase of Streptomyces sp. MC1 is a constitutive enzyme which was mainly associated with biomass and required NAD(P)H as an electron donor. It was active over a broad temperature (19-39 degrees C) and pH (5-8) range, and optimum conditions were 30 degrees C and pH 7. The enzyme was present in supernatant, pellet and cell free extract. Bioremediation with the enzyme was observed in non-compatible cell reproduction systems, conditions frequently found in contaminated environments. PMID:20339215

  19. Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

    PubMed Central

    Prunotto, Marco; Carnevali, Maria Luisa; Candiano, Giovanni; Murtas, Corrado; Bruschi, Maurizio; Corradini, Emilia; Trivelli, Antonella; Magnasco, Alberto; Petretto, Andrea; Santucci, Laura; Mattei, Silvia; Gatti, Rita; Scolari, Francesco; Kador, Peter; Allegri, Landino

    2010-01-01

    Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti–aldose reductase (AR) and anti–manganese superoxide dismutase (SOD2) IgG4 in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG4 from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG4 and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression. PMID:20150532

  20. Inhibition of Aldose Reductase by Gentiana lutea Extracts

    PubMed Central

    Akileshwari, Chandrasekhar; Muthenna, Puppala; Nastasijević, Branislav; Joksić, Gordana; Petrash, J. Mark; Reddy, Geereddy Bhanuprakash

    2012-01-01

    Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications. PMID:22844269

  1. Inhibition of aldose reductase by Gentiana lutea extracts.

    PubMed

    Akileshwari, Chandrasekhar; Muthenna, Puppala; Nastasijević, Branislav; Joksić, Gordana; Petrash, J Mark; Reddy, Geereddy Bhanuprakash

    2012-01-01

    Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications.

  2. Intermediate hyperhomocysteinemia resulting from compound heterozygosity of methylenetetrahydrofolate reductase mutations.

    PubMed Central

    Kang, S S; Wong, P W; Bock, H G; Horwitz, A; Grix, A

    1991-01-01

    Four subjects with thermolabile methylenetetrahydrofolate reductase (MTHFR) were discovered among 16 "obligate" heterozygotes for severe MTHFR deficiency and their family members. All four subjects had less than 25% of normal mean MTHFR specific activity in lymphocyte extracts. Three of them with normal serum folate and cyanocobalamin had intermediate hyperhomocysteinemia, and one with high serum folate and cyanocobalamin had no excessive accumulation of serum homocysteine. The biochemical features in these four subjects are distinguishable from subjects homozygous for the thermolabile MTHFR, whose specific activity is approximately 50% of the normal mean, and from heterozygotes for severe MTHFR deficiency, in whom the enzyme is thermostable and has a specific activity of about 50% of the normal mean. We propose that these four subjects are genetic compounds of the allele for the severe mutation and the allele for thermolabile mutation of the MTHFR gene. It is postulated that subjects with this genetic compound are more susceptible to the development of intermediate hyperhomocysteinemia despite normal folate and B12 levels. Nonetheless, hyperhomocysteinemia due to this compound heterozygosity is correctable by oral folic acid therapy. PMID:1998340

  3. Correlated Protein Motion Measurements of Dihydrofolate Reductase Crystals

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2014-03-01

    We report the first direct measurements of the long range structural vibrational modes in dihydrofolate reductase (DHFR). DHFR is a universal housekeeping enzyme that catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetra-hydrofolate, with the aid of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). This crucial enzymatic role as the target for anti-cancer [methotrexate (MTX)], and other clinically useful drugs, has made DHFR a long-standing target of enzymological studies. The terahertz (THz) frequency range (5-100 cm-1), corresponds to global correlated protein motions. In our lab we have developed Crystal Anisotropy Terahertz Microscopy (CATM), which directly measures these large scale intra-molecular protein vibrations, by removing the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for mouse DHFR with the ligand binding of NADPH and MTX single crystals as well as Escherichia coli DHFR with the ligand binding of NADPH and MTX single crystals. This work is supported by NSF grant MRI2 grant DBI2959989.

  4. Ribonucleotide reductases: divergent evolution of an ancient enzyme.

    PubMed

    Torrents, Eduard; Aloy, Patrick; Gibert, Isidre; Rodríguez-Trelles, Francisco

    2002-08-01

    Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron-sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family. PMID:12107591

  5. A second target of benzamide riboside: dihydrofolate reductase.

    PubMed

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth. PMID:22954684

  6. Stimulation of dihydrofolate reductase promoter activity by antimetabolic drugs.

    PubMed Central

    Eastman, H B; Swick, A G; Schmitt, M C; Azizkhan, J C

    1991-01-01

    Dihydrofolate reductase (DHFR; EC 1.5.1.3) is required in folate metabolism for the synthesis of purines, thymidine, and glycine. Although there have been several reports of induction of DHFR enzyme by methotrexate (MTX), a drug that competitively inhibits DHFR, there are no studies reported that examine the effect of MTX on DHFR gene transcription. We have examined the effect of MTX and other inhibitors of DNA synthesis on DHFR transcription using a transient expression assay. MTX stimulates transient expression in a concentration-dependent manner from a hamster DHFR promoter construct containing 150 base pairs 5' to the start of transcription. Addition of either tetrahydrofolate or hypoxanthine plus thymidine prevents the promoter induction in response to MTX, suggesting that stimulation by MTX results from inhibition of these metabolites. Furthermore, two other antimetabolic drugs--fluorodeoxyuridine and hydroxyurea--also stimulate the DHFR promoter in a concentration-dependent manner. In contrast, aphidicolin, which blocks cell growth through inhibition of DNA polymerase alpha, has no effect on the DHFR promoter. The potential relevance of these results to cross-resistance to chemotherapeutic agents and to the process of gene amplification is discussed. Images PMID:1833762

  7. Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine.

    PubMed

    O'Riordan, Avril A; Morales, Veronica Athie; Mulligan, Linda; Faheem, Nazia; Windle, Henry J; Kelleher, Dermot P

    2012-06-01

    Helicobacter pylori (H. pylori) is the most important etiological agent of chronic active gastritis, peptic ulcer disease and gastric cancer. The aim of this study was to evaluate the efficacy of alkyl hydroperoxide reductase (AhpC) and mannosylated AhpC (mAhpC) as candidate vaccines in the C57BL/6J mouse model of H. pylori infection. Recombinant AhpC was cloned, over-expressed and purified in an unmodified form and was also engineered to incorporate N and C-terminal mannose residues when expressed in the yeast Pichia pastoris. Mice were immunized systemically and mucosally with AhpC and systemically with mAhpC prior to challenge with H. pylori. Serum IgG responses to AhpC were determined and quantitative culture was used to determine the efficacy of vaccination strategies. Systemic prophylactic immunization with AhpC/alum and mAhpC/alum conferred protection against infection in 55% and 77.3% of mice, respectively. Mucosal immunization with AhpC/cholera toxin did not protect against infection and elicited low levels of serum IgG in comparison with systemic immunization. These data support the use of AhpC as a potential vaccine candidate against H. pylori infection. PMID:22512976

  8. Direct electrochemistry of nitrate reductase from the fungus Neurospora crassa.

    PubMed

    Kalimuthu, Palraj; Ringel, Phillip; Kruse, Tobias; Bernhardt, Paul V

    2016-09-01

    We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39μM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained. PMID:27060250

  9. 5alpha-reductase: history and clinical importance.

    PubMed

    Marks, Leonard S

    2004-01-01

    The treatment of men with symptomatic benign prostatic hyperplasia (BPH) has shifted dramatically from surgery to drug therapy over the past decade. The revolution in BPH treatment began with the discovery of congenital 5alpha-reductase (5AR) deficiency, leading to the appreciation of 2 different androgenic hormones: testosterone, which mediates overt masculinization in the adult male, and dihydrotestosterone (DHT), which mediates prostatic growth, acne, facial beard, and male pattern baldness. Inhibition of DHT in adults results in prostatic shrinkage and symptomatic relief in many men, without the side effects seen with conventional androgen-deprivation therapy. The 5AR inhibitor drugs (finasteride and the dual inhibitor, dutasteride) are able to ablate the accumulation of intraprostatic DHT, the mechanism most responsible for prostate growth and maintenance. Not only may these drugs relieve symptoms, but they may also alter the natural history of the BPH process. Future indications for the 5ARI drugs could include chemoprevention of prostate cancer, prophylaxis of BPH-related complications, and treatment of BPH-associated hematuria. PMID:16985920

  10. Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

    PubMed

    Chu, Y X; Chen, H R; Wu, A Z; Cai, R; Pan, J S

    2015-01-01

    Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside. PMID:25966276

  11. Fasciola gigantica thioredoxin glutathione reductase: Biochemical properties and structural modeling.

    PubMed

    Gupta, Ankita; Kesherwani, Manish; Velmurugan, Devadasan; Tripathi, Timir

    2016-08-01

    Platyhelminth thioredoxin glutathione reductase (TGR) is a multifunctional enzyme that crosstalk between the conventional thioredoxin (Trx) and glutathione (GSH) system. It has been validated as a potential drug target in blood flukes. In the present study, we have performed a biochemical study on Fasciola gigantica TGR with substrates DTNB and GSSG. The Michaelis constant (Km) with DTNB was found to be 4.34±0.12μM while it was 61.15±1.50μM with GSSG. The kinetic results were compared with the TGR activities of other helminths. FgTGR showed typical hysteretic behavior with GSSG as other TGRs. We also described a homology-based structure of FgTGR. The cofactors (NADPH and FAD) and substrates (GSSG and DTNB) were docked, and two possible binding sites for substrates were identified in a single chain. The substrates were found to bind more favorably in the second site of TrxR domains. We also presented the first report on binding interaction of DTNB with a TGR. DTNB forms H-bond with His204 and Arg450 of chain A, Sec597, and Gly598 from chain B, salt-bridge with Lys124, and numerous other hydrophobic interactions. Helminth TGR represents an important enzyme in the redox and antioxidant system; hence, its inhibition can be used as an effective strategy against liver flukes. PMID:27112978

  12. Cheminformatics Models for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase

    PubMed Central

    Gaba, Sonam; Jamal, Salma; Open Source Drug Discovery Consortium

    2014-01-01

    Schistosomiasis is a neglected tropical disease caused by a parasite Schistosoma mansoni and affects over 200 million annually. There is an urgent need to discover novel therapeutic options to control the disease with the recent emergence of drug resistance. The multifunctional protein, thioredoxin glutathione reductase (TGR), an essential enzyme for the survival of the pathogen in the redox environment has been actively explored as a potential drug target. The recent availability of small-molecule screening datasets against this target provides a unique opportunity to learn molecular properties and apply computational models for discovery of activities in large molecular libraries. Such a prioritisation approach could have the potential to reduce the cost of failures in lead discovery. A supervised learning approach was employed to develop a cost sensitive classification model to evaluate the biological activity of the molecules. Random forest was identified to be the best classifier among all the classifiers with an accuracy of around 80 percent. Independent analysis using a maximally occurring substructure analysis revealed 10 highly enriched scaffolds in the actives dataset and their docking against was also performed. We show that a combined approach of machine learning and other cheminformatics approaches such as substructure comparison and molecular docking is efficient to prioritise molecules from large molecular datasets. PMID:25629082

  13. Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

    PubMed Central

    Reddish, Michael J.; Vaughn, Morgan B.; Fu, Rong; Dyer, R. Brian

    2016-01-01

    Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not. PMID:26901612

  14. Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase.

    PubMed

    French, C E; Rosser, S J; Davies, G J; Nicklin, S; Bruce, N C

    1999-05-01

    Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems. To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives. Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds. Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings. This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil. PMID:10331811

  15. Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency.

    PubMed

    Froese, D Sean; Huemer, Martina; Suormala, Terttu; Burda, Patricie; Coelho, David; Guéant, Jean-Louis; Landolt, Markus A; Kožich, Viktor; Fowler, Brian; Baumgartner, Matthias R

    2016-05-01

    Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations. PMID:26872964

  16. The superoxide reductase from the early diverging eukaryote Giardia intestinalis.

    PubMed

    Testa, Fabrizio; Mastronicola, Daniela; Cabelli, Diane E; Bordi, Eugenio; Pucillo, Leopoldo P; Sarti, Paolo; Saraiva, Lígia M; Giuffrè, Alessandro; Teixeira, Miguel

    2011-10-15

    Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(•-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(•-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

  17. Solvent effects on catalysis by Escherichia coli dihydrofolate reductase.

    PubMed

    Loveridge, E Joel; Tey, Lai-Hock; Allemann, Rudolf K

    2010-01-27

    Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties.

  18. Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis

    SciTech Connect

    Li Mei; Chen Zhiwei; Zhang Pingfeng; Pan Xiaowei; Jiang Chengying; An Xiaomin; Liu Shuangjiang; Chang Wenrui

    2008-05-09

    Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.

  19. Atypical features of Thermus thermophilus succinate:quinone reductase.

    PubMed

    Kolaj-Robin, Olga; Noor, Mohamed R; O'Kane, Sarah R; Baymann, Frauke; Soulimane, Tewfik

    2013-01-01

    The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His(8)-SdhB and rcII-SdhB-His(6)) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form. Only the trimer exhibited positive cooperativity at high temperatures. The EPR signal of the [2Fe-2S] cluster was sensitive to the presence of substrate and showed an increased rhombicity in the presence of succinate in the native and in all recombinant forms of the enzyme. The detailed analysis of the shape of this signal as a function of pH, substrate concentration and in the presence of various inhibitors and quinones is presented, leading to a model for the molecular mechanism that underlies the influence of succinate on the rhombicity of the EPR signal of the proximal iron-sulfur cluster.

  20. Isoquinoline alkaloids from Tinospora cordifolia inhibit rat lens aldose reductase.

    PubMed

    Patel, Mayurkumar B; Mishra, Shrihari

    2012-09-01

    The inhibitory activity of Tinospora cordifolia stem-derived alkaloids was evaluated against lens aldose reductase (AR) isolated from male Wistar rats. Anticataract potential of the alkaloids of T. cordifolia was evaluated in vitro in rat lenses, considering the activity of normal rat lenses as 100%. The biologically active constituents of T. cordifolia extract were characterized as the isoquinoline alkaloids, jatrorrhizine, palmatine and magnoflorine, by spectral analysis. The inhibitory effects varied with all chemicals and concentrations used. The inhibitory concentration (IC₅₀) values of jatrorrhizine, palmatine and magnoflorine are 3.23, 3.45 and 1.25 µg/mL respectively. The concentration of maximum activity was selected for its effect on galactose-induced polyol accumulation in vitro. The percentage inhibition of galactose-induced polyol accumulation was 62.6, 58.8 and 27.7% in the presence of jatrorrhizine, palmatine and magnoflorine, respectively. Magnoflorine may be useful as lead compounds and new agents for AR inhibition. PMID:22294283

  1. Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase

    SciTech Connect

    French, C.E.; Bruce, N.C.; Nicklin, S.

    1998-08-01

    Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.

  2. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

    SciTech Connect

    Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

    2006-12-01

    Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

  3. Studies on aldose reductase inhibitors from natural products. IV. Constituents and aldose reductase inhibitory effect of Chrysanthemum morifolium, Bixa orellana and Ipomoea batatas.

    PubMed

    Terashima, S; Shimizu, M; Horie, S; Morita, N

    1991-12-01

    The hot water extracts of Chrysanthemum morifolium, Bixa orellana and Ipomoea batatas, were found to have potent inhibitory activity towards lens aldose reductase (AR). Ellagic acid (4) was isolated from C. morifolium and I. batatas, isoscutellarein (7) from B. orellana and 3,5-dicaffeoylquinic acid (10) from I. batatas, respectively, as potent inhibitors. PMID:1814628

  4. Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency

    SciTech Connect

    Goyette, P.; Frosst, P.; Rosenblatt, D.S.; Rozen. R.

    1995-05-01

    5-Methyltetrahydrofolate, the major form of folate in plasma, is a carbon donor for the remethylation of homocysteine to methionine. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. Patients with an autosomal recessive severe deficiency of MTHFR have homocystinuria and a wide range of neurological and vascular disturbances. We have recently described the isolation of a cDNA for MTHFR and the identification of two mutations in patients with severe MTHFR deficiency. We report here the characterization of seven novel mutations in this gene: six missense mutations and a 5{prime} splice-site defect that activates a cryptic splice in the coding sequence. We also present a preliminary analysis of the relationship between genotype and phenotype for all nine mutations identified thus far in this gene. A nonsense mutation and two missense mutations (proline to leucine and threonine to methionine) in the homozygous state are associated with extremely low activity (0%-3%) and onset of symptoms within the 1st year of age. Other missense mutations (arginine to cysteine and arginine to glutamine) are associated with higher enzyme activity and later onset of symptoms. 19 refs., 4 figs., 2 tabs.

  5. 5α-Reductase Type 3 Expression in Human Benign and Malignant Tissues: A Comparative Analysis During Prostate Cancer Progression

    PubMed Central

    Godoy, Alejandro; Kawinski, Elzbieta; Li, Yun; Oka, Daizo; Alexiev, Borislav; Azzouni, Faris; Titus, Mark A.; Mohler, James L.

    2015-01-01

    BACKGROUND A third isozyme of human 5α-steroid reductase, 5α-reductase-3, was identified in prostate tissue at the mRNA level. However, the levels of 5α-reductase-3 protein expression and its cellular localization in human tissues remain unknown. METHODS A specific monoclonal antibody was developed, validated, and used to characterize for the first time the expression of 5α-reductase-3 protein in 18 benign and 26 malignant human tissue types using immunostaining analyses. RESULTS AND CONCLUSIONS In benign tissues, 5α-reductase-3 immunostaining was high in conventional androgen-regulated human tissues, such as skeletal muscle and prostate. However, high levels of expression also were observed in non-conventional androgen-regulated tissues, which suggest either multiples target tissues for androgens or different functions of 5α-reductase-3 among human tissues. In malignant tissues, 5α-reductase-3 immunostaining was ubiquitous but particularly over-expressed in some cancers compared to their benign counterparts, which suggests a potential role for 5α-reductase-3 as a biomarker of malignancy. In benign prostate, 5α-reductase-3 immunostaining was localized to basal epithelial cells, with no immunostaining observed in secretory/luminal epithelial cells. In high-grade prostatic intraepithelial neoplasia (HGPIN), 5α-reductase-3 immunostaining was localized in both basal epithelial cells and neoplastic epithelial cells characteristic of HGPIN. In androgen-stimulated and castration-recurrent prostate cancer (CaP), 5α-reductase-3 immunostaining was present in most epithelial cells and at similar levels, and at levels higher than observed in benign prostate. Analyses of expression and functionality of 5α-reductase-3 in human tissues may prove useful for development of treatment for benign prostatic enlargement and prevention and treatment of CaP. PMID:21557268

  6. ADP-ribosylation of dinitrogenase reductase from Clostridium pasteurianum prevents its inhibition of nitrogenase from Azotobacter vinelandii.

    PubMed

    Murrell, S A; Lowery, R G; Ludden, P W

    1988-04-15

    The effect of ADP-ribosylation of dinitrogenase reductase on its binding to dinitrogenase was investigated. Dinitrogenase reductase from Clostridium pasteurianum (Cp2) was a substrate for the ADP-ribosyltransferase and the dinitrogenase-reductase-activating glycohydrolase from Rhodospirillum rubrum. ADP-ribosylation inactivated Cp2 and prevented its formation of a tight complex with dinitrogenase from Azotobacter vinelandii (Av1). The complex between Cp2 and Av1 could not be ADP-ribosylated once it formed.

  7. The C-terminal loop of aldehyde reductase determines the substrate and inhibitor specificity.

    PubMed

    Barski, O A; Gabbay, K H; Bohren, K M

    1996-11-12

    Human aldehyde reductase has a preference for carboxyl group-containing negatively charged substrates. It belongs to the NADPH-dependent aldo-keto reductase superfamily whose members are in part distinguished by unique C-terminal loops. To probe the role of the C-terminal loops in determining substrate specificities in these enzymes, two arginine residues, Arg308 and Arg311, located in the C-terminal loop of aldehyde reductase, and not found in any other C-terminal loop, were replaced with alanine residues. The catalytic efficiency of the R311A mutant for aldehydes containing a carboxyl group is reduced 150-250-fold in comparison to that of the wild-type enzyme, while substrates not containing a negative charge are unaffected. The R311A mutant is also significantly less sensitive to inhibition by dicarboxylic acids, indicating that Arg311 interacts with one of the carboxyl groups. The inhibition pattern indicates that the other carboxyl group binds to the anion binding site formed by Tyr49, His112, and the nicotinamide moiety of NADP+. The correlation between inhibitor potency and the length of the dicarboxylic acid molecules suggests a distance of approximately 10 A between the amino group of Arg311 and the anion binding site in the aldehyde reductase molecule. The sensitivity of inhibition of the R311A mutant by several commercially available aldose reductase inhibitors (ARIs) was variable, with tolrestat and zopolrestat becoming more potent inhibitors (30- and 5-fold, respectively), while others remained the same or became less potent. The catalytic properties, substrate specificity, and susceptibility to inhibition of the R308A mutant remained similar to that of the wild-type enzyme. The data provide direct evidence for C-terminal loop participation in determining substrate and inhibitor specificity of aldo-keto reductases and specifically identifies Arg311 as the basis for the carboxyl-containing substrate preference of aldehyde reductase. PMID:8916913

  8. Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

    PubMed Central

    Gallego, Oriol; Belyaeva, Olga V.; Porté, Sergio; Ruiz, F. Xavier; Stetsenko, Anton V.; Shabrova, Elena V.; Kostereva, Natalia V.; Farrés, Jaume; Parés, Xavier; Kedishvili, Natalia Y.

    2006-01-01

    Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low Km values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low Km values for retinoids (0.12–1.1 μM), whilst they strongly differ in their kcat values, which range from 0.35 min−1 for AKR1B1 to 302 min−1 for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo. PMID:16787387

  9. The two-domain structure of 5'-adenylylsulfate (APS) reductase from Enteromorpha intestinalis is a requirement for efficient APS reductase activity.

    PubMed

    Kim, Sung-Kun; Gomes, Varinnia; Gao, Yu; Chandramouli, Kala; Johnson, Michael K; Knaff, David B; Leustek, Thomas

    2007-01-16

    5'-Adenylylsulfate (APS) reductase from Enteromorpha intestinalis (EiAPR) is composed of two domains that function together to reduce APS to sulfite. The carboxyl-terminal domain functions as a glutaredoxin that mediates the transfer of electrons from glutathione to the APS reduction site on the amino-terminal domain. To study the basis for the interdomain interaction, a heterologous system was constructed in which the C domain of EiAPR was fused to the carboxyl terminus of the APS reductase from Pseudomonas aeruginosa (PaAPR), an enzyme that normally uses thioredoxin as an electron donor and is incapable of using glutathione for this function. The hybrid enzyme, which retains the [4Fe-4S] cluster from PaAPR, was found to use both thioredoxin and glutathione as an electron donor for APS reduction. The ability to use glutathione was enhanced by the addition of Na2SO4 to the reaction buffer, a property that the hybrid enzyme shares with EiAPR. When the C domain was added as a separate component, it was much less efficient in conferring PaAPR with the ability to use glutathione as an electron donor, despite the fact that the separately expressed C domain functioned in two activities that are typical for glutaredoxins, hydroxyethyl disulfide reduction and electron donation to ribonucleotide reductase. These results suggest that the physical connection of the reductase and C domain on a single polypeptide is critical for the electron-transfer reaction. Moreover, the effect of Na2SO4 suggests that a water-ordering component of the reaction milieu is critical for the catalytic function of plant-type APS reductases by promoting the interdomain interaction.

  10. Syntheses and luminescence study for La1-xEux[B5O8(OH)2]·1.5H2O (0≤x≤0.40) and the dehydrated products β-La1-xEuxB5O9 (0≤x≤0.15)

    NASA Astrophysics Data System (ADS)

    Sun, Xiaorui; Zhou, Zhengyang; Yang, Haixia; Gao, Wenliang; Cong, Rihong; Yang, Tao

    2016-05-01

    La1-xEux[B5O8(OH)2]·1.5H2O with the Eu3+-doping upper limit of 40 atom% were synthesized hydrothermally. Thereafter, thermal treatments at 710 °C were applied to obtain β-La1-xEuxB5O9. The solid solution range is even narrower, i.e. 0≤x≤0.15, due to the mismatch between La3+ and Eu3+. The host borate system shows a typical concentration quenching effect at x=0.20 under CT excitation, and this is postponed to x=0.30 under the f-f excitation. β-La1-xEuxB5O9 shows a very intense absorption of charge transfer, and gives strong red emissions at 615 nm with large R/O ratios (1.9-2.4). The saturation effect appears at x=0.11, which is probably due to the lattice distortion. Eu3+ luminescence was applied as the structural probe to study the local coordination environment change during the dehydration and re-crystallization processes of La0.93Eu0.07[B5O8(OH)2]·1.5H2O.

  11. Overexpression of Aldose Reductase Render Mouse Hepatocytes More Sensitive to Acetaminophen Induced Oxidative Stress and Cell Death.

    PubMed

    Ahmed, Munzir M E; Al-Obosi, J A S; Osman, H M; Shayoub, M E

    2016-04-01

    Acetaminophen (APAP) a commonly used drug for decrease the fever and pain but is capable to induced hepatotoxicity at over dose. This study was carried out to investigate the effect of APAP on the expression of anti-apoptotic and antioxidative defense genes, and whether aldose reductase over-expressing plasmid capable to protect against APAP-induced oxidative stress and cell death. APAP treatment induced oxidative stress and hepatotoxicity, and significantly increased aldose reductase mRNA and protein expression in mouse hepatocyte (AML-12). Unexpectedly, AML-12 cells over-expressing aldose reductase augmented APAP-induced reduction in cell viability, reactive oxygen species (ROS) production, glutathione (GSH) depletion and glutathione S-transferase A2 expression. Moreover, over-expression of aldose reductase potentiated APAP induced reduction on proliferating cell nuclear antigen, B cell lymphoma-extra large (bcl-xL), catalase, glutathione peroxidase-1 (GPx-1) and abolished APAP-induced B-cell lymphoma 2 (bcl-2) inductions. Further, over-expression of aldose reductase significantly abolished AMP activated protein kinase (AMPK) activity in APAP-treated cells and induced p53 expression. This results demonstrate that APAP induced toxicity in AML-12, increased aldose reductase expression, and over-expression of aldose reductase render this cell more susceptible to APAP induced oxidative stress and cell death, this probably due to inhibition AMPK or bcl-2 activity, or may due to competition between aldose reductase and glutathione reductase for NADPH. PMID:27069324

  12. Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

    PubMed

    Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu

    2014-01-01

    Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

  13. Mechanistic studies of ribonucleoside triphosphate reductase from Lactobacillus leichmannii

    SciTech Connect

    Harris, G.M.

    1984-01-01

    The mechanism of action of the adenosylcobalamin (AdoCbl)-dependent ribonucleoside triphosphate reductase (RTPR) was investigated using isotope effect and substrate specificity studies. These experiments were conducted on RTPR purified by a new method from Lactobacillus leichmannii. Isotope effect studies using (3{prime}-{sup 3}H)UTP and (3{prime}-{sup 3}H)ATP demonstrated that the 3{prime} C-H bond of the nucleotide is cleaved in order to cleave the 2{prime} C-OH bond. AdoCbl does not act as a direct H abstractor from the 3{prime} position of the substrate, but instead is thought to act as a radical chain initiator to generate an amino acid radical on the enzyme. Further support for this enzyme mediated cleavage of the 3{prime} C-H bond of the nucleotide and the novel role of AdoCbl came from studies using (3{prime}{sup 3}H)2{prime}-chloro-2{prime}-deoxyuridine 5{prime}-triphosphate ((3{prime}-{sup 3}H)CIUTP). Evidence is presented that during the course of this reaction, the {sup 3}H abstracted from the 3{prime} position of (3{prime}-{sup 3}H)CIUTP was either exchanged with the solvent or returned to the {beta} face of the 2{prime} position to produce (2{prime}{sup 3}H)-2{prime}-deoxy-3{prime}-ketoUTP. This result demonstrates that RTPR is capable of catalyzing a rearrangement reaction. The significance of the RTPR-catalyzed rearrangement with respect to the AdoCbl-dependent enzymes which catalyze rearrangements is discussed.

  14. Differing views of the role of selenium in thioredoxin reductase

    PubMed Central

    Ruggles, Erik L.

    2010-01-01

    This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pKa and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the “chemico-enzymatic” function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the “chemico-biological” function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO2−, seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO2− to Cys-SH). PMID:20397034

  15. Rapid Identification of Aldose Reductase Inhibitory Compounds from Perilla frutescens

    PubMed Central

    Paek, Ji Hun; Shin, Kuk Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

    2013-01-01

    The ethyl acetate (EtOAc) soluble fraction of methanol extracts of Perilla frutescens (P. frutescens) inhibits aldose reductase (AR), the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural analyses of the active compounds were performed by LC-MSn. The main AR inhibiting compounds were tentatively identified as chlorogenic acid and rosmarinic acid by LC-MSn. A two-step high speed counter current chromatography (HSCCC) isolation method was developed with a solvent system of n-hexane-ethyl acetate-methanol-water at 1.5 : 5 : 1 : 5, v/v and 3 : 7 : 5 : 5, v/v. The chemical structures of the isolated compounds were determined by 1H- and 13C-nuclear magnetic resonance spectrometry (NMR). The main compounds inhibiting AR in the EtOAc fraction of methanol extracts of P. frutescens were identified as chlorogenic acid (2) (IC50 = 3.16 μM), rosmarinic acid (4) (IC50 = 2.77 μM), luteolin (5) (IC50 = 6.34 μM), and methyl rosmarinic acid (6) (IC50 = 4.03 μM). PMID:24308003

  16. Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase*

    PubMed Central

    Wu, Sheng-Yi; Rothery, Richard A.; Weiner, Joel H.

    2015-01-01

    We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser719, NarG-His1163, and NarG-His1184); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His1092 and NarG-His1098). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of −88 and −36 mV, respectively). Ala variants of His1092 and His1098 also elicit large ΔEm values of −143 and −101 mV, respectively. An Arg variant of His1092 elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis. PMID:26297003

  17. Short-chain dehydrogenases/reductases in cyanobacteria.

    PubMed

    Kramm, Anneke; Kisiela, Michael; Schulz, Rüdiger; Maser, Edmund

    2012-03-01

    The short-chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)-dependent or NADP(H)-dependent oxidoreductases. They display a wide substrate spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three-dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so-called 'homologous' proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis  elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria. PMID:22251568

  18. Aldo-Keto Reductases 1B in Endocrinology and Metabolism

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Volat, Fanny; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2012-01-01

    The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers. PMID:22876234

  19. The Reaction Mechanism of Methyl-Coenzyme M Reductase

    PubMed Central

    Wongnate, Thanyaporn; Ragsdale, Stephen W.

    2015-01-01

    Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mm). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(NiI)·CH3SCoM) is highly favored (Kd = 79 μm). Only then can the chemical reaction occur (kobs = 20 s−1 at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(NiII)·CoB7S−·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates. PMID:25691570

  20. The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.

    PubMed

    Francis, Kevin; Sapienza, Paul J; Lee, Andrew L; Kohen, Amnon

    2016-02-23

    Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction's transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with (13)C, (15)N, and (2)H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD's conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general.