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Sample records for nadh-cytochrome b5 reductase

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

    PubMed

    Sacco, James C; Trepanier, Lauren A

    2010-01-01

    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. Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. 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. These studies indicate that although novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low-frequency cSNPs seem to only 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.

  2. Reductive Detoxication of Arylhydroxylamine Carcinogens by Human NADH Cytochrome b5 Reductase and Cytochrome b5

    PubMed Central

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

    2008-01-01

    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 inter-individual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. Based on our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxication 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 to 346-fold higher in the purified system compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (Km 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 (∼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 arylhydroxylamine carcinogens

  3. 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

  4. 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

  5. Structure and properties of the recombinant NADH-cytochrome b5 reductase of Physarum polycephalum.

    PubMed

    Ikegami, Terumi; Kameyama, Eiji; Yamamoto, Shin-ya; Minami, Yoshiko; Yubisui, Toshitsugu

    2007-03-01

    A cDNA for NADH-cytochrome b(5) reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5'- and 3'-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K(m) values of 2 microM and 14 microM for NADH and cytochrome b(5) respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b(5) reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.

  6. Distribution of valence electrons of the flavin cofactor in NADH-cytochrome b5 reductase

    PubMed Central

    Takaba, Kiyofumi; Takeda, Kazuki; Kosugi, Masayuki; Tamada, Taro; Miki, Kunio

    2017-01-01

    Flavin compounds such as flavin adenine dinucleotide (FAD), flavin mononucleotide and riboflavin make up the active centers in flavoproteins that facilitate various oxidoreductive processes. The fine structural features of the hydrogens and valence electrons of the flavin molecules in the protein environment are critical to the functions of the flavoproteins. However, information on these features cannot be obtained from conventional protein X-ray analyses at ordinary resolution. Here we report the charge density analysis of a flavoenzyme, NADH-cytochrome b5 reductase (b5R), at an ultra-high resolution of 0.78 Å. Valence electrons on the FAD cofactor as well as the peptide portion, which are clearly visualized even after the conventional refinement, are analyzed by the multipolar atomic model refinement. The topological analysis for the determined electron density reveals the valence electronic structure of the isoalloxazine ring of FAD and hydrogen-bonding interactions with the protein environment. The tetrahedral electronic distribution around the N5 atom of FAD in b5R is stabilized by hydrogen bonding with CαH of Tyr65 and amide-H of Thr66. The hydrogen bonding network leads to His49 composing the cytochrome b5-binding site via non-classical hydrogen bonds between N5 of FAD and CαH of Tyr65 and O of Tyr65 and CβH of His49. PMID:28225078

  7. Inhibitory effects of dietary flavonoids on purified hepatic NADH-cytochrome b5 reductase: structure-activity relationships.

    PubMed

    Çelik, Haydar; Koşar, Müberra

    2012-05-30

    The structure-activity relationships of flavonoids with regard to their inhibitory effects on NADH-cytochrome b5 reductase (E.C. 1.6.2.2), a clinically and toxicologically important enzyme, are not known. In the present study, the inhibitory effects of fourteen selected flavonoids of variable structure on the activity of purified bovine liver cytochrome b5 reductase, which shares a high degree of homology with the human counterpart, were investigated and the relationship between structure and inhibition was examined. Of all the compounds tested, the flavone luteolin was the most potent in inhibiting b5 reductase with an IC50 value of 0.11 μM, whereas naringenin, naringin and chrysin were inactive within the concentration range tested. Most of the remaining flavonoids (morin, quercetin, quercitrin, myricetin, luteolin-7-O-glucoside, (-)-epicatechin, and (+)-catechin) produced a considerable inhibition of enzyme activity with IC50 values ranging from 0.81 to 4.5 μM except apigenin (36 μM), rutin (57 μM) and (+)-taxifolin (IC50 not determined). The magnitude of inhibition was found to be closely related to the chemical structures of flavonoids. Analysis of structure-activity data revealed that flavonoids containing two hydroxyl groups in ring B and a carbonyl group at C-4 in combination with a double bond between C-2 and C-3 produced a much stronger inhibition, whereas substitution of a hydroxyl group at C-3 was associated with a less inhibitory effect. The physiologically relevant IC50 values for most of the flavonoids tested regarding b5 reductase inhibition indicate a potential for significant flavonoid-drug and/or flavonoid-xenobiotic interactions which may have important therapeutic and toxicological outcomes for certain drugs and/or xenobiotics. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  8. Visinin-Like Protein-3 Modulates the Interaction Between Cytochrome b 5 and NADH-Cytochrome b 5 Reductase in a Ca(2+)-Dependent Manner.

    PubMed

    Oikawa, Kensuke; Odero, Gary L; Nafez, Solmaz; Ge, Ning; Zhang, Dali; Kobayashi, Hiroya; Sate, Keisuke; Kimura, Shoji; Tateno, Masatoshi; Albensi, Benedict C

    2016-12-01

    Visinin-like proteins (VILIPs) belong to the calcium sensor protein family. VILIP-1 has been examined as a cerebrospinal fluid biomarker and as a potential indicator for cognitive decline in Alzheimer's disease (AD). However, little is known about VILIP-3 protein biochemistry. We performed co-immunoprecipitation experiments to examine whether VILIP-3 can interact with reduced nicotine adenine dinucleotide (NADH)-cytochrome b 5 reductase. We also evaluated the specificity of cytochrome b 5 within the visinin-like protein subfamily and identified cytochrome P450 isoforms in the brain. In this study, we show that cytochrome b 5 has an affinity for hippocalcin, neurocalcin-δ, and VILIP-3, but not visinin-like protein-1. VILIP-3 was also shown to interact with NADH-cytochrome b 5 reductase in a Ca(2+)-dependent manner. These results suggest that VILIP-3, hippocalcin, and neurocalcin-δ provide a Ca(2+)-dependent modulation to the NADH-dependent microsomal electron transport. The results also suggest that future therapeutic strategies that target calcium-signaling pathways and VILIPs may be of value.

  9. 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.

  10. Evaluation of data in terms of two-dimensional random walk model: interaction between NADH-cytochrome b5 reductase and cytochrome b5.

    PubMed

    Tonegawa, Yoshihiro; Umeda, Noriaki; Hayakawa, Tohru; Ishibashi, Teruo

    2005-10-01

    Normally, bimolecular reactions are analyzed in terms of the Smoluchowski theory. However, when one attempts to generalize this analysis to cases where diffusion proceeds in two other than in three dimensions, one soon encounters severe conceptual difficulties. Although kinetic studies of membrane enzymes are generally difficult because the usual kinetic formalism refers to nonaggregated homogenous solutions, a major goal of our research is to define the molecular mechanism(s) by which alterations in membrane-bound substrate contents affect the enzyme activity in the same membrane. For that purpose, a simplified random-walk model was adopted in the present work. The enzyme reaction in the two-dimensional membrane could be calculated theoretically by applying the classical analysis of heat equation. As a result, the theoretical rate equation well accounting experimental findings was derived on the model of the liver microsomal NADH-cytochrome b5 reductase reaction. Furthermore, it was found that the modification of the simple rigid-sphere collision theory by including a term called the steric factor was not necessary in this derived equation.

  11. Evaluation of bioreductive activation of anticancer drugs idarubicin and mitomycin C by NADH-cytochrome b5 reductase and cytochrome P450 2B4.

    PubMed

    Celik, Haydar; Arinç, Emel

    2013-03-01

    This study attempted to investigate the ability of microsomal NADH-cytochrome b5 reductase and cytochrome P450 2B4 to reductively activate idarubicin and mitomycin C. In vitro plasmid DNA damage experiments and assays using purified hepatic enzymes were employed to examine their respective roles in the metabolic activation of anticancer drugs. Mitomycin C was found to be not a good substrate for microsomal b5 reductase unlike P450 reductase. It produced low amounts of strand breaks in DNA when incubated with b5 reductase and its one-electron reduction by purified enzyme was found as negligible. Our findings revealed that P450 reductase-mediated metabolism of idarubicin resulted in a large increase in single-strand DNA breaks, whereas, b5 reductase neither catalyzed the reduction of idarubicin nor mediated the formation of DNA damage in the presence of idarubicin. The reconstitution studies, on the other hand, have identified rabbit liver CYP2B4 isozyme as being a potential candidate enzyme for reductive bioactivation of idarubicin and mitomycin C. Thus, the present novel findings strongly suggest that while b5 reductase could not play a key role in the cytotoxic and/or antitumor effects of idarubicin and mitomycin C, CYP2B4 could potentiate their activity in combination with P450 reductase.

  12. A role for N-myristoylation in protein targeting: NADH-cytochrome b5 reductase requires myristic acid for association with outer mitochondrial but not ER membranes

    PubMed Central

    1996-01-01

    N-myristoylation is a cotranslational modification involved in protein- protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. The mammalian myristoylated flavoenzyme NADH-cytochrome b5 reductase is integrated into ER and mitochondrial outer membranes via an anchor containing a stretch of 14 uncharged amino acids downstream to the NH2- terminal myristoylate glycine. Since previous studies suggested that the anchoring function could be adequately carried out by the 14 uncharged residues, we investigated a possible role for myristic acid in reductase targeting. The wild type (wt) and a nonmyristoylatable reductase mutant (gly2-->ala) were stably expressed in MDCK cells, and their localization was investigated by immunofluorescence, immuno-EM, and cell fractionation. By all three techniques, the wt protein localized to ER and mitochondria, while the nonmyristoylated mutant was found only on ER membranes. Pulse-chase experiments indicated that this altered steady state distribution was due to the mutant's inability to target to mitochondria, and not to its enhanced instability in that location. Both wt and mutant reductase were resistant to Na2CO3 extraction and partitioned into the detergent phase after treatment of a membrane fraction with Triton X-114, demonstrating that myristic acid is not required for tight anchoring of reductase to membranes. Our results indicate that myristoylated reductase localizes to ER and mitochondria by different mechanisms, and reveal a novel role for myristic acid in protein targeting. PMID:8978818

  13. 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. Copyright 2001 Wiley-Liss, Inc.

  14. 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

  15. 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.

  16. In vitro effects of myricetin, morin, apigenin, (+)-taxifolin, (+)-catechin, (-)-epicatechin, naringenin and naringin on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase.

    PubMed

    Çelik, Haydar; Koşar, Müberra; Arinç, Emel

    2013-06-07

    The microsomal NADH-dependent electron transport system consisting of cytochrome b5 reductase and cytochrome b5 participates in a number of physiologically important processes including lipid metabolism as well as is involved in the metabolism of various drug and xenobiotics. In the present study, we assessed the inhibitory effects of eight dietary flavonoids representing five distinct chemical classes on cytochrome b5 reduction by purified cytochrome b5 reductase. From the flavonoids tested, myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC50 value of 0.35μM. Myricetin inhibited b5 reductase noncompetitively with a Ki of 0.21μM with respect to cofactor NADH, and exhibited a non-linear relationship indicating non-Michaelis-Menten kinetic binding with respect to cytochrome b5. In contrast to the potent inhibitory activity of myricetin, (+)-taxifolin was found to be a weak inhibitor (IC50=9.8μM). The remaining flavonoids were inactive within the concentration range tested (1-50μM). Analysis of structure-activity data suggested that simultaneous presence of three OH groups in ring B is a primary structural determinant for a potent enzyme inhibition. Our results suggest that inhibition of the activity of this system by myricetin or myricetin containing diets may influence the metabolism of therapeutic drugs as well as detoxification of xenobiotics. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  17. 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

  18. 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.

  19. Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

    PubMed

    Barber, M J; Quinn, G B

    2001-11-01

    A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities

  20. Identification and expression analysis of NADH-cytochrome b₅ reductase gene in the cotton bollworm, Helicoverpa armigera.

    PubMed

    Zhao, Chunqing; Tang, Tao; Liu, Jiqin; Feng, Xiaoyun; Qiu, Lihong

    2012-12-10

    NADH-cytochrome b(5) reductase (CBR) is one of the most important components of cytochrome P450s, which play an essential role in the detoxification of xenobiotics as well as insecticide resistance in insect pest. In the present study, two novel full-length cDNAs of CBR of the cotton bollworm, Helicoverpa armigera (Hübner) were amplified by means of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The sequencing results showed that the transcripts were 1809bp and 1518bp for HaCBR1 and HaCBR2, respectively, including 969bp and 939bp of complete open reading frame (ORF), which encoded 322 and 312 amino acids respectively. The putative structure and function of HaCBR1 and HaCBR2 were preliminarily analyzed by SMART program. HaCBR1 and HaCBR2 (GenBank accession numbers: HQ638220 and HQ190046HQ638220HQ190046) showed high identities with CBRs of other species. The expression of HaCBR1 and HaCBR2 mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) in most developmental stages of H. armigera with the exception of eggs, as well as in tissues such as cuticle, fatbody and midgut. The expression level of the two genes was significantly induced by phenobarbital (PB). These results would contribute to the understanding of CBR function in H. armigera and provide information for further study on the interactions of different components of cytochrome P450 enzyme systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Cytochrome b5 Reductase Encoded by CBR1 Is Essential for a Functional Male Gametophyte in Arabidopsis[C][W

    PubMed Central

    Wayne, Laura L.; Wallis, James G.; Kumar, Rajesh; Markham, Jonathan E.; Browse, John

    2013-01-01

    In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the gene encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 heterozygotes segregated 1:1 heterozygous:wild type, indicating a gametophyte defect. Homozygous cbr1-2 seeds were deformed and required Suc for successful germination and seedling establishment. Vegetative growth of cbr1-2 plants was relatively normal, and they produced abundant flowers, but very few seeds. The pollen produced in cbr1-2 anthers was viable, but when germinated on cbr1-2 or wild-type stigmas, most of the resulting pollen tubes did not extend into the transmitting tract, resulting in a very low frequency of fertilization. These results indicate that cytochrome b5 reductase is not essential during vegetative growth but is required for correct pollen function and seed maturation. PMID:23995085

  2. A novel point mutation in a 3{prime} splice site of the NADH-cytochrome b{sub 5} reductase gene results in immunologically undetectable enzyme and impaired NADH-dependent ascorbate regeneration in cultured fibroblasts of a patient with type II hereditary methemoglobinemia

    SciTech Connect

    Shirabe, Komie; Takeshita, Masazumi; Landi, M.T.

    1995-08-01

    Hereditary methemoglobinemia with generalized deficiency of NADH-cytochrome b{sub 5} reductase (b{sub 5}R) (type II) is a rare disease characterized by severe developmental abnormalities, which often lead to premature death. Although the molecular relationship between the symptoms of this condition and the enzyme deficit are not understood, it is thought that an important cause is the loss of the lipid metabolizing activities of the endoplasmic reticulum-located reductase. However, the functions of the form located on outer mitochondrial membranes have not been considered previously. In this study, we have analyzed the gene of an Italian patient and identified a novel G{r_arrow}T transversion at the splice-acceptor site of the 9th exon, which results in the complete absence of immunologically detectable b{sub 5}R in blood cells and skin fibroblasts. In cultured fibroblasts of the patient, NADH-dependent cytochrome c reductase, ferricyanide reductase, and semidehydroascorbate reductase activities were severely reduced. The latter activity is known to be due to b{sub 5}R located on outer mitochondrial membranes. Thus, our results demonstrate that the reductase in its two membrane locations, endoplasmic reticulum and outer mitochondrial membranes, is the product of the same gene and suggest that a defect in ascorbate regeneration may contribute to the phenotype of hereditary methemoglobinemia of generalized type. 37 refs., 5 figs., 2 tabs.

  3. Structure of Physarum polycephalum cytochrome b5 reductase at 1.56 A resolution.

    PubMed

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

    2007-04-01

    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 A. The molecular structure was compared with that of human cytochrome b(5) reductase, which had previously been determined at 1.75 A 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.

  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. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Isolation of ascorbate free radical reductase from rabbit lens soluble fraction.

    PubMed

    Bando, Masayasu; Inoue, Takashi; Oka, Mikako; Nakamura, Kayako; Kawai, Kenji; Obazawa, Hajime; Kobayashi, Shizuko; Takehana, Makoto

    2004-12-01

    Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8.4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of pI 7.0-8.6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.

  6. Cloning and characterization of a maize cytochrome-b5 reductase with Fe3+-chelate reduction capability.

    PubMed

    Bagnaresi, P; Thoiron, S; Mansion, M; Rossignol, M; Pupillo, P; Briat, J F

    1999-03-01

    We previously purified an NADH-dependent Fe3+-chelate reductase (NFR) from maize roots with biochemical features of a cytochrome-b5 reductase (b5R) [Sparla, Bagnaresi, Scagliarini and Trost (1997) FEBS Lett. 414, 571-575]. We have now cloned a maize root cDNA that, on the basis of sequence information, calculated parameters and functional assay, codes for NFR. Maize NFR has 66% and 65% similarity to mammal and yeast b5R respectively. It has a deduced molecular mass of 31.17 kDa and a pI of 8.53. An uncharged region is observed at its N-terminus but no myristoylation consensus site is present. Taken together, these results, coupled with previous biochemical evidence, prove that NFR belongs to the b5R class and document b5R from a plant at the molecular level for the first time. We have also identified a putative Arabidopsis thaliana NFR gene. Its organization (nine exons) closely resembles mammalian b5Rs. Several NFR isoforms are expected to exist in maize. They are probably not produced by alternative translational mechanisms as occur in mammals, because of specific constraints observed in the maize NFR cDNA sequence. In contrast with yeast and mammals, tissue-specific and various subcellular localizations of maize b5R isoforms could result from differential expression of the various members of a multigene family. The first molecular characterization of a plant b5R indicates an overall remarkable evolutionary conservation for these versatile reductase systems. In addition, the well-characterized Fe3+-chelate reduction capabilities of NFR, in addition to known Fe3+-haemoglobin reduction roles for mammal b5R isoforms, suggest further and more generalized roles for the b5R class in endocellular iron reduction.

  7. Cytochrome b5 reductase and the control of lipid metabolism and healthspan.

    PubMed

    Martin-Montalvo, Alejandro; Sun, Yaning; Diaz-Ruiz, Alberto; Ali, Ahmed; Gutierrez, Vincent; Palacios, Hector H; Curtis, Jessica; Siendones, Emilio; Ariza, Julia; Abulwerdi, Gelareh A; Sun, Xiaoping; Wang, Annie X; Pearson, Kevin J; Fishbein, Kenneth W; Spencer, Richard G; Wang, Miao; Han, Xianlin; Scheibye-Knudsen, Morten; Baur, Joe A; Shertzer, Howard G; Navas, Placido; Villalba, Jose Manuel; Zou, Sige; Bernier, Michel; de Cabo, Rafael

    2016-01-01

    Cytochrome b5 reductases (CYB5R) are required for the elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders. However, the physiological role of CYB5R in the context of metabolism, healthspan and aging remains ill-defined. We generated CYB5R-overexpressing flies (CYB5R-OE) and created a transgenic mouse line overexpressing CYB5R3 (CYB5R3-Tg) in the C57BL/6J background to investigate the function of this class of enzymes as regulators of metabolism and age-associated pathologies. Gender- and/or stage-specific induction of CYB5R, and pharmacological activation of CYB5R with tetrahydroindenoindole extended fly lifespan. Increased expression of CYB5R3 was associated with significant improvements in several metabolic parameters that resulted in modest lifespan extension in mice. Diethylnitrosamine-induced liver carcinogenesis was reduced in CYB5R3-Tg mice. Accumulation of high levels of long-chain polyunsaturated fatty acids, improvement in mitochondrial function, decrease in oxidative damage and inhibition of chronic pro-inflammatory pathways occurred in the transgenic animals. These results indicate that CYB5R represents a new target in the study of genes that regulate lipid metabolism and healthspan.

  8. Cytochrome b5 reductase and the control of lipid metabolism and healthspan

    PubMed Central

    Martin-Montalvo, Alejandro; Sun, Yaning; Diaz-Ruiz, Alberto; Ali, Ahmed; Gutierrez, Vincent; Palacios, Hector H; Curtis, Jessica; Siendones, Emilio; Ariza, Julia; Abulwerdi, Gelareh A; Sun, Xiaoping; Wang, Annie X; Pearson, Kevin J; Fishbein, Kenneth W; Spencer, Richard G; Wang, Miao; Han, Xianlin; Scheibye-Knudsen, Morten; Baur, Joe A; Shertzer, Howard G; Navas, Placido; Villalba, Jose Manuel; Zou, Sige; Bernier, Michel; de Cabo, Rafael

    2016-01-01

    Cytochrome b5 reductases (CYB5R) are required for the elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders. However, the physiological role of CYB5R in the context of metabolism, healthspan and aging remains ill-defined. We generated CYB5R-overexpressing flies (CYB5R-OE) and created a transgenic mouse line overexpressing CYB5R3 (CYB5R3-Tg) in the C57BL/6J background to investigate the function of this class of enzymes as regulators of metabolism and age-associated pathologies. Gender- and/or stage-specific induction of CYB5R, and pharmacological activation of CYB5R with tetrahydroindenoindole extended fly lifespan. Increased expression of CYB5R3 was associated with significant improvements in several metabolic parameters that resulted in modest lifespan extension in mice. Diethylnitrosamine-induced liver carcinogenesis was reduced in CYB5R3-Tg mice. Accumulation of high levels of long-chain polyunsaturated fatty acids, improvement in mitochondrial function, decrease in oxidative damage and inhibition of chronic pro-inflammatory pathways occurred in the transgenic animals. These results indicate that CYB5R represents a new target in the study of genes that regulate lipid metabolism and healthspan. PMID:28721264

  9. Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b5 in the upper airways in horse.

    PubMed

    Tydén, E; Olsén, L; Tallkvist, J; Tjälve, H; Larsson, P

    2008-08-01

    Gene and protein expression as well as catalytic activity of cytochrome P450 (CYP) 3A were studied in the nasal olfactory and respiratory mucosa and the tracheal mucosa of the horse. We also examined the activity of NADPH cytochrome P450 reductase (NADPH P450 reductase), the amount of cytochrome b(5) and the total CYP content in these tissues. Comparative values for the above were obtained using liver as a control. The CYP3A related catalytic activity in the tissues of the upper airways was considerably higher than in the liver. The CYP3A gene and protein expression, on the other hand, was higher in the liver than in the upper airway tissues. Thus, the pattern of CYP3A metabolic activity does not correlate with the CYP3A gene and protein expression. Our results showed that the activity of NADPH P450 reductase and the level of cytochrome b(5) in the relation to the gene and protein expression of CYP3A were higher in the tissues of the upper airways than in the liver. It is concluded that CYP3A related metabolism in horse is not solely dependent on the expression of the enzyme but also on adequate levels of NADPH P450 reductase and cytochrome b(5).

  10. Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis.

    PubMed

    Ming, Huixin; Lan, Ying; He, Feng; Xiao, Xue; Zhou, Xiaoying; Zhang, Zhe; Li, Ping; Huang, Guangwu

    2015-08-15

    Cytochrome b5 reductase 2 (CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma (NPC). Inactivation of CYB5R2 is associated with lymph node metastasis in NPC. This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2. Polymerase chain reaction (PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1. NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2, and data was validated by real-time PCR. A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis. An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor (VEGF). In CYB5R2-transfected NPC cells, PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor (FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), interferon beta 1 (IFNB1), and cyclin-dependent kinase inhibitor 2A (CDKN2A) and down-regulated levels of integrin beta 5 (ITGB5), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), transforming growth factor beta receptor 1 (TGFBR1), and VEGF. The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited. Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model. CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis, thereby suppressing tumor formation.

  11. Discovery and characterization of a cytochrome b5 variant in humans with impaired hydroxylamine reduction capacity.

    PubMed

    Kurian, Joseph R; Longlais, Brett J; Trepanier, Lauren A

    2007-08-01

    We have shown that cytochrome b5 (cyt b5), along with its reductase, NADH cytochrome b5 reductase (b5R), is capable of direct xenobiotic biotransformation. We hypothesized that functionally significant genetic variability in cyt b5 could be found in healthy individuals. Cyt b5 cDNAs were prepared from peripheral blood mononuclear cells from 63 individuals. One individual was heterozygous for a sequence variant in cyt b5 (A178G), with a predicted amino acid substitution of T60A. This variant, when expressed in Escherichia. coli, maintained a similar Vmax for the hydroxylamines of sulfamethoxazole, 4-aminobiphenyl, and 2-amino-l-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), compared with wild type cyt b5, with a modestly increased Km (2 to 3.5-fold) for each substrate. When expressed in a mammalian system (HeLa cells), however, T60A was associated with a 70% reduction in cyt b5 protein expression compared with wild type. mRNA expression for both isoforms were comparable in HeLa cells, and translation of these mRNAs in a rabbit reticulocyte lysate system with inhibited proteasomal machinery were also similar. Incubation of these translated enzymes with uninhibited rabbit reticulocyte lysate, however, indicated greater susceptibility of T60A to proteasomal degradation. These data indicate that a naturally occurring variant in cyt b5, T60A, leads to modestly altered affinity for hydroxylamine substrates and dramatically reduced cyt b5 expression. Work is underway to determine the prevalence of this and other variants in cyt b5 or b5R in a larger population, and to determine the association of such variants with differences in hydroxylamine reduction in vivo.

  12. Soluble ascorbate free radical reductase in the human lens.

    PubMed

    Bando, M; Obazawa, H

    1994-01-01

    A major and a minor ascorbate free radical (AFR) reductase were separated from the soluble fraction in the human lens cortex by DEAE-cellulose ion-exchange column chromatography. These AFR reductases also exhibited diaphorase activity using dichlorophenolindophenol and ferricyanide as electron acceptors. The major AFR reductase was partially purified by 5'AMP-Sepharose 4B affinity column chromatography. This partially purified AFR reductase showed a single band of diaphorase activity in native polyacrylamide disc gel electrophoresis. This activity band corresponded to the major protein observed in protein staining by Coomassie Brilliant Blue. However, the protein staining by Coomassie Brilliant Blue showed this activity band surrounded by diffused staining. Molecular weight of the partially purified AFR reductase was determined to be 32 kDa by gel filtration, and the apparent Km value for AFR was about 15 microM. This major lens AFR reductase could be distinguished from soluble Neurospora, Euglena and cucumber AFR reductases, and from two ubiquitous enzymes with reduction activity of AFR and/or foreign compounds, ie, NADH-cytochrome b5 reductase and DT-diaphorase, by their molecular weights, Km values and/or ion-exchange chromatographic behaviors.

  13. Long-term Treatment with Methylene Blue in a Dog with Hereditary Methemoglobinemia Caused by Cytochrome b5 Reductase Deficiency.

    PubMed

    Jaffey, J A; Harmon, M R; Villani, N A; Creighton, E K; Johnson, G S; Giger, U; Dodam, J R

    2017-09-29

    A juvenile male mixed breed dog was presented for lethargy, exercise intolerance, and aggression when touched on the head. Cyanosis, tachycardia, and tachypnea were observed and persisted during oxygen supplementation. Arterial blood gas analysis by co-oximetry identified an increased methemoglobin concentration (27%; normal, <2%) with normal arterial oxygen tension. The methemoglobinemia and associated clinical signs resolved after administration of methylene blue (1 mg/kg) IV, and the dog was discharged. The affected dog's whole-genome sequence contained 2 potentially causal heterozygous CYB5R3 missense mutations suggesting that cytochrome b5 reductase deficiency was responsible for the methemoglobinemia. This hypothesis was confirmed by enzyme analysis that identified cytochrome b5 reductase activity in the affected dog's erythrocytes to only approximately 6% of that in a control sample. Clinical signs recurred 11 days after discharge but normalized and the methemoglobin concentration decreased with methylene blue administration PO (1.5 mg/kg, initially daily and then every other day). Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  14. The Interaction of Microsomal Cytochrome P450 2B4 with its Redox Partners, Cytochrome P450 Reductase and Cytochrome b5

    PubMed Central

    Im, Sang-Choul; Waskell, Lucy

    2010-01-01

    1 Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b5. In both instances, product formation occurs. When the second electron is donated by cytochrome b5, catalysis (product formation) is ∼ 10 to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼ 15% the coupling of NADPH consumption to product formation. Cytochrome b5 has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b5 on cytochrome P450 2B4 reactivity can explain how cytochrome b5 is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b5 to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochome b5 to cytochrome P450 reductase, cytochrome b5 inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b5 are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b5 stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase. PMID:21055385

  15. 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

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

    PubMed

    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.

  17. Comparative Studies on the Induction and Inactivation of Nitrate Reductase in Corn Roots and Leaves 1

    PubMed Central

    Aslam, Muhammad; Oaks, Ann

    1976-01-01

    A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH2-nitrate reductase and NO3−-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO3− was supplied. WO4= suppressed the induction of NADH- and FMNH2-nitrate reductase activities in root tips and leaves. The NO3−-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO4=. In leaves, on the other hand, there was a marked superinduction of the NO3−-induced NADH-cytochrome c reductase in the presence of WO4=. The half-life values of NADH-nitrate reductase and FMNH2-nitrate reductase measured by removing NO3− and adding WO4= to the medium, were 4 hours in root tips and 6 hours in excised leaves. Addition of NO3− in the induction medium together with WO4= gave partial protection of NADH-nitrate reductase and FMNH2-nitrate reductase activities in both root tips and leaves with a t0.5 of 6 and 8 hours, respectively. NO3− also reduced the loss of nitrate reductase activity from mature root sections. In the presence of cycloheximide, both NADH-nitrate reductase and NO3−-induced NADH-cytochrome c reductase activities were lost at similar rates in root tips. NO3− protected the loss of NO3−-induced NADH-cytochrome c reductase to the same extent as that of NADH-nitrate reductase. PMID:16659529

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

    PubMed

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

    2006-01-01

    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/K(m)) 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 A (1 A=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.

  19. 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

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

    PubMed

    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.

  1. 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

  2. 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.

  3. Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

    PubMed

    Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

    2012-02-15

    In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. 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.

  5. 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

    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.

  6. Protonation of the Hydroperoxo Intermediate of Cytochrome P450 2B4 Is Slower in the Presence of Cytochrome P450 Reductase Than in the Presence of Cytochrome b5.

    PubMed

    Pearl, Naw May; Wilcoxen, Jarett; Im, Sangchoul; Kunz, Ryan; Darty, Joseph; Britt, R David; Ragsdale, Stephen W; Waskell, Lucy

    2016-11-29

    Microsomal cytochromes P450 (P450) require two electrons and two protons for the oxidation of substrates. Although the two electrons can be provided by cytochrome P450 reductase, the second electron can also be donated by cytochrome b5 (b5). The steady-state activity of P450 2B4 is increased up to 10-fold by b5. To improve our understanding of the molecular basis of the stimulatory effect of b5 and to test the hypothesis that b5 stimulates catalysis by more rapid protonation of the anionic ferric hydroperoxo heme intermediate of P450 (Fe(3+)OOH)(-) and subsequent formation of the active oxidizing species (Fe(+4)═O POR(•+)), we have freeze-quenched the reaction mixture during a single turnover following reduction of oxyferrous P450 2B4 by each of its redox partners, b5 and P450 reductase. The electron paramagnetic resonance spectra of the freeze-quenched reaction mixtures lacked evidence of a hydroperoxo intermediate when b5 was the reductant presumably because hydroperoxo protonation and catalysis occurred within the dead time of the instrument. However, when P450 reductase was the reductant, a hydroperoxo P450 intermediate was observed. The effect of b5 on the enzymatic efficiency in D2O and the kinetic solvent isotope effect under steady-state conditions are both consistent with the ability of b5 to promote rapid protonation of the hydroperoxo species and more efficient catalysis. In summary, by binding to the proximal surface of P450, b5 stimulates the activity of P450 2B4 by enhancing the rate of protonation of the hydroperoxo intermediate and formation of Compound I, the active oxidizing species, which allows less time for side product formation.

  7. 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

  8. Proteolytic processing of CmPP36, a protein from the cytochrome b(5) reductase family, is required for entry into the phloem translocation pathway.

    PubMed

    Xoconostle-Cázares, B; Ruiz-Medrano, R; Lucas, W J

    2000-12-01

    Cucurbita maxima (pumpkin) phloem sap contains a 31 kDa protein that cross-reacts with antibodies directed against the red clover necrotic mosaic virus movement protein (RCNMV MP). Microsequence data from phloem-purified 31 kDa protein were used to isolate a complementary DNA: the open reading frame encodes a 36 kDa protein belonging to the cytochrome b(5) reductase (Cb5R) family; the gene was termed CmPP36. Western analyses established that CmPP36, RCNMV MP and CmPP16 (Xoconostle-Cázares et al., 1999, Science 283, 94-98) are immunologically related, probably due to a common epitope, represented by the NADH(+)-binding domain of CmPP36. An N-terminal 5 kDa membrane-targeting domain is cleaved to produce the 31 kDa Delta N-CmPP36 detected in the phloem sap. Microinjection experiments established that Delta N-CmPP36, but not CmPP36, is able to interact with plasmodesmata to mediate its cell-to-cell transport. Thus, intercellular movement of CmPP36 requires proteolytic processing in the companion cell to produce a soluble, movement-competent, protein. In contrast to RCNMV and CmPP16, Delta N-CmPP36 interacts with but does not mediate the trafficking of RNA. Northern and in situ RT-PCR studies established that CmPP36 mRNA is present in all plant organs, being highly abundant within vascular tissues. In roots of hydroponically grown pumpkin plants, CmPP36 mRNA levels respond to changes in available iron in the culture solution. Finally, enzymatic assays established that both CmPP36 and Delta N-CmPP36 could reduce Fe(3+)-citrate and Fe(3+)-EDTA in the presence of NADH(+). These findings are discussed in terms of the possible roles played by CmPP36 in phloem function.

  9. 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.

  10. 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.

  11. Ascorbate free radical reductases and diaphorases in soluble fractions of the human lens.

    PubMed

    Bando, M; Obazawa, H

    1995-12-01

    Major and minor ascorbate free radical (AFR) reductases, with diaphorase activity, and three other diaphorases were separated from the human lens soluble fraction by DEAE-cellulose ion-exchange column chromatography. They were characterized for adsorptivity to ion-exchange and 5'AMP-Sepharose 4B affinity columns, kinetic properties, and substrate specificity. The latter diaphorases were closely correlated with NADH-cytochrome beta 5 reductase. The major and minor AFR reductases were regarded as a major diaphorase group different from two ubiquitous diaphorases, i.e., NADH-cytochrome beta 5 reductase and DT-diaphorase. A major AFR reductase was partially purified approximately 50 fold over the lens soluble fraction by ion-exchange, affinity, and gel filtration (Sephacryl S-200 HR) column chromatography. From the partially purified enzyme, 2 bands, one sharp and one diffuse, were obtained by native polyacrylamide gel electrophoresis. Two proteins, of 20 and 24 kDa, were identified in the active enzyme bands by SDS-polyacrylamide gel electrophoresis. This suggests that the 20 and/or 24 kDa proteins may be components of the major AFR reductase.

  12. Limited proteolysis of the nitrate reductase from spinach leaves.

    PubMed

    Kubo, Y; Ogura, N; Nakagawa, H

    1988-12-25

    The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.

  13. 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.

  14. Reduction of sulfamethoxazole and dapsone hydroxylamines by a microsomal enzyme system purified from pig liver and pig and human liver microsomes.

    PubMed

    Clement, Bernd; Behrens, Detlef; Amschler, Juliane; Matschke, Katrin; Wolf, Stephanie; Havemeyer, Antje

    2005-05-27

    Biotransformation involving nitrogen are of pharmacological and toxicological relevance. In principle, nitrogen containing functional groups can undergo all the known biotransformation processes such as oxidation, reduction, hydrolysis and formation of conjugates. For the N-reduction of benzamidoxime an oxygen-insensitive liver microsomal enzyme system that required cytochrome b5, NADH-cytochrome b5 reductase and a cytochrome P450 isoenzyme of the subfamily 2D has been described. In previous studies it was demonstrated that N-hydroxylated derivates of strongly basic functional groups are easily reduced by this enzyme system. The N-hydroxylation of sulfonamides such sulfamethoxazole (SMX) and dapsone (DDS) to sulfamethoxazole-hydroxylamine (SMX-HA) and dapsone-hydroxylamine (DDS-N-OH), respectively is the first step in the formation of reactive metabolites. Therefore it seemed reasonable to study the potential of cytochrome b5, NADH-cytochrome b5 reductase and CYP2D to detoxify these N-hydroxylated metabolites by N-reduction. Metabolites were analysed by HPLC analysis. SMX-HA and DDS-N-OH are reduced by cytochrome b5, NADH-cytochrome b5 reductase and CYP2D but also only by cytochrome b5 and NADH-cytochrome b5 reductase without addition of CYP2D. The reduction rate for SMX-HA by cytochrome b5, NADH-cytochrome b5 reductase and CYP2D was 0,65 +/- 0,1 nmol SMX/min/mg protein. The reduction rate by b5 and b5 reductase was 0,37 +/- 0,15 nmol SMX/min/mg protein. For DDS-N-OH the reduction rate by cytochrome b5, NADH-cytochrome b5 reductase and CYP2D was 1.79 +/- 0.85 nmol DDS/min/mg protein and by cytochrome b5 and NADH-cytochrome b5 reductase 1.25 +/- 0.15 nmol DDS/min/mg protein. Cytochrome b5, NADH-cytochrome b5 reductase are therefore involved in the detoxification of these reactive hydroxylamines and CYP2D increased the N-reduction.

  15. Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA 1

    PubMed Central

    Campbell, Wilbur H.

    1992-01-01

    A cDNA clone was isolated from a maize (Zea mays L. cv W64A×W183E) scutellum λgt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction. ImagesFigure 2Figure 3 PMID:16668941

  16. NADH-coenzyme Q reductase (complex I) deficiency: heterogeneity in phenotype and biochemical findings.

    PubMed

    Pitkänen, S; Feigenbaum, A; Laframboise, R; Robinson, B H

    1996-01-01

    Twelve patient cell lines with biochemically proven complex I deficiency were compared for clinical presentation and outcome, together with their sensitivity to galactose and menadione toxicity. Each patient had elevated lactate to pyruvate ratios demonstrable in fibroblast cultures. Each patient also had decreased rotenone-sensitive NADH-cytochrome c reductase (complexes I and III) with normal succinate cytochrome c reductase (complexes II and III) and cytochrome oxidase (complex IV) activity in cultured skin fibroblasts, indicating a deficient NADH-coenzyme Q reductase (complex I) activity. The patients fell into five categories: severe neonatal lactic acidosis; Leigh disease; cardiomyopathy and cataracts; hepatopathy and tubulopathy; and mild symptoms with lactic acidaemia. Cell lines from 4 out of the 12 patients were susceptible to both galactose and menadione toxicity and 3 of these also displayed low levels of ATP synthesis in digitonin-permeabilized skin fibroblasts from a number of substrates. This study highlights the heterogeneity of complex I deficiency at the clinical and biochemical level.

  17. Pantothenic acid (Vitamin B5)

    MedlinePlus

    ... B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin/niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin ... to colds and other infections, retarded growth, shingles, skin disorders, stimulating adrenal glands, chronic fatigue syndrome, salicylate ...

  18. NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions.

    PubMed Central

    Reed, C J; Lock, E A; De Matteis, F

    1986-01-01

    The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue. Images Fig. 1. PMID:3101674

  19. Natural mutations lead to enhanced proteasomal degradation of human Ncb5or, a novel flavoheme reductase.

    PubMed

    Kálmán, Fanni S; Lizák, Beáta; Nagy, Szilvia K; Mészáros, Tamás; Zámbó, Veronika; Mandl, József; Csala, Miklós; Kereszturi, Eva

    2013-07-01

    NADH cytochrome b5 oxidoreductase (Ncb5or) protects β-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to β-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  20. 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. The...

  1. 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. The...

  2. 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 finds...

  3. 32 CFR 806b.5 - Personal notes.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 6 2011-07-01 2011-07-01 false Personal notes. 806b.5 Section 806b.5 National... Overview of the Privacy Act Program § 806b.5 Personal notes. The Privacy Act does not apply to personal notes on individuals used as memory aids. Personal notes may become Privacy Act records if they are...

  4. 32 CFR 806b.5 - Personal notes.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 6 2014-07-01 2014-07-01 false Personal notes. 806b.5 Section 806b.5 National... Overview of the Privacy Act Program § 806b.5 Personal notes. The Privacy Act does not apply to personal notes on individuals used as memory aids. Personal notes may become Privacy Act records if they are...

  5. 32 CFR 806b.5 - Personal notes.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 false Personal notes. 806b.5 Section 806b.5 National... Overview of the Privacy Act Program § 806b.5 Personal notes. The Privacy Act does not apply to personal notes on individuals used as memory aids. Personal notes may become Privacy Act records if they are...

  6. 32 CFR 806b.5 - Personal notes.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 6 2012-07-01 2012-07-01 false Personal notes. 806b.5 Section 806b.5 National... Overview of the Privacy Act Program § 806b.5 Personal notes. The Privacy Act does not apply to personal notes on individuals used as memory aids. Personal notes may become Privacy Act records if they are...

  7. 32 CFR 242b.5 - Voting.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 2 2014-07-01 2014-07-01 false Voting. 242b.5 Section 242b.5 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS GENERAL... SCIENCES § 242b.5 Voting. (a) The concurrence of a majority of the Regents present at a meeting shall be...

  8. 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 Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS GENERAL... SCIENCES § 242b.5 Voting. (a) The concurrence of a majority of the Regents present at a meeting shall be...

  9. 32 CFR 242b.5 - Voting.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 2 2012-07-01 2012-07-01 false Voting. 242b.5 Section 242b.5 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS GENERAL... SCIENCES § 242b.5 Voting. (a) The concurrence of a majority of the Regents present at a meeting shall be...

  10. 32 CFR 242b.5 - Voting.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 2 2013-07-01 2013-07-01 false Voting. 242b.5 Section 242b.5 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS GENERAL... SCIENCES § 242b.5 Voting. (a) The concurrence of a majority of the Regents present at a meeting shall be...

  11. 45 CFR 73b.5 - Hearings.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 45 Public Welfare 1 2014-10-01 2014-10-01 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 is...

  12. 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.... Orders of Investigation will outline the basis for the investigation, the matters to be investigated, the...

  13. 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.... Orders of Investigation will outline the basis for the investigation, the matters to be investigated, the...

  14. 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.... Orders of Investigation will outline the basis for the investigation, the matters to be investigated, the...

  15. 32 CFR 806b.5 - Personal notes.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Personal notes. 806b.5 Section 806b.5 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR FORCE ADMINISTRATION PRIVACY ACT PROGRAM... notes on individuals used as memory aids. Personal notes may become Privacy Act records if they...

  16. Thioredoxin reductase.

    PubMed Central

    Mustacich, D; Powis, G

    2000-01-01

    The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. PMID:10657232

  17. 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... notation voting, by voting on material circulated to Regents individually or serially, or by polling of...

  18. Effect of L-carnitine supplementation on xenobiotic-metabolizing hepatic enzymes exposed to methanol.

    PubMed

    Olszowy, Zofia; Plewka, Andrzej; Czech, Ewa; Nowicka, Joanna; Plewka, Danuta; Nowaczyk, Grazyna; Kamiński, Marcin

    2006-07-01

    The study aimed to evaluate the effect of L-carnitine on hepatic cytochrome P450-dependent monooxygenases exposed to methanol. Male Spraque-Dawley rats were given methanol (1/4 LD50 and 1/2 LD50) together with L-carnitine (1g/kg body weight). The parameters of microsome electron transport chains I and II and the levels of CYP2E1, CYP2B1/2 and CYP1A2 were measured 8, 12, 24, 48, 72 and 96 h after exposure. L-carnitine did not affect cytochrome P450 but it significantly increased at 72 and 96 h NADPH-cytochrome P450 reductase. It stimulated cytochrome b5 at 48 and 96 h and NADH-cytochrome b5 reductase activity at 12, 72 and 96 h. Methanol, especially the lower dose, inhibited cytochrome P450 after 48 h, but the higher methanol dose inhibited NADH-cytochrome b5 reductase activity in this time. L-carnitine, combined with the lower dose of methanol, stimulated NADPH-cytochrome P450 reductase after 48 h and cytochrome b5 and NADH-cytochrome b5 reductase over the whole period of observation. L-carnitine stimulated CYP2B1/2 but not CYP2E1 and CYP1A2. Methanol stimulated CYP2E1 at 24 h, but CYP1A2 at 96 h in the studied doses. CYP2B1/2 was induced by the lower dose of methanol at 24 h but by the higher one at 96 h. When given together, L-carnitine and methanol (1/2 LD50) significantly stimulated CYP2E1 up to 170% at 24 h and 145% at 96 h.

  19. Cytochrome b5 from Giardia lamblia.

    PubMed

    Alam, Samiah; Yee, Janet; Couture, Manon; Takayama, Shin-ichi J; Tseng, Wan-Hsin; Mauk, A Grant; Rafferty, Steven

    2012-12-01

    The protozoan intestinal parasite Giardia lamblia lacks mitochondria and the ability to make haem yet encodes several putative haem-binding proteins, including three of the cytochrome b(5) family. We cloned one of these (gCYTb5-I) and expressed it within Escherichia coli as a soluble holoprotein. UV-visible and resonance Raman spectra of gCYTb5-I resemble those of microsomal cytochrome b(5), and homology modelling supports a structure in which a pair of invariant histidine residues act as axial ligands to the haem iron. The reduction potential of gCYTb5-I is -165 mV vs. SHE and is relatively low compared to most values (-110 to +80 mV) for this class of protein. The amino- and carboxy-terminal sequences that flank the central haem-binding core of the Giardia cytochromes are highly charged and differ from those of other family members. A core gCYTb5-I variant lacking these flanking sequences was also able to bind haem. The presence of one actual and two probable functional cytochromes b(5) in Giardia is evidence of uncharacterized cytochrome-mediated metabolic processes within this medically important protist.

  20. 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).

  1. 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.

  2. Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons.

    PubMed Central

    Smith, M A; Cross, A R; Jones, O T; Griffiths, W T; Stymne, S; Stobart, K

    1990-01-01

    The major cytochrome in microsomal membrane preparations from developing seeds of safflower (Carthamus tinctorius, var High Linoleate), has a reduced-minus-oxidized difference spectrum characteristic of a b-type cytochrome, and was identified from its midpoint-potential (E'7.2) value as cytochrome b5. Cytochromes P-450 and P-420 were also present. The cytochrome b5 content of microsomal preparations from a number of oilseed species was found to be in the order of 200-300 pmol/mg of protein. The cytochrome b5 was reduced in the membrane preparations by NADH, demonstrating the presence of an NADH: cytochrome b5 reductase; NADPH was a less effective donor. Microsomal membranes catalysed the NAD(P)H-dependent conversion of radioactive oleate into linoleate, indicating acyl-CoA: lysophosphatidylcholine acyltransferase and 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) activity. Desaturation of oleate to linoleate was unaffected by CO, but inhibited by CN-. The addition of oleoyl-CoA to the NADH-reduced membranes resulted in the CN(-)-sensitive partial re-oxidation of cytochrome b5, indicating that electrons from NADH were transferred to the site of desaturation via this cytochrome. The delta 12-desaturase in safflower, therefore, is CN(-)-sensitive and appears to require cytochrome b5 and NADH: cytochrome b5 reductase for activity. PMID:2264826

  3. 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

  4. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2010 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)...

  5. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2010 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...

  6. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2010 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)...

  7. 17 CFR 260.10b-5 - Content.

    Code of Federal Regulations, 2010 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)...

  8. 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 and...

  9. 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 and...

  10. 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 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 and...

  11. 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 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 and...

  12. 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 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 and...

  13. 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. ...

  14. 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 polyethylene...

  15. 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 polyethylene...

  16. 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 polyethylene...

  17. 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 polyethylene...

  18. 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.

  19. 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) Each application for a stay of a trustee's duty...

  20. 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) Each application for a stay of a trustee's duty...

  1. 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) Each application for a stay of a trustee's duty...

  2. Cross-linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5

    PubMed Central

    Zhao, Chunsheng; Gao, Qiuxia; Roberts, Arthur G.; Shaffer, Scott A.; Doneanu, Catalin E.; Xue, Song; Goodlett, David R.; Nelson, Sidney D.; Atkins, William M.

    2012-01-01

    Cytochrome b5 (cyt b5) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached on the underlying mechanism of cyt b5 modulation of CYP catalysis. Both cyt b5 and apo b5, are reported to stimulate the activity of several P450 isoforms. In the present study, the surface interactions of both holo and apo b5 with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the interaction models of holo/apo b5 with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b5 and apo b5 were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B’ loop of CYP3A4, a substrate recognition site (SRS). Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127 and Lys421) are of functional importance. Mutation of these residues reduced or abolished cyt b5 binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b5 and/or cytochrome P450 reductase (CPR) was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that the apo cyt b5 can dock with CYP3A4 in a manner analogous to holo cyt b5 so electron transfer from cyt b5 is not required for its effects. PMID:23150942

  3. Cross-linking mass spectrometry and mutagenesis confirm the functional importance of surface interactions between CYP3A4 and holo/apo cytochrome b(5).

    PubMed

    Zhao, Chunsheng; Gao, Qiuxia; Roberts, Arthur G; Shaffer, Scott A; Doneanu, Catalin E; Xue, Song; Goodlett, David R; Nelson, Sidney D; Atkins, William M

    2012-11-27

    Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.

  4. 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...

  5. Comparison of cytochromes b5 from insects and vertebrates.

    PubMed

    Wang, Lijun; Cowley, Aaron B; Terzyan, Simon; Zhang, Xuejun; Benson, David R

    2007-05-01

    We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.

  6. Catalytic mechanism and substrate selectivity of aldo-keto reductases: insights from structure-function studies of Candida tenuis xylose reductase.

    PubMed

    Kratzer, Regina; Wilson, David K; Nidetzky, Bernd

    2006-09-01

    Aldo-keto reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)-dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure-function relationship studies for xylose reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the epsilon-NH3+ group of Lys is a key source of catalytic power of xylose reductase. A molecular-level analysis of the substrate binding pocket of xylose reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel reductases with improved specificities for biocatalytic applications.

  7. Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.).

    PubMed Central

    Smith, M A; Jonsson, L; Stymne, S; Stobart, K

    1992-01-01

    The major b-type cytochrome in microsomal membrane preparations from developing endosperm of castor bean (Ricinus communis) was cytochrome b5. Cytochrome P-450 was also present. The microsomal membranes had delta 12-hydroxylase activity and catalysed the NAD(P)H-dependent hydroxylation of oleate to yield ricinoleic acid. CO had no effect on the hydroxylase activity. Rabbit polyclonal antibodies were raised against the hydrophilic cytochrome b5 fragment purified from cauliflower (Brassica oleracea) floret microsomes. The anti-(cytochrome b5) IgG inhibited delta 12-hydroxylase, delta 12-desaturase and cytochrome c reductase activity in the microsomes. The results indicate that electrons from NAD(P)H were transferred to the site of hydroxylation via cytochrome b5 and that cytochrome P-450 was not involved. Images Fig. 1. PMID:1417766

  8. 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.

  9. 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

  10. Selective modification of rat hepatic microsomal fatty acid chain elongation and desaturation by fibrates: relationship with peroxisome proliferation.

    PubMed Central

    Alegret, M; Cerqueda, E; Ferrando, R; Vázquez, M; Sánchez, R M; Adzet, T; Merlos, M; Laguna, J C

    1995-01-01

    1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-, palmitoleoyl- and gamma-linolenoyl-CoA elongase, delta-9, delta-6 and delta-5 desaturase activities, and microsomal electron transport chains, as well as the correlation with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. As reported in our previous work, the three drugs behave as peroxisomal proliferators (the order of potency was BFB > CFB > or = GFB) and induced a clear reduction in both plasma cholesterol and triglyceride levels. 3. Palmitoyl-CoA elongation activity was increased by the three drugs (BFB = GFB > CFB), whereas palmitoleoyl-CoA elongation activity was only enhanced by GFB. Elongation activity was not modified by fibrates when gamma-linolenoyl-CoA was used as substrate. These results are in accordance with the existence of three different elongation systems for saturated, mono- and polyunsaturated fatty acids. 4. delta-9, delta-6 and delta-5 desaturase activities were increased by the three fibrates, with an order of potency BFB > CFB = GFB for delta-9 and delta-5, and GFB > BFB = CFB for delta-6. 5. Of the enzyme activities integrated in the microsomal electron transport chains, NADH cytochrome b5 reductase was not affected by fibrate treatment, NADPH cytochrome c reductase activity was enhanced (BFB = GFB > CFB), whereas NADH cytochrome c reductase activity was reduced by CFB and BFB.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7606338

  11. Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine

    PubMed Central

    Bhusari, Sachin; Abouraya, Mahmoud; Padilla, Marcia L.; Pinkerton, Marie E.; Drescher, Nicholas J.; Sacco, James C.; Trepanier, Lauren A.

    2010-01-01

    Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b5 (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity towards SMX-NO, and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue drug-adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b5, as well as significant decreases in SMX-HA reduction in SMX-NO treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in AIDS patients with antioxidant depletion. PMID:20221587

  12. Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine.

    PubMed

    Bhusari, Sachin; Abouraya, Mahmoud; Padilla, Marcia L; Pinkerton, Marie E; Drescher, Nicholas J; Sacco, James C; Trepanier, Lauren A

    2010-08-01

    Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T-cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b (5) (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity toward SMX-NO and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO-related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue-drug adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b (5), as well as significant decreases in SMX-HA reduction in SMX-NO-treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in patients with AIDS with antioxidant depletion.

  13. Low reduction potential cytochrome b5 isotypes of Giardia intestinalis.

    PubMed

    Pazdzior, Robert; Yang, Zhen Alice; Mesbahuddin, Mirfath Sultana; Yee, Janet; van der Est, Art; Rafferty, Steven

    2015-10-01

    Despite lacking mitochondria and a known pathway for heme biosynthesis the micro-aerotolerant anaerobic protozoan parasite Giardia intestinalis encodes four members of the cytochrome b5 family of electron transfer proteins, three of which are small, single-domain proteins. While these are similar in size and fold to their better-known mammalian counterparts the Giardia proteins have distinctly lower reduction potentials, ranging from -140 to -171 mV compared to +6 mV for the bovine microsomal protein. This difference is accounted for by a more polar heme environment in the Giardia proteins, as mutation of a conserved heme pocket tyrosine residue to phenylalanine in the Giardia cytochrome b5 isotype-I (gCYTb5-I Y61F) raises its reduction potential by nearly 100 mV. All three isotypes have UV-visible spectra consistent with axial coordination of the heme by a pair of histidine residues, but electron paramagnetic spectroscopy indicates that the planes of their imidazole rings are nearly perpendicular rather than coplanar as observed in mammalian cytochrome b5, which may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia proteins. Although no function has yet to be ascribed to any Giardia cytochrome b5, the presence of similar sequences in many other eukaryotes indicates that these represent an under-characterized class of low reduction potential family members.

  14. Rate of alteration of hepatic mixed-function oxidase system in rats fed different dietary fats.

    PubMed

    Ammouche, A; Dinh, L; Youyou, A; Clément, M; Bourre, J M

    1993-01-01

    Studies were carried out to evaluate and relate the rate of alteration in mixed-function oxidase system with the changes of the fatty acid composition of rat microsomes induced by different dietary lipids. Male weanling rats were fed from day 21 to 120 with a commercial rat diet or a semisynthetic diet containing no fat or 10% fat consisting of peanut-rapeseed oil, sunflower oil, or salmon oil. In rats fed a fat-free diet, the cytochrome P-450 concentration and aniline hydroxylase, aminopyrine N-demethylase, and NADPH-cytochrome-c reductase activities of liver microsomes at 120 days were, respectively, 26, 16, 10, and 24% lesser than those of rats fed the control diet. However, cytochrome b5 concentration and NADH-cytochrome-b5 reductase activity were, respectively, 33 and 43% higher than those of the control group at the same time. When rats were fed the sunflower oil diet, the cytochrome P-450 concentration and NADH-cytochrome-b5 reductase activity at 120 days were, respectively, 11 and 23% lesser than those of control group. But the cytochrome b5 concentration was 10% higher than that of the control group. In rats fed the fish oil diet, the cytochrome P-450 concentration and NADPH-cytochrome-c reductase, aniline hydroxylase, and aminopyrine N-demethylase activities at 120 days were, respectively, 30, 48, 41, and 31% higher than those of rats fed the control diet. These enzymes were correlated very well (0.84 < r < 0.93), P < 0.05 with dietary sigma polyunsaturated fatty acids (n-3).(ABSTRACT TRUNCATED AT 250 WORDS)

  15. 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

  16. 22 CFR 9b.5 - Temporary Department of State press building passes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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 media... pass valid for one day....

  17. Electron transfer properties and catalytic competence of cytochrome b5 in the fusion protein Hmwb5-EGFP in reactions catalyzed by cytochrome P450 3A4.

    PubMed

    Yantsevich, A V; Gilep, A A; Usanov, S A

    2009-08-01

    In the present paper we describe studies on molecular mechanisms of protein-protein interactions between cytochrome P450 3A4 (CYP3A4) and cytochrome b(5), the latter being incorporated into the artificial recombinant protein Hmwb(5)-EGFP containing full-length cytochrome b(5) (functional module) and a mutant form of the green fluorescent protein EGFP (signal module) fused into a single polypeptide chain. It is shown that cytochrome b(5) within the fusion protein Hmwb(5)-EGFP can be reduced by NADPH-cytochrome P450 reductase in the presence of NADPH, the rate of reduction being dependent on solution ionic strength, indicating that the signal module does not prevent the interaction of the flavo- and hemeproteins. Interaction of cytochrome P450 3A4 and Hmwb(5)-EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb(5)-EGFP, as well as based on steady-state fluorescence anisotropy of the EGFP component of the fusion protein in the presence of CYP3A4. The engineering of chimeric protein Hmwb(5)-EGFP gives an independent method to determine dissociation constant for the complex of cytochrome P450 and cytochrome b(5) that is less sensitive to environmental factors compared to spectrophotometric titration used before. Reconstitution of catalytic activity of cytochrome P450 3A4 in the reaction of testosterone 6beta-hydroxylation in the presence of Hmwb(5)-EGFP indicates that cytochrome b(5) in the fusion protein is able to stimulate the hydroxylation reaction. Using other fusion proteins containing either cytochrome b(5) or its hydrophilic domain to reconstitute catalytic activity of cytochrome P450 3A4 showed that the hydrophobic domain of cytochrome b(5) participates not only in hemeprotein interaction, but also in electron transfer from cytochrome b(5) to cytochrome P450.

  18. Hydrocarbon biomarkers responses in the bivalve, Tivela mactroides, exposed to polluted sediments.

    PubMed

    Sardi, A E; Ramos, R; García, E M

    2013-01-01

    The objective of this work was to evaluate the potential use of the bivalve Tivela mactroides, as a Caribbean sentinel species. Organisms were collected from a relatively clean sandy beach, and were exposed to a gradient of petroleum hydrocarbon-polluted sediments for periods of 3, 4 and 6 days, after which their activity levels of biotransformation enzymes were measured. NADPH-cytochrome c reductase, glutathione transferase and cytochrome b5 activities increased according to total hydrocarbon concentration, whereas NADH-cytochrome ferricyanide reductase activity decreased. Catalase activity was constant while superoxide dismutase activity was inhibited. The results indicated that T. mactroides may serve as a sentinel species. However, further studies are recommended on the influence of biotic and abiotic variables on basal activities of enzymes, and their responses to other pollutants.

  19. Reduction of Hexavalent Chromium by Human Cytochrome b5: Generation of Hydroxyl Radical and Superoxide

    PubMed Central

    Borthiry, Griselda R.; Antholine, William E.; Kalyanaraman, B.; Myers, Judith M.; Myers, Charles R.

    2007-01-01

    The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b5 and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure 53Cr(VI). When 5-Diethoxyphos-phoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO•) adduct was observed as well as a smaller signal for the superoxide (O2•−) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 μM). NADPH, Cr(VI), O2 and the PLs were all required for significant HO• generation. Superoxide dismutase eliminated the O2• − adduct and resulted in a 30% increase in the HO• adduct. Catalase largely diminished the HO• adduct signal indicating its dependence on H2O2. Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H2O2 was not needed, indicating that it was generated by the PLs. Adding exogenous H2O2, however, did increase the amount of DEPMPO/HO• adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethylsulfoxide (DMSO) plus the spin trap α-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO•. Quantification of the various species over time was consistent with a stoichiometric excess of HO• relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b5 in the generation of HO•. Overall, the simultaneous generation of Cr(V) and H2O2 by the PLs and the resulting generation of HO• at low Cr

  20. Effects of membrane mimetics on cytochrome P450-cytochrome b5 interactions characterized by NMR spectroscopy.

    PubMed

    Zhang, Meng; Huang, Rui; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2015-05-15

    Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb5 interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb5 at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb5 resonances characterize CYP2B4-cytb5 interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb5, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb5 imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb5 complex formation by orienting tr-CYP2B4 for efficient contact with cytb5. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb5 in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb5 complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Characterization of a Putative Ancestor of Coxsackievirus B5

    PubMed Central

    Gullberg, Maria; Tolf, Conny; Jonsson, Nina; Mulders, Mick N.; Savolainen-Kopra, Carita; Hovi, Tapani; Van Ranst, Marc; Lemey, Philippe; Hafenstein, Susan; Lindberg, A. Michael

    2010-01-01

    Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines. PMID:20631132

  2. 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.

  3. 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.

  4. Histochemical localization of nitrate reductase.

    PubMed

    Vaughn, K C; Duke, S O

    1981-01-01

    NADH-dependent nitrate reductase (E.C. 1.6.6.1) was ultrastructurally localized in norflurazon-treated and control soybean cotyledons [Glycine max (L.) Merr.] by a method based upon the increase in osmiophilia due to the formation of an azo dye. The reaction product was observed in small vesicles throughout the cytoplasm. An apparent transport of nitrite to the plastid, the site of nitrite reduction, may occur through fusion of the nitrite-containing vesicles with the chloroplast envelope. Plants grown in tungstate lacked nitrate reductase activity as measured by standard assay procedures, and showed no increase in osmiophilia, suggesting a degree of specificity of this cytochemical procedure.

  5. Effect of magnesium ions on the activity of the cytosolic NADH/cytochrome c electron transport system.

    PubMed

    La Piana, Gianluigi; Gorgoglione, Vincenza; Laraspata, Daniela; Marzulli, Domenico; Lofrumento, Nicola E

    2008-12-01

    Cytochrome c (cyto-c), added to isolated mitochondria, activates the oxidation of extramitochondrial NADH and the generation of a membrane potential, both linked to the activity of the cytosolic NADH/cyto-c electron transport pathway. The data presented in this article show that the protective effect of magnesium ions on the permeability of the mitochondrial outer membrane, supported by previously published data, correlates with the finding that, in hypotonic but not isotonic medium, magnesium promotes a differential effect on both the additional release of endogenous cyto-c and on the increased rate of NADH oxidation, depending on whether it is added before or after the mitochondria. At the same time, magnesium prevents or almost completely removes the binding of exogenously added cyto-c. We suggest that, in physiological low-amplitude swelling, magnesium ions may have the function, together with other factors, of modulating the amount of cyto-c molecules transferred from the mitochondrial intermembrane space into the cytosol, required for the correct execution of the apoptotic programme and/or the activation of the NADH/cyto-c electron transport pathway.

  6. Stimulation by pro-apoptotic valinomycin of cytosolic NADH/cytochrome c electron transport pathway-Effect of SH reagents.

    PubMed

    Lofrumento, Dario Domenico; La Piana, Gianluigi; Palmitessa, Valeria; Abbrescia, Daniela Isabel; Lofrumento, Nicola Elio

    2016-07-01

    Intrinsic and extrinsic apoptosis are both characterised by the presence of cytochrome c (cyto-c) in the cytosol. We present data on the extra-mitochondrial NADH oxidation catalysed by exogenous (cytosolic) cyto-c, as a possible answer to the paradox of apoptosis being an energy-dependent program but characterized by the impairment of the respiratory chain. The reduction of molecular oxygen induced by the cytosolic NADH/cyto-c pathway is coupled to the generation of an electrochemical proton gradient available for ATP synthesis. Original findings show that SH reagents inhibit the NADH/cyto-c system with a conformational change mechanism. The mitochondrial integrity-test of sulfite oxidase unequivocally demonstrates that this enzyme (120kDa) can be released outside but exogenous cyto-c (12.5kDa) does not permeate into mitochondria. Valinomycin at 2nM stimulates both the energy-dependent reversible mitochondrial swelling and the NADH/cyto-c oxidation pathway. The pro-apoptotic activity of valinomycin, as well as to the dissipation of membrane potential, can be also ascribed to the increased activity of the NADH/cyto-c oxidation pathway useful as an additional source of energy for apoptosis. It can be speculated that the activation of the NADH/cyto-c system coupled to valinomycin-induced mitochondrial osmotic swelling may represent a strategy to activate apoptosis in confined solid tumours. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. 22 CFR 9b.5 - Temporary Department of State press building passes.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 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...

  8. 17 CFR 240.10b-5 - Employment of manipulative and deceptive devices.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... deceptive devices. 240.10b-5 Section 240.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... Under the Securities Exchange Act of 1934 Manipulative and Deceptive Devices and Contrivances § 240.10b-5 Employment of manipulative and deceptive devices. It shall be unlawful for any person, directly...

  9. 17 CFR 240.10b-5 - Employment of manipulative and deceptive devices.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... deceptive devices. 240.10b-5 Section 240.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... Under the Securities Exchange Act of 1934 Manipulative and Deceptive Devices and Contrivances § 240.10b-5 Employment of manipulative and deceptive devices. It shall be unlawful for any person, directly...

  10. 17 CFR 240.10b-5 - Employment of manipulative and deceptive devices.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... deceptive devices. 240.10b-5 Section 240.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... Under the Securities Exchange Act of 1934 Manipulative and Deceptive Devices and Contrivances § 240.10b-5 Employment of manipulative and deceptive devices. It shall be unlawful for any person, directly...

  11. 17 CFR 240.10b-5 - Employment of manipulative and deceptive devices.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... deceptive devices. 240.10b-5 Section 240.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... Under the Securities Exchange Act of 1934 Manipulative and Deceptive Devices and Contrivances § 240.10b-5 Employment of manipulative and deceptive devices. It shall be unlawful for any person, directly...

  12. 17 CFR 240.10b-5 - Employment of manipulative and deceptive devices.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... deceptive devices. 240.10b-5 Section 240.10b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... Under the Securities Exchange Act of 1934 Manipulative and Deceptive Devices and Contrivances § 240.10b-5 Employment of manipulative and deceptive devices. It shall be unlawful for any person, directly...

  13. 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 Section 1.410(b)-5 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.410(b)-5 Average benefit percentage test. (a) General rule....

  14. 22 CFR 9b.5 - Temporary Department of State press building passes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 22 Foreign Relations 1 2014-04-01 2014-04-01 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...

  15. 22 CFR 9b.5 - Temporary Department of State press building passes.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 22 Foreign Relations 1 2013-04-01 2013-04-01 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...

  16. 22 CFR 9b.5 - Temporary Department of State press building passes.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 22 Foreign Relations 1 2012-04-01 2012-04-01 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...

  17. 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 with...

  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. Neurometabolic disorder with microcephaly, dystonia, and central cyanosis masquerading as cerebral palsy.

    PubMed

    Devadathan, Kalpana; Sreedharan, Mini; Sarasam, Sanuja; Colah, Roshan B; Kunju, P A Mohammed

    2014-11-01

    Many neurodegenerative diseases can be misdiagnosed as cerebral palsy. The correct diagnosis is reached when the condition recurs in families or when there are specific clinical signs. The clinical and imaging features of 3 children, from 2 unrelated families, presenting with global developmental delay and dystonia are described, in whom the presence of cyanosis and methemoglobinemia confirmed the diagnosis of recessive hereditary methemoglobinemia type 2. Magnetic resonance imaging showed significant cerebellar atrophy in 2 of the 3 babies. In dark-skinned children, this condition is underdiagnosed, as mild cyanosis is difficult to detect. Screening for methemoglobinemia in children with dystonia, microcephaly, and progressive cerebellar atrophy can be helpful in identifying more cases. As there is no curative treatment for this autosomal recessive condition, the exact diagnosis offers the best chance for prenatal screening, by detecting deficient NADH--cytochrome b5 reductase enzyme activity or by identifying the specific mutation in cultured amniotic fluid cells. © The Author(s) 2014.

  20. Tumor necrosis factor microsatellite markers TNFa5b5 and TNFa6b5 influence adverse reactions to parenteral gold in Caucasians.

    PubMed

    Evans, T I; Small, R E; Redford, T W; Han, J; Moxley, G

    1999-11-01

    To investigate which HLA haplotypes identified by DRB1 or tumor necrosis factor (TNF) microsatellite markers are associated with adverse reactions to parenteral gold injections. We retrospectively studied 193 Caucasian subjects with rheumatoid arthritis (RA) who had received parenteral gold injections from a university faculty outpatient practice (n = 163) and outpatient clinics at a Department of Veterans Affairs Medical Center (n = 30). DRB1 typing was done by several DNA based techniques. TNF microsatellite genotypes were derived by polymerase chain reaction amplification, sequencing-type gel electrophoresis, and silver staining. Seventy-six subjects had experienced adverse reactions to gold injections (other than nitritoid reactions), 18 of whom had 2 concurrent toxicities. The numbers with adverse reactions included: mucocutaneous (57), proteinuria (25), hematuria without proteinuria (5), thrombocytopenia (3), and miscellaneous (11). By frequency comparisons, no DR was associated with adverse reactions to parenteral gold (chi-squared 4.7, 6 df, NS). Specifically, there was no increased risk of proteinuria or mucocutaneous side effects in the DR3 positive RA group, almost all of whom had the DRB1 allele *0301. By logistic regression modeling controlling for sex and onset age, DR12 and the TNF microsatellite markers a5b5 and a6b5 were associated with mucocutaneous reactions (p < 0.05 for each). The odds ratios favoring mucocutaneous adverse reactions were 3.72 with TNFa5b5 and 2.03 with TNFa6b5. TNFa5b5 was commonly found on the HLA haplotypes bearing DRB1*0101, and TNFa6b5 was on the ones bearing DRB1 alleles of the DR1, DR2, DR3, DR5, or DR6 groups or the DRB1*0401 allele. HLA haplotypes conferring risk of gold induced mucocutaneous reactions were better identified by certain HLA class III markers, namely TNFa5b5 and TNFa6b5, than by any previously associated DR groups.

  1. Deficiency of subunits of Complex I and mitochondrial encephalomyopathy.

    PubMed

    Ichiki, T; Tanaka, M; Nishikimi, M; Suzuki, H; Ozawa, T; Kobayashi, M; Wada, Y

    1988-03-01

    Enzymic activities of the respiratory chain and content of immunochemically detectable subunits in NADH-ubiquinone oxidoreductase (Complex I) were measured in mitochondria from the skeletal muscles of 4 patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). The rotenone-sensitive NADH-cytochrome c reductase activity was extremely decreased, ranging from 0% to 27% of the control value. In all patients, the content of subunits of Complex I was also reduced in parallel with the rotenone-sensitive NADH-cytochrome c reductase activity. It is suggested that the variation in the degree of deficiency of Complex I subunits could explain the clinical heterogeneity of patients with MELAS.

  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. Cytochrome b5 promotes the synthesis of delta 16-C19 steroids by homogeneous cytochrome P-450 C21 side-chain cleavage from pig testis.

    PubMed

    Nakajin, S; Takahashi, M; Shinoda, M; Hall, P F

    1985-10-30

    Conversion of progesterone to 17 alpha-hydroxyprogesterone plus androstenedione (17 alpha-hydroxylation) and to androstadienone (delta 16 synthetase activity) by microsomes from neonatal pig testis, were both inhibited by antibodies raised against homogeneous cytochrome P-450 C21 side-chain cleavage. Inhibition of the two activities showed the same relationship to the concentration of antibody added. Analogous results were obtained with pregnenolone as substrate. In a reconstituted enzyme system consisting of the homogeneous cytochrome P-450 C21 side-chain cleavage enzyme, P-450 reductase and NADPH, addition of cytochrome b5 resulted in the synthesis of the corresponding delta 16-C19-steroid from progesterone (androstadienone) and pregnenolone (androstadienol). The effect of cytochrome b5 was concentration-dependent and prevented by anti-cytochrome b5. It is concluded that the cytochrome P-450 C21 side-chain cleavage enzyme from pig testicular microsomes is also capable of synthesizing delta 16-C19-steroids and is, therefore, likely to be responsible for the large amounts of the pherormone androstadienone produced by male pigs.

  4. Role of cytochrome b5 in the modulation of the enzymatic activities of cytochrome P450 17α-hydroxylase/17,20-lyase (P450 17A1).

    PubMed

    Bhatt, Megh Raj; Khatri, Yogan; Rodgers, Raymond J; Martin, Lisandra L

    2017-06-01

    Cytochrome b5 (cyt b5) is a small hemoprotein that plays a significant role in the modulation of activities of an important steroidogenic enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (P450 17A1, CYP17A1). Located in the zona fasciculata and zona reticularis of the adrenal cortex and in the gonads, P450 17A1 catalyzes two different reactions in the steroidogenic pathway; the 17α-hydroxylation and 17,20-lyase, in the endoplasmic reticulum of these respective tissues. The activities of P450 17A1 are regulated by cyt b5 that enhances the 17,20-lyase reaction by promoting the coupling of P450 17A1 and cytochrome P450 reductase (CPR), allosterically. Cyt b5 can also act as an electron donor to enhance the 16-ene-synthase activity of human P450 17A1. In this review, we discuss the many roles of cyt b5 and focus on the modulation of CYP17A1 activities by cyt b5 and the mechanisms involved. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. 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

  6. 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.

  7. 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

  8. 40 CFR Table B-5 to Subpart B of... - Symbols and Abbreviations

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 6 2014-07-01 2014-07-01 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...

  9. 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...

  10. 40 CFR Table B-5 to Subpart B of... - Symbols and Abbreviations

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 6 2012-07-01 2012-07-01 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...

  11. 17 CFR 240.12b-5 - Determination of affiliates of banks.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... securities of the bank held by a corporation all of the stock of which is directly owned by the United States... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Determination of affiliates of banks. 240.12b-5 Section 240.12b-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION...

  12. 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. ...

  13. 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. ...

  14. 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. ...

  15. 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. ...

  16. 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. ...

  17. 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.

  18. SerpinB5 interacts with KHDRBS3 and FBXO32 in gastric cancer cells.

    PubMed

    Lei, Ke-Feng; Liu, Bing-Ya; Wang, Yan-Fang; Chen, Xue-Hua; Yu, Bei-Qin; Guo, Yan; Zhu, Zheng-Gang

    2011-11-01

    Mammary serine protease inhibitor B5 (SerpinB5) is a potential oncogene in gastric cancer (GC); however, the molecular mechanism by which SerpinB5 promotes oncogenesis remains elusive. In this study, SerpinB5-associated proteins were selected based on yeast two-hybrid screening and microarray analysis after RNA interference and were validated using co-immunoprecipitation (Co-IP) and RNA Co-IP. The expression profiles of the interacting proteins were analyzed by Western blotting and immunohistochemistry. The effects of SerpinB5 on KHDRBS3 and FBXO32 expression in GC cells were analyzed using real-time PCR and Western blotting after the expression of SerpinB5 was modified. By yeast two-hybrid screening and microarray analysis, FBXO32 and KHDRBS3 were found to be SerpinB5-interacting proteins. The interactions were confirmed by Co-IP. An RNA co-immunoprecipitation assay found that KHDRBS3 interacted with FBXO32 mRNA. The expression of SerpinB5 was much stronger in the nucleus of GC cells. FBXO32 was expressed at higher levels in the cytoplasm of GC cells. KHDRBS3 was primarily detected in the nucleus of normal mucosal cells. SerpinB5 expression was modified in GC cells, KHDRBS3 mRNA levels remained stable, however, FBXO32 mRNA levels changed 24 h after changes in KHDRBS3 protein levels were detected. In conclusion, SerpinB5 interacts with KHDRBS3 and FBXO32, and KHDRBS3 can interact with FBXO32 mRNA.

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

    PubMed

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

    2014-01-24

    Uranium is harmful to human health due to its radiation damage and the ability of uranyl ion (UO2(2+)) 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 UO2(2+) 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.

  20. Human aldose reductase and human small intestine aldose reductase are efficient retinal reductases: consequences for retinoid metabolism.

    PubMed

    Crosas, Bernat; Hyndman, David J; Gallego, Oriol; Martras, Sílvia; Parés, Xavier; Flynn, T Geoffrey; Farrés, Jaume

    2003-08-01

    Aldo-keto reductases (AKRs) are NAD(P)H-dependent oxidoreductases that catalyse the reduction of a variety of carbonyl compounds, such as carbohydrates, aliphatic and aromatic aldehydes and steroids. We have studied the retinal reductase activity of human aldose reductase (AR), human small-intestine (HSI) AR and pig aldehyde reductase. Human AR and HSI AR were very efficient in the reduction of all- trans -, 9- cis - and 13- cis -retinal ( k (cat)/ K (m)=1100-10300 mM(-1).min(-1)), constituting the first cytosolic NADP(H)-dependent retinal reductases described in humans. Aldehyde reductase showed no activity with these retinal isomers. Glucose was a poor inhibitor ( K (i)=80 mM) of retinal reductase activity of human AR, whereas tolrestat, a classical AKR inhibitor used pharmacologically to treat diabetes, inhibited retinal reduction by human AR and HSI AR. All- trans -retinoic acid failed to inhibit both enzymes. In this paper we present the AKRs as an emergent superfamily of retinal-active enzymes, putatively involved in the regulation of retinoid biological activity through the assimilation of retinoids from beta-carotene and the control of retinal bioavailability.

  1. Neuroprotective role for carbonyl reductase?

    PubMed

    Maser, Edmund

    2006-02-24

    Oxidative stress is increasingly implicated in neurodegenerative disorders including Alzheimer's, Parkinson's, Huntington's, and Creutzfeld-Jakob diseases or amyotrophic lateral sclerosis. Reactive oxygen species seem to play a significant role in neuronal cell death in that they generate reactive aldehydes from membrane lipid peroxidation. Several neuronal diseases are associated with increased accumulation of abnormal protein adducts of reactive aldehydes, which mediate oxidative stress-linked pathological events, including cellular growth inhibition and apoptosis induction. Combining findings on neurodegeneration and oxidative stress in Drosophila with studies on the metabolic characteristics of the human enzyme carbonyl reductase (CR), it is clear now that CR has a potential physiological role for neuroprotection in humans. Several lines of evidence suggest that CR represents a significant pathway for the detoxification of reactive aldehydes derived from lipid peroxidation and that CR in humans is essential for neuronal cell survival and to confer protection against oxidative stress-induced brain degeneration.

  2. Observation of heme transfer from cytochrome b5 to DNA aptamer.

    PubMed

    Lin, Ying-Wu; Sun, Mei-Hui; Wan, Dun; Liao, Li-Fu

    2012-10-01

    Heme transfer is commonly observed from one heme protein to the other such as from cytochrome b(5) (cyt b(5)) to apo-myoglobin. In this study, instead of to another heme protein, we observed the heme transfer from wild-type (WT) cyt b(5), H39C cyt b(5) with heme axial ligand His39 mutated to Cys39, and DME cyt b(5) with heme replaced by protoporphyrin IX dimethyl ester, to a heme DNA aptamer, PS2.M, respectively, with a different rate constant. The heme transfer was further confirmed by the enhancement of peroxidase activity of the cyt b(5)s-PS2.M system due to the formation of catalytic PS2.M-heme complex. This study provides valuable insights into both cyt b(5)-heme and PS2.M-heme interactions and shows that heme transfer from heme protein to heme-aptamer can be used to evaluate the relative stability of heme proteins. In addition, this study sheds light on the maturation of heme proteins in vivo by interacting with DNA/RNA enzymes. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  3. Effect of the sarcosine residue on sequence scrambling in peptide b(5) ions.

    PubMed

    Harrison, Alex G

    2014-02-01

    The effect of N-methylation on sequence scrambling in the fragmentation of b5 ions has been investigated by studying a variety of peptides containing sarcosine (N-methylglycine). The product ion mass spectra for the b5 ions derived from Sar-A-A-A-Y-A and Sar-A-A-Y-A-A show only minor signals for non-direct sequence ions the major fragmentation reactions occurring from the unrearranged structures. This is in contrast to the b5 ions where the Sar residue is replaced by Ala and sequence scrambling occurs. The b5 ion derived from Y-Sar-A-A-A-A shows a product ion mass spectrum essentially identical to the spectrum of the b5 ion derived from Sar-A-A-A-Y-A, indicating that in the former case macrocyclization has occurred but the macrocyclic form shows a strong preference to reopen to put the Sar residue in the N-terminal position. Similar results were obtained in the comparison of b5 ions derived from A-Sar-A-A-Y-A and Sar-A-A-Y-A-A. The product ion mass spectra of the MH(+) ions of Y-Sar-A-A-A-A and A-Sar-A-A-Y-A show substantial signals for non-direct sequence ions indicating that fragmentation of the MH(+) ions channels extensively through the respective b5 ions and further fragmentation of these species.

  4. Faster Interprotein Electron Transfer in a [Myoglobin, b5] Complex with a Redesigned Interface

    PubMed Central

    Xiong, Peng; Nocek, Judith M.; Vura-Weis, Josh; Lockard, Jenny V.; Wasielewski, Michael R.; Hoffman, Brian M.

    2014-01-01

    Direct measurements of electron transfer (ET) within a protein-protein complex with a redesigned interface formed by physiological partner proteins myoglobin (Mb) and cytochrome b5 (b5) reveal interprotein ET rates comparable to those observed within the photosynthetic reaction center. Brownian dynamics simulations show that Mb in which three surface acid residues are mutated to lysine binds b5 in an ensemble of configurations distributed around a reactive most-probable structure. Correspondingly, charge-separation ET from a photoexcited singlet zinc porphyrin incorporated within Mb to the heme of b5 and the follow-up charge-recombination exhibit distributed kinetics, with median rate constants, kfs=2.1×109second−1 and kbs=4.3×1010second−1, respectively. The latter approaches that for the initial step in photosynthetic charge separation, k = 3.3 × 1011 second−1. PMID:21097931

  5. Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

    PubMed Central

    Sohma, Youhei; Hua, Qing-xin; Liu, Ming; Phillips, Nelson B.; Hu, Shi-Quan; Whittaker, Jonathan; Whittaker, Linda J.; Ng, Aubree; Roberts, Charles T.; Arvan, Peter; Kent, Stephen B. H.; Weiss, Michael A.

    2010-01-01

    Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, HisB5 → Thr markedly destabilizes the hormone (ΔΔGu 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution ThrB5 → His (residue 4) specifies a unique structure with native 1H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, HisB5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that HisB5 does not significantly enhance thermodynamic stability (ΔΔGu 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of ThrB5-insulin is decreased 5-fold, HisB5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of ThrB5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of HisB5 in insulin highlights its critical role in insulin biosynthesis. PMID:19959476

  6. Determination of the hemoglobin surface domains that react with cytochrome b5.

    PubMed

    Naito, N R; Hui, H L; Noble, R W; Hoffman, B M

    2001-02-20

    We have compared the photoinitiated electron-transfer (ET) reaction between cytochrome b(5) (b(5)) and zinc mesoporphyrin-substituted hemoglobin [(ZnM)Hb] and Hb variants in order to determine whether b(5) binds to the subunit surface of either or both Hb chains, or to sites which span the dimer--dimer interface. Because the dimer--dimer interface would be disrupted for monomers or alpha beta dimers, we studied the reaction of b(5) with alpha ZnM chains and (ZnM)Hb beta W37E, which exists as alpha beta dimers in solution. Triplet quenching titrations of the ZnHb proteins with Fe(3+)b(5) show that the binding affinity and ET rate constants for the alpha-chains are the same when they are incorporated into a Hb tetramer or dimer, or exist as monomers. Likewise, the parameters for beta-chains in tetramers and dimers differ minimally. In parallel, we have modified the surface of the Hb chains by neutralizing the heme propionates through the preparation of zinc deuterioporphyrin dimethyl ester hemoglobin, (ZnD-DME)Hb. The charge neutralization increases the ET rate constants 100-fold for the alpha-chains and 40-fold for the beta-chains (but has has little effect on the affinity of either chain type for b(5), similar to earlier results for myoglobin). Together, these results indicate that b(5) binds to sites at the subunit surface of each chain rather than to sites which span the dimer-dimer interface. The charge-neutralization results further suggest that b(5) binds over a broad area of the subunit face, but reacts only in a minority population of binding geometries.

  7. Tumor suppressor role of miR-3622b-5p in ERBB2-positive cancer.

    PubMed

    Lu, Mingjie; Wang, Tongshan; He, Mingfeng; Cheng, Wenfang; Yan, Ting; Huang, Zebo; Zhang, Lan; Zhang, Huo; Zhu, Wei; Zhu, Yichao; Liu, Ping

    2017-02-01

    Over-expression or amplification of ERBB2 is observed in multifarious carcinomas. However, the molecular mechanism of ERBB2 downregulation in ERBB2-positive cancers remains obscure. This experiment investigated the suppressive role of miR-3622b-5p in ERBB2-positive breast and gastric cancers. The luciferase activity of ERBB2 3'-untranslated region-based reporters constructed in HEK-293T, SK-BR-3 and MCF-10A cells suggested that ERBB2 was the target gene of miR-3622b-5p. Over-expressed miR-3622b-5p reduced the protein level of ERBB2, weakened the activation of mTORC1/S6, and induced the apoptosis of ERBB2-positive cancer cells. MiR-3622b-5p was significantly down-regulated in breast and gastric cancer tissues. This down-regulation in ERBB2-positive breast and gastric cancer tissues was more obvious than that in ERBB2-negative breast and gastric cancer tissues. MiR-3622b-5p turned ERBB2-positive cancer cells more vulnerable to the apoptosis induced by cisplatin and 5-fluorouracil. Taken together, miR-3622b-5p is involved in the proliferation and apoptosis of human ERBB2-positive cancer cells via targeting ERBB2/mTORC1 signaling pathway.

  8. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... About half of these individuals adopt a male gender role in adolescence or early adulthood. Related Information ... 1730-5. Citation on PubMed Cohen-Kettenis PT. Gender change in 46,XY persons with 5alpha-reductase- ...

  9. 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.

  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. Characterization of thyroidal glutathione reductase

    SciTech Connect

    Raasch, R.J.

    1989-01-01

    Glutathione levels were determined in bovine and rat thyroid tissue by enzymatic conjugation with 1-chloro-2,4-dinitrobenzene using glutathione S-transferase. Bovine thyroid tissue contained 1.31 {+-} 0.04 mM reduced glutathione (GSH) and 0.14 {+-} 0.02 mM oxidized glutathione (GSSG). In the rat, the concentration of GSH was 2.50 {+-} 0.05 mM while GSSG was 0.21 {+-} 0.03 mM. Glutathione reductase (GR) was purified from bovine thyroid to electrophoretic homogeneity by ion exchange, affinity and molecular exclusion chromatography. A molecular weight range of 102-109 kDa and subunit size of 55 kDa were determined for GR. Thyroidal GR was shown to be a favoprotein with one FAD per subunit. The Michaelis constants of bovine thyroidal GR were determined to be 21.8 {mu}M for NADPH and 58.8 {mu}M for GSSG. The effect of thyroid stimulating hormone (TSH) and thyroxine (T{sub 4}) on in vivo levels of GR and glucose 6-phosphate dehydrogenase were determined in rat thyroid homogenates. Both enzymes were stimulated by TSH treatment and markedly reduced following T{sub 4} treatment. Lysosomal hydrolysis of ({sup 125}I)-labeled and unlabeled thyroglobulin was examined using size exclusion HPLC.

  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. To Analyze the Amelioration of Phenobarbital Induced Oxidative Stress by Erucin, as Indicated by Biochemical and Histological Alterations.

    PubMed

    Arora, Rohit; Bhushan, Sakshi; Kumar, Rakesh; Mannan, Rahul; Kaur, Pardeep; Singh, Bikram; Sharma, Ritika; Vig, Adarsh Pal; Singh, Balbir; Singh, Amrit Pal; Arora, Saroj

    2016-01-01

    Phenobarbital is a commonly employed antidepressant and anti-epileptic drug. The cancer promoting activity of this genotoxic xenobiotic is often ignored. It is responsible for oxidative stress leading to modulation in xenobiotic and antioxidative enzymes. Glucosinolates and more specifically their hydrolytic products are known for their antioxidative and anticancer activities. The present study involves the analysis of hepatoprotective effect of erucin (isolated from Eruca sativa (Mill.) Thell.) against phenobarbital mediated hepatic damage in male wistar rats. The liver homogenate was analyzed for oxidative stress (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and lactate dehydrogenase), other oxidative parameters (thiobarbituric acid reactive species, conjugated dienes and lipid hydroperoxide), phase I enzymes (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, cytochrome P420, cytochrome P450 and cytochrome b5), phase II enzymes (γ-glutamyl transpeptidase, DT-diaphorase and glutathione-S-transferase), serum parameters (alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin and total bilirubin) and certain histological parameters. Erucin accorded protection from phenobarbital induced hepatic damage by normalizing antioxidative enzymes, other oxidative parameters, phase I, II, and serum parameters. Erucin, an analogue of sulforaphane has the potential to act as an anticancer agent by regulating various biochemical parameters.

  14. Reasons for reduced activities of 17 alpha-hydroxylase and C17-C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60Co.

    PubMed

    Inano, H; Ishii-Ohba, H; Suzuki, K; Ikeda, K

    1990-05-01

    Pregnant rats received whole body irradiation with 2.6 Gy gamma-ray from a 60Co source at Day 20 of gestation. When pups were 4 months old, activities of electron transport system and steroid monooxygenase in tests were assayed. The content of total cytochrome P-450 in the irradiated testes had increased to 170% of that in non-irradiated rats, but NADPH-cytochrome P-450 reductase activity had reduced to 36% of the control. Also, amounts of cytochrome b5 in testicular microsomal fraction were decreased markedly after irradiation, but no significant change of NADH-cytochrome b5 reductase activity was observed in the treated pups. Because both 17 alpha-hydroxylase and C17-C20 lyase activities tended to be decreased by fetal irradiation, testosterone production from progesterone and 17 alpha-hydroxyprogesterone was reduced to about 30% of the control. From these results, it has been suggested that the testicular cytochrome P-450 is radioresistant but steroid monooxygenase activities are reduced after the fetal irradiation. We propose that the discrepancy arises from the marked decrement of NADPH-cytochrome P-450 reductase activity.

  15. 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

  16. Mitochondrial DNA haplogroup B5 confers genetic susceptibility to Alzheimer's disease in Han Chinese.

    PubMed

    Bi, Rui; Zhang, Wen; Yu, Dandan; Li, Xiao; Wang, Hui-Zhen; Hu, Qiu-Xiang; Zhang, Chen; Lu, Weihong; Ni, Jianliang; Fang, Yiru; Li, Tao; Yao, Yong-Gang

    2015-03-01

    Mitochondrial dysfunction has been widely reported in psychiatric and neurodegenerative diseases. We aimed to investigate the association between matrilineal structures of Han Chinese populations and Alzheimer's disease (AD) by a 2-stage case-control study: A total of 341 AD patients and 435 normal individuals from Southwest China were analyzed for mitochondrial DNA sequence variations and were classified into respective haplogroups. A total of 371 AD patients and 470 normal individuals from East China, as validation samples, were genotyped for the variants defining the risk haplogroup. Haplogroup B5 had a significantly higher frequency in AD patients (7.33%) than in control subjects (3.68%) from Southwest China, and we found a similar pattern of higher frequency of B5 in patients in the case-control sample from East China. In the combined population, association of haplogroup B5 with AD risk was strengthened (p = 0.02; odds ratio = 1.74; 95% confidence interval = 1.10-2.76). In lymphoblastoid cell lines belonging to haplogroup B5a, we observed significantly increased reactive oxygen species and decreased mitochondrial mass. Hela cells with stable expression of the MT-ATP6 gene with B5-defining variant m.8584G>A also showed a significantly decreased mitochondrial function. Taken together, our results indicated that haplogroup B5 conferred genetic susceptibility to AD in Han Chinese, and this effect was most likely mediated by ancient variant m.8584G>A. The predisposing effect of B5 to AD is consistent with the ancestral-susceptibility model of complex diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. 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

  18. [Degradation of halogenated compounds by haloalkane dehalogenase DadA from Alcanivorax dieselolei B-5 ].

    PubMed

    Li, Anzhang; Shao, Zongze

    2014-09-04

    [OBJECTIVE] Alcanivorax dieselolei B-5 is an important oil-degrading bacterium. We studied its substrate range and degradation of halogenated compounds. [METHODS] Growth capability of B-5 was examined with different halogenated substrates as sole carbon source. A putative haloalkane dehalogenase (HLD) gene named dadA was found from the genome of strain B-5 and analyzed by sequence alignment, phylogenetic analysis and homologous modeling. After heterologous expression in Escherichia coli and purification, the activity of DadA towards 46 substrates was determined. [RESULTS] Strain B-5 was capable of utilizing various halogenated compounds (C3-C,8) as the sole carbon source. DadA had typical catalytic pentad residues of HLD-II subfamily, but it was independent from other members of this subfamily according to phylogenetic analysis. Activity assay showed that DadA has higher specificity and narrower substrate range than other characterized HLDs and it only showed activity toward 1,2,3-tribromopropane, 1,2-dibromo-3-chloropropane and 2,3-dichloroprop-1-ene among 46 tested substrates. [CONCLUSIONS] Strain B-5 and its HLD DadA can degrade halogenated aliphatic pollutants although.

  19. 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.

  20. Immunodiagnosis of tumors in vivo using radiolabeled monoclonal antibody A2B5

    SciTech Connect

    Reintgen, D.S.; Shimizu, K.; Coleman, E.; Briner, W.; Kitzmiller, J.; Eisenbarth, G.; Seigler, H.F.

    1983-07-01

    Recently a murine monoclonal antibody (A2B5) has been described that reacts with a membrane associated GQ ganglioside common to peptide secreting normal cells and tumors. In vitro binding data demonstrated the presence of this ganglioside on neurons, adrenal medulla, and pancreatic islets, along with neuroendocrine tumors such as insulinomas, pheochromocytomas, melanomas and neuroblastomas. Negative binding has previously been shown for tissue sections from liver, kidney, colon, lung, stomach, and tumors not derived from the neural crest. Because of the specificity at A2B5 in vitro, this monoclonal antibody was labeled with /sup 131/I for in vivo tumor localization studies. Daily radionuclear scans were obtained in 5 KX rats bearing the radiation induced rat insulinoma with disappearance of the label from the blood pool and concentration in the tumor so that by the fourth day, the only activity present by scan was in the insulinoma. In addition A2B5 also localized to five different human melanoma cells lines grown in nude mice with high tumor/blood levels compared to normal tissues, while no localization is seen in nudes carrying osteosarcomas, colon, bladder, and renal cell carcinomas. In addition antibody A2B5 did not concentrate in any normal tissue though the antigen is present on several. The finding that A2B5 reacts across species lines (mouse, rat, man) lends itself to obvious diagnostic and therapeutic possibilities.

  1. 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.

  2. Chicken muscle aldose reductase: purification, properties and relationship to other chicken aldo/keto reductases.

    PubMed

    Murphy, D G; Davidson, W S

    1986-01-01

    An enzyme that catalyzes the NADPH-dependent reduction of a wide range of aromatic and hydroxy-aliphatic aldehydes was purified from chicken breast muscle. This enzyme shares many properties with mammalian aldose reductases including molecular weight, relative substrate specificity, Michaelis constants, an inhibitor specificity. Therefore, it seems appropriate to call this enzyme an aldose reductase (EC 1.1.1.21). Chicken muscle aldose reductase appears to be kinetically identical to an aldose reductase that has been purified from chicken kidney (Hara et al., Eur. J. Biochem. 133, 207-214) and to hen muscle L-glycol dehydrogenase (Bernado et al., Biochim. biophys. Acta 659, 189-198). The association of this aldose reductase with muscular dystrophy in the chick is discussed.

  3. Mode of antibacterial action of totarol, a diterpene from Podocarpus nagi.

    PubMed

    Haraguchi, H; Oike, S; Muroi, H; Kubo, I

    1996-04-01

    The antimicrobial mechanism of totarol was studied using Pseudomonas aeruginosa IFO 3080. This diterpene inhibited oxygen consumption and respiratory-driven proton translocation in whole cells, and oxidation of NADH in membrane preparation. NADH-cytochrome c reductase was inhibited by totarol while cytochrome c oxidase was not. NADH-DPIP reductase and NADH-CoQ reductase were also inhibited. The site of respiratory inhibition of totarol was thought to be near CoQ in the bacterial electron transport chain.

  4. Luminescence properties of novel NaSrB5O9:Eu3+ phosphor

    NASA Astrophysics Data System (ADS)

    Dillip, G. R.; Prasad Raju, B. Deva

    2013-02-01

    Europium (III) ions doped NaSrB5O9 phosphor was prepared first time via a one-step conventional solid state reaction method. The prepared phosphors structure was examined by X-ray diffraction (XRD). It reveals that the undoped and Eu3+ doped NaSrB5O9 phosphors are in single crystalline phase. The room-temperature photoluminescence (PL) spectrum of NaSrB5O9:Eu3+ phosphor has shown strong red emission at 618 nm (5D0→7F2) with near UV an excitation wavelength λexc = 394 nm (7F0→5L6). The calculated color coordinates are lies in the orange region. Therefore, emission and excitation characterization of synthesized phosphor shows that the prepared phosphor may be a promising red component for near ultraviolet white light emitting diodes (NUV WLEDs).

  5. Immunodiagnosis of tumors in vivo using radiolabeled monoclonal antibody A2B5.

    PubMed

    Reintgen, D S; Shimizu, K; Coleman, E; Briner, W; Kitzmiller, J; Eisenbarth, G; Seigler, H F

    1983-07-01

    Recently a murine monoclonal antibody (A2B5) has been described that reacts with a membrane associated GQ ganglioside common to peptide secreting normal cells and tumors. In vitro binding data demonstrated the presence of this ganglioside on neurons, adrenal medulla, and pancreatic islets, along with neuroendocrine tumors such as insulinomas, pheochromocytomas, melanomas and neuroblastomas. Negative binding has previously been shown for tissue sections from liver, kidney, colon, lung, stomach, and tumors not derived from the neural crest. Because of the specificity at A2B5 in vitro, this monoclonal antibody was labeled with 131I for in vivo tumor localization studies. Daily radionuclear scans were obtained in 5 KX rats bearing the radiation induced rat insulinoma with disappearance of the label from the blood pool and concentration in the tumor so that by the fourth day, the only activity present by scan was in the insulinoma. Tissue-counting data showed tumor/blood ratios (av +/- SE, 1.29 +/- 0.25) of A2B5 activity two to ten times the average activity found in other organs (0.28 +/- 0.05). No tumor concentration of the control nonspecific monoclonal antibody P3X63 was evident (0.27 +/- 0.04). In addition A2B5 also localized to five different human melanoma cells lines grown in nude mice with high tumor/blood levels (1.04 +/- 0.27) compared to normal tissues (0.32 +/- 0.05) (P = .0005), while no localization is seen in nudes carrying osteosarcomas, colon, bladder, and renal cell carcinomas. In addition antibody A2B5 did not concentrate in any normal tissue though the antigen is present on several. The finding that A2B5 reacts across species lines (mouse, rat, man) lends itself to obvious diagnostic and therapeutic possibilities.

  6. 78 FR 30961 - Notice of Receipt of Petition for Decision that Nonconforming 2005-2007 Alpina B5 Passenger Cars...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-23

    ... Nonconforming 2005-2007 Alpina B5 Passenger Cars Manufactured Before September 1, 2006 Are Eligible for... petition for a decision that nonconforming 2005-2007 Alpina B5 passenger cars manufactured before September... nonconforming 2005-2007 Alpina B5 Series passenger cars manufactured before September 1, 2006 are eligible...

  7. 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.

  8. 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.

  9. 16 CFR 1101.62 - Statutory exceptions to section 6(b)(5) requirements.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...) requirements. 1101.62 Section 1101.62 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION CONSUMER PRODUCT... Information Submitted Pursuant to Section 15(b) of the CPSA § 1101.62 Statutory exceptions to section 6(b)(5... under section 12 (see § 1101.42); (2) Information with respect to a consumer product which...

  10. 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...

  11. 16 CFR 1101.62 - Statutory exceptions to section 6(b)(5) requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Statutory exceptions to section 6(b)(5) requirements. 1101.62 Section 1101.62 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION CONSUMER PRODUCT SAFETY ACT REGULATIONS INFORMATION DISCLOSURE UNDER SECTION 6(b) OF THE CONSUMER PRODUCT SAFETY ACT Information Submitted Pursuant to...

  12. A summary of staphylococcal C-terminal SH3b_5 cell wall binding domains.

    USDA-ARS?s Scientific Manuscript database

    Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset of these proteins contain a C-terminal SH3b_5 cell wall binding domain that has been shown for some to be essential for accurate cell wall recognition and subsequent staphylolytic activity, propert...

  13. The tyrosyl free radical in ribonucleotide reductase.

    PubMed Central

    Gräslund, A; Sahlin, M; Sjöberg, B M

    1985-01-01

    The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical. PMID:3007085

  14. Detection of cytochrome b5 from the house-fly, Musca domestica: comparison of immunological and spectrophotometric methods.

    PubMed

    Wheelock, G D; Scott, J G

    1994-06-01

    Spectrophotometric assay of microsomal cytochrome b5 in house-flies produces different results depending on whether sodium dithionite or NADH is used as the reducing agent and whether or not detergent is present. Microsomes assayed for cytochrome b5 with dithionite in the presence of detergent gave the highest values, followed by dithionite alone, NADH plus detergent, and then NADH alone. Isopropanol treatment of microsomes extracted cytochrome b5 free of spectrophotometrically interfering cytochrome P-450. Studies using immunoblotting and rocket immunoelectrophoresis with polyclonal antisera raised against the purified cytochrome b5 showed that isopropanol treatment quantitatively extracted cytochrome b5.

  15. 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

  16. Dynamic docking and electron transfer between Zn-myoglobin and cytochrome b(5).

    PubMed

    Liang, Zhao-Xun; Nocek, Judith M; Huang, Kai; Hayes, Ryan T; Kurnikov, Igor V; Beratan, David N; Hoffman, Brian M

    2002-06-19

    We present a broad study of the effect of neutralizing the two negative charges of the Mb propionates on the interaction and electron transfer (ET) between horse Mb and bovine cyt b(5), through use of Zn-substituted Mb (ZnMb, 1) to study the photoinitiated reaction, ((3)ZnP)Mb + Fe(3+)cyt b(5) --> (ZnP)(+)Mb + Fe(2+)cyt b(5). The charge neutralization has been carried out both by replacing the Mb heme with zinc-deuteroporphyrin dimethylester (ZnMb(dme), 2), which replaces the charges by small neutral hydrophobic patches, and also by replacement with the newly prepared zinc-deuteroporphyrin diamide (ZnMb(diamide), 3), which converts the charged groups to neutral, hydrophilic ones. The effect of propionate neutralization on the conformation of the zinc-porphyrin in the Mb heme pocket has been studied by multinuclear NMR with an (15)N labeled zinc porphyrin derivative (ZnMb((15)N-diamide), 4). The rates of photoinitiated ET between the Mb's (1-3) and cyt b(5) have been measured over a range of pH values and ionic strengths. Isothermal titration calorimetry (ITC) and NMR methods have been used to independently investigate the effect of charge neutralization on Mb/b(5) binding. The neutralization of the two heme propionates of ZnMb by formation of the heme diester or, for the first time, the diamide increases the second-order rate constant of the ET reaction between ZnMb and cyt b(5) by as much as several 100-fold, depending on pH and ionic strength, while causing negligible changes in binding affinity. Brownian dynamic (BD) simulations and ET pathway calculations provide insight into the protein docking and ET process. The results support a new "dynamic docking" paradigm for protein-protein reactions in which numerous weakly bound conformations of the docked complex contribute to the binding of cyt b(5) to Mb and Hb, but only a very small subset of these are ET active, and this subset does not include the conformations most favorable for binding; the Mb surface is a

  17. Isolation, sequence identification and tissue expression profile of two novel soybean (glycine max) genes-vestitone reductase and chalcone reductase.

    PubMed

    Liu, G Y

    2009-09-01

    The complete mRNA sequences of two soybean (glycine max) genes-vestitone reductase and chalcone reductase, were amplified using the rapid amplification of cDNA ends methods. The sequence analysis of these two genes revealed that soybean vestitone reductase gene encodes a protein of 327 amino acids which has high homology with the vestitone reductase of Medicago sativa (77%). The soybean chalcone reductase gene encodes a protein of 314 amino acids that has high homology with the chalcone reductase of kudzu vine (88%) and medicago sativa (83%). The expression profiles of the soybean vestitone reductase and chalcone reductase genes were studied and the results indicated that these two soybean genes were differentially expressed in detected soybean tissues including leaves, stems, roots, inflorescences, embryos and endosperm. Our experiment established the foundation for further research on these two soybean genes.

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

    PubMed Central

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

    1976-01-01

    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

  19. 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

  20. Fumarate Reductase Activity of Streptococcus faecalis

    PubMed Central

    Aue, B. J.; Diebel, R. H.

    1967-01-01

    Some characteristics of a fumarate reductase from Streptococcus faecalis are described. The enzyme had a pH optimum of 7.4; optimal activity was observed when the ionic strength of the phosphate buffer was adjusted to 0.088. The Km value of the enzyme for reduced flavin mononucleotide was 2 × 10−4 m as determined with a 26-fold preparation. In addition to fumarate, the enzyme reduced maleate and mesaconate. No succinate dehydrogenase activity was detected, but succinate did act as an inhibitor of the fumarate reductase activity. Other inhibitors were malonate, citraconate, and trans-, trans-muconate. Metal-chelating agents did not inhibit the enzyme. A limited inhibition by sulfhydryl-binding agents was observed, and the preparations were sensitive to air oxidation and storage. Glycine, alanine, histidine, and possibly lysine stimulated fumarate reductase activity in the cell-free extracts. However, growth in media supplemented with glycine did not enhance fumarate reductase activity. The enzymatic activity appears to be constitutive. PMID:4960892

  1. Post-translational Regulation of Nitrate Reductase

    USDA-ARS?s Scientific Manuscript database

    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...

  2. Synthesis of symmetric disulfides as potential alternative substrates for trypanothione reductase and glutathione reductase: Part 1.

    PubMed

    Jaouhari, R; Besheya, T; McKie, J H; Douglas, K T

    1995-12-01

    The synthesis of a series of symmetrical disulfides as potential substrates of trypanothione reductase and glutathione reductase was described. The key intermediate in the synthetic approach was the choice of S-(t)butylmercapto-L-cysteine (1). The spermidine ring in the native substrate, trypanothione disulfide (TSST), was replaced with 3-dimethyl-aminopropylamine (DMAPA), while theγ-Glu moiety was replaced by phenylalanyl or tryptophanyl residues. The same modifications in theγ-Glu moiety of glutathione disulfide (GSSG) were applied.

  3. Selective modification of rat hepatic microsomal fatty acid chain elongation and desaturation by fibrates: relationship with peroxisome proliferation.

    PubMed

    Alegret, M; Cerqueda, E; Ferrando, R; Vázquez, M; Sánchez, R M; Adzet, T; Merlos, M; Laguna, J C

    1995-04-01

    1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-, palmitoleoyl- and gamma-linolenoyl-CoA elongase, delta-9, delta-6 and delta-5 desaturase activities, and microsomal electron transport chains, as well as the correlation with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. As reported in our previous work, the three drugs behave as peroxisomal proliferators (the order of potency was BFB > CFB > or = GFB) and induced a clear reduction in both plasma cholesterol and triglyceride levels. 3. Palmitoyl-CoA elongation activity was increased by the three drugs (BFB = GFB > CFB), whereas palmitoleoyl-CoA elongation activity was only enhanced by GFB. Elongation activity was not modified by fibrates when gamma-linolenoyl-CoA was used as substrate. These results are in accordance with the existence of three different elongation systems for saturated, mono- and polyunsaturated fatty acids. 4. delta-9, delta-6 and delta-5 desaturase activities were increased by the three fibrates, with an order of potency BFB > CFB = GFB for delta-9 and delta-5, and GFB > BFB = CFB for delta-6. 5. Of the enzyme activities integrated in the microsomal electron transport chains, NADH cytochrome b5 reductase was not affected by fibrate treatment, NADPH cytochrome c reductase activity was enhanced (BFB = GFB > CFB), whereas NADH cytochrome c reductase activity was reduced by CFB and BFB. 6. The increase in Delta-9 and Delta-5 desaturase activities was highly dependent on the peroxisomal proliferation phenomena, whereas the increase in Delta-6 desaturase activity and the decrease in NADH cytochromec reductase was mainly independent. The modifications of palmitoyl-CoA elongase and NADPH cytochrome c reductase activities, as well as plasma lipid levels, were partially correlated with peroxisomal beta-oxidation, but the r2 values obtained point to the existence of additional

  4. 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.

  5. 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.

  6. In vitro membrane-inserted conformation of the cytochrome b(5) tail.

    PubMed Central

    Hanlon, M R; Begum, R R; Newbold, R J; Whitford, D; Wallace, B A

    2000-01-01

    The cytochrome b(5) tail is a 43-residue membrane-embedded domain that is responsible for anchoring the catalytic domain of cytochrome b(5) to the endoplasmic reticulum membrane. Different models for the structure of the membrane domain of cytochrome b(5) have been proposed, including a helical hairpin and a single transmembrane helix. In the present study, CD spectroscopy was used to investigate the conformation of the tail in different environments, and as a function of temperature, with the goal of understanding the nature of the membrane-bound conformation. Whereas residue property profiling indicates that bending of a helix in the middle of the peptide might be possible, the experimental results in small unilamellar vesicles and lysophosphatidylcholine micelles are more consistent with a single transmembrane helix. Furthermore, although there is evidence for some refolding of the polypeptide with temperature, this is not consistent with a hairpin-to-transmembrane transition. Rather, it appears to represent an increase in helical content in fluid lipid environments, perhaps involving residues at the ends of the transmembrane segment. PMID:11062064

  7. Modulating effect of Liv.100, an ayurvedic formulation on antituberculosis drug-induced alterations in rat liver microsomes.

    PubMed

    Saraswathy, S D; Shyamala Devi, C S

    2001-09-01

    The influence of Liv.100 on the hepatotoxicity of antituberculosis drugs [isoniazid (INH), rifampicin (RMP) pyrazinamide (PZA)] was studied in male albino rats. INH, RMP, and PZA were proved to be the most hepatotoxic. Rats were treated with antituberculosis drugs daily for a period of 6 weeks by intragastric administration. The combined use of antituberculosis drugs elevated the levels of cytochrome P-450 and cytochrome-b5. A significant increase was observed in the levels of NADPH-cytochrome P-450 reductase and NADH-cytochrome-b5 reductases after antitubercular drug administration. During antitubercular drug treatment a significant decrease was also observed in the activity of glucose-6-phosphatase. The extent of NADPH-induced and ascorbic acid-induced lipid peroxides were marked in antitubercular drug treatment, when compared with normal control animals. Oral Liv.100 co-administration, for the same period, modulated the alterations in the xenobiotic metabolizing system and microsomal lipid peroxidation in experimental animals. The results are discussed with reference to drug metabolizing enzymes, lipid peroxidation and the hepatoprotective nature of Liv.100. Copyright 2001 John Wiley & Sons, Ltd.

  8. 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.

  9. Heterogeneous distribution of enzymes in submicrosomal membrane fragments.

    PubMed

    Dallman, P R; Dallner, G; Bergstrand, A; Ernster, L

    1969-05-01

    Microsomal membranes are postulated to contain either a homogeneous arrangement of individual enzymes or groupings of functionally related enzymes. In the present study we attempt to distinguish between these hypotheses in subfractions of rough microsomes from rat liver. After sonication, the individual vesicles that make up the rough-membrane fraction average less than 1/100 of their previous mass. The vesicles in the sonicated suspension are fractionated roughly according to size on a continuous sucrose gradient. Enzyme activity or concentration in fractions of the gradient is expressed on a phospholipid basis. Fractions containing primarily small vesicles differ from those containing larger vesicles in a manner suggesting a certain degree of separation of NADH-linked from NADPH-linked enzymes. NADH-ferricyanide reductase, NADH-cytochrome c reductase and cytochrome b(5) are most concentrated within the large vesicles in the lowest third of the gradient. In contrast, NADPH-cytochrome c reductase and cytochrome P-450 are found in highest concentration in the small vesicles that make up the upper third of the gradient. The results suggest a nonrandom distribution of these two enzyme groups in the membranes of the endoplasmic reticulum.

  10. Organ and tissue-dependent effect of resveratrol and exercise on antioxidant defenses of old mice.

    PubMed

    Tung, Bui Thanh; Rodriguez-Bies, Elisabet; Thanh, Hai Nguyen; Le-Thi-Thu, Huong; Navas, Plácido; Sanchez, Virginia Motilva; López-Lluch, Guillermo

    2015-12-01

    Oxidative stress has been considered one of the causes of aging. For this reason, treatments based on antioxidants or those capable of increasing endogenous antioxidant activity have been taken into consideration to delay aging or age-related disease progression. In this paper, we determine if resveratrol and exercise have similar effect on the antioxidant capacity of different organs in old mice. Resveratrol (6 months) and/or exercise (1.5 months) was administered to old mice. Markers of oxidative stress (lipid peroxidation and glutathione) and activities and levels of antioxidant enzymes (SOD, catalase, glutathione peroxidase, glutathione reductase and transferase and thioredoxin reductases, NADH cytochrome B5-reductase and NAD(P)H-quinone acceptor oxidoreductase) were determined by spectrophotometry and Western blotting in different organs: liver, kidney, skeletal muscle, heart and brain. Both interventions improved antioxidant activity in the major organs of the mice. This induction was accompanied by a decrease in the level of lipid peroxidation in the liver, heart and muscle of mice. Both resveratrol and exercise modulated several antioxidant activities and protein levels. However, the effect of resveratrol, exercise or their combination was organ dependent, indicating that different organs respond in different ways to the same stimulus. Our data suggest that physical activity and resveratrol may be of great importance for the prevention of age-related diseases, but that their organ-dependent effect must be taken into consideration to design a better intervention.

  11. Toxic dark effects of protoporphyrin on the cytochrome P-450 system in rat liver microsomes.

    PubMed Central

    Williams, M; Van der Zee, J; Van Steveninck, J

    1992-01-01

    In erythropoietic protoporphyria, accumulation of protoporphyrin has been found in various tissues and liver cirrhosis occurs frequently in this disease, probably due to toxic dark effects of protoporphyrin. We have studied the effect of porphyrins on various enzymic functions in rat liver microsomes. Incubation of microsomes with protoporphyrin resulted in a concentration-dependent inhibition of the oxidation of 7-ethoxycoumarin and aminopyrine by the cytochrome P-450 system. Kinetic analysis showed a decrease in Vmax., whereas the Km was not affected (non-competitive inhibition). Furthermore, reduction of cytochrome c by the NADPH-cytochrome P-450 reductase and by the NADH-cytochrome b5 reductase was inhibited. However, the activity of the reductases was only affected when the microsomes were pre-incubated with protoporphyrin, and it was found that the inhibition was dependent on the duration of the pre-incubation. Kinetic analysis again revealed non-competitive inhibition. When these experiments were repeated with uroporphyrin, no inhibition could be observed. With Stern-Volmer plots it was demonstrated that this was most likely caused by the localization of the porphyrins: protoporphyrin is localized in the membrane, whereas uroporphyrin remains in solution. From these results it is concluded that accumulation of protoporphyrin in the liver may markedly affect the cytochrome P-450 system and thus its detoxification function. PMID:1332695

  12. 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.

  13. Augmentation of CFTR maturation by S-nitrosoglutathione reductase

    PubMed Central

    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.

    2015-01-01

    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. PMID:26637637

  14. miR-450b-5p induced by oncogenic KRAS is required for colorectal cancer progression

    PubMed Central

    Deng, Dan-ling; Jiao, Hong-Li; Li, Ting-Ting; Wang, Shu-Yang; Wang, Yong-Xia; Xiao, Zhi-Yuan; Wei, Wen-ting; Chen, Yan-Ru; Qiu, Jun-Feng; Yang, Run-Wei; Lin, Jie; Liang, Li; Liao, Wen-Ting; Ding, Yan-Qing

    2016-01-01

    The development and progression of CRC are regarded as a complicated network and progressive event including genetic and/or epigenetic alterations. Recent researches revealed that MicroRNAs are biomarkers and regulators of CRC progression. Analyses of published microarray datasets revealed that miR-450b-5p was highly up-regulated in CRC tissues. In addition, high expression of miR-450b-5p was significantly associated with KRAS mutation. However, the role of miR-450b-5p in the progression of CRC remains unknown. Here, we sought to validate the expression of miR-450b-5p in CRC tissues and investigate the role and underlying mechanism of miR-450b-5p in the progression of CRC. The results revealed that miR-450b-5p was up-regulated in CRC tissues, high expression level of miR-450b-5p was positively associated with poor differentiation, advanced TNM classification and poor prognosis. Moreover, miR-450b-5p was especially high in KRAS-mutated cell lines and could be up-regulated by KRAS/AP-1 signaling. Functional validation revealed that overexpression of miR-450b-5p promoted cell proliferation and tumor growth while inhibited apoptosis of CRC cells. Furthermore, we demonstrated that miR-450b-5p directly bound the 3′-UTRs of SFRP2 and SIAH1, and activated Wnt/β-Catenin signaling. In conclusion, miR-450b-5p induced by oncogenic KRAS is required for colorectal cancer progression. Collectively, our work helped to understand the precise role of miR-450b-5p in the progression of CRC, and might promote the development of new therapeutic strategies against CRC. PMID:27494869

  15. Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

    PubMed

    Eltis, L D; Herbert, R G; Barker, P D; Mauk, A G; Northrup, S H

    1991-04-16

    The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.

  16. Efficient synthesis of tyrosol galactosides by the β-galactosidase from Enterobacter cloacae B5.

    PubMed

    Qi, Tingting; Gu, Guofeng; Xu, Li; Xiao, Min; Lu, Lili

    2017-03-30

    In this work, the β-galactosidase from Enterobacter cloacae B5 (BgaB5) exhibited excellent transglycosylation activity toward tyrosol (p-hydroxyphenethyl alcohol) when using lactose as the glycosyl donor, generating a series of tyrosol glycosides with potential pharmacological properties. The effects of substrate concentration, temperature, pH, and reaction time on the transglycosylation reaction catalyzed by the enzyme BgaB5 were studied in detail. Three tyrosol derivatives were produced in a total high yield of 50.0% when incubating the enzyme with 250 mM tyrosol and 1000 mM lactose (pH 7.5) at 50 °C for 5 min. These derivatives were subsequently purified by column chromatography and preparative thin-layer chromatography. MS analysis of the purified compounds suggested one monogalactoside (M r 300) and two digalactoside derivatives (M r 462). The following NMR analysis further identified them to be p-hydroxyphenethyl β-D-galactopyranoside, p-hydroxyphenethyl β-D- galactopyranosyl-(1 → 3')-β-D-galactopyranoside, and p-hydroxyphenethyl β-D- galactopyranosyl-(1 → 6')-β-D-galactopyranoside, respectively. The yield of the tyrosol monogalactoside which was known to possess potent bioactivities reached 39.4%, higher than other enzymatic yields reported so far. The two digalactosides, which were expected to have potential applications for novel drug screening and discovery, were artificially obtained with 10.6% yield for the first time.

  17. FRUCTOSE-6-PHOSPHATE REDUCTASE FROM SALMONELLA GALLINARUM

    PubMed Central

    Zancan, Glaci T.; Bacila, Metry

    1964-01-01

    Zancan, Glaci T. (Universidade do Paraná, Curitiba, Paraná, Brazil), and Metry Bacila. Fructose-6-phosphate reductase from Salmonella gallinarum. J. Bacteriol. 87:614–618. 1964.—A fructose-6-phosphate reductase present in cell-free extracts of Salmonella gallinarum was purified approximately 42 times. The optimal pH for this enzyme is 8.0. The enzyme is specific for fructose-6-phosphate and reduced nicotinamide adenine dinucleotide (NADH). The dissociation constants are 1.78 × 10−4m for fructose-6-phosphate and 8.3 × 10−5m for NADH. The Q10, reaction order, and equilibrium constant were determined. The enzyme is sensitive to p-chloromercuribenzoic acid, but not to o-iodosobenzoic acid nor to N-ethylmaleimide. PMID:14127579

  18. Characterization of human platelet glutathione reductase.

    PubMed

    Moroff, G; Kosow, D P

    1978-12-08

    Glutathione reductase (NAD(P)h:oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified 1000-fold from the cytoplasmic fraction of human platelets. Salts, including the heretofore unreported effect of sodium citrate, activate the NADPH-dependent reduction of oxidized glutathione. Sodium citrate and monovalent salt activation appears to involve multiple sites having different binding affinities. At sub-saturating sodium phosphate, non-linear double reciprocal plots indicative of substrate activation by oxidized glutathione were observed. Initial velocity double reciprocal plots at sub-saturating and saturating concentrations of phosphate generate a family of converging lines. NADP+ is a partial inhibitor, indicating that the reduction of oxidized glutathione can proceed by more than one pathway. FMN, FAD, and riboflavin inhibit platelet glutathione reductase by influencing only the V while nitrofurantoin inhibition is associated with an increase Koxidized glutathione and a decreased V.

  19. 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.

  20. 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.

  1. Characterization of erythrose reductases from filamentous fungi

    PubMed Central

    2013-01-01

    Proteins with putative erythrose reductase activity have been identified in the filamentous fungi Trichoderma reesei, Aspergillus niger, and Fusarium graminearum by in silico analysis. The proteins found in T. reesei and A. niger had earlier been characterized as glycerol dehydrogenase and aldehyde reductase, respectively. Corresponding genes from all three fungi were cloned, heterologously expressed in Escherichia coli, and purified. Subsequently, they were used to establish optimal enzyme assay conditions. All three enzymes strictly require NADPH as cofactor, whereas with NADH no activity could be observed. The enzymatic characterization of the three enzymes using ten substrates revealed high substrate specificity and activity with D-erythrose and D-threose. The enzymes from T. reesei and A. niger herein showed comparable activities, whereas the one from F. graminearum reached only about a tenth of it for all tested substrates. In order to proof in vivo the proposed enzyme function, we overexpressed the erythrose reductase-encoding gene in T. reesei. An increased production of erythritol by the recombinant strain compared to the parental strain could be detected. PMID:23924507

  2. Migration of epithelial cells in the small intestine of mice perorally infected with coxsackievirus B5.

    PubMed

    Shadoff, N; Loria, R M; Kibrick, S; Broitman, S A

    1979-03-01

    The rate of cell migration in the small intestine during enteric viral infections has not been assessed previously. CD-1 mice (33 days old) were infected perorally with 1.0 X 10(8) plague-forming units of coxsackievirus B5 and 12 hr later were injected intraperitoneally with 2 micron Ci of [3H]thymidine/g of body weight. After 2, 12, 24, 48, 60, and 72 hr, mice were killed, and the small intestine was removed. Specimens obtained at each interval were examined by radioautography; similar specimens were titrated for virus by plaque assay in HeLa cells. In mice perorally infected with coxsackievirus B5, epithelial cells migrated from crypt to villus tip in 60 hr, as compared with 48 hr in uninfected control mice and 24 hr previously reported for mice perorally infected with enteric bacteria (e.g., Salmonella typhimurium). Virus was recovered from intestinal tissue, but no inflammatory response in the limina propria was apparent. These observations are consistent with previous report that substrate absorption rates may be altered during viral and bacterial enteric infection.

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

    PubMed Central

    2015-01-01

    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. PMID:26536144

  4. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. The molecular characterization of a gamma-ray induced mutation resulting in altered HLA-B5 gene expression

    SciTech Connect

    Bowers, A.C.

    1988-01-01

    The preliminary analysis of LCL 721.53 revealed the presence of the HLA-class I B5 gene at the DNA level, the marked reduction of HLA-B5 expression at the mRNA level, and the absence of the HLA-B5 product at the protein level. The HLA-B5 gene of 721.53 was thus chosen as a model for the molecular characterization of the effects of gamma-irradiation on HLA-class I gene expression. The 6.8 kb EcoRI fragment containing the HLA-B5 gene of 721.53 was cloned into {lambda}gt11. Hybrids of the HLA-B5 gene cloned from 721.53 and 721 were constructed, transfected into mouse L cells, and assayed for the presence of HLA-B5 mRNA. The mutation resulting in the absence of steady-state HLA-B5 mRNA in 721.53 lay within the first 1.6 kb of the gene. Sanger dideoxy sequencing of the 1.6 kb EcoRI-KpnI fragment of both 721.53 and 721 revealed a deletion of 22 bp in the {alpha}1 coding region, encompassing amino acids 76 through 83. This frameshift mutation in 721.53 results in the formation of three in frame stop codons in the {alpha}2 exon.

  6. Methionine sulfoxide reductase contributes to meeting dietary methionine requirements

    PubMed Central

    Zhao, Hang; Kim, Geumsoo; Levine, Rodney L.

    2012-01-01

    Methionine sulfoxide reductases are present in all aerobic organisms. They contribute to antioxidant defenses by reducing methionine sulfoxide in proteins back to methionine. However, the actual in vivo roles of these reductases are not well defined. Since methionine is an essential amino acid in mammals, we hypothesized that methionine sulfoxide reductases may provide a portion of the dietary methionine requirement by recycling methionine sulfoxide. We used a classical bioassay, the growth of weanling mice fed diets varying in methionine, and applied it to mice genetically engineered to alter the levels of methionine sulfoxide reductase A or B1. Mice of all genotypes were growth retarded when raised on chow containing 0.10% methionine instead of the standard 0.45% methionine. Retardation was significantly greater in knockout mice lacking both reductases. We conclude that the methionine sulfoxide reductases can provide methionine for growth in mice with limited intake of methionine, such as may occur in the wild. PMID:22521563

  7. Growth, dielectric and nonlinear optical properties of Li3Cs2B5O10 single crystals

    NASA Astrophysics Data System (ADS)

    Sukumar, M.; Ramesh Babu, R.; Ramamurthi, K.

    2017-01-01

    Lithium cesium borate (Li3Cs2B5O10), an alkali metal borate, single crystals were grown by Czochralski method. Chemical etching was performed on grown Li3Cs2B5O10 crystal at various regions. The observed dislocation densities are varied at different regions of the grown Li3Cs2B5O10 crystal. Dielectric behavior of Li3Cs2B5O10 crystal at different temperatures is studied. The third-order nonlinear optical parameters of lithium cesium borate crystal are determined by Z-scan technique. The nonlinear refractive index ( n 2) value is estimated to be -7.272 × 10-11 cm2/W, and the corresponding third-order nonlinear susceptibility ( χ 3) is estimated to be 4.19 × 10-9 esu. The measured nonlinear refractive indices reveal the self-defocusing effect of Li3Cs2B5O10 crystal.

  8. Kinetics of flavin semiquinone reduction of the components of the cytochrome c-cytochrome b5 complex.

    PubMed

    Eltis, L; Mauk, A G; Hazzard, J T; Cusanovich, M A; Tollin, G

    1988-07-26

    The kinetics of flavin semiquinone reduction of the components of the 1:1 complex formed by cytochrome c with either cytochrome b5 or a derivative of cytochrome b5 in which the heme propionates are esterified (DME-cytochrome b5) have been studied. The rate constant for the reduction of horse heart cytochrome c by the electrostatically neutral lumiflavin semiquinone (LfH) is unaffected by complexation with native cytochrome b5 at pH 7. However, complex formation with DME-cytochrome b5 (pH 7) decreases by 35% the rate constant for cytochrome c reduction by LfH. At pH 8, complex formation with native cytochrome b5 decreases the rate constant for cytochrome c reduction by LfH markedly, whereas the rate constant for cytochrome c reduction, either unbound or in the complex formed with DME-cytochrome b5, is increased 2-fold relative to pH 7. These results indicate that the accessibility of the cytochrome c heme is not the same in the complexes formed with the two cytochrome b5 derivatives and that the docking geometry of the complex formed by the two native cytochromes is pH dependent. Binding of horse heart and tuna cytochromes c to native and DME-cytochromes b5 decreases the rate constants for reduction of cytochrome c by the negatively charged flavin mononucleotide semiquinone (FMNH) by approximately 30% and approximately 40%, respectively. This finding is attributed to substantial neutralization of the positive electrostatic potential surface of cytochrome c that occurs when it binds to either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5*

    PubMed Central

    Peng, Hwei-Ming; Liu, Jiayan; Forsberg, Sarah E.; Tran, Hong T.; Anderson, Sean M.; Auchus, Richard J.

    2014-01-01

    Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: 84EVLIKK89-b5: 53EQAGGDATENFEDVGHSTDAR73 and CYP17A1-R347K: 341TPTISDKNR349-b5: 40FLEEHPGGEEVLR52. Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis. PMID:25315771

  10. Structural Elucidation of Chalcone Reductase and Implications for Deoxychalcone Biosynthesis

    PubMed Central

    Bomati, Erin K.; Austin, Michael B.; Bowman, Marianne E.; Dixon, Richard A.; Noel, Joseph P.

    2010-01-01

    4,2′,4′,6′-tetrahydroxychalcone (chalcone) and 4,2′,4′-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/ketoreductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase. PMID:15970585

  11. Whole Genome Sequence of the Heterozygous Clinical Isolate Candida krusei 81-B-5.

    PubMed

    Cuomo, Christina A; Shea, Terrance; Yang, Bo; Rao, Reeta; Forche, Anja

    2017-09-07

    Candida krusei is a diploid, heterozygous yeast that is an opportunistic fungal pathogen in immunocompromised patients. This species also is utilized for fermenting cocoa beans during chocolate production. One major concern in the clinical setting is the innate resistance of this species to the most commonly used antifungal drug fluconazole. Here, we report a high-quality genome sequence and assembly for the first clinical isolate of C. krusei, strain 81-B-5, into 11 scaffolds generated with PacBio sequencing technology. Gene annotation and comparative analysis revealed a unique profile of transporters that could play a role in drug resistance or adaptation to different environments. In addition, we show that, while 82% of the genome is highly heterozygous, a 2.0 Mb region of the largest scaffold has undergone loss of heterozygosity. This genome will serve as a reference for further genetic studies of this pathogen. Copyright © 2017 Cuomo et al.

  12. Stellar UV classification: Some critical remarks and examples taken from A5 to B5 stars

    NASA Astrophysics Data System (ADS)

    Praderie, F.

    1982-10-01

    Parameters needed to classify normal stars, parameters which govern parts of the UV spectrum, and ways of distinguishing abnormal stars are reviewed. The use of photospheric spectral features for UV classification is advocated because the major part of the energy radiating from the star originates from the photosphere. Temperature can be obtained by observing longwave flux in the black body curve. Observational criteria can be derived from continua by connecting the UV to the visible spectrum, and by producing pure UV indices. The first method is well suited to A5 to B5 stars. Classification from UV lines should select local thermodynamic equilibrium photospheric lines such that (line center absorption coefficient)/(continuum opacity at the line (Kc)) is small, and Kc not much continuum opacity at 5000 A. Analysis of AP to BP stars shows that temperature and opacity effects are mixed in the UV, prohibiting the use of pure UV indices as temperature indicators.

  13. Low-energy Be4+/B5+ - H2 cross sections

    NASA Astrophysics Data System (ADS)

    Saha, Bidhan

    2000-06-01

    Single electron capture cross sections from molecular hydrogen by Be4+/B5+ have been calculated using the Molecular Orbital formalism in the semiclassical close-coupling scheme. The important interactions leading to state selective charge transfer are confined at large internuclear seperation. We have found that freezing the molecular details of the target turns out to be a convenient strategy [1]. In our investigation we treat H2 as a pseudo-atom with ionization potential 16.1 eV. The results will be presented in the conference. This work is supported by Research Corporation, NSF CREST and Army High Performance Computing Research. [1] A. Kumar and B C Saha, Phys Rev A 59,1273 (1999).

  14. State-selective charge transfer in slow collisions of B5+ with H2

    NASA Astrophysics Data System (ADS)

    Dwayne, Joseph; Saha, Bidhan

    2004-05-01

    State selective charge transfer from H2 by B5+ in the low energy region (0.01 < E <2 keV/amu) has been investigated employing the semi-classical, impact parameter, close-coupling method based on a molecular expansion augmented with the plane wave electron translation factor. Freezing the molecular features of H2, the colliding system is approximated[1] by a pseudo-one-electron entity. The method of pseudo-potential is used to account for the binding of the electron in the transient diatomic quasi-molecule. A large number of coupled equations are solved to obtain converged cross sections. Comparisons are made with other theoretical and experimental results. [1] A. kumar and B. C. Saha, J. Phys. B 31, L937 (1998); A. Kumar and B. C. Saha, Phys. Rev. A 59, 1273 (1999).

  15. 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.

  16. Molecular evolution, characterization and expression profiling of uterine aldoketoreductase 1B5 gene in endometrium of goat (Capra hircus).

    PubMed

    Kumar, Rohit; Ramteke, P W; Sharma, Sanjeev Kumar; Mitra, Abhijit

    2015-01-01

    Aldoketoreductase 1B5 (AKR1B5), a member of the Aldoketoreductase family, is involved in the production of Prostaglandin F2α (PGF2α) as one of vital prostaglandin F synthase (PGFS). PGs (Prostaglandins) play a crucial role in female reproductive system. In the present study, we cloned and characterized the full-length open reading frame of AKR1B5 gene in Black Bengal (BB) goat. The complete coding sequence of AKR1B5 comprises an entire open reading frame of 951 bp, encoding 316 amino acid (AA) residues. BB AKR1B5 showed >82.9% identity with that of cattle, rabbit, human, and rat at nucleotide and amino acid levels, respectively. Further, a systematic study of AKR1B5 sequence evolution was also conducted using Phylogenetic Analysis by Maximum Likelihood (PAML), entropy plot, and Blossum 62 in a phylogenetic context. Analysis of nonsynonymous to synonymous nucleotide substitution rate ratios (Ka/Ks) revealed that negative selection may have been operating on this gene during evolution in goat, cattle, rabbit, human, and rat, which showed its conservation across species. Further, expression of AKR1B5 was determined by quantitative real-time PCR in goat endometrial tissues at different stages of the estrous cycle and early pregnancy. Our results indicated its high expression at luteolytic phase (stage III; day 16-21) during the estrous cycle. However, during early (day ∼30-40) pregnancy the expression was highest as compared to estrous cycle.

  17. 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.

  18. The orphan protein bis-γ-glutamylcystine reductase joins the pyridine nucleotide-disulfide reductase family

    PubMed Central

    Kim, Juhan; Copley, Shelley D.

    2014-01-01

    Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still “orphan” enyzmes whose activity is known but the genes that encode them have not been identified. Identification of the genes encoding orphan enzymes is important because it allows correct annotation of genes of unknown function or with mis-assigned function. Bis-γ-glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis-γ-glutamylcystine. γ-Glutamylcysteine (γ-Glu-Cys) is the major low molecular weight thiol in halobacteria. We purified GCR from Halobacterium sp. NRC-1 and identified the sequence of 23 tryptic peptides by NanoLC electrospray ionization tandem mass spectrometry. These peptides cover 62% of the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that is annotated as mercuric reductase. GCR and mercuric reductase activities were assayed using enzyme that was expressed in E. coli and re-folded from inclusion bodies. The enzyme had robust GCR activity, but no mercuric reductase activity. The genomes of most, but not all, halobacteria for which whole genome sequences are available have close homologs of GCR, suggesting that there is more to be learned about the low molecular weight thiols used in halobacteria. PMID:23560638

  19. The role of cytochrome b5 in delta 12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.).

    PubMed

    Kearns, E V; Hugly, S; Somerville, C R

    1991-02-01

    The electron donors for the membrane-bound fatty acid desaturases of higher plants have not previously been identified. In order to assess the participation of cytochrome b5 in microsomal fatty acid desaturation, the cytoplasmic domain of microsomal cytochrome b5 was purified from Brassica oleracea, and murine polyclonal antibodies were prepared. The IgG fraction from ascites fluid inhibited 62% of NADH-dependent cytochrome c reduction in safflower (Carthamus tinctorius L.) microsomes. These antibodies also blocked desaturation of oleic acid to linoleic acid in lipids of C. tinctorius microsomes by 93%, suggesting that cytochrome b5 is the electron donor for the delta 12 desaturase.

  20. Microwave dielectric properties of (A2+(1/3)B5+(2/3))0.5Ti0(0.5)O2 (A2+ = Zn, Mg, B5+ = Nb, Ta) ceramics.

    PubMed

    Kim, E S; Kang, D H

    2008-05-01

    Dielectric properties of (A(2+)(1/3)B(5+)(2/3))(0.5)Ti0(0.5)O(2) (A(2+) = Zn, Mg, B(5+) = Nb, Ta) ceramics were investigated at microwave frequencies. A single phase with tetragonal rutile structure was obtained through the entire compositions. Dielectric properties were strongly dependent on the structural characteristics. The specimens with B(5+) = Nb showed a larger dielectric constant than those with B(5+) = Ta due to the decrease of bond valence. Quality factors (Qf) of the specimens with B(5+) = Ta were larger than those with B(5+) = Nb. Temperature coefficient of the resonant frequencies (TCF) of (Zn(1/3)Nb(2/3) )0(0.5)Ti0(0.5)O(2) was larger than that of (Mg(1/3)Ta(2/3))0(0.5)Ti0(0.5)O(2). These results could be attributed to the changes of the temperature coefficient of dielectric constant and the degree of oxygen octahedral distortion.

  1. 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

    1985-10-05

    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

  2. 3-Methyleneoxindole Reductase of Peas 1

    PubMed Central

    Moyed, H. S.; Williamson, Valerie

    1967-01-01

    A 100-fold purification of a reduced triphosphopyridine nucleotide/3-methyleneoxindole reductase of peas has been achieved using conventional protein fractionation procedures. Reduced diphosphopyridine nucleotide is 25-fold less effective than reduced triphosphopyridine nucleotide as the reductant. The preparation is free of other reductase activities including those linking the oxidation of reduced pyridine nucleotide coenzymes to the reduction of cytochrome c; vitamins K1, K2, and K3; O2; nitrate; oxidized glutathione; and thiazolyl blue tetrazolium. The affinity of the enzyme for 3-methyleneoxindole (Ks = 0.5 mm 3-methyleneoxindole) is relatively high. It is, therefore, reasonable to assume that 3-methyleneoxindole is the normal substrate. The enzyme is inhibited by indole-3-acetic acid, indole-3-aldehyde, and by l-naph-thaleneacetic acid. While these are not especially powerful inhibitors (K1 = 1.9-4.0 mm) the competitive relationship with 3-methyleneoxindole indicates that significant inhibition might occur at low intracellular concentrations of the substrate. PMID:6042360

  3. Enzyme toolbox: novel enantiocomplementary imine reductases.

    PubMed

    Scheller, Philipp N; Fademrecht, Silvia; Hofelzer, Sebastian; Pleiss, Jürgen; Leipold, Friedemann; Turner, Nicholas J; Nestl, Bettina M; Hauer, Bernhard

    2014-10-13

    Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine-chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of C=O and activated C=C bonds. However, stereoselective enzymatic reduction of C=N bonds is still in its infancy-it was only recently described after the discovery of enzymes capable of imine reduction. In our work, we increased the spectrum of imine-reducing enzymes by database analysis. By combining the currently available knowledge about the function of imine reductases with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference were identified along with amino acids important for catalysis. Furthermore, their reducing capability was demonstrated by the reduction of the pharmaceutically relevant prochiral imine 2-methylpyrroline. These novel enzymes exhibited comparable to higher catalytic efficiencies than previously described enzymes, and their biosynthetic potential is highlighted by the full conversion of 2-methylpyrroline in whole cells with excellent selectivities.

  4. SPR and electrochemical analyses of interactions between CYP3A4 or 3A5 and cytochrome b5

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

    The combination of SPR biosensor with electrochemical analysis was used for the study of protein-protein interaction between cytochromes CYP3A4 or 3А5 and cytochromes b5: the microsomal, mitochondrial forms of this protein, and 2 ≪chimeric≫ proteins. Kinetic constants of CYP3A4 and CYP3А5 complex formation with cytochromes b5 were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was observed upon their interactions with mitochondrial cytochrome b5. The electrochemical analysis of CYP3A4, CYP3A5, and cytochromes b5 immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 to -0.350 V (vs. Ag/AgCl).

  5. Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

    PubMed Central

    Kalb, Daniel; Lackner, Gerald

    2014-01-01

    A key step in fungal l-lysine biosynthesis is catalyzed by adenylate-forming l-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized l-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes. PMID:25085485

  6. 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

  7. Structural and biochemical characterization of cinnamoyl-coa reductases

    USDA-ARS?s Scientific Manuscript database

    Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a str...

  8. Tracing hepatitis B virus (HBV) genotype B5 (formerly B6) evolutionary history in the circumpolar Arctic through phylogeographic modelling.

    PubMed

    Bouckaert, Remco; Simons, Brenna C; Krarup, Henrik; Friesen, T Max; Osiowy, Carla

    2017-01-01

    Indigenous populations of the circumpolar Arctic are considered to be endemically infected (>2% prevalence) with hepatitis B virus (HBV), with subgenotype B5 (formerly B6) unique to these populations. The distinctive properties of HBV/B5, including high nucleotide diversity yet no significant liver disease, suggest virus adaptation through long-term host-pathogen association. To investigate the origin and evolutionary spread of HBV/B5 into the circumpolar Arctic, fifty-seven partial and full genome sequences from Alaska, Canada and Greenland, having known location and sampling dates spanning 40 years, were phylogeographically investigated by Bayesian analysis (BEAST 2) using a reversible-jump-based substitution model and a clock rate estimated at 4.1 × 10(-5) substitutions/site/year. Following an initial divergence from an Asian viral ancestor approximately 1954 years before present (YBP; 95% highest probability density interval [1188, 2901]), HBV/B5 coalescence occurred almost 1000 years later. Surprisingly, the HBV/B5 ancestor appears to locate first to Greenland in a rapid coastal route progression based on the landscape aware geographic model, with subsequent B5 evolution and spread westward. Bayesian skyline plot analysis demonstrated an HBV/B5 population expansion occurring approximately 400 YBP, coinciding with the disruption of the Neo-Eskimo Thule culture into more heterogeneous and regionally distinct Inuit populations throughout the North American Arctic. HBV/B5 origin and spread appears to occur coincident with the movement of Neo-Eskimo (Inuit) populations within the past 1000 years, further supporting the hypothesis of HBV/host co-expansion, and illustrating the concept of host-pathogen adaptation and balance.

  9. Major Neutralizing Sites on Vaccinia Virus Glycoprotein B5 Are Exposed Differently on Variola Virus Ortholog B6▿

    PubMed Central

    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.

    2007-01-01

    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. PMID:17522205

  10. 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.

  11. miR-135b-5p inhibits LPS-induced TNFα production via silencing AMPK phosphatase Ppm1e

    PubMed Central

    Li, Ping; Fan, Jian-bo; Gao, Yanxia; Zhang, Ming; Zhang, Li; Yang, Ning; Zhao, Xiaojing

    2016-01-01

    AMPK activation in monocytes could suppress lipopolysaccharide (LPS)-induced tissue-damaging TNFa production. We are set to provoke AMPK activation via microRNA (“miRNA”) downregulating its phosphatase Ppm1e. In human U937 and THP-1 monocytes, forced expression of microRNA-135b-5p (“miR-135b-5p”) downregulated Ppm1e and activated AMPK signaling. Further, LPS-induced TNFα production in above cells was dramatically attenuated. Ppm1e shRNA knockdown in U937 cells also activated AMPK and inhibited TNFα production by LPS. AMPK activation is required for miR-135b-induced actions in monocytes, AMPKα shRNA knockdown or T172A dominant negative mutation almost abolished miR-135b-5p's suppression on LPS-induced TNFα production. Significantly, miR-135b-5p inhibited LPS-induced reactive oxygen species (ROS) production, NFκB activation and TNFα mRNA expression in human macrophages. AMPKα knockdown or mutation again abolished above actions by miR-135b-5p. We conclude that miR-135b-5p expression downregulates Ppm1e to activate AMPK signaling, which inhibits LPS-induced TNFα production via suppressing ROS production and NFκB activation. PMID:27793001

  12. Cytochrome b5 Expression in Gonadectomy-induced Adrenocortical Neoplasms of the Domestic Ferret (Mustela putorius furo)

    PubMed Central

    Wagner, S.; Kiupel, M.; Peterson, R.A.; Heikinheimo, M.; Wilson, D.B.

    2008-01-01

    Whereas the adrenal glands of healthy ferrets produce only limited amounts of androgenic steroids, adrenocortical neoplasms that arise in neutered ferrets typically secrete androgens or their derivative, estrogen. The 17,20-lyase activity of cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17) must increase to permit androgen biosynthesis in neoplastic adrenal tissue. We screened ferret adrenocortical tumor specimens for expression of cytochrome b5 (cyt b5), an allosteric regulator that selectively enhances the 17,20-lyase activity of P450c17. Cyt b5 immunoreactivity was evident in 24 of 25 (96 %) adrenocortical adenomas/carcinomas from ferrets with signs of ectopic sex steroid production. Normal adrenocortical cells lacked cyt b5, which may account for the low production of adrenal androgens in healthy ferrets. Other markers characteristic of gonadal somatic cells, such as luteinizing hormone receptor, aromatase, and GATA4, were co-expressed with cyt b5 in some of the tumors. We conclude that cyt b5 is upregulated during gonadectomy-induced adrenocortical neoplasia and is a marker of androgen synthetic potential in these tumors. PMID:18587089

  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. Microencapsulation of an anti-VE-cadherin antibody secreting 1B5 hybridoma cells.

    PubMed

    Orive, G; Hernández, R M; Gascón, A R; Igartua, M; Rojas, A; Pedraz, J L

    2001-12-01

    Accumulating experimental evidence demonstrates that tumor growth and lethality are dependent on angiogenesis. Based on this concept, there is growing interest in the use of antiangiogenesis agents to inhibit tumor expansion. Compelling data implicate vascular endothelium (VE)-cadherin (an endothelium specific protein) as a key factor in the last step of angiogenesis, where the endothelial cells join one to each other and form microtubules (future blood vessels). We propose a novel approach to the inhibition of angiogenesis by immobilizing VE-cadherin-secreting hybridoma cells in alginate-agarose microcapsules. Hybridoma cells can be protected with biocompatible and semipermeable membranes that permit exit of anti-VE-cadherin monoclonal antibodies but not entry of cellular immune mediators. Stability studies were performed to select the suitable microcapsule for cell immobilization. Alginate and agarose solid beads coated with poly-L-lysine and alginate were chosen according to their stability and diffusional properties. 1B5 hybridoma cells were grown within the microcapsules and secreted anti-VE-cadherin antibodies during the 9 days of culture, reaching a cumulative concentration of 1.7 microg/mL. This antibody concentration inhibited microtubule formation (87%) in the in vitro angiogenesis Matrigel assay. Moreover, the antiangiogenic effect observed was antibody concentration related. These findings open a new alternative for the inhibition or prevention of angiogenesis and demonstrates the feasibility of using microencapsulated cells as a control-drug delivery system. Copyright 2001 John Wiley & Sons, Inc.

  15. Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5.

    PubMed

    Liu, Chenli; Wang, Wanpeng; Wu, Yehui; Zhou, Zhongwen; Lai, Qiliang; Shao, Zongze

    2011-05-01

    Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.

  16. Plasmodium yoelii vitamin B5 pantothenate transporter candidate is essential for parasite transmission to the mosquito.

    PubMed

    Hart, Robert J; Lawres, Lauren; Fritzen, Emma; Ben Mamoun, Choukri; Aly, Ahmed S I

    2014-07-11

    In nearly all non-photosynthetic cells, pantothenate (vitamin B5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages.

  17. 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.

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

    PubMed

    Penning, Trevor M

    2015-06-05

    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

  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.

  1. 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.

  2. A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

    SciTech Connect

    McNally, N.; Liu, Xiang Yang; Choudary, P.V.

    1997-01-01

    The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also be used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.

  3. Association of cytochrome b5 with ETR1 ethylene receptor signaling through RTE1 in Arabidopsis

    PubMed Central

    Chang, Jianhong; Clay, John M.; Chang, Caren

    2014-01-01

    Summary Ethylene plays important roles in plant growth, development and stress responses and is perceived by a family of receptors that repress ethylene responses when ethylene is absent. Repression by the ethylene receptor ETR1 depends on an integral membrane protein, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), which acts upstream of ETR1 in the endoplasmic reticulum (ER) membrane and Golgi apparatus. To investigate RTE1 function, we screened for RTE1-interacting proteins using the yeast split ubiquitin assay, which yielded the ER-localized cytochrome b5 (Cb5) isoform D. Cb5s are small hemoproteins that carry out electron transfer reactions in all eukaryotes, but their roles in plants are relatively uncharacterized. Using bimolecular fluorescence complementation (BiFC), we found that all four ER-localized Arabidopsis Cb5 isoforms (AtCb5-B, -C, -D and –E) can interact with RTE1 in plant cells. In support of this interaction, atcb5 mutants exhibited phenotypic parallels with rte1 mutants in Arabidopsis. Phenotypes included partial suppression of etr1-2 ethylene insensitivity and no suppression of RTE1-independent ethylene receptor isoforms. Single loss-of-function mutants, atcb5-b, -c and -d, appeared similar to the wild type, but double mutant combinations displayed a slight ethylene hypersensitivity. Overexpression of AtCb5-D conferred reduced ethylene sensitivity similar to that conferred by RTE1 overexpression, and genetic analyses suggested that AtCb5-D acts upstream of RTE1 in ethylene response. These findings uncover an unexpected role for Cb5, in which Cb5 and RTE1 are functional partners in promoting ETR1-mediated repression of ethylene signaling. PMID:24635651

  4. 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.

  5. Thermodynamics of interactions between mammalian cytochromes P450 and b5.

    PubMed

    Yablokov, Evgeny; Florinskaya, Anna; Medvedev, Alexei; Sergeev, Gennady; Strushkevich, Natallia; Luschik, Alexander; Shkel, Tatsiana; Haidukevich, Irina; Gilep, Andrei; Usanov, Sergey; Ivanov, Alexis

    2017-04-01

    Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Electrostatic analysis of the interaction of cytochrome c with native and dimethyl ester heme substituted cytochrome b5.

    PubMed

    Mauk, M R; Mauk, A G; Weber, P C; Matthew, J B

    1986-11-04

    The stability of the complex formed between cytochrome c and dimethyl ester heme substituted cytochrome b5 (DME-cytochrome b5) has been determined under a variety of experimental conditions to evaluate the role of the cytochrome b5 heme propionate groups in the interaction of the two native proteins. Interaction between cytochrome c and the modified cytochrome b5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8 [25 degrees C (HEPPS), I = 5 mM], DME-cytochrome b5 and cytochrome c form a 1:1 complex with an association constant KA of 3 (1) X 10(6) M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DME-cytochrome b5 and cytochrome c is strongly dependent on ionic strength with KA ranging from 2.4 X 10(7) M-1 at I = 1 mM to 8.2 X 10(4) M-1 at I = 13 mM [pH 8.0 (HEPPS), 25 degrees C]. Calculations for the native, trypsin-solubilized form of cytochrome b5 and cytochrome c confirm that the intermolecular complex proposed by Salemme [Salemme, F. R. (1976) J. Mol. Biol. 102, 563] describes the protein-protein orientation that is electrostatically favored at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. MicroRNA-125b-5p inhibits proliferation and promotes adipogenic differentiation in 3T3-L1 preadipocytes.

    PubMed

    Ouyang, Dan; Ye, Yaqiong; Guo, Dongguang; Yu, Xiaofang; Chen, Jian; Qi, Junjie; Tan, Xiaotong; Zhang, Yuan; Ma, Yongjiang; Li, Yugu

    2015-05-01

    Previous evidence has indicated that the microRNA-125b (miR-125b) family plays important roles in the regulation of cancer cell growth, development, differentiation, and apoptosis. However, whether they contribute to the process of adipocyte differentiation remains unclear. In the present study, we revealed that the expression level of miR-125b-5p, a member of miR-125b family, was dramatically up-regulated during differentiation of 3T3-L1 preadipocyte into mature adipocyte. Supplement of miR-125b-5p into 3T3-L1 cells promoted adipogenic differentiation as evidenced by increased lipid droplets and mRNA levels of adipocyte-specific molecular markers, including peroxisome proliferators-activated receptor γ, CCAAT/enhancer-binding protein α, fatty acid-binding protein 4, and lipoprotein lipase, and by triglyceride accumulation. CCK-8 assay showed that miR-125b-5p supplementation significantly inhibited cell proliferation. Flow cytometry analysis showed that miR-125b-5p impaired G1/S phase transition as well as the mRNA and protein expression of G1/S-related genes, such as Cyclin D2, Cyclin D3, and CDK4. Nevertheless, it had no effect on apoptosis. Additionally, by target gene prediction, we demonstrated that smad4 may be a potential target of miR-125b-5p in mouse 3T3-L1 preadipocytes, accounting for some of miR-125b-5p's functions. Taken together, these data indicated that miR-125b-5p may serve as an important positive regulator in adipocyte differentiation, at least partially through down-regulating smad4.

  8. Evolving the [myoglobin, cytochrome b(5)] complex from dynamic toward simple docking: charging the electron transfer reactive patch.

    PubMed

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

    2012-10-30

    We describe photoinitiated electron transfer (ET) from a suite of Zn-substituted myoglobin (Mb) variants to cytochrome b(5) (b(5)). An electrostatic interface redesign strategy has led to the introduction of positive charges into the vicinity of the heme edge through D/E → K charge-reversal mutation combinations at "hot spot" residues (D44, D60, and E85), augmented by the elimination of negative charges from Mb or b(5) by neutralization of heme propionates. These variations create an unprecedentedly large range in the product of the ET partners' total charges (-5 < -q(Mb)q(b(5)) < 40). The binding affinity (K(a)) increases 1000-fold as -q(Mb)q(b(5)) increases through this range and exhibits a surprisingly simple, exponential dependence on -q(Mb)q(b(5)). 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 -q(Mb)q(b(5)) increases, the complex undergoes a transition from fast to slow-exchange dynamics on the triplet ET time scale, with a correlated progression in the rate constants for intracomplex (k(et)) and bimolecular (k(2)) 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 its application to the extensive body of data for the ET complex between the photosynthetic reaction center and cytochrome c(2). Deviations from this model also are discussed.

  9. Kinetic mechanism of pulmonary carbonyl reductase.

    PubMed

    Matsuura, K; Nakayama, T; Nakagawa, M; Hara, A; Sawada, H

    1988-05-15

    The kinetic mechanism of guinea-pig lung carbonyl reductase was studied at pH 7 in the forward reaction with five carbonyl substrates and NAD(P)H and in the reverse reaction with propan-2-ol and NAD(P)+. In each case the enzyme mechanism was sequential, and product-inhibition studies were consistent with a di-iso ordered bi bi mechanism, in which NAD(P)H binds to the enzyme first and NAD(P)+ leaves last and the binding of cofactor induces isomerization. The kinetic and binding studies of the cofactors and several inhibitors such as pyrazole, benzoic acid, Cibacron Blue and benzamide indicate that the cofactor and Cibacron Blue bind to the free enzyme whereas the other inhibitors bind to the binary and/or ternary complexes.

  10. Kinetic mechanism of pulmonary carbonyl reductase.

    PubMed Central

    Matsuura, K; Nakayama, T; Nakagawa, M; Hara, A; Sawada, H

    1988-01-01

    The kinetic mechanism of guinea-pig lung carbonyl reductase was studied at pH 7 in the forward reaction with five carbonyl substrates and NAD(P)H and in the reverse reaction with propan-2-ol and NAD(P)+. In each case the enzyme mechanism was sequential, and product-inhibition studies were consistent with a di-iso ordered bi bi mechanism, in which NAD(P)H binds to the enzyme first and NAD(P)+ leaves last and the binding of cofactor induces isomerization. The kinetic and binding studies of the cofactors and several inhibitors such as pyrazole, benzoic acid, Cibacron Blue and benzamide indicate that the cofactor and Cibacron Blue bind to the free enzyme whereas the other inhibitors bind to the binary and/or ternary complexes. PMID:3048244

  11. Methylenetetrahydrofolate reductase: biochemical characterization and medical significance.

    PubMed

    Trimmer, Elizabeth E

    2013-01-01

    Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate). The enzyme employs a noncovalently-bound flavin adenine dinucleotide (FAD), which accepts reducing equivalents from NAD(P)H and transfers them to CH2-H4folate. The reaction provides the sole source of CH3-H4folate, which is utilized by methionine synthase in the synthesis of methionine from homocysteine. MTHFR plays a key role in folate metabolism and in the homeostasis of homocysteine; mutations in the enzyme lead to hyperhomocyst(e)inemia. A common C677T polymorphism in MTHFR has been associated with an increased risk for the development of cardiovascular disease, Alzheimer's disease, and depression in adults, and of neural tube defects in the fetus. The mutation also confers protection for certain types of cancers. This review presents the current knowledge of the enzyme, its biochemical characterization, and medical significance.

  12. Characterization of in vitro biotransformation of new, orally active, direct thrombin inhibitor ximelagatran, an amidoxime and ester prodrug.

    PubMed

    Clement, Bernd; Lopian, Katrin

    2003-05-01

    N-Hydroxylated amidines (amidoximes) can be used as prodrugs of amidines. The prodrug principle was developed in our laboratory for pentamidine and had been applied to several other drug candidates. One of these compounds is melagatran, a novel, synthetic, low molecular weight, direct thrombin inhibitor. To increase the poor oral bioavailability due to its strong basic amidine functionality selected to fit the arginine side pocket of thrombin, the less basic N-hydroxylated amidine was used in addition to an ethyl ester-protecting residue. The objective of this investigation was to study the reduction and the hydrolytic metabolism of ximelagatran via two mono-prodrugs (N-hydroxy-melagatran and ethyl-melagatran) to melagatran by in vitro experiments. New high-performance liquid chromatography methods were developed to analyze all four compounds. The biotransformation of ximelagatran to melagatran involving the reduction of the amidoxime function and the ester cleavage could be demonstrated in vitro by microsomes and mitochondria from liver and kidney of pig and human, and the kinetic parameters were determined. So far, one enzyme system capable of reducing N-hydroxylated structures has been identified in pig liver microsomes, consisting of cytochrome b(5), NADH-cytochrome b(5) reductase, and a P450 isoenzyme of the subfamily 2D. This enzyme system also reduces ximelagatran and N-hydroxy-melagatran. The participation of recombinant human CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 with cytochrome b(5) and b(5) reductase in the reduction can be excluded. In summary, ximelagatran and N-hydroxy-melagatran are easily reduced by several enzyme systems located in microsomes and mitochondria of different organs.

  13. 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. 2011 Elsevier B.V. All rights reserved.

  14. 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

  15. Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity.

    PubMed

    Vaidyanathan, Ramanathan; Gopalram, Shubaash; Kalishwaralal, Kalimuthu; Deepak, Venkataraman; Pandian, Sureshbabu Ram Kumar; Gurunathan, Sangiliyandi

    2010-01-01

    Nanostructure materials are attracting a great deal of attention because of their potential for achieving specific processes and selectivity, especially in biological and pharmaceutical applications. The generation of silver nanoparticles using optimized nitrate reductase for the reduction of Ag(+) with the retention of enzymatic activity in the complex is being reported. This report involves the optimization of enzyme activity to bring about enhanced nanoparticle synthesis. Response surface methodology and central composite rotary design (CCRD) were employed to optimize a fermentation medium for the production of nitrate reductase by Bacillus licheniformis at pH 8. The four variables involved in the study of nitrate reductase were Glucose, Peptone, Yeast extract and KNO(3). Glucose had a significant effect on nitrate reductase production. The optimized medium containing (%) Glucose: 1.5, Peptone: 1, Yeast extract: 0.35 and KNO(3): 0.35 resulted in a nitrate reductase activity of 452.206 U/ml which is same as that of the central level. The medium A (showing least nitrate reductase activity) and the medium B (showing maximum nitrate reductase activity) were compared for the synthesis. Spectrophotometric analysis revealed that the particles exhibited a peak at 431 nm and the A(431) for the medium B was 2-fold greater than that of the medium A. The particles were also characterized using TEM. The particles synthesized using the optimized enzyme activity ranged from 10 to 80 nm and therefore can be extended to various medicinal applications.

  16. Microstructure and magnetic viscosity of bulk amorphous Nd60Fe20Al5Co10B5 alloy

    NASA Astrophysics Data System (ADS)

    Tan, X. H.; Xu, H.; Man, H.; Tang, Y. J.; Yang, L. P.; Bai, Q.

    2011-04-01

    The microstructure and magnetic viscosity of bulk amorphous Nd60Fe20Al5Co10B5,prepared by suction casting the molten alloy into a copper mold under an argon atmosphere, have been investigated. The results show that clusters with size 3-5 nm are found to be embedded in the amorphous matrix of as-cast bulk amorphous Nd60Fe20Al5Co10B5 alloy. The Nd60Fe20Al5Co10B5 alloy shows hard magnetic behavior at room temperature, with an intrinsic coercivity of 360 kA/m and a remanence of 69.39 mT. The magnetic viscosity of bulk amorphous Nd60Fe20Al5Co10B5 has been investigated on the major hysteresis loop, and simple logarithmic time dependence is observed. A value for the fluctuation field of 8.24 kA/m is obtained. Analysis based on a Henkel plot is used to confirm the existence of magnetic interaction among clusters. The coercivity mechanism responsible for the hard magnetic behavior of bulk amorphous Nd60Fe20Al5Co10B5 alloy is also discussed.

  17. Synthesis, structure, and properties of the noncentrosymmetric hydrated borate Na(2)B(5)O(8)(OH).2H(2)O.

    PubMed

    Wang, Yongjiang; Pan, Shilie; Tian, Xuelin; Zhou, Zhongxiang; Liu, Gang; Wang, Jide; Jia, Dianzeng

    2009-08-17

    Single crystal of hydrated sodium borate Na(2)B(5)O(8)(OH).2H(2)O has been grown with sizes up to 5 x 5 x 3 mm(3) under mild hydrothermal conditions at 180 degrees C. The structure is determined by single-crystal X-ray diffraction and further characterized by IR and TG analyses. It crystallizes in the orthorhombic space group Pna2(1), with a = 11.967(2) A, b = 6.5320(13) A, c = 11.126(2) A, Z = 4, R1 = 0.0183, and wR2 = 0.0483. The crystal structure of Na(2)B(5)O(8)(OH).2H(2)O is made up of Na-O polyhedra, and [B(5)O(8)(OH)](2-) polyborate anions. Transmittance spectrum is performed on the Na(2)B(5)O(8)(OH).2H(2)O crystal, which shows an absorption edge less than 190 nm in the UV region. The powder second-harmonic generation intensity measured by the Kurtz-Perry method indicates that Na(2)B(5)O(8)(OH).2H(2)O is about half that of KH(2)PO(4) (KDP).

  18. 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.

  19. Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase

    PubMed Central

    Erb, Tobias J.; Brecht, Volker; Fuchs, Georg; Müller, Michael; Alber, Birgit E.

    2009-01-01

    Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO2. Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis. PMID:19458256

  20. The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

    PubMed

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2002-07-16

    Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

  1. Dopamine suppresses neuronal activity of Helisoma B5 neurons via a D2-like receptor, activating PLC and K channels.

    PubMed

    Zhong, L R; Artinian, L; Rehder, V

    2013-01-03

    Dopamine (DA) plays fundamental roles as a neurotransmitter and neuromodulator in the central nervous system. How DA modulates the electrical excitability of individual neurons to elicit various behaviors is of great interest in many systems. The buccal ganglion of the freshwater pond snail Helisoma trivolvis contains the neuronal circuitry for feeding and DA is known to modulate the feeding motor program in Helisoma. The buccal neuron B5 participates in the control of gut contractile activity and is surrounded by dopaminergic processes, which are expected to release DA. In order to study whether DA modulates the electrical activity of individual B5 neurons, we performed experiments on physically isolated B5 neurons in culture and on B5 neurons within the buccal ganglion in situ. We report that DA application elicited a strong hyperpolarization in both conditions and turned the electrical activity from a spontaneously firing state to an electrically silent state. Using the cell culture system, we demonstrated that the strong hyperpolarization was inhibited by the D2 receptor antagonist sulpiride and the phospholipase C (PLC) inhibitor U73122, indicating that DA affected the membrane potential of B5 neurons through the activation of a D2-like receptor and PLC. Further studies revealed that the DA-induced hyperpolarization was inhibited by the K channel blockers 4-aminopyridine and tetraethylammonium, suggesting that K channels might serve as the ultimate target of DA signaling. Through its modulatory effect on the electrical activity of B5 neurons, the release of DA in vivo may contribute to a neuronal output that results in a variable feeding motor program.

  2. 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.

  3. 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.

  4. M3 subtype of muscarinic acetylcholine receptor promotes cardioprotection via the suppression of miR-376b-5p.

    PubMed

    Pan, Zhenyu; Guo, Yueping; Qi, Hanping; Fan, Kai; Wang, Shu; Zhao, Hua; Fan, Yuhua; Xie, Jing; Guo, Feng; Hou, Yunlong; Wang, Ning; Huo, Rong; Zhang, Yong; Liu, Yan; Du, Zhimin

    2012-01-01

    The M(3) subtype of muscarinic acetylcholine receptors (M(3)-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M(3)-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M(3)-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M(3)-mAChR. In H9c2 cells, the viability, intracellular free Ca(2+) concentration ([Ca(2+)]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M(3)-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M(3)-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H(2)O(2)-induced H9c2 cell injuries measured by cells viability, [Ca(2+)]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M(3)-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M(3)-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M(3)-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M(3)-mAChR provides cardioprotection against myocardial ischemia injury.

  5. 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.

  6. 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.

  7. Exploration of Nitrate Reductase Metabolic Pathway in Corynebacterium pseudotuberculosis

    PubMed Central

    Abreu, Vinícius; Diniz, Carlos; Dorneles, Elaine M. S.; Barh, Debmalya

    2017-01-01

    Based on the ability of nitrate reductase synthesis, Corynebacterium pseudotuberculosis is classified into two biovars: Ovis and Equi. Due to the presence of nitrate reductase, the Equi biovar can survive in absence of oxygen. On the other hand, Ovis biovar that does not have nitrate reductase is able to adapt to various ecological niches and can grow on certain carbon sources. Apart from these two biovars, some other strains are also able to carry out the reduction of nitrate. The enzymes that are involved in electron transport chain are also identified by in silico methods. Findings about pathogen metabolism can contribute to the identification of relationship between nitrate reductase and the C. pseudotuberculosis pathogenicity, virulence factors, and discovery of drug targets. PMID:28316974

  8. Exploration of Nitrate Reductase Metabolic Pathway in Corynebacterium pseudotuberculosis.

    PubMed

    Almeida, Sintia; Sousa, Cassiana; Abreu, Vinícius; Diniz, Carlos; Dorneles, Elaine M S; Lage, Andrey P; Barh, Debmalya; Azevedo, Vasco

    2017-01-01

    Based on the ability of nitrate reductase synthesis, Corynebacterium pseudotuberculosis is classified into two biovars: Ovis and Equi. Due to the presence of nitrate reductase, the Equi biovar can survive in absence of oxygen. On the other hand, Ovis biovar that does not have nitrate reductase is able to adapt to various ecological niches and can grow on certain carbon sources. Apart from these two biovars, some other strains are also able to carry out the reduction of nitrate. The enzymes that are involved in electron transport chain are also identified by in silico methods. Findings about pathogen metabolism can contribute to the identification of relationship between nitrate reductase and the C. pseudotuberculosis pathogenicity, virulence factors, and discovery of drug targets.

  9. Enantioselective imine reduction catalyzed by imine reductases and artificial metalloenzymes.

    PubMed

    Gamenara, Daniela; Domínguez de María, Pablo

    2014-05-21

    Adding value to organic synthesis. Novel imine reductases enable the enantioselective reduction of imines to afford optically active amines. Likewise, novel bioinspired artificial metalloenzymes can perform the same reaction as well. Emerging proof-of-concepts are herein discussed.

  10. Purification and characterization of assimilatory nitrite reductase from Candida utilis.

    PubMed

    Sengupta, S; Shaila, M S; Rao, G R

    1996-07-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

  11. 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

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

    PubMed

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

    1997-09-15

    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

  13. Hepatic dysfunction induced by 7, 12-dimethylbenz(α)anthracene and its obviation with erucin using enzymatic and histological changes as indicators.

    PubMed

    Arora, Rohit; Bhushan, Sakshi; Kumar, Rakesh; Mannan, Rahul; Kaur, Pardeep; Singh, Amrit Pal; Singh, Bikram; Vig, Adarsh P; Sharma, Deepika; Arora, Saroj

    2014-01-01

    The toxicity induced by 7, 12-dimethylbenz(α)anthracene (DMBA) has been widely delineated by a number of researchers. This potent chemical damages many internal organs including liver, by inducing the production of reactive oxygen species, DNA-adduct formation and affecting the activities of phase I, II, antioxidant and serum enzymes. Glucosinolate hydrolytic products like isothiocyanates (ITCs) are well known for inhibiting the DNA-adduct formation and modulating phase I, II enzymes. Sulforaphane is ITC, currently under phase trials, is readily metabolized and inter-converted into erucin upon ingestion. We isolated erucin from Eruca sativa (Mill.) Thell. evaluated its hepatoprotective role in DMBA induced toxicity in male wistar rats. The rats were subjected to hepatic damage by five day regular intraperitoneal doses of DMBA. At the end of the protocol, the rats were euthanized, their blood was collected and livers were processed. The liver homogenate was analyzed for phase I (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, cytochrome P450, cytochrome P420 and cytochrome b5), phase II (DT diaphorase, glutathione-S-transferase and γ-glutamyl transpeptidase) and antioxidant enzymes (superoxide dismutase, catalase, guaiacol peroxidise, ascorbate peroxidise, glutathione reductase and lactate dehydrogenase). The level of thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes and reduced glutathione in the liver homogenate was also analyzed. The serum was also analyzed for markers indicating hepatic damage (alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin and total bilirubin). Erucin provided significant protection against DMBA induced damage by modulating the phase I, II and antioxidant enzymes. The histological evaluation of liver tissue was also conducted, which showed the hepatoprotective role of erucin.

  14. Hepatic Dysfunction Induced by 7, 12-Dimethylbenz(α)anthracene and Its Obviation with Erucin Using Enzymatic and Histological Changes as Indicators

    PubMed Central

    Arora, Rohit; Bhushan, Sakshi; Kumar, Rakesh; Mannan, Rahul; Kaur, Pardeep; Singh, Amrit Pal; Singh, Bikram; Vig, Adarsh P.; Sharma, Deepika; Arora, Saroj

    2014-01-01

    The toxicity induced by 7, 12-dimethylbenz(α)anthracene (DMBA) has been widely delineated by a number of researchers. This potent chemical damages many internal organs including liver, by inducing the production of reactive oxygen species, DNA-adduct formation and affecting the activities of phase I, II, antioxidant and serum enzymes. Glucosinolate hydrolytic products like isothiocyanates (ITCs) are well known for inhibiting the DNA-adduct formation and modulating phase I, II enzymes. Sulforaphane is ITC, currently under phase trials, is readily metabolized and inter-converted into erucin upon ingestion. We isolated erucin from Eruca sativa (Mill.) Thell. evaluated its hepatoprotective role in DMBA induced toxicity in male wistar rats. The rats were subjected to hepatic damage by five day regular intraperitoneal doses of DMBA. At the end of the protocol, the rats were euthanized, their blood was collected and livers were processed. The liver homogenate was analyzed for phase I (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, cytochrome P450, cytochrome P420 and cytochrome b5), phase II (DT diaphorase, glutathione-S-transferase and γ-glutamyl transpeptidase) and antioxidant enzymes (superoxide dismutase, catalase, guaiacol peroxidise, ascorbate peroxidise, glutathione reductase and lactate dehydrogenase). The level of thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes and reduced glutathione in the liver homogenate was also analyzed. The serum was also analyzed for markers indicating hepatic damage (alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin and total bilirubin). Erucin provided significant protection against DMBA induced damage by modulating the phase I, II and antioxidant enzymes. The histological evaluation of liver tissue was also conducted, which showed the hepatoprotective role of erucin. PMID:25390337

  15. [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.

  16. [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.

  17. Carbon-carbon double-bond reductases in nature.

    PubMed

    Huang, Minmin; Hu, Haihong; Ma, Li; Zhou, Quan; Yu, Lushan; Zeng, Su

    2014-08-01

    Reduction of C = C bonds by reductases, found in a variety of microorganisms (e.g. yeasts, bacteria, and lower fungi), animals, and plants has applications in the production of metabolites that include pharmacologically active drugs and other chemicals. Therefore, the reductase enzymes that mediate this transformation have become important therapeutic targets and biotechnological tools. These reductases are broad-spectrum, in that, they can act on isolation/conjugation C = C-bond compounds, α,β-unsaturated carbonyl compounds, carboxylic acids, acid derivatives, and nitro compounds. In addition, several mutations in the reductase gene have been identified, some associated with diseases. Several of these reductases have been cloned and/or purified, and studies to further characterize them and determine their structure in order to identify potential industrial biocatalysts are still in progress. In this study, crucial reductases for bioreduction of C = C bonds have been reviewed with emphasis on their principal substrates and effective inhibitors, their distribution, genetic polymorphisms, and implications in human disease and treatment.

  18. Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

    PubMed

    Takeda, Kouji; Nishiyama, Yoshitaka; Yoda, Koji; Watanabe, Toshihiro; Nimura-Matsune, Kaori; Mura, Kiyoshi; Tokue, Chiyoko; Katoh, Tetzuya; Kawasaki, Shinji; Niimura, Youichi

    2004-01-01

    Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

  19. Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

    PubMed

    Heine, Thomas; Tucker, Kathryn; Okonkwo, Nonye; Assefa, Berhanegebriel; Conrad, Catleen; Scholtissek, Anika; Schlömann, Michael; Gassner, George; Tischler, Dirk

    2017-04-01

    The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s(-1). This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

  20. Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

    PubMed Central

    Iuchi, S; Lin, E C

    1987-01-01

    In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector. PMID:3301812

  1. [Expression of HoxB5, SPC and AQP5 in neonatal rats with hyperoxia-induced chronic lung disease].

    PubMed

    Xu, Wei; Fu, Jian-Hua; Xue, Xin-Dong

    2009-01-01

    Alveolar epithelium impairment is one of pathological changes associated with chronic lung disease (CLD). Hoxb5 is one of the few homeobox genes strongly expressed in the developing lung. This study investigated the expression of HoxB5, SPC and AQP5 in rats with CLD in order to explore the role of Hoxb-5 in impairment and reparation of alveolar epithelium. Eighty neonatal rats were randomly exposed to hyperoxia (model group) or to room air (control group) (n=40 each). The CLD model was induced by hyperoxia exposure. The expression of HoxB5, SPC and AQP5 protein and mRNA in the lung tissue was detected by immunohistochemistry and RT-PCR 1, 3, 7, 14 and 21 days after exposure. In the model group HoxB5 expression significantly decreased 7, 14 and 21 days after hyperoxia exposure. SPC expression decreased 3 days after hyperoxia exposure but increased significantly 7, 14 and 21 days after hyperoxia exposure as compared to the control group. AQP5 expression was progressively reduced with prolonged hyperoxia exposure. Hyperoxia exposure may lead to alveolar epithelial cell (AEC) damage in neonatal rats. The increased SPC expression and decreased AQP5 expression suggested that the ability of differentiation and transformation of AECII into AECI decreased in neonatal rats with CLD. The decreased HoxB5 expression following hyperoxia exposure might contribute to a decreased ability of differentiation of AECII.

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... allowable under section 2056(b)(5), relating to the exercise of a general power of appointment by the... and thenceforth it, and the investments made with it, must be so segregated or accounted for as to...,000 to C, as trustee. C is to invest the money and pay all of the income from the investments to W...

  3. Microsecond subdomain folding in dihydrofolate reductase.

    PubMed

    Arai, Munehito; Iwakura, Masahiro; Matthews, C Robert; Bilsel, Osman

    2011-07-08

    The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR--in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse--emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.

  4. Aldose reductase mediates retinal microglia activation

    SciTech Connect

    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{sup GFP} mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR{sup 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. - Highlights: • AR inhibition prevents retinal microglial activation. • Endotoxin-induced ocular cytokine production is reduced in AR null mice. • Overexpression of AR spontaneously induces retinal microglial activation.

  5. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  6. Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo.

    PubMed

    Henderson, Colin J; McLaughlin, Lesley A; Scheer, Nico; Stanley, Lesley A; Wolf, C Roland

    2015-04-01

    The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and >80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by >50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol α-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC0-8 h) of triazolam were increased 4- and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC0-8 h being increased ∼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.

  7. 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

  8. Activity-guided isolation of constituents of Tephrosia purpurea with the potential to induce the phase II enzyme, quinone reductase.

    PubMed

    Chang, L C; Gerhäuser, C; Song, L; Farnsworth, N R; Pezzuto, J M; Kinghorn, A D

    1997-09-01

    An isoflavone, 7,4'-dihydroxy-3',5'-dimethoxyisoflavone (1), and a chalcone, (+)-tephropurpurin (2), both novel compounds, as well as six constituents of known structure, (+)-purpurin (3), pongamol (4), lanceolatin B (5), (-)-maackiain (6), (-)-3-hydroxy-4-methoxy-8,9-methylene-dioxypterocarpan (7), and (-)-medicarpin (8), were obtained as active compounds from Tephrosia purpurea, using a bioassay based on the induction of quinone reductase (QR) activity with cultured Hepa 1c1c7 mouse hepatoma cells. Additionally, three inactive compounds of known structure, 3'-methoxydaidzein, desmoxyphyllin B, and 3,9-dihydroxy-8-methoxycoumestan, were isolated and identified. The structure elucidation of compounds 1 and 2 was carried out by spectral data interpretation.

  9. Activation and detoxification metabolism of urban air pollutants 2-nitrobenzanthrone and carcinogenic 3-nitrobenzanthrone by rat and mouse hepatic microsomes.

    PubMed

    Stiborova, Marie; Cechova, Tereza; Borek-Dohalska, Lucie; Moserova, Michaela; Frei, Eva; Schmeiser, Heinz H; Paca, Jan; Arlt, Volker M

    2012-01-01

    2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. Understanding which enzymes are involved in metabolism of these toxicants is important in the assessment of individual susceptibility. Here, metabolism of 2-NBA and 3-NBA by rat and mouse hepatic microsomes containing cytochromes P450 (CYPs), their reductase (NADPH:CYP reductase), and NADH:cytochrome b5 reductase was investigated under anaerobic and aerobic conditions. In addition, using the same microsomal systems, 2-NBA and 3-NBA were evaluated to be enzymatically activated under anaerobic conditions to species generating 2-NBA- and 3-NBA-derived DNA adducts. High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed for the separation and characterization of 2-NBA and 3-NBA metabolites formed by hepatic microsomes of rats and mice under the anaerobic and aerobic conditions. Microsomal systems isolated from the liver of the control (untreated) rats and rats pretreated with Sudan I, β-naphthoflavone (β-NF), phenobarbital (PB), ethanol and pregnenolon 16α-carbonitrile (PCN), the inducers of cytochromes P450 (CYP) 1A1, 1A1/2, 2B, 2E1 and 3A, respectively, were used in this study. Microsomes of mouse models, a control mouse line (wild-type, WT) and Hepatic Cytochrome P450 Reductase Null (HRN) mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in their livers, were also employed. To detect and quantify the 2-NBA- and 3-NBA-derived DNA adducts, the 32P postlabeling technique was used. Both reductive metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), found to be formed predominantly under the anaerobic conditions, and two 3-NBA oxidative metabolites, whose structures have not yet been investigated, were formed by several microsomal systems used in the study. Whereas a 3-NBA reductive metabolite

  10. Interfacial Hydration, Dynamics and Electron Transfer: Multi-Scale ET Modeling of the Transient [Myoglobin, Cytochrome b5] Complex

    PubMed Central

    Keinan, Shahar; Nocek, Judith M.; Beratan, David N.; Hoffman, Brian M.

    2012-01-01

    Formation of a transient [myoglobin (Mb), cytochrome b5 (cyt b5)] complex is required for the reductive repair of inactive ferri-Mb to its functional ferro-Mb state. The [Mb, cyt b5] complex exhibits dynamic docking (DD), with its cyt b5 partner in rapid exchange at multiple sites on the Mb surface. A triple mutant (Mb(3M)) was designed as part of efforts to shift the electron-transfer process to the simple docking (SD) regime, in which reactive binding occurs at a restricted, reactive region on the Mb surface that dominates the docked ensemble.1 An electrostatically-guided Brownian dynamics (BD) docking protocol was used to generate an initial ensemble of reactive configurations of the complex between unrelaxed partners. This ensemble samples a broad and diverse array of heme-heme distances and orientations. These configurations seeded all-atom constrained molecular dynamics simulations (MD) to generate relaxed complexes for the calculation of electron tunneling matrix elements (TDA) through tunneling-pathway analysis. This procedure for generating an ensemble of relaxed complexes combines the ability of BD calculations to sample the large variety of available conformations and interprotein distances, with the ability of MD to generate the atomic level information, especially regarding the structure of water molecules at the protein-protein interface, that defines electron-tunneling pathways. We used the calculated TDA values to compute ET rates for the [Mb(wt), cyt b5] complex and for the complex with a mutant that has a binding free energy strengthened by three D/E → K charge-reversal mutations, [Mb(3M), cyt b5]. The calculated rate constants are in agreement with the measured values, and the mutant complex ensemble has many more geometries with higher TDA values than does the wild-type Mb complex. Interestingly, water plays a double role in this electron-transfer system, lowering the tunneling barrier as well as inducing protein interface remodeling that

  11. Sulfite reductase protects plants against sulfite toxicity.

    PubMed

    Yarmolinsky, Dmitry; Brychkova, Galina; Fluhr, Robert; Sagi, Moshe

    2013-02-01

    Plant sulfite reductase (SiR; Enzyme Commission 1.8.7.1) catalyzes the reduction of sulfite to sulfide in the reductive sulfate assimilation pathway. Comparison of SiR expression in tomato (Solanum lycopersicum 'Rheinlands Ruhm') and Arabidopsis (Arabidopsis thaliana) plants revealed that SiR is expressed in a different tissue-dependent manner that likely reflects dissimilarity in sulfur metabolism between the plant species. Using Arabidopsis and tomato SiR mutants with modified SiR expression, we show here that resistance to ectopically applied sulfur dioxide/sulfite is a function of SiR expression levels and that plants with reduced SiR expression exhibit higher sensitivity than the wild type, as manifested in pronounced leaf necrosis and chlorophyll bleaching. The sulfite-sensitive mutants accumulate applied sulfite and show a decline in glutathione levels. In contrast, mutants that overexpress SiR are more tolerant to sulfite toxicity, exhibiting little or no damage. Resistance to high sulfite application is manifested by fast sulfite disappearance and an increase in glutathione levels. The notion that SiR plays a role in the protection of plants against sulfite is supported by the rapid up-regulation of SiR transcript and activity within 30 min of sulfite injection into Arabidopsis and tomato leaves. Peroxisomal sulfite oxidase transcripts and activity levels are likewise promoted by sulfite application as compared with water injection controls. These results indicate that, in addition to participating in the sulfate assimilation reductive pathway, SiR also plays a role in protecting leaves against the toxicity of sulfite accumulation.

  12. Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice

    PubMed Central

    Wang, Xiu Jun; Chamberlain, Mark; Vassieva, Olga; Henderson, Colin J.; Wolf, C. Roland

    2005-01-01

    Cytochrome P450 reductase is the unique electron donor for microsomal cytochrome P450s; these enzymes play a major role in the metabolism of endogenous and xenobiotic compounds. In mice with a liver-specific deletion of cytochrome P450 reductase, hepatic cytochrome P450 activity is ablated, with consequent changes in bile acid and lipid homoeostasis. In order to gain insights into the metabolic changes resulting from this phenotype, we have analysed changes in hepatic mRNA expression using microarray analysis and real-time PCR. In parallel with the perturbations in bile acid levels, changes in the expression of key enzymes involved in cholesterol and lipid homoeostasis were observed in hepatic cytochrome P450 reductase null mice. This was characterized by a reduced expression of Cyp7b1, and elevation of Cyp7a1 and Cyp8b1 expression. The levels of mRNAs for other cytochrome P450 genes, including Cyp2b10, Cyp2c29, Cyp3a11 and Cyp3a16, were increased, demonstrating that endogenous factors play a role in regulating the expression of these proteins and that the increases are due, at least in part, to altered levels of transcripts. In addition, levels of mRNAs encoding genes involved in glycolysis and lipid transport were also increased; the latter may provide an explanation for the increased hepatic lipid content observed in the hepatic null mice. Serum testosterone and oestradiol levels were lowered, accompanied by significantly decreased expression of Hsd3b2 (3β-hydroxy-Δ5-steroid dehydrogenase-2), Hsd3b5 (3β-hydroxy-Δ5-steroid dehydrogenase-5) and Hsd11b1 (11β-hydroxysteroid dehydrogenase type 1), key enzymes in steroid hormone metabolism. These microarray data provide important insights into the control of metabolic pathways by the cytochrome system. PMID:15717863

  13. Comparative sequence analysis of B5R gene of zoonotic buffalo pox virus isolates with other orthopoxviruses.

    PubMed

    Chandranaik, B M; Singh, Raj Kumar; Hosamani, Mahusudan; Krishnappa, Giriappa; Harish, Balur R; Chethana, C S; Renukaprasad, C

    2011-02-01

    The present paper describes the isolation of buffalo pox virus from scab lesions and its molecular characterization through B5R gene sequencing. During our study, pustular pox lesions were observed on the teats and mammary parenchyma of cattle and buffaloes, and the disease was of significant zoonotic importance since similar lesions were produced on the hands, legs, and face of people in close contact with the affected animals. The collected scab materials were subjected for virus isolation in 9-11-day-old chicken embryos by the chorioallontoic membrane route and in the Vero cell line. The virus was confirmed by a sensitive and rapid diagnostic polymerase chain reaction using the primers that amplify "A type inclusion" gene, and further, B5R gene of the virus was sequenced and compared with the corresponding sequences of other orthopoxviruses. The results showed high sequence homology of our isolates with other orthopoxviruses.

  14. Valproic acid downregulates RBP4 and elicits hypervitaminosis A-teratogenesis--a kinetic analysis on retinol/retinoic acid homeostatic system.

    PubMed

    Chuang, Chao-Ming; Chang, Chi-Huang; Wang, Hui-Er; Chen, Kuan-Chou; Peng, Chiung-Chi; Hsieh, Chiu-Lan; Peng, Robert Y

    2012-01-01

    Valproic acid (VPA) is an antiepileptic and anti-migraine prophylactic drug. VPA exhibits two severe side effects, namely acute liver toxicity and teratogenicity. These side effects are usually seen at the genetic and somatic levels. The cited action mechanisms involve inhibition of histone deacetylase, hypofolatenemia, hyperhomocysteinemia, and reactive oxidative stress. The proteomic information associated with VPA teratogenicity is still unavailable. We hypothesized that proteomic analysis might help us identify functional proteins that could be relevantly affected by VPA, and this phenomenon could be very sensitive in early embryonic stage, resulting in VPA teratogenicity. Proteomic analysis on the chicken embryos at Hamburger and Hamilton (HH) stage 28 showed that there were significant downregulations of ovotransferrins, carbonic anhydrase-2, retinol binding protein-4 (RBP4), NADH cytochrome b5 reductase 2 (CYB5R2), apolipoprotein A1, and protein SET, together with upregulation of 60S ribosomal protein L22. Among these, RBP4 was the most significantly downregulated (-32%). Kinetic analysis suggested that this situation could trigger hypervitaminosis A (+39.3%), a condition that has been well known to induce teratogenesis.. This is the first report showing that VPA dowregulates RBP4. Our finding not only has led to a possible mechanism of VPA teratogenesis, but also has initiated new preventive strategies for avoiding VPA teratogeneis.

  15. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

    PubMed Central

    Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

    2011-01-01

    The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

  17. 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.

  18. Valproic Acid Downregulates RBP4 and Elicits Hypervitaminosis A-Teratogenesis—A Kinetic Analysis on Retinol/Retinoic Acid Homeostatic System

    PubMed Central

    Chuang, Chao-Ming; Chang, Chi-Huang; Wang, Hui-Er; Chen, Kuan-Chou; Peng, Chiung-Chi; Hsieh, Chiu-Lan; Peng, Robert Y.

    2012-01-01

    Background Valproic acid (VPA) is an antiepileptic and anti-migraine prophylactic drug. VPA exhibits two severe side effects, namely acute liver toxicity and teratogenicity. These side effects are usually seen at the genetic and somatic levels. The cited action mechanisms involve inhibition of histone deacetylase, hypofolatenemia, hyperhomocysteinemia, and reactive oxidative stress. The proteomic information associated with VPA teratogenicity is still unavailable. We hypothesized that proteomic analysis might help us identify functional proteins that could be relevantly affected by VPA, and this phenomenon could be very sensitive in early embryonic stage, resulting in VPA teratogenicity. Methodology/Principal Findings Proteomic analysis on the chicken embryos at Hamburger and Hamilton (HH) stage 28 showed that there were significant downregulations of ovotransferrins, carbonic anhydrase-2, retinol binding protein-4 (RBP4), NADH cytochrome b5 reductase 2 (CYB5R2), apolipoprotein A1, and protein SET, together with upregulation of 60S ribosomal protein L22. Among these, RBP4 was the most significantly downregulated (−32%). Kinetic analysis suggested that this situation could trigger hypervitaminosis A (+39.3%), a condition that has been well known to induce teratogenesis.. Conclusions/Significance This is the first report showing that VPA dowregulates RBP4. Our finding not only has led to a possible mechanism of VPA teratogenesis, but also has initiated new preventive strategies for avoiding VPA teratogeneis. PMID:23028466

  19. Heme synthase (ferrochelatase) catalyzes the removal of iron from heme and demetalation of metalloporphyrins.

    PubMed

    Taketani, Shigeru; Ishigaki, Mutsumi; Mizutani, Atsushi; Uebayashi, Masashi; Numata, Masahiro; Ohgari, Yoshiko; Kitajima, Sakihito

    2007-12-25

    The red pigments in meat products, including cooked cured ham, arise from the reaction of myoglobin with nitric oxide generated from exogenous nitrite. Since carcinogenic nitrosoamines may be generated by the treatment of meats with nitrite, the production of nitrite-free meat products is an attractive alternative. Raw dry-cured (Parma) hams are produced by the treatment of meats with salts other than nitrite. Analysis of pigments in raw dry-cured hams reveals that the main pigment is zinc protoporphyrin, suggesting that the conversion of heme to zinc protoporphyrin occurs via an iron-removal reaction from myoglobin heme during the processing of raw hams. Purification of the iron-removal enzyme showed that it was identical to ferrochelatase. Recombinant ferrochelatase in combination with NADH-cytochrome b5 reductase catalyzed NADH-dependent iron-removal reaction from hemin and hemoproteins. Metal ions such as zinc and cobalt were also removed from the corresponding metalloporphyrins. The addition of zinc ions led to the formation of zinc protoporphyrin. In cultured cells, the conversion of zinc mesoporphyrin to mesoheme was observed to be dependent on ferrochelatase and could be markedly induced during erythroid differentiation. This is the first demonstration of a new enzyme reaction, the reverse reaction of ferrochelatase, which may contribute to a new route of the recycling of protoporphyrin and heme in cells.

  20. Over-expression of miR-196b-5p is significantly associated with the progression of myelodysplastic syndrome.

    PubMed

    Wen, Jing; Huang, Ying; Li, Hongying; Zhang, Xupai; Cheng, Peng; Deng, Donghong; Peng, Zhigang; Luo, Jun; Zhao, Weihua; Lai, Yongrong; Liu, Zhenfang

    2017-02-21

    Myelodysplastic syndrome (MDS) is a clonal stem cell disorder characterized by ineffective hematopoiesis with a high risk of transformation to acute myeloid leukemia (AML). miRNAs function as tumor suppressors and oncogenes in various cancers and regulate the differentiation potential of hematopoietic stem and progenitor cells (HSPCs). It has been suggested that miRNAs may play an important role in progression of MDS. We analyzed bone marrow samples collected from MDS patients according to different risk stratification indicated by the International Prognostic Scoring System (IPSS). We demonstrated that miR-196b-5p was up-regulated in intermediate II and higher groups, and in secondary AML (s-AML) patients in particular (P < 0.01) compared with healthy controls, suggesting that the higher expression levels are associated with increased risk of the development of MDS. We observed changes in proliferation and apoptosis in MDS-L cells following transfection with miR-196-5p mimics or inhibitors. After up-regulating the expression of miR-196b-5p, proliferation of MDS-L cells was up-regulated, whereas apoptosis was down-regulated (P < 0.05). In contrast, down-regulation of miR-196b-5p expression decreased cell proliferation and increased apoptosis (P < 0.05). We concluded that over-expression of miR-196b-5p may be closely associated with the risk of transformation to leukemia in MDS patients.

  1. Effect of Carbonitriding on Corrosion Resistance of Steel 30MnB5 in Two Acidic Environments

    NASA Astrophysics Data System (ADS)

    Yazici, Aysel; Sadrettin Zeybek, M.; Güler, Hüseyin; Koç, Muhterem; Göksel Pekýtkan, F.

    2017-07-01

    Corrosion resistance of steel 30MnB5 is studied after carbonitriding, after conventional quenching, and without treatment. Corrosion tests are performed for from 24 to 168 h in two acidic environments (in a hydrochloric acid solution with 10% mole fraction and in fertilizer-containing soil). It is shown that the rate of corrosion of the steel in both environments after carbonitriding is lower than after quenching).

  2. Purification and properties of proline reductase from Clostridium sticklandii.

    PubMed

    Seto, B; Stadtman, T C

    1976-04-25

    Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.

  3. Potential use of aldose reductase inhibitors to prevent diabetic complications.

    PubMed

    Zenon, G J; Abobo, C V; Carter, B L; Ball, D W

    1990-06-01

    Reviewed are (1) the biochemical basis and pathophysiology of diabetic complications and (2) the structure-activity relationships, pharmacology, pharmacokinetics, clinical trials, and adverse effects of aldose reductase inhibitors (ARIs). ARIs are a new class of drugs potentially useful in preventing diabetic complications, the most widely studied of which have been cataracts and neuropathy. ARIs inhibit aldose reductase, the first, rate-limiting enzyme in the polyol metabolic pathway. In nonphysiological hyperglycemia the activity of hexokinase becomes saturated while that of aldose reductase is enhanced, resulting in intracellular accumulation of sorbitol. Because sorbitol does not readily penetrate the cell membrane it can persist within cells, which may lead to diabetic complications. ARIs are a class of structurally dissimilar compounds that include carboxylic acid derivatives, flavonoids, and spirohydantoins. The major pharmacologic action of an ARI involves competitive binding to aldose reductase and consequent blocking of sorbitol production. ARIs delay cataract formation in animals, but the role of aldose reductase in cataract formation in human diabetics has not been established. The adverse effects of ARIs include hypersensitivity reactions. Although the polyol pathway may not be solely responsible for diabetic complications, studies suggest that therapy with ARIs could be beneficial. Further research is needed to determine the long-term impact and adverse effects of ARIs in the treatment of diabetic complications.

  4. Uterine glutathione reductase activity: modulation by estrogens and progesterone.

    PubMed

    Díaz-Flores, M; Baiza-Gutman, L A; Pedrón, N N; Hicks, J J

    1999-10-29

    The aim of this study was to determine whether glutathione reductase activity in uterine tissue is regulated by sex hormones. In spayed rats uterine glutathione reductase was significantly increased by exogenous estrogen (P< 0.01), progesterone (P< 0.01) or estrogen plus progesterone (P<0.01). When enzyme activity is expressed per mg protein, daily administration of estrogen or progesterone induces a progressive increase of this enzyme between 24 to 48 h or 24 to 72 h of treatment, respectively. Whereas the combination of both steroids causes an earlier and higher increase in glutathione reductase activity at 24 h of treatment. Estradiol singly or in combination with progesterone induced the highest protein concentration in the uterus. Whereas uterine DNA concentration is only significantly affected by estradiol. Our results suggest that uterine glutathione reductase is regulated by estradiol and progesterone and may be involved in maintaining levels of reduced glutathione in the uterus. This compound may be required for control of the redox state of thiol groups and in detoxification reactions involving H2O2 and electrophylic substances. The antioxidant action of estrogens is partially due to the stimulation of glutathione reductase.

  5. Bacterial morphinone reductase is related to Old Yellow Enzyme.

    PubMed Central

    French, C E; Bruce, N C

    1995-01-01

    Morphinone reductase, produced by Pseudomonas putida M10, catalyses the NADH-dependent saturation of the carbon-carbon double bond of morphinone and codeinone, and is believed to be involved in the metabolism of morphine and codeine. The structural gene encoding morphinone reductase, designated morB, was cloned from Ps. putida M10 genomic DNA by the use of degenerate oligonucleotide probes based on elements of the amino acid sequence of the purified enzyme. Sequence analysis and structural characteristics indicated that morphinone reductase is related to the flavoprotein alpha/beta-barrel oxidoreductases, and is particularly similar to Old Yellow Enzyme of Saccharomyces spp. and the related oestrogen-binding protein of Candida albicans. Expressed sequence tags from several plant species show high homology to these enzymes, suggesting the presence of a family of enzymes conserved in plants and fungi. Although related bacterial proteins are known, morphinone reductase appears to be more similar to the eukaryotic proteins. Morphinone reductase was overexpressed in Escherichia coli, and has potential applications for the industrial preparation of semisynthetic opiates. Images Figure 1 Figure 5 PMID:8554504

  6. HMG-CoA reductase guides migrating primordial germ cells.

    PubMed

    Van Doren, M; Broihier, H T; Moore, L A; Lehmann, R

    1998-12-03

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is best known for catalysing a rate-limiting step in cholesterol biosynthesis, but it also participates in the production of a wide variety of other compounds. Some clinical benefits attributed to inhibitors of HMG-CoA reductase are now thought to be independent of any serum cholesterol-lowering effect. Here we describe a new cholesterol-independent role for HMG-CoA reductase, in regulating a developmental process: primordial germ cell migration. We show that in Drosophila this enzyme is highly expressed in the somatic gonad and that it is necessary for primordial germ cells to migrate to this tissue. Misexpression of HMG-CoA reductase is sufficient to attract primordial germ cells to tissues other than the gonadal mesoderm. We conclude that the regulated expression of HMG-CoA reductase has a critical developmental function in providing spatial information to guide migrating primordial germ cells.

  7. Dynamic Control of Electron Transfers in Diflavin Reductases

    PubMed Central

    Aigrain, Louise; Fatemi, Fataneh; Frances, Oriane; Lescop, Ewen; Truan, Gilles

    2012-01-01

    Diflavin reductases are essential proteins capable of splitting the two-electron flux from reduced pyridine nucleotides to a variety of one electron acceptors. The primary sequence of diflavin reductases shows a conserved domain organization harboring two catalytic domains bound to the FAD and FMN flavins sandwiched by one or several non-catalytic domains. The catalytic domains are analogous to existing globular proteins: the FMN domain is analogous to flavodoxins while the FAD domain resembles ferredoxin reductases. The first structural determination of one member of the diflavin reductases family raised some questions about the architecture of the enzyme during catalysis: both FMN and FAD were in perfect position for interflavin transfers but the steric hindrance of the FAD domain rapidly prompted more complex hypotheses on the possible mechanisms for the electron transfer from FMN to external acceptors. Hypotheses of domain reorganization during catalysis in the context of the different members of this family were given by many groups during the past twenty years. This review will address the recent advances in various structural approaches that have highlighted specific dynamic features of diflavin reductases. PMID:23203109

  8. [Methylenetetrahydrofolate reductase and methionine synthase reductase gene polymorphisms in ethnic Han women from Linyi].

    PubMed

    Zhang, Yan-li; Lu, Yan-qiang; Li, Hua-feng; Rui, Xin-yi; Zhang, Li-jun; Wu, Chuan-ye; Fang, Ai-min; Wang, Gui-xi

    2012-12-01

    To explore the distribution of genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) 677C/T, 1298A/C and methionine synthase reductase (MTRR) 66A/G among ethnic Han females from Linyi, and to correlate it with serum level of homocysteine (Hcy). A cross-sectional study was carried out. Oral epithelial cell samples were collected from 825 subjects. MTHFR and MTRR gene polymorphisms were determined with a Taqman-Minor Groove Binder (MGB) method. Distribution of gene polymorphisms was analyzed and compared with others regions of China including Weifang, Zhengzhou, Deyang and Hainan. A biochemical assay was also carried out to determine the total Hcy in plasma of 281 subjects. The reductase activity of MTHFR was classified into decreased and stable groups according to genetic polymorphism of MTHFR. Correlation between MTHFR groups and total Hcy level were also explored. (1) The frequencies of MTHFR677CC, CT and TT genotypes of the selected subjects were 16.7%, 48.3% and 35.0%, respectively. The frequencies of MTHFR 1298AA, AC and CC genotypes were 76.0%, 21.6% and 2.4%, respectively. And those of MTRR 66AA, AG and GG genotypes were 54.7%, 39.4% and 5.9%, respectively. For the selected subjects, their frequency of MTHFR 677TT genotype was higher than that of Deyang and Hainan (P< 0.01), whilst the frequency of MTHFR 1298CC genotype was lower than that of Deyang and Hainan (P < 0.01), and the frequency of MTRR 66 GG genotype was lower than that of Hainan (P< 0.01). (2) The Hcy level for those with decreased MTHFR activity was significantly higher than those with stable MTHFR activity (P< 0.05). MTHFR gene 677C/T, 1298A/C and MTRR 66A/G polymorphisms in ethnic Han women from Linyi have differed significantly from other regions of China. Decreased MTHFR activity caused by genetic polymorphisms is a risk factor for raised Hcy level.

  9. Identification by proton nuclear magnetic resonance of the histidines in cytochrome b5 modified by diethyl pyrocarbonate

    SciTech Connect

    Altman, J.; Lipka, J.J.; Kuntz, I.; Waskell, L. )

    1989-09-19

    Diethyl pyrocarbonate (DEP) is an electrophilic reagent that is used to modify reversibly the histidine residues of proteins. Unfortunately, the lability of the acylated histidine adduct usually does not permit the isolation and identification of the modified histidine. By use of 500-MHz proton NMR spectroscopy, it has been possible to identify the C-H resonances of the nonaxial histidines of trypsin-solubilized bovine, rabbit, and porcine cytochrome b5 and therefore observe the interaction of DEP with specific histidine residues of cytochrome b5. In addition, the pKa of the peripheral histidines of bovine and rabbit cytochrome b5 have been measured in D2O. In the bovine protein it was found that the histidines are modified sequentially with increasing DEP concentration in the order His-26 greater than His-15 greater than His-80. This order is maintained in the rabbit protein with the following additions: His-26 approximately His-27 greater than His-15 greater than or equal to His-17 greater than His-80. The relative reactivity of the peripheral histidines with DEP was rationalized by considering three of their characteristics: (1) the pKa of the histidine, (2) the fraction of the side chain exposed to the solvent, and (3) the hydrogen-bond interactions of the imidazole ring.

  10. 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.

  11. The luminescence and structural characteristics of Eu3+- doped NaSrB5O9 phosphor

    NASA Astrophysics Data System (ADS)

    Dillip, G. R.; Mallikarjuna, K.; Dhoble, S. J.; Raju, B. Deva Prasad

    2014-01-01

    A red-emitting phosphor NaSrB5O9:Eu3+ was synthesized by employing a solid-state reaction (SSR) method. The structures of the phosphors were analyzed by X-ray diffraction (XRD), Fourier-transform infrared (FTIR) and Raman studies. The band at ~282 nm in the excitation spectra indicated the charge transfer band (CTB) of B-O in the host, whereas the CTB of Eu-O was observed at ~275 nm for the NaSrB5O9:Eu3+ (Eu3+=1 at.%) phosphor, which was supported by diffuse reflectance spectroscopy (DRS) measurements. The photoluminescence (PL) measurements exhibited a strong red emission band centered at about 616 nm (5D0→7F2) under an excitation wavelength of 394 nm (7F0→5L6). Upon host excitation at 282 nm, the pristine NaSrB5O9 exhibited a broad UV emission centered at ~362 nm. The energy transfer from host to Eu3+ ions was confirmed from luminescence spectra, excited with a 355 nm Nd:YAG laser. In addition, the asymmetric ratios indicate a higher local symmetry around the Eu3+ ion in the host. The calculated CIE (Commission International de l'Eclairage) coordinates displayed excellent color purity efficiencies (around 99.7%) compared to other luminescent materials.

  12. A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane.

    PubMed Central

    Pedrazzini, E; Villa, A; Borgese, N

    1996-01-01

    Many resident membrane proteins of the endoplasmic reticulum (ER) do not have known retrieval sequences. Among these are the so-called tail-anchored proteins, which are bound to membranes by a hydrophobic tail close to the C terminus and have most of their sequence as a cytosolically exposed N-terminal domain. Because ER tail-anchored proteins generally have short (< or = 17 residues) hydrophobic domains, we tested whether this feature is important for localization, using cytochrome b5 as a model. The hydrophobic domain of cytochrome b5 was lengthened by insertion of five amino acids (ILAAV), and the localization of the mutant was analyzed by immunofluorescence in transiently transfected mammalian cells. While the wild-type cytochrome was localized to the ER, the mutant was relocated to the surface. This relocation was not due to the specific sequence introduced, as demonstrated by the ER localization of a second mutant, in which the original length of the membrane anchor was restored, while maintaining the inserted ILAAV sequence. Experiments with brefeldin A and with cycloheximide demonstrated that the extended anchor mutant reached the plasma membrane by transport along the secretory pathway. We conclude that the short membrane anchor of cytochrome b5 is important for its ER residency, and we discuss the relevance of this finding for other ER tail-anchored proteins. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8633042

  13. Cytochrome b5 and cytokeratin 17 are biomarkers in bronchoalveolar fluid signifying onset of acute lung injury.

    PubMed

    Ménoret, Antoine; Kumar, Sanjeev; Vella, Anthony T

    2012-01-01

    Acute lung injury (ALI) is characterized by pulmonary edema and acute inflammation leading to pulmonary dysfunction and potentially death. Early medical intervention may ameliorate the severity of ALI, but unfortunately, there are no reliable biomarkers for early diagnosis. We screened for biomarkers in a mouse model of ALI. In this model, inhalation of S. aureus enterotoxin A causes increased capillary permeability, cell damage, and increase protein and cytokine concentration in the lungs. We set out to find predictive biomarkers of ALI in bronchoalveolar lavage (BAL) fluid before the onset of clinical manifestations. A cutting edge proteomic approach was used to compare BAL fluid harvested 16 h post S. aureus enterotoxin A inhalation versus BAL fluid from vehicle alone treated mice. The proteomic PF 2D platform permitted comparative analysis of proteomic maps and mass spectrometry identified cytochrome b5 and cytokeratin 17 in BAL fluid of mice challenged with S. aureus enterotoxin A. Validation of cytochrome b5 showed tropic expression in epithelial cells of the bronchioles. Importantly, S. aureus enterotoxin A inhalation significantly decreased cytochrome b5 during the onset of lung injury. Validation of cytokeratin 17 showed ubiquitous expression in lung tissue and increased presence in BAL fluid after S. aureus enterotoxin A inhalation. Therefore, these new biomarkers may be predictive of ALI onset in patients and could provide insight regarding the basis of lung injury and inflammation.

  14. Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase

    PubMed Central

    2013-01-01

    Background The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. Results To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Conclusion Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo. PMID:24308601

  15. Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

    PubMed

    Liu, Yi; Shi, Zi; Maximova, Siela; Payne, Mark J; Guiltinan, Mark J

    2013-12-05

    The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

  16. 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

  17. Selenium in thioredoxin reductase: a mechanistic perspective.

    PubMed

    Lacey, Brian M; Eckenroth, Brian E; Flemer, Stevenson; Hondal, Robert J

    2008-12-02

    Most high M(r) thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys(1)-Cys(2)) which forms an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys(2) position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the eight-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from Drosophila melanogaster and Caenorhabditis elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group pK(a) as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower pK(a) of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to

  18. 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

  19. Isatin-induced increase in the affinity of human ferrochelatase and adrenodoxin reductase interaction.

    PubMed

    Ershov, Pavel; Mezentsev, Yuri; Gilep, Andrey; Usanov, Sergey; Buneeva, Olga; Medvedev, Alexei; Ivanov, Alexis

    2017-09-14

    Isatin (indol-2,3-dione) is an endogenous non-peptide regulator exhibiting a wide range of biological and pharmacological activities, which are poorly characterized in terms of their molecular mechanisms. Identification of many isatin-binding proteins in the mammalian brain and liver suggests that isatin may influence their functions. We have hypothesized that besides direct action on particular protein targets, isatin can act as a regulator of protein-protein interactions (PPIs). In this surface plasmon resonance-based biosensor study we have found that physiologically relevant concentrations of isatin (25-100 μM) increase affinity of interactions between human recombinant ferrochelatase (FECH) and NADPH-dependent adrenodoxin reductase (ADR). In the presence of increasing concentrations of isatin the Kd values demonstrated a significant (up to 6-fold) decrease. It is especially important that the interaction of isatin with each individual protein (FECH, ADR) was basically negligible and therefore could not contribute to the observed effect. This effect was specific only for the FECH/ADR complex formation and was not observed for other protein complexes studied: FECH/cytochrome b5(CYB5A) and FECH/SMAD4. © 2017 The Protein Society.

  20. Do cytochromes function as oxygen sensors in the regulation of nitrate reductase biosynthesis?

    PubMed Central

    MacGregor, C H; Bishop, C W

    1977-01-01

    The observation that oxygen represses nitrate reductase biosynthesis in a hemA mutant grown aerobically with or without delta-aminolevulinic acid indicates that cytochromes are not responsible for nitrate reductase repression in aerobically grown cells. PMID:326768

  1. Studies on the regulation of assimilatory nitrate reductase in Ankistrodesmus braunii.

    PubMed

    Diez, J; Chaparro, A; Vega, J M; Relimpio, A

    1977-01-01

    In the green alga Ankistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH2-or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg(2+), presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH.

  2. 17 CFR 240.10b5-1 - Trading “on the basis of” material nonpublic information in insider trading cases.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Trading âon the basis ofâ material nonpublic information in insider trading cases. 240.10b5-1 Section 240.10b5-1 Commodity and... Deceptive Devices and Contrivances § 240.10b5-1 Trading “on the basis of” material nonpublic information...

  3. 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.

  4. 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.

  5. The expression of HoxB5 and SPC in neonatal rat lung after exposure to fluoxetine.

    PubMed

    Taghizadeh, Razieh; Taghipour, Zahra; Karimi, Akbar; Shamsizadeh, Ali; Taghavi, Mohammad Mohsen; Shariati, Mahdi; Shabanizadeh, Ahmad; Jafari Naveh, Hamid Reza; Bidaki, Reza; Aminzadeh, Fariba

    2016-01-01

    Approximately 10% of pregnant women suffer from pregnancy-associated depression. Fluoxetine, as a selective serotonin reuptake inhibitor, is being employed as a therapy for depressive disorders. The present study aimed to determine the effects of fluoxetine on neonatal lung development. Thirty pregnant Wistar rats (weighing 200-250 g) were treated daily with 7 mg/kg fluoxetine from gestation day 0 to gestation day 21, via gavage. The control group received a similar volume of distilled water only. Following delivery, the newborns and their lungs were immediately weighed in both of the groups. The right lung was fixed for histological assessments while the left lung was used for evaluation of the expression of SPC and HoxB5 by the real-time polymerase chain reaction method. Results have indicated that even though the body weight and the number of neonatal rats in both groups were the same, the lung weight of neonates exposed to fluoxetine was significantly different compared to the control group (P<0.05). Expression of both genes was increased, nonetheless, only elevation of HoxB5 was significant (P<0.05). Histological studies demonstrated that lung tissue in the fluoxetine treatment group morphologically appears to be similar to the pseudoglandular phase, whereas the control group lungs experienced more development. According to the upregulated expression of HoxB5 concerning histological findings, results of the present study showed that fluoxetine can influence lung growth and may in turn lead to delay in lung development. So establishment of studies to identify the effects of antidepressant drugs during pregnancy is deserved.

  6. The expression of HoxB5 and SPC in neonatal rat lung after exposure to fluoxetine

    PubMed Central

    Taghizadeh, Razieh; Taghipour, Zahra; Karimi, Akbar; Shamsizadeh, Ali; Taghavi, Mohammad Mohsen; Shariati, Mahdi; Shabanizadeh, Ahmad; Jafari Naveh, Hamid Reza; Bidaki, Reza; Aminzadeh, Fariba

    2016-01-01

    Objective Approximately 10% of pregnant women suffer from pregnancy-associated depression. Fluoxetine, as a selective serotonin reuptake inhibitor, is being employed as a therapy for depressive disorders. The present study aimed to determine the effects of fluoxetine on neonatal lung development. Methods Thirty pregnant Wistar rats (weighing 200–250 g) were treated daily with 7 mg/kg fluoxetine from gestation day 0 to gestation day 21, via gavage. The control group received a similar volume of distilled water only. Following delivery, the newborns and their lungs were immediately weighed in both of the groups. The right lung was fixed for histological assessments while the left lung was used for evaluation of the expression of SPC and HoxB5 by the real-time polymerase chain reaction method. Results Results have indicated that even though the body weight and the number of neonatal rats in both groups were the same, the lung weight of neonates exposed to fluoxetine was significantly different compared to the control group (P<0.05). Expression of both genes was increased, nonetheless, only elevation of HoxB5 was significant (P<0.05). Histological studies demonstrated that lung tissue in the fluoxetine treatment group morphologically appears to be similar to the pseudoglandular phase, whereas the control group lungs experienced more development. Conclusion According to the upregulated expression of HoxB5 concerning histological findings, results of the present study showed that fluoxetine can influence lung growth and may in turn lead to delay in lung development. So establishment of studies to identify the effects of antidepressant drugs during pregnancy is deserved. PMID:27843293

  7. 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

  8. Dysregulation of MicroRNA-196b-5p and MicroRNA-375 in Gastric Cancer

    PubMed Central

    Lee, Seung Woo; Park, Ki Cheol; Kim, Jeong Goo; Moon, Sung Jin; Kang, Sang Bum; Lee, Dong Soo; Sul, Hae Joung; Ji, Jeong Seon

    2016-01-01

    Purpose Dysregulated microRNAs (miRNAs) can contribute to cancer development by leading to abnormal proliferation of cells, apoptosis, and differentiation. Although several miRNAs that are related to gastric cancer have been identified, the reported results have been inconsistent. The aim of this study was to determine miRNA expression profiles and validate miRNAs up- and down-regulated in gastric cancer. Materials and Methods We evaluated 34 primary gastric cancer tissues and paired adjacent nontumorous gastric tissues. Total RNA was extracted, and low-molecular-weight RNAs (<200 nucleotides) were isolated for further analysis. Two pairs of tissues were processed for GeneChip microarray analysis, and the identified up- and down-regulated miRNAs were validated by real-time quantitative polymerase chain reaction (qPCR). Results In the set of differentially expressed miRNAs, 5 were overexpressed by more than 2 fold, and 5 were reduced by 2 fold or less in gastric cancer tissues compared with normal gastric tissues. Four of these miRNAs (miR-196b-5p, miR-375, miR-483-5p, and miR-486-5p) were then validated by qPCR, and the relative expression levels of 2 miRNAs (miR-196b-5p and miR-375) were significantly different between cancer and normal tissues. Conclusions Our results revealed that the expression of miR-196b-5p and miR-375 significantly correlates with gastric cancer. These miRNAs could therefore serve as diagnostic biomarkers of gastric cancer. PMID:28053808

  9. Protective effect of p-methoxycinnamic acid, an active phenolic acid against 1,2-dimethylhydrazine-induced colon carcinogenesis: modulating biotransforming bacterial enzymes and xenobiotic metabolizing enzymes.

    PubMed

    Gunasekaran, Sivagami; Venkatachalam, Karthikkumar; Jeyavel, Kabalimoorthy; Namasivayam, Nalini

    2014-09-01

    Objective of the study is to evaluate the modifying potential of p-methoxycinnamic acid (p-MCA), an active rice bran phenolic acid on biotransforming bacterial enzymes and xenobiotic metabolizing enzymes in 1,2-dimethylhydrazine-induced rat colon carcinogenesis. 48 male albino wistar rats were divided into six groups. Group1 (control) received modified pellet diet and 0.1 % carboxymethylcellulose; group2 received modified pellet diet along with p-MCA (80 mg/kg b.wt. p.o.) everyday for 16 weeks; groups 3-6 received 1,2-dimethylhydrazine (DMH) (20 mg/kg b.wt.) subcutaneous injection once a week for the first 4 weeks, while groups 4-6 received p-MCA at three different doses of 20, 40 and 80 mg/kg b.wt. p.o. everyday for 16 weeks. A significant increase in carcinogen-activating enzymes (cytochrome P450, cytochrome b5, cytochrome P4502E1, NADH-cytochrome-b5-reductase and NADPH-cytochrome-P450 reductase) with concomitant decrease in phaseII enzymes, DT-Diaphorase, glutathione S-transferase, UDP-glucuronyl-transferase and gamma glutamyltransferase were observed in group3 compared to control. DMH treatment significantly increased the activities of feacal and colonic bacterial enzymes (β-glucosidase, β-galactosidase, β-glucuronidase, nitroreductase, sulphatase and mucinase). p-MCA supplementation (40 mg/kg b.wt) to carcinogen exposed rats inhibited these enzymes, which were near those of control rats. The formation of dysplastic aberrant crypt foci in the colon and the histopathological observations of the liver also supports our biochemical findings. p-MCA (40 mg/kg b.wt.) offers remarkable modulating efficacy of biotransforming bacterial and xenobiotic metabolizing enzymes in colon carcinogenesis.

  10. Diminution of Hepatic Response to 7, 12-dimethylbenz(α)anthracene by Ethyl Acetate Fraction of Acacia catechu Willd. through Modulation of Xenobiotic and Anti-Oxidative Enzymes in Rats

    PubMed Central

    Kumar, Rakesh; Kaur, Rajbir; Singh, Amrit Pal; Arora, Saroj

    2014-01-01

    Background Liver is the primary metabolizing site of body and is prone to damage by exogenous as well as endogenous intoxicants. Polycyclic aromatic hydrocarbons such as 7, 12- dimethylbenz(α)anthracene (DMBA) is an exogenous hepatotoxin, which is well known for modulating phase I, II and anti-oxidative enzymes of liver. Plants contain plethora of polyphenolic compounds which can reverse the damaging effect of various xenobiotics. The present study investigated protective role of the ethyl acetate fraction of Acacia catechu Willd. (EAF) against DMBA induced alteration in hepatic metabolizing and anti-oxidative enzymes in rats. Methodology and Principal Findings The rats were subjected to hepatic damage by treating with DMBA for 7 weeks on alternative days and treatment schedule was terminated at the end of 14 weeks. The rats were euthanized at the end of protocol and livers were homogenized. The liver homogenates were used to analyse phase I (NADPH-cytochrome P450 reducatse, NADH-cytochrome b5 reductase, cytochrome P420, cytochrome b5), phase II (glutathione-S-transferase, DT diaphorase and γ-Glutamyl transpeptidase) and antioxidative enzymes (catalase, superoxide dismutase, ascorbate peroxidase, glutathione reductase, guiacol peroxidase and lactate dehydrogenase). Furthermore, other oxidative stress parameters (thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes and reduced glutathione) and liver marker enzymes (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and alkaline phosphatase) were also studied. The DMBA induced significant changes in activity of hepatic enzymes that was reversed by treatment with three dose levels of EAF. Conclusion It is concluded that EAF affords hepato-protection against DMBA in rats through modulation of phase I, II and anti-oxidative enzymes. PMID:24587216

  11. Dihydrofolate Reductase Activity in Strains of Streptococcus faecium var. durans Resistant to Methasquin and Amethopterin1

    PubMed Central

    Rader, Jeanne I.; Hutchison, Dorris J.

    1972-01-01

    Resistance to the antifolates methasquin and amethopterin has been studied in new strains of Streptococcus faecium var. durans. Two methasquin-resistant strains (SF/MQ, SF/MQT) and an amethopterin-resistant strain (SF/AM) were selected independently from the wild-type S. faecium var. durans (SF/O). SF/MQT is a thymine auxotroph. Total dihydrofolate reductase activity was elevated in each of the resistant strains. The greatest increase (36-fold) was observed in extracts of SF/AM. The methasquin-resistant strains, SF/MQ and SF/MQT, had 29-fold and 8-fold, respectively, more dihydrofolate reductase activity than the parental strain. Total dihydrofolate reductase activity of SF/O was separable by gel filtration into two components: a folate reductase (11%) and a specific dihydrofolate reductase (89%). Folate reductase activity was associated with 88% of the total dihydrofolate reductase activity of SF/MQT, with specific dihydrofolate reductase activity accounting for the remaining 12%. In SF/MQ and SF/AM, folate reductase activity was associated with 97% of the total dihydrofolate reductase activity. Studies of the inhibition by methasquin and amethopterin of partially purified folate reductase and specific dihydrofolate reductase of the mutant strains suggested that resistance was not accompanied by changes in the affinities of these enzymes for either antifolate. PMID:4401600

  12. 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...

  13. 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...

  14. 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 Glutathione...

  15. 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 Glutathione...

  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 Glutathione...

  17. 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 Glutathione...

  18. Obtaining partial purified xylose reductase from Candida guilliermondii

    PubMed Central

    Tomotani, Ester Junko; de Arruda, Priscila Vaz; Vitolo, Michele; de Almeida Felipe, Maria das Graças

    2009-01-01

    The enzymatic bioconversion of xylose into xylitol by xylose reductase (XR) is an alternative for chemical and microbiological processes. The partial purified XR was obtained by using the following three procedures: an agarose column, a membrane reactor or an Amicon Ultra-15 50K Centrifugal Filter device at yields of 40%, 7% and 67%, respectively. PMID:24031408

  19. 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.

  20. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases.

    PubMed

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

    2015-09-28

    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.

  1. Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria

    PubMed Central

    Casciotti, Karen L.; Ward, Bess B.

    2001-01-01

    The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of β-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria. PMID:11319103

  2. 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

  3. 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...

  4. 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)

  5. Dihydrofolate reductase: A potential drug target in trypanosomes and leishmania

    NASA Astrophysics Data System (ADS)

    Zuccotto, Fabio; Martin, Andrew C. R.; Laskowski, Roman A.; Thornton, Janet M.; Gilbert, Ian H.

    1998-05-01

    Dihydrofolate reductase has successfully been used as a drug target in the area of anti-cancer, anti-bacterial and anti-malarial chemotherapy. Little has been done to evaluate it as a drug target for treatment of the trypanosomiases and leishmaniasis. A crystal structure of Leishmania major dihydrofolate reductase has been published. In this paper, we describe the modelling of Trypanosoma cruzi and Trypanosoma brucei dihydrofolate reductases based on this crystal structure. These structures and models have been used in the comparison of protozoan, bacterial and human enzymes in order to highlight the different features that can be used in the design of selective anti-protozoan agents. Comparison has been made between residues present in the active site, the accessibility of these residues, charge distribution in the active site, and the shape and size of the active sites. Whilst there is a high degree of similarity between protozoan, human and bacterial dihydrofolate reductase active sites, there are differences that provide potential for selective drug design. In particular, we have identified a set of residues which may be important for selective drug design and identified a larger binding pocket in the protozoan than the human and bacterial enzymes.

  6. Studies on Marek's Disease Virus Encoded Ribonucleotide Reductase

    USDA-ARS?s Scientific Manuscript database

    Ribonucleotide reductase (RR) is an essential enzyme for the conversion of ribonucleotides to deoxyribonucleotides in prokaryotic and eukaryotic cells. The enzyme consists of two subunits namely RR1 and RR2, both of which associate to form an active holoenzyme. Herpesviruses express a functional R...

  7. [Malate oxidation by mitochondrial succinate:ubiquinone-reductase].

    PubMed

    Belikova, Iu O; Kotliar, A B

    1988-04-01

    Succinate:ubiquinone reductase was shown to catalyze the oxidation of L- and D-stereoisomers of malate by artificial electron acceptors and ubiquinone. The rate of malate oxidation by succinate:ubiquinone reductase is by two orders of magnitude lower than that for the natural substrate--succinate. The values of kinetic constants for the oxidation of D- and L-stereoisomers of malate are equal to: V infinity = 0.1 mumol/min/mg protein, Km = 2 mM and V infinity = 0.05 mumol/min/mg protein, Km = 2 mM, respectively. The malate dehydrogenase activity is fully inhibited by the inhibitors of the dicarboxylate-binding site of the enzyme, i.e., N-ethylmaleimide and malonate and is practically insensitive to carboxin, a specific inhibitor of the ubiquinone-binding center. The enol form of oxaloacetate was shown to be the product of malate oxidation by succinate:ubiquinone reductase. The kinetics of inhibition of the enzyme activity by the ketone and enol forms of oxaloacetate was studied. Both forms of oxaloacetate effectively inhibit the succinate:ubiquinone reductase reaction.

  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. Molybdenum-containing nitrite reductases: Spectroscopic characterization and redox mechanism.

    PubMed

    Wang, Jun; Keceli, Gizem; Cao, Rui; Su, Jiangtao; Mi, Zhiyuan

    2017-01-01

    This review summarizes the spectroscopic results, which will provide useful suggestions for future research. In addition, the fields that urgently need more information are also advised. Nitrite-NO-cGMP has been considered as an important signaling pathway of NO in human cells. To date, all the four known human molybdenum-containing enzymes, xanthine oxidase, aldehyde oxidase, sulfite oxidase, and mitochondrial amidoxime-reducing component, have been shown to function as nitrite reductases under hypoxia by biochemical, cellular, or animal studies. Various spectroscopic techniques have been applied to investigate the structure and catalytic mechanism of these enzymes for more than 20 years. We summarize the published data on the applications of UV-vis and EPR spectroscopies, and X-ray crystallography in studying nitrite reductase activity of the four human molybdenum-containing enzymes. UV-vis has provided useful information on the redox active centers of these enzymes. The utilization of EPR spectroscopy has been critical in determining the coordination and redox status of the Mo center during catalysis. Despite the lack of substrate-bound crystal structures of these nitrite reductases, valuable structural information has been obtained by X-ray crystallography. To fully understand the catalytic mechanisms of these physiologically/pathologically important nitrite reductases, structural studies on substrate-redox center interaction are needed.

  10. 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)

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. [Inhibition of aldose reductase by Chinese herbal medicine].

    PubMed

    Mao, X M; Zhang, J Q

    1993-10-01

    Seven Chinese herbal drugs were screened for experimental inhibition of lens aldose reductase activity, among which quercetin exhibited potent enzyme-inhibitory activities in vitro. Its IC50 value was 3.44 x 10(-7) mol/L. It may be helpful in the prophylaxis and treatment of diabetic complications.

  16. 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.

  17. The polymorphisms in methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase, and the risk of colorectal cancer.

    PubMed

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results.

  18. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    PubMed Central

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results. PMID:22719222

  19. 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.

  20. Circular RNA GLI2 promotes osteosarcoma cell proliferation, migration, and invasion by targeting miR-125b-5p.

    PubMed

    Li, Ji-Feng; Song, Yu-Ze

    2017-07-01

    Circular RNAs are novel identified type of endogenous non-coding RNAs, which exert vital functions in human and animals. However, the in-depth role of circular RNAs in the progression of tumorigenesis, especially osteosarcoma, is still undefined. Our preliminary study had found that cir-GLI2 was significantly upregulated in osteosarcoma tissues compared to adjacent non-tumor tissue. Moreover, cir-GLI2 silencing could effectively suppress the proliferation, migration, and invasion capacity of osteosarcoma cells, indicating the tumor-promoting role. Besides, bioinformatics analysis and luciferase reporter assay predicted the direct binding to miR-125b-5p, which has been reported to function as a tumor suppressor in osteosarcoma. Furthermore, functional experiments validated that cir-GLI2 exerted the tumor-promoting effects on osteosarcoma cells via negatively targeting miR-125b-5p. In conclusion, our study demonstrated that cir-GLI2 acts as an oncogenic circular RNA in osteosarcoma genesis, providing a novel diagnostic and therapeutic target for osteosarcoma.

  1. Measurement of nitrous oxide reductase activity in aquatic sediments

    SciTech Connect

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

    1986-01-01

    Denitrification in aquatic sediments was measured by an N/sub 2/O reductase assay. Sediments consumed small added quantities of N/sub 2/O over short periods (a few hours). In experiments with sediment slurries, N/sub 2/O reductase activity was inhibited by 0/sub 2/, C/sub 2/H/sub 2/, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 ..mu..M) did not influence activity, and moderate levels (about 150 ..mu..M) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N/sub 2/O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater, estuarine, and alkaline-saline environments. An in situ assay was devised in which a solution of N/sub 2/O 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 N/sub 2/O per m/sup 2/ per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N/sub 2/O 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 N/sub 2/O per m/sup 2/ per h made with the acetylene block assay.

  2. 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.

  3. Identification of 5α-reductase isoenzymes in canine skin.

    PubMed

    Bernardi de Souza, Lucilene; Paradis, Manon; Zamberlam, Gustavo; Benoit-Biancamano, Marie-Odile; Price, Christopher

    2015-10-01

    Alopecia X in dogs is a noninflammatory alopecia that may be caused by a hormonal dysfunction. It may be similar to androgenic alopecia in men that is caused by the effect of dihydrotestosterone (DHT). The 5α-reductase isoenzymes, 5αR1 and 5αR2, and a recently described 5αR3, are responsible for the conversion of testosterone into DHT. However, which 5α-reductases are present in canine skin has not yet been described. The main objective of this study was to determine the pattern of expression of 5α-reductase genes in canine skin. Skin biopsies were obtained from healthy, intact young-mature beagles (three males, four females) at three anatomical sites normally affected by alopecia X (dorsal neck, back of thighs and base of tail) and two sites generally unaffected (dorsal head and ventral thorax). Prostate samples (n = 3) were collected as positive controls for 5α-reductase mRNA abundance measurement by real-time PCR. We detected mRNA encoding 5αR1 and 5αR3 but not 5αR2. There were no significant differences in 5αR1 and 5αR3 mRNA levels between the different anatomical sites, irrespective of gender (P > 0.05). Moreover, the mean mRNA abundance in each anatomical site did not differ between males and females (P > 0.05). To the best of the authors' knowledge, this is the first study demonstrating the expression of 5α-reductases in canine skin and the expression of 5αR3 in this tissue. These results may help to elucidate the pathogenesis of alopecia X and to determine more appropriate treatments for this disorder. © 2015 ESVD and ACVD.

  4. Leukemia L1210 cell lines resistant to ribonucleotide reductase inhibitors.

    PubMed

    Cory, J G; Carter, G L

    1988-02-15

    Leukemia L1210 cell lines, ED1 and ED2, were generated which were resistant to the cytotoxic effects of deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine and deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine plus 2,3-dihydro-1H-pyrazole[2,3a]imidazole/Desferal, respectively. The ED1 and ED2 were characterized to show that these cell lines had increased levels of ribonucleotide reductase as measured by CDP reduction. The reductase activity in crude cell-free extracts from the ED1 and ED2 cells was not inhibited by dATP. For CDP reductase, the activation by adenylylimido diphosphate and inhibition by dGTP and dTTP in these extracts from the ED1 and ED2 cells were the same as for the wild-type L1210 cells. The ED1 and ED2 cells were highly cross-resistant, as measured by growth inhibition, to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine, and 2-fluoroadenine arabinoside. While the ED2 cells showed resistance to 2,3-dihydro-1H-pyrazole-[2,3a]-imidazole/Desferal (6-fold), the ED1 and ED2 cell lines showed less resistance to hydroxyurea, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, and the dialdehyde of inosine. These data indicate that the mechanisms of resistance to the ribonucleotide reductase inhibitors are related to the increased level of ribonucleotide reductase activity and to the decreased sensitivity of the effector-binding subunit to dATP.

  5. Assimilatory nitrate reductase from the green alga Ankistrodesmus braunii.

    PubMed

    De la Rosa, M A

    1983-01-01

    Assimilatory nitrate reductase (NAD(P)H-nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii can be purified to homogeneity by dye-ligand chromatography on blue-Sepharose. The purified enzyme, whose turnover number is 623 s-1, presents an optimum pH of 7.5 and Km values of 13 microM, 23 microM and 0.15 mM for NADH, NADPH and nitrate, respectively. The NADH-nitrate reductase activity exhibits an iso ping pong bi bi kinetic mechanism. The molecular weight of the native nitrate reductase is 467 400, while that of its subunits is 58 750. These values suggest an octameric structure for the enzyme, which has been confirmed by electron microscopy. As deduced from spectrophotometric and fluorimetric studies, the enzyme contains FAD and cytochrome b-557 as prosthetic groups. FAD is not covalently bound to the protein and is easily dissociated in diluted solutions from the enzyme. Its apparent Km value is 4 nM, indicative of a high affinity of the enzyme for FAD. The results of the quantitative analyses of prosthetic groups indicate that nitrate reductase contains four molecules of flavin, four heme irons, and two atoms of molybdenum. The three components act sequentially transferring electrons from reduced pyridine nucleotides to nitrate, thus forming a short electron transport chain along the protein. A mechanism is proposed for the redox interconversion of the nitrate reductase activity. Inactivation seems to occur by formation of a stable complex of reduced enzyme with cyanide or superoxide, while reactivation is a consequence of reoxidation of the inactive enzyme. Both reactions imply the transfer of only one electron.

  6. Treatment of hirsutism with 5 alpha-reductase inhibitors.

    PubMed

    Brooks, J R

    1986-05-01

    Much os the evidence gathered from studies of 5 alpha-reductase activity levels and androgen metabolism in the skin of hirsute women and the excretion of androgen metabolites by hirsute women indicates that 5 alpha-reduced androgens are probably of primary importance in hirsutism. Unfortunately, until very recently, the lack of a suitable 5 alpha-reductase inhibitor made it very difficult to adequately test the hypothesis that such an inhibitor might be useful in the treatment of hirsutism and certain other androgen-related diseases. No substance was available which had good, unambiguous activity in vivo as a 5 alpha-reductase inhibitor. A number of 4-azasteroids have now been found to possess excellent 5 alpha-reductase inhibitory activity both in vitro and in vivo. Among other properties, several of these compounds show little or no affinity for the androgen receptor of rat prostate cytosol, they attenuate the growth promoting effect of T, but not DHT, on the ventral prostate of castrated male rats, they cause a marked reduction in prostatic DHT concentration in acutely treated rats and dogs and they bring about a significant decline in prostate size in chronically treated rats and dogs. It is expected that, in the near future, one or more of these highly active 5 alpha-reductase inhibitors will be tested in the clinic as a treatment for hirsutism. The results of those studies will be awaited with a great deal of interest since they should considerably advance our understanding of this disease and possibly contribute to its control.

  7. 26 CFR 31.3121(b)(5)-1 - Services in employ of an instrumentality of the United States specifically exempted from the...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... instrumentality is exempt from the employer tax imposed by section 3111 by virtue of any other provision of law... United States specifically exempted from the employer tax. 31.3121(b)(5)-1 Section 31.3121(b)(5)-1... employ of an instrumentality of the United States specifically exempted from the employer tax. Services...

  8. 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.; hide

    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.

  9. 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.

  10. 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.

  11. Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii.

    PubMed

    Díez, J; López-Ruiz, A

    1989-02-01

    The effects of different culture conditions on nitrate reductase activity and nitrate reductase protein from Monoraphidium braunii have been studied, using two different immunological techniques, rocket immunoelectrophoresis and an enzyme-linked immunosorbent assay, to determine nitrate reductase protein. The nitrogen sources ammonium and glutamine repressed nitrate reductase synthesis, while nitrite, alanine, and glutamate acted as derepressors. There was a four- to eightfold increase of nitrate reductase activity and a twofold increase of nitrate reductase protein under conditions of nitrogen starvation versus growth on nitrate. Nitrate reductase synthesis was repressed in darkness. However, when Monoraphidium was grown under heterotrophic conditions with glucose as the carbon and energy source, the synthesis of nitrate reductase was maintained. With ammonium or darkness, changes in nitrate reductase activity correlated fairly well with changes in nitrate reductase protein, indicating that in both cases loss of activity was due to repression and not to inactivation of the enzyme. Experiments using methionine sulfoximine, to inhibit ammonium assimilation, showed that ammonium per se and not a product of its metabolism was the corepressor of the enzyme. The appearance of nitrate reductase activity after transferring the cells to induction media was prevented by cycloheximide and by 6-methylpurine, although in this latter case the effect was observed only in cells preincubated with the inhibitor for 1 h before the induction period.

  12. 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.

  13. 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.

  14. 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.

  15. Line identifications in the ultraviolet spectra of Tau Herculis, B5 IV, and Zeta Draconis, B6 III

    NASA Technical Reports Server (NTRS)

    Underhill, A. B.; Adelman, S. J.

    1976-01-01

    Tables of the lines found on two tracings each of the ultraviolet spectrum of Tau Her, B5 IV, and Zeta Dra, B6 III, made by the Copernicus satellite and possible identifications are given. The ranges 1025-1451A for Tau Her and 1035 to 1425A for Zeta Dra are covered by the U2 spectrometer at a resolution of 0.2A; the ranges 2028 to 2959A for Tau Her and 2000 to 3000A for Zeta Dra are covered by the V2 spectrometer at a resolution of 0.4A. The observed density of lines in the U2 region is 1.1 lines/A for Tau Her and 1.7 lines/A for Zeta Dra. In the V2 region it is 0.8 lines/A for Tau Her and 0.9 lines/A for Zeta Dra.

  16. Macrolactins from Marine-Derived Bacillus subtilis B5 Bacteria as Inhibitors of Inducible Nitric Oxide and Cytokines Expression

    PubMed Central

    Yan, Xia; Zhou, Yun-Xia; Tang, Xi-Xiang; Liu, Xiu-Xiu; Yi, Zhi-Wei; Fang, Mei-Juan; Wu, Zhen; Jiang, Fu-Quan; Qiu, Ying-Kun

    2016-01-01

    In order to find new natural products with anti-inflammatory activity, chemical investigation of a 3000-meter deep-sea sediment derived bacteria Bacillus subtilis B5 was carried out. A new macrolactin derivative was isolated and identified as 7,13-epoxyl-macrolactin A (1). Owing to the existence of the epoxy ring, 1 exhibited a significant inhibitory effect on the expression of inducible nitric oxide and cytokines, compared with previously isolated known macrolactins (2–5). Real-time Polymerase Chain Reaction (PCR) analysis showed that the new compound significantly inhibited the mRNA expressions of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Reverse transcription-PCR analysis demonstrated that the new compound reduced the mRNA expression level of IL-1β in a concentration-dependent manner. PMID:27792158

  17. A Genetic Analysis of the Rose-Gespleten Region (68c8-69b5) of Drosophila Melanogaster

    PubMed Central

    Hoogwerf, A. M.; Akam, M.; Roberts, D.

    1988-01-01

    We describe a genetic analysis of the region 68C8-69B5 defined by Df(3L)vin-7. We have induced 35 new lethal mutations in this region, which together with 20 existing lethal mutations, visible mutations, genes identified by protein products and one gene deduced from complementation data fall into 37 complementation groups in this 35-band interval. Using existing and newly induced deficiencies we have assigned these to 11 intervals defined by deficiency breakpoints. Those mutations which fell in the same breakpoint interval as the Lsp-2 gene, which codes for the abundant larval serum protein 2, were the subject of detailed study. None was rescued by the active Lsp-2 gene transformed on to chromosome II and we conclude that, as yet, we have no lethal mutations of Lsp-2. PMID:3130292

  18. 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.

  19. 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.

  20. Warm neutral halos around molecular clouds. IV - H I and continuum: Aperture synthesis observations towards the molecular cloud B5

    NASA Technical Reports Server (NTRS)

    Andersson, B.-G.; Roger, R. S.; Wannier, Peter G.

    1992-01-01

    We present aperture synthesis observations of H I (21 cm) line radiation and continuum emission at 408 and 1420 MHz towards a field centered on the molecular cloud B5. The H I emission shows an extended atomic halo around the molecular cloud. The opacity of the halo is derived using H I absorption toward several background sources and a simple source model is presented. The model indicates that the halo is not gravitationally bound to the molecular cloud and that it is in fact expanding away from it. Approximately 350 solar masses are contained in the H I halo. Flux densities and spectral indices for the sources detected in both of the continuum bands are given.

  1. The coupled thermo-mechanical-microstructural finite element modeling of hot stamping process in 22MnB5 steel

    NASA Astrophysics Data System (ADS)

    Chen, Xiangjun; Xiao, Namin; Li, Dianzhong; Li, Guangyao; Sun, Guangyong

    2013-12-01

    In this study, a coupled thermo-mechanical-microstructural finite element model based on the subroutine of commercial software ABAQUS is developed to predict the hot stamping process in 22MnB5 steel. The Johnson-Mehl-Avrami-Kolmogorov type model with Scheil' additivity rule and Koistinen-Marburger model are adopted to simulate the diffusional phase transformation and diffusionless one respectively. During the calculation of temperature and stress/strain field, the contributions of microstructure evolution, e.g. transformation latent heat, transformation strain, and transformation plasticity are taken into account, which give more insight of the material response. The model allows to evaluate the transient stress and strain distributions, the final microstructure constituent and the final distortion of the sheet part during the press quenching process. The effect of transformation plasticity on the geometry precision and the residual stress are discussed.

  2. 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.

  3. 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.

  4. MiR-30b-5p acts as a tumor suppressor, repressing cell proliferation and cell cycle in human hepatocellular carcinoma.

    PubMed

    Qin, Xian; Chen, Jing; Wu, Long; Liu, Zhisu

    2017-03-05

    MicroRNAs have been verified to participate in various biological behaviors of different tumors, via multiple signaling pathways. Many kinds of microRNAs in hepatocellular carcinoma have been researched. However, miR-30b-5p hasn't been included. Our study aim at the impacts of miR-30b-5p on HCC and the pathway it mediating. The results showed miR-30b-5p was significant downregulated in HCC tissues and cell lines. With clinical data, we've discovered miR-30b-5p was correlated with several clinical pathological characteristics, such as survival time, tumor size, HBV infected, pathological stage, differentiation and intrahepatic metastasis. Also we illustrated miR-30b-5p repressed cell proliferation and cell cycle of HCC cell lines. For a further study, we figured out that miR-30b-5p mediated DNMT3A to repress proliferation, meanwhile it targeted USP37 for decelerating cell cycle. This discovery inferred miR-30b-5p a potential favorable biomarker and therapeutic target for HCC diagnosis and treatment.

  5. Stabilization of cytochrome b5 by a conserved tyrosine in the secondary sphere of heme active site: A spectroscopic and computational study

    NASA Astrophysics Data System (ADS)

    Hu, Shan; He, Bo; Wang, Xiao-Juan; Gao, Shu-Qin; Wen, Ge-Bo; Lin, Ying-Wu

    2017-03-01

    Heme proteins perform a large array of biological functions, with the heme group bound non-covalently or covalently. To probe the stabilization role of conserved tyrosine residue in the secondary sphere of heme site in heme proteins, we herein used cytochrome b5 (Cyt b5) as a model protein, and mutated Tyr30 to Phe or His by removal of Tyr30 associated H-bond network and hydrophobic interaction. We performed thermal-induced unfolding studies for the two mutants, Y30F Cyt b5 and Y30H Cyt b5, as monitored by both UV-Vis and CD spectroscopy, as well as heme transfer studies from these proteins to apo-myoglobin, with wild-type Cyt b5 under the same conditions for comparison. The reduced stability of both mutants indicates that both the H-bonding and hydrophobic interactions associated with Tyr30 contribute to the protein stability. Moreover, we performed molecular modeling studies, which revealed that the hydrophobic interaction in the local region of Y30F Cyt b5 was well-remained, whereas Y30H Cyt b5 formed an H-bond network. These observations suggest that the conserved Tyr30 in Cyt b5 is not replaceable due to the presence of both the H-bond network and hydrophobic interaction in the secondary sphere of the heme active site. As demonstrated here for Cyt b5, it may be of practical importance for design of artificial heme proteins by engineering a Tyr in the secondary sphere with improved properties and functions.

  6. miR-125b-5p enhances chemotherapy sensitivity to cisplatin by down-regulating Bcl2 in gallbladder cancer

    PubMed Central

    Yang, Dong; Zhan, Ming; Chen, Tao; Chen, Wei; Zhang, Yunhe; Xu, Sunwang; Yan, Jinchun; Huang, Qihong; Wang, Jian

    2017-01-01

    Gallbladder cancer represents the most common malignancy of the biliary tract and is highly lethal with less than 5% overall 5-year survival rate. Chemotherapy remains the major treatment for late-stage patients. However, insensitivity to these chemotherapeutic agents including cisplatin is common. MicroRNAs (miRNAs) have been shown as modulators of drug resistance in many cancer types. We used genome-wide gene expression analysis in clinical samples to identify miR-125b-5p down-regulated in gallbladder cancer. miR-125b-5p up-regulation promoted cell death in gallbladder cancer cells in the presence of cisplatin. In contrast, knockdown of miR-125b-5p reduced cell death in gallbladder cancer cells treated with cisplatin. Up-regulation of miR-125b-5p significantly decreased tumor growth in combination with cisplatin in a mouse model. We identified Bcl2 as a direct target of miR-125b-5p which mediates the function of miR-125b-5p in gallbladder cancer. In clinical samples, miR-125b-5p was down-regulated in gallbladder cancer whereas Bcl2 was up-regulated and their expression was inversely correlated. Moreover, low miR-125b-5p expression or high expression of Bcl2 is correlated with poor prognosis in gallbladder cancer. Taken together, our findings indicate that miR-125b-5p is a potent chemotherapy sensitizer and may function as a new biomarker for the prognosis of gallbladder cancer patients. PMID:28256505

  7. Insights into the Role of Substrates on the Interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Le Clair, Stéphanie V.; Huang, Rui; Ahuja, Shivani; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2015-02-01

    Mammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process.

  8. MiR-181b-5p downregulates NOVA1 to suppress proliferation, migration and invasion and promote apoptosis in astrocytoma.

    PubMed

    Zhi, Feng; Wang, Qiang; Deng, Danni; 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.

  9. 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.

  10. Ranirestat as a therapeutic aldose reductase inhibitor for diabetic complications.

    PubMed

    Giannoukakis, Nick

    2008-04-01

    There are currently very few drugs available to directly treat diabetic complications. Those that are indicated clinically provide symptomatic relief and do not address the underlying biochemical problems. The involvement of the sorbitol pathway in complications has provided mechanistic insights into the biochemistry of complications and the key enzyme, aldose reductase, has become an attractive pharmacologic target. Among the aldose reductase inhibitors, the most promising is ranirestat. This review outlines the studies with ranirestat and compares its efficacy with other similar inhibitors. A survey of in vitro and in vivo studies was conducted, and with publicly available data from clinical trials, ranirestat efficacy was compared with other similar agents. Ranirestat is safe, exhibits some efficacy and is perhaps the only agent advanced enough in clinical trials to warrant further consideration for diabetic complications.

  11. Pyrroline-5-Carboxylate Reductase in Soybean Nodules 1

    PubMed Central

    Chilson, Oscar P.; Kelly-Chilson, Anne E.; Schneider, Julie D.

    1992-01-01

    Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD+-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP+ and proline. The K1 for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable. PMID:16668837

  12. Characterization of 12-Oxo-Phytodienoic Acid Reductase in Corn

    PubMed Central

    Vick, Brady A.; Zimmerman, Don C.

    1986-01-01

    12-Oxo-phytodienoic acid reductase, an enzyme of the biosynthetic pathway that converts linolenic acid to jasmonic acid, has been characterized from the kernel and seedlings of corn (Zea mays L.). The molecular weight of the enzyme, estimated by gel filtration, was 54,000. Optimum enzyme activity was observed over a broad pH range, from pH 6.8 to 9.0. The enzyme had a Km of 190 micromolar for its substrate, 12-oxo-phytodienoic acid. The preferred reductant was NADPH, for which the enzyme exhibited a Km of 13 micromolar, compared with 4.2 millimolar for NADH. Reductase activity was low in the corn kernel but increased five-fold by the fifth day after germination and then gradually declined. PMID:16664582

  13. [Properties of a nitrite reductase inhibitor protein from Pseudomonas aeruginosa].

    PubMed

    Karapetian, A V; Nalbandian, R M

    1993-08-01

    The amino acid composition and major physico-chemical properties of the "nonblue" copper protein isolated earlier from Pseudomonas aeruginosa have been determined. It has been found that the azurin oxidase, cytochrome c551 oxidase and superoxide dismutase activities of the enzyme are inhibited by this protein. The inhibition seems to be due to the protein interaction with the electron-accepting center of nitrite reductase.

  14. Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala)

    PubMed Central

    Herrera-Juárez, Álvaro Miguel; Martínez-González, José de Jesús; del Arenal Mena, Irene Patricia; Flores-Herrera, Óscar

    2017-01-01

    A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life. PMID:28787021

  15. 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.

  16. Autoregulation of the Synthesis of Nitrate Reductase in Aspergillus nidulans

    PubMed Central

    Cove, D. J.; Pateman, J. A.

    1969-01-01

    In Aspergillus nidulans, the syntheses of nitrate and nitrite reductases are induced by nitrate, and are repressed by ammonium. It is possible in wild-type strains to overcome partially the repressive effect of ammonium, by the addition of high concentrations of nitrate to the growth medium. Mutations which lead to the production of abnormal nitrate reductase affect in addition the control of the synthesis of the nitrate-metabolizing enzymes, which in these strains are produced constitutively. That this is not due to the accumulation of an internal inducer has now been shown, as these mutants have been found to be unable to respond to nitrate induction in the presence of ammonium in the same way as do wild-type strains. To explain these findings, we propose that the nitrate reductase molecule provides the recognition site for nitrate in the control system, such that when it is not complexed with nitrate it acts as a co-repressor, and, when it is complexed, as a co-inducer. PMID:5776531

  17. The effect of quercetin and galangin on glutathione reductase.

    PubMed

    Paulíková, Helena; Berczeliová, Elena

    2005-12-01

    Quercetin and galangin can change the activity of glutathione reductase. Quercetin (a catechol structure in the B-ring) and galangin (any hydroxyl group in the B-ring) have different biological activities but, both possess high antioxidant abilities. Quercetin during the antioxidative action, is converted into an oxidized products (o-semiquinone and o-quinone), and subsequently glutathionyl adducts may be formed or SH-enzyme can be inhibited. We have tried to see whether inhibition of glutathione reductase (GR) can be influenced by preincubation of enzyme with NADPH (a creation of reduced form of enzyme, GRH(2)) and whether diaphorase activity of the enzyme is decreased by these flavonoids. The results confirmed that quercetin inhibits GRH(2) and inhibition is reduced by addition of EDTA or N-acetylcysteine. Both of flavonoids have no effect on diaphorase activity of glutathione reductase and this enzyme could increase the production of free radicals by catalysis of reduction of o-quinone during action of quercetin in vivo.

  18. 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.

  19. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues*

    PubMed Central

    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.

    2016-01-01

    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 SeO32− 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

  20. Rat liver thioredoxin and thioredoxin reductase: purification and characterization.

    PubMed

    Luthman, M; Holmgren, A

    1982-12-21

    A reproducible scheme has been developed for the preparation of rat liver thioredoxin and thioredoxin reductase (EC 1.6.4.5) by using assays based on reduction of insulin and 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Both proteins were purified to homogeneity, as judged from polyacrylamide gel electrophoresis. Thioredoxin had a molecular weight of 12 000 and contained about 110 amino acids including 4 half-cystines and an NH2-terminal valine. Peptide maps of reduced and carboxymethylated thioredoxin showed that the protein had the active center sequence -Cys-Gly-Pro-Cys-Lys-Met- characteristic of thioredoxins also from procaryotes. Prolonged air oxidation of fully reduced thioredoxin created inactive, aggregated disulfide-containing molecules. Thioredoxin reductase showed a subunit molecular weight of 58 000 and a native molecular weight of 116 000. The enzyme was highly specific for NADPH with a Km of 6 microM. It contained FAD as prosthetic group and was sensitive to inhibition by arsenite. Thioredoxin reductase had a Km of 2.5 microM for rat and calf liver thioredoxin and a Kcat of 3000 min-1.

  1. Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis.

    PubMed

    Gahloth, Deepankar; Dunstan, Mark S; Quaglia, Daniela; Klumbys, Evaldas; Lockhart-Cairns, Michael P; Hill, Andrew M; Derrington, Sasha R; Scrutton, Nigel S; Turner, Nicholas J; Leys, David

    2017-09-01

    Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation-PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP-reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.

  2. Early diagnosis and management of 5 alpha-reductase deficiency.

    PubMed Central

    Odame, I; Donaldson, M D; Wallace, A M; Cochran, W; Smith, P J

    1992-01-01

    Two siblings of Pakistani origin, karyotype 46 XY, were born with predominantly female external genitalia with minute phallus, bifid scrotum, urogenital sinus, and palpable gonads. The older sibling at the age of 8 days showed an adequate testosterone response to human chorionic gonadotrophin (hCG) stimulation. The diagnosis of 5 alpha-reductase deficiency was made at age 6 years when no 5 alpha-reduced glucocorticoid metabolites were detectable in urine even after tetracosactrin (Synacthen) stimulation. In the younger sibling the diagnosis of 5 alpha-reductase deficiency was provisionally made at the early age of 3 days on the basis of high urinary tetrahydrocortisol (THF)/allotetrahydrocortisol (5 alpha-THF) ratio and this ratio increased with age confirming the diagnosis. Plasma testosterone: dihydrotestosterone (DHT) ratio before and after hCG stimulation was within normal limits at age 3 days but was raised at age 9 months. Topical DHT cream application to the external genitalia promoted significant phallic growth in both siblings and in the older sibling corrective surgery was facilitated. In prepubertal male pseudohermaphrodites with normal or raised testosterone concentrations, phallic growth in response to DHT cream treatment could be an indirect confirmation of 5 alpha-reductase deficiency. Images Figure 1 PMID:1626992

  3. Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase

    NASA Astrophysics Data System (ADS)

    Lay, Angelina J.; Jiang, Xing-Mai; Kisker, Oliver; Flynn, Evelyn; Underwood, Anne; Condron, Rosemary; Hogg, Philip J.

    2000-12-01

    Disulphide bonds in secreted proteins are considered to be inert because of the oxidizing nature of the extracellular milieu. An exception to this rule is a reductase secreted by tumour cells that reduces disulphide bonds in the serine proteinase plasmin. Reduction of plasmin initiates proteolytic cleavage in the kringle 5 domain and release of the tumour blood vessel inhibitor angiostatin. New blood vessel formation or angiogenesis is critical for tumour expansion and metastasis. Here we show that the plasmin reductase isolated from conditioned medium of fibrosarcoma cells is the glycolytic enzyme phosphoglycerate kinase. Recombinant phosphoglycerate kinase had the same specific activity as the fibrosarcoma-derived protein. Plasma of mice bearing fibrosarcoma tumours contained several-fold more phosphoglycerate kinase, as compared with mice without tumours. Administration of phosphoglycerate kinase to tumour-bearing mice caused an increase in plasma levels of angiostatin, and a decrease in tumour vascularity and rate of tumour growth. Our findings indicate that phosphoglycerate kinase not only functions in glycolysis but is secreted by tumour cells and participates in the angiogenic process as a disulphide reductase.

  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. 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

  6. Quinone Reductase Induction as a Biomarker for Cancer Chemoprevention⊥

    PubMed Central

    Cuendet, Muriel; Oteham, Carol P.; Moon, Richard C.; Pezzuto, John M.

    2007-01-01

    Chemoprevention involves the use of natural or synthetic substances to reduce the risk of developing cancer. Strategies for protecting cells from initiation events include decreasing metabolic enzymes responsible for generating reactive species (phase I enzymes) while increasing phase II enzymes that can deactivate radicals and electrophiles known to intercede in normal cellular processes. Reduction of electrophilic quinones by quinone reductase is an important detoxification pathway. Following evaluation of approximately 3000 plant and marine organism extracts, the number characterized as “active” was established in the range of 12% of the total, and over 60 active compounds have been isolated as quinone reductase inducers. One of them, isoliquiritigenin (1), isolated from tonka bean, was shown to be a monofunctional inducer by having similar quinone reductase inducing ability in wild-type Hepa 1c1c7 cells and two mutant cell lines. To further investigate the mechanism of induction, HepG2 human hepatoma cells stably transfected with ARE-luciferase plasmid were used. Isoliquiritigenin (1) significantly induced the luciferase activity in a dose-dependent manner. On the basis of these results, a full-term cancer chemoprevention study was conducted with 7,12-dimethylbenz[a]anthracene (DMBA)-treated female Sprague-Dawley rats. Dietary administration of 1 increased tumor latency. Based on these promising preliminary results, additional mechanistic studies are underway, as well as full-term carcinogenesis studies with chronic administration schedules. PMID:16562858

  7. The existence and significance of a mitochondrial nitrite reductase.

    PubMed

    Nohl, Hans; Staniek, Katrin; Kozlov, Andrey V

    2005-01-01

    The physiological functions of nitric oxide (NO) are well established. The finding that the endothelium-derived relaxing factor (EDRF) is NO was totally unexpected. It was shown that NO is a reaction product of an enzymatically catalyzed, overall, 5-electron oxidation of guanidinium nitrogen from L-arginine followed by the release of the free radical species NO. NO is synthesized by a single protein complex supported by cofactors, coenzymes (such as tetrahydrobiopterin) and cytochrome P450. The latter can uncouple from substrate oxidation producing O2*- radicals. The research groups of Richter [Ghafourifar P, Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett 1997; 418: 291-296.] and Boveris [Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mitochondria. J Biol Chem 1998; 273: 11038-11043.] identified a mitochondrial NO synthase (NOS). There are, however, increasing reports demonstrating that mitochondrial NO is derived from cytosolic NOS belonging to the Ca2+-dependent enzymes. NO was thought to control cytochrome oxidase. This assumption is controversial due to the life-time of NO in biological systems (millisecond range). We found a nitrite reductase in mitochondria which is of major interest. Any increase of nitrite in the tissue which is the first oxidation product of NO, for instance following NO donors, will stimulate NO-recycling via mitochondrial nitrite reductase. In this paper, we describe the identity and the function of mitochondrial nitrite reductase and the consequences of NO-recycling in the metabolic compartment of mitochondria.

  8. 17 CFR 240.10b5-1 - Trading “on the basis of” material nonpublic information in insider trading cases.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Deceptive Devices and Contrivances § 240.10b5-1 Trading “on the basis of” material nonpublic information in... in any other respect. (a) General. The “manipulative and deceptive devices” prohibited by Section...

  9. 17 CFR 240.10b5-1 - Trading “on the basis of” material nonpublic information in insider trading cases.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Deceptive Devices and Contrivances § 240.10b5-1 Trading “on the basis of” material nonpublic information in... in any other respect. (a) General. The “manipulative and deceptive devices” prohibited by Section...

  10. 17 CFR 240.10b5-1 - Trading “on the basis of” material nonpublic information in insider trading cases.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Deceptive Devices and Contrivances § 240.10b5-1 Trading “on the basis of” material nonpublic information in... in any other respect. (a) General. The “manipulative and deceptive devices” prohibited by Section...

  11. 17 CFR 240.10b5-1 - Trading “on the basis of” material nonpublic information in insider trading cases.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Deceptive Devices and Contrivances § 240.10b5-1 Trading “on the basis of” material nonpublic information in... in any other respect. (a) General. The “manipulative and deceptive devices” prohibited by Section...

  12. Unique structure of Ascaris suum b5-type cytochrome: an additional α-helix and positively charged residues on the surface domain interact with redox partners

    PubMed Central

    Yokota, Takehiro; Nakajima, Yoshitaka; Yamakura, Fumiyuki; Sugio, Shigetoshi; Hashimoto, Muneaki; Takamiya, Shinzaburo

    2005-01-01

    Cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a soluble protein that lacks a C-terminal membrane-anchoring domain, but possesses an N-terminal pre-sequence of 30 amino acids. During the maturation of cytochrome b5, the N-terminal pre-sequence is proteolytically cleaved to form the mature protein of 82 amino acid residues. A. suum cytochrome b5 is a basic protein containing more lysine residues and exhibiting a higher midpoint redox potential than its mammalian counterparts. We developed an expression system for the production of the recombinant nematode cytochrome b5, which is chemically and functionally identical with the native protein. Using this recombinant protein, we have determined the X-ray crystal structure of A. suum cytochrome b5 at 1.8 Å (1 Å=0.1 nm) resolution, and we have shown that this protein is involved in the reduction of nematode body-wall metmyoglobin. The crystal structure of A. suum cytochrome b5 consists of six α-helices and five β-strands. It differs from its mammalian counterparts by having a head-to-tail disulphide bridge, as well as a four-residue insertion in the vicinity of the sixth ligating histidine, which forms an additional α-helix, α4A, between helices α4 and α5. A. suum cytochrome b5 exists predominantly as a haem-orientation B isomer. Furthermore, the haem plane is rotated approx. 80° relative to the axis formed by haem-Fe and Nϵ atoms of the two histidine residues that are ligated to haem-Fe. The charge distribution around the haem crevice of A. suum cytochrome b5 is remarkably different from that of mammalian cytochrome b5 in that the nematode protein bears positively charged lysine residues surrounding the haem crevice. Using immunohistochemistry, we found that A. suum cytochrome b5 is present in the nematode hypodermis. Based on this histochemical and structural information, the physiological function of A. suum cytochrome b5 and its interaction with nematode

  13. miR-196b-5p Regulates Colorectal Cancer Cell Migration and Metastases through Interaction with HOXB7 and GALNT5.

    PubMed

    Stiegelbauer, Verena; Vychytilova-Faltejskova, Petra; Karbiener, Michael; Pehserl, Anna-Maria; Reicher, Andreas; Resel, Margit; Heitzer, Ellen; Ivan, Cristina; Bullock, Marc; Ling, Hui; Deutsch, Alexander; Wulf-Goldenberg, Annika; Adiprasito, Jan Basri; Stoeger, Herbert; Haybaeck, Johannes; Svoboda, Marek; Stotz, Michael; Hoefler, Gerald; Slaby, Ondrej; Calin, George Adrian; Gerger, Armin; Pichler, Martin

    2017-09-01

    Purpose: miR-196b-5p has been previously implicated in malignant transformation; however, its role in colorectal cancer has not been fully explored. In this study, we examine the clinical and biological relevance of miR-196b-5p, and the molecular pathways regulated by miR-196b-5p in colorectal cancer.Experimental Design: miR-196b-5p expression was quantitated by qRT-PCR in 2 independent cohorts composed of 292 patients with colorectal cancer in total, to explore its biomarker potential. Transient and stable gain- and loss-of-function experiments were conducted in a panel of colorectal cancer cell lines and mice, to evaluate the impact of miR-196b-5p on proliferation, chemosensitivity, migration/invasion, and metastases formation in vitro and in vivo The molecular pathways influenced by miR-196b-5p were characterized using whole transcriptome profiling, in silico target prediction tools, luciferase interaction assays, and phenocopy/rescue gene knockdown experiments.Results: Low miR-196b-5p expression was significantly associated with metastases and poor outcomes in 2 independent colorectal cancer patient cohorts (P < 0.05, log-rank test). miR-196b-5p inhibition led to significantly increased colorectal cancer cell migration/invasion and metastases formation in mice, whereas ectopic overexpression showed the opposite phenotype. Molecular profiling and target confirmation identified an interaction between miR-196b-5p and HOXB7 and GALNT5, which in turn regulated colorectal cancer cell migration.Conclusions: The association of low levels of miR-196b-5p and poor prognosis in patients with colorectal cancer can be explained by its influence on cancer cell migration and metastases formation. miR-196b-5p has an impact on colorectal cancer progression pathways through direct interaction with genes involved in cancer cell migration. Clin Cancer Res; 23(17); 5255-66. ©2017 AACR. ©2017 American Association for Cancer Research.

  14. Methionine Sulfoxide Reductases Are Essential for Virulence of Salmonella Typhimurium

    PubMed Central

    Rouf, Syed Fazle; Kitowski, Vera; Böhm, Oliver M.; Rhen, Mikael; Jäger, Timo; Bange, Franz-Christoph

    2011-01-01

    Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H2O2, and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H2O2, as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium. PMID:22073230

  15. Kinetic characteristics of ZENECA ZD5522, a potent inhibitor of human and bovine lens aldose reductase.

    PubMed

    Cook, P N; Ward, W H; Petrash, J M; Mirrlees, D J; Sennitt, C M; Carey, F; Preston, J; Brittain, D R; Tuffin, D P; Howe, R

    1995-04-18

    Aldose reductase (aldehyde reductase 2) catalyses the conversion of glucose to sorbitol, and methylglyoxal to acetol. Treatment with aldose reductase inhibitors (ARIs) is a potential approach to decrease the development of diabetic complications. The sulphonylnitromethanes are a recently discovered class of aldose reductase inhibitors, first exemplified by ICI215918. We now describe enzyme kinetic characterization of a second sulphonylnitromethane, 3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide (ZD5522), which is at least 10-fold more potent against bovine lens aldose reductase in vitro and which also has a greater efficacy for reduction of rat nerve sorbitol levels in vivo (ED95 = 2.8 mg kg-1 for ZD5522 and 20 mg kg-1 for ICI 215918). ZD5522 follows pure noncompetitive kinetics against bovine lens aldose reductase when either glucose or methylglyoxal is varied (K(is) = K(ii) = 7.2 and 4.3 nM, respectively). This contrasts with ICI 215918 which is an uncompetitive inhibitor (K(ii) = 100 nM) of bovine lens aldose reductase when glucose is varied. Against human recombinant aldose reductase, ZD5522 displays mixed noncompetitive kinetics with respect to both substrates (K(is) = 41 nM, K(ii) = 8 nM with glucose and K(is) = 52 nM, K(ii) = 3.8 nM with methylglyoxal). This is the first report of the effects of a sulphonylnitromethane on either human aldose reductase or utilization of methylglyoxal. These results are discussed with reference to a Di Iso Ordered Bi Bi mechanism for aldose reductase, where the inhibitors compete with binding of both the aldehyde substrate and alcohol product. This model may explain why aldose reductase inhibitors follow noncompetitive or uncompetitive kinetics with respect to aldehyde substrates, and X-ray crystallography paradoxically locates an ARI within the substrate binding site. Aldehyde reductase (aldehyde reductase 1) is closely related to aldose reductase. Inhibition of bovine kidney aldehyde reductase by ZD5522

  16. TLR2-dependent modulation of dendritic cells by LT-IIa-B5, a novel mucosal adjuvant derived from a type II heat-labile enterotoxin

    PubMed Central

    Lee, Chang Hoon; Masso-Welch, Patricia; Hajishengallis, George; Connell, Terry D.

    2011-01-01

    A host of human pathogens invades the body at mucosal surfaces. Yet, strong, protective mucosal immune responses directed against those pathogens routinely cannot be induced without the use of adjuvants. Although the strongest mucosal adjuvants are members of the family of HLTs, the inherent toxicities of HLT holotoxins preclude their clinical use. Herein, it is shown that LT-IIa-B5 enhances mucosal immune responses by modulating activities of DCs. i.n. immunization of mice with OVA in the presence of LT-IIa-B5 recruited DCs to the NALT and significantly increased uptake of OVA by those DCs. Furthermore, LT-IIa-B5 increased expression of CCR7 by DCs, which mediated enhanced migration of the cells from the NALT to the draining CLNs. LT-IIa-B5 also enhanced maturation of DCs, as revealed by increased surface expression of CD40, CD80, and CD86. Ag-specific CD4+ T cell proliferation was augmented in the CLNs of mice that had received i.n. LT-IIa-B5. Finally, when used as an i.n. adjuvant, LT-IIa-B5 dramatically increased the levels of OVA-specific salivary IgA and OVA-specific serum IgG. Strikingly, each of the activities induced by LT-IIa-B5 was strictly TLR2-dependent. The data strongly suggest that the immunomodulatory properties of LT-IIa-B5 depend on the productive modulation of mucosal DCs. Notably, this is the first report for any HLT to demonstrate in vivo the elicitation of strong, TLR2-dependent modulatory effects on DCs with respect to adjuvanticity. PMID:21791597

  17. PARP inhibitor increases chemosensitivity by upregulating miR-664b-5p in BRCA1-mutated triple-negative breast cancer

    PubMed Central

    Song, Wei; Tang, Lin; Xu, Yumei; Xu, Jing; Zhang, Wenwen; Xie, Hui; Wang, Shui; Guan, Xiaoxiang

    2017-01-01

    Emerging evidence has shown that adding poly(ADP-ribose) polymerase (PARP) inhibitors to chemotherapy regimens is superior to the control regimens alone in BRCA1-mutated triple-negative breast cancer (TNBC) patients, but their underlying mechanisms have not been fully elucidated. In this study, using miRNA microarray analysis of two BRCA1-mutated TNBC cell lines, we found that miR-664b-5p expression was increased after adding a PARP inhibitor, olaparib, to a carboplatin (CBP) plus gemcitabine (GEM) therapy regimen. Functional assays showed miR-664b-5p overexpression inhibited proliferation, migration and invasion in BRCA1-mutated TNBC cells. CCNE2 was identified as a novel functional target of miR-664b-5p, and CCNE2 knockdown revealed effects similar to those observed with miR-664b-5p overexpression. Both CCNE2 knockdown and miR-664b-5p overexpression significantly increased the chemosensitivity of BRCA1-mutated TNBC cells. In addition, in vivo studies indicated that miR-664b-5p inhibited tumour growth compared with the control in tumour xenograft models, and we also found that CCNE2 expression was inversely correlated with miR-664b-5p expression in 90 TNBC patient samples. In conclusion, miR-664b-5p functions as a tumour suppressor and has an important role in the regulation of PARP inhibitors to increase chemosensitivity by targeting CCNE2. This may be one of the possible mechanisms by which PARP inhibitors increase chemosensitivity in BRCA1-mutated TNBC. PMID:28176879

  18. PARP inhibitor increases chemosensitivity by upregulating miR-664b-5p in BRCA1-mutated triple-negative breast cancer.

    PubMed

    Song, Wei; Tang, Lin; Xu, Yumei; Xu, Jing; Zhang, Wenwen; Xie, Hui; Wang, Shui; Guan, Xiaoxiang

    2017-02-08

    Emerging evidence has shown that adding poly(ADP-ribose) polymerase (PARP) inhibitors to chemotherapy regimens is superior to the control regimens alone in BRCA1-mutated triple-negative breast cancer (TNBC) patients, but their underlying mechanisms have not been fully elucidated. In this study, using miRNA microarray analysis of two BRCA1-mutated TNBC cell lines, we found that miR-664b-5p expression was increased after adding a PARP inhibitor, olaparib, to a carboplatin (CBP) plus gemcitabine (GEM) therapy regimen. Functional assays showed miR-664b-5p overexpression inhibited proliferation, migration and invasion in BRCA1-mutated TNBC cells. CCNE2 was identified as a novel functional target of miR-664b-5p, and CCNE2 knockdown revealed effects similar to those observed with miR-664b-5p overexpression. Both CCNE2 knockdown and miR-664b-5p overexpression significantly increased the chemosensitivity of BRCA1-mutated TNBC cells. In addition, in vivo studies indicated that miR-664b-5p inhibited tumour growth compared with the control in tumour xenograft models, and we also found that CCNE2 expression was inversely correlated with miR-664b-5p expression in 90 TNBC patient samples. In conclusion, miR-664b-5p functions as a tumour suppressor and has an important role in the regulation of PARP inhibitors to increase chemosensitivity by targeting CCNE2. This may be one of the possible mechanisms by which PARP inhibitors increase chemosensitivity in BRCA1-mutated TNBC.

  19. MicroRNA-106b-5p regulates cisplatin chemosensitivity by targeting polycystic kidney disease-2 in non-small-cell lung cancer.

    PubMed

    Yu, Shaorong; Qin, Xiaobing; Chen, Tingting; Zhou, Leilei; Xu, Xiaoyue; Feng, Jifeng

    2017-09-01

    Systemic therapy with cytotoxic agents remains one of the main treatment methods for non-small-cell lung cancer (NSCLC). Cisplatin is a commonly used chemotherapeutic agent, that, when combined with other drugs, is an effective treatment for NSCLC. However, effective cancer therapy is hindered by a patient's resistance to cisplatin. Unfortunately, the potential mechanism underlying such resistance remains unclear. In this study, we explored the mechanism of microRNA-106b-5p (miR-106b-5p), which is involved in the resistance to cisplatin in the A549 cell line of NSCLC. Quantitative real-time PCR was used to test the expression of miR-106-5p in the A549 and the A549/DDP cell line of NSCLC. The cell counting kit-8 assay was used to detect cell viability. Flow cytometry was used to measure cell cycle and cell apoptosis. Luciferase reporter assays and western blot were performed to confirm whether polycystic kidney disease-2 (PKD2) is a direct target gene of miR-106b-5p. Immunohistochemistry was performed to examine the distribution of PKD2 expression in patients who are sensitive and resistant to cisplatin. The experiments indicated that the expression of miR-106b-5p was significantly decreased in A549/DDP compared with that in A549. MiR-106b-5p affected the tolerance of cells to cisplatin by negatively regulating PKD2. Upregulation of miR-106b-5p or downregulation of PKD2 expression can cause A549/DDP cells to become considerably more sensitive to cisplatin. The results showed that miR-106b-5p enhanced the sensitivity of A549/DDP cells to cisplatin by targeting the expression of PKD2. These findings suggest that the use of miR-106b-5p may be a promising clinical strategy in the treatment of NSCLC.

  20. [Purification and characterization of Linoleoyl-CoA desaturase from rat liver microsomes (author's transl)].

    PubMed

    Okayasu, T

    1981-01-01

    Rat liver microsomes catalyzed the oxidative delta 6-desaturation of linoleoyl-CoA (C18: 2, delta 9.12.) to gamma-linolenoyl-CoA (c18: 3, delta 6.9.12.) by using molecular oxygen and NADH or NADPH as the electron donors. The antibodies against cytochrome b5 inhibited markedly the delta 6-desaturation in the intact microsomes of the rat liver, suggesting that cytochrome b5 participated in the delta 6-desaturation. These experimental results led us to the hypothesis that the delta 6-desaturation of linoleoyl-CoA followed the scheme. (See formula in text). Terminal "delta 6-desaturase" was purified from rat liver microsomes for the first time by Triton X-100 solubilization, DEAE-cellulose, CM-Sephadex and cytochrome b5-Sepharose chromatography using its high affinity for cytochrome b5. The final enzyme preparation was homogeneous when applied to sodium dodecyl sulfate disc gel electrophoresis. delta 6-desaturase appeared as a single polypeptide of 66,000 daltons containing 49% nonpolar amino acid residues and one atom of non-heme iron. We confirmed that delta 6-desaturase differed from delta 9-desaturase, which converted stearoyl-CoA to oleoyl-CoA. The delta 6-desaturase activity required NADH (or NADPH), linoleoyl-CoA, oxygen, lipid or detergent and three enzymes, such as NADH-cytochrome b5 reductase (or NADPH-cytochrome P -450 reductase), cytochrome b5, and delta 6-desaturase. The reconstituted system of these components also confirmed the electron flow represented in Scheme 1. The delta 6-desaturase activity was inhibited by iron chelators, cyanine and p-chloromercuriphenyl sulfonate. In the reconstituted system of Km value for linoleoyl-CoA was 47 micro M, the maximal velocity was 83nmol/min/mg protein of delta 6-desaturase and the optimal pH was 7.0. Catalase, superoxide dismutase and t-butanol showed supportive effects on the delta 6-desaturation of the reconstituted system when purified enzymes were employed.

  1. 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

  2. Association of the 17-hydroxysteroid dehydrogenase type 5 gene polymorphism (-71A/G HSD17B5 SNP) with hyperandrogenemia in polycystic ovary syndrome (PCOS).

    PubMed

    Marioli, Dimitra J; Saltamavros, Alexandros D; Vervita, Vasiliki; Koika, Vasiliki; Adonakis, George; Decavalas, George; Markou, Kostas B; Georgopoulos, Neoklis A

    2009-08-01

    To evaluate the association of an activating single-nucleotide polymorphism (SNP) at position -71 of the promoter of 17beta-hydroxysteroid dehydrogenase type 5 gene (-71A/G HSD17B5 SNP) and polycystic ovary syndrome (PCOS) in a well characterized cohort of caucasian PCOS women with biochemical hyperandrogenemia. The PCOS patients and unrelated healthy control subjects were genotyped for the -71A/G HSD17B5 SNP. The acquired genotypic data was tested for association with PCOS and other quantitative phenotypic traits of the syndrome in PCOS patients. Subjects were recruited from the Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, at the University Hospital of Patras, Greece. Genotyping and biochemical determinations took place at the Laboratory of Molecular Endrocinology, University of Patras Medical School, Rion, Greece. Participants comprised 150 caucasian Greek PCOS women with biochemical hyperandrogenism and chronic anovulation and polycystic ovarian morphology on ultrasound and 51 healthy control subjects. HSD17B5 genotype, serum testosterone, serum androstenedione. No association of the -71A/G HSD17B5 SNP with PCOS was detected. However, the -71G HSD17B5 variant was associated with increased serum testosterone levels and decreased androstenedione/testosterone ratio. The -71G HSD17B5 variant is not a major component of the molecular pathogenetic mechanisms of PCOS, although it might contribute to the severity of hyperandrogenemia in women with PCOS and biochemical hyperandrogenism.

  3. cMyc/miR-125b-5p Signalling Determines Sensitivity to Bortezomib in Preclinical Model of Cutaneous T-Cell Lymphomas

    PubMed Central

    Willumsgaard, Ayalah; Skov, Anne Guldhammer; Palmieri, Dario; Gasparini, Pierluigi; Laganá, Alessandro; Woetmann, Anders; Ødum, Niels; Croce, Carlo Maria; Gniadecki, Robert

    2013-01-01

    Successful/effective cancer therapy in low grade lymphoma is often hampered by cell resistance to anti-neoplastic agents. The crucial mechanisms responsible for this phenomenon are poorly understood. Overcoming resistance of tumor cells to anticancer agents, such as proteasome inhibitors, could improve their clinical efficacy. Using cutaneous T-cell lymphoma (CTCL) as a model of the chemotherapy-resistant peripheral lymphoid malignancy, we demonstrated that resistance to proteasome inhibition involved a signaling between the oncogene cMyc and miR-125b-5p. Bortezomib repressed cMyc and simultaneously induced miR-125b-5p that exerted a cytoprotective effect through the downmodulation of MAD4. Overexpression of cMyc repressed miR-125b-5p transcription and sensitized lymphoma cells to bortezomib. The central role of miR-125b-5p was further confirmed in a mouse model of T-cell lymphoma, where xenotransplantation of human CTCL cells overexpressing miR-125b-5p resulted in enhanced tumor growth and a shorter median survival. Our findings describe a novel mechanism through which miR-125b-5p not only regulates tumor growth in vivo, but also increases cellular resistance to proteasome inhibitors via modulation of MAD4. PMID:23527180

  4. The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H

    PubMed Central

    Zhang, Qi; Cao, Chunyang; Wang, Zhi-Qiang; Wang, Yun-Hua; Wu, Houming; Huang, Zhong-Xian

    2004-01-01

    A comparative study on the solution structures of bovine microsomal cytochrome b5 (Tb5) and the mutant V45H has been achieved by 1D and 2D 1H-NMR spectroscopy to clarify the differences in the solution conformations between these two proteins. The results reveal that the global folding of the V45H mutant in solution is unchanged, but the subtle changes exist in the orientation of the axial ligand His39, and heme vinyl groups. The side chain of His45 in V45H mutant extends to the outer edge of the heme pocket leaving a cavity at the site originally occupied by the inner methyl group of Val45 residue. In addition, the imidazole ring of axial ligand His39 rotates counterclockwise by ~3° around the His-Fe-His axis, and the 4-heme vinyl group turns to the space vacated by the removed side chain due to the mutation. Furthermore, the helix III of the heme pocket undergoes outward displacement, while the linkage between helix II and III is shifted leftward. These observations are not only consistent with the pattern of the pseudocontact shifts of the heme protons, but also well account for the lower stability of V45H mutant against heat and urea. PMID:15273310

  5. Superbroad visible to NIR photoluminescence from Bi+ evidenced in Ba2B5O9Cl: Bi crystal.

    PubMed

    Zheng, Jiayu; Tan, Linling; Wang, Liping; Peng, Mingying; Xu, Shanhui

    2016-02-08

    The nature of bismuth NIR luminescence is essential to develop the bismuth doped laser materials with high efficiency and desirable emission wavelength, and it, thereby, receives rising interests. Our previous work reported the Bi(0) luminescence from Ba2B5O9Cl: Bi with a lifetime of ~30 μs and the conversion of Bi(2+) to Bi(0). This work found indeed the conversion could be enabled in the compound by an in situ reduction technique and it, however, happens via an intermediate state of Bi(+). Once the ion of Bi(+) is stabilized and built into the compound, it can luminesce in a super broad spectral range from 600 to 1200 nm with a lifetime longer than 1 ms, due to the cascade transitions from (3)P2 and (3)P1 to (3)P0. This is completely different from Bi(0) and Bi(2+) in the compound, and it has never been noticed before. We believe this work can help us better understand the complex nature of bismuth luminescence.

  6. An investigation of laser cutting quality of 22MnB5 ultra high strength steel using response surface methodology

    NASA Astrophysics Data System (ADS)

    Tahir, Abdul Fattah Mohd; Aqida, Syarifah Nur

    2017-07-01

    In hot press forming, changes of mechanical properties in boron steel blanks have been a setback in trimming the final shape components. This paper presents investigation of kerf width and heat affected zone (HAZ) of ultra high strength 22MnB5 steel cutting. Sample cutting was conducted using a 4 kW Carbon Dioxide (CO2) laser machine with 10.6 μm wavelength with the laser spot size of 0.2 mm. A response surface methodology (RSM) using three level Box-Behnken design of experiment was developed with three factors of peak power, cutting speed and duty cycle. The parameters were optimised for minimum kerf width and HAZ formation. Optical evaluation using MITUTOYO TM 505 were conducted to measure the kerf width and HAZ region. From the findings, laser duty cycle was crucial to determine cutting quality of ultra-high strength steel; followed by cutting speed and laser power. Meanwhile, low power intensity with continuous wave contributes the narrowest kerf width formation and least HAZ region.

  7. [The effect of isatin on protein-protein interactions between cytochrome b5 and cytochromes P450].

    PubMed

    Ershov, P V; Yablokov, E O; Mezentsev, Yu V; Kalushskiy, L A; Florinskaya, A V; Veselovsky, A V; Gnedenko, O V; Gilep, A A; Usanov, S A; Medvedev, A E; Ivanov, A S

    2017-03-01

    Cytochromes P450 (CYP) are involved in numerous biochemical processes including metabolism of xenobiotics, biosynthesis of cholesterol, steroid hormones etc. Since some CYP catalyze indol oxidation to isatin, we have hypothesized that isatin can regulate protein-protein interactions (PPI) between components of the CYP system thus representing a (negative?) feedback mechanism. The aim of this study was to investigate a possible effect of isatin on interaction of human CYP with cytochrome b5 (CYB5A). Using the optical biosensor test system employing surface plasmon resonance (SPR) we have investigated interaction of immobilized CYB5A with various CYP in the absence and in the presence of isatin. The SPR-based experiments have shown that a high concentration of isatin (270 mM) increases Kd values for complexes CYB5A/CYP3А5 and CYB5A/CYP3A4 (twofold and threefold, respectively), but has no influence on complex formation between CYB5A and other CYP (including indol-metabolizing CYP2C19 and CYP2E1). Isatin injection to the optical biosensor chip with the preformed molecular complex CYB5A/CYP3A4 caused a 30%-increase in its dissociation rate. Molecular docking manipulations have shown that isatin can influence interaction of CYP3А5 or CYP3A4 with CYB5A acting at the contact region of CYB5A/CYP.

  8. 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.

  9. ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER

    PubMed Central

    Beaufay, Henri; Amar-Costesec, Alain; Thinès-Sempoux, Denise; Wibo, Maurice; Robbi, Mariette; Berthet, Jacques

    1974-01-01

    Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b5, and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, β-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity. PMID:4150490

  10. Human cytochrome-P450 enzymes metabolize N-(2-methoxyphenyl)hydroxylamine, a metabolite of the carcinogens o-anisidine and o-nitroanisole, thereby dictating its genotoxicity.

    PubMed

    Naiman, Karel; Martínková, Markéta; Schmeiser, Heinz H; Frei, Eva; Stiborová, Marie

    2011-12-24

    N-(2-Methoxyphenyl)hydroxylamine is a component in the human metabolism of two industrial and environmental pollutants and bladder carcinogens, viz. 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole), and it is responsible for their genotoxicity. Besides its capability to form three deoxyguanosine adducts in DNA, N-(2-methoxyphenyl)-hydroxylamine is also further metabolized by hepatic microsomal enzymes. To investigate its metabolism by human hepatic microsomes and to identify the major microsomal enzymes involved in this process are the aims of this study. N-(2-Methoxyphenyl)hydroxylamine is metabolized by human hepatic microsomes predominantly to o-anisidine, one of the parent carcinogens from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome-b(5) reductase were used to characterize human liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute the main activity for this metabolic step in human liver to CYP3A4, 2E1 and 2C (more than 90%). The enzymes CYP2D6 and 2A6 also partake in this N-(2-methoxyphenyl)hydroxylamine metabolism in human liver, but only to ∼6%. Among the human recombinant CYP enzymes tested in this study, human CYP2E1, followed by CYP3A4, 1A2, 2B6 and 2D6, were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. The results found in this study indicate that genotoxicity of N-(2-methoxyphenyl)hydroxylamine is dictated by its spontaneous decomposition to nitrenium/carbenium ions generating DNA adducts, and by its susceptibility to metabolism by CYP enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine: An anticancer agent targeting hypoxic cells

    PubMed Central

    Seow, Helen A.; Penketh, Philip G.; Shyam, Krishnamurthy; Rockwell, Sara; Sartorelli, Alan C.

    2005-01-01

    To target malignant cells residing in hypoxic regions of solid tumors, we have designed and synthesized prodrugs generating the cytotoxic alkylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) after bioreductive activation. We postulate that one of these agents, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), requires enzymatic nitro reduction to produce 90CE, whereas another agent, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(4-nitrobenzyloxy)carbonyl]hydrazine (PNBC), can also be activated by nucleophilic attack by thiols such as glutathione (GSH)/GST. We demonstrated that these agents selectively kill hypoxic EMT6 mouse mammary carcinoma and CHO cells. In hypoxia, 50 μM KS119 produced 5 logs of kill of EMT6 cells without discernable cytotoxicity in air; similar effects were observed with CHO cells. PNBC was less efficacious against hypoxic tumor cells and also had some toxicity to aerobic cells, presumably because of GST/thiol activation, making PNBC less interesting as a selective hypoxic-cell cytotoxin. BALB/c mice with established EMT6 solid tumors were used to demonstrate that KS119 could reach and kill hypoxic cells in solid tumors. To gain information on bioreductive enzymes involved in the activation of KS119, cytotoxicity was measured in CHO cell lines overexpressing NADH:cytochrome b5 reductase (NBR), NADPH:cytochrome P450 reductase (NPR), or NAD(P)H: quinone oxidoreductase 1 (NQO1). Increased cytotoxicity occurred in cells overexpressing NBR and NPR, whereas overexpressed NQO1 had no effect. These findings were supported by enzymatic studies using purified NPR and xanthine oxidase to activate KS119. KS119 has significant potential as a hypoxia-selective tumor-cell cytotoxin and is unlikely to cause major toxicity to well oxygenated normal tissues. PMID:15964988

  12. Detecting subtle functional differences in ketopantoate reductase and related enzymes using a rule-based approach with sequence-structure homology recognition scores.

    PubMed

    Mondal, Sukanta; Nagao, Chioko; Mizuguchi, Kenji

    2010-11-01

    Ketopatoate reductase (KPR) is the second enzyme in the pantothenate (vitamin B(5)) biosynthesis pathway, an essential metabolic pathway identified as a potential target for new antimicrobials. The sequence similarity among putative KPRs is limited and KPR itself belongs to a large superfamily of 6-phosphogluconate dehydrogenases. Therefore, it is necessary to discriminate between true and other enzymes. In this paper, we describe a systematic analysis of putative KPRs in the context of this superfamily. Detailed structural analysis allowed us to define key residues for KPR activity and we classified eight structural genomics structures of the KPR family into four functional subclasses. We proposed a semi-automatic protocol, using sequence-structure homology recognition scores, for assigning KPR and related proteins to these subclasses and applied it to a representative set of 103 completely sequenced bacterial genomes. A similar approach can be applied to other enzyme families, which would aid the correct identification of drug targets and help design novel specific inhibitors.

  13. Synthesis of 17beta-N-substituted 19-Nor-10-azasteroids as inhibitors of human 5alpha-reductases I and II.

    PubMed

    Scarpi, Dina; Occhiato, Ernesto G; Danza, Giovanna; Serio, Mario; Guarna, Antonio

    2002-11-01

    The synthesis of 17beta-[N-(phenyl)methyl/phenyl-amido] substituted 10-azasteroids has been accomplished by either the TiCl4- or TMSOTf-catalysed reaction of carbamates 11 and 12 with Danishefsky's diene. The reaction provided 5alpha-H isomers 3a-5a and 5beta-H isomers 3b-5b depending on the reaction conditions. Both epimers of each compound were tested against human 5alpha-reductase types I and II. Unexpectedly, 5beta-H compounds were found more active than their 5alpha-H counterparts, the best inhibitors being 3b (IC50=279 and 2000 nM toward isoenzyme I and II, respectively) and 5b (IC50=913 and 247 nM toward isoenzymes I and II, respectively).

  14. Structure of the Molybdenum Site of EEcherichia Coli Trimethylamine N-Oxide Reductase

    SciTech Connect

    Zhang, L.; Nelson, K.Johnson; Rajagopalan, K.V.; George, G.N.

    2009-05-28

    We report a structural characterization of the molybdenum site of recombinant Escherichia coli trimethylamine N-oxide (TMAO) reductase using X-ray absorption spectroscopy. The enzyme active site shows considerable similarity to that of dimethyl sulfoxide (DMSO) reductase, in that, like DMSO reductase, the TMAO reductase active site can exist in multiple forms. Examination of the published crystal structure of TMAO oxidase from Shewanella massilia indicates that the postulated Mo coordination structure is chemically impossible. The presence of multiple active site structures provides a potential explanation for the anomalous features reported from the crystal structure.

  15. A flavone from Manilkara indica as a specific inhibitor against aldose reductase in vitro.

    PubMed

    Haraguchi, Hiroyuki; Hayashi, Ryosuke; Ishizu, Takashi; Yagi, Akira

    2003-09-01

    Isoaffinetin (5,7,3',4',5'-pentahydroxyflavone-6-C-glucoside) was isolated from Manilkara indica as a potent inhibitor of lens aldose reductase by bioassay-directed fractionation. This C-glucosyl flavone showed specific inhibition against aldose reductases (rat lens, porcine lens and recombinant human) with no inhibition against aldehyde reductase and NADH oxidase. Kinetic analysis showed that isoaffinetin exhibited uncompetitive inhibition against both dl-glyceraldehyde and NADPH. A structure-activity relationship study revealed that the increasing number of hydroxy groups in the B-ring contributes to the increase in aldose reductase inhibition by C-glucosyl flavones.

  16. Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

    PubMed Central

    Hoffmann, Tamara; Frankenberg, Nicole; Marino, Marco; Jahn, Dieter

    1998-01-01

    During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation. PMID:9422613

  17. Regulation of Nitrate Reductase Activity in Corn (Zea mays L.) Seedlings by Endogenous Metabolites 1

    PubMed Central

    Schrader, L. E.; Hageman, R. H.

    1967-01-01

    Primary and secondary metabolites of inorganic nitrogen metabolism were evaluated as inhibitors of nitrate reductase (EC 1.6.6.1) induction in green leaf tissue of corn seedlings. Nitrite, nitropropionic acid, ammonium ions, and amino acids were not effective as inhibitors of nitrate reductase activity or synthesis. Increasing α-amino nitrogen and protein content of intact corn seedlings by culture techniques significantly enhanced rather than decreased the potential for induction of nitrate reductase activity in excised seedlings. Secondary metabolites, derived from phenylalanine and tyrosine, were tested as inhibitors of induction of nitrate reductase. Of the 9 different phenylpropanoid compounds tested, only coumarin, trans-cinnamic and trans-o-hydroxycinnamic acids inhibited induction of nitrate reductase. While coumarin alone exhibited a relatively greater inhibitory effect on enzyme induction than on general protein synthesis (the latter measured by incorporation of labeled amino acids), this differential effect may have been dependent upon unequal rates of synthesis and accumulation with respect to the initial levels of nitrate reductase and general proteins. Because of the short half-life of nitrate reductase, inhibitors of protein synthesis in general could still achieve differential regulation of nitrogen metabolism. Coumarin did not inhibit nitrate reductase activity when added directly to the assay mixture at 5 mm. Carbamyl phosphate and its chemical derivative, cyanate, were found to be competitive (with nitrate) inhibitors of nitrate reductase. The data suggest that cyanate is the active inhibitor in the carbamyl phosphate preparations. PMID:16656715

  18. 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. Copyright © 2014 Elsevier GmbH. All rights reserved.

  19. 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

  20. Components of glycine reductase from Eubacterium acidaminophilum. Cloning, sequencing and identification of the genes for thioredoxin reductase, thioredoxin and selenoprotein PA.

    PubMed

    Lübbers, M; Andreesen, J R

    1993-10-15

    The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (grdA) and the first 23 amino acids of the large subunit of protein PC of glycine reductase (grdC) belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklandii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.

  1. Predictive In Silico Studies of Human 5-hydroxytryptamine Receptor Subtype 2B (5-HT2B) and Valvular Heart Disease

    PubMed Central

    Reid, Terry-Elinor; Kumar, Krishna

    2014-01-01

    Serotonin (5-HT) receptors are neuromodulator neurotransmitter receptors which when activated generate a signal transduction pathway within cells resulting in cell-cell communication. 5-hydroxytryptamine (serotonin) receptor 2B (5-HT2B) is a subtype of the seven members of 5-hydroxytrytamine (5-HT) family of receptors which is the largest member of the super family of 7-transmembrane G-protein coupled receptors (GPCRs). Not only do 5-HT receptors play physiological roles in the cardiovascular system, gastrointestinal and endocrine function and the central nervous, but they also play a role in behavioral functions. In particular 5-HT2B receptor is wide spread with regards to its distribution throughout bodily tissues and is expressed at high levels in the lungs, peripheral tissues, liver, kidney and prostate just to name a few. Hence 5-HT2B participates in multiple biological functions including CNS regulation, regulation of gastrointestinal motality, cardiovascular regulation and 5-HT transport system regulation. While 5-HT2B is a viable drug target and has therapeutic indications for treating obesity, psychotherapy, Parkinson’s disease etc. there is a growing concern regarding adverse drug reactions, specifically valvulopathy associated with 5-HT2B agonists. Due to the sequence homology experienced by 5-HT2 subtypes there is also a concern regarding the off target effects of 5-HT2A and 5-HT2C agonists. The concept of subtype selectivity is of paramount importance and can be tackled with the aid of in silico studies, specifically cheminformatics, to develop models to predict valvulopathy associated toxicity of drug candidates prior to clinical trials. This review has highlighted three in silico approaches thus far that have been successful in either predicting 5-HT2B toxicity of molecules or identifying important interactions between 5-HT2B and drug molecules that bring about valvulopathy related toxicities. PMID:23675941

  2. Granulin-epithelin precursor and ATP-dependent binding cassette (ABC)B5 regulate liver cancer cell chemoresistance.

    PubMed

    Cheung, Siu Tim; Cheung, Phyllis F Y; Cheng, Christine K C; Wong, Nicholas C L; Fan, Sheung Tat

    2011-01-01

    Chemotherapy is used to treat unresectable liver cancer with marginal efficacy; this might result from hepatic cancer cells with stem cell and chemoresistant features. Gene expression profiling studies have shown that hepatic cancer cells express granulin-epithelin precursor (GEP); we investigated its role in hepatic cancer stem cell functions and chemoresistance. The effects of GEP and drug transporter signaling on chemoresistance were investigated in hepatic cancer stem cells. We analyzed the expression patterns of 142 clinical samples from liver tumors, adjacent nontumorous liver tissue, and liver tissue from patients who did not have cancer. GEP regulated the expression of the adenosine triphosphate-dependent binding cassette (ABC)B5 drug transporter in liver cancer cells. Chemoresistant cells that expressed GEP had increased levels of ABCB5; suppression of ABCB5 sensitized the cells to doxorubicin uptake and apoptosis. Most cells that expressed GEP and ABCB5 also expressed the hepatic cancer stem cell markers CD133 and EpCAM; blocking ABCB5 reduced their expression. Expression levels of GEP and ABCB5 were correlated in human liver tumor samples. ABCB5 levels were increased in liver cancer cells compared with nontumor liver tissue from patients with cirrhosis or hepatitis, or normal liver tissue. ABCB5 expression was associated with the recurrence of hepatocellular carcinoma after partial hepatectomy. Expression of GEP and ABCB5 in liver cancer stem cells is associated with chemoresistance and reduced survival times of patients with hepatocellular carcinoma. Reagents designed to target these proteins might be developed as therapeutics and given in combination with chemotherapy to patients with liver cancer. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  3. Processing of chimeric mammalian cytochrome b5 precursors in Escherichia coli: reaction specificity of signal peptidase and identification of an aminopeptidase in post-translocational processing.

    PubMed Central

    Harding, V; Karim, A; Kaderbhai, N; Jones, A; Evans, A; Kaderbhai, M A

    1993-01-01

    A chimeric precursor interlinked by an arginine residue between the full-length signal sequence of alkaline phosphatase and the eukaryotic cytoplasmic cytochrome b5 was constructed. Expression of the chimeric precursor protein in Escherichia coli resulted in efficient export of spectrally authentic cytochrome b5 into the periplasm [Karim, Harding, Evans, Kaderbhai and Kaderbhai (1993) Bio/Technology 11, 612-618]. On sequencing, the apparent absence of arginine at the N-terminus of the secreted cytochrome b5 implied that the chimera was either miscleaved by signal peptidase or further processed following signal excision by an uncharacterized peptidase. The influence of the N-terminal region of cytochrome b5 on the unusual processing of the chimeric precursor was investigated by engineering a number of variant forms in which the region between Arg+1 and the mature portion of cytochrome b5 was extended and varied. Observations of the in vivo processed patterns of these variant cytochrome b5 forms exported into the periplasm revealed that the absence of arginine was due to neither miscleavage of the translocated precursor by the signal peptidase nor the nature of the early region of cytochrome b5. In fact, the selective excision of the arginine residue occurred subsequent to signal sequence deletion by an aminopeptidase which was sensitive to the metal chelator o-phenanthroline. We show that this aminopeptidase also participates in the trimming of the N-terminal arginine residue of the bacterial alkaline phosphatase to generate the three isoenzymes in the periplasm. Images Figure 4 Figure 5 Figure 6 PMID:8352742

  4. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases.

    PubMed

    Sattler, Steven A; Walker, Alexander M; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

    2017-02-01

    Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species.

  6. Thioredoxin Glutathione Reductase-Dependent Redox Networks in Platyhelminth Parasites

    PubMed Central

    Bonilla, Mariana; Gladyshev, Vadim N.

    2013-01-01

    Abstract Significance: Platyhelminth parasites cause chronic infections that are a major cause of disability, mortality, and economic losses in developing countries. Maintaining redox homeostasis is a major adaptive problem faced by parasites and its disruption can shift the biochemical balance toward the host. Platyhelminth parasites possess a streamlined thiol-based redox system in which a single enzyme, thioredoxin glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase (TR) domains, supplies electrons to oxidized glutathione (GSSG) and thioredoxin (Trx). TGR has been validated as a drug target for schistosomiasis. Recent Advances: In addition to glutathione (GSH) and Trx reduction, TGR supports GSH-independent deglutathionylation conferring an additional advantage to the TGR redox array. Biochemical and structural studies have shown that the TR activity does not require the Grx domain, while the glutathione reductase and deglutathionylase activities depend on the Grx domain, which receives electrons from the TR domains. The search for TGR inhibitors has identified promising drug leads, notably oxadiazole N-oxides. Critical Issues: A conspicuous feature of platyhelminth TGRs is that their Grx-dependent activities are temporarily inhibited at high GSSG concentrations. The mechanism underlying the phenomenon and its biological relevance are not completely understood. Future Directions: The functional diversity of Trxs and Grxs encoded in platyhelminth genomes remains to be further assessed to thoroughly understand the TGR-dependent redox network. Optimization of TGR inhibitors and identification of compounds targeting other parasite redox enzymes are good options to clinically develop relevant drugs for these neglected, but important diseases. Antioxid. Redox Signal. 19, 735–745. PMID:22909029

  7. Purification and properties of nitrate reductase from Mitsuokella multiacidus.

    PubMed

    Yamamoto, I; Shimizu, H; Tsuji, T; Ishimoto, M

    1986-03-01

    Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).

  8. 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). Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Galangin, a dietary flavonol inhibits tumor initiation during experimental pulmonary tumorigenesis by modulating xenobiotic enzymes and antioxidant status.

    PubMed

    Devadoss, Dinesh; Ramar, Manikandan; Chinnasamy, Arulvasu

    2014-02-05

    The aim of present study was to elucidate anti-initiating efficacy of galangin against benzo(a)pyrene (B(a)P)-induced lung carcinogenesis in male Swiss albino mice. Therefore, the activities of xenobiotic metabolic enzymes such as phase I and II were examined in lung as well as liver tissues (to compare the effects between target and non-target organs). Besides, the activities/levels of tissue marker enzymes, antioxidants, lipid peroxidation (LPO), cytochrome P450 1A1 (CYP1A1) expressions and histological observation of lungs were also analyzed. B(a)P (50 mg/kg body weight) was administered to male Swiss albino mice (20-25 g) to experimentally induce lung cancer. B(a)P-induced animals showed increased activity of phase I (Cytochrome P450, Cytochrome b5, NADPH Cytochrome P450 redcutase and NADH Cytochrome b5 reductase) drug metabolic enzymes, LPO levels, tissue marker enzymes and decreased activity of phase II metabolic enzymes (glutathione-S-transferase, DT-diaphorase and UDP-glucuronyl transferase) as well as antioxidant levels. Histological examination of lungs revealed severe alveolar and bronchiolar damages in B(a)P-induced mice. Immunohistochemical and western blot analysis of CYP1A1 increased significantly in lung tissues of B(a)P-induced animals. Treatment with galangin (20 mg/kg body weight) efficiently counteracted all the above anomalies and restored cellular homeostasis. Our results demonstrate that galangin can modify xenobiotic enzymes in murine model of pulmonary tumorigenesis.

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

    PubMed Central

    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. PMID:24555099

  11. 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.

  12. 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.

  13. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

    SciTech Connect

    Resch, Michael G.; Linger, Jeffrey; McGeehan, John; Tyo, Keith; Beckham, Gregg

    2016-05-26

    Carboxylic acid reductases (CARs) are recently emerging reductive enzymes for the direct production of aldehydes from biologically-produced carboxylic acids. Recent work has demonstrated that these powerful enzymes are able to reduce a very broad range of volatile- to long-chain fatty acids as well as aromatic acids. Here, we express four CAR enzymes from different fungal origins to test their activity against fatty acids commonly produced in oleaginous microbes. These in vitro results will inform metabolic engineering strategies to conduct mild biological reduction of carboxylic acids in situ, which is conventionally done via hydrotreating catalysis at high temperatures and hydrogen pressures.

  14. The dynamic energy landscape of dihydrofolate reductase catalysis.

    PubMed

    Boehr, David D; McElheny, Dan; Dyson, H Jane; Wright, Peter E

    2006-09-15

    We used nuclear magnetic resonance relaxation dispersion to characterize higher energy conformational substates of Escherichia coli dihydrofolate reductase. Each intermediate in the catalytic cycle samples low-lying excited states whose conformations resemble the ground-state structures of preceding and following intermediates. Substrate and cofactor exchange occurs through these excited substates. The maximum hydride transfer and steady-state turnover rates are governed by the dynamics of transitions between ground and excited states of the intermediates. Thus, the modulation of the energy landscape by the bound ligands funnels the enzyme through its reaction cycle along a preferred kinetic path.

  15. 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. © The Author(s) 2015.

  16. PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

    PubMed Central

    Auvergne, R; Wu, C; Connell, A; Au, S; Cornwell, A; Osipovitch, M; Benraiss, A; Dangelmajer, S; Guerrero-Cazares, H; Quinones-Hinojosa, A; Goldman, SA

    2016-01-01

    Glioblastoma (GBM) remains the most common and lethal intracranial tumor. In a comparison of gene expression by A2B5-defined tumor-initiating progenitor cells (TPCs) to glial progenitor cells derived from normal adult human brain, we found that the F2R gene encoding PAR1 was differentially overexpressed by A2B5-sorted TPCs isolated from gliomas at all stages of malignant development. In this study, we asked if PAR1 is causally associated with glioma progression. Lentiviral knockdown of PAR1 inhibited the expansion and self-renewal of human GBM-derived A2B5+ TPCs in vitro, while pharmacological inhibition of PAR 1 similarly slowed both the growth and migration of A2B5+ TPCs in culture. In addition, PAR1 silencing potently suppressed tumor expansion in vivo, and significantly prolonged the survival of mice following intracranial transplantation of human TPCs. These data strongly suggest the importance of PAR1 to the self-renewal and tumorigenicity of A2B5-defined glioma TPCs; as such, the abrogation of PAR1-dependent signaling pathways may prove a promising strategy for gliomas. PMID:26616854

  17. miR-15b-5p induces endoplasmic reticulum stress and apoptosis in human hepatocellular carcinoma, both in vitro and in vivo, by suppressing Rab1A

    PubMed Central

    Yang, Yang; Hou, Ni; Wang, Xiaofei; Wang, Lumin; Chang, Su'e; He, Kang; Zhao, Zhenghao; Zhao, Xiaoge; Song, Tusheng; Huang, Chen

    2015-01-01

    In human hepatocellular carcinoma (HCC), aberrant expression of miRNAs correlates with tumor cell proliferation, apoptosis, invasion, and migration by targeting downstream proteins. miR-15b levels are reported increased in HCC tissues; however, they negatively correlate to HCC recurrence. Our aim was to understand the reason for this phenomenon. We used the reverse transcription-polymerase chain reaction (RT-PCR) to measure miR-15b-5p expression in both HCC tissues and HCC cell lines. Our results were consistent with the report. Using bioinformatics and luciferase reporter assays, we identified Rab1A as a novel and direct target of miR-15b-5p. Inhibiting the function of Rab1A with shRab1A also inhibited the growth of HCC cells and induced endoplasmic reticulum stress (ERS) and apoptosis. Similarly, suppressing Rab1A by overexpression of miR-15b-5p also inhibited cell growth and induced ERS and apoptosis. Moreover, re-expression of Rab1A rescued the miR-15b-5p -induced ERS, apoptosis, and growth inhibition in HCC cells. In vivo assays were further performed to testify them. Taken together, our data suggest that miR-15b-5p induces ERS, apoptosis, and growth inhibition by targeting and suppressing Rab1A, acting as a tumor suppressor gene in HCC. This finding suggests a novel relation among Rabs, miRNAs, and apoptosis. PMID:26023735

  18. 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

  19. The role of small heat-shock protein αB-crystalline (HspB5) in COPD pathogenesis

    PubMed Central

    Cherneva, Radostina V; Georgiev, Ognian B; Petrova, Daniela S; Trifonova, Nedka L; Stamenova, Maria; Ivanova, Vesela; Vlasov, Veselin I

    2012-01-01

    Background αB-crystallin (HspB5) is a chaperone whose role as a marker of innate immunity activation as well as its therapeutic potential have recently been investigated in several inflammatory diseases: multiple sclerosis, myocardial ischemia, and Guillain–Barré syndrome. Aim The aim of this study is to determine the role of αB-crystallin in chronic obstructive pulmonary disease (COPD) pathogenesis and inflammation. Materials Plasma levels of αB-crystallin were studied in 163 patients: 52 healthy non-COPD smokers; 20 COPD smokers in Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I–II; 43 COPD smokers in GOLD stages III-IV. Forty-eight patients were diagnosed with acute inflammatory respiratory disease. The plasma levels of αB-crystallin antibodies were determined by an enzyme-linked immunosorbent assay (Calbiochem), and were confirmed with Western blotting. Tissue expression of the protein was compared in three different groups of patients: COPD smokers, COPD nonsmokers, and in patients with age-related emphysema. Results The mean level of anti-αB-crystallin antibodies in non-COPD smokers was 0.291nm. In COPD smokers it was 0.352 nm and, in patients with inflammatory lung diseases, 0.433 nm. There was a statistically significant difference between COPD smokers and healthy non-COPD smokers (P = 0.010). The same could be observed comparing the group of patients with acute inflammation and non-COPD healthy smokers (P = 0.007). There was no statistically significant difference between patients with mild/moderate inflammation and those with severe COPD. Tissue detection of the protein showed that it was significantly overexpressed in COPD smokers in comparison to COPD nonsmokers and was only slightly expressed in patients with age-related emphysema. Conclusion αB-crystallin is increased in patients with inflammatory lung diseases. Though unspecific, it could be used in a panel of markers discerning COPD smokers from healthy nonsmokers

  20. The role of small heat-shock protein αB-crystalline (HspB5) in COPD pathogenesis.

    PubMed

    Cherneva, Radostina V; Georgiev, Ognian B; Petrova, Daniela S; Trifonova, Nedka L; Stamenova, Maria; Ivanova, Vesela; Vlasov, Veselin I

    2012-01-01

    αB-crystallin (HspB5) is a chaperone whose role as a marker of innate immunity activation as well as its therapeutic potential have recently been investigated in several inflammatory diseases: multiple sclerosis, myocardial ischemia, and Guillain-Barré syndrome. The aim of this study is to determine the role of αB-crystallin in chronic obstructive pulmonary disease (COPD) pathogenesis and inflammation. Plasma levels of αB-crystallin were studied in 163 patients: 52 healthy non-COPD smokers; 20 COPD smokers in Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I-II; 43 COPD smokers in GOLD stages III-IV. Forty-eight patients were diagnosed with acute inflammatory respiratory disease. The plasma levels of αB-crystallin antibodies were determined by an enzyme-linked immunosorbent assay (Calbiochem), and were confirmed with Western blotting. Tissue expression of the protein was compared in three different groups of patients: COPD smokers, COPD nonsmokers, and in patients with age-related emphysema. The mean level of anti-αB-crystallin antibodies in non-COPD smokers was 0.291 nm. In COPD smokers it was 0.352 nm and, in patients with inflammatory lung diseases, 0.433 nm. There was a statistically significant difference between COPD smokers and healthy non-COPD smokers (P = 0.010). The same could be observed comparing the group of patients with acute inflammation and non-COPD healthy smokers (P = 0.007). There was no statistically significant difference between patients with mild/moderate inflammation and those with severe COPD. Tissue detection of the protein showed that it was significantly overexpressed in COPD smokers in comparison to COPD nonsmokers and was only slightly expressed in patients with age-related emphysema. αB-crystallin is increased in patients with inflammatory lung diseases. Though unspecific, it could be used in a panel of markers discerning COPD smokers from healthy nonsmokers. As αB-crystallin is a regulator of innate

  1. 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.

  2. Potential-dependent surface-enhanced resonance Raman spectroscopy at nanostructured TiO2 : a case study on cytochrome b5.

    PubMed

    Han, Xiao Xia; Köhler, Christopher; Kozuch, Jacek; Kuhlmann, Uwe; Paasche, Lars; Sivanesan, Arumugam; Weidinger, Inez M; Hildebrandt, Peter

    2013-12-20

    Nanostructured titanium dioxide (TiO2 ) electrodes, prepared by anodization of titanium, are employed to probe the electron-transfer process of cytochrome b5 (cyt b5 ) by surface-enhanced resonance Raman (SERR) spectroscopy. Concomitant with the increased nanoscopic surface roughness of TiO2 , achieved by raising the anodization voltage from 10 to 20 V, the enhancement factor increases from 2.4 to 8.6, which is rationalized by calculations of the electric field enhancement. Cyt b5 is immobilized on TiO2 under preservation of its native structure but it displays a non-ideal redox behavior due to the limited conductivity of the electrode material. The electron-transfer efficiency which depends on the crystalline phase of TiO2 has to be improved by appropriate doping for applications in bioelectrochemistry. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions

    PubMed Central

    1976-01-01

    The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product. PMID:791954

  4. ReBe2B5O11 (Re = Y, Gd): rare-earth beryllium borates as deep-ultraviolet nonlinear-optical materials.

    PubMed

    Yan, Xue; Luo, Siyang; Lin, Zheshuai; Yao, Jiyong; He, Ran; Yue, Yinchao; Chen, Chuangtian

    2014-02-17

    Two novel rare-earth beryllium borates ReBe2B5O11 (Re = Y, Gd) have been discovered. These materials possess a unique structural feature with a platelike infinite ∞(2)[Be2B5O11]3– superlayer, which is first found in beryllium borates. The superlayer can be seen as sandwich-shaped with ∞(1)[B4O8]4– chains linking up with a ∞(2)[Be2BO5]3– sublayer above and below via the B–O–Be bond. Each ∞(2)[Be2B5O11]3– layer is further connected to the neighboring layer through Re3+ cations coordinating with O atoms. Both of these two crystals have very short cutoff wavelengths below 200 nm and exhibit relatively large nonlinear-optical (NLO) effects, indicating their promising applications as good deep-UV NLO crystals.

  5. Oxidation of NADH by a rotenone and antimycin-sensitive pathway in the mitochondrion of procyclic Trypanosoma brucei brucei.

    PubMed

    Beattie, D S; Obungu, V H; Kiaira, J K

    1994-03-01

    The pathway of NADH oxidation in the procyclic Trypanosoma brucei brucei was investigated in a crude mitochondrial membrane fraction and in whole cells permeabilized with digitonin. NADH:cytochrome c reductase activity was 75% inhibited by concentrations of antimycin that inhibited 95% succinate:cytochrome c reductase activity suggesting that the major pathway for NADH oxidation in the mitochondria involved the cytochrome bc1 complex of the electron transfer chain. Both NADH:cytochrome c and NADH:ubiquinone reductase activities were inhibited 80-90% by rotenone indicating the presence of a complex I-like NADH dehydrogenase in the mitochondrion of trypanosomes. In w