Sample records for b-type cytochromes specific

  1. Cytochrome b6 arginine 214 of Synechococcus sp. PCC 7002, a key residue for quinone-reductase site function and turnover of the cytochrome bf complex.

    PubMed

    Nelson, Matthew E; Finazzi, Giovanni; Wang, Qing Jun; Middleton-Zarka, Kelly A; Whitmarsh, John; Kallas, Toivo

    2005-03-18

    Quinone-reductase (Q(i)) domains of cyanobacterial/chloroplast cytochrome bf and bacterial/mitochondrial bc complexes differ markedly, and the cytochrome bf Q(i) site mechanism remains largely enigmatic. To investigate the bf Q(i) domain, we constructed the mutation R214H, which substitutes histidine for a conserved arginine in the cytochrome b(6) polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew approximately 2.5-fold more slowly than the wild type. Slower growth arose from correspondingly slower overall turnover of the bf complex. Specifically, as shown in single flash turnover experiments of cytochrome b(6) reduction and oxidation, the R214H mutation partially blocked electron transfer to the Q(i) site, mimicking the effect of the Q(i) site inhibitor 2-N-4-hydroxyquinoline-N-oxide. The kinetics of cytochrome b(6) oxidation were largely unaffected by hydrogen-deuterium exchange in the mutant but were slowed considerably in the wild type. This suggests that although protonation events influenced the kinetics of cytochrome b(6) oxidation at the Q(i) site in the wild type, electron flow limited this reaction in the R214H mutant. Redox titration of membranes revealed midpoint potentials (E(m,7)) of the two b hemes similar to those in the wild type. Our data define cytochrome b(6) Arg(214) as a key residue for Q(i) site catalysis and turnover of the cytochrome bf complex. In the recent cytochrome bf structures, Arg(214) lies near the Q(i) pocket and the newly discovered c(i) or x heme. We propose a model for Q(i) site function and a role for Arg(214) in plastoquinone binding.

  2. Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane.

    PubMed

    Canova-Davis, E; Chiang, J Y; Waskell, L

    1985-06-01

    Cytochrome b5 has recently been shown to be required in the reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane [E. Canova-Davis and L. A. Waskell, J. biol. Chem. 259, 2541 (1984)]. To determine whether this observation in the reconstituted system was merely dependent on the particular ratios of the various components or some other fortuitous, unknown factor, or whether cytochrome b5 plays a role in the liver microsomal metabolism of methoxyflurane, the following studies were undertaken. Antibody to rabbit holocytochrome b5 was raised in guinea pigs. The antibody to cytochrome b5 was able to inhibit 75% of the microsomal metabolism of methoxyflurane. This same antibody also inhibited methoxyflurane metabolism in the reconstituted system. When the antibody to cytochrome b5 was treated with purified cytochrome b5 before addition to the microsomes, it did not inhibit methoxyflurane metabolism. Furthermore, the antibody to cytochrome b5 did not inhibit the microsomal metabolism of benzphetamine. This suggests that cytochrome b5 was required for the microsomal metabolism of methoxyflurane. It is possible that cytochrome b5 functioned in the metabolism of methoxyflurane by retaining a specific conformation of cytochrome P-450 and not by transferring the second electron to cytochrome P-450. To explore this possibility, cytochrome b5 was reconstituted with Mn3+-protoporphyrin IX. The Mn3+-protoporphyrin IX derivative retained the conformation of cytochrome b5 but not its electron transfer properties. This manganese derivative of cytochrome b5 was unable to stimulate the metabolism of methoxyflurane. The study demonstrated that cytochrome b5 was obligatory for the microsomal metabolism of methoxyflurane, whereas it was not required for the microsomal N-demethylation of benzphetamine. Moreover, the heme moiety of cytochrome b5 functioned to transfer electrons in this reaction.

  3. Kinetic studies on the oxidation of cytochrome b(5) Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes.

    PubMed

    Yao, Ping; Wang, Yun-Hua; Sun, Bing-Yun; Xie, Yi; Hirota, Shun; Yamauchi, Osamu; Huang, Zhong-Xian

    2002-04-01

    To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.

  4. Defining the in Vivo Role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5.

    PubMed

    Finn, Robert D; McLaughlin, Lesley A; Ronseaux, Sebastien; Rosewell, Ian; Houston, J Brian; Henderson, Colin J; Wolf, C Roland

    2008-11-14

    In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.

  5. A mutation in yeast mitochondrial DNA results in a precise excision of the terminal intron of the cytochrome b gene.

    PubMed

    Hill, J; McGraw, P; Tzagoloff, A

    1985-03-25

    The yeast nuclear gene CBP2 was previously proposed to code for a protein necessary for processing of the terminal intron in the cytochrome b pre-mRNA (McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chem. 258, 9459-9468). In the present study we describe a mitochondrial mutation capable of suppressing the respiratory deficiency of cbp2 mutants. The mitochondrial suppressor mutation has been shown to be the result of a precise excision of the last intervening sequence from the cytochrome b gene. Strains with the altered mitochondrial DNA have normal levels of mature cytochrome b mRNA and of cytochrome b and exhibit wild type growth on glycerol. These results confirm that CBP2 codes for a protein specifically required for splicing of the cytochrome b intron and further suggest that absence of the intervening sequence does not noticeably affect the expression of respiratory function in mitochondria.

  6. Identification of a c-Type Cytochrome Specific for Manganese Dioxide (MnO2) Reduction in Anaeromyxobacter dehalogenans Strain 2CP-C

    NASA Astrophysics Data System (ADS)

    Pfiffner, S. M.; Nissen, S.; Liu, X.; Chourey, K.; Vishnivetskaya, T. A.; Hettich, R.; Loeffler, F.

    2014-12-01

    Anaeromyxobacter dehalogenans is a metabolically versatile Deltaproteobacterium and conserves energy from the reduction of various electron acceptors, including insoluble MnO2 and ferric oxides/oxyhydroxides (FeOOH). The goal of this study was to identify c-type cytochromes involved in electron transfer to MnO2. The characterization of deletion mutants has revealed a number of c-type cytochromes involved in electron transfer to solid metal oxides in Shewanella spp. and Geobacter spp; however, a genetic system for Anaeromyxobacter is not available. The A. dehalogenans str. 2CP-C genome encodes 68 putative c-type cytochromes, which all lack functional assignments. To identify c-type cytochromes involved in electron transfer to solid MnO2, protein expression profiles of A. dehalogenans str. 2CP-C cells grown with acetate as electron donor and MnO2, ferric citrate, FeOOH, nitrate or fumarate as electron acceptors were compared. Whole cell proteomes were analyzed after trypsin proteolysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Distinct c-type cytochrome expression patterns were observed with cells grown with different electron acceptors. A. dehalogenans str. 2CP-C grown with MnO2 expressed 25 out of the 68 c-type cytochromes encoded on the genome. The c-type cytochrome Adeh_1278 was only expressed in strain 2CP-C grown with MnO2. Reverse transcription PCR confirmed that the Adeh_1278 gene was transcribed in MnO2-grown cells but not in cells grown with other terminal electron acceptors. The expression of the Adeh_1278 gene correlated with Mn(IV) reduction activity. Adeh_1278 has three heme binding motifs and is predicted to be located in the periplasm. The identification of Adeh_1278 as a protein uniquely expressed when MnO2 serves as electron acceptor suggests its utility as a biomarker for MnO2 reduction. This example demonstrates the value of the LC-MS/MS approach for identifying specific proteins of interest and making functional assignments

  7. Assembly of the mitochondrial membrane system. XVIII. Genetic loci on mitochondrial DNA involved in cytochrome b biosynthesis.

    PubMed

    Tzagoloff, A; Foury, F; Akai, A

    1976-11-24

    1. Fourteen cytoplasmic mutants of Saccharomyces cerevisiae with a specific deficiency of cytochrome b have been studied. The mutations have been shown to occur in two separate genetic loci, COB 1 and COB 2. These loci can be distinguished by mit- X mit- crosses. Pairwise crosses of cytochrome b mutants belonging to different loci yield 4-6% wild type recombinants corresponding to recombinational frequencies of 8-12%. In intra-locus crosses, the recombinational frequencies range from 1% to less than 0.01%. The two loci can also be distinguished by mit- X rho- crosses. Twenty rho- testers have been isolated of which ten preferentially restore mutations in COB 1 and ten others in COB 2. 2. The COB 1 and COB 2 loci have been localized on mitochondrial DNA between the two antibiotic resistance loci OLI 1 and OLI 2 in the order OLI 2-COB 2-COB 1-OLI 1. The results of mit- X mit- and mit- X rho- crosses have also been used to map the cytochrome b mutations relative to each other. The maps obtained by the two independent methods are in good agreement. 3. Mutations in COB 1 have been found to be linked to the OLI1 locus in some but not in other strains of S. cervisiae. This evidence suggests that there may be a spacer region between the two loci whose length varies from strain to strain. 4. Two mutations in COB 2 have been found to cause a loss of a mitochondrial translation product corresponding to the cytochrome b apoprotein. Instead of the wild type protein the mutants have a new low-molecular weight product which is probably a fragment of cytochrome b. The fact that the mutations revert suggests that they are nonsense mutations in the structural gene of cytochrome b.

  8. Methoxyflurane acts at the substrate binding site of cytochrome P450 LM2 to induce a dependence on cytochrome b5.

    PubMed

    Lipka, J J; Waskell, L A

    1989-01-01

    Rabbit cytochrome P450 isozyme 2 requires cytochrome b5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541-2546]. To determine whether the requirement for cytochrome b5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b5, we found that methoxyflurane did not create a cytochrome b5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b5 dependence of the mixed function oxidase cytochrome P450 LM2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM2 and suggest the focus of future studies of this question.

  9. A hypothetical complex between crystalline flavocytochrome b2 and cytochrome c.

    PubMed

    Tegoni, M; White, S A; Roussel, A; Mathews, F S; Cambillau, C

    1993-08-01

    Flavocytochrome b2 and cytochrome c are physiological electron transfer partners in yeast mitochondria. The formation of a stable complex between them has been demonstrated both in solution and in the crystalline state. On the basis of the three-dimensional structures, using molecular modeling and energy minimization, we have generated a hypothetical model for the interaction of these redox partners in the crystal lattice. General criteria such as good charge and surface complementarity, plausible orientation, and separation distance of the prosthetic groups, as well as more specific criteria such as the stoichiometry determined in the crystal, and the involvement of both domains and of more than one subunit of flavocytochrome b2 led us to discriminate between several possible interaction sites. In the hypothetical model we present, four cytochrome c molecules interact with a tetramer of flavocytochrome b2. The b2 and c hemes are coplanar, with an edge-to-edge distance of 14 A. The contact surface area is ca. 800 A2. Several electrostatic interactions involving the flavin and the heme domains of flavocytochrome b2 stabilize the binding of cytochrome c.

  10. Identification of fraud (with pig stuffs) in chicken-processed meat through information of mitochondrial cytochrome b.

    PubMed

    Yacoub, Haitham A; Sadek, Mahmoud A

    2017-11-01

    This study was conducted to find out the fraud in chicken-processed meat ingredients to protect consumers from commercial adulteration and authentication through a reliable way: direct amplification of conserved segment of cytochrome b gene of mitochondrial DNA, in addition, using species-specific primer assay for a certain cytochrome b. The results reported that chicken-processed meats were identified as a chicken meat based on amplification of conserved cytochrome b gene of mtDNA, while different fragments sizes were produced after the application of species-specific primer as follows: 227, 157, 274, 331, 389 and 439 bp for raw meat of chicken, goat, cattle, sheep, pig and horse, respectively. The results revealed that all chicken meat products are produced with 227 bp in size. While, an adulteration with pork stuffs was observed in some of the chicken meat products using a species-specific primer of cytochrome b gene, namely, chicken luncheon and chicken burger. This study represents a reliable technique that could be used to provide a promising solution for identifying the commercial adulteration and substitutions in processed meat in retail markets.

  11. Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c.

    PubMed

    Samhan-Arias, Alejandro K; Fortalezas, Sofia; Cordas, Cristina M; Moura, Isabel; Moura, José J G; Gutierrez-Merino, Carlos

    2018-05-01

    In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b 5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b 5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b 5 reductase was measured. Complex formation between both proteins suggests that cytochrome b 5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b 5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  12. 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

  13. Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

    PubMed

    Papa, S; Lorusso, M; Izzo, G; Capuano, F

    1981-02-15

    1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi

  14. Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

    PubMed

    Amino, Hisako; Osanai, Arihiro; Miyadera, Hiroko; Shinjyo, Noriko; Tomitsuka, Eriko; Taka, Hikari; Mineki, Reiko; Murayama, Kimie; Takamiya, Shinzaburo; Aoki, Takashi; Miyoshi, Hideto; Sakamoto, Kimitoshi; Kojima, Somei; Kita, Kiyoshi

    2003-05-01

    We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

  15. The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5.

    PubMed

    Canova-Davis, E; Waskell, L

    1984-02-25

    Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.

  16. 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.

  17. Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center.

    PubMed

    Gray, K A; Grooms, M; Myllykallio, H; Moomaw, C; Slaughter, C; Daldal, F

    1994-03-15

    The facultative phototrophic bacterium Rhodobacter capsulatus is capable of growth in a wide range of environmental conditions using a highly branched electron-transfer chain. During respiratory growth of this organism reducing equivalents are conveyed to oxygen via two terminal oxidases, previously called "cyt b410" (cytochrome c oxidase) and "cyt b260" (quinol oxidase). The cytochrome c oxidase was purified to homogeneity from a semiaerobically grown R. capsulatus strain. The purified enzyme consumes oxygen at a rate of 600 s-1, oxidizes reduced equine cyt c and R. capsulatus cyt c2, and has high sensitivity to cyanide. The complex is composed of three major polypeptides of apparent molecular masses 45, 32, and 28 kDa on SDS-PAGE. The 32- and 28-kDa proteins also stain with tetramethylbenzidine, indicating that they are c-type cytochromes. Partial amino acid sequences obtained from each of the subunits reveal significant homology to the fixN, fixO, and fixP gene products of Bradyrhizobium japonicum and Rhizobium meliloti. The reduced enzyme has an optical absorption spectrum with distinct features near 550 and 560 nm and an asymmetric Soret band centered at 418 nm, indicating the presence of both c- and b-type cytochromes. Two electrochemically distinct cyt c are apparent, with redox midpoint potentials (Em7) of 265 and 320 mV, while the low-spin cyt b has an Em7 value of 385 mV. The enzyme binds carbon monoxide, and the CO difference spectrum indicates that CO binds to a high-spin cyt b. Pyridine hemochrome and HPLC analyses suggest that the complex contains 1 mol of heme C to 1 mol of protoheme and that neither heme O nor heme A is present.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Structure-Function of the Cytochrome b 6f Complex of Oxygenic Photosynthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cramer, W. A.; Yamashita, E.; Baniulis, D.

    2014-03-20

    Structure–function of the major integral membrane cytochrome b 6f complex that functions in cyanobacteria, algae, and green plants to transfer electrons between the two reaction center complexes in the electron transport chain of oxygenic photosynthesis is discussed in the context of recently obtained crystal structures of the complex and soluble domains of cytochrome f and the Rieske iron–sulfur protein. The energy-transducing function of the complex, generation of the proton trans-membrane electrochemical potential gradient, centers on the oxidation/reduction pathways of the plastoquinol/plastoquinone (QH 2/Q), the proton donor/acceptor within the complex. These redox reactions are carried out by five redox prosthetic groupsmore » embedded in each monomer, the high potential two iron–two sulfur cluster and the heme of cytochrome f on the electropositive side (p) of the complex, two noncovalently bound b-type hemes that cross the complex and the membrane, and a covalently bound c-type heme (c n) on the electronegative side (n). These five redox-active groups are organized in high- (cyt f/[2Fe–2S] and low-potential (hemes b p, b n, c n) electron transport pathways that oxidize and reduce the quinol and quinone on the p- and n-sides in a Q-cycle-type mechanism, while translocating as many as 2 H + to the p-side aqueous side for every electron transferred through the high potential chain to the photosystem I reaction center. The presence of heme c n and the connection of the n-side of the membrane and b 6f complex to the cyclic electron transport chain indicate that the Q cycle in the oxygenic photosynthetic electron transport chain differs from those connected to the bc 1 complex in the mitochondrial respiratory chain and the chain in photosynthetic bacteria. Inferences from the structure and C2 symmetry of the complex for the pathway of QH 2/Q transfer within the complex, problems posed by the presence of lipid in the inter-monomer cavity, and the narrow

  19. Photooxidation of the cytochrome b-559 in the presence of various substituted 2-anilinothiophenes and of some other compounds, in Chlamydomonas reinhardtii.

    PubMed

    Maroc, J; Garnier, J

    1979-11-08

    Five substituted 2-anilinothiophenes and two substituted carbonylcyanide-phenylhydrazones were comparatively studied with respect to their capacities for inducing photooxidation of the cytochrome b-559 in chloroplast fragments and in whole cells of Chlamydomonas reinhardtii (wild type and P-700-lacking mutant Fl 5). In addition, some other compounds: antimycin A, picric acid, tetraphenylboron and NH4Cl were also tested. Cytochrome b-559 photooxidations were clearly observed in the presence of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), 2-(3,4,5-trichloro)anilino-3,5-dinitrothiophene (ANT 2s), 2-(4-chloro)anilino-3,5-dinitrothiophene and, with greater amplitudes, in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone and carbonylcyanide-m-chlorophenylhydrazone, both in whole cells and in chloroplast fragments. Picric acid, antimycin A and tetraphenylboron were also able to induce cytochrome b-559 photooxidation in chloroplast fragments, but not in whole cells. In the wild type, the highest photoinduced redox changes were 1.1 (carbonylcyanide-p-trifluoromethoxyphenylhydrazone, carbonylcyanide-m-chlorophenyl-hydrazone) and 0.6 (ANT 2p, ANT 2s) mumol of oxidized cytochrome b-559/1 mmol of chlorophyll, corresponding to 40% and 23% of the redox changes which could be induced chemically. All these cytochrome b-559 photooxidations, the greater part of which was inhibited by 3-(3,4-dichloropheny)-1,1-dimethylurea and occurred in the mutant Fl 5, appeared to be mainly Photosystem II-dependent reactions. But 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive Photosystem I-dependent photooxidations of cytochrome b-559 occurred also in the wild type. On the other hand, 2-(4-dimethylamine)-anilino-3,5-dinitrothiophene, 2-N-methyl-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene and NH4Cl did not induce any cytochrome b-559 photooxidation. These results were discussed taking in consideration the nature of the molecular substitutions

  20. Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

    NASA Astrophysics Data System (ADS)

    Novianty, E.; Kartikasari, L. R.; Lee, J. H.; Cahyadi, M.

    2017-04-01

    Meat based food products have a big opportunity to mix and adulterated with other meats. Muslim communities are prohibited to consume pork-containing product or other pig derivatives in food. Therefore, the high sensitivity, fast, cheap and accurate approach is needed to detect pig contamination in raw meat and meat-processed product such as meatball. The aim of this study was to identify pork contamination in meatball using genetic marker of mitochondrial DNA cytochrome b gene by duplex-PCR. Samples were prepared and designed by following the proportions 0, 1, 5, 10, 25% of pork in meatballs, respectively. The DNA genome was extracted from meatballs and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA cytochrome b gene. The results showed that the DNA genome was successfully isolated from pork, beef, and contaminated meatballs. Furthermore, 2% agarose gels was able to visualize of duplex-PCR to identify pork contamination in meatballs up to very small proportion (1%). It can be concluded that duplex-PCR of mt-DNA cytochrome b gene was very sensitive to identify pork contamination in meatball with the presence of specific 398 bp DNA band.

  1. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    PubMed Central

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-01-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative. Images PMID:3186722

  2. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    PubMed

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-11-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative.

  3. Polymorphisms of cytochrome b gene in Leishmania parasites and their relation to types of cutaneous leishmaniasis lesions in Pakistan.

    PubMed

    Myint, Chomar Kaung; Asato, Yutaka; Yamamoto, Yu-ichi; Kato, Hirotomo; Bhutto, Abdul M; Soomro, Farooq R; Memon, Muhamad Z; Matsumoto, Jun; Marco, Jorge D; Oshiro, Minoru; Katakura, Ken; Hashiguchi, Yoshihisa; Uezato, Hiroshi

    2008-02-01

    The exact species and/or strains of Leishmania parasites involved strongly influence the clinical and epidemiological features of leishmaniasis, and current knowledge of those influences and relationships is inadequate. We report that cytochrome b (cyt b) gene sequencing identified causal Leishmania parasites of 69 cutaneous leishmaniasis cases in Pakistan over a 3-year period. Of 21 cases in highland areas (Quetta city, Balochistan province), 16 (76.2%) were identified as Leishmania (L.) tropica and five (23.8%) as Leishmania (L.) major. Of 48 cases from lowland areas, cities/villages in Indus valley in Sindh and Balochistan provinces, 47 (97.9%) were identified as L. (L.) major and one (2.1%) as L. (L.) tropica. Statistical analysis (Fisher's exact test) revealed a significant difference (P < 0.0001) in the distribution of the two species by altitude; L. (L.) major is predominant in lowland and L. (L.) tropica at highland areas. The present result enriched our earlier finding, based on the first year's cultured parasite data, that only L. (L.) tropica was found in highland areas and only L. (L.) major in lowland areas. Among Leishmania samples analyzed, three types of cyt b polymorphism of L. (L.) major were found, including 45 (86.5%) cases of type I, six (11.5%) of type II and one (2%) of type III. We report for the first time on the presence of polymorphisms in L. (L.) major (types I, II and III) based on species identification using cyt b gene sequencing from clinical samples. Moreover, we found no correlation between clinical presentation (wet-, dry- and/or mixed-types of cutaneous lesions) and causal Leishmania parasites.

  4. RFLP and sequence analysis of the cytochrome b gene of selected animals and man: methodology and forensic application.

    PubMed

    Zehner, R; Zimmermann, S; Mebs, D

    1998-01-01

    To identify common animal species by analysis of the cytochrome b gene a method has been developed to obtain PCR products of a large domain of the cytochrome b gene (981 bp out of 1140 bp) in humans, selected mammals and birds using the same specifically designed primers. Species-specific RFLP patterns are generated by co-restriction with the restriction endonucleases ALU I and NCO I. The RFLP patterns obtained are conclusive even in mixtures of two or more species. The results were confirmed by sequence analysis which in addition explained intraspecies variations in the RFLP patterns. The method has been applied to forensic casework studies where the origin of roasted meat, stomach contents and a bone sample has been successfully identified.

  5. OnpA, an Unusual Flavin-Dependent Monooxygenase Containing a Cytochrome b5 Domain

    PubMed Central

    Xiao, Yi; Liu, Ting-Ting; Dai, Hui; Zhang, Jun-Jie; Liu, Hong; Tang, Huiru; Leak, David J.

    2012-01-01

    ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b5 domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b5 domain were associated with binding of the heme, and the cytochrome b5 domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b5 domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b5 and flavin-dependent monooxygenases. PMID:22267507

  6. Contribution of Electrostatics to the Kinetics of Electron Transfer from NADH-Cytochrome b5 Reductase to Fe(III)-Cytochrome b5.

    PubMed

    Kollipara, Sireesha; Tatireddy, Shivakishore; Pathirathne, Thusitha; Rathnayake, Lasantha K; Northrup, Scott H

    2016-08-25

    Brownian dynamics (BD) simulations provide here a theoretical atomic-level treatment of the reduction of human ferric cytochrome b5 (cyt b5) by NADH-cytochrome b5 reductaste (cyt b5r) and several of its mutants. BD is used to calculate the second-order rate constant of electron transfer (ET) between the proteins for direct correlation with experiments. Interestingly, the inclusion of electrostatic forces dramatically increases the reaction rate of the native proteins despite the overall negative charge of both proteins. The role played by electrostatic charge distribution in stabilizing the ET complexes and the role of mutations of several amino acid residues in stabilizing or destabilizing the complexes are analyzed. The complex with the shortest ET reaction distance (d = 6.58 Å) from rigid body BD is further subjected to 1 ns of molecular dynamics (MD) in a periodic box of TIP3P water to produce a more stable complex allowed by flexibility and with a shorter average reaction distance d = 6.02 Å. We predict a docking model in which the following ion-ion interactions are dominant (cyt b5r/cyt b5): Lys162-Heme O1D/Lys163-Asp64/Arg91-Heme O1A/Lys125-Asp70.

  7. Comparative c-type cytochrome expression analysis in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C grown with soluble and insoluble oxidised metal electron acceptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nissen, Silke; Liu, Xiaoxin; Chourey, Karuna

    2012-01-01

    The genomes of Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C encode 40 and 69 putative c-type cytochrome genes, respectively. Deletion mutant and biochemical studies have assigned specific functions to a few c-type cytochromes involved in electron transfer to oxidised metals in Shewanella oneidensis strain MR-1. Although promising, the genetic approach is limited to gene deletions that produce a distinct phenotype, and organism for which a genetic system is available. To more comprehensively investigate and compare c-type cytochrome expression in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C, proteomic measurements were used to characterise lysates of cells grownmore » with soluble Fe(III) (as ferric citrate) and insoluble Mn(IV) (as MnO2) as electron acceptors. Strain MR-1 expressed 19 and 20, and strain 2CP-C expressed 27 and 25 c-type cytochromes when grown with Fe(III) and Mn(IV), respectively. The majority of c-type cytochromes (77% for strain MR-1 and 63% for strain 2CP-C) were expressed under both growth conditions; however, the analysis also revealed unique c-type cytochromes that were specifically expressed in cells grown with soluble Fe(III) or insoluble Mn(IV). Proteomic characterisation proved to be a promising approach for determining the c-type cytochrome complement expressed under different growth conditions, and will help elucidating the specific functions of more c-type cytochromes that are the basis for Shewanella and Anaeromyxobacter respiratory versatility.« less

  8. Haloarcula marismortui cytochrome b-561 is encoded by the narC gene in the dissimilatory nitrate reductase operon.

    PubMed

    Yoshimatsu, Katsuhiko; Araya, Osamu; Fujiwara, Taketomo

    2007-01-01

    The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was -27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.

  9. Proton transfer in the K-channel analog of B-type Cytochrome c oxidase from Thermus thermophilus.

    PubMed

    Woelke, Anna Lena; Wagner, Anke; Galstyan, Gegham; Meyer, Tim; Knapp, Ernst-Walter

    2014-11-04

    A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers.

  10. Proton Transfer in the K-Channel Analog of B-Type Cytochrome c Oxidase from Thermus thermophilus

    PubMed Central

    Woelke, Anna Lena; Wagner, Anke; Galstyan, Gegham; Meyer, Tim; Knapp, Ernst-Walter

    2014-01-01

    A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers. PMID:25418102

  11. Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

    USGS Publications Warehouse

    Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

    1997-01-01

    The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

  12. Application of cytochrome b DNA sequences for the authentication of endangered snake species.

    PubMed

    Wong, Ka-Lok; Wang, Jun; But, Paul Pui-Hay; Shaw, Pang-Chui

    2004-01-06

    In order to enforce the conservation program and curbing the illegal trading and consumption of endangered snake species, the value of cytochrome b sequence in the authentication of snake species was evaluated. As an illustration, DNA was extracted, selected cytochrome b DNA sequences amplified and sequenced from six snakes commonly consumed in Hong Kong. Cataloging with sequences available in public, a cytochrome b database containing 90 species of snakes was constructed. In this database, sequence homology between snakes ranged from 70.68 to 95.11%. On the other hand, intraspecific variation of three tested snakes was 0-0.98%. Using the database, we were able to determine the identity of six meat samples confiscated by the Agriculture, Fisheries and Conservation Department, HKSAR.

  13. Evolution of cytochrome bc complexes: from membrane-anchored dehydrogenases of ancient bacteria to triggers of apoptosis in vertebrates

    PubMed Central

    Dibrova, Daria V.; Cherepanov, Dmitry A.; Galperin, Michael Y.; Skulachev, Vladimir P.; Mulkidjanian, Armen Y.

    2013-01-01

    This review traces the evolution of the cytochrome bc complexes from their early spread among prokaryotic lineages and up to the mitochondrial cytochrome bc1 complex (complex III) and its role in apoptosis. The results of phylogenomic analysis suggest that the bacterial cytochrome b6f-type complexes with short cytochromes b were the ancient form that preceded in evolution the cytochrome bc1-type complexes with long cytochromes b. The common ancestor of the b6f-type and the bc1-type complexes probably resembled the b6f-type complexes found in Heliobacteriaceae and in some Planctomycetes. Lateral transfers of cytochrome bc operons could account for the several instances of acquisition of different types of bacterial cytochrome bc complexes by archaea. The gradual oxygenation of the atmosphere could be the key evolutionary factor that has driven further divergence and spread of the cytochrome bc complexes. On one hand, oxygen could be used as a very efficient terminal electron acceptor. On the other hand, auto-oxidation of the components of the bc complex results in the generation of reactive oxygen species (ROS), which necessitated diverse adaptations of the b6f-type and bc1-type complexes, as well as other, functionally coupled proteins. A detailed scenario of the gradual involvement of the cardiolipin-containing mitochondrial cytochrome bc1 complex into the intrinsic apoptotic pathway is proposed, where the functioning of the complex as an apoptotic trigger is viewed as a way to accelerate the elimination of the cells with irreparably damaged, ROS-producing mitochondria. PMID:23871937

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

    PubMed

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

    2010-09-24

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

  15. Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

    PubMed

    Gray, K A; Dutton, P L; Daldal, F

    1994-01-25

    Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

  16. Structural insights into electron transfer in caa3-type cytochrome oxidase

    PubMed Central

    Lyons, Joseph A.; Aragão, David; Slattery, Orla; Pisliakov, Andrei V.; Soulimane, Tewfik; Caffrey, Martin

    2012-01-01

    Summary Paragraph Cytochrome c oxidase is a member of the heme copper oxidase superfamily (HCO)1. HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme’s function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c2–4. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented. PMID:22763450

  17. New data on epizootiology and genetics of piroplasms based on sequences of small ribosomal subunit and cytochrome b genes.

    PubMed

    Criado, A; Martinez, J; Buling, A; Barba, J C; Merino, S; Jefferies, R; Irwin, P J

    2006-12-20

    As a continuation of our studies on molecular epizootiology of piroplasmosis in Spain and other countries, we present in this contribution the finding of new hosts for some piroplasms, as well as information on their 18S rRNA gene sequences. Genetic data were complemented with sequences of apocytochrome b gene (whenever possible). The following conclusions were drawn from these molecular studies: Theileria annulata is capable of infecting dogs, since it was diagnosed in a symptomatic animal. According to cytochrome b sequences, isolates from cows and dog present slight differences. The same isolates showed, however, identical sequence in the 18S rRNA gene. This exemplifies well the usefulness of the mitochondrial gene for examining infra-specific variation. Babesia bovis is an occasional parasite of equines, since it was detected in two symptomatic horses. We found evidence of genetic polymorphism occurring in the 18S rRNA gene of Spanish T. equi-like and B. ovis isolates. B. bennetti from Spanish seagull is loosely related to B. ovis, and might represent a genetically distinct branch of babesids. A partial sequence of a cytochrome b pseudogene was obtained for the first time in Babesia canis rossi from South Africa. The pseudogene is distantly related to B. bigemina cytochrome b gene. These new findings confirm the ability of some piroplasms to infect multiple hosts, as well as the existence of a relatively wide genetic polymorphisms with respect to the cytochrome b gene. On the other hand, the existence of mtDNA-like pseudogenes of possible nuclear location in piroplasms is interesting due to their possible impact on molecular phylogeny studies.

  18. Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus[C][W

    PubMed Central

    Rexroth, Sascha; Rexroth, Dorothea; Veit, Sebastian; Plohnke, Nicole; Cormann, Kai U.; Nowaczyk, Marc M.; Rögner, Matthias

    2014-01-01

    The cyanobacterial cytochrome b6f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b6f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700+ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b6f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b6f complexes with different functions that might be correlated with supercomplex formation. PMID:25139006

  19. Overlapping and lineage-specific roles for the type-B response regulators of monocots and dicots.

    PubMed

    Kim, Hyo Jung; Kieber, Joseph J; Schaller, G Eric

    2012-09-01

    Cytokinins are plant hormones with profound roles in growth and development. Cytokinin signaling is mediated through a 'two-component' signaling system composed of histidine kinases, histidine-containing phosphotransfer proteins, and response regulators. Phylogenetic analysis of two-component signaling elements from the monocot rice and the dicot Arabidopsis reveals lineage-specific expansions of the type-B response regulators, transcription factors that act as positive regulators for the cytokinin signal. We recently reported in Plant Physiology on a functional analysis of rice type-B response regulators. A type-B response regulator from a subfamily comprised of both monocot and dicot type-B response regulators complemented an Arabidopsis type-B response regulator mutant, but a type-B response regulator from a monocot-specific subfamily generally did not. Here, we extend this analysis to demonstrate that the promoter of an Arabidopsis cytokinin primary response gene is induced by type-B response regulators from a shared subfamily, but not by one from a lineage-specific subfamily. These results support a model in which the type-B response regulators of monocots and dicots share conserved roles in the cytokinin signaling pathway but have also diverged to take on lineage-specific roles.

  20. Redox active molecules cytochrome c and vitamin C enhance heme-enzyme peroxidations by serving as non-specific agents for redox relay

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gade, Sudeep Kumar; Bhattacharya, Subarna; Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer At low concentrations, cytochrome c/vitamin C do not catalyze peroxidations. Black-Right-Pointing-Pointer But low levels of cytochrome c/vitamin C enhance diverse heme peroxidase activities. Black-Right-Pointing-Pointer Enhancement positively correlates to the concentration of peroxide in reaction. Black-Right-Pointing-Pointer Reducible additives serve as non-specific agents for redox relay in the system. Black-Right-Pointing-Pointer Insight into electron transfer processes in routine and oxidative-stress states. -- Abstract: We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome cmore » and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding - (1) the promiscuous role of cytochrome b{sub 5} in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.« less

  1. Evidence from cytochrome b sequences and allozymes for a new species of alcid: The long-billed murrelet (Brachyramphus perdix)

    USGS Publications Warehouse

    Friesen, Vicki L.; Piatt, John F.; Baker, Allan J.

    1996-01-01

    Marbled Murrelets (Brachyramphus marmoratus) are coastal seabirds that breed predominantly in old-growth forest throughout the North Pacific. Presently they are classified into two phenotypically distinct subspecies: one in North America (B. m. marmoratus) and one in Asia (B. m. perdix). The Asian form was classified as a separate species in 1811, but was lumped with B. marmoratus during the 20th century. Populations of both types are considered threatened or endangered and information about the extent of genetic differentiation among birds from different sites is required for their conservation. We compared variation in 1,045 base pairs of the mitochondrial cytochrome b gene and 39 allozyme loci among Marbled Murrelets and the closely related Kittlitz's Murrelets (B. brevirostris) from throughout the North Pacific. All analyses indicted that North American and Asian Marbled Murrelets are genetically distinct: cytochrome b sequences were highly divergent, fixed allele differences occurred at two allozyme loci, and estimated gene flow was essentially zero. Phylogenetic analyses of cytochrome b sequences and allozymes both provided strong support for a monophyletic relationship among North American Marbled Murrelets and Kittlitz's Murrelets, with Long-billed Murrelets forming the basal lineage. Long-billed and North American Marbled Murrelets clearly represent distinct species by any definition, and must be managed independently. Significant genetic differentiation also was found among both Marbled and Kittlitz's Murrelets from different sites within North America.

  2. Water exit pathways and proton pumping mechanism in B-type cytochrome c oxidase from molecular dynamics simulations.

    PubMed

    Yang, Longhua; Skjevik, Åge A; Han Du, Wen-Ge; Noodleman, Louis; Walker, Ross C; Götz, Andreas W

    2016-09-01

    Cytochrome c oxidase (CcO) is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. While proton uptake channels as well as water exit channels have been identified for A-type CcOs, the means by which water and protons exit B-type CcOs remain unclear. In this work, we investigate potential mechanisms for proton transport above the dinuclear center (DNC) in ba3-type CcO of Thermus thermophilus. Using long-time scale, all-atom molecular dynamics (MD) simulations for several relevant protonation states, we identify a potential mechanism for proton transport that involves propionate A of the active site heme a3 and residues Asp372, His376 and Glu126(II), with residue His376 acting as the proton-loading site. The proposed proton transport process involves a rotation of residue His376 and is in line with experimental findings. We also demonstrate how the strength of the salt bridge between residues Arg225 and Asp287 depends on the protonation state and that this salt bridge is unlikely to act as a simple electrostatic gate that prevents proton backflow. We identify two water exit pathways that connect the water pool above the DNC to the outer P-side of the membrane, which can potentially also act as proton exit transport pathways. Importantly, these water exit pathways can be blocked by narrowing the entrance channel between residues Gln151(II) and Arg449/Arg450 or by obstructing the entrance through a conformational change of residue Tyr136, respectively, both of which seem to be affected by protonation of residue His376. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Absence of sugars in electrophoretically purified cytochrome b5 demonstrated by combined gas chromatography-mass spectrometry

    PubMed Central

    1981-01-01

    The problem of determining small but significant amounts of carbohydrates, in purified proteins, has been studied using the membrane protein, cytochrome b5. A newly developed method that involves direct gas chromatography-mass spectrometry of sugars obtained by hydrolysis of proteins purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) allows the identification and determination of small amounts of carbohydrates (e.g., 20 micrograms of glycoprotein containing a minimum of 0.1% monosaccharide), even in the presence of relatively high amounts of impurities. Application of this method to cytochrome b5 fragments obtained by tryptic digestion from rat liver microsomes and purified by combined gel filtration and ion exchange chromatography, followed by SDS PAGE, has consistently yielded values below 0.07 mol of the individual sugars and aminosugars per mole cytochrome b5. It is concluded that cytochrome b5, at least its trypsin-released major amino- terminal fragment, is not constitutively glycosylated. PMID:7251667

  4. Autoantigen-specific B-cell depletion overcomes failed immune tolerance in type 1 diabetes.

    PubMed

    Henry, Rachel A; Kendall, Peggy L; Thomas, James W

    2012-08-01

    Eliminating autoantigen-specific B cells is an attractive alternative to global B-cell depletion for autoimmune disease treatment. To identify the potential for targeting a key autoimmune B-cell specificity in type 1 diabetes, insulin-binding B cells were tracked within a polyclonal repertoire using heavy chain B-cell receptor (BCR) transgenic (VH125Tg) mice. Insulin-specific B cells are rare in the periphery of nonautoimmune VH125Tg/C57BL/6 mice and WT/NOD autoimmune mice, whereas they clearly populate 1% of mature B-cell subsets in VH125Tg/NOD mice. Autoantigen upregulates CD86 in anti-insulin B cells, suggesting they are competent to interact with T cells. Endogenous insulin occupies anti-insulin BCR beginning with antigen commitment in bone marrow parenchyma, as identified by a second anti-insulin monoclonal antibody. Administration of this monoclonal antibody selectively eliminates insulin-reactive B cells in vivo and prevents disease in WT/NOD mice. Unexpectedly, developing B cells are less amenable to depletion, despite increased BCR sensitivity. These findings exemplify how a critical type 1 diabetes B-cell specificity escapes immune tolerance checkpoints. Disease liability is corrected by eliminating this B-cell specificity, providing proof of concept for a novel therapeutic approach for autoimmune disease.

  5. 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).

  6. Cytochrome bc1 complexes of microorganisms.

    PubMed Central

    Trumpower, B L

    1990-01-01

    The cytochrome bc1 complex is the most widely occurring electron transfer complex capable of energy transduction. Cytochrome bc1 complexes are found in the plasma membranes of phylogenetically diverse photosynthetic and respiring bacteria, and in the inner mitochondrial membrane of all eucaryotic cells. In all of these species the bc1 complex transfers electrons from a low-potential quinol to a higher-potential c-type cytochrome and links this electron transfer to proton translocation. Most bacteria also possess alternative pathways of quinol oxidation capable of circumventing the bc1 complex, but these pathways generally lack the energy-transducing, protontranslocating activity of the bc1 complex. All cytochrome bc1 complexes contain three electron transfer proteins which contain four redox prosthetic groups. These are cytochrome b, which contains two b heme groups that differ in their optical and thermodynamic properties; cytochrome c1, which contains a covalently bound c-type heme; and a 2Fe-2S iron-sulfur protein. The mechanism which links proton translocation to electron transfer through these proteins is the proton motive Q cycle, and this mechanism appears to be universal to all bc1 complexes. Experimentation is currently focused on understanding selected structure-function relationships prerequisite for these redox proteins to participate in the Q-cycle mechanism. The cytochrome bc1 complexes of mitochondria differ from those of bacteria, in that the former contain six to eight supernumerary polypeptides, in addition to the three redox proteins common to bacteria and mitochondria. These extra polypeptides are encoded in the nucleus and do not contain redox prosthetic groups. The functions of the supernumerary polypeptides of the mitochondrial bc1 complexes are generally not known and are being actively explored by genetically manipulating these proteins in Saccharomyces cerevisiae. Images PMID:2163487

  7. [Cytochrome b-559 photooxidation in the presence of carbonyl cyanide p-trifluorometh-oxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or p-benzoquinone in three non-photosynthetic mutants of Chlamydomonas reinhardti (author's transl)].

    PubMed

    Maroc, J; Garnier, J

    1975-04-14

    Studies of absorbance related to the cytochrome b-559 photooxidation induced by FCCP, with and without addition of 3-p-chlorophenyl-1, 1-dimethylurea (CMU), DBMIB or p-benzoquinone, in whole cells and in chloroplast fragments of Chlamydomonas reinhardti, were carried out. In addition to the wild type, three strains of non-photosynthetic mutants were used: Fl 5, which lacks P 700; Fl 9 and Fl 15, which are deficient in bound cytochrome c-553 and in cytochrome b-563. In the presence of FCCP, whole cells and chloroplast fragments of the four strains showed a System II-dependent photooxidation of cytochrome b-559. This photooxidation was inhibited by CMU but it occurred again in presence of FCCP, CMU and DBMIB. In chloroplast fragments, cytochrome b-559 photooxidation was also inhibited by an excess of FCCP; it was recovered, likewise, by addition of DBMIB. In whole cells, the highest measured redox changes were: 1 mu mol oxidized cytochrome b-559 per 1 mmol chlorophyll, corresponding approximately to about one seventh (wild type, Fl5) or one fifth (Fl 9, Fl 15) of the total amount of this cytochrome. Another kind of cytochrome b-559 photooxidation, CMU-insensitive, also occurred in the mutants Fl 9 and Fl 15 and in the wild type, but not in the mutant Fl 5. This latter kind of photooxidation was observed with chloroplast fragments in the presence of FCCP and CMU and also with whole cells in the presence of FCCP, CMU and p-benzoquinone. These reactions can be attributed to the Photosystem I; they do not require the intervention of the cytochrome c-553. A high-potential form of cytochrome b-559, hydroquinone-reducible, was involved in these two kinds of photooxidation. In addition, a lower potential form, reducible only by ascorbate, appeared to be able to interfere also. An interpretation is attempted, taking into consideration the various effects of FCCP and DBMIB, at different concentrations, on photosynthetic electron transport.

  8. Cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiology.

    PubMed

    Jennings, Brett L; Sahan-Firat, Seyhan; Estes, Anne M; Das, Kanak; Farjana, Nasreen; Fang, Xiao R; Gonzalez, Frank J; Malik, Kafait U

    2010-10-01

    Hypertension is the leading cause of cardiovascular diseases, and angiotensin II is one of the major components of the mechanisms that contribute to the development of hypertension. However, the precise mechanisms for the development of hypertension are unknown. Our recent study showing that angiotensin II-induced vascular smooth muscle cell growth depends on cytochrome P450 1B1 led us to investigate its contribution to hypertension caused by this peptide. Angiotensin II was infused via miniosmotic pump into rats (150 ng/kg per minute) or mice (1000 μg/kg per day) for 13 days resulting in increased blood pressure, increased cardiac and vascular hypertrophy, increased vascular reactivity to vasoconstrictor agents, increased vascular reactive oxygen species production, and endothelial dysfunction in both species. The increase in blood pressure and associated pathophysiological changes were minimized by the cytochrome P450 1B1 inhibitor 2,3',4,5'-tetramethoxystilbene in both species and was markedly reduced in Cyp1b1(-/-) mice. These data suggest that cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiological changes. Moreover, 2,3',4,5'-tetramethoxystilbene, which prevents both cytochrome P450 1B1-dependent and -independent components of angiotensin II-induced hypertension and inhibits associated pathophysiological changes could be clinically useful in the treatment of hypertension and associated cardiovascular and inflammatory diseases.

  9. CYTOCHROME P450 1B1 CONTRIBUTES TO ANGIOTENSIN II-INDUCED HYPERTENSION AND ASSOCIATED PATHOPHYSIOLOGY

    PubMed Central

    Jennings, Brett L.; Sahan-Firat, Seyhan; Estes, Anne M.; Das, Kanak; Farjana, Nasreen; Fang, Xiao R.; Gonzalez, Frank J.; Malik, Kafait U.

    2010-01-01

    Hypertension is the leading cause of cardiovascular diseases, and angiotensin II is one of the major components of the mechanisms that contribute to the development of hypertension. However, the precise mechanisms for the development of hypertension are unknown. Our recent study that angiotensin II-induced vascular smooth muscle cell growth is dependent on cytochrome P450 1B1 led us to investigate its contribution to hypertension caused by this peptide. Angiotensin II was infused via miniosmotic pump into rats (150 ng/kg/min) or mice (1000 μg/kg/day) for 13 days resulting in increased blood pressure, increased cardiac and vascular hypertrophy, increased vascular reactivity to vasoconstrictor agents, increased reactive oxygen species production, and endothelial dysfunction in both species. The increase in blood pressure and associated pathophysiological changes were minimized by the cytochrome P450 1B1 inhibitor, 2,3′,4,5′-tetramethoxystilbene in both species and was markedly reduced in Cyp1b1-/- mice. These data suggest that cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiological changes. Moreover, 2,3′,4,5′-tetramethoxystilbene which prevents both cytochrome P450 1B1-dependent and independent components of angiotensin II-induced hypertension and inhibits associated pathophysiological changes could be clinically useful in the treatment of hypertension and associated cardiovascular and inflammatory diseases. PMID:20805442

  10. Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation

    PubMed Central

    Yun, Jiae; Malvankar, Nikhil S; Ueki, Toshiyuki; Lovley, Derek R

    2016-01-01

    Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface. PMID:26140532

  11. Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.

    PubMed

    Yun, Jiae; Malvankar, Nikhil S; Ueki, Toshiyuki; Lovley, Derek R

    2016-02-01

    Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

  12. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12.

    PubMed

    Morishima, Nobuhiro; Nakanishi, Keiko; Takenouchi, Hiromi; Shibata, Takehiko; Yasuhiko, Yukuto

    2002-09-13

    Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98-103), yet the functional consequences of caspase-12 activation have been unclear. We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts. The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor. Although cytochrome c released from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress. These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c. To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation. The binding protein protects procaspase-12 from processing in vitro. Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner.

  13. 77 FR 67678 - Content Specifications and Shielding Evaluations for Type B Transportation Packages

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-13

    ...The U.S. Nuclear Regulatory Commission (NRC or the Commission) is issuing for public comment Draft Regulatory Issue Summary (RIS) 2012-XX, ``Content Specifications and Shielding Evaluations for Type B Transportation Packages.'' This RIS clarifies the NRC's use of staff guidance in NUREG-1609, ``Standard Review Plan for Transport Packages for Radioactive Material,'' for the review of content specifications and shielding evaluations included in the Certificates of Compliance (CoC) and safety analysis reports (SARs) for Type B transportation packages.

  14. 78 FR 26090 - Content Specifications and Shielding Evaluations for Type B Transportation Packages

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-03

    ...The U.S. Nuclear Regulatory Commission (NRC) is issuing Regulatory Issue Summary (RIS) 2013-04, ``Content Specifications and Shielding Evaluations for Type B Transportation Packages.'' This RIS clarifies the NRC's use of staff guidance in NUREG-1609, ``Standard Review Plan for Transport Packages for Radioactive Material,'' for the review of content specifications and shielding evaluations included in the Certificates of Compliance (CoC) and safety analysis reports (SARs) for Type B transportation packages. The RIS does not impose any additional regulatory requirements on NRC licensees.

  15. Effect of retinal impulse blockage on cytochrome oxidase-poor interpuffs in the macaque striate cortex: quantitative EM analysis of neurons.

    PubMed

    Wong-Riley, M T; Trusk, T C; Kaboord, W; Huang, Z

    1994-09-01

    One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase-rich puffs in its supragranular layers. Neurons in puffs have been classified as type A, B, and C in ascending order of cytochrome oxidase content, with type C cells being the most vulnerable to retinal impulse blockade. The present study aimed at analysing cytochrome oxidase-poor interpuffs with reference to their metabolic cell types and the effect of intraretinal tetrodotoxin treatment. The same three metabolic types were found in interpuffs, except that type B and C neurons were smaller and less cytochrome oxidase-reactive in interpuffs than in puffs. Type A neurons had small perikarya, low levels of cytochrome oxidase, and received exclusively symmetric axosomatic synapses. The largest neurons were pyramidal, type B cells with moderate cytochrome oxidase activity and were also contacted exclusively by symmetric axosomatic synapses. Type C cells medium-sized with a rich supply of large, darkly reactive mitochondria and possessed all the characteristics of GABAergic neurons. They were the only cell type that received both symmetric and asymmetric axosomatic synapses. Two weeks of monocular tetrodotoxin blockade in adult monkeys caused all three major cell types in deprived interpuffs to suffer a significant downward shift in the size and cytochrome oxidase reactivity of their mitochondria, but the effects were more severe in type B and C neurons. In nondeprived interpuffs, all three cell types gained both in size and absolute number of mitochondria, and type A cells also had an elevated level of cytochrome oxidase, indicating that they might be functioning at a competitive advantage over cells in deprived columns. However, type B and C neurons showed a net loss of darkly reactive mitochondria, indicating that these cells became less active. Thus, mature interpuff neurons remained vulnerable to retinal impulse blockade and the metabolic capacity of these cells remains tightly

  16. Cytochrome P460 Genes from the Methanotroph Methylococcus capsulatus Bath†

    PubMed Central

    Bergmann, David J.; Zahn, James A.; Hooper, Alan B.; DiSpirito, Alan A.

    1998-01-01

    P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231–244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879–5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457–460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium. PMID

  17. c-Type Cytochrome-Dependent Formation of U(IV) Nanoparticles by Shewanella oneidensis

    PubMed Central

    Marshall, Matthew J; Dohnalkova, Alice C; Kennedy, David W; Shi, Liang; Wang, Zheming; Boyanov, Maxim I; Lai, Barry; Kemner, Kenneth M; McLean, Jeffrey S; Reed, Samantha B; Culley, David E; Bailey, Vanessa L; Simonson, Cody J; Saffarini, Daad A; Romine, Margaret F; Zachara, John M

    2006-01-01

    Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI) and formation of extracelluar UO 2 nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO 2 nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO 2-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO 2 nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO 2 nanoparticles. In the environment, such association of UO 2 nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O 2 or transport in soils and sediments. PMID:16875436

  18. In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

    PubMed

    Pearson, Josh T; Siu, Sophia; Meininger, David P; Wienkers, Larry C; Rock, Dan A

    2010-03-30

    Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

  19. 10 CFR 33.14 - Requirements for the issuance of a Type B specific license of broad scope.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... of broad scope. 33.14 Section 33.14 Energy NUCLEAR REGULATORY COMMISSION SPECIFIC DOMESTIC LICENSES OF BROAD SCOPE FOR BYPRODUCT MATERIAL Specific Licenses of Broad Scope § 33.14 Requirements for the issuance of a Type B specific license of broad scope. An application for a Type B specific license of broad...

  20. 10 CFR 33.14 - Requirements for the issuance of a Type B specific license of broad scope.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... of broad scope. 33.14 Section 33.14 Energy NUCLEAR REGULATORY COMMISSION SPECIFIC DOMESTIC LICENSES OF BROAD SCOPE FOR BYPRODUCT MATERIAL Specific Licenses of Broad Scope § 33.14 Requirements for the issuance of a Type B specific license of broad scope. An application for a Type B specific license of broad...

  1. 10 CFR 33.14 - Requirements for the issuance of a Type B specific license of broad scope.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... of broad scope. 33.14 Section 33.14 Energy NUCLEAR REGULATORY COMMISSION SPECIFIC DOMESTIC LICENSES OF BROAD SCOPE FOR BYPRODUCT MATERIAL Specific Licenses of Broad Scope § 33.14 Requirements for the issuance of a Type B specific license of broad scope. An application for a Type B specific license of broad...

  2. 10 CFR 33.14 - Requirements for the issuance of a Type B specific license of broad scope.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... of broad scope. 33.14 Section 33.14 Energy NUCLEAR REGULATORY COMMISSION SPECIFIC DOMESTIC LICENSES OF BROAD SCOPE FOR BYPRODUCT MATERIAL Specific Licenses of Broad Scope § 33.14 Requirements for the issuance of a Type B specific license of broad scope. An application for a Type B specific license of broad...

  3. 10 CFR 33.14 - Requirements for the issuance of a Type B specific license of broad scope.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... of broad scope. 33.14 Section 33.14 Energy NUCLEAR REGULATORY COMMISSION SPECIFIC DOMESTIC LICENSES OF BROAD SCOPE FOR BYPRODUCT MATERIAL Specific Licenses of Broad Scope § 33.14 Requirements for the issuance of a Type B specific license of broad scope. An application for a Type B specific license of broad...

  4. Nitrite reduction in paracoccus halodenitrificans: Evidence for the role of a cd-type cytochrome in ammonia formation

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Cronin, S. E.

    1984-01-01

    Cell-free extracts prepared from Paracoccus halodenitrificans catalyzed the reduction of nitrate to ammonia in the presence of dithionite and methyl viologen. Enzyme activity was located in the soluble fraction and was associated with a cytochrome whose spectral properties resembled those of a cd-type cytochrome. Unlike the sissimilatory cd-cytochrome nitrate reductase associated with the membrane fraction of P. halodenitrificans, this soluble cd-cytochrome did not reduce nitrite to nitrous oxide.

  5. Supercapacitors based on c-type cytochromes using conductive nanostructured networks of living bacteria.

    PubMed

    Malvankar, Nikhil S; Mester, Tünde; Tuominen, Mark T; Lovley, Derek R

    2012-02-01

    Supercapacitors have attracted interest in energy storage because they have the potential to complement or replace batteries. Here, we report that c-type cytochromes, naturally immersed in a living, electrically conductive microbial biofilm, greatly enhance the device capacitance by over two orders of magnitude. We employ genetic engineering, protein unfolding and Nernstian modeling for in vivo demonstration of charge storage capacity of c-type cytochromes and perform electrochemical impedance spectroscopy, cyclic voltammetry and charge-discharge cycling to confirm the pseudocapacitive, redox nature of biofilm capacitance. The biofilms also show low self-discharge and good charge/discharge reversibility. The superior electrochemical performance of the biofilm is related to its high abundance of cytochromes, providing large electron storage capacity, its nanostructured network with metallic-like conductivity, and its porous architecture with hydrous nature, offering prospects for future low cost and environmentally sustainable energy storage devices. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Biogenesis of the yeast cytochrome bc1 complex.

    PubMed

    Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

    2009-01-01

    The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

  7. A role for cytochrome b5 in the in vivo disposition of anti-cancer and cytochrome P450 probe drugs in mice

    PubMed Central

    Henderson, Colin J.; McLaughlin, Lesley A.; Finn, Robert D.; Ronseaux, Sebastien; Kapelyukh, Yury; Wolf, C. Roland

    2014-01-01

    The role of microsomal cytochrome b5 (Cyb5) in defining the rate of drug metabolism and disposition has been intensely debated for several decades. Recently we described mouse models involving the hepatic or global deletion of Cyb5, demonstrating its central role in in vivo drug disposition. We have now used the cytochrome b5 complete null (BCN) model to determine the role of Cyb5 in the metabolism of ten pharmaceuticals metabolised by a range of cytochrome P450s, including five anti-cancer drugs, in vivo and in vitro. The extent to which metabolism was significantly affected by the absence of Cyb5 was substrate-dependent, with AUC increased (75-245%), and clearance decreased (35-72%), for phenacetin, metoprolol and chlorzoxazone. Tolbutamide disposition was not significantly altered by Cyb5 deletion, while for midazolam clearance was decreased by 66%. The absence of Cyb5 had no effect on gefitinib and paclitaxel disposition, while significant changes in the in vivo pharmacokinetics of cyclophosphamide were measured (Cmax and terminal half-life increased 55% and 40%, respectively), tamoxifen (AUClast and Cmax increased 370% and 233%, respectively) and anastrozole (AUC and terminal half-life increased 125% and 62%, respectively; clearance down 80%). These data from provide strong evidence that both hepatic and extra-hepatic Cyb5 levels are an important determinant of in vivo drug disposition catalysed by a range of cytochrome P450s, including currently-prescribed anti-cancer agents, and that individuality in Cyb5 expression could be a significant determinant in rates of drug disposition in man. PMID:24115751

  8. Taxonomic relationships among Phenacomys voles as inferred by cytochrome b

    USGS Publications Warehouse

    Bellinger, M.R.; Haig, S.M.; Forsman, E.D.; Mullins, T.D.

    2005-01-01

    Taxonomic relationships among red tree voles (Phenacomys longicaudus longicaudus, P. l. silvicola), the Sonoma tree vole (P. pomo), the white-footed vole (P. albipes), and the heather vole (P. intermedius) were examined using 664 base pairs of the mitochondrial cytochrome b gene. Results indicate specific differences among red tree voles, Sonoma tree voles, white-footed voles, and heather voles, but no clear difference between the 2 Oregon subspecies of red tree voles (P. l. longicaudus and P. l. silvicola). Our data further indicated a close relationship between tree voles and albipes, validating inclusion of albipes in the subgenus Arborimus. These 3 congeners shared a closer relationship to P. intermedius than to other arvicolids. A moderate association between porno and albipes was indicated by maximum parsimony and neighbor-joining phylogenetic analyses. Molecular clock estimates suggest a Pleistocene radiation of the Arborimus clade, which is concordant with pulses of diversification observed in other murid rodents. The generic rank of Arborimus is subject to interpretation of data.

  9. Cell type-specific translational repression of Cyclin B during meiosis in males.

    PubMed

    Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

    2015-10-01

    The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I. © 2015. Published by The Company of Biologists Ltd.

  10. Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase.

    PubMed

    Balodite, Elina; Strazdina, Inese; Galinina, Nina; McLean, Samantha; Rutkis, Reinis; Poole, Robert K; Kalnenieks, Uldis

    2014-09-01

    The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc1 complex via cytochrome c552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase. © 2014 The Authors.

  11. Carbon monoxide inhibits omega-oxidation of leukotriene B4 by human polymorphonuclear leukocytes: evidence that catabolism of leukotriene B4 is mediated by a cytochrome P-450 enzyme.

    PubMed

    Shak, S; Goldstein, I M

    1984-09-17

    Carbon monoxide significantly inhibits omega-oxidation of exogenous leukotriene B4 to 20-OH-leukotriene B4 and 20-COOH-leukotriene B4 by unstimulated polymorphonuclear leukocytes as well as omega-oxidation of leukotriene B4 that is generated when cells are stimulated with the calcium ionophore, A23187. Inhibition of omega-oxidation by carbon monoxide is concentration-dependent, completely reversible, and specific. Carbon monoxide does not affect synthesis of leukotriene B4 by stimulated polymorphonuclear leukocytes or other cell functions (i.e., degranulation, superoxide anion generation). These findings suggest that a cytochrome P-450 enzyme in human polymorphonuclear leukocytes is responsible for catabolizing leukotriene B4 by omega-oxidation.

  12. Development of multiplex PCR assay for authentication of Cornu Cervi Pantotrichum in traditional Chinese medicine based on cytochrome b and C oxidase subunit 1 genes.

    PubMed

    Gao, Lijun; Xia, Wei; Ai, Jinxia; Li, Mingcheng; Yuan, Guanxin; Niu, Jiamu; Fu, Guilian; Zhang, Lihua

    2016-07-01

    This study describes a method for discriminating the true Cervus antlers from its counterfeits using multiplex PCR. Bioinformatics were carried out to design the specific alleles primers for mitochondrial (mt) cytochrome b (Cyt b) and cytochrome C oxidase subunit 1 (Cox 1) genes. The mt DNA and genomic DNA were extracted from Cervi Cornu Pantotrichum through the modified alkaline and the salt-extracting method in addition to its counterfeits, respectively. Sufficient DNA templates were extracted from all samples used in two methods, and joint fragments of 354 bp and 543 bp that were specifically amplified from both of true Cervus antlers served as a standard control. The data revealed that the multiplex PCR-based assays using two primer sets can be used for forensic and quantitative identification of original Cervus deer products from counterfeit antlers in a single step.

  13. Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

    PubMed

    Jeřábek, Petr; Florián, Jan; Stiborová, Marie; Martínek, Václav

    2014-10-28

    Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

  14. Cytochrome b 5 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 b 5 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.

  15. Effects of Muscle-Specific Oxidative Stress on Cytochrome c Release and Oxidation-Reduction Potential Properties.

    PubMed

    Ke, Yiling; Mitacek, Rachel M; Abraham, Anupam; Mafi, Gretchen G; VanOverbeke, Deborah L; DeSilva, Udaya; Ramanathan, Ranjith

    2017-09-06

    Mitochondria play a significant role in beef color. However, the role of oxidative stress in cytochrome c release and mitochondrial degradation is not clear. The objective was to determine the effects of display time on cytochrome c content and oxidation-reduction potential (ORP) of beef longissimus lumborum (LL) and psoas major (PM) muscles. PM discolored by day 3 compared with LL. On day 0, mitochondrial content and mitochondrial oxygen consumption were greater in PM than LL. However, mitochondrial content and oxygen consumption were lower (P < 0.05) in PM than LL by day 7. Conversely, cytochrome c content in sarcoplasm was greater on days 3 and 7 for PM than LL. There were no significant differences in ORP for LL during display, but ORP increased for PM on day 3 when compared with day 0. The results suggest that muscle-specific oxidative stress can affect cytochrome c release and ORP changes.

  16. Maturation of the unusual single-cysteine (XXXCH) mitochondrial c-type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway

    PubMed Central

    Allen, James W. A.; Ginger, Michael L.; Ferguson, Stuart J.

    2004-01-01

    The c-type cytochromes are characterized by the covalent attachment of haem to the polypeptide via thioether bonds formed from haem vinyl groups and, normally, the thiols of two cysteines in a CXXCH motif. Intriguingly, the mitochondrial cytochromes c and c1 from two euglenids and the Trypanosomatidae contain only a single cysteine within the haem-binding motif (XXXCH). There are three known distinct pathways by which c-type cytochromes are matured post-translationally in different organisms. The absence of genes encoding any of these c-type cytochrome biogenesis machineries is established here by analysis of six trypanosomatid genomes, and correlates with the presence of single-cysteine cytochromes c and c1. In contrast, we have identified a comprehensive catalogue of proteins required for a typical mitochondrial oxidative phosphorylation apparatus. Neither spontaneous nor catalysed maturation of the single-cysteine Trypanosoma brucei cytochrome c occurred in Escherichia coli. However, a CXXCH variant was matured by the E. coli cytochrome c maturation machinery, confirming the proposed requirement of the latter for two cysteines in the haem-binding motif and indicating that T. brucei cytochrome c can accommodate a second cysteine in a CXXCH motif. The single-cysteine haem attachment conserved in cytochromes c and c1 of the trypanosomatids is suggested to be related to their cytochrome c maturation machinery, and the environment in the mitochondrial intermembrane space. Our genomic and biochemical studies provide very persuasive evidence that the trypanosomatid mitochondrial cytochromes c are matured by a novel biogenesis system. PMID:15500440

  17. The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.

    PubMed

    Gruenke, L D; Konopka, K; Cadieu, M; Waskell, L

    1995-10-20

    The complete stoichiometry of the metabolism of the cytochrome b5 (cyt b5)-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b5 for its metabolism. Cyt b5 invariably improved the efficiency of product formation. That is, in the presence of cyt b5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b5. Under some experimental conditions cyt b5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b5 depends on the net effect of the stimulatory and inhibitory effects of cyt b5. When total NADPH consumption is inhibited by cyt b5, the rapidly metabolized, highly coupled (approximately equal to 50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (approximately equal to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.

  18. [Comparative studies of serological typing and HLA-A, B antigen genotyping with PCR using sequence-specific primers].

    PubMed

    Wu, Da-lin; Ling, Han-xin; Tang, Hao

    2004-11-01

    To evaluate the accuracy of PCR with sequence-specific primers (PCR-SSP) for HLA-I genotyping and analyze the causes of the errors occurring in the genotyping. DNA samples and were obtained from 34 clinical patients, and serological typing with monoclonal antibody (mAb) and HLA-A and, B antigen genotyping with PCR-SSP were performed. HLA-A and, B alleles were successfully typed in 34 clinical samples by mAb and PCR-SSP. No false positive or false negative results were found, and the erroneous and missed diagnosis rates were obviously higher in serological detection, being 23.5% for HLA-A and 26.5% for HLA-B. Error or confusion was more likely to occur in the antigens of A2 and A68, A32 and A33, B5, B60 and B61. DNA typing for HLA-I class (A, B antigens) by PCR-SSP has high resolution, high specificity, and good reproducibility, which is more suitable for clinical application than serological typing. PCR-SSP may accurately detect the alleles that are easily missed or mistaken in serological typing.

  19. Phylogenetic analysis of different breeds of domestic chickens in selected area of Peninsular Malaysia inferred from partial cytochrome b gene information and RAPD markers.

    PubMed

    Yap, Fook Choy; Yan, Yap Jin; Loon, Kiung Teh; Zhen, Justina Lee Ning; Kamau, Nelly Warau; Kumaran, Jayaraj Vijaya

    2010-10-01

    The present investigation was carried out in an attempt to study the phylogenetic analysis of different breeds of domestic chickens in Peninsular Malaysia inferred from partial cytochrome b gene information and random amplified polymorphic DNA (RAPD) markers. Phylogenetic analysis using both neighbor-joining (NJ) and maximum parsimony (MP) methods produced three clusters that encompassed Type-I village chickens, the red jungle fowl subspecies and the Japanese Chunky broilers. The phylogenetic analysis also revealed that majority of the Malaysian commercial chickens were randomly assembled with the Type-II village chickens. In RAPD assay, phylogenetic analysis using neighbor-joining produced six clusters that were completely distinguished based on the locality of chickens. High levels of genetic variations were observed among the village chickens, the commercial broilers, and between the commercial broilers and layer chickens. In this study, it was found that Type-I village chickens could be distinguished from the commercial chickens and Type-II village chickens at the position of the 27th nucleotide of the 351 bp cytochrome b gene. This study also revealed that RAPD markers were unable to differentiate the type of chickens, but it showed the effectiveness of RAPD in evaluating the genetic variation and the genetic relationships between chicken lines and populations.

  20. 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

  1. NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

    PubMed Central

    Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

    2013-01-01

    This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

  2. Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities.

    PubMed

    Ferrer-Sevillano, Francisco; Fernández-Cañón, José M

    2007-03-01

    Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

  3. Novel phacB-Encoded Cytochrome P450 Monooxygenase from Aspergillus nidulans with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities▿

    PubMed Central

    Ferrer-Sevillano, Francisco; Fernández-Cañón, José M.

    2007-01-01

    Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (ΔphacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B). PMID:17189487

  4. A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activation.

    PubMed

    Meyer, Mark B; Benkusky, Nancy A; Kaufmann, Martin; Lee, Seong Min; Onal, Melda; Jones, Glenville; Pike, J Wesley

    2017-10-20

    The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D 3 to its hormonal form, 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1 , are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH) 2 D 3 -mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH) 2 D 3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1 We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH) 2 D 3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activation

    PubMed Central

    Meyer, Mark B.; Benkusky, Nancy A.; Kaufmann, Martin; Lee, Seong Min; Onal, Melda; Jones, Glenville; Pike, J. Wesley

    2017-01-01

    The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D3 to its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1, are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH)2D3-mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH)2D3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1. We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH)2D3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease. PMID:28808057

  6. Cytochrome oxidase assembly does not require catalytically active cytochrome C.

    PubMed

    Barrientos, Antoni; Pierre, Danielle; Lee, Johnson; Tzagoloff, Alexander

    2003-03-14

    Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

  7. Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy

    NASA Astrophysics Data System (ADS)

    Forman, C. J.; Wang, N.; Yang, Z. Y.; Mowat, C. G.; Jarvis, S.; Durkan, C.; Barker, P. D.

    2013-05-01

    Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.

  8. Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter

    PubMed Central

    Reed, James R.; dela Cruz, Albert Leo N.; Lomnicki, Slawo M.; Backes, Wayne L.

    2015-01-01

    Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO-Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ≥ 230°C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200-209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50°C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR•P450 complex. PMID:25817938

  9. Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing.

    PubMed

    Wiseman, Alan

    2003-04-01

    Cytochromes P450 (EC 1.14.14.1) are mixed function oxidases (oxygenases) that can catalyse redox bioconversions of food components. Also, efficacious removal of undesirable components can be achieved using solid-support immobilised enzyme (IME) of a selection from 2700 isoforms of cytochromes P450 (CYP). Cytochromes P450 co-immobilised with other enzymes, or protein receptors, may be used to confer a secondary order of regio- or stereo-specificity of chiral bioconversion: these can be predictable in silico by utilisation of QSARs (quantitative structure/activity relationships).

  10. Molecular phylogeny of some avian species using Cytochrome b gene sequence analysis

    PubMed Central

    Awad, A; Khalil, S. R; Abd-Elhakim, Y. M

    2015-01-01

    Veritable identification and differentiation of avian species is a vital step in conservative, taxonomic, forensic, legal and other ornithological interventions. Therefore, this study involved the application of molecular approach to identify some avian species i.e. Chicken (Gallus gallus), Muskovy duck (Cairina moschata), Japanese quail (Coturnix japonica), Laughing dove (Streptopelia senegalensis), and Rock pigeon (Columba livia). Genomic DNA was extracted from blood samples and partial sequence of the mitochondrial cytochrome b gene (358 bp) was amplified and sequenced using universal primers. Sequences alignment and phylogenetic analyses were performed by CLC main workbench program. The obtained five sequences were deposited in GenBank and compared with those previously registered in GenBank. The similarity percentage was 88.60% between Gallus gallus and Coturnix japonica and 80.46% between Gallus gallus and Columba livia. The percentage of identity between the studied species and GenBank species ranged from 77.20% (Columba oenas and Anas platyrhynchos) to 100% (Gallus gallus and Gallus sonneratii, Coturnix coturnix and Coturnix japonica, Meleagris gallopavo and Columba livia). Amplification of the partial sequence of mitochondrial cytochrome b gene proved to be practical for identification of an avian species unambiguously. PMID:27175180

  11. Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

    PubMed

    Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

    1989-03-15

    Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

  12. 6β-HYDROXYTESTOSTERONE, A CYTOCHROME P450 1B1-TESTOSTERONE-METABOLITE, MEDIATES ANGIOTENSIN II-INDUCED RENAL DYSFUNCTION IN MALE MICE

    PubMed Central

    Pingili, Ajeeth K.; Thirunavukkarasu, Shyamala; Kara, Mehmet; Brand, David; Katsurada, Akemi; Majid, Dewan S. A.; Navar, L. Gabriel; Gonzalez, Frank J.; Malik, Kafait U.

    2016-01-01

    6β-hydroxytestosterone, a cytochrome P450 1B1-derived metabolite of testosterone, contributes to the development of angiotensin II-induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the maintenance of renal homeostasis, development of hypertension and end organ damage, this study was conducted to determine the contribution of 6β-hydroxytestosterone to angiotensin II actions on water consumption and renal function in male Cyp1b1+/+ and Cyp1b1−/− mice. Castration of Cyp1b1+/+ mice or Cyp1b1−/− gene disruption minimized the angiotensin II-induced increase in water consumption, urine output, proteinuria, and sodium excretion and decreases in urine osmolality. 6β-hydroxytestosterone did not alter angiotensin II-induced increases in water intake, urine output, proteinuria, and sodium excretion or decreases in osmolality in Cyp1b1+/+ mice, but restored these effects of angiotensin II in Cyp1b1−/− or castrated mice Cyp1b1+/+ mice. Cyp1b1 gene disruption or castration prevented angiotensin II-induced renal fibrosis, oxidative stress, inflammation, urinary excretion of angiotensinogen, expression of angiotensin II type 1 receptor, and angiotensin converting enzyme. 6β-hydroxytestosterone did not alter angiotensin II-induced renal fibrosis, inflammation, oxidative stress, urinary excretion angiotensinogen, expression of angiotensin II type 1 receptor, or angiotensin converting enzyme in Cyp1b1+/+ mice; however, in Cyp1b1−/− or castrated mice Cyp1b1+/+ mice, it restored these effects of angiotensin II. These data indicate that 6β-hydroxytestosterone contributes to increased thirst, impairment of renal function, and end organ injury associated with angiotensin II-induced hypertension in male mice and that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension in males. PMID:26928804

  13. 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.

  14. Induction of tumor necrosis factor alpha by the group- and type-specific polysaccharides from type III group B streptococci.

    PubMed Central

    Mancuso, G; Tomasello, F; von Hunolstein, C; Orefici, G; Teti, G

    1994-01-01

    Previous studies suggested that circulating tumor necrosis factor alpha (TNF-alpha) may have a pathophysiologic role in experimental neonatal sepsis induced by group B streptococci (GBS). This study was undertaken to investigate the ability of the type III and group-specific polysaccharides of GBS to induce TNF-alpha production and TNF-alpha-dependent lethality in neonatal rats. The cytokine was detected in plasma samples by the L929 cytotoxicity assay. Intracardiac injections of either polysaccharide induced dose-dependent, transient elevations in plasma TNF-alpha levels that returned to baseline values after 5 h. The group-specific antigen induced significantly higher mean peak TNF-alpha levels than the type III antigen (125 +/- 47 versus 44 +/- 15 U/ml with 70 mg/kg of body weight). Glycogen (70 mg/kg), used as a negative control, did not induce TNF-alpha. The lipopolysaccharide-neutralizing agent polymyxin B did not decrease TNF-alpha levels induced by either polysaccharide, ruling out contamination with endotoxin as a possible cause of TNF-alpha induction. Fifty percent lethal doses of the type III and group-specific antigens given as intracardiac injections were 105 and 16 mg/kg, respectively. Salmonella endotoxin, used as a positive control, had a 50% lethal dose of 0.1 mg/kg. The lethal activities of GBS polysaccharides, as well as endotoxin, were completely prevented by pretreatment of neonatal rats with the respective specific antibodies or anti-murine TNF-alpha serum. To assess the relative importance of the type-specific substance in TNF-alpha induction by whole bacteria, two unrelated GBS transposon mutants devoid of only the type-specific capsular polysaccharide (COH1-13 and COH31-15) were employed. Each of the heat-killed unencapsulated mutants was able to produce plasma TNF-alpha level elevations or TNF-alpha-dependent lethality but was significantly less efficient in these activities than the corresponding encapsulated wild-type strain. These data

  15. Site directed mutagenesis of the heme axial ligands of cytochrome b559 affects the stability of the photosystem II complex.

    PubMed Central

    Pakrasi, H B; De Ciechi, P; Whitmarsh, J

    1991-01-01

    Cytochrome (cyt) b559, an integral membrane protein, is an essential component of the photosystem II (PSII) complex in the thylakoid membranes of oxygenic photosynthetic organisms. Cyt b559 has two subunits, alpha and beta, each with one predicted membrane spanning alpha-helical domain. The heme cofactor of this cytochrome is coordinated between two histidine residues. Each of the two subunit polypeptides of cyt b559 has one His residue. To investigate the influence of these His residues on the structure of cyt b559 and the PSII complex, we used a site directed mutagenesis approach to replace each His residue with a Leu residue. Introduction of these missense mutations in the transformable unicellular cyanobacterium, Synechocystis 6803, resulted in complete loss of PSII activity. Northern blot analysis showed that these mutations did not affect the stability of the polycistronic mRNA that encompasses both the psbE and the psbF genes, encoding the alpha and the beta subunits, respectively. Moreover, both of the single His mutants showed the presence of the alpha subunit which was 1.5 kd smaller than the same polypeptide in wild type cells. A secondary effect of such a structural change was that D1 and D2, two proteins that form the catalytic core (reaction center) of PSII, were also destabilized. Our results demonstrate that proper axial coordination of the heme cofactor in cyt b559 is important for the structural integrity of the reaction center of PSII. Images PMID:1904816

  16. Cell-type-specific role of ΔFosB in nucleus accumbens in modulating inter-male aggression.

    PubMed

    Aleyasin, Hossein; Flanigan, Meghan E; Golden, Sam A; Takahashi, Aki; Menard, Caroline; Pfau, Madeline L; Multer, Jacob; Pina, Jacqueline; McCabe, Kathryn A; Bhatti, Naemal; Hodes, Georgia E; Heshmati, Mitra; Neve, Rachael L; Nestler, Eric J; Heller, Elizabeth A; Russo, Scott J

    2018-06-11

    A growing number of studies implicate the brain's reward circuitry in aggressive behavior. However, the cellular and molecular mechanisms within brain reward regions that modulate the intensity of aggression as well as motivation for it have been underexplored. Here, we investigate the cell-type-specific influence of ΔFosB, a transcription factor known to regulate a range of reward and motivated behaviors, acting in the nucleus accumbens (NAc)-a key reward region-in male aggression in mice. We show that ΔFosB is specifically increased in dopamine D1 receptor (Drd1) expressing medium spiny neurons (D1-MSNs) in NAc after repeated aggressive encounters. Viral-mediated induction of ΔFosB selectively in D1-MSNs of NAc intensifies aggressive behavior, without affecting the preference for the aggression-paired context in a conditioned place preference (CPP) assay. In contrast, ΔFosB induction selectively in D2-MSNs reduces the time spent exploring the aggression-paired context during CPP without affecting the intensity of aggression per se. These data strongly support a dissociable cell-type-specific role for ΔFosB in the NAc in modulating aggression and aggression reward. Significance Statement: Aggressive behavior is associated with several neuropsychiatric disorders and can be disruptive for the individuals as well as their victims. Studies have shown a positive reinforcement mechanism underlying aggressive behavior that shares many common features with drug addiction. Here, we explore the cell-type-specific role of the addiction-associated transcription factor ΔFosB in the nucleus accumbens (NAc) in aggression. We found that ΔFosB expression promotes aggressive behavior, effects that are dissociable from its effects on aggression reward. This finding is a significant first step in identifying therapeutic targets for the reduction of aggressive behavior across a range of neuropsychiatric illnesses. Copyright © 2018 the authors.

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

    PubMed

    Chen, Xi'en; Zhang, Yalin

    2015-03-10

    NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

    PubMed

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

    2013-11-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons. © 2013 International Society for Neurochemistry.

  19. Comparison of Vertebrate Cytochrome b and Prepronociceptin for Blood Meal Analyses in Culicoides

    PubMed Central

    Hadj-Henni, Leila; De Meulemeester, Thibaut; Depaquit, Jérôme; Noël, Philippe; Germain, Adeline; Helder, Remi; Augot, Denis

    2015-01-01

    To date, studies on host preferences and blood meal identification have been conducted for Culicoides species using molecular-based methods such as PCR techniques to amplify only a fragment from universal vertebrate mitochondrial genes such as cytochrome c oxidase subunit I or cytochrome b (Cyt b). The vertebrate prepronociceptin gene (PNOC) was also tested in this field. However, the choice of molecular marker to identify blood meal is critical. The objective of our study is to compare the ability of Cyt b and PNOC as molecular markers for blood meal identification depending on the stage of blood meal digestion. In order to determine whether these Cyt b and PNOC could provide a positive result, 565 blood-fed females of Culicoides spp were collected and morphologically identified. The samples were collected between 2012 and 2014, in two localities in France. The collection localities were near either livestock or a forest. To catch the specimens, we used UV CDC miniature light traps. PNOC sequence of donkeys (Equus asinus) was sequenced and submitted because it was missing in GenBank. Our findings emphasize that the PNOC marker is not suitable to separate closely related Equid species such as horses and donkeys. The Cyt b marker was able to identify 204 more samples when compared to PNOC (99.55% of specimens). Cyt b appears to be better able to detect the origin of blood meals from females with digested blood in their abdomens. We conclude that Cyt b is a good marker as it increases the accuracy of blood meal identification of engorged females containing digested blood in their abdomens. The host opportunist behavior of Culicoides, especially that of C. obsoletus and C. scoticus, the main vectors of BTV in Europe was also highlighted. PMID:26664944

  20. Abundance of the multiheme c-type cytochrome OmcB increases in outer biofilm layers of electrode-grown Geobacter sulfurreducens.

    PubMed

    Stephen, Camille S; LaBelle, Edward V; Brantley, Susan L; Bond, Daniel R

    2014-01-01

    When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB) and cytoplasmic acetate kinase (AckA) were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode). Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10-20 µm distant from the electrode surface compared to inner layers (0-10 µm). In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.

  1. Evidence for involvement of multiple forms of cytochrome P-450 in aflatoxin B sup 1 metabolism in human liver

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Forrester, L.M.; Wolf, C.R.; Neal, G.E.

    Liver cancer is a major cause of premature death in many areas of Africa and Asia and its incidence is strongly correlated with exposure to aflatoxin B{sub 1} (AFB{sub 1}). Because AFB{sub 1} requires metabolic activation to achieve a biological response, there is a need for detailed knowledge of the mechanism of activation to assess individual risk. The authors carried out an extensive study using a total of 19 human liver samples to determine the individual variability in the metabolism of the toxin to mutagenic or detoxification products and to identify the specific cytochrome P-450 forms involved in these processes.more » Metabolism to the toxic 8,9-epoxide or to products mutagenic in the Ames test was found to exhibit very large individual variation. These data demonstrate that, although P450IIIA probably plays an important role in AFB{sub 1} activation, several other cytochrome P-450 forms have the capacity to activate the toxin. Similar considerations apply to detoxifying metabolism to aflatoxin Q{sub 1} and aflatoxin M{sub 1}. The levels of expression of many of the forms of cytochrome P-450 involved in AFB{sub 1} metabolism are known to be highly sensitive to environmental factors. This indicates that such factors will be an important determinant in individual susceptibility to the tumorigenic action of AFB{sub 1}.« less

  2. Gender and Gonadal Status Differences in Zona Reticularis Expression in Marmoset Monkey Adrenals: Cytochrome b5 Localization With Respect To Cytochrome P450 17,20-Lyase Activity.

    PubMed Central

    Pattison, J Christina; Saltzman, Wendy; Abbott, David H; Hogan, Brynn K; Nguyen, Ann D; Husen, Bettina; Einspanier, Almuth; Conley, Alan J; Bird., Ian M

    2007-01-01

    Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive. PMID:17222503

  3. Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

    PubMed Central

    Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

    2012-01-01

    S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase

  4. c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm

    PubMed Central

    Durand, Anne; Azzouzi, Asma; Bourbon, Marie-Line; Steunou, Anne-Soisig; Liotenberg, Sylviane; Maeshima, Akinori; Astier, Chantal; Argentini, Manuela; Saito, Shingo

    2015-01-01

    ABSTRACT In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the periplasm. Central to these systems, the Cu+ ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The fate of copper in the periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu+ ATPase copA mutant. CopI is induced by copper, localized in the periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the periplasm of the wild-type cells when they are exposed to high copper concentrations. PMID:26396241

  5. Cytochrome b sequences in black-crowned night-herons (Nycticorax nycticorax) from heronries exposed to genotoxic contaminants

    USGS Publications Warehouse

    Dahl, Christopher R.; Bickham, John W.; Wickliffe, Jeffery K.; Custer, Thomas W.

    2001-01-01

    DNA sequence analysis of a 215 base-pair region of the mitochondrial cytochrome b gene was used to examine genetic variation and search for evidence of an increased mutation rate in black-crowned night-herons. We examined five populations exposed to environmental contamination (primarily PAHs and PCBs) and one reference population from the eastern U.S. There was no evidence of a high mutation rate even within populations previously shown to exhibit increased variation in DNA content among somatic cells as a result of petroleum exposure. Three haplotypes were observed among 99 individuals. The low level of variability could be evidence for a genetic bottleneck, or that cytochrome b is too conservative for use in population genetic studies of this species. With the exception of one population from Louisiana, pair-wise Phist estimates were very low, indicative of little population structure and potentially high rates of effective migration among populations.

  6. Shared features of S100B immunohistochemistry and cytochrome oxidase histochemistry in the ventroposterior thalamus and lateral habenula in neonatal rats.

    PubMed

    Muneoka, Katsumasa; Funahashi, Hisayuki; Ogawa, Tetsuo; Whitaker-Azmitia, Patricia M; Shioda, Seiji

    2012-10-01

    The ventroposterior thalamus and the habenular nuclei of the epithalamus are relevant to the monoaminergic system functionally and anatomically. The glia-derived S100B protein plays a critical role in the development of the nervous system including the monoaminergic systems. In this study, we performed an immunohistochemical study of glia-related proteins including S100B, serotonin transporter, and microtubule-associated protein 2, as well as cytochrome oxidase histochemistry in neonatal rats. Results showed the same findings for S100B immunohistochemistry between the ventroposterior thalamus and the lateral habenula at postnatal day 7: intense staining in cell bodies of astrocytes, diffusely spread immunoproduct in the intercellular space, and S100B-free areas as well as a strong reaction to cytochrome oxidase histochemistry. Further common features were the scarcity of glial fibrillary acidic protein-positive astrocytes and the few apoptotic cells observed. The results of the cytochrome oxidase reaction suggested that S100B is released actively into intercellular areas in restricted brain regions showing high neuronal activity at postnatal day 7. Pathology of the ventroposterior thalamus and the habenula is suggested in mental disorders, and S100B might be a key factor for investigations in these areas. Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

  7. Cytochrome components of nitrate- and sulfate-respiring Desulfovibrio desulfuricans ATCC 27774.

    PubMed Central

    Liu, M C; Costa, C; Coutinho, I B; Moura, J J; Moura, I; Xavier, A V; LeGall, J

    1988-01-01

    Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells. PMID:2848008

  8. Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein.

    PubMed

    Islas-Osuna, María A; Silva-Moreno, Begonia; Caceres-Carrizosa, Nidia; García-Robles, Jesús M; Sotelo-Mundo, Rogerio R; Yepiz-Plascencia, Gloria M

    2006-05-01

    Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.

  9. Characterization of the Critical Amino Acids of an Aspergillus parasiticus Cytochrome P-450 Monooxygenase Encoded by ordA That Is Involved in the Biosynthesis of Aflatoxins B1, G1, B2, and G2

    PubMed Central

    Yu, Jiujiang; Chang, Perng-Kuang; Ehrlich, Kenneth C.; Cary, Jeffrey W.; Montalbano, Beverly; Dyer, John M.; Bhatnagar, Deepak; Cleveland, Thomas E.

    1998-01-01

    The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400→Leu-400, Ala-143→Ser-143, and Ile-528→Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 in A. parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee. PMID:9835571

  10. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA

    DOE PAGES

    Liu, Yimo; Wang, Zheming; Liu, Juan; ...

    2014-09-24

    The multiheme, outer membrane c-type cytochrome (c-Cyt) OmcB of Geobacter sulfurreducens was previously proposed to mediate electron transfer across the outer membrane. However, the underlying mechanism has remained uncharacterized. In G. sulfurreducens, the omcB gene is part of two tandem four-gene clusters, each is predicted to encode a transcriptional factor (OrfR/OrfS), a porin-like outer membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (OmaB/OmaC), and an outer membrane c-Cyt (OmcB/OmcC), respectively. Here we showed that OmbB/OmbC, OmaB/OmaC and OmcB/OmcC of G. sulfurreducens PCA formed the porin-cytochrome (Pcc) protein complexes, which were involved in transferring electrons across the outer membrane. The isolated Pccmore » protein complexes reconstituted in proteoliposomes transferred electrons from reduced methyl viologen across the lipid bilayer of liposomes to Fe(III)-citrate and ferrihydrite. The pcc clusters were found in all eight sequenced Geobacter and 11 other bacterial genomes from six different phyla, demonstrating a widespread distribution of Pcc protein complexes in phylogenetically diverse bacteria. Deletion of ombB-omaB-omcB-orfS-ombC-omaC-omcC gene clusters had no impact on the growth of G. sulfurreducens PCA with fumarate, but diminished the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. Finally, complementation with the ombB-omaB-omcB gene cluster restored the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite.« less

  11. 7,12-Dimethylbenzanthracene induces apoptosis in RL95-2 human endometrial cancer cells: Ligand-selective activation of cytochrome P450 1B1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Ji Young; Medical Research Science Center, Dong-A University, Busan 602-714; Lee, Seung Gee

    2012-04-15

    7,12-Dimethylbenzanthracene (DMBA), a polycyclic aromatic hydrocarbon, exhibits mutagenic, carcinogenic, immunosuppressive, and apoptogenic properties in various cell types. To achieve these functions effectively, DMBA is modified to its active form by cytochrome P450 1 (CYP1). Exposure to DMBA causes cytotoxicity-mediated apoptosis in bone marrow B cells and ovarian cells. Although uterine endometrium constitutively expresses CYP1A1 and CYP1B1, their apoptotic role after exposure to DMBA remains to be elucidated. Therefore, we chose RL95-2 endometrial cancer cells as a model system for studying DMBA-induced cytotoxicity and cell death and hypothesized that exposure to DMBA causes apoptosis in this cell type following CYP1A1 and/ormore » CYP1B1 activation. We showed that DMBA-induced apoptosis in RL95-2 cells is associated with activation of caspases. In addition, mitochondrial changes, including decrease in mitochondrial potential and release of mitochondrial cytochrome c into the cytosol, support the hypothesis that a mitochondrial pathway is involved in DMBA-induced apoptosis. Exposure to DMBA upregulated the expression of AhR, Arnt, CYP1A1, and CYP1B1 significantly; this may be necessary for the conversion of DMBA to DMBA-3,4-diol-1,2-epoxide (DMBA-DE). Although both CYP1A1 and CYP1B1 were significantly upregulated by DMBA, only CYP1B1 exhibited activity. Moreover, knockdown of CYP1B1 abolished DMBA-induced apoptosis in RL95-2 cells. Our data show that RL95-2 cells are susceptible to apoptosis by exposure to DMBA and that CYP1B1 plays a pivotal role in DMBA-induced apoptosis in this system. -- Highlights: ► Cytotoxicity-mediated apoptogenic action of DMBA in human endometrial cancer cells. ► Mitochondrial pathway in DMBA-induced apoptosis of RL95-2 endometrial cancer cells. ► Requirement of ligand-selective activation of CYP1B1 in DMBA-induced apoptosis.« less

  12. Novel primers for complete mitochondrial cytochrome b genesequencing in mammals

    USGS Publications Warehouse

    Naidu, Ashwin; Fitak, Robert R.; Munguia-Vega, Adrian; Culver, Melanie

    2011-01-01

    Sequence-based species identification relies on the extent and integrity of sequence data available in online databases such as GenBank. When identifying species from a sample of unknown origin, partial DNA sequences obtained from the sample are aligned against existing sequences in databases. When the sequence from the matching species is not present in the database, high-scoring alignments with closely related sequences might produce unreliable results on species identity. For species identification in mammals, the cytochrome b (cyt b) gene has been identified to be highly informative; thus, large amounts of reference sequence data from the cyt b gene are much needed. To enhance availability of cyt b gene sequence data on a large number of mammalian species in GenBank and other such publicly accessible online databases, we identified a primer pair for complete cyt b gene sequencing in mammals. Using this primer pair, we successfully PCR amplified and sequenced the complete cyt b gene from 40 of 44 mammalian species representing 10 orders of mammals. We submitted 40 complete, correctly annotated, cyt b protein coding sequences to GenBank. To our knowledge, this is the first single primer pair to amplify the complete cyt b gene in a broad range of mammalian species. This primer pair can be used for the addition of new cyt b gene sequences and to enhance data available on species represented in GenBank. The availability of novel and complete gene sequences as high-quality reference data can improve the reliability of sequence-based species identification.

  13. Reaction of cyanide with cytochrome ba3 from Thermus thermophilus: spectroscopic characterization of the Fe(II)a3-CN.Cu(II)B-CN complex suggests four 14N atoms are coordinated to CuB.

    PubMed Central

    Surerus, K K; Oertling, W A; Fan, C; Gurbiel, R J; Einarsdóttir, O; Antholine, W E; Dyer, R B; Hoffman, B M; Woodruff, W H; Fee, J A

    1992-01-01

    Cytochrome ba3 from Thermus thermophilus reacts slowly with excess HCN at pH 7.4 to create a form of the enzyme in which CuA, cytochrome b, and CuB remain oxidized, while cytochrome a3 is reduced by one electron, presumably with the formation of cyanogen. We have examined this form of the enzyme by UV-visible, resonance Raman, EPR, and electron nuclear double resonance spectroscopies in conjunction with permutations of 13C- and 15N-labeled cyanide. The results support a model in which one CN- binds through the carbon atom to ferrous a3, supporting a low-spin (S = 0) configuration on the Fe; bridging by this cyanide to the CuB is weak or absent. Four 14N atoms, presumably donated by histidine residues of the protein, provide a strong equatorial ligand field about CuB; a second CN- is coordinated through the carbon atom to CuB in an axial position. PMID:1314380

  14. Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

    PubMed

    Shak, S; Goldstein, I M

    1985-09-01

    Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.

  15. Cancer Activation and Polymorphisms of Human Cytochrome P450 1B1

    PubMed Central

    Chun, Young-Jin; Kim, Donghak

    2016-01-01

    Human cytochrome P450 enzymes (P450s, CYPs) are major oxidative catalysts that metabolize various xenobiotic and endogenous compounds. Many carcinogens induce cancer only after metabolic activation and P450 enzymes play an important role in this phenomenon. P450 1B1 mediates bioactivation of many procarcinogenic chemicals and carcinogenic estrogen. It catalyzes the oxidation reaction of polycyclic aromatic carbons, heterocyclic and aromatic amines, and the 4-hydroxylation reaction of 17β-estradiol. Enhanced expression of P450 1B1 promotes cancer cell proliferation and metastasis. There are at least 25 polymorphic variants of P450 1B1 and some of these have been reported to be associated with eye diseases. In addition, P450 1B1 polymorphisms can greatly affect the metabolic activation of many procarcinogenic compounds. It is necessary to understand the relationship between metabolic activation of such substances and P450 1B1 polymorphisms in order to develop rational strategies for the prevention of its toxic effect on human health. PMID:27123158

  16. Structure of caa(3) cytochrome c oxidase--a nature-made enzyme-substrate complex.

    PubMed

    Noor, Mohamed Radzi; Soulimane, Tewfik

    2013-05-01

    Aerobic respiration, the energetically most favorable metabolic reaction, depends on the action of terminal oxidases that include cytochrome c oxidases. The latter forms a part of the heme-copper oxidase superfamily and consists of three different families (A, B, and C types). The crystal structures of all families have now been determined, allowing a detailed structural comparison from evolutionary and functional perspectives. The A2-type oxidase, exemplified by the Thermus thermophilus caa(3) oxidase, contains the substrate cytochrome c covalently bound to the enzyme complex. In this article, we highlight the various features of caa(3) enzyme and provide a discussion of their importance, including the variations in the proton and electron transfer pathways.

  17. The coxBAC Operon Encodes a Cytochrome c Oxidase Required for Heterotrophic Growth in the Cyanobacterium Anabaena variabilis Strain ATCC 29413

    PubMed Central

    Schmetterer, Georg; Valladares, Ana; Pils, Dietmar; Steinbach, Susanne; Pacher, Margit; Muro-Pastor, Alicia M.; Flores, Enrique; Herrero, Antonia

    2001-01-01

    Three genes, coxB, coxA, and coxC, found in a clone from a gene library of the cyanobacterium Anabaena variabilis strain ATCC 29413, were identified by hybridization with an oligonucleotide specific for aa3-type cytochrome c oxidases. Deletion of these genes from the genome of A. variabilis strain ATCC 29413 FD yielded strain CSW1, which displayed no chemoheterotrophic growth and an impaired cytochrome c oxidase activity. Photoautotrophic growth of CSW1, however, was unchanged, even with dinitrogen as the nitrogen source. A higher cytochrome c oxidase activity was detected in membrane preparations from dinitrogen-grown CSW1 than from nitrate-grown CSW1, but comparable activities of respiratory oxygen uptake were found in the wild type and in CSW1. Our data indicate that the identified cox gene cluster is essential for fructose-dependent growth in the dark, but not for growth on dinitrogen, and that other terminal respiratory oxidases are expressed in this cyanobacterium. Transcription analysis showed that coxBAC constitutes an operon which is expressed from two transcriptional start points. The use of one of them was stimulated by fructose. PMID:11591688

  18. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

    PubMed Central

    Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  19. The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.

    PubMed

    Haid, A; Schweyen, R J; Bechmann, H; Kaudewitz, F; Solioz, M; Schatz, G

    1979-03-01

    Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).

  20. Detection of species and molecular typing of Leishmania in suspected patients by targeting cytochrome b gene in Zahedan, southeast of Iran.

    PubMed

    Mirahmadi, Hadi; Rezaee, Nasrin; Mehravaran, Ahmad; Heydarian, Peyman; Raeghi, Saber

    2018-05-01

    Cutaneous leishmaniasis (CL) is one of the most important health problems that are capable of involving both tropical and subtropical areas, especially in Iran. This cross-sectional study aimed to differentiate the species that are able to cause CL in Zahedan city by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. It was conducted on 145 suspected CL patients in Zahedan city between 2014 and 2016. The smears were initially prepared, air-dried, fixed with absolute methanol, and stained with 10% Giemsa. Then, we examined the stained samples by a light microscope under 1000× magnifications. PCR assay targeted cytochrome b (cyt b ) gene using LCBF1 and LCBR2 primers and the products digested by Ssp1 enzymes. From 145 suspected CL patients, 76 (52.4%) were positive in microscopic examination. In addition, we detected gene of interest (cyt b ) in 98 (67.5%). The results of PCR-RFLP indicated that 53/98 (54%) cases were Leishmania major and 45/98 (46%) were Leishmania tropica , and the main species in these areas was L. major . We concluded that the microscopic examination is not sensitive enough and is not able to distinguish between different Leishmania species. Instead, molecular methods like PCR-RFLP can be appropriately used with promising results.

  1. Rhodobacter sphaeroides spd mutations allow cytochrome c2-independent photosynthetic growth.

    PubMed Central

    Rott, M A; Donohue, T J

    1990-01-01

    In Rhodobacter sphaeroides, cytochrome c2 (cyt c2) is a periplasmic redox protein required for photosynthetic electron transfer. cyt c2-deficient mutants created by replacing the gene encoding the apoprotein for cyt c2 (cycA) with a kanamycin resistance cartridge are photosynthetically incompetent. Spontaneous mutations that suppress this photosynthesis deficiency (spd mutants) arise at a frequency of 1 to 10 in 10(7). We analyzed the cytochrome content of several spd mutants spectroscopically and by heme peroxidase assays. These suppressors lacked detectable cyt c2, but they contained a new soluble cytochrome which was designated isocytochrome c2 (isocyt c2) that was not detectable in either cycA+ or cycA mutant cells. When spd mutants were grown photosynthetically, isocyt c2 was present at approximately 20 to 40% of the level of cyt c2 found in photosynthetically grown wild type cells, and it was found in the periplasm with cytochromes c' and c554. These spd mutants also had several other pleiotropic phenotypes. Although photosynthetic growth rates of the spd mutants were comparable to those of wild-type strains at all light intensities tested, they contained elevated levels of B800-850 pigment-protein complexes. Several spd mutants contained detectable amounts of isocyt c2 under aerobic conditions. Finally, heme peroxidase assays indicated that, under anaerobic conditions, the spd mutants may contain another new cytochrome in addition to isocyt c2. These pleiotropic phenotypes, the frequency at which the spd mutants arise, and the fact that a frameshift mutagen is very effective in generating the spd phenotype suggest that some spd mutants contain a mutation in loci which regulate cytochrome synthesis. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 PMID:2156806

  2. Engineering of a functional human NADH-dependent cytochrome P450 system

    PubMed Central

    Döhr, Olaf; Paine, Mark J. I.; Friedberg, Thomas; Roberts, Gordon C. K.; Wolf, C. Roland

    2001-01-01

    A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent kcat of W676A is equivalent (90%) to the NADPH-dependent kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent KMNADPH and KMNADH values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2′-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system. PMID:11136248

  3. A complex of cardiac cytochrome c1 and cytochrome c.

    PubMed

    Chiang, Y L; Kaminsky, L S; King, T E

    1976-01-10

    The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of

  4. [Effect of bemethyl on cytochrome P-450-dependent monoxygenases in the human liver and lymphocytes].

    PubMed

    Sorokina, E A; Sibiriak, S V; Sergeeva, S A

    2002-01-01

    Effects of the actoprotector bemithyl (50 mg/kg, p.o.) upon a single or five-fold administration on the cytochrome P-450 and b5 content and the isoform-specific and nonspecific monooxygenase activity [aminopyrine-N-demethylase, aniline-p-hydroxylase, 4-nitroanisole-o-demethylase,2,5-diphenyloxazole-p-hydroxylase, 7-ethoxyresorufin-o-deethylase (EROD), benzyloxyresorufin-o-debenzylase (BROD)] in rat liver were evaluated. In addition, the influence of bemithyl (0.(1)-100 microM) on the development of EROD and BROD activity was studied on the mitogen-stimulated human lymphocytes in vitro. Administered in rats, bemithyl exhibited the properties of a cytochrome P-450 inductor of the mixed type, which was manifested by an increase in the total cytochrome P-450 content in liver microsomes and in the monooxygenase activity related to both Ah-receptor-dependent and -independent isoforms (except for the aniline-p-hydroxylase activity). The induction of the monooxygenase activity realized by Ah-receptor-dependent isoforms (4-nitroanisole-o-demethylase, 2,5-diphenyloxazole-p-hydroxylase, and EROD activity) was more pronounced, reaching maximum upon a single drug administration. Acting upon the human lymphocytes in vitro, high concentrations of bemithyl increased expression of the EROD activity, while low drug concentrations stimulated the BROD activity.

  5. 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.

  6. Differentiation of Trypanosoma cruzi I subgroups through characterization of cytochrome b gene sequences.

    PubMed

    Spotorno O, Angel E; Córdova, Luis; Solari I, Aldo

    2008-12-01

    To identify and characterize chilean samples of Trypanosoma cruzi and their association with hosts, the first 516 bp of the mitochondrial cytochrome b gene were sequenced from eight biological samples, and phylogenetically compared with other known 20 American sequences. The molecular characterization of these 28 sequences in a maximum likelihood phylogram (-lnL = 1255.12, tree length = 180, consistency index = 0.79) allowed the robust identification (bootstrap % > 99) of three previously known discrete typing units (DTU): DTU IIb, IIa, and I. An apparently undescribed new sequence found in four new chilean samples was detected and designated as DTU Ib; they were separated by 24.7 differences, but robustly related (bootstrap % = 97 in 500 replicates) to those of DTU I by sharing 12 substitutions, among which four were nonsynonymous ones. Such new DTU Ib was also robust (bootstrap % = 100), and characterized by 10 unambiguous substitutions, with a single nonsynonymous G to T change at site 409. The fact that two of such new sequences were found in parasites from a chilean endemic caviomorph rodent, Octodon degus, and that they were closely related to the ancient DTU I suggested old origins and a long association to caviomorph hosts.

  7. Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase.

    PubMed Central

    Green, G N; Gennis, R B

    1983-01-01

    A screening procedure was devised which permitted the isolation of a cytochrome d-deficient mutant by its failure to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Cytochrome a1 and probably cytochrome b558 were also missing in the mutant. Growth and oxygen uptake rates were similar for both parent and mutant strains. However, the strain lacking cytochrome d had an increased sensitivity to cyanide, indicating that cytochrome d confers some resistance to this respiratory inhibitor. The gene responsible for these phenotypes has been named cyd and maps between tolA and sucB. PMID:6304009

  8. Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wilderman, P. Ross, E-mail: pwilderman@ucsd.edu; Jang, Hyun-Hee; Malenke, Jael R.

    Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteinsmore » gave the characteristic maximum at 450 nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system. - Highlights: • Three CYP2B enzymes from Neotoma lepida were cloned, engineered, and expressed. • A mix of catalytic and binding assays yields unique results for each enzyme. • Mutational analysis indicates CYP{sub 2}B substrate recognition remains to be clarified. • Reported N. lepida

  9. High-Resolution Melting (HRM) of the Cytochrome B Gene: A Powerful Approach to Identify Blood-Meal Sources in Chagas Disease Vectors

    PubMed Central

    Peña, Victor H.; Fernández, Geysson J.; Gómez-Palacio, Andrés M.; Mejía-Jaramillo, Ana M.; Cantillo, Omar; Triana-Chávez, Omar

    2012-01-01

    Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate. PMID:22389739

  10. Biochemical evaluation of interactions between synergistic molecules and phase I enzymes involved in insecticide resistance in B- and Q-type Bemisia tabaci (Hemiptera: Aleyrodidae).

    PubMed

    Panini, Michela; Tozzi, Francesco; Zimmer, Christoph T; Bass, Chris; Field, Linda; Borzatta, Valerio; Mazzoni, Emanuele; Moores, Graham

    2017-09-01

    Metabolic resistance is an important consideration in the whitefly Bemisia tabaci, where an esterase-based mechanism has been attributed to pyrethroid resistance and over-expression of the cytochrome P450, CYP6CM1, has been correlated to resistance to imidacloprid and other neonicotinoids. In vitro interactions between putative synergists and CYP6CM1, B and Q-type esterases were investigated, and structure-activity relationship analyses allowed the identification of chemical structures capable of acting as inhibitors of esterase and oxidase activities. Specifically, methylenedioxyphenyl (MDP) moieties with a polyether chain were preferable for optimum inhibition of B-type esterase, whilst corresponding dihydrobenzofuran structures were potent for the Q-esterase variation. Potent inhibition of CYP6CM1 resulted from structures which contained an alkynyl chain with a terminal methyl group. Synergist candidates could be considered for field control of B. tabaci, especially to abrogate neonicotinoid resistance. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  11. In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803.

    PubMed

    Luján, María A; Martínez, Jesús I; Alonso, Pablo J; Torrado, Alejandro; Roncel, Mercedes; Ortega, José M; Sancho, Javier; Picorel, Rafael

    2015-11-01

    The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

    PubMed

    Yoshida, Saishu; Kato, Takako; Kanno, Naoko; Nishimura, Naoto; Nishihara, Hiroto; Horiguchi, Kotaro; Kato, Yukio

    2017-10-01

    Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.

  13. RoxB Is a Novel Type of Rubber Oxygenase That Combines Properties of Rubber Oxygenase RoxA and Latex Clearing Protein (Lcp).

    PubMed

    Birke, Jakob; Röther, Wolf; Jendrossek, Dieter

    2017-07-15

    Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), have been described so far. RoxA proteins (RoxAs) are c -type cytochromes of ≈70 kDa produced by Gram-negative rubber-degrading bacteria, and they cleave polyisoprene into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD), a C 15 oligo-isoprenoid, as the major end product. Lcps are common among Gram-positive rubber degraders and do not share amino acid sequence similarities with RoxAs. Furthermore, Lcps have much smaller molecular masses (≈40 kDa), are b -type cytochromes, and cleave polyisoprene to a mixture of C 20 , C 25 , C 30 , and higher oligo-isoprenoids as end products. In this article, we purified a new type of rubber oxygenase, RoxB Xsp (RoxB of Xanthomonas sp. strain 35Y). RoxB Xsp is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c -type hemes). However, RoxB Xsp differs from all currently known RoxAs in having a distinctive product spectrum of C 20 , C 25 , C 30 , and higher oligo-isoprenoids that has been observed only for Lcps so far. Purified RoxB Xsp revealed the highest specific activity of 4.5 U/mg (at 23°C) of all currently known rubber oxygenases and exerts a synergistic effect on the efficiency of polyisoprene cleavage by RoxA Xsp RoxB homologs were identified in several other Gram-negative rubber-degrading species, pointing to a prominent function of RoxB for the biodegradation of rubber in Gram-negative bacteria. IMPORTANCE The enzymatic cleavage of rubber (polyisoprene) is of high environmental importance given that enormous amounts of rubber waste materials are permanently released (e.g., by abrasion of tires). Research from the last decade has discovered rubber oxygenase A, RoxA, and latex clearing protein (Lcp) as being responsible for the primary enzymatic attack on the hydrophobic and water-insoluble biopolymer poly( cis -1,4-isoprene) in Gram

  14. Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

    PubMed

    Kobayashi, Misato; Hoshinaga, Yukiko; Miura, Natsuko; Tokuda, Yuki; Shigeoka, Shigeru; Murai, Atsushi; Horio, Fumihiko

    2014-01-01

    The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

  15. A Protein Structure Initiative Approach to Expression, Purification, and In Situ Delivery of Human Cytochrome b5 to Membrane Vesicles†

    PubMed Central

    Sobrado, Pablo; Goren, Michael A.; James, Declan; Amundson, Carissa K.; Fox, Brian G.

    2008-01-01

    A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of ~18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3 mg per]of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed. PMID:18226920

  16. A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles.

    PubMed

    Sobrado, Pablo; Goren, Michael A; James, Declan; Amundson, Carissa K; Fox, Brian G

    2008-04-01

    A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3mg per g of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.

  17. Ultrafast dynamics of the photo-excited hemes b and cn in the cytochrome b6f complex.

    PubMed

    Agarwal, Rachna; Chauvet, Adrien A P

    2017-01-25

    The dynamics of hemes b and c n within the cytochrome b 6 f complex are investigated by means of ultrafast broad-band transient absorption spectroscopy. On the one hand, the data reveal that, subsequent to visible light excitation, part of the b hemes undergoes pulse-limited photo-oxidation, with the liberated electron supposedly being transferred to one of the adjacent aromatic amino acids. Photo-oxidation is followed by charge recombination in about 8.2 ps. Subsequent to charge recombination, heme b is promoted to a vibrationally excited ground state that relaxes in about 4.6 ps. On the other hand, heme c n undergoes ultrafast ground state recovery in about 140 fs. Interestingly, the data also show that, in contrast to previous beliefs, Chl a is involved in the photochemistry of hemes. Indeed, subsequent to heme excitation, Chl a bleaches and recovers to its ground state in 90 fs and 650 fs, respectively. Chl a bleaching allegedly corresponds to the formation of a short lived Chl a anion. Beyond the previously suggested structural role, this study provides unique evidence that Chl a is directly involved in the photochemistry of the hemes.

  18. High-Molecular-Mass Multi-c-Heme Cytochromes from Methylococcus capsulatus Bath†

    PubMed Central

    Bergmann, David J.; Zahn, James A.; DiSpirito, Alan A.

    1999-01-01

    The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme c concentration was estimated to be 8.2 ± 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94,000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome. PMID:9922265

  19. Docking and QSAR comparative studies of polycyclic aromatic hydrocarbons and other procarcinogen interactions with cytochromes P450 1A1 and 1B1.

    PubMed

    Gonzalez, J; Marchand-Geneste, N; Giraudel, J L; Shimada, T

    2012-01-01

    To obtain chemical clues on the process of bioactivation by cytochromes P450 1A1 and 1B1, some QSAR studies were carried out based on cellular experiments of the metabolic activation of polycyclic aromatic hydrocarbons and heterocyclic aromatic compounds by those enzymes. Firstly, the 3D structures of cytochromes 1A1 and 1B1 were built using homology modelling with a cytochrome 1A2 template. Using these structures, 32 ligands including heterocyclic aromatic compounds, polycyclic aromatic hydrocarbons and corresponding diols, were docked with LigandFit and CDOCKER algorithms. Binding mode analysis highlighted the importance of hydrophobic interactions and the hydrogen bonding network between cytochrome amino acids and docked molecules. Finally, for each enzyme, multilinear regression and artificial neural network QSAR models were developed and compared. These statistical models highlighted the importance of electronic, structural and energetic descriptors in metabolic activation process, and could be used for virtual screening of ligand databases. In the case of P450 1A1, the best model was obtained with artificial neural network analysis and gave an r (2) of 0.66 and an external prediction [Formula: see text] of 0.73. Concerning P450 1B1, artificial neural network analysis gave a much more robust model, associated with an r (2) value of 0.73 and an external prediction [Formula: see text] of 0.59.

  20. 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.

  1. Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

    PubMed

    Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

    1976-03-01

    Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

  2. Characterization of the cydAB-Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

    PubMed Central

    Kana, Bavesh D.; Weinstein, Edward A.; Avarbock, David; Dawes, Stephanie S.; Rubin, Harvey; Mizrahi, Valerie

    2001-01-01

    The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the γ-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 × 102 Pa of pO2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 × 102 Pa of pO2 or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO2 of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that

  3. Cytochrome P450 2B6*5 Increases Relapse after Cyclophosphamide-containing Conditioning and Autologous Transplantation for Lymphoma

    PubMed Central

    Bachanova, Veronika; Shanley, Ryan; Malik, Farhana; Chauhan, Lata; Lamba, Vishal; Weisdorf, Daniel J.; Burns, Linda J.; Lamba, Jatinder Kaur

    2016-01-01

    Cyclophosphamide (Cy) is a prodrug that depends on bioactivation by hepatic cytochrome P450 (CYP) enzymes for its cytotoxicity. We evaluated the influence of single nucleotide polymorphisms (SNPs) of CYP enzymes on the efficacy of autologous hematopoietic cell transplantation (HCT) for lymphoma. SNPs of 22 genes were analyzed in 93 patients with Hodgkin (n=52) and non-Hodgkin lymphoma (n=41) treated with high-dose Cy followed by autologous HCT between 2004–2012. Preparative regimens contained Cy (120mg/kg) combined with carmustine/etoposide (n=61) or Cy (6000mg/m2) with total body irradiation (n=32). Lack of complete remission as measured by pre-transplant positron emission tomography was the sole clinical factor associated with increased risk of relapse (HR 2.1). In genomic analysis, we identified a single SNP rs3211371 in exon 9 (C >T) of the CYP2B6 gene (allele designation 2B6*5) that significantly impacted patient outcomes. After adjusting for disease status and conditioning regimen, patients with CYP2B6*1/*5 genotype had a higher 2-year relapse rate (HR 3.3; 95%CI 1.6–6.5; p=0.041) and decreased overall survival (HR 13.5; 95%CI 3.5–51.9; p=0.008) than patients with wild-type allele. Patients with two hypo-functional CYP2B6 variant genotypes, *5 and *6, experienced 2-year PFS of only 11% (95%CI 1–39%) compared to 67% (95% CI 55–77%) for patients with the wild-type CYP2B6*1 allele in exon 9. Our results suggest that CYP2B6 SNPs influence the efficacy of high-dose Cy and significantly reduce the success of autologous HCT for lymphoma patients with the CYP2B6*5 variant. PMID:25677220

  4. Routine HLA-B genotyping with PCR-sequence-specific oligonucleotides detects a B*52 variant (B*5206).

    PubMed

    Hoelsch, K; Lenggeler, I; Pfannes, W; Knabe, H; Klein, H-G; Woelpl, A

    2005-05-01

    A new human leukocyte antigen (HLA)-B allele was found during routine typing of samples for a German unrelated bone marrow donor registry, the "Aktion Knochenmarkspende Bayern". After first interpretation of data of two independent low-resolution sequence-specific oligonucleotide typing tests, a B*51 variant was suggested. Further analysis via sequence-based typing identified the sequence as new B*52 allele. This new allele officially assigned as B*5206 differs from HLA-B*520102 by one nucleotide exchange in exon 2. The mutation is located at nucleotide position 274, at which a cytosine is substituted by a thymine leading to an amino acid change at protein position 67 from serine (TCC) to phenylalanine (TTC).

  5. Identification, Classification, and Phylogeny of the Pathogenic Species Exophiala jeanselmei and Related Species by Mitochondrial Cytochrome b Gene Analysis

    PubMed Central

    Wang, Li; Yokoyama, Koji; Miyaji, Makoto; Nishimura, Kazuko

    2001-01-01

    We analyzed a 402-bp sequence of the mitochondrial cytochrome b gene of 34 strains of Exophiala jeanselmei and 16 strains representing 12 related species. The strains of E. jeanselmei were classified into 20 DNA types and 17 amino acid types. The differences between these strains were found in 1 to 60 nucleotides and 1 to 17 amino acids. On the basis of the identities and similarities of nucleotide and amino acid sequences, some strains were reidentified: i.e., two strains of E. jeanselmei var. hetermorpha and one strain of E. castellanii as E. dermatitidis (including the type strain), three strains of E. jeanselmei as E. jeanselmei var. lecanii-corni (including the type strain), three strains of E. jeanselmei as E. bergeri (including the type strain), seven strains of E. jeanselmei as E. pisciphila (including the type strain), seven strains of E. jeanselmei as E. jeanselmei var. jeanselmei (including the type strain), one strain of E. jeanselmei as Fonsecaea pedrosoi (including the type strain), and one strain of E. jeanselmei as E. spinifera (including the type strain). Some E. jeanselmei strains showed distinct nucleotide and amino acid sequences. The amino-acid-based UPGMA (unweighted pair group method with the arithmetic mean) tree exhibited nearly the same topology as those of the DNA-based trees obtained by neighbor joining, maximum parsimony, and maximum likelihood methods. PMID:11724862

  6. Patient-Specific Neutralizing Antibody Responses to Herpes Simplex Virus Are Attributed to Epitopes on gD, gB, or Both and Can Be Type Specific

    PubMed Central

    Huang, Zhen-Yu; Gallagher, John R.; Lin, Yixin; Lou, Huan; Whitbeck, J. Charles; Wald, Anna; Cohen, Gary H.; Eisenberg, Roselyn J.

    2015-01-01

    ABSTRACT Herpes simplex virus 1 (HSV-1) and HSV-2 infect many humans and establish a latent infection in sensory ganglia. Although some infected people suffer periodic recurrences, others do not. Infected people mount both cell-mediated and humoral responses, including the production of virus-neutralizing antibodies (Abs) directed at viral entry glycoproteins. Previously, we examined IgGs from 10 HSV-seropositive individuals; all neutralized virus and were directed primarily against gD or gD+gB. Here, we expand our studies and examine 32 additional sera from HSV-infected individuals, 23 of whom had no recurrent disease. Using an Octet RED96 system, we screened all 32 serum samples directly for both glycoprotein binding and competition with known neutralizing anti-gD and -gB monoclonal Abs (MAbs). On average, the recurrent cohort exhibited higher binding to gD and gB and had higher neutralization titers. There were similar trends in the blocking of MAbs to critical gD and gB epitopes. When we depleted six sera of Abs to specific glycoproteins, we found different types of responses, but always directed primarily at gD and/or gB. Interestingly, in one dual-infected person, the neutralizing response to HSV-2 was due to gD2 and gB2, whereas HSV-1 neutralization was due to gD1 and gB1. In another case, virus neutralization was HSV-1 specific, with the Ab response directed entirely at gB1, despite this serum blocking type-common anti-gD and -gB neutralizing MAbs. These data are pertinent in the design of future HSV vaccines since they demonstrate the importance of both serotypes of gD and gB as immunogens. IMPORTANCE We previously showed that people infected with HSV produce neutralizing Abs directed against gD or a combination of gD+gB (and in one case, gD+gB+gC, which was HSV-1 specific). In this more extensive study, we again found that gD or gD+gB can account for the virus neutralizing response and critical epitopes of one or both of these proteins are represented in

  7. Patient-Specific Neutralizing Antibody Responses to Herpes Simplex Virus Are Attributed to Epitopes on gD, gB, or Both and Can Be Type Specific.

    PubMed

    Cairns, Tina M; Huang, Zhen-Yu; Gallagher, John R; Lin, Yixin; Lou, Huan; Whitbeck, J Charles; Wald, Anna; Cohen, Gary H; Eisenberg, Roselyn J

    2015-09-01

    Herpes simplex virus 1 (HSV-1) and HSV-2 infect many humans and establish a latent infection in sensory ganglia. Although some infected people suffer periodic recurrences, others do not. Infected people mount both cell-mediated and humoral responses, including the production of virus-neutralizing antibodies (Abs) directed at viral entry glycoproteins. Previously, we examined IgGs from 10 HSV-seropositive individuals; all neutralized virus and were directed primarily against gD or gD+gB. Here, we expand our studies and examine 32 additional sera from HSV-infected individuals, 23 of whom had no recurrent disease. Using an Octet RED96 system, we screened all 32 serum samples directly for both glycoprotein binding and competition with known neutralizing anti-gD and -gB monoclonal Abs (MAbs). On average, the recurrent cohort exhibited higher binding to gD and gB and had higher neutralization titers. There were similar trends in the blocking of MAbs to critical gD and gB epitopes. When we depleted six sera of Abs to specific glycoproteins, we found different types of responses, but always directed primarily at gD and/or gB. Interestingly, in one dual-infected person, the neutralizing response to HSV-2 was due to gD2 and gB2, whereas HSV-1 neutralization was due to gD1 and gB1. In another case, virus neutralization was HSV-1 specific, with the Ab response directed entirely at gB1, despite this serum blocking type-common anti-gD and -gB neutralizing MAbs. These data are pertinent in the design of future HSV vaccines since they demonstrate the importance of both serotypes of gD and gB as immunogens. We previously showed that people infected with HSV produce neutralizing Abs directed against gD or a combination of gD+gB (and in one case, gD+gB+gC, which was HSV-1 specific). In this more extensive study, we again found that gD or gD+gB can account for the virus neutralizing response and critical epitopes of one or both of these proteins are represented in sera of naturally

  8. Regulation of nitrite resistance of the cytochrome cbb3 oxidase by cytochrome c ScyA in Shewanella oneidensis

    PubMed Central

    Yin, Jianhua; Jin, Miao; Zhang, Haiyan; Ju, Lili; Zhang, Lili; Gao, Haichun

    2015-01-01

    Cytochrome c proteins, as enzymes to exchange electrons with substrates or as pure electron carriers to shuttle electrons, play vital roles in bacterial respiration and photosynthesis. In Shewanella oneidensis, a research model for the respiratory diversity, at least 42 c-type cytochromes are predicted to be encoded in the genome and are regarded to be the foundation of its highly branched electron transport pathways. However, only a small number of c-type cytochromes have been extensively studied. In this study, we identify soluble cytochrome c ScyA as an important factor influencing the nitrite resistance of a strain devoid of the bd oxidase by utilizing a newly developed transposon mutagenesis vector, which enables overexpression of the gene(s) downstream of the insertion site. We show that when in overabundance ScyA facilitates growth against nitrite inhibition by enhancing nitrite resistance of the cbb3 oxidase. Based on the data presented in this study, we suggest two possible mechanisms underlying the observed effect of ScyA: (1) ScyA increases electron flow to the cbb3 oxidase; (2) ScyA promotes nitrite resistance of the cbb3 oxidase, possibly by direct interaction. PMID:25417822

  9. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants.

    PubMed

    Meyer, Thomas; Melin, Frédéric; Richter, Oliver-M H; Ludwig, Bernd; Kannt, Aimo; Müller, Hanne; Michel, Hartmut; Hellwig, Petra

    2015-02-27

    Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn(2+) ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Evidence from Studies with Acifluorfen for Participation of a Flavin-Cytochrome Complex in Blue Light Photoreception for Phototropism of Oat Coleoptiles 12

    PubMed Central

    Leong, Ta-Yan; Briggs, Winslow R.

    1982-01-01

    The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor. PMID:16662593

  11. Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race.

    PubMed

    Tsou, Chung-Yau; Matsunaga, Shigeki; Okada, Shigeru

    2018-01-01

    The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a k m for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a k cat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Interspecies Variation in the Functional Consequences of Mutation of Cytochrome c

    PubMed Central

    Josephs, Tracy M.; Hibbs, Moira E.; Ong, Lily; Morison, Ian M.; Ledgerwood, Elizabeth C.

    2015-01-01

    The naturally occurring human cytochrome c variant (G41S) is associated with a mild autosomal dominant thrombocytopenia (Thrombocytopenia Cargeeg) caused by dysregulation of platelet production. The molecular basis of the platelet production defect is unknown. Despite high conservation of cytochrome c between human and mouse (91.4% identity), introducing the G41S mutation into mouse cytochrome c in a knockin mouse (Cycs G41S/G41S) did not recapitulate the low platelet phenotype of Thrombocytopenia Cargeeg. While investigating the cause of this disparity we found a lack of conservation of the functional impact of cytochrome c mutations on caspase activation across species. Mutation of cytochrome c at residue 41 has distinct effects on the ability of cytochrome c to activate caspases depending on the species of both the cytochrome c and its binding partner Apaf-1. In contrast to our previous results showing the G41S mutation increases the ability of human cytochrome c to activate caspases, here we find this activity is decreased in mouse G41S cytochrome c. Additionally unlike wildtype human cytochrome c, G41S cytochrome c is unable to activate caspases in Xenopus embryo extracts. Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1. This suggests that the electrostatic interaction between cytochrome c and Apaf-1 is not the sole determinant of binding, with additional factors controlling binding specificity and affinity. These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway. PMID:26086723

  13. Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi VI. Electron Transport in Mutant Strains Lacking Either Cytochrome 553 or Plastocyanin 1

    PubMed Central

    Gorman, Donald S.; Levine, R. P.

    1966-01-01

    A mutant strain of Chlamydomonas reinhardi, ac-206, lacks cytochrome 553, at least in an active and detectable form. Chloroplast fragments of this mutant strain are inactive in the photoreduction of NADP when the source of electrons is water, but they are active when the electron source is 2,6-dichlorophenolindophenol and ascorbate. The addition of either cytochrome 553 or plastocyanin, obtained from the wild-type strain, has no effect upon the photosynthetic activities of the mutant strain. Cells of the mutant strain lack both the soluble and insoluble forms of cytochrome 553, but they possess the mitochondrial type cytochrome c. Thus, the loss of cytochrome 553 appears to be specific. Another mutant strain, ac-208, lacks plastocyanin, or possesses it in an inactive and undetectable form. Chloroplast fragments of ac-208 are inactive in the photoreduction of NADP with either water or 2,6-dichlorophenolindophenol and ascorbate as electron donors. However, these reactions are restored upon the addition of plastocyanin. The addition of cytochrome 553 has no effect. The measurement of light-induced absorbance changes with ac-208 reveal that, in the absence of plastocyanin, light fails to sensitize the oxidation of cytochrome 553, but it will sensitize its reduction. However, the addition of plastocyanin restores the light-induced cytochrome oxidation. A third mutant strain, ac-208 (sup.) carries a suppressor mutation that partially restores the wild phenotype. This mutant strain appears to possess a plastocyanin that is less stable than that of the wild-type strain. The observations with the mutant strains are discussed in terms of the sequence of electron transport System II → cytochrome 553 → plastocyanin → System I. PMID:16656453

  14. Calcium transport in vesicles energized by cytochrome oxidase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rosier, Randy N.

    1979-01-01

    Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K + selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K + flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interactionmore » with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.« less

  15. Cardiolipin deficiency causes a dissociation of the b 6 c:caa 3 megacomplex in B. subtilis membranes.

    PubMed

    García Montes de Oca, Led Yered Jafet; Cabellos Avelar, Tecilli; Picón Garrido, Gerardo Ignacio; Chagoya-López, Alicia; González de la Vara, Luis; Delgado Buenrostro, Norma Laura; Chirino-López, Yolanda Irasema; Gómez-Lojero, Carlos; Gutiérrez-Cirlos, Emma Berta

    2016-08-01

    The associations among respiratory complexes in energy-transducing membranes have been established. In fact, it is known that the Gram-negative bacteria Paracoccus denitrificans and Escherichia coli have respiratory supercomplexes in their membranes. These supercomplexes are important for channeling substrates between enzymes in a metabolic pathway, and the assembly of these supercomplexes depends on the protein subunits and membrane lipids, mainly cardiolipin, which is present in both the mitochondrial inner membrane and bacterial membranes. The Gram-positive bacterium Bacillus subtilis has a branched respiratory chain, in which some complexes generate proton motive force whereas others constitute an escape valve of excess reducing power. Some peculiarities of this respiratory chain are the following: a type II NADH dehydrogenase, a unique b 6 c complex that has a b 6 type cytochrome with a covalently bound heme, and a c-type heme attached to the third subunit, which is similar to subunit IV of the photosynthetic b 6 f complex. Cytochrome c oxygen reductase (caa 3 ) contains a c-type cytochrome on subunit I. We previously showed that the b 6 c and the caa 3 complexes form a supercomplex. Both the b 6 c and the caa 3 together with the quinol oxygen reductase aa 3 generate the proton motive force in B. subtilis. In order to seek proof that this supercomplex is important for bacterial growth in aerobic conditions we compared the b 6 c: caa 3 supercomplex from wild type membranes with membranes from two mutants lacking cardiolipin. Both mutant complexes were found to have similar activity and heme content as the wild type. Clear native electrophoresis showed that mutants lacking cardiolipin had b 6 c:caa 3 supercomplexes of lower mass or even individual complexes after membrane solubilization with digitonin. The use of dodecyl maltoside revealed a more evident difference between wild-type and mutant supercomplexes. Here we provide evidence showing that cardiolipin

  16. Identification of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) by using polymerase chain reaction amplification and restriction analysis of the mitochondrial cytochrome b gene.

    PubMed

    Carrera, E; García, T; Céspedes, A; González, I; Sanz, B; Hernández, P E; Martín, R

    1998-04-01

    Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the identification of fresh and smoked samples of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Digestion of the 359-bp PCR product with the endonucleases EcoRV and TaqI yielded specific banding patterns for salmon and trout. This genetic marker can be very useful for detecting fraudulent substitution of the cheaper smoked trout for the more expensive smoked salmon.

  17. On the potential of using peculiarities of the protein intrinsic disorder distribution in mitochondrial cytochrome b to identify the source of animal meats

    PubMed Central

    Yacoub, Haitham A.; Sadek, Mahmoud A.; Uversky, Vladimir N.

    2017-01-01

    ABSTRACT This study was conducted to identify the source of animal meat based on the peculiarities of protein intrinsic disorder distribution in mitochondrial cytochrome b (mtCyt-b). The analysis revealed that animal and avian species can be discriminated based on the proportions of the two groups of residues, Leu+Ile, and Ser+Pro+Ala, in the amino acid sequences of their mtCyt-b. Although levels of the overall intrinsic disorder in mtCyt-b is not very high, the peculiarities of disorder distribution within the sequences of mtCyt-b from different species varies in a rather specific way. In fact, positions and intensities of disorder/flexibility “signals” in the corresponding disorder profiles are relatively unique for avian and animal species. Therefore, it is possible to devise a set of simple rules based on the peculiarities of disorder profiles of their mtCyt-b proteins to discriminate among species. This intrinsic disorder-based analysis represents a new technique that could be used to provide a promising solution for identification of the source of meats. PMID:28331777

  18. Genetic diversities of cytochrome B in Xinjiang Uyghur unveiled its origin and migration history

    PubMed Central

    2013-01-01

    Background Uyghurs are one of the many populations of Central Eurasia that is considered to be genetically related to Eastern and Western Eurasian populations. However, there are some different opinions on the relative importance of the degree of Eastern and Western Eurasian genetic influence. In addition, the genetic diversity of the Uyghur in different geographic locations has not been clearly studied. Results In this study, we are the first to report on the DNA polymorphism of cytochrome B in the Uyghur population located in Xinjiang in northwest China. We observed a total of 102 mutant sites in the 240 samples that were studied. The average number of mutated nucleotides in the samples was 5.126. A total of 93 different haplotypes were observed. The gene diversity and discrimination power were 0.9480 and 0.9440, respectively. There were founder and bottleneck haplotypes observed in Xinjiang Uyghurs. Xinjiang Uyghurs are more genetically related to Chinese population in genetics than to Caucasians. Moreover, there was genetic diversity between Uyghurs from the southern and northern regions. There was significance in genetic distance between the southern Xinjiang Uyghurs and Chinese population, but not between the northern Xinjiang Uyghurs and Chinese. The European vs. East Asian contribution to the ten regional Uyghur groups varies among the groups and the European contribution to the Uyghur increases from north to south geographically. Conclusion This study is the first report on DNA polymorphisms of cytochrome B in the Uyghur population. The study also further confirms that there are significant genetic differences among the Uyghurs in different geographical locations. PMID:24103151

  19. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

    PubMed Central

    Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F

    1988-01-01

    V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

  20. The Cytochrome b 6 f Complex: Biophysical Aspects of Its Functioning in Chloroplasts.

    PubMed

    Tikhonov, Alexander N

    2018-01-01

    This chapter presents an overview of structural properties of the cytochrome (Cyt) b 6 f complex and its functioning in chloroplasts. The Cyt b 6 f complex stands at the crossroad of photosynthetic electron transport pathways, providing connectivity between Photosystem (PSI) and Photosysten II (PSII) and pumping protons across the membrane into the thylakoid lumen. After a brief review of the chloroplast electron transport chain, the consideration is focused on the structural organization of the Cyt b 6 f complex and its interaction with plastoquinol (PQH 2 , reduced form of plastoquinone), a mediator of electron transfer from PSII to the Cyt b 6 f complex. The processes of PQH 2 oxidation by the Cyt b 6 f complex have been considered within the framework of the Mitchell's Q-cycle. The overall rate of the intersystem electron transport is determined by PQH 2 turnover at the quinone-binding site Q o of the Cyt b 6 f complex. The rate of PQH 2 oxidation is controlled by the intrathylakoid pH in , which value determines the protonation/deprotonation events in the Q o -center. Two other regulatory mechanisms associated with the Cyt b 6 f complex are briefly overviewed: (i) redistribution of electron fluxes between alternative (linear and cyclic) pathways, and (ii) "state transitions" related to redistribution of solar energy between PSI and PSII.

  1. Mitochondrial cytochrome b sequence variations and population structure of Siberian chipmunk (Tamias sibiricus) in Northeastern Asia and population substructure in South Korea.

    PubMed

    Lee, Mu-Yeong; Lissovsky, Andrey A; Park, Sun-Kyung; Obolenskaya, Ekaterina V; Dokuchaev, Nikolay E; Zhang, Ya-Ping; Yu, Li; Kim, Young-Jun; Voloshina, Inna; Myslenkov, Alexander; Choi, Tae-Young; Min, Mi-Sook; Lee, Hang

    2008-12-31

    Twenty-five chipmunk species occur in the world, of which only the Siberian chipmunk, Tamias sibiricus, inhabits Asia. To investigate mitochondrial cytochrome b sequence variations and population structure of the Siberian chipmunk in northeastern Asia, we examined mitochondrial cytochrome b sequences (1140 bp) from 3 countries. Analyses of 41 individuals from South Korea and 33 individuals from Russia and northeast China resulted in 37 haplotypes and 27 haplotypes, respectively. There were no shared haplotypes between South Korea and Russia--northeast China. Phylogenetic trees and network analysis showed 2 major maternal lineages for haplotypes, referred to as the S and R lineages. Haplotype grouping in each cluster was nearly coincident with its geographic affinity. In particular, 3 distinct groups were found that mostly clustered in the northern, central and southern parts of South Korea. Nucleotide diversity of the S lineage was twice that of lineage R. The divergence between S and R lineages was estimated to be 2.98-0.98 Myr. During the ice age, there may have been at least 2 refuges in South Korea and Russia--northeast China. The sequence variation between the S and R lineages was 11.3% (K2P), which is indicative of specific recognition in rodents. These results suggest that T. sibiricus from South Korea could be considered a separate species. However, additional information, such as details of distribution, nuclear genes data or morphology, is required to strengthen this hypothesis.

  2. Vanadium(V) Reduction by Shewanella oneidensis MR-1 Requires Menaquinone and Cytochromes from the Cytoplasmic and Outer Membranes

    PubMed Central

    Myers, Judith M.; Antholine, William E.; Myers, Charles R.

    2004-01-01

    The metal-reducing bacterium Shewanella oneidensis MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome. In these studies, several cell components required for the reduction of vanadium(V) were determined. V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB. A partial role for the OM cytochrome OmcA was evident. Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV). V(V) reduction did not support anaerobic growth. This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species. PMID:15006760

  3. Cytochrome and Alternative Pathway Respiration in Green Algae 1

    PubMed Central

    Weger, Harold G.; Guy, Robert D.; Turpin, David H.

    1990-01-01

    Inhibitor titration curves and discrimination against 18O2 by mitochondrial respiration in three strains of green algae (Selenastrum minutum [Naeg.] Collins, and two strains of Chlamydomonas reinhardtii Dangeard) with differing respiratory capabilities were determined. Discrimination for cytochrome pathway respiration ranged from 19.89 to 20.43%. Discrimination for alternative pathway respiration by wild-type C. reinhardtii (measured in the presence of KCN) was 25.46%, while discrimination values for a cytochrome oxidase deficient mutant of C. reinhardtii ranged from 24.24 to 24.96%. In the absence of KCN, the alternative pathway was not engaged in wild-type C. reinhardtii, the only algal strain that possessed both cytochrome and alternative pathway capacities. PMID:16667462

  4. Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution▿ †

    PubMed Central

    Trefzer, Axel; Jungmann, Volker; Molnár, István; Botejue, Ajit; Buckel, Dagmar; Frey, Gerhard; Hill, D. Steven; Jörg, Mario; Ligon, James M.; Mason, Dylan; Moore, David; Pachlatko, J. Paul; Richardson, Toby H.; Spangenberg, Petra; Wall, Mark A.; Zirkle, Ross; Stege, Justin T.

    2007-01-01

    Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin. PMID:17483257

  5. Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan.

    PubMed

    Marco, Jorge D; Bhutto, Abdul M; Soomro, Farooq R; Baloch, Javed H; Barroso, Paola A; Kato, Hirotomo; Uezato, Hiroshi; Katakura, Ken; Korenaga, Masataka; Nonaka, Shigeo; Hashiguchi, Yoshihisa

    2006-08-01

    Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

  6. Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

    PubMed

    Miura, Hiroshi; Mogi, Tatsushi; Ano, Yoshitaka; Migita, Catharina T; Matsutani, Minenosuke; Yakushi, Toshiharu; Kita, Kiyoshi; Matsushita, Kazunobu

    2013-06-01

    Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

  7. Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site.

    PubMed

    Letessier, Anne; Millot, Gaël A; Koundrioukoff, Stéphane; Lachagès, Anne-Marie; Vogt, Nicolas; Hansen, R Scott; Malfoy, Bernard; Brison, Olivier; Debatisse, Michelle

    2011-02-03

    Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.

  8. Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu

    2013-02-15

    The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomicmore » approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed

  9. Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase.

    PubMed

    Paumann, Martina; Feichtinger, Markus; Bernroitner, Margit; Goldfuhs, Judith; Jakopitsch, Christa; Furtmüller, Paul G; Regelsberger, Günther; Peschek, Günter A; Obinger, Christian

    2004-10-08

    Cytochrome c6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b6f to photosystem I. The CuA domain of cytochrome c oxidase, the terminal enzyme which catalyzes the four-electron reduction of molecular oxygen in the respiratory chains of mitochondria and many bacteria, also has a periplasmic location. In order to test whether cytochrome c6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccmA-H gene cluster) and the recombinant soluble CuA domain (i.e., the donor binding and electron entry site) of subunit II of cytochrome c oxidase from Synechocystis PCC 6803. The forward and the reverse electron transfer reactions were studied by the stopped-flow technique and yielded apparent bimolecular rate constants of (3.3 +/- 0.3) x 10(5) M(-1) s(-1) and (3.9 +/- 0.1) x 10(6) M(-1) s(-1), respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 degrees C. This corresponds to an equilibrium constant Keq of 0.085 in the physiological direction (DeltarG'0 = 6.1 kJ/mol). The reduction of the CuA fragment by cytochrome c6 is almost independent on ionic strength, which is in contrast to the reaction of the CuA domain with horse heart cytochrome c, which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c6 as mobile electron carrier in both cyanobacterial electron transport pathways. Copyright 2004 Federation of European Biochemical Societies

  10. Molecular phylogeny of mitochondrial cytochrome b and 12S rRNA sequences in the Felidae: ocelot and domestic cat lineages.

    PubMed

    Masuda, R; Lopez, J V; Slattery, J P; Yuhki, N; O'Brien, S J

    1996-12-01

    Molecular phylogeny of the cat family Felidae is derived using two mitochondrial genes, cytochrome b and 12S rRNA. Phylogenetic methods of weighted maximum parsimony and minimum evolution estimated by neighbor-joining are employed to reconstruct topologies among 20 extant felid species. Sequence analyses of 363 bp of cytochrome b and 376 bp of the 12S rRNA genes yielded average pair-wise similarity values between felids ranging from 94 to 99% and from 85 to 99%, respectively. Phylogenetic reconstruction supports more recent, intralineage associations but fails to completely resolve interlineage relationships. Both genes produce a monophyletic group of Felis species but vary in the placement of the pallas cat. The ocelot lineage represents an early divergence within the Felidae, with strong associations between ocelot and margay, Geoffroy's cat and kodkod, and pampas cat and tigrina. Implications of the relative recency of felid evolution, presence of ancestral polymorphisms, and influence of outgroups in placement of the topological root are discussed.

  11. Mitochondrial Group II Introns, Cytochrome c Oxidase, and Senescence in Podospora anserina†

    PubMed Central

    Begel, Odile; Boulay, Jocelyne; Albert, Beatrice; Dufour, Eric; Sainsard-Chanet, Annie

    1999-01-01

    Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (α) has been thought to play a prominent role in this syndrome. Intron α is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the α sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron α. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron α is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron α plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, “immortality” can be acquired not by the absence of intron α but rather by the lack of active cytochrome c oxidase. PMID:10330149

  12. Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog meat adulteration in meatball formulation.

    PubMed

    Rahman, Md Mahfujur; Ali, Md Eaqub; Hamid, Sharifah Bee Abd; Mustafa, Shuhaimi; Hashim, Uda; Hanapi, Ummi Kalthum

    2014-08-01

    A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was developed. The assay selectively amplified a 100-bp region of canine mitochondrial cytochrome b gene from pure, raw, processed and mixed backgrounds. The specificity of the assay was tested against 11 animals and 3 plants species, commonly available for meatball formulation. The stability of the assay was proven under extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using 0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in processed foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Component identification of electron transport chains in curdlan-producing Agrobacterium sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis.

    PubMed

    Zhang, Hongtao; Setubal, Joao Carlos; Zhan, Xiaobei; Zheng, Zhiyong; Yu, Lijun; Wu, Jianrong; Chen, Dingqiang

    2011-06-01

    Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.

  14. Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47.

    PubMed

    Guerra-Castellano, Alejandra; Díaz-Moreno, Irene; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-04-01

    Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c - namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death - is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II.

    PubMed

    Takagi, Daisuke; Ifuku, Kentaro; Nishimura, Taishi; Miyake, Chikahiro

    2018-05-22

    The light reactions of photosynthesis are known to comprise both linear and cyclic electron flow in order to convert light energy into chemical energy in the form of NADPH and ATP. Antimycin A (AA) has been proposed as an inhibitor of ferredoxin-dependent cyclic electron flow around photosystem I (CEF-PSI) in photosynthesis research. However, its precise inhibitory mechanism and target site had not been elucidated yet. Here we show that AA inhibits the cyclic (alternative) electron flow via cytochrome b 559 (Cyt b 559 ) within photosystem II (CEF-PSII). When AA was applied to thylakoid membranes isolated from spinach leaves, the high potential form of Cyt b 559 , which was reduced in the dark, was transformed into the lower potential forms and readily oxidized by molecular oxygen. In the absence of AA, the reduced Cyt b 559 was oxidized by P680 + upon light illumination and re-reduced in the dark, mainly by the electron from the Q B site on the acceptor side of PSII. In contrast, AA suppressed the oxidation of Cyt b 559 and induced its reduction under the illumination. This inhibition of Cyt b 559 oxidation by AA enhanced photoinhibition of PSII. Based on the above results, we propose caution regarding the use of AA for evaluating CEF-PSI per se and concurrently propose that AA provides for new insights into, and interpretations of, the physiological importance of Cyt b 559 , rather than that of CEF-PSI in photosynthetic organisms.

  16. Homeostatic plasticity shapes cell-type-specific wiring in the retina

    PubMed Central

    Tien, Nai-Wen; Soto, Florentina; Kerschensteiner, Daniel

    2017-01-01

    SUMMARY Convergent input from different presynaptic partners shapes the responses of postsynaptic neurons. Whether developing postsynaptic neurons establish connections with each presynaptic partner independently, or balance inputs to attain specific responses is unclear. Retinal ganglion cells (RGCs) receive convergent input from bipolar cell types with different contrast responses and temporal tuning. Here, using optogenetic activation and pharmacogenetic silencing, we found that type 6 bipolar cells (B6) dominate excitatory input to ONα-RGCs. We generated mice in which B6 cells were selectively removed from developing circuits (B6-DTA). In B6-DTA mice, ONα-RGCs adjusted connectivity with other bipolar cells in a cell-type-specific manner. They recruited new partners, increased synapses with some existing partners, and maintained constant input from others. Patch clamp recordings revealed that anatomical rewiring precisely preserved contrast- and temporal frequency response functions of ONα-RGCs, indicating that homeostatic plasticity shapes cell-type-specific wiring in the developing retina to stabilize visual information sent to the brain. PMID:28457596

  17. Combustion derived ultrafine particles induce cytochrome P-450 expression in specific lung compartments in the developing neonatal and adult rat

    PubMed Central

    Chan, Jackie K. W.; Vogel, Christoph F.; Baek, Jaeeun; Kodani, Sean D.; Uppal, Ravi S.; Bein, Keith J.; Anderson, Donald S.

    2013-01-01

    Vehicle exhaust is rich in polycyclic aromatic hydrocarbons (PAH) and can be a dominant contributor to ultrafine urban particulate matter (PM). Exposure to ultrafine PM is correlated with respiratory infections and asthmatic symptoms in young children. The lung undergoes substantial growth, alveolarization, and cellular maturation within the first years of life, which may be impacted by environmental pollutants such as PM. PAHs in PM can serve as ligands for the aryl hydrocarbon receptor (AhR) that induces expression of certain isozymes in the cytochrome P-450 superfamily, such as CYP1A1 and CYP1B1, localized in specific lung cell types. Although AhR activation and induction has been widely studied, its context within PM exposure and impact on the developing lung is poorly understood. In response, we have developed a replicable ultrafine premixed flame particle (PFP) generating system and used in vitro and in vivo models to define PM effects on AhR activation in the developing lung. We exposed 7-day neonatal and adult rats to a single 6-h PFP exposure and determined that PFPs cause significant parenchymal toxicity in neonates. PFPs contain weak AhR agonists that upregulate AhR-xenobiotic response element activity and expression and are capable inducers of CYP1A1 and CYP1B1 expression in both ages with different spatial and temporal patterns. Neonatal CYP1A1 expression was muted and delayed compared with adults, possibly because of differences in the enzyme maturation. We conclude that the inability of neonates to sufficiently adapt in response to PFP exposure may, in part, explain their susceptibility to PFP and urban ultrafine PM. PMID:23502512

  18. Prevention of dopaminergic toxicity of MPTP in mice by phenylethylamine, a specific substrate of type B monoamine oxidase.

    PubMed Central

    Melamed, E.; Youdim, M. B.

    1985-01-01

    N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is toxic to dopaminergic neurones in several mammalian species including mice. Combined treatment with phenylethylamine prevented in mice the long-term (30 days post-treatment) dopamine depletions in striatum induced by MPTP. Phenylethylamine, a naturally-occurring specific substrate of monoamine oxidase (MAO) type B, probably protects against effects of MPTP by competitively inhibiting the oxidative conversion of MPTP to its toxic metabolite N-methyl-4-phenylpyridinium ion catalysed by MAO-B. PMID:3877535

  19. In vivo detection of peripherin-specific autoreactive B cells during type 1 diabetes pathogenesis1

    PubMed Central

    Garabatos, Nahir; Alvarez, Raimon; Carrillo, Jorge; Carrascal, Jorge; Izquierdo, Cristina; Chapman, Harold D.; Presa, Maximiliano; Mora, Conchi; Serreze, David V.; Verdaguer, Joan; Stratmann, Thomas

    2014-01-01

    Summary Autoreactive B cells are essential for the pathogenesis of type 1 diabetes. The genesis and dynamics of autoreactive B cells remain unknown. Here, we analyzed the immune response in the NOD mouse model to the neuronal protein peripherin (PRPH), a target antigen of islet-infiltrating B cells. PRPH autoreactive B cells recognized a single linear epitope of this protein, in contrast to the multiple epitope recognition commonly observed during autoreactive B cell responses. Autoantibodies to this epitope were also detected in the disease-resistant NOR and C57BL/6 strains. To specifically detect the accumulation of these B cells, we developed a novel approach, octameric peptide display, to follow the dynamics and localization of anti-PRPH B cell during disease progression. Before extended insulitis established, anti-PRPH B cells preferentially accumulated in the peritoneum. Anti-PRPH B cells were likewise detected in C57BL/6 mice, albeit at lower frequencies. As disease unfolded in NOD mice, anti-PRPH B cells invaded the islets and increased in number at the peritoneum of diabetic but not pre-diabetic mice. Isotype switched B cells were only detected in the peritoneum. Anti-PRPH B cells represent a heterogeneous population composed of both B1 and B2 subsets. In the spleen, anti-PRPH B cell were predominantly in the follicular subset. Therefore, anti-PRPH B cells represent a heterogeneous population that is generated early in life but proliferates as diabetes establishes. These findings on the temporal and spatial progression of autoreactive B cells should be relevant for our understanding of B cell function in diabetes pathogenesis. PMID:24610011

  20. Exceptional longevity and exceptionally high metabolic rates in anthropoid primates are linked to a major modification of the ubiquinone reduction site of cytochrome b.

    PubMed

    Rottenberg, Hagai

    2014-10-01

    The maximal lifespan of Anthropoid primates (monkeys, apes and humans) exceed the lifespan of most other mammals of equal body mass. Unexpectedly, their exceptional longevity is associated with exceptionally high metabolic rates, in apparent contradiction to the Free Radical Theory of Aging. It was therefore suggested that in anthropoid primates (and several other taxa of mammals and birds) the mitochondrial electron transport complexes evolved to modify the relationship between basal electron transport and superoxide generation to allow for the evolution of exceptional longevity. Cytochrome b, the core protein of the bc1 complex is a major source of superoxide. The amino-acid sequence of cytochrome b evolved much faster in anthropoid than in prosimian primates, and most other mammals, resulting in a large change in the amino-acids composition of the protein. As a result of these changes cytochrome b in anthropoid primates is significantly less hydrophobic and contains more polar residues than other primates and most other mammals. Most of these changes are clustered around the reduction site of uboiquinone. In particular a key positively charged residue, arginine 313, that interacts with propionate D of heme bH, and thus raises its redox potential, is substituted in anthropoid primates with the neutral residue glutamine, most likely resulting in a lower redox potential of heme bH and faster reduction of ubiquinone at high proton motive force. It is suggested that these changes contribute to the observed increased rates of basal metabolism and reduce the rates of superoxide production, thus allowing for increased lifespan.

  1. Cloning, cell-type specificity, and regulatory function of the mouse alpha(1B)-adrenergic receptor promoter.

    PubMed

    Zuscik, M J; Piascik, M T; Perez, D M

    1999-12-01

    The functionality of a 3422-base pair promoter fragment from the mouse alpha(1B)-adrenergic receptor (alpha(1B)AR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat alpha(1B)AR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known alpha(1B)AR-expressing BC(3)H1, NB41A3, and DDT(1)MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known alpha(1B)AR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that alpha(1)ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-forming cells, which are known to be alpha(1B)AR negative, showed minimal CAT expression. Indicating regulatory function through cis-acting elements, RAT-1, R3T3, NB41A3, BC(3)H1, and DDT(1)MF2 cells transfected with the promoter-CAT construct all showed increased CAT production when challenged with forskolin or hypoxic conditions. Additionally, tissue-specific regulation of the promoter was observed when cells were simultaneously challenged with both forskolin and hypoxia. These results collectively demonstrate that a 3.4-kb PvuII fragment of the murine alpha(1B)AR gene promoter can: 1) drive tissue-specific production of a CAT reporter in both clonal and primary cell lines; and 2) confer tissue-specific regulation

  2. Incidental Detection of Type B2 Thymoma on 68Ga-Labeled Prostate-Specific Membrane Antigen PET/CT Imaging.

    PubMed

    Krishnaraju, Venkata Subramanian; Basher, Rajender Kumar; Singh, Harmandeep; Singh, Shrawan Kumar; Bal, Amanjit; Mittal, Bhagwant Rai

    2018-05-01

    Ga-labeled prostate-specific membrane antigen is a novel radiotracer for imaging of prostate cancer. We report a hormonally treated patient with prostate carcinoma, presenting with lower urinary tract symptoms and rising prostate-specific antigen levels, who underwent Ga-labeled prostate-specific membrane antigen PET/CT for suspected recurrence. No tracer avid lesion was noted in the prostate gland and locoregional area. However, intense tracer avid heterogeneously enhancing soft tissue lesion with cystic areas and coarse calcifications was seen in the anterior mediastinum. PET/CT-guided biopsy from the mediastenal lesion revealed type B2 thymoma.

  3. Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

    PubMed

    Kurdrid, Pavinee; Subudhi, Sanjukta; Cheevadhanarak, Supapon; Tanticharoen, Morakot; Hongsthong, Apiradee

    2007-12-01

    When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

  4. Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

    PubMed

    Pinto, L F; Moraes, E; Albano, R M; Silva, M C; Godoy, W; Glisovic, T; Lang, M A

    2001-11-01

    N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.

  5. Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris

    USGS Publications Warehouse

    Lovley, D.R.; Widman, P.K.; Woodward, J.C.; Phillips, E.J.P.

    1993-01-01

    The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium- contaminated waters and waste streams.

  6. Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b6f Complex from Nostoc sp. PCC 7120*

    PubMed Central

    Baniulis, Danas; Yamashita, Eiki; Whitelegge, Julian P.; Zatsman, Anna I.; Hendrich, Michael P.; Hasan, S. Saif; Ryan, Christopher M.; Cramer, William A.

    2009-01-01

    The crystal structure of the cyanobacterial cytochrome b6f complex has previously been solved to 3.0-Å resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b6f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b6f complex. Purified b6f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b6f complex, determined to a resolution of 3.0Å (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme bp that is rotated 180° about the α- and γ-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme cn is similar to that previously found in the b6f complex from other sources. PMID:19189962

  7. Presence of a new cytochrome b - like pigment with a peak at 567 nm in various aerobic bacteria.

    PubMed

    Jacobs, N J; O'Hara, J; Gray, C T

    1983-09-01

    Several physiological groups of bacteria were examined for the presence of a cytochrome b - like pigment which is demonstrable in dithionite-reduced minus substrate-reduced difference spectra. This pigment is characterized by an unusually high alpha band at 567 nm, a low concentration relative to conventional cytochromes, and an inability to be fully reduced by endogenous substrates or NADH. Previous studies with one denitrifying and nondenitrifying species of the genus Pseudomonas, in Paracoccus denitrificans, in Alcaligenes faecalis, in Azotobacter vinelandii, in Branhamella catarrhalis, and in Neisseria lactamicus. In all these organisms, the peak of the 567-nm pigment is accompanied by a peak of about equal height at approximately 559 nm, which exhibits similar properties to the 567-nm pigment. The 567-nm pigment was not demonstrable by this technique in Gluconobacter oxydans subspecies suboxydans, Bacillus subtilis, Bacillus licheniformis, Aeromonas hydrophilia, Escherichia coli, a Klebsiella species, Moraxella osloensis, Aquaspirillum itersonii, Micrococcus lysodeikticus, Micrococcus luteus, Agrobacterium tumefaciens, or Rhizobium meliloti.

  8. [Estimation of time detection limit for human cytochrome b in females of Lutzomyia evansi].

    PubMed

    Vergara, José Gabriel; Verbel-Vergara, Daniel; Montesino, Ana Milena; Pérez-Doria, Alveiro; Bejarano, Eduar Elías

    2017-03-29

    Molecular biology techniques have allowed a better knowledge of sources of blood meals in vector insects. However, the usefulness of these techniques depends on both the quantity of ingested blood and the digestion process in the insect. To identify the time limit for detection of the human cytochrome b (Cyt b) gene in experimentally fed females of Lutzomyia evansi. Eight groups of L. evansi females were fed on human blood and sacrificed at intervals of 24 hours post-ingestion. Total DNA was extracted from each female and a segment of 358 bp of Cyt b was amplified. In order to eliminate false positives, amplification products were subjected to a restriction fragment length polymorphism (RFLP) analysis. The human Cyt b gene segment was detected in 86% (49/57) of the females of L. evansi, from 0 to 168 hours after blood ingestion. In 7% (4/57) of the individuals we amplified insect DNA, while in the remaining 7%, the band of interest was not amplified. We did not find any statistical differences between groups of females sacrificed at different times post-blood meal regarding the amplification of the human Cyt b gene segment or the number of samples amplified. The human Cyt b gene segment was detectable in L. evansi females up to 168 hours after blood ingestion.

  9. Ultrafast hydrogen exchange reveals specific structural events during the initial stages of folding of cytochrome c.

    PubMed

    Fazelinia, Hossein; Xu, Ming; Cheng, Hong; Roder, Heinrich

    2014-01-15

    Many proteins undergo a sharp decrease in chain dimensions during early stages of folding, prior to the rate-limiting step in folding. However, it remains unclear whether compact states are the result of specific folding events or a general hydrophobic collapse of the poly peptide chain driven by the change in solvent conditions. To address this fundamental question, we extended the temporal resolution of NMR-detected H/D exchange labeling experiments into the microsecond regime by adopting a microfluidics approach. By observing the competition between H/D exchange and folding as a function of labeling pH, coupled with direct measurement of exchange rates in the unfolded state, we were able to monitor hydrogen-bond formation for over 50 individual backbone NH groups within the initial 140 microseconds of folding of horse cytochrome c. Clusters of solvent-shielded amide protons were observed in two α-helical segments in the C-terminal half of the protein, while the N-terminal helix remained largely unstructured, suggesting that proximity in the primary structure is a major factor in promoting helix formation and association at early stages of folding, while the entropically more costly long-range contacts between the N- and C-terminal helices are established only during later stages. Our findings clearly indicate that the initial chain condensation in cytochrome c is driven by specific interactions among a subset of α-helical segments rather than a general hydrophobic collapse.

  10. Cytochrome c-553 is not required for photosynthetic activity in the cyanobacterium Synechococcus.

    PubMed Central

    Laudenbach, D E; Herbert, S K; McDowell, C; Fork, D C; Grossman, A R; Straus, N A

    1990-01-01

    In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins. A deletion mutant was constructed that had no detectable cytochrome c-553 based on spectral analyses and tetramethylbenzidine-hydrogen peroxide staining of proteins resolved by polyacrylamide gel electrophoresis. The mutant strain was not impaired in overall photosynthetic activity. However, this mutant exhibited a decreased efficiency of cytochrome f oxidation. These results indicate that cytochrome c-553 is not an absolute requirement for reducing Photosystem I reaction centers in Synechococcus sp. PCC 7942. PMID:1967057

  11. Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea).

    PubMed

    Xu, Xiao-Lu; Wu, Xiao-Qin; Ye, Jian-Ren; Huang, Lin

    2015-03-06

    Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

  12. Three novel B-type mannose-specific lectins of Cynoglossus semilaevis possess varied antibacterial activities against Gram-negative and Gram-positive bacteria.

    PubMed

    Sun, Yuan-yuan; Liu, Li; Li, Jun; Sun, Li

    2016-02-01

    Lectins are a group of sugar-binding proteins that are important factors of the innate immune system. In this study, we examined, in a comparative manner, the expression and function of three Bulb-type (B-type) mannose-specific lectins (named CsBML1, CsBML2, and CsBML3) from tongue sole. All three lectins possess three repeats of the conserved mannose binding motif QXDXNXVXY. Expression of CsBML1, CsBML2, and CsBML3 was most abundant in liver and upregulated by bacterial infection. Recombinant (r) CsBML1, CsBML2, and CsBML3 bound to a wide arrange of bacteria in a dose-dependent manner and with different affinities. All three lectins displayed mannose-specific and calcium-dependent agglutinating capacities but differed in agglutinating profiles. rCsBML1 and rCsBML2, but not rCsBML3, killed target bacteria in vitro and inhibited bacterial dissemination in fish tissues in vivo. These results indicate for the first time that in teleost, different members of B-type mannose-specific lectins likely play different roles in antibacterial immunity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Cytochrome P450 CYP716A254 catalyzes the formation of oleanolic acid from β-amyrin during oleanane-type triterpenoid saponins biosynthesis in Anemone flaccida.

    PubMed

    Zhan, Chuansong; Ahmed, Shakeel; Hu, Sheng; Dong, Shuang; Cai, Qian; Yang, Tewu; Wang, Xuekui; Li, Xiaohua; Hu, Xuebo

    2018-01-01

    Anemone flaccida Fr. Shmidt (Ranunculaceae), known as 'Di Wu' in China, is a perennial herb which has long been used to treat arthritis. The rhizome of A. flaccida contains pharmacologically active components i.e. oleanane-type triterpenoid saponins. Oleanolic acid is natural triterpenoid in plants with diverse biological activities. The biosynthesis of oleanolic acid involves cyclization of 2,3-oxidosqualene to the oleanane-type triterpenoid skeleton, followed by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenase (CYP450). Previously, we identified four possible cytochrome P450 genes belonging to CYP716A subfamily from the transcriptome of A. flaccida. In this study, we identified one of those genes "CYP716A254" encoding a cytochrome P450 monooxygenase from A. flaccida that catalyzes the conversion of the β-amyrin into oleanolic acid. The heterologous expression of CYP716A254 in yeast resulted in oxidation of β-amyrin at the C-18 position to oleanolic acid production. These results provide an important basis for further studies of oleanane-type triterpenoid saponins synthesis in A. flaccida. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product.

    PubMed

    Tae, G S; Black, M T; Cramer, W A; Vallon, O; Bogorad, L

    1988-12-27

    Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis.

    PubMed

    Duggal, Ruchia; Liu, Yilin; Gregory, Michael C; Denisov, Ilia G; Kincaid, James R; Sligar, Stephen G

    2016-08-19

    Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second CC lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal FeS vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to

  16. Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens.

    PubMed

    Zacharoff, Lori; Chan, Chi Ho; Bond, Daniel R

    2016-02-01

    The respiration of metals by the bacterium Geobacter sulfurreducens requires electrons generated by metabolism to pass from the interior of the cell to electron acceptors beyond the cell membranes. The G. sulfurreducens inner membrane multiheme c-type cytochrome ImcH is required for respiration to extracellular electron acceptors with redox potentials greater than -0.1 V vs. SHE, but ImcH is not essential for electron transfer to lower potential acceptors. In contrast, deletion of cbcL, encoding an inner membrane protein consisting of b-type and multiheme c-type cytochrome domains, severely affected reduction of low potential electron acceptors such as Fe(III)-oxides and electrodes poised at -0.1 V vs. SHE. Catalytic cyclic voltammetry of a ΔcbcL strain growing on poised electrodes revealed a 50 mV positive shift in driving force required for electron transfer out of the cell. In non-catalytic conditions, low-potential peaks present in wild type biofilms were absent in ∆cbcL mutants. Expression of cbcL in trans increased growth at low redox potential and restored features to cyclic voltammetry. This evidence supports a model where CbcL is a component of a second electron transfer pathway out of the G. sulfurreducens inner membrane that dominates when redox potential is at or below -0.1 V vs. SHE. Copyright © 2015. Published by Elsevier B.V.

  17. Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex.

    PubMed

    Firsov, Alexander M; Kotova, Elena A; Korepanova, Evgeniya A; Osipov, Anatoly N; Antonenko, Yuri N

    2015-03-01

    Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Autoantigen-specific regulatory T cells induced in patients with Type 1 Diabetes Mellitus by Insulin B-chain immunotherapy

    PubMed Central

    Orban, Tihamer; Farkas, Klara; Jalahej, Heyam; Kis, Janos; Treszl, Andras; Falk, Ben; Reijonen, Helena; Wolfsdorf, Joseph; Ricker, Alyne; Matthews, Jeffrey B.; Tchao, Nadio; Sayre, Peter; Bianchine, Pete

    2009-01-01

    There is a growing body of evidence to suggest that the autoimmunity observed in type 1 diabetes mellitus (T1DM) is the result of an imbalance between autoaggressive and regulatory cell subsets. Therapeutics that supplement or enhance the existing regulatory subset are therefore a much sought after goal in this indication. Here, we report the results of a double blind, placebo controlled, phase I clinical trial of a novel antigen-specific therapeutic in 12 subjects with recently diagnosed T1DM. Our primary objective was to test its safety. The study drug, human insulin B-chain in incomplete Freund’s adjuvant (IFA) was administered as a single intramuscular injection, with subjects followed for 2 years. All subjects completed therapy and all follow-up visits. The therapy was generally safe and well-tolerated. Mixed meal stimulated C-peptide responses, measured every 6 months, showed no statistical differences between arms. All patients vaccinated with the autoantigen, but none who received placebo, developed robust insulin-specific humoral and T cell responses. Up to two years following the single injection, in peripheral blood from subjects in the experimental arm, but not the control arm, insulin B-chain-specific CD4+ T cells could be isolated and cloned that showed phenotypic and functional characteristics of regulatory T cells. The induction of a lasting, robust immune response generating autoantigen-specific regulatory T cells provides strong justification for further testing of this therapy in type 1 diabetes. (clinicaltrials.gov identifier NCT00057499). PMID:19931408

  19. Cytochrome P450-mediated metabolism of vitamin D

    PubMed Central

    Jones, Glenville; Prosser, David E.; Kaufmann, Martin

    2014-01-01

    The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field. PMID:23564710

  20. Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific B cells.

    PubMed Central

    Villiers, M B; Villiers, C L; Jacquier-Sarlin, M R; Gabert, F M; Journet, A M; Colomb, M G

    1996-01-01

    Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells. Images Figure 2 PMID:8958046

  1. Membrane Proteomics of Arabidopsis Glucosinolate Mutants cyp79B2/B3 and myb28/29

    PubMed Central

    Mostafa, Islam; Yoo, Mi-Jeong; Zhu, Ning; Geng, Sisi; Dufresne, Craig; Abou-Hashem, Maged; El-Domiaty, Maher; Chen, Sixue

    2017-01-01

    Glucosinolates (Gls) constitute a major group of natural metabolites represented by three major classes (aliphatic, indolic and aromatic) of more than 120 chemical structures. In our previous work, soluble proteins and metabolites in Arabidopsis mutants deficient of aliphatic (myb28/29) and indolic Gls (cyp79B2B3) were analyzed. Here we focus on investigating the changes at the level of membrane proteins in these mutants. Our LC/MS-MS analyses of tandem mass tag (TMT) labeled peptides derived from the cyp79B2/B3 and myb28/29 relative to wild type resulted in the identification of 4,673 proteins, from which 2,171 are membrane proteins. Fold changes and statistical analysis showed 64 increased and 74 decreased in cyp79B2/B3, while 28 increased and 17 decreased in myb28/29. As to the shared protein changes between the mutants, one protein was increased and eight were decreased. Bioinformatics analysis of the changed proteins led to the discovery of three cytochromes in glucosinolate molecular network (GMN): cytochrome P450 86A7 (At1g63710), cytochrome P450 71B26 (At3g26290), and probable cytochrome c (At1g22840). CYP86A7 and CYP71B26 may play a role in hydroxyl-indolic Gls production. In addition, flavone 3′-O-methyltransferase 1 represents an interesting finding as it is likely to participate in the methylation process of the hydroxyl-indolic Gls to form methoxy-indolic Gls. The analysis also revealed additional new nodes in the GMN related to stress and defense activity, transport, photosynthesis, and translation processes. Gene expression and protein levels were found to be correlated in the cyp79B2/B3, but not in the myb28/29. PMID:28443122

  2. Identification of human cytochrome P450s as autoantigens.

    PubMed

    Manns, M P; Johnson, E F

    1991-01-01

    Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

  3. Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b[subscript 6]f Complex from Nostoc sp. PCC 7120

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baniulis, Danas; Yamashita, Eiki; Whitelegge, Julian P.

    2009-06-08

    The crystal structure of the cyanobacterial cytochrome b{sub 6}f complex has previously been solved to 3.0-{angstrom} resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b{sub 6}f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical tomore » those in the M. laminosus b{sub 6}f complex. Purified b{sub 6}f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b{sub 6}f complex, determined to a resolution of 3.0{angstrom} (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme b{sub p} that is rotated 180 deg. about the {alpha}- and {gamma}-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme cn is similar to that previously found in the b{sub 6}f complex from other sources.« less

  4. Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

    PubMed

    Biniek, Catherine; Heyno, Eiri; Kruk, Jerzy; Sparla, Francesca; Trost, Paolo; Krieger-Liszkay, Anja

    2017-04-01

    The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2 •- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2 •- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2 •- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

  5. In vivo low-level light therapy increases cytochrome oxidase in skeletal muscle.

    PubMed

    Hayworth, Christopher R; Rojas, Julio C; Padilla, Eimeira; Holmes, Genevieve M; Sheridan, Eva C; Gonzalez-Lima, F

    2010-01-01

    Low-level light therapy (LLLT) increases survival of cultured cells, improves behavioral recovery from neurodegeneration and speeds wound healing. These beneficial effects are thought to be mediated by upregulation of mitochondrial proteins, especially the respiratory enzyme cytochrome oxidase. However, the effects of in vivo LLLT on cytochrome oxidase in intact skeletal muscle have not been previously investigated. We used a sensitive method for enzyme histochemistry of cytochrome oxidase to examine the rat temporalis muscle 24 h after in vivo LLLT. The findings showed for the first time that in vivo LLLT induced a dose- and fiber type-dependent increase in cytochrome oxidase in muscle fibers. LLLT was particularly effective at enhancing the aerobic capacity of intermediate and red fibers. The findings suggest that LLLT may enhance the oxidative energy metabolic capacity of different types of muscle fibers, and that LLLT may be used to enhance the aerobic potential of skeletal muscle.

  6. Elastic rotation of Escherichia coli F(O)F(1) having ε subunit fused with cytochrome b(562) or flavodoxin reductase.

    PubMed

    Oka, Hideyuki; Hosokawa, Hiroyuki; Nakanishi-Matsui, Mayumi; Dunn, Stanley D; Futai, Masamitsu; Iwamoto-Kihara, Atsuko

    2014-04-18

    Intra-molecular rotation of FOF1 ATP synthase enables cooperative synthesis and hydrolysis of ATP. In this study, using a small gold bead probe, we observed fast rotation close to the real rate that would be exhibited without probes. Using this experimental system, we tested the rotation of FOF1 with the ε subunit connected to a globular protein [cytochrome b562 (ε-Cyt) or flavodoxin reductase (ε-FlavR)], which is apparently larger than the space between the central and the peripheral stalks. The enzymes containing ε-Cyt and ε-FlavR showed continual rotations with average rates of 185 and 148 rps, respectively, similar to the wild type (172 rps). However, the enzymes with ε-Cyt or ε-FlavR showed a reduced proton transport. These results indicate that the intra-molecular rotation is elastic but proton transport requires more strict subunit/subunit interaction. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Estrogen and Cytochrome P450 1B1 Contribute to Both Early- and Late-Stage Head and Neck Carcinogenesis

    PubMed Central

    Shatalova, Ekaterina G.; Klein-Szanto, Andres J.P.; Devarajan, Karthik; Cukierman, Edna; Clapper, Margie L.

    2010-01-01

    Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer in the U.S. The goal of this study was to evaluate the contribution of estrogens to the development of HNSCCs. Various cell lines derived from early- and late-stage head and neck lesions were used to: characterize the expression of estrogen synthesis and metabolism genes, including cytochrome P450 (CYP)1B1, examine the effect of estrogen on gene expression and evaluate the role of CYP1B1 and/or estrogen in cell motility, proliferation and apoptosis. Estrogen metabolism genes (CYP1B1, CYP1A1, catechol-o-methyltransferase, UDP-glucuronosyltransferase 1A1, and glutathione-S-transferase P1) and estrogen receptor (ER)β were expressed in cell lines derived from both premalignant (MSK-Leuk1) and malignant (HNSCC) lesions. Exposure to estrogen induced CYP1B1 2.3 to 3.6 fold relative to vehicle-treated controls (P=0.0004) in MSK-Leuk1 cells but not in HNSCC cells. CYP1B1 knockdown by shRNA reduced the migration and proliferation of MSK-Leuk1 cells by 57% and 45%, respectively. Exposure of MSK-Leuk1 cells to estrogen inhibited apoptosis by 26%, while supplementation with the antiestrogen fulvestrant restored estrogen-dependent apoptosis. Representation of the estrogen pathway in human head and neck tissues from 128 patients was examined using tissue microarrays. The majority of the samples exhibited immunohistochemical staining for ERβ (91.9%), CYP1B1 (99.4%) and 17β-estradiol (88.4%). CYP1B1 and ERβ were elevated in HNSCCs relative to normal epithelium (P=0.024 and 0.008, respectively). These data provide novel insight into the mechanisms underlying head and neck carcinogenesis and facilitate the identification new targets for chemopreventive intervention. PMID:21205741

  8. The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar1[OPEN

    PubMed Central

    Blaukopf, Markus; Yuen, Macaire M.S.; Withers, Stephen G.; Mattsson, Jim; Russell, John H.; Bohlmann, Jörg

    2015-01-01

    Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures in the foliage of WRC. Thujones are produced from (+)-sabinene by sabinol and sabinone. Using metabolite analysis, enzyme assays with WRC tissue extracts, cloning, and functional characterization of cytochrome P450 monooxygenases, we established that trans-sabin-3-ol but not cis-sabin-3-ol is the intermediate in thujone biosynthesis in WRC. Based on transcriptome analysis, full-length complementary DNA cloning, and characterization of expressed P450 proteins, we identified CYP750B1 and CYP76AA25 as the enzymes that catalyze the hydroxylation of (+)-sabinene to trans-sabin-3-ol. Gene-specific transcript analysis in contrasting WRC genotypes producing high and low amounts of monoterpenoids, including a glandless low-terpenoid clone, as well as assays for substrate specificity supported a biological role of CYP750B1 in α- and β-thujone biosynthesis. This P450 belongs to the apparently gymnosperm-specific CYP750 family and is, to our knowledge, the first member of this family to be functionally characterized. In contrast, CYP76AA25 has a broader substrate spectrum, also converting the sesquiterpene farnesene and the herbicide isoproturon, and its transcript profiles are not well correlated with thujone accumulation. PMID:25829465

  9. The cytochrome b6f complex at the crossroad of photosynthetic electron transport pathways.

    PubMed

    Tikhonov, Alexander N

    2014-08-01

    Regulation of photosynthetic electron transport at the level of the cytochrome b6f complex provides efficient performance of the chloroplast electron transport chain (ETC). In this review, after brief overview of the structural organization of the chloroplast ETC, the consideration of the problem of electron transport control is focused on the plastoquinone (PQ) turnover and its interaction with the b6f complex. The data available show that the rates of plastoquinol (PQH2) formation in PSII and its diffusion to the b6f complex do not limit the overall rate of electron transfer between photosystem II (PSII) and photosystem I (PSI). Analysis of experimental and theoretical data demonstrates that the rate-limiting step in the intersystem chain of electron transport is determined by PQH2 oxidation at the Qo-site of the b6f complex, which is accompanied by the proton release into the thylakoid lumen. The acidification of the lumen causes deceleration of PQH2 oxidation, thus impeding the intersystem electron transport. Two other mechanisms of regulation of the intersystem electron transport have been considered: (i) "state transitions" associated with the light-induced redistribution of solar energy between PSI and PSII, and (ii) redistribution of electron fluxes between alternative pathways (noncyclic electron transport and cyclic electron flow around PSI). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  10. Cytochrome b5 Reductase 1 Triggers Serial Reactions that Lead to Iron Uptake in Plants.

    PubMed

    Oh, Young Jun; Kim, Hanul; Seo, Sung Hee; Hwang, Bae Geun; Chang, Yoon Seok; Lee, Junho; Lee, Dong Wook; Sohn, Eun Ju; Lee, Sang Joon; Lee, Youngsook; Hwang, Inhwan

    2016-04-04

    Rhizosphere acidification is essential for iron (Fe) uptake into plant roots. Plasma membrane (PM) H(+)-ATPases play key roles in rhizosphere acidification. However, it is not fully understood how PM H(+)-ATPase activity is regulated to enhance root Fe uptake under Fe-deficient conditions. Here, we present evidence that cytochrome b5 reductase 1 (CBR1) increases the levels of unsaturated fatty acids, which stimulate PM H(+)-ATPase activity and thus lead to rhizosphere acidification. CBR1-overexpressing (CBR1-OX) Arabidopsis thaliana plants had higher levels of unsaturated fatty acids (18:2 and 18:3), higher PM H(+)-ATPase activity, and lower rhizosphere pH than wild-type plants. By contrast, cbr1 loss-of-function mutant plants showed lower levels of unsaturated fatty acids and lower PM H(+)-ATPase activity but higher rhizosphere pH. Reduced PM H(+)-ATPase activity in cbr1 could be restored in vitro by addition of unsaturated fatty acids. Transcript levels of CBR1, fatty acids desaturase2 (FAD2), and fatty acids desaturase3 (FAD3) were increased under Fe-deficient conditions. We propose that CBR1 has a crucial role in increasing the levels of unsaturated fatty acids, which activate the PM H(+)-ATPase and thus reduce rhizosphere pH. This reaction cascade ultimately promotes root Fe uptake. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  11. Multilayered Polyelectrolyte Microcapsules: Interaction with the Enzyme Cytochrome C Oxidase

    PubMed Central

    Pastorino, Laura; Dellacasa, Elena; Noor, Mohamed R.; Soulimane, Tewfik; Bianchini, Paolo; D'Autilia, Francesca; Antipov, Alexei; Diaspro, Alberto; Tofail, Syed A. M.; Ruggiero, Carmelina

    2014-01-01

    Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties. PMID:25372607

  12. An orphan cbb3-type cytochrome oxidase subunit supports Pseudomonas aeruginosa biofilm growth and virulence

    PubMed Central

    Jo, Jeanyoung; Cortez, Krista L; Cornell, William Cole; Price-Whelan, Alexa

    2017-01-01

    Hypoxia is a common challenge faced by bacteria during associations with hosts due in part to the formation of densely packed communities (biofilms). cbb3-type cytochrome c oxidases, which catalyze the terminal step in respiration and have a high affinity for oxygen, have been linked to bacterial pathogenesis. The pseudomonads are unusual in that they often contain multiple full and partial (i.e. ‘orphan’) operons for cbb3-type oxidases and oxidase subunits. Here, we describe a unique role for the orphan catalytic subunit CcoN4 in colony biofilm development and respiration in the opportunistic pathogen Pseudomonas aeruginosa PA14. We also show that CcoN4 contributes to the reduction of phenazines, antibiotics that support redox balancing for cells in biofilms, and to virulence in a Caenorhabditis elegans model of infection. These results highlight the relevance of the colony biofilm model to pathogenicity and underscore the potential of cbb3-type oxidases as therapeutic targets. PMID:29160206

  13. Consequences of structural modifications in cytochrome b559 on the electron acceptor side of Photosystem II.

    PubMed

    Nakamura, Makoto; Boussac, Alain; Sugiura, Miwa

    2018-05-19

    Cytb 559 in Photosystem II is a heterodimeric b-type cytochrome. The subunits, PsbE and PsbF, consist each in a membrane α-helix. Mutants were previously designed and studied in Thermosynechococcus elongatus (Sugiura et al., Biochim Biophys Acta 1847:276-285, 2015) either in which an axial histidine ligand of the haem-iron was substituted for a methionine, the PsbE/H23M mutant in which the haem was lacking, or in which the haem environment was modified, the PsbE/Y19F and PsbE/T26P mutants. All these mutants remained active showing that the haem has no structural role provided that PsbE and PsbF subunits are present. Here, we have carried on the characterization of these mutants. The following results were obtained: (i) the Y19F mutation hardly affect the E m of Cytb 559 , whereas the T26P mutation converts the haem into a form with a E m much below 0 mV (so low that it is likely not reducible by Q B - ) even in an active enzyme; (ii) in the PsbE/H23M mutant, and to a less extent in PsbE/T26P mutant, the electron transfer efficiency from Q A - to Q B is decreased; (iii) the lower E m of the Q A /Q A - couple in the PsbE/H23M mutant correlates with a higher production of singlet oxygen; (iv) the superoxide and/or hydroperoxide formation was not increased in the PsbE/H23M mutant lacking the haem, whereas it was significantly larger in the PsbE/T26P. These data are discussed in view of the literature to discriminate between structural and redox roles for the haem of Cytb 559 in the production of reactive oxygen species.

  14. Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.

    PubMed Central

    Sibbesen, O; Koch, B; Halkier, B A; Møller, B L

    1994-01-01

    The cytochrome P-450 enzyme (hemethiolate enzyme) that catalyzes the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of the cyanogenic glucoside dhurrin, has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The cytochrome P-450 enzyme was solubilized with the detergents Renex 690, reduced Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and isolated by ion-exchange (DEAE-Sepharose) and dye (Cibacron blue and reactive red 120) column chromatography. To prevent irreversible aggregation of the cytochrome P-450 enzyme, the isolation procedure was designed without any concentration step--i.e., with dilution of the ion-exchange gel with gel filtration material. The isolated enzyme, which we designate the cytochrome P-450TYR enzyme, gives rise to the specific formation of a type I substrate binding spectrum in the presence of L-tyrosine. The microsomal preparation contains 0.2 nmol of total cytochrome P-450/mg of protein. The cytochrome P-450TYR enzyme is estimated to constitute approximately 20% of the total cytochrome P-450 content of the microsomal membranes and about 0.2% of their total protein content. The apparent molecular mass of the cytochrome P-450TYR enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450TYR enzyme is specific as monitored by Western blot analysis and inhibits the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile catalyzed by the microsomal system. The cytochrome P-450TYR enzyme exhibits high substrate specificity and acts as an N-hydroxylase on a single endogenous substrate. The reported isolation procedure based on dye columns constitutes a gentle isolation method for cytochrome P-450 enzymes and is of general use as indicated by its ability to separate cytochrome P-450TYR from the cytochrome P-450 enzyme catalyzing the C

  15. EXOGENOUS CYTOCHROME C RESTORES MYOCARDIAL CYTOCHROME OXIDASE ACTIVITY INTO THE LATE PHASE OF SEPSIS

    PubMed Central

    Piel, David A.; Deutschman, Clifford S.; Levy, Richard J.

    2009-01-01

    Mitochondrial dysfunction is thought to play a role in the pathogenesis of a variety of disease states, including sepsis. An acquired defect in oxidative phosphorylation potentially causes sepsis-induced organ dysfunction. Cytochrome oxidase (CcOX), the terminal oxidase of the respiratory chain, is competitively inhibited early in sepsis and progresses, becoming noncompetitive during the late phase. We have previously demonstrated that exogenous cytochrome c can overcome myocardial CcOX competitive inhibition and improve cardiac function during murine sepsis at the 24-h point. Here, we evaluate the effect of exogenous cytochrome c on CcOX activity and survival in mice at the later time points. Exogenous cytochrome c (800 μg) or saline was intravenously injected 24 h after cecal ligation and puncture (CLP) or sham operation. Steady-state mitochondrial cytochrome c levels and heme c content increased significantly 48 h post-CLP and remained elevated at 72 h in cytochrome c-injected mice compared with saline injection. Cecal ligation and puncture inhibited CcOX at 48 h in saline-injected mice. However, cytochrome c injection abrogated this inhibition and restored CcOX kinetic activity to sham values at 48 h. Survival after CLP to 96 h after cytochrome c injection approached 50% compared with only 15% after saline injection. Thus, a single injection of exogenous cytochrome c 24 h post-CLP repletes mitochondrial substrate levels for up to 72 h, restores myocardial COX activity, and significantly improves survival. PMID:18414235

  16. A mitochondrial DNA hypomorph of cytochrome oxidase specifically impairs male fertility in Drosophila melanogaster

    PubMed Central

    Patel, Maulik R; Miriyala, Ganesh K; Littleton, Aimee J; Yang, Heiko; Trinh, Kien; Young, Janet M; Kennedy, Scott R; Yamashita, Yukiko M; Pallanck, Leo J; Malik, Harmit S

    2016-01-01

    Due to their strict maternal inheritance in most animals and plants, mitochondrial genomes are predicted to accumulate mutations that are beneficial or neutral in females but harmful in males. Although a few male-harming mtDNA mutations have been identified, consistent with this ‘Mother’s Curse’, their effect on females has been largely unexplored. Here, we identify COIIG177S, a mtDNA hypomorph of cytochrome oxidase II, which specifically impairs male fertility due to defects in sperm development and function without impairing other male or female functions. COIIG177S represents one of the clearest examples of a ‘male-harming’ mtDNA mutation in animals and suggest that the hypomorphic mtDNA mutations like COIIG177S might specifically impair male gametogenesis. Intriguingly, some D. melanogaster nuclear genetic backgrounds can fully rescue COIIG177S -associated sterility, consistent with previously proposed models that nuclear genomes can regulate the phenotypic manifestation of mtDNA mutations. DOI: http://dx.doi.org/10.7554/eLife.16923.001 PMID:27481326

  17. The Chemically Inducible Plant Cytochrome P450 CYP76B1 Actively Metabolizes Phenylureas and Other Xenobiotics1

    PubMed Central

    Robineau, Tiburce; Batard, Yannick; Nedelkina, Svetlana; Cabello-Hurtado, Francisco; LeRet, Monique; Sorokine, Odile; Didierjean, Luc; Werck-Reichhart, Danièle

    1998-01-01

    Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas. CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds. Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation. PMID:9808750

  18. The Kinetic Mechanism for Cytochrome P450 Metabolism of Type II Binding Compounds: Evidence Supporting Direct Reduction

    PubMed Central

    Pearson, Joshua; Dahal, Upendra P.; Rock, Daniel; Peng, Chi-Chi; Schenk, James O.; Joswig-Jones, Carolyn; Jones, Jeffrey P.

    2011-01-01

    The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at subsaturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; 1) Dead-end type II binding, 2) a rapid equilibrium between type I and II binding modes before reduction, and 3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1. PMID:21530484

  19. Higher platelet cytochrome oxidase specific activity in surviving than in non-surviving septic patients

    PubMed Central

    2014-01-01

    Introduction In a previous study with 96 septic patients, we found that circulating platelets in 6-months surviving septic patients showed higher activity and quantity of cytochrome c oxidase (COX) normalized by citrate synthase (CS) activity at moment of severe sepsis diagnosis than non-surviving septic patients. The objective of this study was to estimate whether COX specific activity during the first week predicts 1-month sepsis survival in a larger cohort of patients. Methods Using a prospective, multicenter, observational study carried out in six Spanish intensive care units with 198 severe septic patients, we determined COX activity per proteins (COXact/Prot) in circulating platelets at day 1, 4 and 8 of the severe sepsis diagnosis. Endpoints were 1-month and 6-months mortality. Results Survivor patients (n = 130) showed higher COXact/Prot (P < 0.001) than non-survivors (n = 68) at day 1, 4 and 8 of severe sepsis diagnosis. More than a half of the 6-months survivor patients showed an increase in their COXact/Prot from day 1 to 8. However, most of the 1-month non-survivors exhibited a decrease in their COXact/Prot from day 1 to 8. Multiple logistic regression analyses showed that of platelet COXact/Prot > 0.30 mOD/min/mg at day 1 (P = 0.002), 4 (P = 0.006) and 8 (P = 0.02) was associated independently with 1-month mortality. Area under the curve of COXact/Prot at day 1, 4 and 8 to predict 30-day survival were 0.70 (95% CI = 0.63-0.76; P < 0.001), 0.71 (95% CI = 0.64-0.77; P < 0.001) and 0.71 (95% CI = 0.64-0.78; P < 0.001), respectively. Conclusions The new findings of our study, to our knowledge the largest series reporting data about mitochondrial function during follow-up in septic patients, were that septic patients that survive 1-month have a higher platelet cytochrome oxidase activity at moment of sepsis diagnosis and during the first week than non-survivors, and that platelet cytochrome oxidase

  20. Membrane cytochromes of Escherichia coli chl mutants.

    PubMed Central

    Hackett, N R; Bragg, P D

    1983-01-01

    The cytochromes present in the membranes of Escherichia coli cells having defects in the formate dehydrogenase-nitrate reductase system have been analyzed by spectroscopic, redox titration, and enzyme fractionation techniques. Four phenotypic classes differing in cytochrome composition were recognized. Class I is represented by strains with defects in the synthesis or insertion of molybdenum cofactor. Cytochromes of the formate dehydrogenase-nitrate reductase pathway are present. Class II strains map in the chlC-chlI region. The cytochrome associated with nitrate reductase (cytochrome bnr) is absent in these strains, whereas that associated with formate dehydrogenase (cytochrome bfdh) is the major cytochrome in the membranes. Class III strains lack both cytochromes bfdh and bnr but overproduce cytochrome d of the aerobic pathway even under anaerobic conditions in the presence of nitrate. Class III strains have defects in the regulation of cytochrome synthesis. An fdhA mutant produced cytochrome bnr but lacked cytochrome bfdh. These results support the view that chlI (narI) is the structural gene for cytochrome bnr and that chlC (narG) and chlI(narI) are in the same operon, and they provide evidence of the complexity of the regulation of cytochrome synthesis. PMID:6302081

  1. Enhanced hepatic and kidney cytochrome p-450 activities in nandrolone decanoate treated albino mice.

    PubMed

    Acharjee, B K; Mahanta, R

    2009-04-01

    Anabolic androgenic steroids are the xenobiotic substrates that are metabolized in the body by the protective enzyme systems. Mixed function oxygenase enzymes include a group of enzymes which play an essential role in the metabolism of a broad range of xenobiotics including endogenous and exogenous substrates. Cytochrome P-450, a member of mixed function oxygenase enzymes, plays an important role in oxidative metabolism of drugs and xenobiotics entering human body. Various anabolic steroids are found either to increase or decrease the activity of cytochrome P-450. However, effect of nandrolone decanoate, most commonly abused anabolic steroid, on cytochrome P-450 activity is still fragmentary. In the present study, albino mice were administered intramuscular 2.5 mg of nandrolone decanoate injection at 15 days interval. Cytochrome P-450 activity is determined by following the method of Omura and Sato (1964) in liver and kidney tissues of both normal and experimental groups upto 90 days. Investigation shows a significant (p <0.01) increase of cytochrome P-450 (nmol/mg) activity in liver tissue as compared to that of kidney tissues. A tissue specific and dose specific increase of cytochrome P-450 activity is observed. Mean cytochrome P-450 is found highest in liver tissue on 45(th) day whereas the activity in kidney tissue is noticed on 90(th) day of treatment. From the above observation, nandrolone decanoate can be suggested as a potent inducer of cytochrome P-450 activity like other anabolic steroids.

  2. The Folding Energy Landscape and Free Energy Excitations of Cytochrome c

    PubMed Central

    Weinkam, Patrick; Zimmermann, Jörg; Romesberg, Floyd E.

    2014-01-01

    The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Due to the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case that follows different principles than those used to describe folding of proteins without cofactors. Recent investigations, however, demonstrate that models which are commonly used to describe folding for many proteins work well for cytochrome c when heme is explicitly introduced and generally provide results that agree with experimental observations. We will first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Since attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as “perfectly funneled.” In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are sources of frustration which cause specific transient intermediates to be populated. Native structure-based models do include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold in order for folding to proceed. The ability of native structure-based models to capture these type of motions is in part responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe well the sequence of folding events for cytochrome c inferred from hydrogen exchange experiments thereby justifying their use as a starting point. At low pH, the folding sequence of cytochrome c deviates from that at pH 7 and from those predicted from models with perfectly funneled energy landscapes. Alternate folding pathways are a result of

  3. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O- dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black- crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross- reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p'-DDE were detected in embryos from Cat Island. Cytochrome P450- associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

  4. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu-g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than five-fold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O-dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p, p'-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

  5. Suicide inactivation of cytochrome P-450 by methoxsalen. Evidence for the covalent binding of a reactive intermediate to the protein moiety

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Labbe, G.; Descatoire, V.; Beaune, P.

    Incubation of rat liver microsomes with (3H)methoxsalen and NADPH resulted in the covalent binding of a methoxsalen intermediate to proteins comigrating with cytochromes P-450 UT-A, PB-B/D, ISF-G and PCN-E. Binding was increased by pretreatments with phenobarbital, beta-naphthoflavone (beta NF) and dexamethasone. Such pretreatments also increased the loss of CO-binding capacity either after administration of methoxsalen, or after incubation of hepatic microsomes with methoxsalen and NADPH. Immunoprecipitation of the methoxsalen metabolite-protein adducts in phenobarbital-induced microsomes was moderate with anti-UT-A antibodies, but marked with anti-PB-B/D and anti-PCN-E antibodies. Immunoprecipitation was observed also with anti-ISF-G (anti-beta NF-B) antibodies in beta NF-induced microsomes. Methoxsalenmore » (0.25 mM) inhibited markedly the benzphetamine demethylase activity of phenobarbital-induced microsomes and the erythromycin demethylase activity of dexamethasone-induced microsomes. Whereas methoxsalen itself did not produce any binding spectrum, in contrast either in vivo administration of methoxsalen or incubation in vitro with methoxsalen and NADPH resulted in a low-to-high spin conversion of cytochrome P-450 as suggested by the appearance of a spectrum analogous to a type I binding spectrum. This low-to-high spin conversion was apparently due to a methoxsalen intermediate (probably, covalently bound to the protein and preventing partial sixth ligation of the iron). We conclude that suicide inactivation of cytochrome P-450 by methoxsalen is related to the covalent binding of a methoxsalen intermediate to the protein moiety of several cytochrome P-450 isoenzymes (including UT-A, PB-B/D, PCN-E as well as ISF-G and/or beta NF-B).« less

  6. Cytochrome P450 family member CYP704B2 catalyzes the {omega}-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice.

    PubMed

    Li, Hui; Pinot, Franck; Sauveplane, Vincent; Werck-Reichhart, Danièle; Diehl, Patrik; Schreiber, Lukas; Franke, Rochus; Zhang, Ping; Chen, Liang; Gao, Yawei; Liang, Wanqi; Zhang, Dabing

    2010-01-01

    The anther cuticle and microspore exine act as protective barriers for the male gametophyte and pollen grain, but relatively little is known about the mechanisms underlying the biosynthesis of the monomers of which they are composed. We report here the isolation and characterization of a rice (Oryza sativa) male sterile mutant, cyp704B2, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. These defects are caused by a mutation in a cytochrome P450 family gene, CYP704B2. The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of omega -hydroxylated fatty acids with 16 and 18 carbon chains. Our results provide insights into the biosynthesis of the two biopolymers sporopollenin and cutin. Specifically, our study indicates that the omega -hydroxylation pathway of fatty acids relying on this ancient CYP704B family, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development.

  7. Albendazole sulfonation by rat liver cytochrome P-450c.

    PubMed

    Souhaili-El Amri, H; Mothe, O; Totis, M; Masson, C; Batt, A M; Delatour, P; Siest, G

    1988-08-01

    The metabolism of albendazole (ABZ) was studied in perfused livers from control and ABZ-treated rats (10.6 mg/kg, per os, each day for 10 days). In the perfusion fluid, the concentration of ABZ-sulfoxide (SO-ABZ) remained unchanged in treated, as compared to control animals, whereas ABZ-sulfone (SO2-ABZ) was increased in treated animals. In bile, only SO-ABZ was present. The transformation kinetics of SO-ABZ to SO2-ABZ in microsomes from rats treated with ABZ, 3-methylcholanthrene, Aroclor and isosafrole were biphasic. This suggests that enzyme activity was a consequence of two enzyme systems, one characterized by low affinity and high capacity, the other by high affinity and low capacity, the latter could be induced by 3-methylcholanthrene, ABZ, Aroclor and isosafrole. Cytochrome P-450c was induced potently in vivo by ABZ as proven by increased monooxygenase (7-ethoxyresorufin and 7-ethoxycoumarin-O-deethylase) activities and by Elisa test (a 5-fold increase in hemoprotein concentration was observed). Purified and reconstituted cytochrome P-450c from 3-methylcholanthrene or ABZ-treated rat liver were able to produce SO2-ABZ (2.01 and 1.70 nmol/mg/15 min, respectively, whereas cytochrome P-450b produced 10 times less SO2-ABZ). Immunological assays, as well as activity measurements showed a relationship between cytochrome P-450c-3-methylcholanthrene and cytochrome P-450c-ABZ. We conclude that induction of cytochrome P-450c by ABZ is the probable explanation for the enhanced formation of SO2-ABZ in vivo.

  8. Interfacial hydration, dynamics and electron transfer: multi-scale ET modeling of the transient [myoglobin, cytochrome b5] complex.

    PubMed

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

    2012-10-28

    Formation of a transient [myoglobin (Mb), cytochrome b(5) (cyt b(5))] complex is required for the reductive repair of inactive ferri-Mb to its functional ferro-Mb state. The [Mb, cyt b(5)] complex exhibits dynamic docking (DD), with its cyt b(5) 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. 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 (T(DA)) 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 T(DA) values to compute ET rates for the [Mb(wt), cyt b(5)] 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 b(5)]. The calculated rate constants are in agreement with the measured values, and the mutant complex ensemble has many more geometries with higher T(DA) 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

  9. Mutations of Cytochrome b559 and PsbJ on and near the QC Site in Photosystem II Influence the Regulation of Short-Term Light Response and Photosynthetic Growth of the Cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Huang, Jine-Yung; Chiu, Yi-Fang; Ortega, José M; Wang, Hsing-Ting; Tseng, Tien-Sheng; Ke, Shyue-Chu; Roncel, Mercedes; Chu, Hsiu-An

    2016-04-19

    The characteristic features of two types of short-term light adaptations of the photosynthetic apparatus of the cyanobacterium Synechocystis sp. PCC 6803, state transition and blue-green light-induced fluorescence quenching, were compared in wild-type and cytochrome b559 and PsbJ mutant cells with mutations on and near the QC site in photosystem II (PSII). All mutant cells grew photoautotrophically and assembled stable PSII. Thermoluminescence emission experiments showed a decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the A16FJ, S28Aβ, and V32Fβ mutant cells. When dark-adapted wild-type and mutant cells were illuminated by medium-intensity blue light, the increase in the PSII fluorescence yield (indicating a transition to state 1) was more prominent in mutant than wild-type cells. Strong blue-light conditions induced a quenching of fluorescence corresponding to nonphotochemical fluorescence quenching (NPQ). The extension of NPQ decreased significantly in the mutants, and the kinetics appeared to be affected. When similar measures were repeated on an orange carotenoid protein (OCP)-deficient background, little or no quenching was observed, which confirms that the decrease in fluorescence under strong blue light corresponded to the OCP-dependent NPQ. Immunoblot results showed that the attenuated effect of blue light-induced NPQ in mutant cells was not due to a lack of OCP. Photosynthetic growth and biomass production were greater for A16FJ, S28Aβ, and V32Fβ mutant cells than for wild-type cells under normal growth conditions. Our results suggest that mutations of cytochrome b559 and PsbJ on and near the QC site of PSII may modulate the short-term light response in cyanobacteria.

  10. Actinomycin D Inhibition of the Zinc-induced Formation of Cytochrome c in Ustilago1

    PubMed Central

    Brown, D. H.; Cappellini, R. A.; Price, C. A.

    1966-01-01

    As reported earlier by Grimm & Allen, the addition of zinc to the sporidia of the smut fungus, Ustilago sphaerogena, evokes the formation of large amounts of cytochrome c. This occurs under conditions where the rates of increase of dry weight, RNA, and DNA remain unaffected. Actinomycin D added with zinc specifically abolishes the formation of cytochrome c. The system behaves as if cytochrome c were formed de novo. PMID:5956845

  11. Comparative study of the affinity and metabolism of type I and type II binding quinoline carboxamide analogs by cytochrome P450 3A4

    PubMed Central

    Dahal, Upendra P.; Joswig-Jones, Carolyn; Jones, Jeffrey P.

    2011-01-01

    Compounds that coordinate to the heme-iron of cytochrome P450 (CYP) enzymes are assumed to increase metabolic stability. However, recently we observed that the type II binding quinoline carboxamide (QCA) compounds were metabolically less stable. To test if the higher intrinsic clearance of type II binding compounds relative to type I binding compounds is general for other metabolic transformations, we synthesized a library of QCA compounds that could undergo N-dealkylation, O-dealkylation, benzylic hydroxylation and aromatic hydroxylation. The results demonstrated that type II binding QCA analogs were metabolically less stable (2 to 12 fold) at sub-saturating concentration compared to type I binding counterparts for all the transformations. When the rates of different metabolic transformations between type I and type II binding compounds were compared, they were found to be in the order of N-demethylation>benzylic hydroxylation> O-demethylation> aromatic hydroxylation. Finally, for the QCA analogs with aza-heteroaromatic rings, we did not detect metabolism in aza-aromatic rings (pyridine, pyrazine, pyrimidine) indicating electronegativity of the nitrogen can change regioselectivity in CYP metabolism. PMID:22087535

  12. An Isotopic Labelling Strategy to Study Cytochrome P450 Oxidations of Terpenes.

    PubMed

    Rinkel, Jan; Litzenburger, Martin; Bernhardt, Rita; Dickschat, Jeroen Sidney

    2018-04-26

    The cytochrome P450 monooxygenase CYP267B1 from Sorangium cellulosum was applied for enzymatic oxidation of the sesquiterpene alcohols T-muurolol and isodauc-8-en-11-ol. Various isotopically labelled geranyl and farnesyl diphosphates were used for product identification from micro-scale reactions, for determination of the absolute configurations of unknown compounds, to follow the stereochemical course of a cytochrome P450-catalysed hydroxylation step, and to investigate kinetic isotope effects. Overall, this study demonstrates that isotopically labelled terpene precursors are highly useful to follow cytochrome P450 dependent oxidations of terpenes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Binding Specificity Determines the Cytochrome P450 3A4 Mediated Enantioselective Metabolism of Metconazole.

    PubMed

    Zhuang, Shulin; Zhang, Leili; Zhan, Tingjie; Lu, Liping; Zhao, Lu; Wang, Haifei; Morrone, Joseph A; Liu, Weiping; Zhou, Ruhong

    2018-01-25

    Cytochrome P450 3A4 (CYP3A4) is a promiscuous enzyme, mediating the biotransformations of ∼50% of clinically used drugs, many of which are chiral molecules. Probing the interactions between CYP3A4 and chiral chemicals is thus essential for the elucidation of molecular mechanisms of enantioselective metabolism. We developed a stepwise-restrained-molecular-dynamics (MD) method to model human CYP3A4 in a complex with cis-metconazole (MEZ) isomers and performed conventional MD simulations with a total simulation time of 2.2 μs to probe the molecular interactions. Our current study, which employs a combined experimental and theoretical approach, reports for the first time on the distinct conformational changes of CYP3A4 that are induced by the enantioselective binding of cis-MEZ enantiomers. CYP3A4 preferably metabolizes cis-RS MEZ over the cis-SR isomer, with the resultant enantiomer fraction for cis-MEZ increasing rapidly from 0.5 to 0.82. cis-RS MEZ adopts a more extended structure in the active pocket with its Cl atom exposed to the solvent, whereas cis-SR MEZ sits within the hydrophobic core of the active pocket. Free-energy-perturbation calculations indicate that unfavorable van der Waals interactions between the cis-MEZ isomers and the CYP3A4 binding pocket predominantly contribute to their binding-affinity differences. These results demonstrate that binding specificity determines the cytochrome P450 3A4 mediated enantioselective metabolism of cis-MEZ.

  14. Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b-Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties

    PubMed Central

    Birke, Jakob; Röther, Wolf

    2015-01-01

    Specific polyisoprene-cleaving activities of 1.5 U/mg and 4.6 U/mg were determined for purified Strep-tagged latex clearing protein (Lcp) of Streptomyces sp. strain K30 at 23°C and 37°C, respectively. Metal analysis revealed the presence of approximately one atom of iron per Lcp molecule. Copper, which had been identified in Lcp1VH2 of Gordonia polyisoprenivorans previously, was below the detection limit in LcpK30. Heme was identified as a cofactor in purified LcpK30 by (i) detection of characteristic α-, β-, and γ (Soret)-bands at 562 nm, 532 nm, and 430 nm in the visible spectrum after chemical reduction, (ii) detection of an acetone-extractable porphyrin molecule, (iii) determination of a heme b-type-specific absorption maximum (556 nm) after chemical conversion of the heme group to a bipyridyl-heme complex, and (iv) detection of a b-heme-specific m/z value of 616.2 via mass spectrometry. Spectroscopic analysis showed that purified Lcp as isolated contains an oxidized heme-Fe3+ that is free of bound dioxygen. This is in contrast to the rubber oxygenase RoxA, a c-type heme-containing polyisoprene-cleaving enzyme present in Gram-negative rubber degraders, in which the covalently bound heme firmly binds a dioxygen molecule. LcpK30 also differed from RoxA in the lengths of the rubber degradation cleavage products and in having a higher melting point of 61.5°C (RoxA, 54.3°C). In summary, RoxA and Lcp both are equipped with a heme cofactor and catalyze an oxidative C-C cleavage reaction but differ in the heme subgroup type and in several biochemical and biophysical properties. These findings suggest differences in the catalytic reaction mechanisms. PMID:25819959

  15. Thermodynamic Characterization of a Triheme Cytochrome Family from Geobacter sulfurreducens Reveals Mechanistic and Functional Diversity

    PubMed Central

    Morgado, Leonor; Bruix, Marta; Pessanha, Miguel; Londer, Yuri Y.; Salgueiro, Carlos A.

    2010-01-01

    Abstract A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c7 from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e−/H+ transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e−/H+ coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G

  16. Mitochondrial cytochrome b DNA sequence variations: an approach to fish species identification in processed fish products.

    PubMed

    Pepe, Tiziana; Trotta, Michele; di Marco, Isolina; Cennamo, Paola; Anastasio, Aniello; Cortesi, Maria Luisa

    2005-02-01

    The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.

  17. Mapping the fine specificity of ABO monoclonal reagents with A and B type-specific function-spacer-lipid constructs in kodecytes and inkjet printed on paper.

    PubMed

    Barr, Katie; Korchagina, Elena; Ryzhov, Ivan; Bovin, Nicolai; Henry, Stephen

    2014-10-01

    Monoclonal (MoAb) reagents are routinely used and are usually very reliable for the serologic determination of ABO blood types. However, the fine specificity and cross-reactivity of these reagents are often unknown, particularly against synthetic antigens used in some diagnostic assays. If nonserologic assays or very sensitive techniques other than those specifically prescribed by the manufacturer are used, then there is a risk of incorrect interpretation of results. Forty-seven MoAbs and two polyclonal ABO reagents were tested against red blood cell (RBC) kodecytes prepared with A trisaccharide, A Type 1, A Type 2, A Type 3, A Type 4, B trisaccharide, B Type 1, B Type 2, acquired B trisaccharide, and Le(a) trisaccharide function-spacer-lipid (FSL) constructs. Natural RBCs were tested in parallel. In addition these FSL constructs were printed onto paper with a desktop inkjet printer and used in a novel immunoassay that identifies reactivity through the appearance of alphanumeric characters. Mapping of MoAbs with kodecytes and printed FSL constructs revealed a series of broad recognition patterns. All ABO MoAbs tested were reactive with the RBC dominant Type 2 ABO antigens. Unexpectedly some anti-A reagents were reactive against the B Type 1 antigen, while others were poorly reactive with trisaccharide antigens. All ABO MoAbs detect the RBC dominant Type 2 ABO antigens; however, some reagents may show minor reactivity with inappropriate blood group antigens, which needs to be considered when using these reagents in alternative or highly sensitive analytic systems. © 2014 AABB.

  18. Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics.

    PubMed

    Reidy, G F; Mehta, I; Murray, M

    1989-05-01

    The anti-parkinsonian agent orphenadrine has been shown to form an in vitro metabolic intermediate (MI) complex in hepatic microsomes isolated from phenobarbital (PB)-treated rats. The present study was undertaken to assess the cytochrome P-450 isozyme specificity of inhibition and MI complexation. Spectral studies with untreated and PB-induced rat hepatic microsomes confirmed earlier reports on the selectivity of P-450 complexation by orphenadrine; MI complex formation was only observed with PB-induced microsomes. Inhibition studies with the P-450 substrates androst-4-ene-3,17-dione (androstenedione) and 7-pentoxyresorufin revealed selective inhibition of P-450 PB-B/D-associated monooxygenase activity. Thus, in microsomes from untreated male rats, orphenadrine failed to significantly inhibit (less than 50% inhibition up to a concentration of 300 microM) any of the major pathways of P-450-associated androstenedione metabolism. Preincubation of these microsomal fractions with orphenadrine and NADPH was not associated with increased inhibition of androstenedione metabolism. However, in PB-induced microsomes, P-450 PB-B/D-specific androstenedione 16 beta-hydroxylase activity was significantly and selectively inhibited (IC50 = 90 microM). Preincubation of orphenadrine with NADPH-supplemented PB-induced microsomes for 2, 4, or 8 min before androstenedione addition resulted in increased inhibition toward 16 beta-hydroxylase activity, lowering the observed IC50 to 6.6, 0.47, and 0.06 microM), respectively. Preincubation did not affect the selectivity of inhibition. In the absence of preincubation, orphenadrine appeared to be a potent mixed (competitive/noncompetitive)-type inhibitor of P-450 PB-B/D-associated pentoxyresorufin O-depentylation (Ki = 3.8 microM). Preincubation of orphenadrine with NADPH-supplemented microsomal fractions for 4 min resulted in a 30-fold lowering of the apparent inhibitor constant (Ki = 0.13 microM) and a change in the apparent inhibition

  19. Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B.

    PubMed

    Fukasawa, Kayoko M; Hirose, Junzo; Hata, Toshiyuki; Ono, Yukio

    2006-09-26

    Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data

  20. Role of Outer Membrane C-Type Cytochromes MtrC and OmcA in Shewanella Oneidensis MR-1 Cell Production, Accumulation, and Detachment During Respiration on Hematite

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mitchell, Andrew C.; Peterson, L.; Reardon, Catherine L.

    2012-07-01

    Solid phase iron oxides are considered to be important terminal electron acceptors for microbial respiration in many anoxic environments. Besides the knowledge that cells attach to and reduce these substrates, other aspects of surface-associated cell behavior and the related cell surface components that influence cell-mineral interactions are not well understood. In the present study, wild-type cells of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 formed thin biofilms one-to-two cell layers in thickness when respiring on natural specular hematite under flow conditions similar to those which exist in aquatic sediments and subsurface environments. The distribution of cells within the biofilm indicatedmore » that direct contact was not required for electron transfer from cells to the mineral surface. Detached biomass in the form of single cells represented >99% of the surface-associated wild-type cell production from respiration on hematite over the biofilm life cycle. A mutant deficient in the outer membrane c35 type cytochrome OmcA, while still able to respire and replicate on hematite, established a lower steady-state cell density on the mineral surface than that of the wild-type strain. A mutant deficient in MtrC, another outer membrane c-type cytochrome, and a mutant deficient in both cytochromes were unable to reduce sufficient amounts of hematite to support detectable growth on the mineral surface. When considered in the context of previous work, the results support a growing body of evidence that the relative importance of OmcA and MtrC to cell respiration and replication depends on the form of iron oxide available as terminal electron acceptor.« less

  1. Mitochondrial ATPase Subunit 6 and Cytochrome B Gene Variations in Obese Turkish Children

    PubMed Central

    Demir, Durkadın; Türkkahraman, Doğa; Samur, Anıl Aktaş; Lüleci, Güven; Akçurin, Sema; M. Alper, Özgül

    2014-01-01

    Objective: Due to the importance of energy metabolism in mitochondria, mitochondrial genome variations are evaluated in energy-related diseases such as obesity. To date, several nuclear genes were found to be related to obesity. Our aim in this study was to investigate the presence of polymorphisms in mitochondrial ATPase subunit 6 (mt-ATP6) and cytochrome b (mt-CytB) genes that may be associated with childhood obesity. Methods: The mt-ATP6 and mt-CytB genes were amplified and entirely sequenced in a series of 100 obese and in an equal number of healthy Turkish children aged between 6-14 years. Results: A total of 118 synonymous and nonsynonymous variations were detected in the obese and control groups. Only two previously reported synonymous substitutions (mt.8614T>C and mt.8994G>A) in the mt-ATP6 gene were found to be significantly higher in the obese group compared to the control group (p<0.05). In the mt-ATP6 gene, one novel nonsynonymous substitution (mt.8726C>T) and one novel synonymous substitution (mt.9108A>T) were found. In the mt-CytB gene, one nonsynonymous substitution (mt.14880T>C) and two synonymous substitutions (mt.14891C>T and mt.15091C>T) were novel substitutions. Conclusion: Two synonymous substitutions (mt.8614T>C and mt.8994G>A) in the mt-ATP6 gene may be associated with childhood obesity. Our study provides the first data about mitochondrial genome variations in a Turkish obese population and also the first in obese children. More cases should be screened in obese groups in order to understand the effects of mitochondrial polymorphisms in the development of obesity. PMID:25541891

  2. Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

    PubMed

    Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

    2006-10-01

    The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

  3. The novel primers for mammal species identification-based mitochondrial cytochrome b sequence: implication for reserved wild animals in Thailand and endangered mammal species in Southeast Asia.

    PubMed

    Muangkram, Yuttamol; Wajjwalku, Worawidh; Amano, Akira; Sukmak, Manakorn

    2018-01-01

    We presented the powerful techniques for species identification using the short amplicon of mitochondrial cytochrome b gene sequence. Two faecal samples and one single hair sample of the Asian tapir were tested using the new cytochrome b primers. The results showed a high sequence similarity with the mainland Asian tapir group. The comparative sequence analysis of the reserved wild mammals in Thailand and the other endangered mammal species from Southeast Asia comprehensibly verified the potential of our novel primers. The forward and reverse primers were 94.2 and 93.2%, respectively, by the average value of the sequence identity among 77 species sequences, and the overall mean distance was 35.9%. This development technique could provide rapid, simple, and reliable tools for species confirmation. Especially, it could recognize the problematic biological specimens contained less DNA material from illegal products and assist with wildlife crime investigation of threatened species and related forensic casework.

  4. Spectroscopic Studies of Abiotic and Biological Nanomaterials: Silver Nanoparticles, Rhodamine 6G Adsorbed on Graphene, and c-Type Cytochromes and Type IV Pili in Geobacter sulfurreducens

    NASA Astrophysics Data System (ADS)

    Thrall, Elizabeth S.

    This thesis describes spectroscopic studies of three different systems: silver nanoparticles, the dye molecule rhodamine 6G adsorbed on graphene, and the type IV pili and c-type cytochromes produced by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens. Although these systems are quite different in some ways, they can all be considered examples of nanomaterials. A nanomaterial is generally defined as having at least one dimension below 100 nm in size. Silver nanoparticles, with sub-100 nm size in all dimensions, are examples of zero-dimensional nanomaterials. Graphene, a single atomic layer of carbon atoms, is the paradigmatic two-dimensional nanomaterial. And although bacterial cells are on the order of 1 μm in size, the type IV pili and multiheme c-type cytochromes produced by G. sulfurreducens can be considered to be one- and zero-dimensional nanomaterials respectively. A further connection between these systems is their strong interaction with visible light, allowing us to study them using similar spectroscopic tools. The first chapter of this thesis describes research on the plasmon-mediated photochemistry of silver nanoparticles. Silver nanoparticles support coherent electron oscillations, known as localized surface plasmons, at resonance frequencies that depend on the particle size and shape and the local dielectric environment. Nanoparticle absorption and scattering cross-sections are maximized at surface plasmon resonance frequencies, and the electromagnetic field is amplified near the particle surface. Plasmonic effects can enhance the photochemistry of silver particles alone or in conjunction with semiconductors according to several mechanisms. We study the photooxidation of citrate by silver nanoparticles in a photoelectrochemical cell, focusing on the wavelength-dependence of the reaction rate and the role of the semiconductor substrate. We find that the citrate

  5. Production, purification and detergent exchange of isotopically labeled Bacillussubtilis cytochrome b₅₅₈ (SdhC).

    PubMed

    Baureder, Michael; Hederstedt, Lars

    2011-11-01

    Cytochrome b₅₅₈ of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b₅₅₈. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b₅₅₈ produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque.

    PubMed

    Hu, Minlu; Zhou, Tian; Pearlman, Andrew P; Paton, Dorothy L; Rohan, Lisa C

    2016-01-01

    This manuscript summarizes our recent progress in examine the CYP1A1 and CYP1B1 as well as a number of nuclear receptors in the female genital and colorectal tissues of human and pigtailed macaque. Understanding the nuclear receptor mediated regulation of CYP1A1 and 1B1 expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. However, there is a lack of systematic and comparative analysis of the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and clinically relevant animal models. The current study aims to fill this gap. We found CYP1A1, CYP1B1 and a number of nuclear receptors were expressed in the female genital and colorectal tissues of human and macaque. However, the mRNA level and protein localization of these CYP enzymes and NRs depended on the type of tissue examined. Cytochrome P450 (CYP) 1A1 and CYP1B1 activate hormonal and environmental procarcinogens, and are associated with carcinogenesis in female genital and colorectal tissues. Understanding the nuclear receptor (NR) mediated regulation of CYP expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. The study aims to analyze the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and pigtailed macaques. We found that compared to the liver, human CYP1A1 mRNA level in the genital and colorectal tissues was significantly lower, while the CYP1B1 level was significantly higher. CYP1A1 protein was mainly localized in the plasma membrane of the uterine and endocervical epithelial cells. The CYP1B1 protein was concentrated in the nucleus of genital and colorectal tissues. Fourteen NRs in the genital tract and 12 NRs in colorectal tissue were expressed at levels similar to or higher than the liver. The expression and localization of CYP1A1, CYP1B1, and NRs in

  7. Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production

    Treesearch

    Nian-Qing Shi; Brian Davis; Fred Sherman; Jose Cruz; Thomas W. Jeffries

    1999-01-01

    The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization...

  8. Identification of three cytochrome P450 genes in the Chagas' disease vector Triatoma infestans: Expression analysis in deltamethrin susceptible and resistant populations.

    PubMed

    Grosso, Carla G; Blariza, María J; Mougabure-Cueto, Gastón; Picollo, María I; García, Beatriz A

    2016-10-01

    Cytochrome P450 monooxygenases play a predominant role in the metabolism of insecticides. Many insect P450 genes have frequently been associated with detoxification processes allowing the insect to become tolerant or resistant to insecticides. The increases of expression of P450 genes at transcriptional level are often consider responsible for increasing the metabolism of insecticides and seems to be a common phenomenon in the evolution of resistance development in insects. As pyrethroid resistance has been detected in Triatoma infestans, it was of interest to analyze genes associated with resistance to insecticides such as those encoding for cytochromes P450. With this purpose, the cDNA sequences of three cytochrome P450 genes (CYP4EM7, CYP3085B1, and CYP3092A6) were identified in this species. Primers and specific Taqman probes were designed from these sequences to determine their expression by quantitative PCR. The mRNA levels of the cytochrome P450 genes identified were determined from total RNA extracted from pools of fat body collected from individuals of different resistant and susceptible strains of T. infestans, and at different interval times after the topical application of the lethal doses 50% (LD50) of deltamethrin on the ventral abdomen of insects belonging to the different populations analyzed. It was detected overexpression of the CYP4EM7 gene in the most resistant strain of T. infestans and the expression of the three cytochrome P450 genes isolated was induced by deltamethrin in the susceptible and resistant populations included in this study. These results suggest that these genes would be involved in the detoxification of deltamethrin and support the hypothesis that considers to the cytochrome P450 genes of importance in the development of pyrethroid resistance. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis1

    PubMed Central

    Zhang, Haoming; Gay, Sean C.; Shah, Manish; Foroozesh, Maryam; Liu, Jiawang; Osawa, Yoichi; Zhang, Qinghai; Stout, C. David; Halpert, James R.; Hollenberg, Paul F.

    2013-01-01

    The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min−1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by ESI-LC/MS, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206 leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low affinity site being the catalytic site and the high affinity site on the protein periphery. Computer-aided docking and MD simulations with one or two ligands bound revealed that the high affinity site is situated at the entrance of a substrate access channel surrounded by the F’ helix, β1/β2 loop and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302. PMID:23276288

  10. Cytochrome P450 Family Member CYP704B2 Catalyzes the ω -Hydroxylation of Fatty Acids and Is Required for Anther Cutin Biosynthesis and Pollen Exine Formation in Rice[W][OA

    PubMed Central

    Li, Hui; Pinot, Franck; Sauveplane, Vincent; Werck-Reichhart, Danièle; Diehl, Patrik; Schreiber, Lukas; Franke, Rochus; Zhang, Ping; Chen, Liang; Gao, Yawei; Liang, Wanqi; Zhang, Dabing

    2010-01-01

    The anther cuticle and microspore exine act as protective barriers for the male gametophyte and pollen grain, but relatively little is known about the mechanisms underlying the biosynthesis of the monomers of which they are composed. We report here the isolation and characterization of a rice (Oryza sativa) male sterile mutant, cyp704B2, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. These defects are caused by a mutation in a cytochrome P450 family gene, CYP704B2. The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of ω -hydroxylated fatty acids with 16 and 18 carbon chains. Our results provide insights into the biosynthesis of the two biopolymers sporopollenin and cutin. Specifically, our study indicates that the ω -hydroxylation pathway of fatty acids relying on this ancient CYP704B family, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development. PMID:20086189

  11. Bloodmeal Identification in Field-Collected Sand Flies From Casa Branca, Brazil, Using the Cytochrome b PCR Method.

    PubMed

    Carvalho, G M L; Rêgo, F D; Tanure, A; Silva, A C P; Dias, T A; Paz, G F; Andrade Filho, J D

    2017-07-01

    PCR-based identification of vertebrate host bloodmeals has been performed on several vectors species with success. In the present study, we used a previously published PCR protocol followed by DNA sequencing based on primers designed from multiple alignments of the mitochondrial cytochrome b gene used to identify avian and mammalian hosts of various hematophagous vectors. The amplification of a fragment encoding a 359 bp sequence of the Cyt b gene yielded recognized amplification products in 192 female sand flies (53%), from a total of 362 females analyzed. In the study area of Casa Branca, Brazil, blood-engorged female sand flies such as Lutzomyia longipalpis (Lutz & Neiva, 1912), Migonemyia migonei (França, 1924), and Nyssomyia whitmani (Antunes & Coutinho, 1939) were analyzed for bloodmeal sources. The PCR-based method identified human, dog, chicken, and domestic rat blood sources. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Human Hepatic Cytochrome P450-Specific Metabolism of the Organophosphorus Pesticides Methyl Parathion and Diazinon

    PubMed Central

    Tian, Yuan; Knaak, James B.; Kostyniak, Paul J.; Olson, James R.

    2012-01-01

    Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (Km and Vmax) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (Km = 1.25 μM; Vmax = 9.78 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 1.03 μM; Vmax = 4.67 nmol · min−1 · nmol P450−1), and CYP1A2 (Km = 1.96 μM; Vmax = 5.14 nmol · min−1 · nmol P450−1), and the bioactivation of diazinon was mediated primarily by CYP1A1 (Km = 3.05 μM; Vmax = 2.35 nmol · min−1 · nmol P450−1), CYP2C19 (Km = 7.74 μM; Vmax = 4.14 nmol · min−1 · nmol P450−1), and CYP2B6 (Km = 14.83 μM; Vmax = 5.44 nmol · min−1 · nmol P450−1). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (Km = 16.8 μM; Vmax = 1.38 nmol · min−1 · nmol P450−1) and 3A4 (Km = 104 μM; Vmax = 5.15 nmol · min−1 · nmol P450−1), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (Km = 5.04 μM; Vmax = 5.58 nmol · min−1 · nmol P450−1). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure. PMID:21969518

  13. Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.

    PubMed

    Gay, Sean C; Shah, Manish B; Talakad, Jyothi C; Maekawa, Keiko; Roberts, Arthur G; Wilderman, P Ross; Sun, Ling; Yang, Jane Y; Huelga, Stephanie C; Hong, Wen-Xu; Zhang, Qinghai; Stout, C David; Halpert, James R

    2010-04-01

    The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

  14. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    PubMed

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Specific expression of Neu2 type B in mouse thymus and the existence of a membrane-bound form in COS cells.

    PubMed

    Koda, Toshiaki; Kijimoto-Ochiai, Shigeko; Uemura, Satoshi; Inokuchi, Jin-ichi

    2009-10-02

    Neu2 mRNA from the mouse thymus, as we have reported [K. Kotani, A. Kuroiwa, T. Saito, Y. Matsuda, T. Koda, S. Kijimoto-Ochiai, Cloning, chromosomal mapping, and characteristic 5'-UTR sequence of murine cytosolic sialidase, Biochem. Biophys. Res. Commun. 286 (2001) 250-258], has a novel sequence at the 5' terminus that shows the ability to encode 6 extra amino acids in the N-terminus than that of the muscle. In this paper, we analyzed the cDNA and EST database and found the five types of alternative splicing of Neu2 mRNA: A, B, C, D and N. We studied the expression of these types in the immune tissues and found that the thymus expressed only type B. We constructed 2 types of plasmid that encode long (B) or short (C) form of Neu2 protein, and transfected them into COS7 cells to study them under the same conditions. We found that 30-40% of the both forms of Neu2 activity was located in the crude membrane-fraction, and hydrolyzed ganglioside effectively, while both soluble fraction showed particular behavior with substrate specificity. Microscopic study by active staining with X-NANA showed that they located not only in the cytoplasm but also in areas surrounding the nucleus and in the peripheral ruffled spot.

  16. Cytochrome and alternative pathway respiration in green algae : measurements using inhibitors and o(2) discrimination.

    PubMed

    Weger, H G; Guy, R D; Turpin, D H

    1990-05-01

    Inhibitor titration curves and discrimination against (18)O(2) by mitochondrial respiration in three strains of green algae (Selenastrum minutum [Naeg.] Collins, and two strains of Chlamydomonas reinhardtii Dangeard) with differing respiratory capabilities were determined. Discrimination for cytochrome pathway respiration ranged from 19.89 to 20.43%. Discrimination for alternative pathway respiration by wild-type C. reinhardtii (measured in the presence of KCN) was 25.46%, while discrimination values for a cytochrome oxidase deficient mutant of C. reinhardtii ranged from 24.24 to 24.96%. In the absence of KCN, the alternative pathway was not engaged in wild-type C. reinhardtii, the only algal strain that possessed both cytochrome and alternative pathway capacities.

  17. On the Monoheme Character of Cytochromes c′

    PubMed Central

    Kennel, S. J.; Meyer, T. E.; Kamen, M. D.; Bartsch, R. G.

    1972-01-01

    Interpretations of data bearing on structures of cytochromes cc′—a class of variant c-type heme proteins from bacteria—in support of a diheme-bearing single chain as a basic structural unit, appear to be invalid in the light of recent studies. These reveal that nearly all members of this class exist as dimers that can be dissociated into, if they do not already exist as, monoheme-bearing monomers. The particular case of the Chromatium protein, held to be the source of a peptic-“core” peptide containing two covalently-bonded heme groups, has been re-examined by preparation of tryptic and chymotryptic peptides derived from the heme-bearing region of the protein, as well as by repetition of experiments on peptic digestion, with more rigorous purification of the resultant peptides than was previously done. It is shown that this protein can also be dissociated into identical subunits, bearing a single heme prosthetic group, and accounting essentially for all its heme content. Thus, the previous terminology—cytochromes cc′—based on supposition of heme group heterogeneity, is inconsistent with these findings and should be replaced by cytochromes c′. PMID:4343972

  18. Heterologous expression of Helicoverpa armigera cytochrome P450 CYP6B7 in Pichia pastoris and interactions of CYP6B7 with insecticides.

    PubMed

    Zhao, Chunqing; Song, Genmiao; Duan, Hongxia; Tang, Tao; Wang, Chen; Qiu, Lihong

    2017-09-01

    Previous studies indicated that constitutive over-expression of cytochrome P450 CYP6B7 was involved in fenvalerate resistance in Helicoverpa armigera. In this study, the CYP6B7 gene from H. armigera (namely HaCYP6B7), was heterologously expressed in Pichia pastoris GS115. A vector pPICZA-HaCYP6B7 was constructed and transformed into P. pastoris GS115, the transformant of pPICZA-HaCYP6B7-GS115 was then cultured and induced by 1% (v/v) methanol and the heterologous expression of HaCYP6B7 protein in P. pastoris was confirmed by SDS-PAGE and western blot. Microsomes containing the expressed HaCYP6B7 showed activities against model substrate p-nitroanisole and 7-ethoxycoumarin, with p-nitroanisole O-demethylation (PNOD) and 7-ethoxycoumarin O-deethylation (ECOD) activities of 15.66- and 4.75-fold of the control, respectively. Moreover, it showed degradation activities against the insecticides bifenthrin, fenvalerate and chlorpyrifos, with clearance activities of 6.88-, 1.49- and 2.27-fold of the control, respectively. The interactions of HaCYP6B7 with insecticides were further confirmed by molecular docking in silico with binding scores of 5.450, 5.295 and 2.197 between putative HaCYP6B7 protein and bifenthrin, fenvalerate and chlorpyrifos, respectively. The results of present study provided more direct and important evidence on the role of HaCYP6B7 conferring pyrethroid resistance in H. armigera. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  19. Structural basis for cyclization specificity of two Azotobacter type III polyketide synthases: a single amino acid substitution reverses their cyclization specificity.

    PubMed

    Satou, Ryutaro; Miyanaga, Akimasa; Ozawa, Hiroki; Funa, Nobutaka; Katsuyama, Yohei; Miyazono, Ken-ichi; Tanokura, Masaru; Ohnishi, Yasuo; Horinouchi, Sueharu

    2013-11-22

    Type III polyketide synthases (PKSs) show diverse cyclization specificity. We previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp-281 of ArsB corresponded to Gly-284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 Å, respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase.

  20. Equine cytochrome P450 2B6 — Genomic identification, expression and functional characterization with ketamine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Peters, L.M.; Demmel, S.; Pusch, G.

    2013-01-01

    Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug–drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drugmore » metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V{sub max} for S-/and R-norketamine formation was 0.49 and 0.45 nmol/h/mg cellular protein and K{sub m} was 3.41 and 2.66 μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC{sub 50} of 5.63 and 6.26 μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a

  1. Bicarbonate stabilizes isolated D1/D2/cytochrome b559 complex of photosystem 2 against thermoinactivation.

    PubMed

    Pobeguts, O V; Smolova, T N; Klimov, V V

    2012-02-01

    It has been shown that thermoinactivation of the isolated D1/D2/cytochrome b(559) complex (RC) of photosystem 2 (PS-2) from pea under anaerobic conditions at 35°C in 20 mM Tris-HCl buffer (pH 7.2) depleted of HCO(3)(-), with 35 mM NaCl and 0.05% n-dodecyl-β-maltoside, results in a decrease in photochemical activity measured by photoreduction of the PS-2 primary electron acceptor, pheophytin (by 50% after 3 min of heating), which is accompanied by aggregation of the D1 and D2 proteins. Bicarbonate, formate, and acetate anions added to the sample under these conditions differently influence the maintenance of photochemical activity: a 50% loss of photochemical activity occurs in 11.5 min of heating in the presence of bicarbonate and in 4 and 4.6 min in the presence of formate and acetate, respectively. The addition of bicarbonate completely prevents aggregation of the D1 and D2 proteins as opposed to formate and acetate (their presence has no effect on the aggregation during thermoinactivation). Since the isolated RCs have neither inorganic Mn/Ca-containing core of the water-oxidizing complex nor nonheme Fe(2+), it is supposed that bicarbonate specifically interacts with the hydrophilic domains of the D1 and D2 proteins, which prevents their structural modification that is a signal for aggregation of these proteins and the loss of photochemical activity.

  2. Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis.

    PubMed

    Kato, Hirotomo; Gomez, Eduardo A; Martini-Robles, Luiggi; Muzzio, Jenny; Velez, Lenin; Calvopiña, Manuel; Romero-Alvarez, Daniel; Mimori, Tatsuyuki; Uezato, Hiroshi; Hashiguchi, Yoshihisa

    2016-07-01

    A countrywide epidemiological study was performed to elucidate the current geographic distribution of causative species of cutaneous leishmaniasis (CL) in Ecuador by using FTA card-spotted samples and smear slides as DNA sources. Putative Leishmania in 165 samples collected from patients with CL in 16 provinces of Ecuador were examined at the species level based on the cytochrome b gene sequence analysis. Of these, 125 samples were successfully identified as Leishmania (Viannia) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, and L. (Leishmania) mexicana. Two dominant species, L. (V.) guyanensis and L. (V.) braziliensis, were widely distributed in Pacific coast subtropical and Amazonian tropical areas, respectively. Recently reported L. (V.) naiffi and L. (V.) lainsoni were identified in Amazonian areas, and L. (L.) mexicana was identified in an Andean highland area. Importantly, the present study demonstrated that cases of L. (V.) braziliensis infection are increasing in Pacific coast areas.

  3. Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zacharoff, Lori; Chan, Chi Ho; Bond, Daniel R.

    2015-09-05

    The respiration of metals by the bacterium Geobacter sulfurreducens requires electrons generated by metabolism to pass from the interior of the cell to electron acceptors beyond the cell membranes. The G. sulfurreducens inner membrane multiheme c-type cytochrome ImcH is required for respiration to extracellular electron acceptors with redox potentials greater than - 0.1 V vs. SHE, but ImcH is not essential for electron transfer to lower potential acceptors. In contrast, deletion of cbcL, encoding an inner membrane protein consisting of b-type and multiheme c-type cytochrome domains, severely affected reduction of low potential electron acceptors such as Fe(III)-oxides and electrodes poisedmore » at - 0.1 V vs. SHE. Catalytic cyclic voltammetry of a ΔcbcL strain growing on poised electrodes revealed a 50 mV positive shift in driving force required for electron transfer out of the cell. In non-catalytic conditions, low-potential peaks present in wild type biofilms were absent in ΔcbcL mutants. Expression of cbcL in trans increased growth at low redox potential and restored features to cyclic voltammetry. This evidence supports a model where CbcL is a component of a second electron transfer pathway out of the G. sulfurreducens inner membrane that dominates when redox potential is at or below - 0.1 V vs. SHE.« less

  4. Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

    PubMed

    Fox, Rebecca M; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J

    2013-05-01

    FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

  5. Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA

    PubMed Central

    Fox, Rebecca M.; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J.

    2013-01-01

    FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously. PMID:23578928

  6. TOWARD A MOLECULAR PHYLOGENY FOR PEROMYSCUS: EVIDENCE FROM MITOCHONDRIAL CYTOCHROME-b SEQUENCES

    PubMed Central

    Bradley, Robert D.; Durish, Nevin D.; Rogers, Duke S.; Miller, Jacqueline R.; Engstrom, Mark D.; Kilpatrick, C. William

    2009-01-01

    One hundred DNA sequences from the mitochondrial cytochrome-b gene of 44 species of deer mice (Peromyscus (sensu stricto), 1 of Habromys, 1 of Isthmomys, 2 of Megadontomys, and the monotypic genera Neotomodon, Osgoodomys, and Podomys were used to develop a molecular phylogeny for Peromyscus. Phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference) were conducted to evaluate alternative hypotheses concerning taxonomic arrangements (sensu stricto versus sensu lato) of the genus. In all analyses, monophyletic clades were obtained that corresponded to species groups proposed by previous authors; however, relationships among species groups generally were poorly resolved. The concept of the genus Peromyscus based on molecular data differed significantly from the most current taxonomic arrangement. Maximum-likelihood and Bayesian trees depicted strong support for a clade placing Habromys, Megadontomys, Neotomodon, Osgoodomys, and Podomys within Peromyscus. If Habromys, Megadontomys, Neotomodon, Osgoodomys, and Podomys are regarded as genera, then several species groups within Peromyscus (sensu stricto) should be elevated to generic rank. Isthmomys was associated with the genus Reithrodontomys; in turn this clade was sister to Baiomys, indicating a distant relationship of Isthmomys to Peromyscus. A formal taxonomic revision awaits synthesis of additional sequence data from nuclear markers together with inclusion of available allozymic and karyotypic data. PMID:19924266

  7. Bioenergetics at extreme temperature: Thermus thermophilus ba(3)- and caa(3)-type cytochrome c oxidases.

    PubMed

    Radzi Noor, Mohamed; Soulimane, Tewfik

    2012-04-01

    Seven years into the completion of the genome sequencing projects of the thermophilic bacterium Thermus thermophilus strains HB8 and HB27, many questions remain on its bioenergetic mechanisms. A key fact that is occasionally overlooked is that oxygen has a very limited solubility in water at high temperatures. The HB8 strain is a facultative anaerobe whereas its relative HB27 is strictly aerobic. This has been attributed to the absence of nitrate respiration genes from the HB27 genome that are carried on a mobilizable but highly-unstable plasmid. In T. thermophilus, the nitrate respiration complements the primary aerobic respiration. It is widely known that many organisms encode multiple biochemically-redundant components of the respiratory complexes. In this minireview, the presence of the two cytochrome c oxidases (CcO) in T. thermophilus, the ba(3)- and caa(3)-types, is outlined along with functional considerations. We argue for the distinct evolutionary histories of these two CcO including their respective genetic and molecular organizations, with the caa(3)-oxidase subunits having been initially 'fused'. Coupled with sequence analysis, the ba(3)-oxidase crystal structure has provided evolutionary and functional information; for example, its subunit I is more closely related to archaeal sequences than bacterial and the substrate-enzyme interaction is hydrophobic as the elevated growth temperature weakens the electrostatic interactions common in mesophiles. Discussion on the role of cofactors in intra- and intermolecular electron transfer and proton pumping mechanism is also included. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. HLA-DR4-associated T and B cell responses to specific determinants on the IA-2 autoantigen in type 1 diabetes.

    PubMed

    McLaughlin, Kerry A; Gulati, Kavita; Richardson, Carolyn C; Morgan, Diana; Bodansky, H Jonathan; Feltbower, Richard G; Christie, Michael R

    2014-11-01

    Autoantibodies to IA-2 in type 1 diabetes are associated with HLA-DR4, suggesting influences of HLA-DR4-restricted T cells on IA-2-specific B cell responses. The aim of this study was to investigate possible T-B cell collaboration by determining whether autoantibodies to IA-2 epitopes are associated with T cell responses to IA-2 peptides presented by DR4. T cells secreting the cytokines IFN-γ and IL-10 in response to seven peptides known to elicit T cell responses in type 1 diabetes were quantified by cytokine ELISPOT in HLA-typed patients characterized for Abs to IA-2 epitopes. T cell responses were detected to all peptides tested, but only IL-10 responses to 841-860 and 853-872 peptides were associated with DR4. Phenotyping by RT-PCR of FACS-sorted CD45RO(hi) T cells secreting IL-10 in response to these two peptides indicated that these expressed GATA-3 or T-bet, but not FOXP3, consistent with these being Th2 or Th1 memory T cells rather than of regulatory phenotype. T cell responses to the same two peptides were also associated with specific Abs: those to 841-860 peptide with Abs to juxtamembrane epitopes, which appear early in prediabetes, and those to peptide 853-872 with Abs to an epitope located in the 831-862 central region of the IA-2 tyrosine phosphatase domain. Abs to juxtamembrane and central region constructs were both DR4 associated. This study identifies a region of focus for B and T cell responses to IA-2 in HLA-DR4 diabetic patients that may explain HLA associations of IA-2 autoantibodies, and this region may provide a target for future immune intervention to prevent disease. Copyright © 2014 by The American Association of Immunologists, Inc.

  9. Combination of cytochrome b heteroduplex-assay and sequencing for identification of triatomine blood meals.

    PubMed

    Buitrago, Rosio; Depickère, Stéphanie; Bosseno, Marie-France; Patzi, Edda Siñani; Waleckx, Etienne; Salas, Renata; Aliaga, Claudia; Brenière, Simone Frédérique

    2012-01-01

    The identification of blood meals in vectors contributes greatly to the understanding of interactions between vectors, microorganisms and hosts. The aim of the current work was to complement the validation of cytochrome b (Cytb) heteroduplex assay (HDA) previously described, and to add the sequencing of the Cytb gene of some samples for the identification of blood meals in triatomines. Experimental feedings of reared triatomines helped to clarify the sensitivity of the HDA. Moreover, the sequencing coupled with the HDA, allowed the assessment of the technique's taxonomic level of discrimination. The primers used to produce DNA fragments of Cytb genes for HDA had a very high sensitivity for vertebrate DNAs, rather similar for mammals, birds and reptiles. However, the formation of heteroduplex depended on blood meal's quality rather than its quantity; a correlation was observed between blood meals' color and the positivity of HDA. HDA electrophoresis profiles were reproducible, and allowed the discrimination of blood origins at the species level. However, in some cases, intraspecific variability of Cytb gene generated different HDA profiles. The HDA based on comparison of electrophoresis profiles is a very useful tool for screening large samples to determine blood origins; the subsequent sequencing of PCR products of Cytb corresponding to different HDA profiles allowed the identification of species whatever the biotope in which the vectors were captured. Copyright © 2011. Published by Elsevier B.V.

  10. Insights into Hydrocarbon Assimilation by Eurotialean and Hypocrealean Fungi: Roles for CYP52 and CYP53 Clans of Cytochrome P450 Genes.

    PubMed

    Huarte-Bonnet, Carla; Kumar, Suresh; Saparrat, Mario C N; Girotti, Juan R; Santana, Marianela; Hallsworth, John E; Pedrini, Nicolás

    2018-03-01

    Several filamentous fungi are able to concomitantly assimilate both aliphatic and polycyclic aromatic hydrocarbons that are the biogenic by-products of some industrial processes. Cytochrome P450 monooxygenases catalyze the first oxidation reaction for both types of substrate. Among the cytochrome P450 (CYP) genes, the family CYP52 is implicated in the first hydroxylation step in alkane-assimilation processes, while genes belonging to the family CYP53 have been linked with oxidation of aromatic hydrocarbons. Here, we perform a comparative analysis of CYP genes belonging to clans CYP52 and CYP53 in Aspergillus niger, Beauveria bassiana, Metarhizium robertsii (formerly M. anisopliae var. anisopliae), and Penicillium chrysogenum. These species were able to assimilate n-hexadecane, n-octacosane, and phenanthrene, exhibiting a species-dependent modification in pH of the nutrient medium during this process. Modeling of the molecular docking of the hydrocarbons to the cytochrome P450 active site revealed that both phenanthrene and n-octacosane are energetically favored as substrates for the enzymes codified by genes belonging to both CYP52 and CYP53 clans, and thus appear to be involved in this oxidation step. Analyses of gene expression revealed that CYP53 members were significantly induced by phenanthrene in all species studied, but only CYP52X1 and CYP53A11 from B. bassiana were highly induced with n-alkanes. These findings suggest that the set of P450 enzymes involved in hydrocarbon assimilation by fungi is dependent on phylogeny and reveal distinct substrate and expression specificities.

  11. Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen.

    PubMed

    Castresana, J; Lübben, M; Saraste, M; Higgins, D G

    1994-06-01

    Cytochrome oxidase is a key enzyme in aerobic metabolism. All the recorded eubacterial (domain Bacteria) and archaebacterial (Archaea) sequences of subunits 1 and 2 of this protein complex have been used for a comprehensive evolutionary analysis. The phylogenetic trees reveal several processes of gene duplication. Some of these are ancient, having occurred in the common ancestor of Bacteria and Archaea, whereas others have occurred in specific lines of Bacteria. We show that eubacterial quinol oxidase was derived from cytochrome c oxidase in Gram-positive bacteria and that archaebacterial quinol oxidase has an independent origin. A considerable amount of evidence suggests that Proteobacteria (Purple bacteria) acquired quinol oxidase through a lateral gene transfer from Gram-positive bacteria. The prevalent hypothesis that aerobic metabolism arose several times in evolution after oxygenic photosynthesis, is not sustained by two aspects of the molecular data. First, cytochrome oxidase was present in the common ancestor of Archaea and Bacteria whereas oxygenic photosynthesis appeared in Bacteria. Second, an extant cytochrome oxidase in nitrogen-fixing bacteria shows that aerobic metabolism is possible in an environment with a very low level of oxygen, such as the root nodules of leguminous plants. Therefore, we propose that aerobic metabolism in organisms with cytochrome oxidase has a monophyletic and ancient origin, prior to the appearance of eubacterial oxygenic photosynthetic organisms.

  12. Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

    PubMed

    Driscoll, William J; Chaturvedi, Shalini; Mueller, Gregory P

    2007-08-03

    Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

  13. Fine Specificity and Cross-Reactions of Monoclonal Antibodies to Group B Streptococcal Capsular Polysaccharide Type III

    PubMed Central

    Pincus, Seth H.; Moran, Emily; Maresh, Grace; Jennings, Harold J.; Pritchard, David G.; Egan, Marianne L.; Blixt, Ola

    2012-01-01

    Group B streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. Despite aggressive campaigns using antenatal prophylactic antibiotic therapy, infections continue. Developing an effective maternal vaccine is a public health priority. Antibody (Ab) to the capsular polysaccharide (CPS) is considered the dominant “protective” immune mediator. Here we study the fine specificity and potential host reactivity of a panel of well-characterized murine monoclonal Abs against the type III CPS by examining the binding of the Abs to intact and neuraminidase-digested GBS, purified CPS, synthetic carbohydrate structures, and cells. The results showed marked differences in the fine specificity among these mAbs to a single carbohydrate structure. Cross-reactions with synthetic GD3 and GT3 carbohydrates, representing structures found on surfaces of neural and developing cells, were demonstrated using carbohydrate array technology. The anti-CPSIII mAbs did not react with cells expressing GD3 and GT3, nor did mAbs specific for the host carbohydrates cross-react with GBS, raising questions about the physiological relevance of this cross-reaction. But in the process of these investigations, we serendipitously demonstrated cross-reactions of some anti-CPSIII mAbs with antigens, likely carbohydrates, found on human leukocytes. These studies suggest caution in the development of a maternal vaccine to prevent infection by this important human pathogen. PMID:22634296

  14. Fine specificity and cross-reactions of monoclonal antibodies to group B streptococcal capsular polysaccharide type III.

    PubMed

    Pincus, Seth H; Moran, Emily; Maresh, Grace; Jennings, Harold J; Pritchard, David G; Egan, Marianne L; Blixt, Ola

    2012-07-06

    Group B streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. Despite aggressive campaigns using antenatal prophylactic antibiotic therapy, infections continue. Developing an effective maternal vaccine is a public health priority. Antibody (Ab) to the capsular polysaccharide (CPS) is considered the dominant "protective" immune mediator. Here we study the fine specificity and potential host reactivity of a panel of well-characterized murine monoclonal Abs against the type III CPS by examining the binding of the Abs to intact and neuraminidase-digested GBS, purified CPS, synthetic carbohydrate structures, and cells. The results showed marked differences in the fine specificity among these mAbs to a single carbohydrate structure. Cross-reactions with synthetic GD3 and GT3 carbohydrates, representing structures found on surfaces of neural and developing cells, were demonstrated using carbohydrate array technology. The anti-CPS(III) mAbs did not react with cells expressing GD3 and GT3, nor did mAbs specific for the host carbohydrates cross-react with GBS, raising questions about the physiological relevance of this cross-reaction. But in the process of these investigations, we serendipitously demonstrated cross-reactions of some anti-CPS(III) mAbs with antigens, likely carbohydrates, found on human leukocytes. These studies suggest caution in the development of a maternal vaccine to prevent infection by this important human pathogen. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Evidence that cytochrome b{sub 5} acts as a redox donor in CYP17A1 mediated androgen synthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Duggal, Ruchia; Liu, Yilin; Gregory, Michael C.

    2016-08-19

    Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second C−C lyase step, at the expense of glucocorticoid production. Cytochrome b{sub 5} (cyt b{sub 5}) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b{sub 5} increases androgen biosynthesis. Cyt b{sub 5} could act as an allosteric effector, binding to CYP17A1 and eithermore » changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b{sub 5} could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b{sub 5}, we generated a redox-inactive form of cyt b{sub 5}, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b{sub 5}), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b{sub 5} were enhanced ∼5 fold as compared to reactions in the absence of cyt b{sub 5}. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b{sub 5}. Upon addition of Mn-b{sub 5} to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b{sub 5}-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the

  16. Ecology of malaria parasites infecting Southeast Asian macaques: evidence from cytochrome b sequences

    PubMed Central

    Putaporntip, Chaturong; Jongwutiwes, Somchai; Thongaree, Siriporn; Seethamchai, Sunee; Grynberg, Priscila; Hughes, Austin L.

    2010-01-01

    Although malaria parasites infecting non-human primates are important models for human malaria, little is known of the ecology of infection by these parasites in the wild. We extensively sequenced cytochrome b (cytb) of malaria parasites (Apicomplexa: Haemosporida) from free-living Southeast Asian monkeys Macaca nemestrina and M. fascicularis. The two most commonly observed taxa were P. inui and Hepatocystis sp., but certain other sequences did not cluster closely with any previously sequenced species. Most of the major clades of parasites were found in both Macaca species; and the two most commonly occurring parasite infected the two Macaca species at approximately equal levels. However, P. inui showed evidence of genetic differentiation between the populations infecting the two Macaca species, suggesting limited movement of this parasite among hosts. Moreover, coinfection with Plasmodium and Hepatocystis species occurred significantly less frequently than expected on the basis of the rates of infection with either taxon alone, suggesting the possibility of competitive exclusion. The results revealed unexpectedly complex communities of Plasmodium and Hepatocystis taxa infecting wild Southeast Asian monkeys. Parasite taxa differed with respect to both the frequency of between-host movement and their frequency of coinfection. PMID:20646216

  17. Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque

    PubMed Central

    Hu, Minlu; Zhou, Tian; Pearlman, Andrew P; Paton, Dorothy L; Rohan, Lisa C

    2017-01-01

    Summary This manuscript summarizes our recent progress in examine the CYP1A1 and CYP1B1 as well as a number of nuclear receptors in the female genital and colorectal tissues of human and pigtailed macaque. Understanding the nuclear receptor mediated regulation of CYP1A1 and 1B1 expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. However, there is a lack of systematic and comparative analysis of the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and clinically relevant animal models. The current study aims to fill this gap. We found CYP1A1, CYP1B1 and a number of nuclear receptors were expressed in the female genital and colorectal tissues of human and macaque. However, the mRNA level and protein localization of these CYP enzymes and NRs depended on the type of tissue examined. Cytochrome P450 (CYP) 1A1 and CYP1B1 activate hormonal and environmental procarcinogens, and are associated with carcinogenesis in female genital and colorectal tissues. Understanding the nuclear receptor (NR) mediated regulation of CYP expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. The study aims to analyze the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and pigtailed macaques. We found that compared to the liver, human CYP1A1 mRNA level in the genital and colorectal tissues was significantly lower, while the CYP1B1 level was significantly higher. CYP1A1 protein was mainly localized in the plasma membrane of the uterine and endocervical epithelial cells. The CYP1B1 protein was concentrated in the nucleus of genital and colorectal tissues. Fourteen NRs in the genital tract and 12 NRs in colorectal tissue were expressed at levels similar to or higher than the liver. The expression and localization of CYP1A1, CYP1B1, and

  18. Contribution of cytochrome P450 1B1 to hypertension and associated pathophysiology: a novel target for antihypertensive agents.

    PubMed

    Malik, Kafait U; Jennings, Brett L; Yaghini, Fariborz A; Sahan-Firat, Seyhan; Song, Chi Young; Estes, Anne M; Fang, Xiao R

    2012-08-01

    The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone (DOCA)-salt-, and N(ω)-nitro-L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3',5'-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Contribution of Cytochrome P450 1B1 to Hypertension and Associated Pathophysiology: A Novel Target for Antihypertensive Agents

    PubMed Central

    Malik, Kafait U.; Jennings, Brett L.; Yaghini, Fariborz A.; Sahan-Firat, Seyhan; Song, Chi Young; Estes, Anne M.; Fang, Xiao R.

    2012-01-01

    The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone Nω-nitro-(DOCA)-salt-, and L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3′,5′-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src. PMID:22210049

  20. Cell-secreted flavins bound to membrane cytochromes dictate electron transfer reactions to surfaces with diverse charge and pH.

    PubMed

    Okamoto, Akihiro; Kalathil, Shafeer; Deng, Xiao; Hashimoto, Kazuhito; Nakamura, Ryuhei; Nealson, Kenneth H

    2014-07-11

    The variety of solid surfaces to and from which microbes can deliver electrons by extracellular electron transport (EET) processes via outer-membrane c-type cytochromes (OM c-Cyts) expands the importance of microbial respiration in natural environments and industrial applications. Here, we demonstrate that the bifurcated EET pathway of OM c-Cyts sustains the diversity of the EET surface in Shewanella oneidensis MR-1 via specific binding with cell-secreted flavin mononucleotide (FMN) and riboflavin (RF). Microbial current production and whole-cell differential pulse voltammetry revealed that RF and FMN enhance EET as bound cofactors in a similar manner. Conversely, FMN and RF were clearly differentiated in the EET enhancement by gene-deletion of OM c-Cyts and the dependency of the electrode potential and pH. These results indicate that RF and FMN have specific binding sites in OM c-Cyts and highlight the potential roles of these flavin-cytochrome complexes in controlling the rate of electron transfer to surfaces with diverse potential and pH.

  1. Flower colour and cytochromes P450.

    PubMed

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-02-19

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

  2. Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis

    PubMed Central

    Kato, Hirotomo; Gomez, Eduardo A.; Martini-Robles, Luiggi; Muzzio, Jenny; Velez, Lenin; Calvopiña, Manuel; Romero-Alvarez, Daniel; Mimori, Tatsuyuki; Uezato, Hiroshi; Hashiguchi, Yoshihisa

    2016-01-01

    A countrywide epidemiological study was performed to elucidate the current geographic distribution of causative species of cutaneous leishmaniasis (CL) in Ecuador by using FTA card-spotted samples and smear slides as DNA sources. Putative Leishmania in 165 samples collected from patients with CL in 16 provinces of Ecuador were examined at the species level based on the cytochrome b gene sequence analysis. Of these, 125 samples were successfully identified as Leishmania (Viannia) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, and L. (Leishmania) mexicana. Two dominant species, L. (V.) guyanensis and L. (V.) braziliensis, were widely distributed in Pacific coast subtropical and Amazonian tropical areas, respectively. Recently reported L. (V.) naiffi and L. (V.) lainsoni were identified in Amazonian areas, and L. (L.) mexicana was identified in an Andean highland area. Importantly, the present study demonstrated that cases of L. (V.) braziliensis infection are increasing in Pacific coast areas. PMID:27410039

  3. The role of Sox6 in zebrafish muscle fiber type specification.

    PubMed

    Jackson, Harriet E; Ono, Yosuke; Wang, Xingang; Elworthy, Stone; Cunliffe, Vincent T; Ingham, Philip W

    2015-01-01

    The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification. The GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres. Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine. The Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation

  4. Bioenergetics and the role of soluble cytochromes C for alkaline adaptation in gram-negative alkaliphilic Pseudomonas.

    PubMed

    Matsuno, T; Yumoto, I

    2015-01-01

    Very few studies have been conducted on alkaline adaptation of Gram-negative alkaliphiles. The reversed difference of H(+) concentration across the membrane will make energy production considerably difficult for Gram-negative as well as Gram-positive bacteria. Cells of the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21(T) grown at pH 10 under low-aeration intensity have a soluble cytochrome c content that is 3.6-fold higher than that of the cells grown at pH 7 under high-aeration intensity. Cytochrome c-552 content was higher (64% in all soluble cytochromes c) than those of cytochrome c-554 and cytochrome c-551. In the cytochrome c-552-dificient mutant grown at pH 10 under low-aeration intensity showed a marked decrease in μ max⁡ [h(-1)] (40%) and maximum cell turbidity (25%) relative to those of the wild type. Considering the high electron-retaining abilities of the three soluble cytochromes c, the deteriorations in the growth of the cytochrome c-552-deficient mutant could be caused by the soluble cytochromes c acting as electron storages in the periplasmic space of the bacterium. These electron-retaining cytochromes c may play a role as electron and H(+) condenser, which facilitate terminal oxidation at high pH under air-limited conditions, which is difficult to respire owing to less oxygen and less H(+).

  5. Bioenergetics and the Role of Soluble Cytochromes c for Alkaline Adaptation in Gram-Negative Alkaliphilic Pseudomonas

    PubMed Central

    Matsuno, T.; Yumoto, I.

    2015-01-01

    Very few studies have been conducted on alkaline adaptation of Gram-negative alkaliphiles. The reversed difference of H+ concentration across the membrane will make energy production considerably difficult for Gram-negative as well as Gram-positive bacteria. Cells of the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21T grown at pH 10 under low-aeration intensity have a soluble cytochrome c content that is 3.6-fold higher than that of the cells grown at pH 7 under high-aeration intensity. Cytochrome c-552 content was higher (64% in all soluble cytochromes c) than those of cytochrome c-554 and cytochrome c-551. In the cytochrome c-552-dificient mutant grown at pH 10 under low-aeration intensity showed a marked decrease in μ max⁡ [h−1] (40%) and maximum cell turbidity (25%) relative to those of the wild type. Considering the high electron-retaining abilities of the three soluble cytochromes c, the deteriorations in the growth of the cytochrome c-552-deficient mutant could be caused by the soluble cytochromes c acting as electron storages in the periplasmic space of the bacterium. These electron-retaining cytochromes c may play a role as electron and H+ condenser, which facilitate terminal oxidation at high pH under air-limited conditions, which is difficult to respire owing to less oxygen and less H+. PMID:25705691

  6. Gene structure of CYP3A4, an adult-specific form of cytochrome P450 in human livers, and its transcriptional control.

    PubMed

    Hashimoto, H; Toide, K; Kitamura, R; Fujita, M; Tagawa, S; Itoh, S; Kamataki, T

    1993-12-01

    CYP3 A4 is the adult-specific form of cytochrome P450 in human livers [Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. & Kamataki, T. (1990) Biochemistry 29, 4430-4433]. The sequences of three genomic clones for CYP3A4 were analyzed for all exons, exon-intron junctions and the 5'-flanking region from the major transcription site to nucleotide position -1105, and compared with those of the CYP3A7 gene, a fetal-specific form of cytochrome P450 in humans. The results showed that the identity of 5'-flanking sequences between CYP3A4 and CYP3A7 genes was 91%, and that each 5'-flanking region had characteristic sequences termed as NFSE (P450NF-specific element) and HFLaSE (P450HFLa specific element), respectively. A basic transcription element (BTE) also lay in the 5'-flanking region of the CYP3A4 gene as seen in many CYP genes [Yanagida, A., Sogawa, K., Yasumoto, K. & Fujii-Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470-1475]. The BTE binding factor (BTEB) was present in both adult and fetal human livers. To examine the transcriptional activity of the CYP3A4 gene, DNA fragments in the 5'-flanking region of the gene were inserted in front of the simian virus 40 promoter and the chloramphenicol acetyltransferase structural gene, and the constructs were transfected in HepG2 cells. The analysis of the chloramphenicol acetyltransferase activity indicated that (a) specific element(s) which could bind with a factor(s) in livers was present in the 5'-flanking region of the CYP3A4 gene to show the transcriptional activity.

  7. Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species.

    PubMed

    Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

    2012-03-16

    The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

  8. Strain-specific reverse transcriptase PCR assay: means to distinguish candidate vaccine from wild-type strains of respiratory syncytial virus.

    PubMed Central

    Zheng, H; Peret, T C; Randolph, V B; Crowley, J C; Anderson, L J

    1996-01-01

    Candidate live-virus vaccines for respiratory syncytial virus are being developed and are beginning to be evaluated in clinical trials. To distinguish candidate vaccine strains from wild-type strains isolated during these trials, we developed PCR assays specific to two sets of candidate vaccine strains. The two sets were a group A strain (3A), its three attenuated, temperature-sensitive variant strains, a group B strain (2B), and its four attenuated, temperature-sensitive variant strains. The PCR assays were evaluated by testing 18 group A wild-type strains, the 3A strains, 9 group B wild-type strains, and the 2B strains. PCR specific to group A wild-type strains amplified only group A wild-type strains, and 3A-specific PCR amplified only 3A strains. PCR specific to group B wild-type strains amplified all group A and group B strains but gave a 688-bp product for group B wild-type strains, a 279-bp product for 2B strains, a 547-bp product for all group A strains, and an additional 688-bp product for some group A strains, including 3A strains. These types of PCR assays can, in conjunction with other methods, be used to efficiently distinguish candidate vaccine strains from other respiratory syncytial virus strains. PMID:8789010

  9. Application of molecular genetics method for differentiating Martes zibellina L. heart from its adulterants in traditional Chinese medicine based on mitochondrial cytochrome b gene.

    PubMed

    Li, Mingcheng; Xia, Wei; Wang, Miao; Yang, Mingyan; Zhang, Lihua; Guo, Jie

    2014-02-01

    The use of Martes zibellina L. heart as a famous kind of traditional Chinese medicine has been documented for many years in China. Identification of its authenticity as raw materials became a key in controlling of herbal preparations. In this study, the characteristics of mitochondrial cytochrome b (Cyt b) gene from four species of Martes were explored, and a specific molecular genetics technique for identifying the heart of M. zibellina L. in addition to some close relatives from their counterfeits was established. The bioinformatics was carried out to design the primers for the Cyt b gene based on the different species of Martes. PCR and sequencing technology were performed. The mt DNA was extracted from all of fresh M. zibellina L., Martes melampus. Martes flavigula. Martes martes heart samples and dry M. zibellina L. heart powder through the modified alkaline extracting method in addition to its counterfeits including the chicken heart, duck heart, goose heart, rabbit heart and Mustela vison. The complete mt DNA was separated from all samples used in the study, and the Cyt b gene with 310 bp segments was amplified only from M. zibellina L. heart as DNA template by the PCR technique. The sequencing indicated that the segment amplified by the PCR was homologous with the species of M. zibellina in GenBank. The data revealed that the primers and selected segment could be used as the genetic markers to identify M. zibellina L. heart from its counterfeits among different animal species.

  10. The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

    PubMed

    Veit, Sebastian; Nagadoi, Aritaka; Rögner, Matthias; Rexroth, Sascha; Stoll, Raphael; Ikegami, Takahisa

    2016-06-01

    PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Electron transfer between cytochrome. alpha. and copper A in cytochrome c oxidase: A perturbed equilibrium study

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morgan, J.E.; Li, P.M.; Jang, D.J.

    1989-08-22

    Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by the perturbed equilibrium method. The authors have prepared a three-electron-reduced, CO-inhibited form of the enzyme in which cytochrome a and copper A are partially reduced and in an intramolecular redox equilibrium. When these samples were irradiated with a nitrogen laser to photodissociate the bound CO, changes in absorbance at 598 and 830 nm were observed which were consistent with a fast electron transfer from cytochrome a to copper A. The absorbance changes at 598 nm gave an apparent rate of 17,000 {plus minus} 2,000 s{sup {minus}1} (1more » {sigma}), at pH 7.0 and 25.5{degree}C. These changes were not observed in either the CO mixed-valence or the CO-inhibited fully reduced forms of the enzyme. The rate was fastest at about pH 8.0, falling off toward both lower and higher pHs. There was a small but clear temperature dependence. The process was also observed in the cytochrome c-cytochrome c oxidase high-affinity complex. The electron equilibration measured between cytochrome {alpha} and copper A is far faster than any rate measured or inferred previously for this process.« less

  12. Humanlike substitutions to Ω-loop D of yeast iso-1-cytochrome c only modestly affect dynamics and peroxidase activity.

    PubMed

    Lei, Haotian; Bowler, Bruce E

    2018-06-01

    Structural studies of yeast iso-1-cytochrome c (L.J. McClelland, T.-C. Mou, M.E. Jeakins-Cooley, S.R. Sprang, B.E. Bowler, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 6648-6653) show that modest movement of Ω-loop D (residues 70-85, average RMSD versus the native structure: 0.81 Å) permits loss of Met80-heme ligation creating an available coordination site to catalyze the peroxidase activity mediated by cytochrome c early in apoptosis. However, Ala81 and Gly83 move significantly (RMSDs of 2.18 and 1.26 Å, respectively). Ala81 and Gly83 evolve to Ile and Val, respectively, in human cytochrome c and peroxidase activity decreases 25-fold relative to the yeast protein at pH 7. To test the hypothesis that these residues evolved to restrict the peroxidase activity of cytochrome c, A81I and G83V variants of yeast iso-1-cytochrome c were prepared. For both variants, the apparent pK a of the alkaline transition increases by 0.2 to 0.3 relative to the wild type (WT) protein and the rate of opening the heme crevice is slowed. The cooperativity of acid unfolding is decreased for the G83V variant. At pH 7 and 8, the catalytic rate constant, k cat , for the peroxidase activity of both variants decreases relative to WT, consistent with the effects on alkaline isomerization. Below pH 7, the loss in the cooperativity of acid unfolding causes k cat for peroxidase activity to increase for the G83V variant relative to WT. Neither variant decreases k cat to the level of the human protein, indicating that other residues also contribute to the low peroxidase activity of human cytochrome c. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Use of P450 cytochrome inhibitors in studies of enokipodin biosynthesis

    PubMed Central

    Ishikawa, Noemia Kazue; Tahara, Satoshi; Namatame, Tomohiro; Farooq, Afgan; Fukushi, Yukiharu

    2013-01-01

    Enokipodins A, B, C, and D are antimicrobial sesquiterpenes isolated from the mycelial culture medium of Flammulina velutipes, an edible mushroom. The presence of a quaternary carbon stereocenter on the cyclopentane ring makes enokipodins A-D attractive synthetic targets. In this study, nine different cytochrome P450 inhibitors were used to trap the biosynthetic intermediates of highly oxygenated cuparene-type sesquiterpenes of F. velutipes. Of these, 1-aminobenzotriazole produced three less-highly oxygenated biosynthetic intermediates of enokipodins A-D; these were identified as (S)-(−)-cuparene-1,4-quinone and epimers at C-3 of 6-hydroxy-6-methyl-3-(1,2,2-trimethylcyclopentyl)-2-cyclohexen-1-one. One of the epimers was found to be a new compound. PMID:24688524

  14. The diheme cytochrome c4 from Vibrio cholerae is a natural electron donor to the respiratory cbb3 oxygen reductase

    PubMed Central

    Chang, Hsin-Yang; Ahn, Young; Pace, Laura A.; Lin, Myat T.; Lin, Yun-Hui; Gennis, Robert B.

    2010-01-01

    The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb3 oxygen reductase. The cbb3 oxygen reductase has been previously isolated and characterized, however the natural mobile electron donor(s) which shuttles electrons between the bc1 complex and the cbb3 oxygen reductase is not known. The most likely candidates are the diheme cytochrome c4 and mono-heme cytochrome c5, which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c4 and c5 from V. cholerae have been cloned and expressed heterologously in E. coli. It is shown that reduced cytochrome c4 is a substrate for the purified cbb3 oxygen reductase and can support steady state oxygen reductase activity of at least 300 e−1/s. In contrast, reduced cytochrome c5 is not a good substrate for the cbb3 oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c4 does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c4 indicates that the apparent lack of saturation is due to the strong dependence of KM and Vmax on the concentration of oxidized cytochrome c4. Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c4 are 240 mV and 340 mV (vs SHE), similar to the electrochemical properties of other c4-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c4-type cytochrome and a cbb3 oxygen reductase within the β- and γ- proteobacterial clades, suggesting that cytochrome c4 is the likely natural electron donor to the cbb3 oxygen reductases within these organisms. These would include the β-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb3 oxygen reductases are the only terminal

  15. 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

    large "target" with a small "bullseye" for the cyt b(5) "arrow". This paradigm differs sharply from the more familiar, "simple" docking within a single, or narrow range of conformations, where binding strength and ET reactivity increase in parallel. Likewise, it is distinct from, although complementary to, the well-known picture of conformational control of ET through "gating", or a related picture of "conformational coupling". The new model describes situations in which tight binding does not correlate with efficient ET reactivity, and explains how it is possible to modulate reactivity without changing affinity. Such "decoupling" of reactivity from binding clearly is of physiological relevance for the reduction of met-Mb in muscle and of met-Hb in a red cell, where tight binding of cyt b(5) to the high concentration of ferrous-Mb/Hb would prevent the cytochrome from finding and reducing the oxidized proteins; it likely is of physiological relevance in other situations, as well.

  16. Immunochemical cross-reactions between type III group B Streptococcus and type 14 Streptococcus pneumoniae.

    PubMed Central

    Crumrine, M H; Fischer, G W; Balk, M W

    1979-01-01

    Serological cross-reactions between certain streptococci and some serotypes of Streptococcus pneumoniae have been reported. These studies detail the serological cross-reactivity observed between hot HCl-extracted group b streptococcus type III (GBS III) antigens and S. pneumoniae type 14 (Pn 14) polysaccharide. Similar electrophoretic migration patterns of GBS III and Pn 14 were observed when either type-specific BGS III antisera or pneumococcal omniserum was utilized to precipitate these antigens. Both the GBS III antigen and the Pn 14 polysaccharide migrated toward the cathode, whereas all other pneumococcal polysaccharides migrated toward the anode. No cross-reactions were observed between GBS III antisera and the 11 other types of pneumococcal polysaccharides. Lines of identity were observed between type-specific GBS III antisera and monospecific Pn 14 antiserum with either GBS III antigens or purified Pn 14 polysaccharide. The cross-reacting antigens of GBS III and Pn 14 appear to be identical by immunodiffusion and immunoelectrophoresis. Images PMID:40876

  17. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-,more » and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.« less

  18. Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

    PubMed

    Arlt, Volker M; Poirier, Miriam C; Sykes, Sarah E; John, Kaarthik; Moserova, Michaela; Stiborova, Marie; Wolf, C Roland; Henderson, Colin J; Phillips, David H

    2012-09-03

    Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  19. Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances.

    PubMed

    Dantas, Joana M; Ferreira, Marisa R; Catarino, Teresa; Kokhan, Oleksandr; Raj Pokkuluri, P; Salgueiro, Carlos A

    2018-05-16

    The bacterium Geobacter sulfurreducens can transfer electrons to the quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH 2 QDS) can also be used as energy source G. sulfurreducens. Such bi-directional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bi-functional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH 2 QDS. Using stopped-flow measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same bi-functional behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for 13 C, 15 N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and 1 H heme methyl group signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex with AQDS through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens. Copyright © 2018. Published by Elsevier B.V.

  20. Streptococcus group B typing: comparison of counter-immunoelectrophoresis with the precipitin method.

    PubMed

    Kubín, V; Jelínková, J; Franêk, J

    1977-07-01

    The method of counter-immunoelectrophoresis (CIE) was tested for its applicability to group B streptococcus typing. The results obtained were compared with the typing by the ring precipitin test. Identical antigens and identical hyperimmune typing serum batches had been used in both methods. A large majority of 75 freshly isolated strains were typed identically by both methods. Five strains with a weak antigenic outfit were untypable by the ring precipitin test but were typed by CIE owing to a higher sensitivity of CIE method. Two strains were typable by the precipitin test but not by CIE; an explanation for this phenomenon is lacking. The CIE method in group B typing is specific, rapid, highly sensitive and relatively simple. It requires strict maintenance of standard conditions. The method is economical with respect to manipulation and material, requires small amounts of diagnostic antisera. Potent antisera may be used diluted. Moreover, sera for CIE typing need not be absorbed to remove group B antibodies. CIE method is practicable for group B streptococcus typing, especially in laboratories carrying out routine large scale type identification.

  1. Genome-wide identification of 52 cytochrome P450 (CYP) genes in the copepod Tigriopus japonicus and their B[α]P-induced expression patterns.

    PubMed

    Han, Jeonghoon; Kim, Duck-Hyun; Kim, Hui-Su; Nelson, David R; Lee, Jae-Seong

    2017-09-01

    Cytochrome P450s (CYPs) are enzymes with a heme-binding domain that are found in all living organisms. CYP enzymes have important roles associated with detoxification of xenobiotics and endogenous compounds (e.g. steroids, fatty acids, and hormones). Although CYP enzymes have been reported in several invertebrates, including insects, little is known about copepod CYPs. Here, we identified the entire repertoire of CYP genes (n=52) from whole genome and transcriptome sequences of the benthic copepod Tigriopus japonicus, including a tandem duplication (CYP3026A3, CYP3026A4, CYP3026A5), and examined patterns of gene expression over various developmental stages and in response to benzo[α]pyrene (B[α]P) exposure. Through phylogenetic analysis, the 52 T. japonicus CYP genes were assigned to five distinct clans: CYP2 (22 genes), CYP3 (19 genes), CYP4 (two genes), CYP20 (one gene), and mitochondrial (eight genes). Developmental stage and gender-specific expression patterns of the 52 T. japonicus CYPs were analyzed. CYP3022A1 was constitutively expressed during all developmental stages. CYP genes in clans 2 and 3 were induced in response to B[α]P, suggesting that these differentially modulated CYP transcripts are likely involved in defense against exposure to B[α]P and other pollutants. This study enhances our understanding of the repertoire of CYP genes in copepods and of their potential role in development and detoxification in copepods. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species*

    PubMed Central

    Paulus, Angela; Rossius, Sebastiaan Gijsbertus Hendrik; Dijk, Madelon; de Vries, Simon

    2012-01-01

    The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species. PMID:22287551

  3. Survey of familial glaucoma shows a high incidence of cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) mutations in non-consanguineous congenital forms in a Spanish population

    PubMed Central

    Millá, Elena; Mañé, Begoña; Duch, Susana; Hernan, Imma; Borràs, Emma; Planas, Ester; Dias, Miguel de Sousa; Carballo, Miguel

    2013-01-01

    Purpose To identify myocilin (MYOC) and cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) mutations in a Spanish population with different clinical forms of familial glaucoma or ocular hypertension (OHT). Methods Index patients from 226 families participated in this study. Patients were diagnosed with familial glaucoma or OHT by complete ophthalmologic examination. Screening for MYOC mutations was performed in 207 index patients: 96 with adult-onset primary open-angle glaucoma (POAG), 21 with primary congenital glaucoma (PCG), 18 with juvenile-onset open-angle glaucoma (JOAG), five with Axenfeld-Rieger syndrome (ARS), and 67 with other types of glaucoma. One hundred two of the families (including all those in whom a MYOC mutation was detected) were also screened for CYP1B1 mutations: 45 POAG, 25 PCG, 21 JOAG, four ARS, and seven others. Results We examined 292 individuals (patients and relatives) with a positive family history of glaucoma or OHT. We identified two novel MYOC variants, p.Lys39Arg and p.Glu218Lys, in two families with POAG, and six previously reported MYOC mutations in seven families with POAG (four), JOAG (one), PCG (one), and normotensive glaucoma (one). CYP1B1 mutations were found in 16 index patients with PCG (nine), POAG (three), JOAG (two), and ARS (two). Conclusions The high percentage (9/25=36%) of mutations in CYP1B1 found in non-consanguineous patients with congenital glaucoma mandates genetic testing. However, the percentage of mutations (9/207=4.4%) in MYOC associated with glaucoma is relatively low in our population. The variable phenotype expression of glaucoma, even in families, cannot be explained with a digenic mechanism between MYOC and CYP1B1. PMID:23922489

  4. Diazotrophic specific cytochrome c oxidase required to overcome light stress in the cyanobacterium Nostoc muscorum.

    PubMed

    Bhargava, Santosh; Chouhan, Shweta

    2016-01-01

    Diazotrophic, filamentous and heterocystous cyanobacterium Nostoc muscorum perform photosynthesis in vegetative whereas nitrogen fixation occurs in heterocyst only. However, despite their metabolic plasticity, respiration takes place both in vegetative cells and heterocysts. The role of the respiratory electron transport system and terminal oxidases under light stress is not evident so far. As compared to the diazotrophically grown cultures, the non-diazotrophically grown cultures of the N. muscorum show a slight decrease in their growth, chlorophyll a contents and photosynthetic O2 evolution under light stress. Whereas respiratory O2 uptake under identical stress condition increases several fold. Likewise, nitrogen fixing enzyme i.e. nitrogenase over-expresses itself under light stress condition. The terminal enzyme of respiratory electron transport chain i.e. cytochrome c oxidase shows more activity under light stress, whilst light stress has no impact on Ca(++)-dependent ATPase activity. This leads to the conclusion that under light stress, cytochrome c oxidase plays a vital role in mitigating given light stress.

  5. Surface multiheme c-type cytochromes from Thermincola potens: Implications for dissimilatory metal reduction by Gram-positive bacteria

    NASA Astrophysics Data System (ADS)

    Carlson, H. K.; Iavarone, A. T.; Gorur, A.; Yeo, B. S.; Tran, R.; Melnyk, R. A.; Mathies, R. A.; Auer, M.; Coates, J. D.

    2011-12-01

    Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they have been shown to be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or the humic substances analog, anthraquinone-2,6-disulfonate (AQDS). The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS and that several MHCs are localized to the cell wall or cell surface of T. potens. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results are the first direct evidence for cell-wall associated cytochromes and MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

  6. Differential tissue-specific function of the Adora2b in cardio-protection

    PubMed Central

    Seo, Seong-wook; Koeppen, Michael; Bonney, Stephanie; Gobel, Merit; Thayer, Molly; Harter, Patrick N.; Ravid, Katya; Eltzschig, Holger K.; Mittelbronn, Michel; Walker, Lori; Eckle, Tobias

    2015-01-01

    The adenosine A2b-receptor (Adora2b) has been implicated in cardio-protection from myocardial ischemia. As such the Adora2b was found to be critical in ischemic preconditioning (IP) or ischemia reperfusion (IR) injury of the heart. While the Adora2b is present on various cells types, the tissue specific role of the Adora2b in cardio-protection is still unknown. To study the tissue specific role of Adora2b signaling on inflammatory cells, endothelia or myocytes during myocardial ischemia in vivo, we intercrossed floxed Adora2b mice with Lyz2-Cre+, VE-Cadherin-Cre+ or Myosin-Cre+ transgenic mice, respectively. Mice were exposed to 60 minutes of myocardial ischemia with or without IP (4×5min) followed by 120 minutes of reperfusion. Cardio-protection by IP was abolished in Adora2bf/f-VE-Cadherin-Cre+ or Adora2bf/f-Myosin-Cre+, indicating that Adora2bs signaling on endothelia or myocytes mediates IP. In contrast, primarily Adora2b signaling on inflammatory cells was necessary to provide cardio-protection in IR injury, indicated by significantly larger infarcts and higher troponin levels in Adora2bf/f-Lyz2-Cre+ mice only. Cytokine profiling of IR injury in Adora2bf/f-Lyz2-Cre+ mice pointed towards PMNs. Analysis of PMNs from Adora2bf/f-Lyz2-Cre+ confirmed PMNs as one source of identified tissue cytokines. Finally, adoptive transfer of Ador2b−/− PMNs revealed a critical role of the Adorab2 on PMNs in cardio-protection from IR-injury. Adora2b signaling mediates different types of cardio-protection in a tissue specific manner. These findings have implications for the use of Adora2b agonists in the treatment or prevention of myocardial injury by ischemia. PMID:26136425

  7. Cytochrome P450-mediated hepatic metabolism of new fluorescent substrates in cats and dogs.

    PubMed

    van Beusekom, C D; Schipper, L; Fink-Gremmels, J

    2010-12-01

    This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities associated with CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured. Cat liver microsomes metabolized all substrates selected for the assessment of cytochrome P450 activity. The activities associated with CYP3A and CYP2B were higher than the activities of the other measured CYPs. Substrate selectivity could be demonstrated by inhibition studies with α-naphthoflavone (CYP1A), tranylcypromine/quercetine (CYP2C), quinidine (CYP2D), diethyldithiocarbamic acid (CYP2E) and ketoconazole (CYP3A) respectively. Other prototypical inhibitors used for characterization of human CYP activities such as furafylline (CYP1A), tranylcypromine (CYP2B) and sulfaphenazole (CYP2C) did not show significant effects in cat and dog liver microsomes. Moreover, IC50-values of cat CYPs differed from dog and human CYPs underlining the interspecies differences. Gender differences were observed in the oxidation of 7-ethoxy-4-trifluoromethylcoumarin (CYP2B) and 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D), which were significantly higher in male cats than in females. Conversely, oxidation of the substrates dibenzylfluorescein (CYP2C) and 7-methoxy-4-trifluoromethylcoumarin (CYP2E) showed significant higher activities in females than in male cats. Overall CYP-activities in cat liver microsomes were lower than in those from dogs or humans, except for CYP2B. The presented difference between feline and canine CYP-activities are useful to establish dose corrections for feline patients of intensively metabolized drugs licensed for dogs or humans. © 2010 Blackwell Publishing Ltd.

  8. Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Osawa, Y.; Coon, M.J.

    1987-08-01

    In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, (1,2,6,7,16,17-3H)progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting (1,7,16-3H)progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of /sup 3/H/submore » 2/O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of (1,7,16-/sup 3/H)progesterone and (4-14C)progesterone was employed.« less

  9. Novel type of VanB2 teicoplanin-resistant hospital-associated Enterococcus faecium.

    PubMed

    Santona, Antonella; Paglietti, Bianca; Al-Qahtani, Ahmed A; Bohol, Marie Fe F; Senok, Abiola; Deligios, Massimo; Rubino, Salvatore; Al-Ahdal, Mohammed N

    2014-08-01

    Seven high-risk clones of vancomycin-resistant Enterococcus faecium (VREF) belonging to clonal complex 17 were identified using multilocus sequence typing (MLST) among clinical isolates from Saudi Arabia. Among these isolates, a new hospital-associated sequence type (ST795), VanB(2)-type teicoplanin-resistant strain was detected. Its unusual phenotype resulted from a new combination of mutations in the ddl, vanS and vanW genes, which confirmed the trend of evolution in VanB-type resistance. Furthermore, characteristics of adaptation and persistence in the hospital environment of ST795 were emphasised by the presence of genes and clusters recognised to be specific for hospital-associated VREF. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  10. Cardiolipin-Specific Peroxidase Reactions of Cytochrome c in Mitochondria During Irradiation-Induced Apoptosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Belikova, Natalia A.; Jiang Jianfei; Tyurina, Yulia Y.

    2007-09-01

    Purpose: To determine whether cytochrome c (cyt c) content and associated cardiolipin oxidation can be determinants of cell sensitivity to irradiation-induced apoptosis. Methods and Materials: The small interfering RNA (siRNA) approach was used to engineer HeLa cells with lowered contents of cyt c (14%, HeLa 1.2 cells). Cells were treated by {gamma}-irradiation (in doses of 5-40 Gy). Lipid oxidation was characterized by electrospray ionization mass spectrometry analysis and fluorescence high-performance liquid chromatography-based Amplex Red assay. Release of a proapoptotic factor (cyt c, Smac/DIABLO) was detected by Western blotting. Apoptosis was revealed by caspase-3/7 activation and phosphatidylserine externalization. Results: Irradiation causedmore » selective accumulation of hydroperoxides in cardiolipin (CL) but not in other phospholipids. HeLa 1.2 cells responded by a lower irradiation-induced accumulation of CL oxidation products than parental HeLa cells. Proportionally decreased release of a proapoptotic factor, Smac/DIABLO, was detected in cyt c-deficient cells after irradiation. Caspase-3/7 activation and phosphatidylserine externalization were proportional to the cyt c content in cells. Conclusions: Cytochrome c is an important catalyst of CL peroxidation, critical to the execution of the apoptotic program. This new role of cyt c in irradiation-induced apoptosis is essential for the development of new radioprotectors and radiosensitizers.« less

  11. Genetic variation of Taenia pisiformis collected from Sichuan, China, based on the mitochondrial cytochrome B gene.

    PubMed

    Yang, Deying; Ren, Yongjun; Fu, Yan; Xie, Yue; Nie, Huaming; Nong, Xiang; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou

    2013-08-01

    Taenia pisiformis is one of the most important parasites of canines and rabbits. T. pisiformis cysticercus (the larval stage) causes severe damage to rabbit breeding, which results in huge economic losses. In this study, the genetic variation of T. pisiformis was determined in Sichuan Province, China. Fragments of the mitochondrial cytochrome b (cytb) (922 bp) gene were amplified in 53 isolates from 8 regions of T. pisiformis. Overall, 12 haplotypes were found in these 53 cytb sequences. Molecular genetic variations showed 98.4% genetic variation derived from intra-region. FST and Nm values suggested that 53 isolates were not genetically differentiated and had low levels of genetic diversity. Neutrality indices of the cytb sequences showed the evolution of T. pisiformis followed a neutral mode. Phylogenetic analysis revealed no correlation between phylogeny and geographic distribution. These findings indicate that 53 isolates of T. pisiformis keep a low genetic variation, which provide useful knowledge for monitoring changes in parasite populations for future control strategies.

  12. Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance.

    PubMed

    Hou, Yuting; An, Jianhong; Deng, Chunyan; Chen, Shu; Xiang, Juan

    2016-07-01

    The interactions between the redox couple of cytochrome c (Cyt c) and cytochrome c oxidase (COX) were investigated at a mimic redox-modulated interface by using an electrochemical surface plasmon resonance (EC-SPR) system. Although early studies of the binding between COX and Cyt c have been conducted using several techniques in homogeneous solutions, a problem still inherent is that ferro-cytochrome c (Cyt c red), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt c ox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt c red is quantitatively electro-generated from Cyt c ox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt c red/Cyt c ox can be evaluated accurately. In this study, the values of association/dissociation rate constants (k a, k d) for both COX/Cyt c red and COX/Cyt c ox were obtained. The dissociation constants, K D, were finally calculated as 3.33 × 10(-8) mol · L(-1) for COX/Cyt c red and 4.25 × 10(-5) mol · L(-1) for COX/Cyt c ox, respectively. In-situ EC-SPR is promising for better mimicking the in vivo condition that COX is embedded in the inner mitochondrial membrane and Cyt c acts as an electron shuttle in the mobile phase. It is an effective method for the investigation of redox-dependent biomolecular interactions. Graphical Abstract Schematic representation of the experimental designs using EC-SPR system. (a) the Au-Cys-COX SPR chip with SAM layers. (b) redox-modulated Cyt c and its binding onto pre-immobilized COX.

  13. Phylogenetic relationships of Malaysia's long-tailed macaques, Macaca fascicularis, based on cytochrome b sequences.

    PubMed

    Abdul-Latiff, Muhammad Abu Bakar; Ruslin, Farhani; Fui, Vun Vui; Abu, Mohd-Hashim; Rovie-Ryan, Jeffrine Japning; Abdul-Patah, Pazil; Lakim, Maklarin; Roos, Christian; Yaakop, Salmah; Md-Zain, Badrul Munir

    2014-01-01

    Phylogenetic relationships among Malaysia's long-tailed macaques have yet to be established, despite abundant genetic studies of the species worldwide. The aims of this study are to examine the phylogenetic relationships of Macaca fascicularis in Malaysia and to test its classification as a morphological subspecies. A total of 25 genetic samples of M. fascicularis yielding 383 bp of Cytochrome b (Cyt b) sequences were used in phylogenetic analysis along with one sample each of M. nemestrina and M. arctoides used as outgroups. Sequence character analysis reveals that Cyt b locus is a highly conserved region with only 23% parsimony informative character detected among ingroups. Further analysis indicates a clear separation between populations originating from different regions; the Malay Peninsula versus Borneo Insular, the East Coast versus West Coast of the Malay Peninsula, and the island versus mainland Malay Peninsula populations. Phylogenetic trees (NJ, MP and Bayesian) portray a consistent clustering paradigm as Borneo's population was distinguished from Peninsula's population (99% and 100% bootstrap value in NJ and MP respectively and 1.00 posterior probability in Bayesian trees). The East coast population was separated from other Peninsula populations (64% in NJ, 66% in MP and 0.53 posterior probability in Bayesian). West coast populations were divided into 2 clades: the North-South (47%/54% in NJ, 26/26% in MP and 1.00/0.80 posterior probability in Bayesian) and Island-Mainland (93% in NJ, 90% in MP and 1.00 posterior probability in Bayesian). The results confirm the previous morphological assignment of 2 subspecies, M. f. fascicularis and M. f. argentimembris, in the Malay Peninsula. These populations should be treated as separate genetic entities in order to conserve the genetic diversity of Malaysia's M. fascicularis. These findings are crucial in aiding the conservation management and translocation process of M. fascicularis populations in Malaysia.

  14. The folding energy landscape and free energy excitations of cytochrome c.

    PubMed

    Weinkam, Patrick; Zimmermann, Jörg; Romesberg, Floyd E; Wolynes, Peter G

    2010-05-18

    The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Because of the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case, following principles different from those used to describe the folding of proteins without cofactors. Recent investigations, however, demonstrate that common models describing folding for many proteins work well for cytochrome c when heme is explicitly introduced, generally providing results that agree with experimental observations. In this Account, we first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Because attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as "perfectly funneled". In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are denoted sources of "frustration", which cause specific transient intermediates to be populated. Native structure-based models do, however, include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold so that folding can proceed. The ability of native structure-based models to capture this kind of motion is partly responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe the sequence of folding events for cytochrome c well (as inferred from hydrogen-exchange experiments), thereby justifying their use as a starting point. At low pH, the experimentally observed folding sequence of cytochrome c deviates from that at pH 7 and from models with perfectly funneled energy landscapes. Here, alternate folding pathways are

  15. Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

    PubMed Central

    Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.; Choi, Vivian; Kasper, Charles B.; Pearl, Naw May; Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy; Kim, Jung-Ja P.

    2011-01-01

    The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners. PMID:21345800

  16. Flower colour and cytochromes P450†

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

  17. Pharmacokinetics and Differential Regulation of Cytochrome P450 Enzymes in Type 1 Allergic Mice.

    PubMed

    Tanino, Tadatoshi; Komada, Akira; Ueda, Koji; Bando, Toru; Nojiri, Yukie; Ueda, Yukari; Sakurai, Eiichi

    2016-12-01

    Type 1 allergic diseases are characterized by elevated production of specific immunoglobulin E (IgE) for each antigen and have become a significant health problem worldwide. This study investigated the effect of IgE-mediated allergy on drug pharmacokinetics. To further understand differential suppression of hepatic cytochrome P450 (P450) activity, we examined the inhibitory effect of nitric oxide (NO), a marker of allergic conditions. Seven days after primary sensitization (PS7) or secondary sensitization (SS7), hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities were decreased to 45%-75% of the corresponding control; however, CYP2D activity was not downregulated. PS7 and SS7 did not change the expression levels of five P450 proteins. Disappearance of CYP1A2 and CYP2D substrates from the plasma was not significantly different between allergic mice and control mice. In contrast, the area under the curve of a CYP1A2-mediated metabolite in PS7 and SS7 mice was reduced by 50% of control values. Total clearances of a CYP2E1 substrate in PS7 and SS7 mice were significantly decreased to 70% and 50% respectively, of the control without altering plasma protein binding. Hepatic amounts of CYP1A2 and CYP2E1 substrates were enhanced by allergic induction, being responsible for each downregulated activity. NO scavenger treatment completely improved the downregulated P450 activities. Therefore, our data suggest that the onset of IgE-mediated allergy alters the pharmacokinetics of major P450-metabolic capacity-limited drugs except for CYP2D drugs. NO is highly expected to participate in regulatory mechanisms of the four P450 isoforms. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Glycosyltransferases A and B: Four Critical Amino Acids Determine Blood Type

    NASA Astrophysics Data System (ADS)

    Rose, Natisha L.; Palcic, Monica M.; Evans, Stephen V.

    2005-12-01

    Human A, B, and O blood type is determined by the presence or absence of distinct carbohydrate structures on red blood cells. Type O individuals have α-fucose(1→2)galactose disaccharides [O(H) structures] on their cell surfaces while in type A or B individuals, the O antigen is capped by the addition of an α- N -acetylgalactosamine or α-galactose residue, respectively. The addition of these monosaccharides is catalyzed by glycosyltransferase A (GTA) or glycosyltransferase B (GTB). These are homologous enzymes differing by only 4 amino acids out of 354 that change the specificity from GTA to GTB. In this review the chemistry of the blood group ABO system and the role of GTA, GTB, and the four critical amino acids in determining blood group status are discussed. See JCE Featured Molecules .

  19. Identification of two new cytochrome P450 genes and RNA interference to evaluate their roles in detoxification of commonly used insecticides in Locusta migratoria.

    PubMed

    Guo, Yanqiong; Zhang, Jianzhen; Yu, Rongrong; Zhu, Kun Yan; Guo, Yaping; Ma, Enbo

    2012-05-01

    Cytochrome P450 monooxygenases (cytochrome P450s), found in virtually all living organisms, play an important role in the metabolism of xenobiotics such as drugs, pesticides, and plant toxins. We have previously evaluated the responses of the oriental migratory locust (Locusta migratoria) to the pyrethroid insecticide deltamethrin and revealed that increased cytochrome P450 enzyme activity was due to increased transcription of multiple cytochrome P450 genes. In this study, we identified for the first time two new cytochrome P450 genes, which belong to two novel cytochrome P450 gene families. CYP409A1 belongs to CYP409 family whereas CYP408B1 belongs to CYP408 family. Our molecular analysis indicated that CYP409A1 was mainly expressed in fatbodies, midgut, gastric caecum, foregut and Malpighian tubules of the third- and fourth-instar nymphs, whereas CYP408B1 was mainly expressed in foregut, hindgut and muscle of the insects at all developmental stages examined. The expression of these two cytochrome P450 genes were differentially affected by three representative insecticides, including carbaryl (carbamate), malathion (organophosphate) and deltamethrin (pyrethroid). The exposure of the locust to carbaryl, malathion and deltamethrin resulted in reduced, moderately increased and significantly increased transcript levels, respectively, of the two cytochrome P450 genes. Our further analysis of their detoxification roles by using RNA interference followed by deltamethrin bioassay showed increased nymph mortalities by 21.1% and 16.7%, respectively, after CYP409A1 and CYP408B1 were silenced. These results strongly support our notion that these two new cytochrome P450 genes play an important role in deltamethrin detoxification in the locust. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Identification of an Extracellular Polysaccharide Network Essential for Cytochrome Anchoring and Biofilm Formation in Geobacter sulfurreducens▿ †

    PubMed Central

    Rollefson, Janet B.; Stephen, Camille S.; Tien, Ming; Bond, Daniel R.

    2011-01-01

    Transposon insertions in Geobacter sulfurreducens GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in trans. The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly-d-lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type G. sulfurreducens which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of c-type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of c-type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the xap (extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the Geobacter outer membrane. PMID:21169487

  1. Type B drum packages

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McCoy, J.C.

    1994-08-01

    The Type B drum packages (TBD) are conceptualized as a family of containers in which a single 208 L or 114 L (55 gal or 30 gal) drum containing Type B quantities of radioactive material (RAM) can be packaged for shipment. The TBD containers are being developed to fill a void in the packaging and transportation capabilities of the U.S. Department of Energy as no container packaging single drums of Type B RAM exists offering double containment. Several multiple-drum containers currently exist, as well as a number of shielded casks, but the size and weight of these containers present manymore » operational challenges for single-drum shipments. As an alternative, the TBD containers will offer up to three shielded versions (light, medium, and heavy) and one unshielded version, each offering single or optional double containment for a single drum. To reduce operational complexity, all versions will share similar design and operational features where possible. The primary users of the TBD containers are envisioned to be any organization desiring to ship single drums of Type B RAM, such as laboratories, waste retrieval activities, emergency response teams, etc. Currently, the TBD conceptual design is being developed with the final design and analysis to be completed in 1995 to 1996. Testing and certification of the unshielded version are planned to be completed in 1996 to 1997 with production to begin in 1997 to 1998.« less

  2. Addition of DNA to CrVI and Cytochrome b5 Containing Proteoliposomes Leads to Generation of DNA Strand Breaks and CrIII Complexes

    PubMed Central

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

    2009-01-01

    Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b5 and NADPH :P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of CrVI (hexavalent chromium, as CrO42− ), and the generation of CrV, superoxide (O2·−) , and hydroxyl radical (HO˙). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O2. CrV is formed under both conditions, so the breaks are not mediated directly by CrV. The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO˙ from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO42− , and plasmid DNA, intense EPR signals at g = 5.7and g = 5.0 were observed. These signals are attributed to specific CrIII complexes with large zero field splitting (ZFS). Without DNA, the signals in the g = 5 region were weak. The large ZFS signals were not seen, when CrIIICl3 was incubated with DNA, suggesting that the CrIII–DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g = 2 is attributed to CrIII complexes with a small ZFS. This g = 2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific CrIII complexes that depend on the presence of plasmid DNA and the manner in which the CrIII is formed. PMID:18729091

  3. Genetic differentiation in blue shark, Prionace glauca, from the central Pacific Ocean, as inferred by mitochondrial cytochrome b region.

    PubMed

    Li, Weiwen; Dai, Xiaojie; Zhu, Jiangfeng; Tian, Siquan; He, Shan; Wu, Feng

    2017-07-01

    Six hundred and ninety-seven base pairs of cytochrome b gene of mtDNA was sequenced and analyzed for 78 blue shark Prionace glauca individuals from three sampled locations in the central Pacific Ocean (CPO). In total, three polymorphic sites were detected which defined four haplotypes. The haplotype diversity (h) ranged from 0.517 to 0.768, and nucleotide diversity (π) was between 0.0007 and 0.0011. Analysis of molecular variance indicated a non-significant differentiation among subpopulations. Furthermore, pairwise F ST score analysis revealed a non-significant differentiation among three sampled regions. Generally, low genetic differences were found between different geographic locations in the CPO. This study suggests a single panmictic population of P. glauca in the CPO.

  4. Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.

    2011-08-05

    Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides themore » reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.« less

  5. Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

    PubMed Central

    Bloomer, J C; Baldwin, S J; Smith, G J; Ayrton, A D; Clarke, S E; Chenery, R J

    1994-01-01

    1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation. PMID:7888294

  6. Serological Studies of Types A, B, and E Botulinal Toxins by Passive Hemagglutination and Bentonite Flocculation

    PubMed Central

    Johnson, H. M.; Brenner, K.; Angelotti, R.; Hall, H. E.

    1966-01-01

    Johnson, H. M. (Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio), K. Brenner, R. Angelotti, and H. E. Hall. Serological studies of types A, B, and E botulinal toxins by passive hemagglutination and bentonite flocculation. J. Bacteriol. 91:967–974. 1966.—Formalinized sheep red blood cells (SRBC), sensitized with types A, B, and E botulinal toxoids and toxins by bis-diazotized benzidine (BDB), were tested against A, B, and E antitoxins prepared in horses and rabbits. Type B antitoxin cross-reacted with A toxoid SRBC, but the reciprocal cross-reaction was not observed. E toxin SRBC were specifically agglutinated by E antitoxin. Flocculation of antigen-sensitized bentonite particles was less sensitive in titration of antitoxin than hemagglutination. Also, reciprocal cross-reactions were observed between types A and B antitoxins. Cross-reactions in both serological tests were eliminated by titration of antitoxins in the presence of the heterologous antigens, with no inhibitory effect on the homologous antitoxins. Generally, equine antitoxins were less suitable for agglutinations, especially of antigen-sensitized bentonite particles. Types A, B, and E antitoxins were specifically inhibited by 43, 39, and 245 mouse ld50 of their respective homologous toxins in the hemagglutination-inhibition test. A, B, and E antitoxins were specifically inhibited by 500, 950, and 1,500 mouse ld50 of their respective homologous toxins in bentonite flocculation inhibitions. Formalinized SRBC sensitized with rabbit types A and B antitoxins by BDB were respectively clumped by as little as 0.75 to 1.3 mouse ld50 of A toxin and 2.3 ld50 of B toxin, whereas bentonite particles sensitized by the same antitoxins were specifically clumped by 150 ld50 of A toxin and 630 ld50 of B toxin. E antitoxin sensitization of SRBC or bentonite particles was not successful. Evidence is presented that indicates that the serological procedures are applicable to the detection of botulinal toxins

  7. Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kolodkin-Gal, I; Elsholz, AKW; Muth, C

    2013-04-29

    Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa(3) and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showedmore » that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.« less

  8. Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

    PubMed Central

    Kolodkin-Gal, Ilana; Elsholz, Alexander K.W.; Muth, Christine; Girguis, Peter R.; Kolter, Roberto; Losick, Richard

    2013-01-01

    Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration. PMID:23599347

  9. Deletion and anergy of polyclonal B cells specific for ubiquitous membrane-bound self-antigen

    PubMed Central

    Taylor, Justin J.; Martinez, Ryan J.; Titcombe, Philip J.; Barsness, Laura O.; Thomas, Stephanie R.; Zhang, Na; Katzman, Shoshana D.; Jenkins, Marc K.

    2012-01-01

    B cell tolerance to self-antigen is critical to preventing antibody-mediated autoimmunity. Previous work using B cell antigen receptor transgenic animals suggested that self-antigen–specific B cells are either deleted from the repertoire, enter a state of diminished function termed anergy, or are ignorant to the presence of self-antigen. These mechanisms have not been assessed in a normal polyclonal repertoire because of an inability to detect rare antigen-specific B cells. Using a novel detection and enrichment strategy to assess polyclonal self-antigen–specific B cells, we find no evidence of deletion or anergy of cells specific for antigen not bound to membrane, and tolerance to these types of antigens appears to be largely maintained by the absence of T cell help. In contrast, a combination of deleting cells expressing receptors with high affinity for antigen with anergy of the undeleted lower affinity cells maintains tolerance to ubiquitous membrane-bound self-antigens. PMID:23071255

  10. Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria

    PubMed Central

    Carlson, Hans K.; Iavarone, Anthony T.; Gorur, Amita; Yeo, Boon Siang; Tran, Rosalie; Melnyk, Ryan A.; Mathies, Richard A.; Auer, Manfred; Coates, John D.

    2012-01-01

    Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium. PMID:22307634

  11. Thermodynamics of camphor migration in cytochrome P450cam by atomistic simulations.

    PubMed

    Rydzewski, J; Nowak, W

    2017-08-10

    Understanding the mechanisms of ligand binding to enzymes is of paramount importance for the design of new drugs. Here, we report on the use of a novel biased molecular dynamics (MD) methodology to study the mechanism of camphor binding to cytochrome P450cam. Microsecond-long MD simulations allowed us to observe reaction coordinates characterizing ligand diffusion from the active site of cytochrome P450cam to solvent via three egress routes. These atomistic simulations were used to estimate thermodynamic quantities along the reaction coordinates and indicate diverse binding configurations. The results suggest that the diffusion of camphor along the pathway near the substrate recognition site (SRS) is thermodynamically preferred. In addition, we show that the diffusion near the SRS is triggered by a transition from a heterogeneous collection of closed ligand-bound conformers to the basin comprising the open conformations of cytochrome P450cam. The conformational change accompanying this switch is characterized by the retraction of the F and G helices and the disorder of the B' helix. These results are corroborated by experimental studies and provide detailed insight into ligand binding and conformational behavior of the cytochrome family. The presented methodology is general and can be applied to other ligand-protein systems.

  12. Comparison of Three Different Hepatitis C Virus Genotyping Methods: 5'NCR PCR-RFLP, Core Type-Specific PCR, and NS5b Sequencing in a Tertiary Care Hospital in South India.

    PubMed

    Daniel, Hubert D-J; David, Joel; Raghuraman, Sukanya; Gnanamony, Manu; Chandy, George M; Sridharan, Gopalan; Abraham, Priya

    2017-05-01

    Based on genetic heterogeneity, hepatitis C virus (HCV) is classified into seven major genotypes and 64 subtypes. In spite of the sequence heterogeneity, all genotypes share an identical complement of colinear genes within the large open reading frame. The genetic interrelationships between these genes are consistent among genotypes. Due to this property, complete sequencing of the HCV genome is not required. HCV genotypes along with subtypes are critical for planning antiviral therapy. Certain genotypes are also associated with higher progression to liver cirrhosis. In this study, 100 blood samples were collected from individuals who came for routine HCV genotype identification. These samples were used for the comparison of two different genotyping methods (5'NCR PCR-RFLP and HCV core type-specific PCR) with NS5b sequencing. Of the 100 samples genotyped using 5'NCR PCR-RFLP and HCV core type-specific PCR, 90% (κ = 0.913, P < 0.00) and 96% (κ = 0.794, P < 0.00) correlated with NS5b sequencing, respectively. Sixty percent and 75% of discordant samples by 5'NCR PCR-RFLP and HCV core type-specific PCR, respectively, belonged to genotype 6. All the HCV genotype 1 subtypes were classified accurately by both the methods. This study shows that the 5'NCR-based PCR-RFLP and the HCV core type-specific PCR-based assays correctly identified HCV genotypes except genotype 6 from this region. Direct sequencing of the HCV core region was able to identify all the genotype 6 from this region and serves as an alternative to NS5b sequencing. © 2016 Wiley Periodicals, Inc.

  13. Transient binding of CO to Cu(B) in cytochrome c oxidase is dynamically linked to structural changes around a carboxyl group: a time-resolved step-scan Fourier transform infrared investigation.

    PubMed Central

    Heitbrink, Dirk; Sigurdson, Håkan; Bolwien, Carsten; Brzezinski, Peter; Heberle, Joachim

    2002-01-01

    The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the mid-infrared range (2300-950 cm(-1)). Difference spectra of the bovine complex were obtained at -20 degrees C with 5 micros time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu(B) and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK(a) of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme. PMID:11751290

  14. Islet-specific monoamine oxidase A and B expression depends on MafA transcriptional activity and is compromised in type 2 diabetes.

    PubMed

    Ganic, Elvira; Johansson, Jenny K; Bennet, Hedvig; Fex, Malin; Artner, Isabella

    2015-12-25

    Lack or dysfunction of insulin producing β cells results in the development of type 1 and type 2 diabetes mellitus, respectively. Insulin secretion is controlled by metabolic stimuli (glucose, fatty acids), but also by monoamine neurotransmitters, like dopamine, serotonin, and norepinephrine. Intracellular monoamine levels are controlled by monoamine oxidases (Mao) A and B. Here we show that MaoA and MaoB are expressed in mouse islet β cells and that inhibition of Mao activity reduces insulin secretion in response to metabolic stimuli. Moreover, analysis of MaoA and MaoB protein expression in mouse and human type 2 diabetic islets shows a significant reduction of MaoB in type 2 diabetic β cells suggesting that loss of Mao contributes to β cell dysfunction. MaoB expression was also reduced in β cells of MafA-deficient mice, a mouse model for β cell dysfunction, and biochemical studies showed that MafA directly binds to and activates MaoA and MaoB transcriptional control sequences. Taken together, our results show that MaoA and MaoB expression in pancreatic islets is required for physiological insulin secretion and lost in type 2 diabetic mouse and human β cells. These findings demonstrate that regulation of monoamine levels by Mao activity in β cells is pivotal for physiological insulin secretion and that loss of MaoB expression may contribute to the β cell dysfunction in type 2 diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. A novel CYP27B1 mutation causes a feline vitamin D-dependent rickets type IA.

    PubMed

    Grahn, Robert A; Ellis, Melanie R; Grahn, Jennifer C; Lyons, Leslie A

    2012-08-01

    A 12-week-old domestic cat presented at a local veterinary clinic with hypocalcemia and skeletal abnormalities suggestive of rickets. Osteomalacia (rickets) is a disease caused by impaired bone mineralization leading to an increased prevalence of fractures and deformity. Described in a variety of species, rickets is most commonly caused by vitamin D or calcium deficiencies owing to both environmental and or genetic abnormalities. Vitamin D-dependent rickets type 1A (VDDR-1A) is a result of the enzymatic pathway defect caused by mutations in the 25-hydroxyvitamin D(3)-1-alpha-hydroxylase gene [cytochrome P27 B1 (CYP27B1)]. Calcitriol, the active form of vitamin D(3), regulates calcium homeostasis, which requires sufficient dietary calcium availability and correct hormonal function for proper bone growth and maintenance. Patient calcitriol concentrations were low while calcidiol levels were normal suggestive of VDDR-1A. The entire DNA coding sequencing of CYP27B1 was evaluated. The affected cat was wild type for previously identified VDDR-1A causative mutations. However, six novel mutations were identified, one of which was a nonsense mutation at G637T in exon 4. The exon 4 G637T nonsense mutation results in a premature protein truncation, changing a glutamic acid to a stop codon, E213X, likely causing the clinical presentation of rickets. The previously documented genetic mutation resulting in feline VDDR-1A rickets, as well as the case presented in this research, result from novel exon 4 CYP27B1 mutations, thus exon 4 should be the initial focus of future sequencing efforts.

  16. Types A and B Niemann-Pick Disease.

    PubMed

    Schuchman, Edward H; Wasserstein, Melissa P

    2016-06-01

    Two distinct metabolic abnormalities are included under the eponym Niemann-Pick disease (NPD). The first is due to the deficient activity of the enzyme acid sphingomyelinase (ASM). Patients with ASM deficiency are classified as having types A and B Niemann-Pick disease (NPD). Type A NPD patients exhibit hepatosplenomegaly, frequent pulmonary infections, and profound central nervous system involvement in infancy. They rarely survive beyond two years of age. Type B patients also have hepatosplenomegaly and progressive alterations of their lungs, but there are usually no central nervous system signs. The age of onset and rate of disease progression varies greatly among type B patients, and they frequently live into adulthood. Recently, patients with phenotypes intermediate between types A and B NPD also have been identified. These individuals represent the expected continuum caused by inheriting different mutations in the ASM gene (SMPD1). Patients in the second category are designated as having type C NPD. Impaired intracellular trafficking of cholesterol causes type C NPD, and two distinct gene defects have been found. In this chapter only types A and B NPD will be discussed.

  17. Diversity of Sarcocystis spp shed by opossums in Brazil inferred with phylogenetic analysis of DNA coding ITS1, cytochrome B, and surface antigens.

    PubMed

    Valadas, Samantha Y O B; da Silva, Juliana I G; Lopes, Estela Gallucci; Keid, Lara B; Zwarg, Ticiana; de Oliveira, Alice S; Sanches, Thaís C; Joppert, Adriana M; Pena, Hilda F J; Oliveira, Tricia M F S; Ferreira, Helena L; Soares, Rodrigo M

    2016-05-01

    Although few species of Sarcocystis are known to use marsupials of the genus Didelphis as definitive host, an extensive diversity of alleles of surface antigen genes (sag2, sag3, and sag4) has been described in samples of didelphid opossums in Brazil. In this work, we studied 25 samples of Sarcocystis derived from gastrointestinal tract of opossums of the genus Didelphis by accessing the variability of sag2, sag3, sag4, gene encoding cytochrome b (cytB) and first internal transcribed spacer (ITS1). Reference samples of Sarcocystis neurona (SN138) and Sarcocystis falcatula (SF1) maintained in cell culture were also analyzed. We found four allele variants of cytB, seven allele variants of ITS1, 10 allele variants of sag2, 13 allele variants of sag3, and 6 allele variants of sag4. None of the sporocyst-derived sequences obtained from Brazilian opossums revealed 100% identity to SN138 at cytB gene, nor to SN138 or SF1 at ITS1 locus. In addition, none of the sag alleles were found identical to either SF1 or SN138 homologous sequences, and a high number of new sag allele types were found other than those previously described in Brazil. Out of ten sag2 alleles, four are novel, while eight out of 13 sag3 alleles are novel and one out of six sag4 alleles is novel. Further studies are needed to clarify if such a vast repertoire of allele variants of Sarcocystis is the consequence of re-assortments driven by sexual exchange, in order to form individuals with highly diverse characteristics, such as pathogenicity, host spectrum, among others or if it only represents allele variants of different species with different biological traits. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Enhanced leaf photosynthesis as a target to increase grain yield: insights from transgenic rice lines with variable Rieske FeS protein content in the cytochrome b6 /f complex.

    PubMed

    Yamori, Wataru; Kondo, Eri; Sugiura, Daisuke; Terashima, Ichiro; Suzuki, Yuji; Makino, Amane

    2016-01-01

    Although photosynthesis is the most important source for biomass and grain yield, a lack of correlation between photosynthesis and plant yield among different genotypes of various crop species has been frequently observed. Such observations contribute to the ongoing debate whether enhancing leaf photosynthesis can improve yield potential. Here, transgenic rice plants that contain variable amounts of the Rieske FeS protein in the cytochrome (cyt) b6 /f complex between 10 and 100% of wild-type levels have been used to investigate the effect of reductions of these proteins on photosynthesis, plant growth and yield. Reductions of the cyt b6 /f complex did not affect the electron transport rates through photosystem I but decreased electron transport rates through photosystem II, leading to concomitant decreases in CO2 assimilation rates. There was a strong control of plant growth and grain yield by the rate of leaf photosynthesis, leading to the conclusion that enhancing photosynthesis at the single-leaf level would be a useful target for improving crop productivity and yield both via conventional breeding and biotechnology. The data here also suggest that changing photosynthetic electron transport rates via manipulation of the cyt b6 /f complex could be a potential target for enhancing photosynthetic capacity in higher plants. © 2015 John Wiley & Sons Ltd.

  19. Chemical property based sequence characterization of PpcA and its homolog proteins PpcB-E: A mathematical approach

    PubMed Central

    Pal Choudhury, Pabitra

    2017-01-01

    Periplasmic c7 type cytochrome A (PpcA) protein is determined in Geobacter sulfurreducens along with its other four homologs (PpcB-E). From the crystal structure viewpoint the observation emerges that PpcA protein can bind with Deoxycholate (DXCA), while its other homologs do not. But it is yet to be established with certainty the reason behind this from primary protein sequence information. This study is primarily based on primary protein sequence analysis through the chemical basis of embedded amino acids. Firstly, we look for the chemical group specific score of amino acids. Along with this, we have developed a new methodology for the phylogenetic analysis based on chemical group dissimilarities of amino acids. This new methodology is applied to the cytochrome c7 family members and pinpoint how a particular sequence is differing with others. Secondly, we build a graph theoretic model on using amino acid sequences which is also applied to the cytochrome c7 family members and some unique characteristics and their domains are highlighted. Thirdly, we search for unique patterns as subsequences which are common among the group or specific individual member. In all the cases, we are able to show some distinct features of PpcA that emerges PpcA as an outstanding protein compared to its other homologs, resulting towards its binding with deoxycholate. Similarly, some notable features for the structurally dissimilar protein PpcD compared to the other homologs are also brought out. Further, the five members of cytochrome family being homolog proteins, they must have some common significant features which are also enumerated in this study. PMID:28362850

  20. The differentiation of tuna (family: Scombridae) products through the PCR-based analysis of the cytochrome b gene and parvalbumin introns.

    PubMed

    Abdullah, Asadatun; Rehbein, Hartmut

    2016-01-30

    In spite of the many studies performed over the years, there are still problems in the authentication of closely related tuna species, not only for canned fish but also for raw products. With the aim of providing screening methods to identify different tuna species and related scombrids, segments of mitochondrial cytochrome b (cyt b) and nuclear parvalbumin genes were amplified and sequenced or subjected to single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) analyses. The nucleotide diagnostic sites in the cyt b gene of five tuna species from Indonesia were determined in this study and used to construct a phylogenetic tree. In addition, the suitability of the nuclear gene that encodes parvalbumin for the differentiation of tuna species was determined by SSCP and RFLP analyses of an intron segment. RFLP differentiated Thunnus albacares and from T. obesus, and fish species in the Thunnus genus could be distinguished from bullet tuna (Auxis rochei) by SSCP. Parvalbumin-based polymerase chain reaction systems could serve as an additional tool in the detection and identification of tuna and other Scombridae fish species for routine seafood control. This reaction can be performed in addition to the cyt b analysis as previously described. © 2015 Society of Chemical Industry.

  1. Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial Cytochrome b targets

    USGS Publications Warehouse

    Schill, W.B.; Mathes, M.V.

    2008-01-01

    We designed and tested real-time PCR probe/primer sets to detect and quantify Cytochrome b sequences of mitochondrial DNA (mtDNA) from nine vertebrate species of pet (dog), farm (cow, chicken, sheep, horse, pig), wildlife (Canada goose, white-tailed deer), and human. Linear ranges of the assays were from 101 to 108 copies/??l. To formally test the performance of the assays, twenty blinded fecal suspension samples were analyzed by real-time PCR to identify the source of the feces. Sixteen of the twenty samples were correctly and unambiguously identified. Average sensitivity was calculated to be 0.850, while average specificity was found to be 0.994. One beef cow sample was not detected, but mtDNA from 11 other beef cattle of both sexes and varying physiological states was found in concentrations similar (3.45 ?? 107 copies/g) to thatfound in human feces (1.1 ?? 107 copies/g). Thus, environmental conditions and sample handling are probably important factors for successful detection of fecal mtDNA. When sewage samples were analyzed, only human mtDNA (7.2 ?? 104 copies/100 mL) was detected. With a detection threshold of 250 copies/reaction, an efficient concentration and purification method resulted in a final detection limit for human feces of 1.8 mg/100 mL water.

  2. Species identification in mixed tuna samples with next-generation sequencing targeting two short cytochrome b gene fragments.

    PubMed

    Kappel, Kristina; Haase, Ilka; Käppel, Christine; Sotelo, Carmen G; Schröder, Ute

    2017-11-01

    Conventional Sanger sequencing of PCR products is the gold standard for species authentication of seafood products. However, this method is inappropriate for the analysis of products that might contain mixtures of species, such as tinned tuna. The purpose of this study was to test whether next-generation sequencing (NGS) can be a solution for the authentication of mixed products. Nine tuna samples containing mixtures of up to four species were prepared and subjected to an NGS approach targeting two short cytochrome b gene (cytb) fragments on the Illumina MiSeq platform. Sequence recovery was precise and admixtures of as low as 1% could be identified, depending on the species composition of the mixtures. Duplicate samples as well as two individual NGS runs produced very similar results. A first test of three commercial tinned tuna samples indicated the presence of different species in the same tin, although this is forbidden by EU law. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Cytochrome oxidase subunit 2 gene allows simultaneous detection and typing of Trypanosoma rangeli and Trypanosoma cruzi.

    PubMed

    de Sá, Amanda Regina Nichi; Steindel, Mário; Demeu, Lara Maria Kalempa; Lückemeyer, Débora Denardin; Grisard, Edmundo Carlos; Neto, Quirino Alves de Lima; de Araújo, Silvana Marques; Toledo, Max Jean de Ornelas; Gomes, Mônica Lúcia

    2013-12-23

    The parasites Trypanosoma rangeli and Trypanosoma cruzi share vectors and hosts over a wide geographical area in Latin America. In this study, we propose a single molecular approach for simultaneous detection and typing of T. rangeli and T. cruzi. A restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase II gene (COII-RFLP) using enzyme AluI and different amounts of DNA from the major genetic groups of T. rangeli and T. cruzi (KP1+/KP1- and DTU-I/DTU-II) was carried out. The same marker was tested on the other T. cruzi DTUs (DTU-III to DTU-VI) and on DNA extracted from gut contents of experimentally infected triatomines. The COII PCR generates a ~400 bp fragment, which after digestion with AluI (COII-RFLP) can be used to distinguish T. rangeli from T. cruzi and simultaneously differentiate the major genetic groups of T. rangeli (KP1+ and KP1-) and T. cruzi (DTU-I and DTU-II). The COII-RFLP generated bands of ~120 bp and ~280 bp for KP1+, whereas for KP1- no amplicon cleavage was observed. For T. cruzi, digestion of COII revealed a ~300 bp band for DTU-I and a ~250 bp band for DTU-II. For DTU-III to DTU-VI, COII-RFLP generated bands ranging from ~310 to ~330 bp, but the differentiation of these DTUs was not as clear as the separation between DTU-I and DTU-II. After AluI digestion, a species-specific fragment of ~80 bp was observed for all DTUs of T. cruzi. No cross-amplification was observed for Leishmania spp., T. vivax or T. evansi. The COII-RFLP allowed simultaneous detection and typing of T. rangeli and T. cruzi strains according to their major genetic groups (KP1+/KP1- and DTU-I/DTU-II) in vitro and in vivo, providing a reliable and sensitive tool for epidemiological studies in areas where T. rangeli and T. cruzi coexist.

  4. Cytochrome oxidase subunit 2 gene allows simultaneous detection and typing of Trypanosoma rangeli and Trypanosoma cruzi

    PubMed Central

    2013-01-01

    Background The parasites Trypanosoma rangeli and Trypanosoma cruzi share vectors and hosts over a wide geographical area in Latin America. In this study, we propose a single molecular approach for simultaneous detection and typing of T. rangeli and T. cruzi. Methods A restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase II gene (COII-RFLP) using enzyme AluI and different amounts of DNA from the major genetic groups of T. rangeli and T. cruzi (KP1+/KP1- and DTU-I/DTU-II) was carried out. The same marker was tested on the other T. cruzi DTUs (DTU-III to DTU-VI) and on DNA extracted from gut contents of experimentally infected triatomines. Results The COII PCR generates a ~400 bp fragment, which after digestion with AluI (COII-RFLP) can be used to distinguish T. rangeli from T. cruzi and simultaneously differentiate the major genetic groups of T. rangeli (KP1+ and KP1-) and T. cruzi (DTU-I and DTU-II). The COII-RFLP generated bands of ~120 bp and ~280 bp for KP1+, whereas for KP1- no amplicon cleavage was observed. For T. cruzi, digestion of COII revealed a ~300 bp band for DTU-I and a ~250 bp band for DTU-II. For DTU-III to DTU-VI, COII-RFLP generated bands ranging from ~310 to ~330 bp, but the differentiation of these DTUs was not as clear as the separation between DTU-I and DTU-II. After AluI digestion, a species-specific fragment of ~80 bp was observed for all DTUs of T. cruzi. No cross-amplification was observed for Leishmania spp., T. vivax or T. evansi. Conclusions The COII-RFLP allowed simultaneous detection and typing of T. rangeli and T. cruzi strains according to their major genetic groups (KP1+/KP1- and DTU-I/DTU-II) in vitro and in vivo, providing a reliable and sensitive tool for epidemiological studies in areas where T. rangeli and T. cruzi coexist. PMID:24360167

  5. Epigenetic regulation of normal human mammary cell type-specific miRNAs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

    2011-08-26

    Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linkedmore » to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.« less

  6. Tributyltin interacts with mitochondria and induces cytochrome c release.

    PubMed Central

    Nishikimi, A; Kira, Y; Kasahara, E; Sato, E F; Kanno, T; Utsumi, K; Inoue, M

    2001-01-01

    Although triorganotins are potent inducers of apoptosis in various cell types, the critical targets of these compounds and the mechanisms by which they lead to cell death remain to be elucidated. There are two major pathways by which apoptotic cell death occurs: one is triggered by a cytokine mediator and the other is by a mitochondrion-dependent mechanism. To elucidate the mechanism of triorganotin-induced apoptosis, we studied the effect of tributyltin on mitochondrial function. We found that moderately low doses of tributyltin decrease mitochondrial membrane potential and induce cytochrome c release by a mechanism inhibited by cyclosporine A and bongkrekic acid. Tributyltin-induced cytochrome c release is also prevented by dithiols such as dithiothreitol and 2,3-dimercaptopropanol but not by monothiols such as GSH, N-acetyl-L-cysteine, L-cysteine and 2-mercaptoethanol. Further studies with phenylarsine oxide agarose revealed that tributyltin interacts with the adenine nucleotide translocator, a functional constituent of the mitochondrial permeability transition pore, which is selectively inhibited by dithiothreitol. These results suggest that, at low doses, tributyltin interacts selectively with critical thiol residues in the adenine nucleotide translocator and opens the permeability transition pore, thereby decreasing membrane potential and releasing cytochrome c from mitochondria, a series of events consistent with established mechanistic models of apoptosis. PMID:11368793

  7. Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity

    PubMed Central

    Perroud, Thomas D.; Bokoch, Michael P.; Zare, Richard N.

    2005-01-01

    We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pH-induced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0–9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5–10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry. PMID:16314563

  8. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

    PubMed Central

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

    2017-01-01

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis. PMID:27758862

  9. Comparison of circulating, hepatocyte specific messenger RNA and microRNA as biomarkers for chronic hepatitis B and C.

    PubMed

    Zhang, Xiaonan; Zhang, Zhanqing; Dai, Fahui; Shi, Bisheng; Chen, Liang; Zhang, Xinxin; Zang, Guoqing; Zhang, Jiming; Chen, Xiaorong; Qian, Fangxing; Hu, Yunwen; Yuan, Zhenghong

    2014-01-01

    Circulating microRNAs have been widely recognized as a novel category of biomarker in a variety of physiological and pathological conditions. Other reports revealed that fragments of organ specific messenger RNAs are also detectable in serum/plasma and can be utilized as sensitive indicators of liver pathology and cancer. In order to assess the sensitivity and reliability of these two class of RNAs as marker of hepatitis B or C induced chronic liver disease, we collected plasma samples from 156 chronic hepatitis B or C patients (HBV active n = 112, HBV carrier n = 19, hepatitis C n = 25) and 22 healthy donors and quantified their circulating mRNA for albumin, HP (haptoglobin), CYP2E1 (cytochrome P450, family 2, subfamily E) and ApoA2 (Apolipoprotein A2) in conjunction with microRNA-122, a well established marker for acute and chronic liver injury. We found that plasma microRNA-122 level is significantly elevated in patients with active HBV but not in HBV carriers. Furthermore, microRNA-122 is not elevated in HCV patients even though their median serum alanine aminotransferase (sALT) was three fold of the healthy donors. Nevertheless, circulating mRNAs, especially albumin mRNA, showed much more sensitivity in distinguishing active hepatitis B, hepatitis B carrier or HCV patients from healthy control. Correlation and multiple linear regression analysis suggested that circulating mRNAs and miRNAs are much more related to HBsAg titre than to sALT. Immunoprecipitation of HBsAg in HBV patients' plasma resulted in enrichment of albumin and HP mRNA suggesting that fragments of liver specific transcripts can be encapsidated into HBsAg particles. Taken together, our results suggest that hepatocyte specific transcripts in plasma like albumin mRNA showed greater sensitivity and specificity in differentiating HBV or HCV induced chronic liver disease than microRNA-122. Circulating mRNA fragments merit more attention in the quest of next generation biomarkers for

  10. Comparison of Circulating, Hepatocyte Specific Messenger RNA and microRNA as Biomarkers for Chronic Hepatitis B and C

    PubMed Central

    Zhang, Xiaonan; Zhang, Zhanqing; Dai, Fahui; Shi, Bisheng; Chen, Liang; Zhang, Xinxin; Zang, Guoqing; Zhang, Jiming; Chen, Xiaorong; Qian, Fangxing; Hu, Yunwen; Yuan, Zhenghong

    2014-01-01

    Circulating microRNAs have been widely recognized as a novel category of biomarker in a variety of physiological and pathological conditions. Other reports revealed that fragments of organ specific messenger RNAs are also detectable in serum/plasma and can be utilized as sensitive indicators of liver pathology and cancer. In order to assess the sensitivity and reliability of these two class of RNAs as marker of hepatitis B or C induced chronic liver disease, we collected plasma samples from 156 chronic hepatitis B or C patients (HBV active n = 112, HBV carrier n = 19, hepatitis C n = 25) and 22 healthy donors and quantified their circulating mRNA for albumin, HP (haptoglobin), CYP2E1 (cytochrome P450, family 2, subfamily E) and ApoA2 (Apolipoprotein A2) in conjunction with microRNA-122, a well established marker for acute and chronic liver injury. We found that plasma microRNA-122 level is significantly elevated in patients with active HBV but not in HBV carriers. Furthermore, microRNA-122 is not elevated in HCV patients even though their median serum alanine aminotransferase (sALT) was three fold of the healthy donors. Nevertheless, circulating mRNAs, especially albumin mRNA, showed much more sensitivity in distinguishing active hepatitis B, hepatitis B carrier or HCV patientsfrom healthy control. Correlation and multiple linear regression analysis suggested that circulating mRNAs and miRNAs are much more related to HBsAg titre than to sALT. Immunoprecipitation of HBsAg in HBV patients’ plasma resulted in enrichment of albumin and HP mRNA suggesting that fragments of liver specific transcripts can be encapsidated into HBsAg particles. Taken together, our results suggest that hepatocyte specific transcripts in plasma like albumin mRNA showed greater sensitivity and specificity in differentiating HBV or HCV induced chronic liver disease than microRNA-122. Circulating mRNA fragments merit more attention in the quest of next generation biomarkers for

  11. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reed, James R., E-mail: rreed@lsuhsc.edu; Cruz, Albert Leo N. dela, E-mail: adelac2@tigers.lsu.edu; Lomnicki, Slawo M., E-mail: slomni1@lsu.edu

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductasemore » and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2–CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • Particulate matter (PM) competitively inhibited CYP1A2 activity. • EPFRs were much more potent CYP1A2 inhibitors than other types of PM. • PM interacts differently with different forms of P450.

  12. Phylogenetic relationships of Malaysia’s long-tailed macaques, Macaca fascicularis, based on cytochrome b sequences

    PubMed Central

    Abdul-Latiff, Muhammad Abu Bakar; Ruslin, Farhani; Fui, Vun Vui; Abu, Mohd-Hashim; Rovie-Ryan, Jeffrine Japning; Abdul-Patah, Pazil; Lakim, Maklarin; Roos, Christian; Yaakop, Salmah; Md-Zain, Badrul Munir

    2014-01-01

    Abstract Phylogenetic relationships among Malaysia’s long-tailed macaques have yet to be established, despite abundant genetic studies of the species worldwide. The aims of this study are to examine the phylogenetic relationships of Macaca fascicularis in Malaysia and to test its classification as a morphological subspecies. A total of 25 genetic samples of M. fascicularis yielding 383 bp of Cytochrome b (Cyt b) sequences were used in phylogenetic analysis along with one sample each of M. nemestrina and M. arctoides used as outgroups. Sequence character analysis reveals that Cyt b locus is a highly conserved region with only 23% parsimony informative character detected among ingroups. Further analysis indicates a clear separation between populations originating from different regions; the Malay Peninsula versus Borneo Insular, the East Coast versus West Coast of the Malay Peninsula, and the island versus mainland Malay Peninsula populations. Phylogenetic trees (NJ, MP and Bayesian) portray a consistent clustering paradigm as Borneo’s population was distinguished from Peninsula’s population (99% and 100% bootstrap value in NJ and MP respectively and 1.00 posterior probability in Bayesian trees). The East coast population was separated from other Peninsula populations (64% in NJ, 66% in MP and 0.53 posterior probability in Bayesian). West coast populations were divided into 2 clades: the North-South (47%/54% in NJ, 26/26% in MP and 1.00/0.80 posterior probability in Bayesian) and Island-Mainland (93% in NJ, 90% in MP and 1.00 posterior probability in Bayesian). The results confirm the previous morphological assignment of 2 subspecies, M. f. fascicularis and M. f. argentimembris, in the Malay Peninsula. These populations should be treated as separate genetic entities in order to conserve the genetic diversity of Malaysia’s M. fascicularis. These findings are crucial in aiding the conservation management and translocation process of M. fascicularis populations

  13. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.

    Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cellsmore » expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.« less

  14. Ultraviolet B radiation induces impaired lifecycle traits and modulates expression of cytochrome P450 (CYP) genes in the copepod Tigriopus japonicus.

    PubMed

    Puthumana, Jayesh; Lee, Min-Chul; Park, Jun Chul; Kim, Hui-Su; Hwang, Dae-Sik; Han, Jeonghoon; Lee, Jae-Seong

    2017-03-01

    To evaluate the effects of ultraviolet B (UV-B) radiation at the developmental, reproductive, and molecular levels in aquatic invertebrates, we measured UV-B-induced acute toxicity, impairments in developmental and reproductive traits, and UV-B interaction with the entire family of cytochrome P450 (CYP) genes in the intertidal benthic copepod Tigriopus japonicus. We found a significant, dose-dependent reduction (P<0.05) in the survival of T. japonicus that began as a developmental delay and decreased fecundity. The 48h LD10 and LD50 were 1.35 and 1.84kJ/m 2 , and the CYP inhibitor (PBO) elevated mortality, confirming the involvement of CYP genes in UV-B induced toxicity. Low-dose UV-B (1.5kJ/m 2 ) induced developmental delays, and higher doses (6-18kJ/m 2 ) caused reproductive impairments in ovigerous females. The significant up-regulation of CYP genes belonging to clans 2/3/MT/4/20 in T. japonicus exposed to UV-B (12kJ/m 2 ) confirmed molecular interaction between UV-B and CYP genes. Moreover, orphan CYPs, such as CYP20A1, provide good insight on the deorphanization of invertebrate CYPs. Overall, these results demonstrate the involvement of UV-B radiation in the expression of all the CYP genes in T. japonicus and their susceptibility to UV-B radiation. This will provide a better understanding of the mechanistic effects of UV-B in copepods through the predicted AhR-mediated up-regulation of CYP genes. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.

    PubMed

    Hirawake, H; Taniwaki, M; Tamura, A; Amino, H; Tomitsuka, E; Kita, K

    1999-08-04

    We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.

  16. The interaction of representative members from two classes of antimycotics--the azoles and the allylamines--with cytochromes P-450 in steroidogenic tissues and liver.

    PubMed

    Schuster, I

    1985-06-01

    Spectrophotometric studies with ketoconazole, clotrimazole and miconazole show strong type-II interactions with several cytochromes P-450, particularly (Ks greater than 10(7)M-1; pH7.4; 25 degrees C) with the 11 beta-hydroxylase of adrenal mitochondria, with the 17 alpha/20 lyase of testis microsomes and with some forms of cytochromes P-450 of liver. A tight binding of the azoles also occurs to the reduced cytochromes, giving rise to an impeded CO binding to the haem iron. The binding of the azoles to 11 beta-hydroxylase and 17 alpha/20 lyase is much tighter than the binding of endogenous substrates, and consequently inhibition of steroidogenesis will occur at these sites. The metabolism of xenobiotic substrates by the cytochromes P-450 of liver will also be severely impeded. In contrast, the allylamines naftifine and SF 86-327 are type-I substrates for a small portion of cytochromes P-450 of liver microsomes only and there is no spectral evidence for binding to the cytochromes P-450 involved in steroid biosynthesis.

  17. Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats.

    PubMed

    Murray, M

    1988-05-01

    Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear

  18. Kunitz-type protease inhibitors group B from Solanum palustre.

    PubMed

    Speransky, Anna S; Cimaglia, Fabio; Krinitsina, Anastasya A; Poltronieri, Palmiro; Fasano, Pasqua; Bogacheva, Anna M; Valueva, Tatiana A; Halterman, Dennis; Shevelev, Alexei B; Santino, Angelo

    2007-11-01

    Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.

  19. Botulinum toxin type A versus botulinum toxin type B for cervical dystonia.

    PubMed

    Duarte, Gonçalo S; Castelão, Mafalda; Rodrigues, Filipe B; Marques, Raquel E; Ferreira, Joaquim; Sampaio, Cristina; Moore, Austen P; Costa, João

    2016-10-26

    , with a higher probability of benefit from BtA treatment. None of the trials were free of for-profit bias, nor did they provide information regarding registered study protocols. All trials evaluated the effect of a single Bt treatment session, and not repeated treatment sessions, using doses from 100 U to 250 U of BtA (all onabotulinumtoxinA, or Botox, formulations) and 5000 U to 10,000 U of BtB (rimabotulinumtoxinB, or Myobloc/Neurobloc).We found no difference between the two types of botulinum toxin in terms of overall efficacy, with a mean difference of -1.44 (95% CI -3.58 to 0.70) points lower on the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) for BtB-treated participants, measured at two to four weeks after injection. The proportion of participants with adverse events was also not different between BtA and BtB (BtB versus BtA risk ratio (RR) 1.40; 95% CI 1.00 to 1.96). However, when compared to BtA, treatment with BtB was associated with an increased risk of one adverse events of special interest, namely treatment-related sore throat/dry mouth (BtB versus BtA RR of 4.39; 95% CI 2.43 to 7.91). Treatment-related dysphagia (swallowing difficulties) was not different between BtA and BtB (RR 2.89; 95% CI 0.80 to 10.41). The two types of botulinum toxin were otherwise clinically non-distinguishable in all the remaining outcomes. The previous version of this review did not include any trials, since these were still ongoing at the time. Therefore, with this update we are able to change the conclusions of this review. There is low quality evidence that a single treatment session of BtA (specifically onabotulinumtoxinA) and a single treatment session of BtB (rimabotulinumtoxinB) are equally effective and safe in the treatment of adults with certain types of cervical dystonia. Treatment with BtB appears to present an increased risk of sore throat/dry mouth, compared to BtA. Overall, there is no clinical evidence from these single-treatment trials to support or

  20. Blarina brevicauda as a biological monitor of polychlorinated biphenyls: Evaluation of hepatic cytochrome p450 induction

    USGS Publications Warehouse

    Russell, J.S.; Halbrook, R.S.; Woolf, A.; French, J.B.; Melancon, M.J.

    2004-01-01

    We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with $-naphthoflavone ($NF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received $NF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

  1. Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction.

    PubMed

    Russell, Julie S; Halbrook, Richard S; Woolf, Alan; French, John B; Melancon, Mark J

    2004-08-01

    We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

  2. Label-free Raman observation of cytochrome c dynamics during apoptosis

    PubMed Central

    Okada, Masaya; Smith, Nicholas Isaac; Palonpon, Almar Flotildes; Endo, Hiromi; Kawata, Satoshi; Sodeoka, Mikiko; Fujita, Katsumasa

    2012-01-01

    We performed label-free observation of molecular dynamics in apoptotic cells by Raman microscopy. Dynamic changes in cytochrome c distribution at the Raman band of 750 cm-1 were observed after adding an apoptosis inducer to the cells. The comparison of mitochondria fluorescence images and Raman images of cytochrome c confirmed that changes in cytochrome c distribution can be distinguished as release of cytochrome c from mitochondria. Our observation also revealed that the redox state of cytochrome c was maintained during the release from the mitochondria. Monitoring mitochondrial membrane potential with JC-1 dye confirmed that the observed cytochrome c release was associated with apoptosis. PMID:22184220

  3. Immunochemical characterization of the "native" type III polysaccharide of group B Streptococcus

    PubMed Central

    1976-01-01

    The type III polysaccharide of -roup B Streptococcus has been isolated and purified by a method that employs washing of intact cells at neutral pH. That the polysaccharide prepared by this procedure is the "native" type III antigen is suggested by its molecular size in excess of 10(6) daltons, its degradation by acid and heat treatment to a fragment with immunologic characteristics of the classical HCl antigen, and its type-specific serologic activity. The type III polysaccharide in native form contains sialic acid, galactose, glucose, glucosamine, heptose, and mannose. It is acidic in nature, is resistant to neuramindiase degradation, contains no O-acetyl groups, and does not share antigenic determinants with capsular type K1 antigen of Escherichia coli or Group B polysaccharide antigen of Neiserria meningitidis. PMID:55450

  4. Regulatory interactions in the dimeric cytochrome bc(1) complex: the advantages of being a twin.

    PubMed

    Covian, Raul; Trumpower, Bernard L

    2008-09-01

    The dimeric cytochrome bc(1) complex catalyzes the oxidation-reduction of quinol and quinone at sites located in opposite sides of the membrane in which it resides. We review the kinetics of electron transfer and inhibitor binding that reveal functional interactions between the quinol oxidation site at center P and quinone reduction site at center N in opposite monomers in conjunction with electron equilibration between the cytochrome b subunits of the dimer. A model for the mechanism of the bc(1) complex has emerged from these studies in which binding of ligands that mimic semiquinone at center N regulates half-of-the-sites reactivity at center P and binding of ligands that mimic catalytically competent binding of ubiquinol at center P regulates half-of-the-sites reactivity at center N. An additional feature of this model is that inhibition of quinol oxidation at the quinone reduction site is avoided by allowing catalysis in only one monomer at a time, which maximizes the number of redox acceptor centers available in cytochrome b for electrons coming from quinol oxidation reactions at center P and minimizes the leakage of electrons that would result in the generation of damaging oxygen radicals.

  5. Characterization of the recognition specificity of BH2, a monoclonal antibody prepared against the HLA-B27 heavy chain.

    PubMed

    Yu, Hui-Chun; Huang, Kuang-Yung; Lu, Ming-Chi; Huang, Hsien-Lu; Liu, Wei-Ting; Lee, Wen-Chien; Liu, Su-Qin; Huang, Hsien-Bin; Lai, Ning-Sheng

    2015-04-13

    BH2, a monoclonal antibody prepared against the denatured human leukocytic antigen-B27 heavy chain (HLA-B27 HC), can immunoprecipitate the misfolded HLA-B27 HC complexed with Bip in the endoplasmic reticulum and recognize the homodimerized HLA-B27 HC that is often observed on the cell membrane of patients suffered from ankylosing spondylitis (AS). However, the recognition specificity of BH2 toward the other molecules of HLA-B type and toward the different types of HLA molecules remained uncharacterized. In this study, we carried out the HLA-typing by using the Luminex Technology to characterize the recognition specificity of BH2 and analyzed the binding domain of HLA-B27 HC by BH2. Our results indicated that BH2 preferably binds to molecules of HLA-B and -C rather than HLA-A and the binding site is located within the α2 domain of HLA-B27 HC.

  6. Engineering Herbicide Metabolism in Tobacco and Arabidopsis with CYP76B1, a Cytochrome P450 Enzyme from Jerusalem Artichoke1

    PubMed Central

    Didierjean, Luc; Gondet, Laurence; Perkins, Roberta; Lau, Sze-Mei Cindy; Schaller, Hubert; O'Keefe, Daniel P.; Werck-Reichhart, Danièle

    2002-01-01

    The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites. PMID:12226498

  7. Redox State of Cytochromes in Frozen Yeast Cells Probed by Resonance Raman Spectroscopy.

    PubMed

    Okotrub, Konstantin A; Surovtsev, Nikolay V

    2015-12-01

    Cryopreservation is a well-established technique used for the long-term storage of biological materials whose biological activity is effectively stopped under low temperatures (suspended animation). Since most biological methods do not work in a low-temperature frozen environment, the mechanism and details of the depression of cellular activity in the frozen state remain largely uncharacterized. In this work, we propose, to our knowledge, a new approach to study the downregulation of the redox activity of cytochromes b and c in freezing yeast cells in a contactless, label-free manner. Our approach is based on cytochrome photobleaching effects observed in the resonance Raman spectra of live cells. Photoinduced and native redox reactions that contributed to the photobleaching rate were studied over a wide temperature range (from -173 to +25 °C). We found that ice formation influences both the rate of cytochrome redox reactions and the balance between the reduced and oxidized cytochromes. We demonstrate that the temperature dependence of native redox reaction rates can be well described by the thermal activation law with an apparent energy of 32.5 kJ/mol, showing that the redox reaction rate is ∼10(15) times slower at liquid nitrogen temperature than at room temperature. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2

    EPA Science Inventory

    EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2. T M Ross1, B P Anderson1, G Zhao2, R A Pegram1 and J W Allis1. 1U.S. EPA, ORD, NHEERL, Research Triangle Park, NC; 2University of North Carolina, Chapel Hill, NC.
    Sponsor: H Barton

    Bromodichlorometh...

  9. Typed Multiset Rewriting Specifications of Security Protocols

    DTIC Science & Technology

    2011-10-01

    to define the type of a tuple as the sequence of the types of its components. Therefore, if A is a principal name and kA is a public key for A, the...tuple (A, kA ) would have type “principal × pubK A” (the Cartesian product symbol “×” is the standard constructor for tuple types). This construction...allows us to associate a generic principal with A’s public key: if B is another principal, then (B, kA ) will have this type as well. We will often need

  10. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    PubMed Central

    2010-01-01

    Background Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is

  11. Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

    PubMed

    Jetzschmann, K J; Yarman, A; Rustam, L; Kielb, P; Urlacher, V B; Fischer, A; Weidinger, I M; Wollenberger, U; Scheller, F W

    2018-04-01

    Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his 6 -tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his 6 -tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode. Copyright © 2018. Published by Elsevier B.V.

  12. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis

    PubMed Central

    Rao, P. S. S.; Kumar, Santosh

    2015-01-01

    High prevalence of cigarette smoking in HIV patients is associated with increased HIV pathogenesis and disease progression. While the effect of smoking on the occurrence of lung cancer has been studied extensively, the association between smoking and HIV pathogenesis is poorly studied. We have recently shown the possible role of cytochrome P450 (CYP) in smoking/nicotine-mediated viral replication. In this review, we focus on the potential role of CYP pathway in polycyclic aromatic hydrocarbons (PAH), important constituents of cigarette smoke, mediated HIV pathogenesis. More specifically, we will discuss the role of CYP1A1 and CYP1B1, which are the major PAH-activating CYP enzymes. Our results have shown that treatment with cigarette smoke condensate (CSC) increases viral replication in HIV-infected macrophages. CSC contains PAH, which are known to be activated by CYP1A1 and CYP1B1 into procarcinogens/toxic metabolites. The expression of these CYPs is regulated by aryl hydrocarbon receptors (AHR), the cellular target of PAH, and an important player in various diseases including cancer. We propose that PAH/AHR-mediated CYP pathway is a novel target to develop new interventions for HIV positive smokers. PMID:26082767

  13. New potent and selective cytochrome P450 2B6 (CYP2B6) inhibitors based on three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis

    PubMed Central

    Korhonen, L E; Turpeinen, M; Rahnasto, M; Wittekindt, C; Poso, A; Pelkonen, O; Raunio, H; Juvonen, R O

    2007-01-01

    Background and purpose: The cytochrome P450 2B6 (CYP2B6) enzyme metabolises a number of clinically important drugs. Drug-drug interactions resulting from inhibition or induction of CYP2B6 activity may cause serious adverse effects. The aims of this study were to construct a three-dimensional structure-activity relationship (3D-QSAR) model of the CYP2B6 protein and to identify novel potent and selective inhibitors of CYP2B6 for in vitro research purposes. Experimental approach: The inhibition potencies (IC50 values) of structurally diverse chemicals were determined with recombinant human CYP2B6 enzyme. Two successive models were constructed using Comparative Molecular Field Analysis (CoMFA). Key results: Three compounds proved to be very potent and selective competitive inhibitors of CYP2B6 in vitro (IC50<1 μM): 4-(4-chlorobenzyl)pyridine (CBP), 4-(4-nitrobenzyl)pyridine (NBP), and 4-benzylpyridine (BP). A complete inhibition of CYP2B6 activity was achieved with 0.1 μM CBP, whereas other CYP-related activities were not affected. Forty-one compounds were selected for further testing and construction of the final CoMFA model. The created CoMFA model was of high quality and predicted accurately the inhibition potency of a test set (n=7) of structurally diverse compounds. Conclusions and implications: Two CoMFA models were created which revealed the key molecular characteristics of inhibitors of the CYP2B6 enzyme. The final model accurately predicted the inhibitory potencies of several structurally unrelated compounds. CBP, BP and NBP were identified as novel potent and selective inhibitors of CYP2B6 and CBP especially is a suitable inhibitor for in vitro screening studies. PMID:17325652

  14. Effects of cysteine introduction into three homologous cytochromes C.

    PubMed

    Kobayashi, Yoshiko; Sonoyama, Takafumi; Takeda, Taku; Sambongi, Yoshihiro

    2009-05-01

    A cysteine residue was systematically introduced into three homologous cytochromes c from Hydrogenobacter thermophilus, Hydrogenophilus thermoluteolus, and Pseudomonas aeruginosa at a conserved position. The H. thermoluteolus variant showed the most decreased thermal stability as compared with the wild type, which might have been due in part to crosslinked polymer formation. The effects of cysteine introduction differed even at the conserved position in these homologous proteins.

  15. Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

    PubMed Central

    Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

    1999-01-01

    Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

  16. In situ detection of microbial c-type cytochrome based on intrinsic peroxidase-like activity using screen-printed carbon electrode.

    PubMed

    Wen, Junlin; He, Daigui; Yu, Zhen; Zhou, Shungui

    2018-08-15

    C-type cytochromes (c-cyts) facilitate microbial extracellular electron transfer and play critical roles in biogeochemical cycling, bioelectricity generation and bioremediation. In this study, a simple and effective method has been developed to detect microbial c-cyts by means of peroxidase mimetic reaction on screen-printed carbon electrode (SPCE). To this end, bacteria cells were immobilized onto the working electrode surface of SPCE by a simple drop casting. After introducing 3,3',5,5'-tetramethylbenzidine (TMB) solution, microbial c-cyts with peroxidase-like activity catalyze the oxidation of TMB in the presence of hydrogen peroxide. The oxidized TMB was electrochemically determined and the current signal was employed to calculate the c-cyts content. This electrochemical method is highly sensitive for microbial c-cyts with a low detection limit of 40.78 fmol and a wide detection range between 51.70 fmol and 6.64 pmol. Moreover, the proposed technique can be universally expanded to detect c-cyts in other bacteria species such as Fontibacter ferrireducens, Pseudomonas aeruginosa, Comamonas guangdongensis and Escherichia coli. Furthermore, the proposed method confers an in situ facile and quantitative c-cyts detection without any destructive sample preparations, complex electrode modifications and expensive enzyme- or metal particle- based signal amplification. The suggested method advances an intelligent strategy for in situ quantification of microbial c-cyts and consequently holds promising application potential in microbiology and environmental science. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Types A and B Niemann-Pick disease.

    PubMed

    Schuchman, Edward H; Wasserstein, Melissa P

    2015-03-01

    Two distinct metabolic abnormalities are encompassed under the eponym Niemann-Pick disease (NPD). The first is due to the deficient activity of the enzyme acid sphingomyelinase (ASM). Patients with ASM deficiency are classified as having types A and B Niemann-Pick disease (NPD). Type A NPD patients exhibit hepatosplenomegaly in infancy and profound central nervous system involvement. They rarely survive beyond two years of age. Type B patients also have hepatosplenomegaly and pathologic alterations of their lungs, but there are usually no central nervous system signs. The age of onset and rate of disease progression varies greatly among type B patients, and they frequently live into adulthood. Recently, patients with phenotypes intermediate between types A and B NPD also have been identified. These individuals represent the expected continuum caused by inheriting different mutations in the ASM gene (SMPD1). Patients in the second NPD category are designated as having types C and D NPD. These patients may have mild hepatosplenomegaly, but the central nervous system is profoundly affected. Impaired intracellular trafficking of cholesterol causes types C and D NPD, and two distinct gene defects have been found. In this chapter only types A and B NPD will be discussed. Copyright © 2014. Published by Elsevier Ltd.

  18. Cytochrome c oxidase rather than cytochrome c is a major determinant of mitochondrial respiratory capacity in skeletal muscle of aged rats: role of carnitine and lipoic acid.

    PubMed

    Tamilselvan, Jayavelu; Sivarajan, Kumarasamy; Anusuyadevi, Muthuswamy; Panneerselvam, Chinnakkannu

    2007-09-01

    The release of mitochondrial cytochrome c followed by activation of caspase cascade has been reported with aging in various tissues, whereas little is known about the caspase-independent pathway involved in mitochondrial dysfunction. To determine the functional impact of cytochrome c loss on mitochondrial respiratory capacity, we monitored NADH redox transitions and oxygen consumption in isolated skeletal muscle mitochondria of 4- and 24-month-old rats in the presence and absence of exogenous cytochrome c; and assessed the efficacy of cosupplementation of carnitine and lipoic acid on age-related alteration in mitochondrial respiration. The loss of mitochondrial cytochrome c with age was accompanied with alteration in respiratory transition, which in turn was not rescued by exogenous addition of cytochrome c to isolated mitochondria. The analysis of mitochondrial and nuclear-encoded cytochrome c oxidase subunits suggests that the decreased levels of cytochrome c oxidase may be attributed for the irresponsiveness to exogenously added cytochrome c on mitochondrial respiratory transitions, possibly through reduction of upstream electron carriers. Oral supplementation of carnitine and lipoic acid to aged rats help to maintaining the mitochondrial oxidative capacity by regulating the release of cytochrome c and improves cytochrome c oxidase transcript levels. Thus, carnitine and lipoic acid supplementation prevents the loss of cytochrome c and their associated decline in cytochrome c oxidase activity; thereby, effectively attenuating any putative decrease in cellular energy and redox status with age.

  19. Dynamically heterogenous partitions and phylogenetic inference: an evaluation of analytical strategies with cytochrome b and ND6 gene sequences in cranes.

    PubMed

    Krajewski, C; Fain, M G; Buckley, L; King, D G

    1999-11-01

    ki ctes over whether molecular sequence data should be partitioned for phylogenetic analysis often confound two types of heterogeneity among partitions. We distinguish historical heterogeneity (i.e., different partitions have different evolutionary relationships) from dynamic heterogeneity (i.e., different partitions show different patterns of sequence evolution) and explore the impact of the latter on phylogenetic accuracy and precision with a two-gene, mitochondrial data set for cranes. The well-established phylogeny of cranes allows us to contrast tree-based estimates of relevant parameter values with estimates based on pairwise comparisons and to ascertain the effects of incorporating different amounts of process information into phylogenetic estimates. We show that codon positions in the cytochrome b and NADH dehydrogenase subunit 6 genes are dynamically heterogenous under both Poisson and invariable-sites + gamma-rates versions of the F84 model and that heterogeneity includes variation in base composition and transition bias as well as substitution rate. Estimates of transition-bias and relative-rate parameters from pairwise sequence comparisons were comparable to those obtained as tree-based maximum likelihood estimates. Neither rate-category nor mixed-model partitioning strategies resulted in a loss of phylogenetic precision relative to unpartitioned analyses. We suggest that weighted-average distances provide a computationally feasible alternative to direct maximum likelihood estimates of phylogeny for mixed-model analyses of large, dynamically heterogenous data sets. Copyright 1999 Academic Press.

  20. The origin and evolution of seahorses (genus Hippocampus): a phylogenetic study using the cytochrome b gene of mitochondrial DNA.

    PubMed

    Casey, Stephen P; Hall, Heather J; Stanley, Helen F; Vincent, Amanda C J

    2004-02-01

    Phylogenetic relationships among 93 specimens of 22 species of seahorses (genus Hippocampus) from the Atlantic and Indo-Pacific Oceans were analysed using cytochrome b gene sequence data. A maximum sequence divergence of 23.2% (Kimura 2-parameter model) suggests a pre-Tethyan origin for the genus. Despite a greater number of seahorse species in the Indo-Pacific than in the Atlantic Ocean, there was no compelling genetic evidence to support an Indo-Pacific origin for the genus Hippocampus. The phylogenetic data suggest that high diversity in the Indo-Pacific results from speciation events dating from the Pleistocene to the Miocene, or earlier. Both vicariance and dispersal events in structuring the current global distribution of seahorses. The results suggested that several species designations need re-evaluating, and further phylogeographic studies are required to determine patterns and processes of seahorse dispersal.

  1. Construction and application of a functional library of cytochrome P450 monooxygenases from the filamentous fungus Aspergillus oryzae.

    PubMed

    Nazir, K H M Nazmul Hussain; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2011-05-01

    A functional library of cytochrome P450 monooxygenases from Aspergillus oryzae (AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

  2. Application of HC-AFW1 Hepatocarcinoma Cells for Mechanistic Studies: Regulation of Cytochrome P450 2B6 Expression by Dimethyl Sulfoxide and Early Growth Response 1.

    PubMed

    Petzuch, Barbara; Groll, Nicola; Schwarz, Michael; Braeuning, Albert

    2015-11-01

    Various exogenous compounds, for example, the drugs bupropione and propofol, but also various cytostatics, are metabolized in the liver by the enzyme cytochrome P450 (P450) CYP2B6. Transcription from the CYP2B6 gene is regulated mainly via the transcription factors constitutive androstane receptor (CAR) and pregnane-X-receptor (PXR). Most hepatic cell lines express no or only low levels of CYP2B6 because of loss of these two regulators. Dimethyl sulfoxide (DMSO) is frequently used in liver cell cultivation and is thought to affect the expression of various P450 isoforms by inducing or preserving cellular differentiation. We studied the effects of up to 1.5% of DMSO as cell culture medium supplement on P450 expression in hepatocarcinoma cells from line HC-AFW1. DMSO did not induce differentiation of the HC-AFW1 cell line, as demonstrated by unaltered levels of selected mRNA markers important for hepatocyte differentiation, and also by the lack of a DMSO effect on a broader spectrum of P450s. By contrast, CYP2B6 mRNA was strongly induced by DMSO. This process was independent of CAR or PXR activation. Interestingly, elevated transcription of CYP2B6 was accompanied by a simultaneous induction of early growth response 1 (EGR1), a transcription factor known to influence the expression of CYP2B6. Expression of wild-type EGR1 or of a truncated, dominant-negative EGR1 mutant was able to mimic or attenuate the DMSO effect, respectively. These findings demonstrate that EGR1 is involved in the regulation of CYP2B6 by DMSO in HC-AFW1 cells. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reed, James R., E-mail: rreed@lsuhsc.edu; The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112; Cawley, George F.

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of severalmore » P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is

  4. Reduction of Heavy Metals by Cytochrome c(3)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    ABDELOUAS,A.; GONG,W.L.; LUTZE,W.

    2000-01-18

    We report on reduction and precipitation of Se(VI), Pb(II), CU(II), U(VI), Mo(VI), and Cr(VI) in water by cytochrome c{sub 3} isolated from Desulfomicrobium baczdatum [strain 9974]. The tetraheme protein cytochrome c{sub 3} was reduced by sodium dithionite. Redox reactions were monitored by UV-visible spectroscopy of cytochrome c{sub 3}. Analytical electron microscopy work showed that Se(VI), Pb(II), and CU(II) were reduced to the metallic state, U(W) and Mo(W) to U(IV) and Mo(IV), respectively, and Cr(VI) probably to Cr(III). U(IV) and Mo(W) precipitated as oxides and Cr(III) as an amorphous hydroxide. Cytochrome c{sub 3} was used repeatedly in the same solution withoutmore » loosing its effectiveness. The results suggest usage of cytochrome c{sub 3} to develop innovative and environmentally benign methods to remove heavy metals from waste- and groundwater.« less

  5. Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

    PubMed

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H; Allain, Fabrice

    2010-01-15

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

  6. Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases*

    PubMed Central

    Deligny, Audrey; Denys, Agnès; Marcant, Adeline; Melchior, Aurélie; Mazurier, Joël; van Kuppevelt, Toin H.; Allain, Fabrice

    2010-01-01

    Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells. PMID:19940140

  7. A stromal region of cytochrome b6f subunit IV is involved in the activation of the Stt7 kinase in Chlamydomonas

    PubMed Central

    Zito, Francesca; Blangy, Stéphanie; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles

    2017-01-01

    The cytochrome (cyt) b6f complex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cyt b6f activates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplast petD gene, coding for subunit IV of the cyt b6f complex, and complementation of a ΔpetD host strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cyt b6f subunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cyt b6f and recombinant Stt7 kinase domain show that cyt b6f enhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cyt b6f complex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane. PMID:29078388

  8. Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

    PubMed Central

    Shi, Liang; Squier, Thomas C; Zachara, John M; Fredrickson, James K

    2007-01-01

    Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope. PMID:17581116

  9. Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

    PubMed

    Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

    2017-02-01

    Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC 50 ) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l -1 ) and acetamiprid (4.96 to 865 mg l -1 ). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

  10. Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hernandez, Juan P.; Chapman, Laura M.; Kretschmer, Xiomara C.

    2006-10-15

    Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ micemore » and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16{alpha}-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females.« less

  11. Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice

    PubMed Central

    Hernandez, Juan P.; Chapman, Laura M.; Kretschmer, Xiomara C.; Baldwin, William S.

    2007-01-01

    Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ mice and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16α-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females. PMID:16828826

  12. Population genetic structure and geographic differentiation in butter catfish, Ompok bimaculatus, from Indian waters inferred by cytochrome b mitochondrial gene.

    PubMed

    Kumar, Ravindra; Pandey, Brijesh Kumar; Sarkar, Uttam Kumar; Nagpure, Naresh Sahebrao; Baisvar, Vishwamitra Singh; Agnihotri, Praveen; Awasthi, Abhishek; Mishra, Abha; Kumar, Narendra

    2017-05-01

    Documentation of genetic differentiation among the populations of a species can provide useful information that has roles in conservation, breeding, and management plans. In the present study, we examined the genetic structure and phylogenetic relationships among the 149 individuals of Ompok bimaculatus belonging to 24 populations, collected from Indian waters, using cytochrome b gene. The combined analyses of data suggested that the Indian O. bimaculatus consist of three distinct mtDNA lineages with star-like haplotypes network, which exhibited high genetic variation and haplotypic diversity. Analysis of molecular variance indicated that most of the observed genetic variation was found among the populations suggesting restricted gene flow. Long-term interruption of gene flow was also evidenced by high overall Fst values (0.82367) that could be favored by the discontinuous distributions of the lineages.

  13. Types A and B Niemann-Pick disease.

    PubMed

    Schuchman, Edward H; Desnick, Robert J

    The eponym Niemann-Pick disease (NPD) refers to a group of patients who present with varying degrees of lipid storage and foam cell infiltration in tissues, as well as overlapping clinical features including hepatosplenomegaly, pulmonary insufficiency and/or central nervous system (CNS) involvement. Due to the pioneering work of Roscoe Brady and co-workers, we now know that there are two distinct metabolic abnormalities that account for NPD. The first is due to the deficient activity of the enzyme acid sphingomyelinase (ASM; "types A & B" NPD), and the second is due to defective function in cholesterol transport ("type C" NPD). Herein only types A and B NPD will be discussed. Type A NPD patients exhibit hepatosplenomegaly in infancy and profound CNS involvement. They rarely survive beyond 2-3years of age. Type B patients also have hepatosplenomegaly and pathologic alterations of their lungs, but there are usually no CNS signs. The age of onset and rate of disease progression varies greatly among type B patients, and they frequently live into adulthood. Intermediate patients also have been reported with mild to moderate neurological findings. All patients with types A and B NPD have mutations in the gene encoding ASM (SMPD1), and thus the disease is more accurately referred to as ASM deficiency (ASMD). Herein we will review the clinical, pathological, biochemical, and genetic findings in types A and B NPD, and emphasize the seminal contributions of Dr. Brady to this disease. We will also discuss the current status of therapy for this disorder. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

    PubMed Central

    Pakrasi, H B; Williams, J G; Arntzen, C J

    1988-01-01

    The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII. Images PMID:3130246

  15. Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs.

    PubMed

    Novak, David; Mojovic, Milos; Pavicevic, Aleksandra; Zatloukalova, Martina; Hernychova, Lenka; Bartosik, Martin; Vacek, Jan

    2018-02-01

    Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electron-transfer properties and ability to regulate the processes involved in homeostasis or apoptosis. Here we report an electrochemical strategy for investigating the electroactivity of cyt c and its analogs with a disrupted heme moiety, i.e. apocytochrome c (acyt c) and porphyrin cytochrome c (pcyt c). The electrochemical data are supplemented with low-temperature and spin-probe electron paramagnetic resonance (EPR) spectroscopy. The main contribution of this report is a complex evaluation of cyt c reduction and oxidation at the level of surface-localized amino acid residues and the heme moiety in a single electrochemical scan. The electrochemical pattern of cyt c is substantially different to both analogs acyt c and pcyt c, which could be applicable in further studies on the redox properties and structural stability of cytochromes and other hemeproteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The cytochrome p450 homepage.

    PubMed

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  17. Co-expression of cytochrome b561 and ascorbate oxidase in leaves of wild watermelon under drought and high light conditions.

    PubMed

    Nanasato, Yoshihiko; Akashi, Kinya; Yokota, Akiho

    2005-09-01

    Despite carrying out C3 photosynthesis, wild watermelon (Citrullus lanatus sp.) exhibits exceedingly good tolerance to severe drought at high light intensities. However, the mechanism(s) by which this plant protects itself from photodamage has yet to be elucidated. In this study, we characterized wild watermelon cytochrome b561 (cyt b561), which potentially mediates regeneration of apoplastic ascorbate by transferring electrons from cytosolic ascorbate across the plasma membrane. Two cDNA species for wild watermelon cyt b561, designated CLb561A and CLb561B, were isolated. Levels of both CLb561A mRNA and protein were significantly elevated in the leaves during drought at a light intensity of 700 micromol photons m(-2) s(-1). The transcript of CLb561B was detected to a much lesser extent, but no CLb561B protein was produced under any condition used in this study. A transient expression assay with the CLb561A::green fluorescent protein fusion construct showed clear fluorescence on the plasma membrane of onion epidermal cells. The CLb561A protein was enriched in the plasma membrane fraction in leaves of transgenic tobacco expressing CLb561A. Moreover, the high activity of apoplastic ascorbate oxidase (AO), which was able to dispose of cyt b561-transferred reducing equivalents, increased in leaves of wild watermelon grown at high light intensity, but not lower light intensities. Taken together, these observations suggest the occurrence of a novel pathway for excess light energy dissipation in wild watermelon leaves, where excessive energy absorbed by chloroplasts can be transported to and dissipated safely in the apoplasts through the cooperative action of cyt b561 and AO.

  18. A novel assay for detecting antibodies to cytochrome P4502D6, the molecular target of liver kidney microsomal antibody type 1.

    PubMed

    Kerkar, N; Ma, Y; Hussain, M; Muratori, L; Targett, C; Williams, R; Bianchi, F B; Mieli-Vergani, G; Vergani, D

    1999-03-04

    Liver Kidney Microsomal type 1 (LKM1) antibody, the diagnostic marker of autoimmune hepatitis type 2, is also found in a proportion of patients with hepatitis C virus infection (HCV). It is detected conventionally by the subjective immunofluorescence technique. Our aim was to establish a simple and objective enzyme-linked immunosorbent assay (ELISA) that measures antibodies to cytochrome P4502D6 (CYP2D6), the target of LKM1. An indirect ELISA using eukaryotically expressed CYP2D6 was designed. Absorbance values obtained against a reference microsomal preparation were subtracted from those obtained against a microsomal preparation over-expressing CYP2D6, thus removing the non-CYP2D6-specific reaction. Sera from 51 LKM1 positive patients (21 autoimmune hepatitis and 30 with HCV infection), 111 LKM1 negative patients with chronic liver disease (including 20 with HCV infection) and 43 healthy controls were tested. Of 51 patients positive by immunofluorescence, 48 were also positive by ELISA while all the 154 LKM1 negative subjects were also negative by ELISA. There was a high degree of association between IFL and ELISA as demonstrated by a kappa reliability value of 0.96. The absorbance values by ELISA correlated with immunofluorescence LKM1 titres both in autoimmune hepatitis (r = 0.74, p < 0.001) and HCV infection (r = 0.67, p < 0.001). The simple, objective ELISA described has the potential to replace the standard immunofluorescence technique.

  19. Unveiling the Structural Basis That Regulates the Energy Transduction Properties within a Family of Triheme Cytochromes from Geobacter sulfurreducens

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dantas, Joana M.; Simões, Telma; Morgado, Leonor

    A family of triheme cytochromes from Geobacter sulfurreducens plays an important role in extracellular electron transfer. In addition to their role in electron transfer pathways, two members of this family (PpcA and PpcD) were also found to be able to couple e(-)/H+ transfer through the redox Bohr effect observed in the physiological pH range, a feature not observed for cytochromes PpcB and PpcE. In attempting to understand the molecular control of the redox Bohr effect in this family of cytochromes, which is highly homologous both in amino acid sequence and structures, it was observed that residue 6 is a conservedmore » leucine in PpcA and PpcD, whereas in the other two characterized members (PpcB and PpcE) the equivalent residue is a phenylalanine. To determine the role of this residue located close to the redox Bohr center, we replaced Leu(6) in PpcA with Phe and determined the redox properties of the mutant, as well as its solution structure in the fully reduced state. In contrast with the native form, the mutant PpcAL6F is not able to couple the e(-)/H+ pathway. We carried out the reverse mutation in PpcB and PpcE (i.e., replacing Phe(6) in these two proteins by leucine) and the mutated proteins showed an increased redox Bohr effect. The results clearly establish the role of residue 6 in the control of the redox Bohr effect in this family of cytochromes, a feature that could enable the rational design of G. sulfurreducens strains that carry mutant cytochromes with an optimal redox Bohr effect that would be suitable for various biotechnological applications.« less

  20. An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1.

    PubMed

    Ovigne, J M; Vermot-Desroches, C; Lecron, J C; Portier, M; Lupker, J; Pecceu, F; Wijdenes, J

    1998-06-01

    The neuropeptide neurotensin (NT) interacts with two types of human receptors (hNTR) termed hNTR-1 and hNTR-2. This study describes a monoclonal antibody (MAb) specific for hNTR-1, B-N6. This MAb binds specifically to hNTR-1, but not to hNTR-2 transfected CHO cells. B-N6 and NT display a reciprocal competition and react in a similar way to trypsin, suggesting that the B-N6 epitope is at or close to the NT binding site on the third extracellular loop. Unlike B-N6, NT induces hNTR-1 internalization. Although neither NT-FITC nor B-N6 binding was detected by flow cytometry on different human cells, specific mRNA expression for hNTR-1 was detected in these cells. In CHO cells expressing hNTR-1 and a luciferase gene coupled to the krox24 reporter, B-N6 and the antagonist SR 48692 inhibited NT-induced intracellular activation of krox24 in a dose-dependent manner. From these results it is concluded that B-N6 is an antagonistic anti-hNTR-1 MAb.

  1. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

    PubMed

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A

    2015-08-11

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

  2. A world of cytochrome P450s

    PubMed Central

    Nelson, David R.

    2013-01-01

    The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450. PMID:23297353

  3. Covalent modification of cytochrome c by reactive metabolites of furan.

    PubMed

    Phillips, Martin B; Sullivan, Mathilde M; Villalta, Peter W; Peterson, Lisa A

    2014-01-21

    Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivity of these two reactive intermediates, cytochrome c was reacted with BDA in the presence and absence of GSH. As judged by MALDI-TOF mass spectrometry, BDA reacts extensively with cytochrome c to form adducts that add 66 Da to the protein, consistent with the formation of pyrrolinone adducts. Addition of GSH to the reaction mixture reduced the overall extent of adduct formation. The mass of the adducted protein was shifted by 355 Da as expected for GSH-BDA-protein cross-link formation. LC-MS/MS analysis of the tryptic digests of the alkylated protein indicated that the majority of adducts occurred on lysine residues, with BDA reacting less selectively than GSH-BDA. Both types of adducts may contribute to the toxic effects of furan.

  4. Covalent Modification of Cytochrome C by Reactive Metabolites of Furan

    PubMed Central

    Phillips, Martin B.; Sullivan, Mathilde M.; Villalta, Peter W.; Peterson, Lisa A.

    2014-01-01

    Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivity of these two reactive intermediates, cytochrome c was reacted with BDA in the presence and absence of GSH. As judged by MALDI-TOF mass spectrometry, BDA reacts extensively with cytochrome c to form adducts that add 66 Da to the protein, consistent with the formation of pyrrolinone adducts. Addition of GSH to the reaction mixture reduced the overall extent of adduct formation. The mass of the adducted protein was shifted by 355 Da as expected for GSH-BDA-protein cross-link formation. LC-MS/MS analysis of the tryptic digests of the alkylated protein indicated that the majority of adducts occurred on lysine residues, with BDA reacting less selectively than GSH-BDA. Both types of adducts may contribute to the toxic effects of furan. PMID:24364757

  5. A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

    PubMed

    Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

    1989-02-28

    1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

  6. Preparation guide for class B software specification documents

    NASA Technical Reports Server (NTRS)

    Tausworthe, R. C.

    1979-01-01

    General conceptual requirements and specific application rules and procedures are provided for the production of software specification documents in conformance with deep space network software standards and class B standards. Class B documentation is identified as the appropriate level applicable to implementation, sustaining engineering, and operational uses by qualified personnel. Special characteristics of class B documents are defined.

  7. Evidence for a Key Role of Cytochrome bo3 Oxidase in Respiratory Energy Metabolism of Gluconobacter oxydans

    PubMed Central

    Richhardt, Janine; Luchterhand, Bettina; Büchs, Jochen

    2013-01-01

    The obligatory aerobic acetic acid bacterium Gluconobacter oxydans oxidizes a variety of substrates in the periplasm by membrane-bound dehydrogenases, which transfer the reducing equivalents to ubiquinone. Two quinol oxidases, cytochrome bo3 and cytochrome bd, then catalyze transfer of the electrons from ubiquinol to molecular oxygen. In this study, mutants lacking either of these terminal oxidases were characterized. Deletion of the cydAB genes for cytochrome bd had no obvious influence on growth, whereas the lack of the cyoBACD genes for cytochrome bo3 severely reduced the growth rate and the cell yield. Using a respiration activity monitoring system and adjusting different levels of oxygen availability, hints of a low-oxygen affinity of cytochrome bd oxidase were obtained, which were supported by measurements of oxygen consumption in a respirometer. The H+/O ratio of the ΔcyoBACD mutant with mannitol as the substrate was 0.56 ± 0.11 and more than 50% lower than that of the reference strain (1.26 ± 0.06) and the ΔcydAB mutant (1.31 ± 0.16), indicating that cytochrome bo3 oxidase is the main component for proton extrusion via the respiratory chain. Plasmid-based overexpression of cyoBACD led to increased growth rates and growth yields, both in the wild type and the ΔcyoBACD mutant, suggesting that cytochrome bo3 might be a rate-limiting factor of the respiratory chain. PMID:23852873

  8. Granzyme B mediated function of Parvovirus B19-specific CD4+ T cells

    PubMed Central

    Kumar, Arun; Perdomo, Maria F; Kantele, Anu; Hedman, Lea; Hedman, Klaus; Franssila, Rauli

    2015-01-01

    A novel conception of CD4+ T cells with cytolytic potential (CD4+ CTL) is emerging. These cells appear to have a part in controlling malignancies and chronic infections. Human parvovirus B19 can cause a persistent infection, yet no data exist on the presence of B19-specific CD4+ CTLs. Such cells could have a role in the pathogenesis of some autoimmune disorders reported to be associated with B19. We explored the cytolytic potential of human parvovirus B19-specific T cells by stimulating peripheral blood mononuclear cell (PBMC) with recombinant B19-VP2 virus-like particles. The cytolytic potential was determined by enzyme immunoassay-based quantitation of granzyme B (GrB) and perforin from the tissue culture supernatants, by intracellular cytokine staining (ICS) and by detecting direct cytotoxicity. GrB and perforin responses with the B19 antigen were readily detectable in B19-seropositive individuals. T-cell depletion, HLA blocking and ICS experiments showed GrB and perforin to be secreted by CD4+ T cells. CD4+ T cells with strong GrB responses were found to exhibit direct cytotoxicity. As anticipated, ICS of B19-specific CD4+ T cells showed expected co-expression of GrB, perforin and interferon gamma (IFN-γ). Unexpectedly, also a strong co-expression of GrB and interleukin 17 (IL-17) was detected. These cells expressed natural killer (NK) cell surface marker CD56, together with the CD4 surface marker. To our knowledge, this is the first report on virus-specific CD4+ CTLs co-expressing CD56 antigen. Our results suggest a role for CD4+ CTL in B19 immunity. Such cells could function within both immune regulation and triggering of autoimmune phenomena such as systemic lupus erythematosus (SLE) or rheumatoid arthritis. PMID:26246896

  9. The Cytochrome P450 Homepage

    PubMed Central

    2009-01-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 (CYP) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described. PMID:19951895

  10. Structural Basis of Resistance to Anti-Cytochrome bc1 Complex Inhibitors: Implication for Drug Improvement

    PubMed Central

    Esser, Lothar; Yu, Chang-An; Xia, Di

    2016-01-01

    The emergence of drug resistance has devastating economic and social consequences, a testimonial of which is the rise and fall of inhibitors against the respiratory component cytochrome bc1 complex, a time tested and highly effective target for disease control. Unfortunately, the mechanism of resistance is a multivariate problem, including primarily mutations in the gene of the cytochrome b subunit but also activation of alternative pathways of ubiquinol oxidation and pharmacokinetic effects. There is a considerable interest in designing new bc1 inhibitors with novel modes of binding and lower propensity to induce the development of resistance. The accumulation of crystallographic data of bc1 complexes with and without inhibitors bound provides the structural basis for rational drug design. In particular, the cytochrome b subunit offers two distinct active sites that can be targeted for inhibition - the quinol oxidation site and the quinone reduction site. This review brings together available structural information of inhibited bc1 by various quinol oxidation- and reduction-site inhibitors, the inhibitor binding modes, conformational changes upon inhibitor binding of side chains in the active site and large scale domain movements of the iron-sulfur protein subunit. Structural data analysis provides a clear understanding of where and why existing inhibitors fail and points towards promising alternatives. PMID:23688079

  11. The photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum.

    PubMed

    Bernal-Bayard, Pilar; Puerto-Galán, Leonor; Yruela, Inmaculada; García-Rubio, Inés; Castell, Carmen; Ortega, José M; Alonso, Pablo J; Roncel, Mercedes; Martínez, Jesús I; Hervás, Manuel; Navarro, José A

    2017-09-01

    The photosynthetic cytochrome c 550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c 550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c 550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c 550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c 550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c 550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c 550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c 550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre.

  12. Luminogenic cytochrome P450 assays.

    PubMed

    Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

    2006-08-01

    Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

  13. Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

    PubMed

    Hildebrandt, P; Greinert, R; Stier, A; Taniguchi, H

    1989-12-08

    The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form

  14. F429 Regulation of Tunnels in Cytochrome P450 2B4: A Top Down Study of Multiple Molecular Dynamics Simulations

    PubMed Central

    Mancini, Giordano; Zazza, Costantino

    2015-01-01

    The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization. PMID:26415031

  15. F429 Regulation of Tunnels in Cytochrome P450 2B4: A Top Down Study of Multiple Molecular Dynamics Simulations.

    PubMed

    Mancini, Giordano; Zazza, Costantino

    2015-01-01

    The root causes of the outcomes of the single-site mutation in enzymes remain by and large not well understood. This is the case of the F429H mutant of the cytochrome P450 (CYP) 2B4 enzyme where the substitution, on the proximal surface of the active site, of a conserved phenylalanine 429 residue with histidine seems to hamper the formation of the active species, Compound I (porphyrin cation radical-Fe(IV) = O, Cpd I) from the ferric hydroperoxo (Fe(III)OOH-, Cpd 0) precursor. Here we report a study based on extensive molecular dynamic (MD) simulations of 4 CYP-2B4 point mutations compared to the WT enzyme, having the goal of better clarifying the importance of the proximal Phe429 residue on CYP 2B4 catalytic properties. To consolidate the huge amount of data coming from five simulations and extract the most distinct structural features of the five species studied we made an extensive use of cluster analysis. The results show that all studied single polymorphisms of F429, with different side chain properties: i) drastically alter the reservoir of conformations accessible by the protein, perturbing global dynamics ii) expose the thiolate group of residue Cys436 to the solvent, altering the electronic properties of Cpd0 and iii) affect the various ingress and egress channels connecting the distal sites with the bulk environment, altering the reversibility of these channels. In particular, it was observed that the wild type enzyme exhibits unique structural features as compared to all mutant species in terms of weak interactions (hydrogen bonds) that generate a completely different dynamical behavior of the complete system. Albeit not conclusive, the current computational investigation sheds some light on the subtle and critical effects that proximal single-site mutations can exert on the functional mechanisms of human microsomal CYPs which should go rather far beyond local structure characterization.

  16. Cytochrome P450 2D6 polymorphism and character traits.

    PubMed

    Suzuki, Eiji; Kitao, Yoshie; Ono, Yutaka; Iijima, Yoshimi; Inada, Toshiya

    2003-06-01

    It has been suggested that cytochrome P450 2D6 (CYP2D6) is involved in dopamine metabolism within the brain. The dopamine system is suggested to play a role in determining normal character. The purpose of this study was to examine whether character traits are dependent on cytochrome P450 2D6 activity. We investigated the association between temperament and CYP2D6 gene polymorphism. The subjects were all Japanese and the polymorphism genotyped in the present study was CYP2D6*10. Character traits were assessed using the Temperament and Character Inventory. There was no overall or specific association between personality traits and the CYP2D6*10 allele and genotype frequencies. The present results do not support the hypothesis that CYP2D6 activity affects temperament and character.

  17. High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

    PubMed

    Tsotsou, Georgia Eleni; Cass, Anthony Edward George; Gilardi, Gianfranco

    2002-01-01

    A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.

  18. Levels of Antibodies Specific to Tetanus Toxoid, Haemophilus influenzae Type b, and Pneumococcal Capsular Polysaccharide in Healthy Children and Adults

    PubMed Central

    Schauer, Uwe; Stemberg, Frank; Rieger, Christian H. L.; Büttner, Wolfgang; Borte, Michael; Schubert, Simone; Möllers, Helga; Riedel, Frank; Herz, Udo; Renz, Harald; Herzog, Wilhelm

    2003-01-01

    Antibody levels specific for capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) and for tetanus toxoid were measured in serum samples of 386 age-stratified subjects. The study group consists of healthy adult blood donors and hospitalized children undergoing elective surgery, excluding individuals with a history of infection. In children, anti-tetanus toxoid antibody levels displayed two peaks of 1.20 IU/ml (20.4 mg/liter) and 1.65 IU/ml (28.1 mg/liter) related to the schedule of routine childhood immunization in the first year and at 8 years of age. Eighty percent of the antibodies are of the immunoglobulin G1 (IgG1) isotype. For pneumococcal capsular polysaccharide (PCP), the specific antibody levels represent the acquisition of natural immunity. The initial concentration of 9.2 mg/liter was low in infancy (0.5 to 1 years of age) and remained low until 3 to 4 years of age (14.6 mg/liter). During this period PCP antibodies were almost 100% of the IgG2 subclass. Thereafter, IgG anti-PCP antibody titers increased steadily to adult levels (59.5 mg/liter). The data are intended to provide reference ranges to aid in the interpretation of specific antibody determinations in the clinical setting. PMID:12626443

  19. CD22 Promotes B-1b Cell Responses to T Cell-Independent Type 2 Antigens.

    PubMed

    Haas, Karen M; Johnson, Kristen L; Phipps, James P; Do, Cardinal

    2018-03-01

    CD22 (Siglec-2) is a critical regulator of B cell activation and survival. CD22 -/- mice generate significantly impaired Ab responses to T cell-independent type 2 (TI-2) Ags, including haptenated Ficoll and pneumococcal polysaccharides, Ags that elicit poor T cell help and activate BCR signaling via multivalent epitope crosslinking. This has been proposed to be due to impaired marginal zone (MZ) B cell development/maintenance in CD22 -/- mice. However, mice expressing a mutant form of CD22 unable to bind sialic acid ligands generated normal TI-2 Ab responses, despite significantly reduced MZ B cells. Moreover, mice treated with CD22 ligand-binding blocking mAbs, which deplete MZ B cells, had little effect on TI-2 Ab responses. We therefore investigated the effects of CD22 deficiency on B-1b cells, an innate-like B cell population that plays a key role in TI-2 Ab responses. B-1b cells from CD22 -/- mice had impaired BCR-induced proliferation and significantly increased intracellular Ca 2+ concentration responses following BCR crosslinking. Ag-specific B-1b cell expansion and plasmablast differentiation following TI-2 Ag immunization was significantly impaired in CD22 -/- mice, consistent with reduced TI-2 Ab responses. We generated CD22 -/- mice with reduced CD19 levels (CD22 -/- CD19 +/- ) to test the hypothesis that augmented B-1b cell BCR signaling in CD22 -/- mice contributes to impaired TI-2 Ab responses. BCR-induced proliferation and intracellular Ca 2+ concentration responses were normalized in CD22 -/- CD19 +/- B-1b cells. Consistent with this, TI-2 Ag-specific B-1b cell expansion, plasmablast differentiation, survival, and Ab responses were rescued in CD22 -/- CD19 +/- mice. Thus, CD22 plays a critical role in regulating TI-2 Ab responses through regulating B-1b cell signaling thresholds. Copyright © 2018 by The American Association of Immunologists, Inc.

  20. Quantum Torus Algebras and B(C)-Type Toda Systems

    NASA Astrophysics Data System (ADS)

    Wang, Na; Li, Chuanzhong

    2017-12-01

    In this paper, we construct a new even constrained B(C)-type Toda hierarchy and derive its B(C)-type Block-type additional symmetry. Also we generalize the B(C)-type Toda hierarchy to the N-component B(C)-type Toda hierarchy which is proved to have symmetries of a coupled \\bigotimes ^NQT_+ algebra ( N-fold direct product of the positive half of the quantum torus algebra QT).

  1. Electrostatic orientation of the electron-transfer complex between plastocyanin and cytochrome c.

    PubMed

    Roberts, V A; Freeman, H C; Olson, A J; Tainer, J A; Getzoff, E D

    1991-07-15

    To understand the specificity and efficiency of protein-protein interactions promoting electron transfer, we evaluated the role of electrostatic forces in precollision orientation by the development of two new methods, computer graphics alignment of protein electrostatic fields and a systematic orientational search of intermolecular electrostatic energies for two proteins at present separation distances. We applied these methods to the plastocyanin/cytochrome c interaction, which is faster than random collision, but too slow for study by molecular dynamics techniques. Significant electrostatic potentials were concentrated on one-fourth (969 A2) of the plastocyanin surface, with the greatest negative potential centered on the Tyr-83 hydroxyl within the acidic patch, and on one-eighth (632 A2) of the cytochrome c surface, with the greatest positive potential centered near the exposed heme edge. Coherent electrostatic fields occurred only over these regions, suggesting that local, rather than global, charge complementarity controls productive recognition. The three energetically favored families of pre-collision orientations all directed the positive region surrounding the heme edge of cytochrome c toward the acidic patch of plastocyanin but differed in heme plane orientation. Analysis of electrostatic fields, electrostatic energies of precollision orientations with 12 and 6 A separation distances, and surface topographies suggested that the favored orientations should converge to productive complexes promoting a single electron-transfer pathway from the cytochrome c heme edge to Tyr-83 of plastocyanin. Direct interactions of the exposed Cu ligand in plastocyanin with the cytochrome c heme edge are not unfavorable sterically or electrostatically but should occur no faster than randomly, indicating that this is not the primary pathway for electron transfer.

  2. Relative importance of driving force and electrostatic interactions in the reduction of multihaem cytochromes by small molecules.

    PubMed

    Quintas, Pedro O; Cepeda, Andreia P; Borges, Nuno; Catarino, Teresa; Turner, David L

    2013-06-01

    Multihaem cytochromes are essential to the energetics of organisms capable of bioremediation and energy production. The haems in several of these cytochromes have been discriminated thermodynamically and their individual rates of reduction by small electron donors were characterized. The kinetic characterization of individual haems used the Marcus theory of electron transfer and assumed that the rates of reduction of each haem by sodium dithionite depend only on the driving force, while electrostatic interactions were neglected. To determine the relative importance of these factors in controlling the rates, we studied the effect of ionic strength on the redox potential and the rate of reduction by dithionite of native Methylophilus methylotrophus cytochrome c″ and three mutants at different pH values. We found that the main factor determining the rate is the driving force and that Marcus theory describes this satisfactorily. This validates the method of the simultaneous fitting of kinetic and thermodynamic data in multihaem cytochromes and opens the way for further investigation into the mechanisms of these proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. THE OXIDATION OF EXOGENOUS AND ENDOGENOUS CYTOCHROME C IN MITOCHONDRIA

    PubMed Central

    Muscatello, Umberto; Carafoli, Ernesto

    1969-01-01

    The effect of the nonionic detergent Lubrol on the oxidation of endogenous and exogenous cytochrome c by cytochrome oxidase in intact and fragmented mitochondria was studied. Mitochondria and mitochondrial fragments from liver, kidney, heart, and skeletal muscle have been used. Negatively stained preparations of intact mitochondria showed the particles of Fernández-Morán on the matrix side of their inner membrane system: under these conditions, the oxidation rate of externally added cytochrome c was very high, and it was stimulated very poorly by Lubrol. Mechanical fragmentation of liver mitochondria yielded vesicles with a smooth external profile: also under these conditions, the oxidation of externally added cytochrome c was very high, and poorly stimulated by Lubrol. The oxidation of endogenous cytochrome c was also unaffected by Lubrol. On the other hand, fragmentation of heart and skeletal muscle mitochondria yielded vesicles having numerous particles of Fernández-Morán on their external profiles. Under these conditions, the oxidation of exogenous cytochrome c was low and was markedly stimulated by Lubrol. On the contrary, no activation of the oxidation of endogenous cytochrome c was induced by the detergent. The results indicate a difference in the permeability properties of the two faces of the inner mitochondrial membrane: a permeability barrier for cytochrome c is suggested to exist at the inner face. PMID:4303915

  4. Cell-Type-Specific Optogenetics in Monkeys.

    PubMed

    Namboodiri, Vijay Mohan K; Stuber, Garret D

    2016-09-08

    The recent advent of technologies enabling cell-type-specific recording and manipulation of neuronal activity spurred tremendous progress in neuroscience. However, they have been largely limited to mice, which lack the richness in behavior of primates. Stauffer et al. now present a generalizable method for achieving cell-type specificity in monkeys. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

    PubMed Central

    Huang, Yong; Lu, Xue-Ping; Wang, Luo-Luo; Wei, Dong; Feng, Zi-Jiao; Zhang, Qi; Xiao, Lin-Fan; Dou, Wei; Wang, Jin-Jun

    2015-01-01

    NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis. PMID:26681597

  6. The effects of detergents DDM and beta-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b6f complex from spinach chloroplasts.

    PubMed

    Chen, XiaoBo; Zhao, XiaoHui; Zhang, JianPing; Li, LiangBi; Kuang, TingYun

    2007-08-01

    The singlet excited state lifetime of the chlorophyll a (Chl a) in cytochrome b(6)f (Cyt b(6)f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b(6)f complexes from different sources ( approximately 200 and approximately 600 ps are the two measured results). The present study demonstrated that the singlet excited state lifetime is associated with the detergents n-dodecyl-beta-D-maltoside (DDM) and n-octyl-beta-D-glucopyranoside (beta-OG), but has nothing to do with the different sources of Cyt b(6)f complexes. Compared with the Cyt b(6)f dissolved in beta-OG, the Cyt b(6)f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, approximately 200 ps, of the Chl a in Cyt b(6)f complex in DDM is closer to the true in vivo.

  7. Relative effect potency estimates of dioxin-like activity for dioxins, furans, and dioxin-like PCBs in adults based on cytochrome P450 1A1 and 1B1 gene expression in blood.

    PubMed

    Wimmerová, Soňa; van den Berg, Martin; Chovancová, Jana; Patayová, Henrieta; Jusko, Todd A; van Duursen, Majorie B M; Palkovičová Murínová, Ľubica; Canton, Rocio F; van Ede, Karin I; Trnovec, Tomáš

    2016-11-01

    In the risk assessment of PCDDs, PCDFs, and dioxin-like (DL) PCBs, regulatory authorities support the use of the toxic equivalency factor (TEF)-scheme derived from a heterogeneous data set of the relative effect potency (REPs) estimates. We sought to determine REPs for dioxin-like compounds (DLCs) using expression of cytochrome P450 (CYP) 1A1 and 1B1 mRNA in human peripheral blood mononuclear cells representing two different pathways. We used a sex and age adjusted regression-based approach comparing the strength of association between each DLC and the cytochrome P450 (CYP) 1A1 and 1B1 mRNA expression in 320 adults residing in an organochlorine-polluted area of eastern Slovakia. We calculated REPs based on CYP1A1 expression for 4 PCDDs, 8 PCDFs, and 1 PCB congener, and based on CYP1B1 expression for 5 PCDFs and 11 PCB congeners. REPs from CYP1A1 correlated with REPs previously derived from thyroid volume (ρ=0.85; p<0.001) and serum FT4 (ρ=0.77; p=0.009). The 13 log REPs from CYP1A1 correlated with log WHO-TEFs (r=0.63; p=0.015) and 11 log PCB REPs with PCB consensus toxicity factors (CTFs) for compounds with WHO-TEFs (r=0.80; p=0.003). The complete set of derived 56 log REPs correlated with the log CTFs (r=0.77; p=0.001) and log WHO-TEFs (r=0.81; p<0.001). REPs calculated from thyroid and cytochrome P450 endpoints realistically reflect human exposure scenarios because they are based on human chronic and low-dose exposures. While the CYP 1A1 seems more suitable for toxicity evaluation of PCDD/Fs, the CYP 1B1 is more apt for PCDFs and PCBs and reflects different pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene.

    PubMed

    Kihara, Takahiro; Hiroe, Ayaka; Ishii-Hyakutake, Manami; Mizuno, Kouhei; Tsuge, Takeharu

    2017-08-01

    Bacillus cereus and Bacillus megaterium both accumulate polyhydroxyalkanoate (PHA) but their PHA biosynthetic gene (pha) clusters that code for proteins involved in PHA biosynthesis are different. Namely, a gene encoding MaoC-like protein exists in the B. cereus-type pha cluster but not in the B. megaterium-type pha cluster. MaoC-like protein has an R-specific enoyl-CoA hydratase (R-hydratase) activity and is referred to as PhaJ when involved in PHA metabolism. In this study, the pha cluster of B. cereus YB-4 was characterized in terms of PhaJ's function. In an in vitro assay, PhaJ from B. cereus YB-4 (PhaJ YB4 ) exhibited hydration activity toward crotonyl-CoA. In an in vivo assay using Escherichia coli as a host for PHA accumulation, the recombinant strain expressing PhaJ YB4 and PHA synthase led to increased PHA accumulation, suggesting that PhaJ YB4 functioned as a monomer supplier. The monomer composition of the accumulated PHA reflected the substrate specificity of PhaJ YB4 , which appeared to prefer short chain-length substrates. The pha cluster from B. cereus YB-4 functioned to accumulate PHA in E. coli; however, it did not function when the phaJ YB4 gene was deleted. The B. cereus-type pha cluster represents a new example of a pha cluster that contains the gene encoding PhaJ.

  9. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c

    PubMed Central

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A.

    2015-01-01

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ’s histone chaperone activity. PMID:26216969

  10. Detection of influenza virus types A and B and type A subtypes (H1, H3, and H5) by multiplex polymerase chain reaction.

    PubMed

    Boonsuk, Pitirat; Payungporn, Sunchai; Chieochansin, Thaweesak; Samransamruajkit, Rujipat; Amonsin, Alongkorn; Songserm, Thaweesak; Chaisingh, Arunee; Chamnanpood, Pornchai; Chutinimitkul, Salin; Theamboonlers, Apiradee; Poovorawan, Yong

    2008-07-01

    Infections with influenza virus type A and B present serious public health problems on a global scale. However, only influenza A virus has been reported to cause fatal pandemic in many species. To provide suitable clinical management and prevent further virus transmission, efficient and effective clinical diagnosis is essential. Therefore, we developed multiplex PCR assays for detecting influenza types A and B and the subtypes of influenza A virus (H1, H3 and H5). Upon performing multiplex PCR assays with type-specific primer sets, the clearly distinguishable products representing influenza A and B virus were separated by agarose gel electrophoresis. In addition, the subtypes of influenza A virus (H1, H3 and H5), which are most common in humans, can be readily distinguished by PCR with subtype-specific primer sets, yielding PCR products of different sizes depending on which subtype has been amplified. This method was tested on 46 influenza virus positive specimens of avian and mammalian (dog and human) origins collected between 2006 and 2008. The sensitivity of this method, tested against known concentrations of each type and subtype specific plasmid, was established to detect 10(3) copies/microl. The method's specificity was determined by testing against other subtypes of influenza A virus (H2, H4 and H6-H15) and respiratory pathogens commonly found in humans. None of them could be amplified, thus excluding cross reactivity. In conclusion, the multiplex PCR assays developed are advantageous as to rapidity, specificity, and cost effectiveness.

  11. A Critical Role for the cccA Gene Product, Cytochrome c2, in Diverting Electrons from Aerobic Respiration to Denitrification in Neisseria gonorrhoeae

    PubMed Central

    Hopper, Amanda C.; Li, Ying

    2013-01-01

    Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c2, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c4 and c5 transfer electrons to cytochrome oxidase cbb3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c4 or c5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc2), cycA (Δc4), cycB (Δc5), and ccoP (ccoP-C368A), we demonstrate that cytochrome c2 is required for electron transfer from cytochrome c4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae. PMID:23543713

  12. Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states.

    PubMed

    Ogura, T; Yoshikawa, S; Kitagawa, T

    1985-12-17

    Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).

  13. Functional mechanism of neuroprotection by inhibitors of type B monoamine oxidase in Parkinson's disease.

    PubMed

    Naoi, Makoto; Maruyama, Wakako

    2009-08-01

    Neuroprotective therapy has been proposed for age-related neurodegenerative disorders, including Parkinson's disease. Inhibitors of type B monoamine oxidase (MAOB-Is), rasagiline and (-)deprenyl, are the most promising candidate neuroprotective drugs. Clinical trials of rasagiline in patients with Parkinson's disease suggest that rasagiline may have some disease-modifying effects. Results using animal and cellular models have proved that the MAOB-Is protect neurons by the intervention of 'intrinsic' mitochondrial apoptotic cascade and the induction of prosurvival antiapoptotic Bcl-2 and neurotrophic factors. Rasagiline-related MAOB-Is prevent mitochondrial permeability transition induced by various insults and activation of subsequent apoptotic cascades: cytochrome c release, casapase activation, and condensation and fragmentation of nuclear DNA. MAOB-Is increase transcription of prosurvival genes through activating the nuclear transcription factor-(NF) system. Rasagiline increases the protein and mRNA levels of GDNF in dopaminergic SH-SY5Y cells, whereas (-)deprenyl increases those of BDNF. Systemic administration of (-)deprenyl and rasagiline increases these neurotrophic factors in the cerebrospinal fluid from patients with Parkinson's disease and nonhuman primates. This review presents recent advances in our understanding of the neuroprotection offered by MAOB-Is and possible evaluation of neuroprotective efficacy in clinical samples is discussed.

  14. Biomonitoring the cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in hair: impact of exposure, hair pigmentation, and cytochrome P450 1A2 phenotype.

    PubMed

    Turesky, Robert J; Liu, Lin; Gu, Dan; Yonemori, Kim M; White, Kami K; Wilkens, Lynne R; Le Marchand, Loïc

    2013-03-01

    Hair is a promising tissue to assess exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen formed in cooked meats. However, an understanding of how dietary exposure to PhIP, cytochrome P450 1A2 activity-a key enzyme involved in PhIP metabolism, and hair pigmentation affect the level of PhIP accrued in hair is required to determine the reliability of the PhIP hair level as a biomarker of exposure to this carcinogen. We examined the impact of PhIP exposure, cytochrome P450 1A2 activity, and hair pigmentation on the levels of PhIP accumulated in the hair of volunteers on a 4-week semicontrolled diet of cooked meat containing known quantities of PhIP. The amount of PhIP in hair increased, on average, 15-fold in light- and dark-haired individuals during consumption of cooked meat. PhIP levels in hair were correlated to PhIP intake (ρ = 0.53; P < 0.001), and the relationship was strengthened when PhIP levels were normalized for the melanin content of hair (ρ = 0.71; P < 0.001). However, PhIP accrual in hair was not correlated to cytochrome P450 1A2 activity, as assessed by the caffeine test, or to the levels of unmetabolized PhIP in urine or to the metabolic ratio of the major urinary metabolite N(2)-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine to unmetabolized PhIP. The use of the PhIP hair biomarker should take hair pigmentation into account for accurate exposure assessment of PhIP. PhIP hair levels can serve as a biomarker in epidemiologic studies investigating the association of heterocyclic aromatic amine (HAA), cooked meat, and cancer risk.

  15. Diagnostic Cytochrome b gene profiles for the identification of paca (Cuniculus paca) bushmeat: implications for the monitoring of illegal hunting and wildlife trade.

    PubMed

    Silva-Neto, A A; Ferreira, P B; Torres, R A; Texeira, R H F; Duarte, J M B; Barbosa, A C; Vargas, R C; Garcia, J E

    2016-02-01

    Paca (Cuniculus paca Linnaeus, 1766) is the second largest rodent found in Brazil. The quality of the meat and a long tradition of hunting have contributed to the decline of the natural populations of this species. Hunting of paca is strictly prohibited in Brazil, but in spite of this restriction, no forensic tools are available for the identification of the meat. We describe an efficient method, based on single nucleotide polymorphisms of the cytochrome b gene, that can be used to differentiate biological material derived from paca from those of domestic species commonly used as sources of meat. The identification of the presence of C. paca in the samples was 100% reliable.

  16. Association of polymorphisms and haplotypes in the cytochrome P450 1B1 gene with uterine leiomyoma: A case control study

    PubMed Central

    SALIMI, SAEEDEH; KHODAMIAN, MARYAM; NAROOIE-NEJAD, MEHRNAZ; HAJIZADEH, AZAM; FAZELI, KIMIA; NAMAZI, LIDA; YAGHMAEI, MINOO

    2015-01-01

    Uterine leiomyoma (UL) is an estrogen-dependent neoplasm of the uterus and estrogen metabolizing enzymes affect its promotion and progression. The aim of the present study was to evaluate the association between four single-nucleotide polymorphisms (SNPs) of the cytochrome P450 1B1 (CYP1B1) gene and UL risk. Four SNPs of the CYP1B1 gene in 105 UL patients and 112 unrelated healthy controls were genotyped using a direct sequencing method. Haplotype analyses were performed with UNPHASED software and linkage disequilibrium (LD) was assessed by Haploview software. There were no associations between Leu432Val (rs1056836), Asp449Asp (rs1056837) and Asn453Ser (rs1800440) polymorphisms of the CYP1B1 gene and UL. Although the genotypic frequencies of the Arg368His (rs79204362) polymorphism did not differ between the two groups, the frequency of A (His) allele was significantly higher in UL females (P=0.02). In addition, the frequency of GTAA haplotype was significantly higher in the controls and played a protective role in UL susceptibility. A strong LD between the three common SNPs (rs1056836, rs1056837 and rs1800440) in the CYP1B1 gene was observed in the population. In conclusion, a higher frequency of the CYP1B1 368His (A) allele was observed in UL females. The frequency of the GTAA haplotype was significantly higher in healthy females and this haplotype played a protective role in UL susceptibility. PMID:26075073

  17. Fluorescence labelling of NADPH-cytochrome P-450 reductase with the monobromomethyl derivative of syn-9,10-dioxabimane.

    PubMed Central

    Vogel, F; Lumper, L

    1983-01-01

    The kinetics of thiol-group alkylation in NADPH-cytochrome P-450 reductase during its inactivation by monobromobimane has been studied using the fluorimetric determination of S-bimane-L-cysteine by high-performance liquid chromatography. Loss of activity during the reaction of NADPH-cytochrome P-450 reductase with monobromobimane is caused by the alkylation of one single critical cysteine residue, which can be protected against thiol-specific reagents by NADP(H). The chemical stability of the bimane group allows the digestion of bimane-labelled NADPH-cytochrome P-450 reductase by CNBr. The critical cysteine residue could be located in a CNBr-cleaved peptide purified to homogeneity with Mr 10 500 +/- 1 000 and valine as N-terminus. Images Fig. 2. PMID:6414464

  18. Genotype analysis, using PCR with type-specific primers, of hepatitis B virus isolates from patients coinfected with hepatitis delta virus genotype II from Miyako Island, Japan.

    PubMed

    Moriyama, Moriyama; Taira, Masaaki; Matsumura, Hiroshi; Aoki, Hiroshi; Mikuni, Morio; Kaneko, Miki; Shioda, Atsuo; Iwaguchi, Kayo; Arai, Shinobu; Ichijima, Sagiri; Iwasaki, Hiroko; Tanaka, Naohide; Abe, Kenji; Arakawa, Yasuyuki

    2003-01-01

    The aims of this study were to determine the hepatitis B virus (HBV) genotypes in hepatitis delta virus (HDV) RNA-positive patients and to characterize the HBV nucleotide sequences that may be found on a distant island of Japan. This study included three patients with chronic hepatitis who were positive for hepatitis B surface antigen (enzyme-linked immunosorbent assay; ELISA), HDV antibody (ELISA) and HDV RNA by polymerase chain reaction (PCR). The HBV genotype was determined by nested PCR using type-specific primers. The first-round PCR products from two patients were sequenced, followed by an investigation of nucleotide homology. Viruses from all three patients in this study were classified as HBV genotype B. Comparison with HBV isolates from geographically neighboring regions revealed that the two HBV isolates had 97.9-98.6% identity at the nucleotide level to a Chinese isolate, 98.3-98.6% identity to the Okinawa isolate and 98.6-98.8% identity to a Japanese isolate of genotype B. On phylogenetic analysis, the HBV isolates from the two patients were classified as HBV genotype B. The HBV isolates of cases 1 and 3 clustered in the same group as isolates from the Chinese mainland and Japanese mainland, which are geographically near Miyako Island. The HBV isolates coinfected with HDV found on Miyako Island were of genotype B. The PCR method based on genotype-specific primers was useful in determining HBV genotypes. Copyright 2003 S. Karger AG, Basel

  19. Effects of thalidomide, cytochrome P-450 and TNF-alpha on angiogenesis in a three-dimensional collagen gel-culture.

    PubMed

    Fujita, Keiko; Asami, Yoshiko; Murata, Eiko; Akita, Masumi; Kaneko, Katsuji

    2002-10-01

    The anti-angiogenic effects of thalidomide were examined in mouse aortae grown in a three-dimensional collagen gel-culture. In our in vitro model, (+/-)-thalidomide and (-)-thalidomide exhibited no anti-angiogenic effects. On the other hand, when the culture was treated with thalidomide plus cytochrome P-450, both types of thalidomides significantly inhibited angiogenesis. Co-administration of 100 microg/ml thalidomide plus 200 microg/ml cytochrome P-450 inhibited angiogenesis more strongly than thalidomide plus cytochrome P-450 at other concentrations (10 microg/ml + 200 microg/ml and 100 microg/ml + 20 microg/ml). To study the relation between the anti-angiogenic effect and TNF-alpha, we also evaluated the concentration of TNF-alpha in the culture medium. We found that the concentration of TNF-alpha was correlated to the strength of the anti-angiogenic effect. The inhibition of angiogenesis by thalidomide and cytochrome P-450 takes place through a suppression of TNF-alpha and involves the metabolism of the thalidomide.

  20. Molecular phylogenetics of finches and sparrows: consequences of character state removal in cytochrome b sequences.

    PubMed

    Groth, J G

    1998-12-01

    The complete mitochondrial cytochrome b genes of 53 genera of oscine passerine birds representing the major groups of finches and some allies were compared. Phylogenetic trees resulting from three levels of character partition removal (no data removed, transitions at third positions of codons removed, and all transitions removed [transversion parsimony]) were generally concordant, and all supported several basic statements regarding relationships of finches and finch-like birds, including: (1) larks (Alaudidae) show no close relationship to any finch group; (2) Peucedramus (olive warbler) is phylogenetically far removed from true wood warblers; (3) a clade consisting of fringillids, passerids, motacillids, and emberizids is supported, and this clade is characterized by evolution of a vestigial 10th wing primary; and (4) Hawaiian honeycreepers are derived from within the cardueline finches. Excluding transition substitutions at third positions of codons resulted in phylogenetic trees similar to, but with greater bootstrap nodal support than, trees derived using either all data (equally weighted) or transversion parsimony. Relative to the shortest trees obtained using all data, the topologies obtained after elimination of third-position transitions showed only slight increases in realized treelength and homoplasy. These increases were negligable compared to increases in overall nodal support; therefore, this partition removal scheme may enhance recovery of deep phylogenetic signal in protein-coding DNA datasets. Copyright 1998 Academic Press.

  1. Mutational analysis of the Saccharomyces cerevisiae cytochrome c oxidase assembly protein Cox11p.

    PubMed

    Banting, Graham S; Glerum, D Moira

    2006-03-01

    Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa(3) peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that Deltacox11, like Deltasco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure.

  2. Mechanisms of m-cresol induced protein aggregation studied using a model protein cytochrome c†

    PubMed Central

    Singh, Surinder M.; Hutchings, Regina L.; Mallela, Krishna M.G.

    2014-01-01

    Multi-dose protein formulations require an effective antimicrobial preservative (AP) to inhibit microbial growth during long-term storage of unused formulations. m-cresol is one such AP, but has been shown to cause protein aggregation. However, the fundamental physical mechanisms underlying such AP-induced protein aggregation are not understood. In this study, we used a model protein cytochrome c to identify the protein unfolding that triggers protein aggregation. m-cresol induced cytochrome c aggregation at preservative concentrations that are commonly used to inhibit microbial growth. Addition of m-cresol decreased the temperature at which the protein aggregated and increased the aggregation rate. However, m-cresol did not perturb the tertiary or secondary structure of cytochrome c. Instead, it populated an “invisible” partially unfolded intermediate where a local protein region around the methionine residue at position 80 was unfolded. Stabilizing the Met80 region drastically decreased the protein aggregation, which conclusively shows that this local protein region acts as an aggregation “hot-spot”. Based on these results, we propose that APs induce protein aggregation by partial rather than global unfolding. Because of the availability of site-specific probes to monitor different levels of protein unfolding, cytochrome c provided a unique advantage in characterizing the partial protein unfolding that triggers protein aggregation. PMID:21229618

  3. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jan, Yi-Hua; Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu; Baker, Angela A.

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling,more » a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.« less

  4. Development of cytochrome P450 2D6-specific LKM-autoantibodies following liver transplantation for Wilson's disease -- possible association with a steroid-resistant transplant rejection episode.

    PubMed

    Lohse, A W; Obermayer-Straub, P; Gerken, G; Brunner, S; Altes, U; Dienes, H P; Manns, M P; Meyer zum Büschenfelde, K H

    1999-07-01

    Antibodies to cytochrome P450 2D6, also known as LKM1-autoantibodies, are characteristic for a subgroup of patients with autoimmune hepatitis, but can also occasionally be found in hepatitis C. We observed the occurrence of LKM1-autoantibodies 4 months after liver transplantation for Wilson's disease, in close association with a steroid-resistant rejection episode, in the absence of evidence for autoimmune hepatitis or hepatitis C. Sera from several time points prior to and following transplantation were tested for LKM-reactivity by immunofluorescence, ELISA and Western blotting. Antigen specificity was confirmed by Western blotting analysis on different cytochrome P450 isoenzymes. The absence of viral hepatitis C and hepatitis G virus infection was confirmed by polymerase chain reaction. The serum of the organ donor was also tested. All the sera prior to transplantation and up to 4 months after transplantation were LKM-negative by all assay systems used. In the course of a steroid-resistant rejection episode at this time, the patient developed LKM antibodies at high titre (70% in inhibition ELISA) and has remained positive since (now more than 4 years). Reactivity was exclusively to the cytochrome isoenzyme 2D6. Hepatitis C infection never occurred, but hepatitis G was transiently present many years prior to transplantation. The donor serum was negative for all autoantibodies and for hepatitis C and G virus infection. We here describe a patient developing LKM1-autoantibodies without evidence of autoimmune or viral hepatitis. The close temporal association with a transplant rejection episode suggests immunological mechanisms of rejection together with hepatocellular injury as a pathogenetic mechanism.

  5. Cytochrome P450 1B1 and catechol-O-methyltransferase genetic polymorphisms and breast cancer risk in Chinese women: results from the shanghai breast cancer study and a meta-analysis.

    PubMed

    Wen, Wanqing; Cai, Qiuyin; Shu, Xiao-Ou; Cheng, Jia-Rong; Parl, Fritz; Pierce, Larry; Gao, Yu-Tang; Zheng, Wei

    2005-02-01

    Cytochrome P450 1B1 (CYP1B1) and catechol-O-methyltransferase (COMT) are important estrogen-metabolizing enzymes and, thus, genetic polymorphisms of these enzymes may affect breast cancer risk. A population-based case-control study was conducted to assess the association of breast cancer risk with CYP1B1 and COMT polymorphisms. A meta-analysis was done to summarize the findings from this and previous studies. Included in this study were 1,135 incident breast cancer cases diagnosed from August 1996 through March 1998 among female residents of Shanghai and 1,235 randomly selected, age frequency-matched controls from the same general population. The common alleles of the CYP1B1 gene were Arg (79.97%) in codon 48, Ala (80.53%) in codon 119, and Leu (86.57%) in codon 432. The Val allele accounted for 72.46% of the total alleles identified in codon 108/158 of the COMT gene. No overall associations of breast cancer risk were found with any of the single nucleotide polymorphisms described above. This finding was supported by a meta-analysis of all previous published studies. No gene-gene interactions were observed between CYP1B1 and COMT genotypes. The associations of breast cancer risk with factors related to endogenous estrogen exposure, such as years of menstruation and body mass index, were not significantly modified by the CYP1B1 and COMT genotypes. We observed, however, that women who carried one copy of the variant allele in CYP1B1 codons 48 or 119 were less likely to have estrogen receptor-positive breast cancer than those who carried two copies of the corresponding wild-type alleles. The results from this study were consistent with those from most previous studies, indicating no major associations of breast cancer risk with CYP1B1 and COMT polymorphisms.

  6. Phylogenetic analysis of Bolivian bat trypanosomes of the subgenus schizotrypanum based on cytochrome B sequence and minicircle analyses.

    PubMed

    García, Lineth; Ortiz, Sylvia; Osorio, Gonzalo; Torrico, Mary Cruz; Torrico, Faustino; Solari, Aldo

    2012-01-01

    The aim of this study was to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats. Eighteen cloned stocks were isolated from 115 bats belonging to Carollia perspicillata (Phyllostomidae) from three Amazonian areas of the Chapare Province of Bolivia and studied by xenodiagnosis using the vectors Rhodnius robustus and Triatoma infestans (Trypanosoma cruzi marenkellei) or haemoculture (Trypanosoma dionisii). The PCR DNA amplified was analyzed by nucleotide sequences of maxicircles encoding cytochrome b and by means of the molecular size of hyper variable regions of minicircles. Ten samples were classified as Trypanosoma cruzi marinkellei and 8 samples as Trypanosoma dionisii. The two species have a different molecular size profile with respect to the amplified regions of minicircles and also with respect to Trypanosoma cruzi and Trypanosoma rangeli used for comparative purpose. We conclude the presence of two species of bat trypanosomes in these samples, which can clearly be identified by the methods used in this study. The presence of these trypanosomes in Amazonian bats is discussed.

  7. Phylogenetic Analysis of Bolivian Bat Trypanosomes of the Subgenus Schizotrypanum Based on Cytochrome b Sequence and Minicircle Analyses

    PubMed Central

    García, Lineth; Ortiz, Sylvia; Osorio, Gonzalo; Torrico, Mary Cruz; Torrico, Faustino; Solari, Aldo

    2012-01-01

    The aim of this study was to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats. Eighteen cloned stocks were isolated from 115 bats belonging to Carollia perspicillata (Phyllostomidae) from three Amazonian areas of the Chapare Province of Bolivia and studied by xenodiagnosis using the vectors Rhodnius robustus and Triatoma infestans (Trypanosoma cruzi marenkellei) or haemoculture (Trypanosoma dionisii). The PCR DNA amplified was analyzed by nucleotide sequences of maxicircles encoding cytochrome b and by means of the molecular size of hyper variable regions of minicircles. Ten samples were classified as Trypanosoma cruzi marinkellei and 8 samples as Trypanosoma dionisii. The two species have a different molecular size profile with respect to the amplified regions of minicircles and also with respect to Trypanosoma cruzi and Trypanosoma rangeli used for comparative purpose. We conclude the presence of two species of bat trypanosomes in these samples, which can clearly be identified by the methods used in this study. The presence of these trypanosomes in Amazonian bats is discussed. PMID:22590570

  8. 18 CFR 1b.4 - Types of 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 Types of investigations. 1b.4 Section 1b.4 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.4 Types of investigations...

  9. 18 CFR 1b.4 - Types of 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 Types of investigations. 1b.4 Section 1b.4 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.4 Types of investigations...

  10. 18 CFR 1b.4 - Types of 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 Types of investigations. 1b.4 Section 1b.4 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.4 Types of investigations...

  11. 18 CFR 1b.4 - Types of 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 Types of investigations. 1b.4 Section 1b.4 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.4 Types of investigations...

  12. HLA-B*5808, a new HLA-B allele characterized by sequence based typing.

    PubMed

    Poli, F; Crespiatico, L; Frison, S; Longhi, E; Marlianici, E; Scalamogna, M

    2003-12-01

    This brief communication describes a new HLA-B allele (HLA-B*5808) detected in an Italian white volunteer bone marrow donor. With serology, this subject was typed as HLA-B15,17, whereas with molecular biology B*15, B*51, B*52 and/or B*58 could be assigned. In order to clarify the results, direct and cloning sequencing of exons 2, 3 and 4 were carried out. This new allele is identical to HLA-B*5801 in exon 2 except for a silent point mutation at nucleotide 141 where a C is substituted by a T; exons 3 and 4 are typical of HLA-B*51, B*52 and B*78. The peculiar sequence of B*5808 could explain the discrepancy between the serological and molecular typing results.

  13. Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology

    PubMed Central

    Hüttemann, Maik; Lee, Icksoo; Gao, Xiufeng; Pecina, Petr; Pecinova, Alena; Liu, Jenney; Aras, Siddhesh; Sommer, Natascha; Sanderson, Thomas H.; Tost, Monica; Neff, Frauke; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Naton, Beatrix; Rathkolb, Birgit; Rozman, Jan; Favor, Jack; Hans, Wolfgang; Prehn, Cornelia; Puk, Oliver; Schrewe, Anja; Sun, Minxuan; Höfler, Heinz; Adamski, Jerzy; Bekeredjian, Raffi; Graw, Jochen; Adler, Thure; Busch, Dirk H.; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Weissmann, Norbert; Doan, Jeffrey W.; Bassett, David J. P.; Grossman, Lawrence I.

    2012-01-01

    Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (−50 and −29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (−12%), reduced total oxygen consumption rate (−8%), improved glucose tolerance, and reduced grip force (−14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction

  14. Interaction of photosystem I from Phaeodactylum tricornutum with plastocyanins as compared with its native cytochrome c6: Reunion with a lost donor.

    PubMed

    Bernal-Bayard, Pilar; Pallara, Chiara; Carmen Castell, M; Molina-Heredia, Fernando P; Fernández-Recio, Juan; Hervás, Manuel; Navarro, José A

    2015-12-01

    In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. HLA-B40, B18, B27, and B37 allele discrimination using group-specific amplification and SSCP method.

    PubMed

    Bannai, M; Tokunaga, K; Lin, L; Ogawa, A; Fujisawa, K; Juji, T

    1996-04-01

    We developed a system for discriminating HLA-B40, B18, B27, and B37 alleles using a two-step PCR method followed by SSCP analysis. Fragments (0.8 kb) including exon 2, intron 2, and exon 3 were amplified in the first PCR. We used two sets of primers, one specific for HLA-B60-related alleles and the other specific for HLA-B61-related, B18, B27, and B37 alleles. No amplifications of other class I genes or pseudogenes were observed. In the second PCR, exon 2 and exon 3 were amplified separately, using diluents of the first PCR products as templates. HLA-B61-related, B18, B27, B37, and B60-related alleles were clearly discriminated in the SSCP analysis of the second PCR products. In a population study in which B61 alleles were analyzed, B*4003 was detected in two Japanese individuals in addition to two B61 alleles previously reported to occur in Japanese, B*4002 and B*4006. The relative frequencies of B*4002, B*4006, and B*4003 in Japanese were 58, 35, and 6%, respectively. The individuals having B*4003 are the first non-South Americans in whom this allele has been detected. The SSCP banding patterns of 18 HLA-B60-positive Japanese population samples were identical to those of a B*40012 sample for both exon 2 and exon 3. We also demonstrated that the B37 allele occurring in some Japanese is B*3701.

  16. Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

    PubMed

    Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

    2017-11-01

    CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. N-terminal pro-B-type Natriuretic Peptides' Prognostic Utility Is Overestimated in Meta-analyses Using Study-specific Optimal Diagnostic Thresholds.

    PubMed

    Potgieter, Danielle; Simmers, Dale; Ryan, Lisa; Biccard, Bruce M; Lurati-Buse, Giovanna A; Cardinale, Daniela M; Chong, Carol P W; Cnotliwy, Miloslaw; Farzi, Sylvia I; Jankovic, Radmilo J; Lim, Wen Kwang; Mahla, Elisabeth; Manikandan, Ramaswamy; Oscarsson, Anna; Phy, Michael P; Rajagopalan, Sriram; Van Gaal, William J; Waliszek, Marek; Rodseth, Reitze N

    2015-08-01

    N-terminal fragment B-type natriuretic peptide (NT-proBNP) prognostic utility is commonly determined post hoc by identifying a single optimal discrimination threshold tailored to the individual study population. The authors aimed to determine how using these study-specific post hoc thresholds impacts meta-analysis results. The authors conducted a systematic review of studies reporting the ability of preoperative NT-proBNP measurements to predict the composite outcome of all-cause mortality and nonfatal myocardial infarction at 30 days after noncardiac surgery. Individual patient-level data NT-proBNP thresholds were determined using two different methodologies. First, a single combined NT-proBNP threshold was determined for the entire cohort of patients, and a meta-analysis conducted using this single threshold. Second, study-specific thresholds were determined for each individual study, with meta-analysis being conducted using these study-specific thresholds. The authors obtained individual patient data from 14 studies (n = 2,196). Using a single NT-proBNP cohort threshold, the odds ratio (OR) associated with an increased NT-proBNP measurement was 3.43 (95% CI, 2.08 to 5.64). Using individual study-specific thresholds, the OR associated with an increased NT-proBNP measurement was 6.45 (95% CI, 3.98 to 10.46). In smaller studies (<100 patients) a single cohort threshold was associated with an OR of 5.4 (95% CI, 2.27 to 12.84) as compared with an OR of 14.38 (95% CI, 6.08 to 34.01) for study-specific thresholds. Post hoc identification of study-specific prognostic biomarker thresholds artificially maximizes biomarker predictive power, resulting in an amplification or overestimation during meta-analysis of these results. This effect is accentuated in small studies.

  18. Biomonitoring the Cooked Meat Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Hair: Impact of Exposure, Pigmentation and Cytochrome P450 1A2 Phenotype

    PubMed Central

    Turesky, Robert J.; Liu, Lin; Gu, Dan; Yonemori, Kim M.; White, Kami K.; Wilkens, Lynne R.; Marchand, Loic Le

    2013-01-01

    Background Hair is a promising tissue to assess exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen formed in cooked meats. However, an understanding of how dietary exposure to PhIP, cytochrome P450 1A2 activity - a key enzyme involved in PhIP metabolism, and hair pigmentation affect the level of PhIP accrued in hair is required in order to determine the reliability of the PhIP hair level as a biomarker of exposure to this carcinogen. Methods We examined the impact of PhIP exposure, cytochrome P450 1A2 activity, and hair pigmentation on the levels of PhIP accumulated in the hair of volunteers on a 4-week semi-controlled diet of cooked meat containing known quantities of PhIP. Results The amount of PhIP in hair increased, on average, 15-fold in light- and dark-haired individuals during consumption of cooked meat. PhIP levels in hair were correlated to PhIP intake (ρ = 0.53; p < 0.001), and the relationship was strengthened when PhIP levels were normalized for the melanin content of hair (ρ = 0.71; p < 0.001). However, PhIP accrual in hair was not correlated to cytochrome P450 1A2 activity, as assessed by the caffeine test, or to the levels of unmetabolized PhIP in urine, or to the metabolic ratio of the major urinary metabolite N2-(ß-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine to unmetabolized PhIP. Conclusions The employment of the PhIP hair biomarker should take hair pigmentation into account for accurate exposure assessment. Impact PhIP hair levels can serve as a biomarker in epidemiological studies investigating the association of HAAs, cooked meat and cancer risk. PMID:23329727

  19. Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

    PubMed Central

    Wang, Ming; Roberts, David L.; Paschke, Rosemary; Shea, Thomas M.; Masters, Bettie Sue Siler; Kim, Jung-Ja P.

    1997-01-01

    Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase. PMID:9237990

  20. Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc1 complex affects the mobility of the [2Fe2S] domain.

    PubMed

    Ghosh, M; Wang, Y; Ebert, C E; Vadlamuri, S; Beattie, D S

    2001-01-16

    Mutating three conserved alanine residues in the tether region of the iron-sulfur protein of the yeast cytochrome bc(1) complex resulted in 22-56% decreases in enzymatic activity [Obungu et al. (2000) Biochim. Biophys. Acta 1457, 36-44]. The activity of the cytochrome bc(1) complex isolated from A86L was decreased 60% compared to the wild-type without loss of heme or protein and without changes in the 2Fe2S cluster or proton-pumping ability. The activity of the bc(1) complex from mutant A92R was identical to the wild-type, while loss of both heme and activity was observed in the bc(1) complex isolated from mutant A90I. Computer simulations indicated that neither mutation A86L nor mutation A92R affects the alpha-helical backbone in the tether region; however, the side chain of the leucine substituted for Ala-86 interacts with the side chain of Leu-89. The Arrhenius plot for mutant A86L was apparently biphasic with a transition observed at 17-19 degrees C and an activation energy of 279.9 kJ/mol below 17 degrees C and 125.1 kJ/mol above 17 degrees C. The initial rate of cytochrome c(1) reduction was lowered 33% in mutant A86L; however, the initial rate of cytochrome b reduction was unaffected, suggesting that movement of the tether region of the iron-sulfur protein is necessary for maximum rates of enzymatic activity. Substituting a leucine for Ala-86 impedes the unwinding of the alpha-helix and hence movement of the tether.

  1. Skeletal Muscle-Specific Overexpression of PGC-1α Induces Fiber-Type Conversion through Enhanced Mitochondrial Respiration and Fatty Acid Oxidation in Mice and Pigs.

    PubMed

    Zhang, Lin; Zhou, Ying; Wu, Wangjun; Hou, Liming; Chen, Hongxing; Zuo, Bo; Xiong, Yuanzhu; Yang, Jinzeng

    2017-01-01

    Individual skeletal muscles in the animal body are heterogeneous, as each is comprised of different fiber types. Type I muscle fibers are rich with mitochondria, and have high oxidative metabolisms while type IIB fibers have few mitochondria and high glycolytic metabolic capacity. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a transcriptional co-activator that regulates mitochondrial biogenesis and respiratory function, is implicated in muscle fiber-type switching. Over-expression of PGC-1α in transgenic mice increased the proportion of red/oxidative type I fiber. During pig muscle growth, an increased number of type I fibers can give meat more red color. To explore the roles of PGC-1α in regulation of muscle fiber type conversion, we generated skeletal muscle-specific PGC-1α transgenic mice and pig. Ectopic over-expression of PGC-1α was detected in both fast and slow muscle fibers. The transgenic animals displayed a remarkable amount of red/oxidative muscle fibers in major skeletal muscle tissues. Skeletal muscles from transgenic mice and pigs have increased expression levels of oxidative fiber markers such as MHC1, MHC2x, myoglobin and Tnni1, and decreased expressions of glycolytic fiber genes (MHC2a, MHC2b, CASQ-1 and Tnni2). The genes responsible for the TCA cycle and oxidative phosphorylation, cytochrome coxidase 2 and 4, and citrate synthase were also increased in the transgenic mice and pigs. These results suggested that transgenic over-expressed PGC-1α significantly increased muscle mitochondrial biogenesis, resulting in qualitative changes from glycolytic to oxidative energy generation. The transgenic animals also had elevated levels of PDK4 and PPARγ proteins in muscle tissue, which can lead to increased glycogen deposition and fatty acid oxidation. Therefore, the results support a significant role of PGC-1α in conversion of fast glycolytic fibers to slow and oxidative fiber through enhanced mitochondrial respiration

  2. Flow Cytometric Human Leukocyte Antigen-B27 Typing with Stored Samples for Batch Testing

    PubMed Central

    Seo, Bo Young

    2013-01-01

    Background Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples. Methods We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio. Results Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used. Conclusions The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method. PMID:23667843

  3. The Trw Type IV Secretion System of Bartonella Mediates Host-Specific Adhesion to Erythrocytes

    PubMed Central

    Vayssier-Taussat, Muriel; Le Rhun, Danielle; Deng, Hong Kuan; Biville, Francis; Cescau, Sandra; Danchin, Antoine; Marignac, Geneviève; Lenaour, Evelyne; Boulouis, Henri Jean; Mavris, Maria; Arnaud, Lionel; Yang, Huanming; Wang, Jing; Quebatte, Maxime; Engel, Philipp; Saenz, Henri; Dehio, Christoph

    2010-01-01

    Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The α-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host

  4. CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

    PubMed Central

    Unterweger, Birgit; Bulach, Dieter M.; Scoble, Judith; Midgley, David J.; Greenfield, Paul; Lyras, Dena; Johanesen, Priscilla

    2016-01-01

    ABSTRACT We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida. Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101

  5. Role of Cytochrome c in Apoptosis: Increased Sensitivity to Tumor Necrosis Factor Alpha Is Associated with Respiratory Defects but Not with Lack of Cytochrome c Release▿

    PubMed Central

    Vempati, Uma D.; Diaz, Francisca; Barrientos, Antoni; Narisawa, Sonoko; Mian, Abdul M.; Millán, José Luis; Boise, Lawrence H.; Moraes, Carlos T.

    2007-01-01

    Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-α)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-α-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways. PMID:17210651

  6. Activation of caspase-dependent apoptosis by intracellular delivery of cytochrome c-based nanoparticles

    PubMed Central

    2014-01-01

    Background Cytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery. Results Cytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4’,6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles. Conclusions Our results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing

  7. Altered Cytochrome c Display Precedes Apoptotic Cell Death in Drosophila

    PubMed Central

    Varkey, Johnson; Chen, Po; Jemmerson, Ronald; Abrams, John M.

    1999-01-01

    Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases. PMID:10037791

  8. Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

    PubMed Central

    Poupin, P.; Truffaut, N.; Combourieu, B.; Besse, P.; Sancelme, M.; Veschambre, H.; Delort, A. M.

    1998-01-01

    A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ 1H nuclear magnetic resonance (1H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and 1H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. 1H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring. PMID:9435074

  9. Selective Infection of Antigen-Specific B Lymphocytes by Salmonella Mediates Bacterial Survival and Systemic Spreading of Infection

    PubMed Central

    de Wit, Jelle; Martinoli, Chiara; Zagato, Elena; Janssen, Hans; Jorritsma, Tineke; Bar-Ephraïm, Yotam E.; Rescigno, Maria; Neefjes, Jacques; van Ham, S. Marieke

    2012-01-01

    Background The bacterial pathogen Salmonella causes worldwide disease. A major route of intestinal entry involves M cells, providing access to B cell-rich Peyer’s Patches. Primary human B cells phagocytose Salmonella typhimurium upon recognition by the specific surface Ig receptor (BCR). As it is unclear how Salmonella disseminates systemically, we studied whether Salmonella can use B cells as a transport device for spreading. Methodology/Principal Findings Human primary B cells or Ramos cell line were incubated with GFP-expressing Salmonella. Intracellular survival and escape was studied in vitro by live cell imaging, flow cytometry and flow imaging. HEL-specific B cells were transferred into C57BL/6 mice and HEL-expressing Salmonella spreading in vivo was analyzed investigating mesenteric lymph nodes, spleen and blood. After phagocytosis by B cells, Salmonella survives intracellularly in a non-replicative state which is actively maintained by the B cell. Salmonella is later excreted followed by reproductive infection of other cell types. Salmonella-specific B cells thus act both as a survival niche and a reservoir for reinfection. Adoptive transfer of antigen-specific B cells before oral infection of mice showed that these B cells mediate in vivo systemic spreading of Salmonella to spleen and blood. Conclusions/Significance This is a first example of a pathogenic bacterium that abuses the antigen-specific cells of the adaptive immune system for systemic spreading for dissemination of infection. PMID:23209805

  10. Altered expression of intestinal duodenal cytochrome b and divalent metal transporter 1 might be associated with cardio-renal anemia syndrome.

    PubMed

    Naito, Yoshiro; Sawada, Hisashi; Oboshi, Makiko; Okuno, Keisuke; Yasumura, Seiki; Okuhara, Yoshitaka; Eguchi, Akiyo; Nishimura, Koichi; Soyama, Yuko; Asakura, Masanori; Ishihara, Masaharu; Tsujino, Takeshi; Masuyama, Tohru

    2017-11-01

    The interaction among heart failure (HF), chronic kidney disease (CKD), and anemia is called cardio-renal anemia syndrome. The mechanism of anemia in cardio-renal anemia syndrome is complex and remains completely unknown. We have previously reported that impaired intestinal iron transporters may contribute to the mechanism of anemia in HF using in vivo HF model rats. In this study, we assessed intestinal iron transporters in CKD model rats to investigate the association of intestinal iron transporters in the mechanism of cardio-renal anemia syndrome. CKD was induced by 5/6 nephrectomy in Sprague-Dawley rats. Sham-operated rats served as a control. After 24-week surgery, CKD rats exhibited normocytic normochromic anemia and normal serum erythropoietin levels despite of anemia. Serum iron levels were decreased in CKD rats compared with the controls. Of interest, intestinal expression of critical iron importers, such as duodenal cytochrome b (Dcyt-b) and divalent metal transporter 1 (DMT-1), was decreased in CKD rats compared with the controls. On the other hand, intestinal expression of ferroportin, an intestinal iron exporter, was not different in the control and CKD groups. Moreover, hepatic expression of hepcidin, a regulator of iron homeostasis, did not differ between the control and CKD groups. These results suggest that impaired intestinal expression of Dcyt-b and DMT-1 might be associated with the reduction of an iron uptake in CKD. Taken together, impaired these intestinal iron transporters may become a novel therapeutic target for cardio-renal anemia syndrome.

  11. The risk for type B aortic dissection in Marfan syndrome.

    PubMed

    den Hartog, Alexander W; Franken, Romy; Zwinderman, Aeilko H; Timmermans, Janneke; Scholte, Arthur J; van den Berg, Maarten P; de Waard, Vivian; Pals, Gerard; Mulder, Barbara J M; Groenink, Maarten

    2015-01-27

    Aortic dissections involving the descending aorta are a major clinical problem in patients with Marfan syndrome. The purpose of this study was to identify clinical parameters associated with type B aortic dissection and to develop a risk model to predict type B aortic dissection in patients with Marfan syndrome. Patients with the diagnosis of Marfan syndrome and magnetic resonance imaging or computed tomographic imaging of the aorta were followed for a median of 6 years for the occurrence of type B dissection or the combined end point of type B aortic dissection, distal aortic surgery, and death. A model using various clinical parameters as well as genotyping was developed to predict the risk for type B dissection in patients with Marfan syndrome. Between 1998 and 2013, 54 type B aortic dissections occurred in 600 patients with Marfan syndrome (mean age 36 ± 14 years, 52% male). Independent variables associated with type B aortic dissection were prior prophylactic aortic surgery (hazard ratio: 2.1; 95% confidence interval: 1.2 to 3.8; p = 0.010) and a proximal descending aorta diameter ≥27 mm (hazard ratio: 2.2; 95% confidence interval: 1.1 to 4.3; p = 0.020). In the risk model, the 10-year occurrence of type B aortic dissection in low-, moderate-, and high-risk patients was 6%, 19%, and 34%, respectively. Angiotensin II receptor blocker therapy was associated with fewer type B aortic dissections (hazard ratio: 0.3; 95% confidence interval: 0.1 to 0.9; p = 0.030). Patients with Marfan syndrome with prior prophylactic aortic surgery are at substantial risk for type B aortic dissection, even when the descending aorta is only slightly dilated. Angiotensin II receptor blocker therapy may be protective in the prevention of type B aortic dissections. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  12. Two reaction pathways for transformation of high potential cytochrome b559 of PS II into the intermediate potential form.

    PubMed

    Kaminskaya, Olga; Shuvalov, Vladimir A; Renger, Gernot

    2007-06-01

    This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized ("O-type" effects) or when it is reduced ("R-type" effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the "O-type" mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an "R-type" mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q(C) which interacts with Cyt b559 and is clearly not the Q(B) site. Binding of compounds L to the Q(C) site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E(m) of HP Cyt b559 with increasing concentration of L (up to 10 K(ox)(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q(C) site when HP Cyt b559 is reduced (described by K(red)(L)) induce a conversion of HP Cyt b559 to lower potential redox forms ("R-type" transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q(C) site are discussed.

  13. Human scavenger receptor class B type I is expressed with cell-specific fashion in both initial and terminal site of reverse cholesterol transport.

    PubMed

    Nakagawa-Toyama, Yumiko; Hirano, Ken-ichi; Tsujii, Ken-ichi; Nishida, Makoto; Miyagawa, Jun-ichiro; Sakai, Naohiko; Yamashita, Shizuya

    2005-11-01

    The reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which high-density lipoprotein (HDL) removes cholesterol from lipid-laden cells and delivers it to the liver. Scavenger receptor class B type I (SR-BI) is a HDL receptor in the liver and adrenal glands and is involved in the selective uptake of cholesteryl ester from HDL, which has been extensively, analyzed using rodent models. However, the expression and regulation of the human homologue of this receptor are not known yet. We previously reported that this receptor is expressed in in vitro differentiated macrophages and its expression is up-regulated by the addition of modified lipoproteins into the medium [Hirano K, Yamashita S, Nakagawa Y, et al. Expression of human scavenger receptor class B type I in cultured human monocyte-derived macrophages and atherosclerotic lesions. Circ Res 1999;85:108-16]. In order to further investigate the physiological significance of this receptor in humans, we have performed extensive immunohistochemical analyses with specimens of the liver and adrenal glands as well as arteries with different stages of atherosclerotic lesions. In human liver and adrenal glands, a positive SR-BI immunoreactivity was detected in both hepatic and adrenal parenchymal cells as well as Kupffer cells. These parenchymal cells had a strong signal on the cell surface, whereas Kupffer cells showed a heterogeneous and punctate pattern. In human aorta and coronary arteries, SR-BI was highly expressed in atherosclerotic plaques, but not in non-atherosclerotic lesions. Double immunostaining revealed that SR-BI was expressed in a subpopulation of macrophages, of which staining pattern was similar to that observed in Kupffer cells. These data clearly demonstrated that SR-BI was expressed with cell-specific fashions in both the initial and terminal step of RCT in humans. Thus, SR-BI might be physiologically relevant and have distinct tissue-specific functions.

  14. Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    PubMed Central

    Taylor, James A.; Pastrana, Cesar L.; Butterer, Annika; Pernstich, Christian; Gwynn, Emma J.; Sobott, Frank; Moreno-Herrero, Fernando; Dillingham, Mark S.

    2015-01-01

    The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed. PMID:25572315

  15. Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde metabolism: deformylation with olefin formation.

    PubMed Central

    Roberts, E S; Vaz, A D; Coon, M J

    1991-01-01

    As we have briefly described elsewhere, cytochrome P-450 catalyzes the oxidative deformylation of cyclohexane carboxaldehyde to yield cyclohexene and formic acid in a reaction believed to involve a peroxyhemiacetal-like adduct formed between the substrate and molecular oxygen-derived hydrogen peroxide. This reaction is a useful model for the demethylation reactions catalyzed by the steroidogenic P-450s, aromatase, and lanosterol demethylase. In the present study, the cytochrome P-450-catalyzed formation of olefinic products from a series of xenobiotic aldehydes has been demonstrated. Isobutyraldehyde and trimethylacetaldehyde, but not propionaldehyde, are converted to the predicted olefinic products, suggesting a requirement for branching at the alpha carbon. In addition, the four C5 aldehydes of similar hydrophobicity were compared for their ability to undergo the reaction. The straight-chain valeraldehyde gave no olefinic products with five different rabbit liver microsomal P-450 isozymes. However, increasing activity was seen with the other isomers in the order of isovaleraldehyde, 2-methylbutyraldehyde, and trimethylacetaldehyde, with all of the P-450 cytochromes. The catalytic rate with trimethylacetaldehyde is highest with antibiotic-inducible P-450 form 3A6, followed by phenobarbital-inducible form 2B4 and ethanol-inducible form 2E1. Citronellal, a beta-branched aldehyde that is found in many essential oils and is widely used as an odorant and a flavorant, was found to undergo the oxidative deformylation reaction to yield 2,6-dimethyl-1,5-heptadiene, but only with P-450 2B4. The oxidative cleavage reaction with olefin formation appears to be widespread, as judged by the variety of aldehydes that serve as substrates and of P-450 cytochromes that serve as catalysts. PMID:1924356

  16. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen speciesmore » compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.« less

  17. Cytochrome P4502D6(193-212): a new immunodominant epitope and target of virus/self cross-reactivity in liver kidney microsomal autoantibody type 1-positive liver disease.

    PubMed

    Kerkar, Nanda; Choudhuri, Kaushik; Ma, Yun; Mahmoud, Ayman; Bogdanos, Dimitrios P; Muratori, Luigi; Bianchi, Francesco; Williams, Roger; Mieli-Vergani, Giorgina; Vergani, Diego

    2003-02-01

    Cytochrome P4502D6 (CYP2D6), target of liver kidney microsomal autoantibody type 1 (LKM1), characterizes autoimmune hepatitis type 2 (AIH2) but is also found in patients with chronic hepatitis C virus (HCV) infection. To provide a complete linear epitope B cell map of CYP2D6, we tested peptides spanning the entire sequence of CYP2D6. In addition to confirming previously described antigenic sites, we identified four new epitopes (193-212, 238-257, 268-287, and 478-497). CYP2D6(193-212) is immunodominant and was the target of 12 of 13 (93%) patients with AIH2 and 5 of 10 (50%) HCV/LKM1-positive patients. Because LKM1 is present in both AIH2 and a viral infection, we tested whether Abs to CYP2D6(193-212) arise through cross-reactive immunity between virus and self. We identified a hexameric sequence "RLLDLA" sharing 5 of 6 aa with "RLLDLS" of HCV(2985-2990) and all 6 aa with CMV(130-135). Of 17 CYP2D6(193-212)-reactive sera, 11 (7 AIH and 4 HCV) reacted by ELISA with the HCV homologue, 8 (5 AIH and 3 HCV) with the CMV homologue, and 8 (5 AIH and 3 HCV) showed double reactivity. Autoantibody binding to CYP2D6(193-212) was inhibited by preincubation with HCV(2977-2996) or CMV(121-140). Recombinant HCV-nonstructural protein 5 and CMV-UL98 proteins also inhibited Ab binding to CYP2D6(193-212). Affinity-purified CYP2D6(193-212)-specific Ab inhibited the metabolic activity of CYP2D6. The demonstrated similarity and cross-reactivity between CYP2D6(193-212) and two unrelated viruses suggests that multiple exposure to viruses mimicking self may represent an important pathway to the development of autoimmunity.

  18. Granzyme B ELISPOT assay to measure influenza-specific cellular immunity.

    PubMed

    Salk, Hannah M; Haralambieva, Iana H; Ovsyannikova, Inna G; Goergen, Krista M; Poland, Gregory A

    2013-12-15

    The immunogenicity and efficacy of influenza vaccination are markedly lower in the elderly. Granzyme B (GrzB), quantified in fresh cell lysates, has been suggested to be a marker of cytotoxic T lymphocyte (CTL) response and a predictor of influenza illness among vaccinated older individuals. We have developed an influenza-specific GrzB ELISPOT assay using cryopreserved PBMCs. This method was tested on 106 healthy older subjects (ages 50-74) at baseline (Day 0) and three additional time points post-vaccination (Day 3, Day 28, Day 75) with influenza A/H1N1-containing vaccine. No significant difference was seen in GrzB response between any of the time points, although influenza-specific GrzB response appears to be elevated at all post-vaccination time points. There was no correlation between GrzB response and hemagglutination inhibition (HAI) titers, indicating no relationship between the cytolytic activity and humoral antibody levels in this cohort. Additionally, a significant negative correlation between GrzB response and age was observed. These results reveal a reduction in influenza-specific GrzB response as one ages. In conclusion, we have developed and optimized an influenza-specific ELISPOT assay for use with frozen cells to quantify the CTL-specific serine protease GrzB, as a measure of cellular immunity after influenza vaccination. © 2013.

  19. [Differentiation of influenza (Flu) type A, type B, and respiratory syncytial virus (RSV) by QuickNavi™-Flu+RSV].

    PubMed

    Kohiyama, Risa; Miyazawa, Takashi; Shibano, Nobuko; Inano, Koichi

    2014-01-01

    Because it is not easy to differentiate Influenza virus (Flu) from RS virus (RSV) just by clinical symptoms, to accurately diagnose those viruses in conjunction with patient's clinical symptoms, rapid diagnostic kits has been used separately for each of those viruses. In our new study, we have developed a new rapid diagnostic kit, QuickNavi™-Flu+RSV. The kit can detect Flu A, Flu B, and RSV antigens with a single sample collection and an assay. Total of 2,873 cases (including nasopharyngeal swabs and nasopharyngeal aspirates specimens) in 2010/2011 and 2011/2012 seasons were evaluated with QuickNavi™-Flu+RSV and a commercially available kit. Sensitivity, specificity, and accuracy of Flu type A, type B, and RSV were above 95% when compared to commercially available kits (QuickNavi™-Flu and QuickNavi™-RSV) and considered to be equivalent to the commercially available kits. In 2011/2012 season, RSV infections increased prior to Flu season and continued during the peak of the Flu season. The kit can contribute to accurate diagnosis of Flu and RSV infections since co-infection cases have also been reported during the 2011/2012 season. QuickNavi™-Flu+RSV is useful for differential diagnosis of respiratory infectious diseases since it can detect Flu type A, type B, and RSV virus antigens with a single sample collection.

  20. Inkjet-printed selective microfluidic biosensor using CNTs functionalized by cytochrome P450 enzyme

    NASA Astrophysics Data System (ADS)

    Krivec, Matic; Leitner, Raimund; Überall, Florian; Hochleitner, Johannes

    2017-05-01

    An additive manufacturing concept, consisting of 3D photopolymer printing and Ag nanoparticle printing, was investigated for the construction of a microfluidic biosensor based on immobilized cytochrome P450 enzyme. An acylate-type microfluidic chamber composed of two parts, i.e. chamber-housing and chamber-lid was printed with a polyjet 3D printer. A 3-electrode sensor structure was inkjet-printed on the lid using a combination of Ag and graphene printing. The working electrode was covered with carbon nanotubes by drop-casting and immobilized with cytochrome P450 2D6 enzyme. The microfluidic sensor shows a significant response to a test xenobiotic, i.e. dextromethorphan; the cyclic voltammetrical measurements show a corresponding oxidation peak at 0.4 V with around 5 μM detection limit.

  1. Cardiovascular responses in type A and type B men to a series of stressors.

    PubMed

    Ward, M M; Chesney, M A; Swan, G E; Black, G W; Parker, S D; Rosenman, R H

    1986-02-01

    Fifty-six healthy adult males were administered the Type A Structured Interview and assessed as exhibiting either Type A (N = 42) or Type B (N = 14) behavior pattern. They were monitored for systolic (SBP) and diastolic blood pressure (DBP) and heart rate (HR) responses during a series of six challenging tasks: Mental Arithmetic, Hypothesis Testing, Reaction Time, Video Game, Handgrip, and Cold Pressor. The results indicated that Type A subjects exhibited greater cardiovascular responses than did Type B subjects during some (Hypothesis Testing, Reaction Time, Video Game and Mental Arithmetic) but not all (Handgrip and Cold Pressor) of the tasks. These results are discussed in terms of previously reported findings on conditions that do and do not produce differences in Type A/B cardiovascular stress responses.

  2. Dose-specific efficacy of Haemophilus influenzae type b conjugate vaccines: a systematic review and meta-analysis of controlled clinical trials

    PubMed Central

    GRIFFITHS, U. K.; CLARK, A.; GESSNER, B.; MINERS, A.; SANDERSON, C.; SEDYANINGSIH, E. R.; MULHOLLAND, K. E.

    2012-01-01

    SUMMARY Global coverage of infant Haemophilus influenzae type b (Hib) vaccination has increased considerably during the past decade, partly due to GAVI Alliance donations of the vaccine to low-income countries. In settings where large numbers of children receive only one or two vaccine doses rather than the recommended three doses, dose-specific efficacy estimates are needed to predict impact. The objective of this meta-analysis is to determine Hib vaccine efficacy against different clinical outcomes after receiving one, two or three doses of vaccine. Studies were eligible for inclusion if a prospective, controlled design had been used to evaluate commercially available Hib conjugate vaccines. Eight studies were included. Pooled vaccine efficacies against invasive Hib disease after one, two or three doses of vaccine were 59%, 92% and 93%, respectively. The meta-analysis provides robust estimates for use in decision-analytical models designed to predict the impact of Hib vaccine. PMID:22583474

  3. 39 CFR 3010.13 - Proceedings for Type 1-A and Type 1-B rate adjustment filings.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 39 Postal Service 1 2010-07-01 2010-07-01 false Proceedings for Type 1-A and Type 1-B rate... (Type 1-A and 1-B Rate Adjustments) § 3010.13 Proceedings for Type 1-A and Type 1-B rate adjustment... include: (1) The general nature of the proceeding; (2) A reference to legal authority to which the...

  4. Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)

    ClinicalTrials.gov

    2017-08-15

    Depression; Depressive Disorder; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Intermediate Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  5. Purification of preparative quantities of group B Streptococcus type III oligosaccharides.

    PubMed

    Paoletti, L C; Johnson, K D

    1995-06-30

    Many bacterial capsular polysaccharides are regularly repeating units of oligosaccharides. Bacterial oligosaccharides have been used in neoglycoconjugate vaccines and as reagents in the study of specific antibody binding. Unfortunately, separation methods have not been adequate for the purification of preparative quantities of bacterial oligosaccharides. Here we describe a size-exclusion procedure that resulted in the resolution of group B Streptococcus type III oligosaccharides composed of 4-25 sugars.

  6. Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.

    PubMed Central

    Bonkovsky, H L; Sinclair, J F; Healey, J F; Sinclair, P R; Smith, E L

    1984-01-01

    The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem

  7. A cadmium-transporting P1B-type ATPase in yeast Saccharomyces cerevisiae.

    PubMed

    Adle, David J; Sinani, Devis; Kim, Heejeong; Lee, Jaekwon

    2007-01-12

    Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense mutation in a predicted ATP-binding residue conserved in P1B-type ATPases. Consistent with a previous report that identifies an equivalent mutation in a copper-transporting P1B-type ATPase of a Wilson disease patient, the PCA1 allele found in laboratory yeast strains is nonfunctional. Overexpression or deletion of the functional allele in yeast demonstrates that PCA1 is a cadmium efflux pump. Cadmium as well as copper and silver, but not other metals examined, dramatically increase PCA1 protein expression through post-transcriptional regulation and promote subcellular localization to the plasma membrane. Our study has revealed a novel metal detoxification mechanism in yeast mediated by a P1B-type ATPase that is unique in structure, substrate specificity, and mode of regulation.

  8. Structure at 1.3 A resolution of Rhodothermus marinus caa(3) cytochrome c domain.

    PubMed

    Srinivasan, Vasundara; Rajendran, Chitra; Sousa, Filipa L; Melo, Ana M P; Saraiva, Lígia M; Pereira, Manuela M; Santana, Margarida; Teixeira, Miguel; Michel, Hartmut

    2005-02-04

    The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold. This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase. The R.marinus caa(3) uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.

  9. Evaluation of pharmacokinetic and pharmacodynamic profiles of liposomes for the cell type-specific delivery of small molecule drugs.

    PubMed

    Dasa, Siva Sai Krishna; Suzuki, Ryo; Mugler, Emily; Chen, Lanlin; Jansson-Löfmark, Rasmus; Michaëlsson, Erik; Lindfors, Lennart; Klibanov, Alexander L; French, Brent A; Kelly, Kimberly A

    2017-11-01

    Liposome-based drug formulations represent an exciting avenue of research as they increase efficacy to toxicity ratios. Current formulations rely on passive accumulation to the disease site where drug is taken up by the cells. Ligand mediated targeting increases the net accumulation of liposomes, however, an unexplored benefit is to potentially refine pharmacodynamics (PD) of a drug specifically to different cell types within diseased tissue. As a model system, we engineered cardiomyocyte- (I-1) and endothelial-targeted (B-40) liposomes to carry a VEGFR2 inhibitor (PTK787), and examined the effect of cell type-specific delivery on both pharmacokinetics (PK) and PD. Neovascularization in post-myocardial infarction was significantly reduced by B-40 liposomes loaded with PTK787 as compared to animals injected with I-1 liposomes, and profoundly more as compared to free PTK787. This study thus shows that the intraorgan targeting of drugs through cell type-specific delivery holds substantial promise towards lowering the minimal efficacious dose administered systemically. Published by Elsevier Inc.

  10. Properties and structure of a low-potential, penta-heme cytochrome c 552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum.

    PubMed

    Chen, Jing-Hua; Yu, Long-Jiang; Boussac, Alain; Wang-Otomo, Zheng-Yu; Kuang, Tingyun; Shen, Jian-Ren

    2018-04-24

    The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c 552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c 552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.

  11. Analysis of the vhoGAC and vhtGAC operons from Methanosarcina mazei strain Gö1, both encoding a membrane-bound hydrogenase and a cytochrome b.

    PubMed

    Deppenmeier, U; Blaut, M; Lentes, S; Herzberg, C; Gottschalk, G

    1995-01-15

    DNA encompassing the structural genes of two membrane-bound hydrogenases from Methanosarcina mazei Gö1 was cloned and sequenced. The genes, arranged in the order vhoG and vhoA as well as vhtG and vhtA, were identified as those encoding the small and the large subunits of the NiFe hydrogenases [Deppenmeier, U., Blaut, M., Schmidt, B. & Gottschalk, G. (1992) Arch. Microbiol. 157, 505-511]. Northern-blot analysis revealed that the structural genes formed part of two operons, both containing one additional open reading frame (vhoC and vhtC) which codes for a cytochrome b. This conclusion was drawn from the homology of the deduced N-terminal amino acid sequences of vhoC and vhtC and the N-terminus of a 27-kDa cytochrome isolated from Ms. mazei C16. VhoC and VhtC contain four tentative hydrophobic segments which might span the cytoplasmic membrane. Hydropathy plots suggest that His23 and His50 are involved in heme coordination. The comparison of the sequencing data of vhoG and vhtG with the experimentally determined N-terminus of the small subunit indicate the presence of a 48-amino-acid leader peptide in front of the polypeptides. VhoA and VhtA contained the conserved sequence DPCXXC in the C-terminal region, which excludes the presence of a selenocysteine residue in these hydrogenases. Promoter sequences were found upstream of vhoG and vhtG, respectively. Downstream of vhoC, a putative terminator sequence was identified. Alignments of the deduced amino acid sequences of the gene clusters vhoGAC and vhtGAC showed 92-97% identity. Only the C-termini of VhoC and VhtC were not similar.

  12. B-Lymphocytes Expressing an Ig Specificity Recognizing the Pancreatic β-Cell Autoantigen Peripherin Are Potent Contributors to Type 1 Diabetes Development in NOD Mice

    PubMed Central

    Leeth, Caroline M.; Racine, Jeremy; Chapman, Harold D.; Arpa, Berta; Carrillo, Jorge; Carrascal, Jorge; Wang, Qiming; Ratiu, Jeremy; Egia-Mendikute, Leire; Rosell-Mases, Estela; Stratmann, Thomas

    2016-01-01

    Although the autoimmune destruction of pancreatic β-cells underlying type 1 diabetes (T1D) development is ultimately mediated by T cells in NOD mice and also likely in humans, B cells play an additional key pathogenic role. It appears that the expression of plasma membrane–bound Ig molecules that efficiently capture β-cell antigens allows autoreactive B cells that bypass normal tolerance induction processes to be the subset of antigen-presenting cells most efficiently activating diabetogenic T cells. NOD mice transgenically expressing Ig molecules recognizing antigens that are (insulin) or are not (hen egg lysozyme [HEL]) expressed by β-cells have proven useful in dissecting the developmental basis of diabetogenic B cells. However, these transgenic Ig specificities were originally selected for their ability to recognize insulin or HEL as foreign, rather than autoantigens. Thus, we generated and characterized NOD mice transgenically expressing an Ig molecule representative of a large proportion of naturally occurring islet-infiltrating B cells in NOD mice recognizing the neuronal antigen peripherin. Transgenic peripherin-autoreactive B cells infiltrate NOD pancreatic islets, acquire an activated proliferative phenotype, and potently support accelerated T1D development. These results support the concept of neuronal autoimmunity as a pathogenic feature of T1D, and targeting such responses could ultimately provide an effective disease intervention approach. PMID:26961115

  13. Insights into Korean red fox (Vulpes vulpes) based on mitochondrial cytochrome b sequence variation in East Asia.

    PubMed

    Yu, Jeong-Nam; Han, Sang-Hoon; Kim, Bang-Hwan; Kryukov, Alexey P; Kim, Soonok; Lee, Byoung-Yoon; Kwak, Myounghai

    2012-11-01

    The red fox (Vulpes vulpes) is the most widely distributed terrestrial carnivore in the world, occurring throughout most of North America, Europe, Asia, and North Africa. In South Korea, however, this species has been drastically reduced due to habitat loss and poaching. Consequently, it is classified as an endangered species in Korea. As a first step of a planned red fox restoration project, preserved red fox museum specimens were used to determine the genetic status of red foxes that had previously inhabited South Korea against red foxes from neighboring countries. Total eighty three mtDNA cytochrome b sequences, including 22 newly obtained East Asian red fox sequences and worldwide red fox sequences from NCBI, were clustered into three clades (i.e., I, II, and III) based on haplotype network and neighbor-joining trees. The mean genetic distance between clades was 2.0%. Clade III contained South Korean and other East Asian samples in addition to Eurasian and North Pacific individuals. In clade III, South Korean individuals were separated into two lineages of Eurasian and North Pacific groups, showing unclear phylogeographic structuring and admixture. This suggests that South Korean red fox populations may have been composed of individuals from these two different genetic lineages.

  14. DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, C.-C.; Lii, C.-K.; Liu, K.-L.

    The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation bymore » n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 {mu}M arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.« less

  15. Electrostatic redesign of the [myoglobin, cytochrome b5] interface to create a well-defined docked complex with rapid interprotein electron transfer.

    PubMed

    Xiong, Peng; Nocek, Judith M; Griffin, Amanda K K; Wang, Jingyun; Hoffman, Brian M

    2009-05-27

    Cyt b(5) is the electron-carrier "repair" protein that reduces met-Mb and met-Hb to their O(2)-carrying ferroheme forms. Studies of electron transfer (ET) between Mb and cyt b(5) revealed that they react on a "Dynamic Docking" (DD) energy landscape on which binding and reactivity are uncoupled: binding is weak and involves an ensemble of nearly isoenergetic configurations, only a few of which are reactive; those few contribute negligibly to binding. We set the task of redesigning the surface of Mb so that its reaction with cyt b(5) instead would occur on a conventional "simple docking" (SD) energy landscape, on which a complex exhibits a well-defined (set of) reactive binding configuration(s), with binding and reactivity thus no longer being decoupled. We prepared a myoglobin (Mb) triple mutant (D44K/D60K/E85K; Mb(+6)) substituted with Zn-deuteroporphyrin and monitored cytochrome b(5) (cyt b(5)) binding and electron transfer (ET) quenching of the (3)ZnMb(+6) triplet state. In contrast, to Mb(WT), the three charge reversals around the "front-face" heme edge of Mb(+6) have directed cyt b(5) to a surface area of Mb adjacent to its heme, created a well-defined, most-stable structure that supports good ET pathways, and apparently coupled binding and ET: both K(a) and k(et) are increased by the same factor of approximately 2 x 10(2), creating a complex that exhibits a large ET rate constant, k(et) = 10(6 1) s(-1), and is in slow exchange (k(off) < k(et)). In short, these mutations indeed appear to have created the sought-for conversion from DD to simple docking (SD) energy landscapes.

  16. Cytochrome P450 diversity in the tree of life.

    PubMed

    Nelson, David R

    2018-01-01

    Sequencing in all areas of the tree of life has produced >300,000 cytochrome P450 (CYP) sequences that have been mined and collected. Nomenclature has been assigned to >41,000 CYP sequences and the majority of the remainder has been sorted by BLAST searches into clans, families and subfamilies in preparation for naming. The P450 sequence space is being systematically explored and filled in. Well-studied groups like vertebrates are covered in greater depth while new insights are being added into uncharted territories like horseshoe crab (Limulus polyphemus), tardigrades (Hypsibius dujardini), velvet worm (Euperipatoides_rowelli), and basal land plants like hornworts, liverworts and mosses. CYPs from the fungi, one of the most diverse groups, are being explored and organized as nearly 800 fungal species are now sequenced. The CYP clan structure in fungi is emerging with 805 CYP families sorting into 32 CYP clans. >3000 bacterial sequences are named, mostly from terrestrial or freshwater sources. Of 18,379 bacterial sequences downloaded from the CYPED database, all are >43% identical to named CYPs. Therefore, they fit in the 602 named P450 prokaryotic families. Diversity in this group is becoming saturated, however 25% of 3305 seawater bacterial P450s did not match known P450 families, indicating marine bacterial CYPs are not as well sampled as land/freshwater based bacterial CYPs. Future sequencing plans of the Genome 10K project, i5k and GIGA (Global Invertebrate Genomics Alliance) are expected to produce more than one million cytochrome P450 sequences by 2020. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Induction of cytochrome P450 enzymes in rat liver by two conazoles, myclobutanil and triadimefon.

    PubMed

    Sun, G; Grindstaff, R D; Thai, S F; Lambert, G R; Tully, D B; Dix, D J; Nesnow, S

    2007-02-01

    This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon, on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague Dawley rats. Rats were dosed with the conazoles at three dose levels by gavage for 14 days: myclobutanil (150, 75, and 10mgkg(-1) body weight day(-1); triadimefon (115, 50, and 10 mg kg(-1) body weight day-'), which included their maximum tolerated dose levels (MTD). Both myclobutanil and triadimefon significantly induced pentoxyresorufin O-depentylase activities at their MTD levels: myclobutanil, 8.1-fold at 150mgkg(-1) body weight day- ; and triadimefon, 18.5-fold at 115mgkg(-1) body weight day-'. Benzyloxyresorufin O-debenzylase activities were similarly increased: myclobutanil, 13.3-fold; triadimefon, 27.7-fold. Quantitative real-time reverse-transcription polymerase chain reaction assays were used to characterize the mRNA expression of specific CYP genes induced by these two conazoles. Myclobutanil and triadimefon treatment at their MTD levels significantly increased rat hepatic mRNA expression of CYP2B1 (14.3- and 54.6-fold), CYP3A23/3A1 (2.2- and 7.3-fold), and CYP3A2 (1.5- and 1.7-fold). Western immunoblots of rat hepatic microsomal proteins identified significantly increased levels of CYP isoforms after myclobutanil or triadimefon treatment at their MTD levels: CYP2BI/2 (4.8- and 5.3-fold), and CYP3A1 (2.2- and 2.9-fold). Triadimefon also increased CYP3A2 immunoreactive protein levels 1.8-fold. These results indicate that triadimefon and myclobutanil, like other triazole-containing conazoles, induced CYP2B and CYP3A families of cytochromes in rat liver.

  18. Applicability of Type A/B alcohol dependence in the general population.

    PubMed

    Tam, Tammy W; Mulia, Nina; Schmidt, Laura A

    2014-05-01

    This study examined the concurrent and predictive validity of Type A/B alcohol dependence in the general population-a typology developed in clinical populations to gauge severity of dependence. Data were drawn from Waves 1 and 2 of the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC). The sample included 1,172 alcohol-dependent drinkers at baseline who were reinterviewed three years later. Latent class analysis was used to derive Type A/B classification using variables replicating the original Type A/B typology. Predictive validity of the Type A/B classification was assessed by multivariable linear and logistic regressions. A two-class solution consistent with Babor's original Type A/B typology adequately fit the data. Type B alcoholics in the general population, compared to Type As, had higher alcohol severity and more co-occurring drug, mental, and physical health problems. In the absence of treatment services utilization, Type B drinkers had two times the odds of being alcohol dependent three years later. Among those who utilized alcohol treatment services, Type B membership was predictive of heavy drinking and drug dependence, but not alcohol dependence, three years later. Findings suggest that Type A/B classification is both generalizable to, and valid within, the US general population of alcohol dependent drinkers. Results highlight the value of treatment for mitigating the persistence of dependence among Type B alcoholics in the general population. Screening for markers of vulnerability to Type B dependence could be of clinical value for health care providers to determine appropriate intervention. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

    PubMed Central

    Glerum, M; Robinson, B H; Spratt, C; Wilson, J; Patrick, D

    1987-01-01

    Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient. PMID:2821802

  20. Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

    PubMed

    Szczesna-Skorupa, E; Chen, C D; Rogers, S; Kemper, B

    1998-12-08

    Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.