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Sample records for radiotracer targeting cytochrome

  1. (68) Ga-labeled Ciprofloxacin Conjugates as Radiotracers for Targeting Bacterial Infection.

    PubMed

    Satpati, Drishty; Arjun, Chanda; Krishnamohan, Repaka; Samuel, Grace; Banerjee, Sharmila

    2016-05-01

    With an aim of developing a bacteria-specific molecular imaging agent, ciprofloxacin has been modified with a propylamine spacer and linked to two common bifunctional chelators, p-SCN-Bz-DOTA and p-SCN-Bz-NOTA. The two ciprofloxacin conjugates, CP-PA-SCN-Bz-DOTA (1) and CP-PA-SCN-Bz-NOTA (2), were radiolabeled with (68)Ga in >90% radiochemical yield and were moderately stable in vitro for 4 h. The efficacy of (68)Ga-1 and (68)Ga-2 has been investigated in vitro in Staphylococcus aureus cells where bacterial binding of the radiotracers (0.9-1.0% for (68)Ga-1 and 1.6-2.3% for (68)Ga-2) could not be blocked in the presence of excess amount of unlabeled ciprofloxacin. However, uptake of radiotracers in live bacterial cells was significantly higher (p < 0.01) than that in non-viable bacterial cells. Bacterial infection targeting efficacy of (68)Ga-1 and (68)Ga-2 was tested in vivo in rats where the infected muscle-to-inflamed muscle ((68)Ga-1: 2 ± 0.2, (68)Ga-2: 3 ± 0.5) and infected muscle-to-normal muscle ratios ((68)Ga-1: 3 ± 0.4, (68)Ga-2: 6.6 ± 0.8) were found to improve at 120 min p.i. Fast blood clearance and renal excretion was observed for both the radiotracers. The two (68)Ga-labeled infection targeting radiotracers could discriminate between bacterial infection and inflammation in vivo and are worthy of further detailed investigation as infection imaging agents at the clinical level. PMID:26647765

  2. Chemistry and biology of radiotracers that target changes in sympathetic and parasympathetic nervous systems in heart disease.

    PubMed

    Eckelman, William C; Dilsizian, Vasken

    2015-06-01

    Following the discovery of the sympathetic and parasympathetic nervous system, numerous adrenoceptor drugs were radiolabeled and potent radioligands were prepared in order to image the β-adrenergic and the muscarinic systems. But the greatest effort has been in preparing noradrenaline analogs, such as norepinephrine, (11)C-metahydroxyephedrine, and (123)I-metaiodobenzylguanidine that measure cardiac sympathetic nerve varicosities. Given the technical and clinical challenges in designing and validating targeted adrenoceptor-binding radiotracers, namely the heavily weighted flow dependence and relatively low target-to-background ratio, both requiring complicated mathematic analysis, and the inability of targeted adrenoceptor radioligands to have an impact on clinical care of heart disease, the emphasis has been on radioligands monitoring the norepinephrine pathway. The chemistry and biology of such radiotracers, and the clinical and prognostic impact of these innervation imaging studies in patients with heart disease, are examined.

  3. Lansoprazole is an antituberculous prodrug targeting cytochrome bc1

    PubMed Central

    Rybniker, Jan; Vocat, Anthony; Sala, Claudia; Busso, Philippe; Pojer, Florence; Benjak, Andrej; Cole, Stewart T.

    2015-01-01

    Better antibiotics capable of killing multi-drug-resistant Mycobacterium tuberculosis are urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity against M. tuberculosis. Ex vivo pharmacokinetics and target identification studies reveal that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochrome bc1 inhibitors is highly active against drug-resistant clinical isolates and spares the human H+K+-ATPase thus providing excellent opportunities for targeting the major pathogen M. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens. PMID:26158909

  4. Preclinical Comparative Study of (68)Ga-Labeled DOTA, NOTA, and HBED-CC Chelated Radiotracers for Targeting PSMA.

    PubMed

    Ray Banerjee, Sangeeta; Chen, Zhengping; Pullambhatla, Mrudula; Lisok, Ala; Chen, Jian; Mease, Ronnie C; Pomper, Martin G

    2016-06-15

    (68)Ga-labeled, low-molecular-weight imaging agents that target the prostate-specific membrane antigen (PSMA) are increasingly used clinically to detect prostate and other cancers with positron emission tomography (PET). The goal of this study was to compare the pharmacokinetics of three PSMA-targeted radiotracers: (68)Ga-1, using DOTA-monoamide as the chelating agent; (68)Ga-2, containing the macrocyclic chelating agent p-SCN-Bn-NOTA; and (68)Ga-DKFZ-PSMA-11, currently in clinical trials, which uses the acyclic chelating agent, HBED-CC. The PSMA-targeting scaffold for all three agents utilized a similar Glu-urea-Lys-linker construct. Each radiotracer enabled visualization of PSMA+ PC3 PIP tumor, kidney, and urinary bladder as early as 15 min post-injection using small animal PET/computed tomography (PET/CT). (68)Ga-2 demonstrated the fastest rate of clearance from all tissues in this series and displayed higher uptake in PSMA+ PC3 PIP tumor compared to (68)Ga-1 at 1 h post-injection. There was no significant difference in PSMA+ PC3 PIP tumor uptake for the three agents at 2 and 3 h post-injection. (68)Ga-DKFZ-PSMA-11 demonstrated the highest uptake and retention in normal tissues, including kidney, blood, spleen, and salivary glands and PSMA-negative PC3 flu tumors up to 3 h post-injection. In this preclinical evaluation (68)Ga-2 had the most advantageous characteristics for PSMA-targeted PET imaging. PMID:27076393

  5. 64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria.

    PubMed

    Zhou, Yang; Kim, Young-Seung; Yan, Xin; Jacobson, Orit; Chen, Xiaoyuan; Liu, Shuang

    2011-08-01

    Enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of (64)Cu-labeled Lissamine rhodamine B (LRB), (64)Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetamido)ethyl)sulfamoyl)benzenesulfonate) as a new radiotracer for imaging tumors in athymic nude mice bearing U87MG human glioma xenografts by positron emission tomography (PET). We also explored its localization mechanism using Cu(DOTA-LRB) as the fluorescent probe in both the U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that (64)Cu(DOTA-LRB) had the highest tumor uptake (6.54 ± 1.50, 6.91 ± 1.26, 5.68 ± 1.13, 7.58 ± 1.96, and 5.14 ± 1.50%ID/g at 0.5, 1, 2, 4, and 24 h postinjection, respectively) among many (64)Cu-labeled organic cations evaluated in the same animal model. The cellular staining study indicated that Cu(DOTA-LRB) was able to localize in mitochondria of U87MG glioma cells due to the enhanced negative mitochondrial potential. This statement is completely supported by the results from decoupling experiment with carbonylcyanide-m-chlorophenylhydrazone (CCCP). MicroPET data showed that the U87MG glioma tumors were clearly visualized as early as 30 min postinjection with (64)Cu(DOTA-LRB). (64)Cu(DOTA-LRB) remained stable during renal excretion, but underwent extensive degradation during hepatobiliary excretion. On the basis of the results from this study, it was concluded that (64)Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors.

  6. Evaluation of 64Cu-labeled acridinium cation: a PET radiotracer targeting tumor mitochondria.

    PubMed

    Zhou, Yang; Kim, Young-Seung; Shi, Jiyun; Jacobson, Orit; Chen, Xiaoyuan; Liu, Shuang

    2011-04-20

    This report presents the synthesis and evaluation of (64)Cu(DO3A-xy-ACR) (DO3A-xy-ACR = 2,6-bis(dimethylamino)-10-(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)acridin-10-ium) as a radiotracer for imaging tumors in athymic nude mice bearing U87MG glioma xenografts by PET (positron emission tomography). The biodistribution data suggested that (64)Cu(DO3A-xy-ACR) was excreted mainly through the renal system with >65% of injected radioactivity being recovered from urine samples at 1 h postinjection (p.i.). The tumor uptake of (64)Cu(DO3A-xy-ACR) was 1.07 ± 0.23, 1.58 ± 0.55, 2.71 ± 0.66, 3.47 ± 1.19, and 3.52 ± 1.72%ID/g at 0.5, 1, 2, 4, and 24 h p.i., respectively. (64)Cu(DO3A-xy-ACR) had very high liver uptake (31.90 ± 3.98, 24.95 ± 5.64, 15.20 ± 4.29, 14.09 ± 6.82, and 8.18 ± 1.27%ID/g at 0.5, 1, 2, 4, and 24 h p.i., respectively) with low tumor/liver ratios. MicroPET studies showed that the tumors were clearly visualized as early as 30 min p.i. in the glioma-bearing mouse administered with (64)Cu(DO3A-xy-ACR). The high liver radioactivity accumulation was also seen. (64)Cu(DO3A-xy-ACR) had a relatively high metabolic stability during excretion via both renal and hepatobiliary routes, but it was completely decomposed in the liver homogenate. We explored the localization mechanism of Cu(DO3A-xy-ACR) using both U87MG human glioma and the cultured primary U87MG glioma cells. The results from the cellular staining assays showed that (64)Cu(DO3A-xy-ACR) is able to localize in the mitochondria of living U87MG glioma cells due to the enhanced negative mitochondrial potential as compared to normal cells. Although (64)Cu(DO3A-xy-ACR) is not an ideal PET radiotracer for tumor imaging due to its high liver uptake, the results from this study strongly suggest that (64)Cu-labeled acridinium cations are indeed able to localize in the energized mitochondria of tumor cells.

  7. PET radiotracer [18F]-P6 selectively targeting COX-1 as a novel biomarker in ovarian cancer: Preliminary investigation

    PubMed Central

    Uddin, Jashim; Vitale, Paola; Panella, Andrea; Crews, Brenda C.; Daniel, Cristina K.; Ghebreselasie, Kebreab; Nickels, Mike; Tantawy, Mohammed N.; Manning, H. Charles; Marnett, Lawrence J.; Scilimati, Antonio

    2015-01-01

    Cyclooxygenase-1 (COX-1), but not COX-2, is expressed at high levels in the early stages of human epithelial ovarian cancer where it seems to play a key role in cancer onset and progression. As a consequence, COX-1 is an ideal biomarker for early ovarian cancer detection. A series of novel fluorinated COX-1-targeted imaging agents derived from P6 was developed by using a highly selective COX-1 inhibitor as a lead compound. Among these new compounds, designed by structural modification of P6, 3-(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-phenylisoxazole ([18/19F]-P6) is the most promising derivative [IC50 = 2.0 μM (purified oCOX-1) and 1.37 μM (hOVCAR-3 cell COX-1)]. Its tosylate precursor was also prepared and, a method for radio[18F]chemistry was developed and optimized. The radiochemistry was carried out using a carrier-free K18F/Kryptofix 2.2.2 complex, that afforded [18F]-P6 in good radio-chemical yield (18%) and high purity (>95%). In vivo PET/CT imaging data showed that the radiotracer [18F]-P6 was selectively taken up by COX-1-expressing ovarian carcinoma (OVCAR 3) tumor xenografts as compared with the normal leg muscle. Our results suggest that [18F]-P6 might be an useful radiotracer in preclinical and clinical settings for in vivo PET-CT imaging of tissues that express elevated levels of COX-1. PMID:24832612

  8. Optimizing tumor targeting of the lipophilic EGFR-binding radiotracer SKI243 using a liposomal nanoparticle delivery system

    PubMed Central

    Pillarsetty, Nagavarakishore; Glekas, Athanasios; Punzalan, Blesida; Longo, Valerie; Gönen, Mithat; Zanzonico, Pat; Smith-Jones, Peter

    2015-01-01

    Positron emission tomography (PET) of epidermal growth factor receptor (EGFR) kinase-specific radiolabeled tracers could provide a means for non-invasively characterizing EGFR expression and signaling activity in patients' tumors before, during, and after therapy with EGFR inhibitors. Towards this goal, our group has developed PET tracers which irreversibly bind to EGFR. However, tumor uptake is relatively low because of both the lipophilicity of such tracers (e.g. the morpholino-[124I]-IPQA [SKI212243]), with octanol-to-water partition coefficients of up to 4, and a short dwell time in the blood and significant hepatobiliary clearance and intestinal reuptake. Liposomal nanoparticle delivery systems may favorably alter the pharmacokinetic profile and improve tumor targeting of highly lipophilic but otherwise promising cancer imaging tracers, such as the EGFR inhibitor SKI243. SKI243 is therefore an interesting model molecule for incorporation into lipid-based nanoparticles, as it would not only improve their solubility but also increase the circulation time, availability and, potentially, targeting of tumors. In the current study, we compared the pharmacokinetics and tumor targeting of the bare EGFR kinase-targeting radiotracer SKI212243 (SKI243) with that of the same tracer embedded in liposomes. SKI243 and liposomal SKI243 are both taken up by tumor xenografts but liposomal SKI243 remained in the blood longer and consequently exhibited a 3- to 6-fold increase in uptake in the tumor among several other organs. PMID:21047536

  9. H-CRRETAWAC-OH, a Lead Structure for the Development of Radiotracer Targeting Integrin α5β1?

    PubMed Central

    Maschauer, Simone; Einsiedel, Jürgen; Eder, Iris E.; Gmeiner, Peter; Virgolini, Irene J.

    2014-01-01

    Imaging of angiogenic processes is of great interest in preclinical research as well as in clinical settings. The most commonly addressed target structure for imaging angiogenesis is the integrin αvβ3. Here we describe the synthesis and evaluation of [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH, a radiolabelled peptide designed to selectively target the integrin α5β1. Conjugation of 4-nitrophenyl-(RS)-2-[18F]fluoropropionate provided [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH in high radiochemical purity (>95%) and a radiochemical yield of approx. 55%. In vitro evaluation showed α5β1 binding affinity in the nanomolar range, whereas affinity to αvβ3 and αIIbβ3 was >50 μM. Cell uptake studies using human melanoma M21 (αvβ3-positive andα5β1-negative), human melanoma M21-L (αvβ3-negative and α5β1-negative), and human prostate carcinoma DU145 (αvβ3-negative and α5β1-positive) confirmed receptor-specific binding. The radiotracer was stable in human serum and showed low protein binding. Biodistribution studies showed tumour uptake ranging from 2.5 to 3.5% ID/g between 30 and 120 min post-injection. However, blocking studies and studies using mice bearing α5β1-negative M21 tumours did not confirm receptor-specific uptake of [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH, although this radiopeptide revealed high affinity and substantial selectivity to α5β1 in vitro. Further experiments are needed to study the in vivo metabolism of this peptide and to develop improved radiopeptide candidates suitable for PET imaging of α5β1 expression in vivo. PMID:25374888

  10. New Radiotracers for Imaging of Vascular Targets in Angiogenesis-related Diseases

    PubMed Central

    Hong, Hao; Chen, Feng; Zhang, Yin; Cai, Weibo

    2014-01-01

    Tremendous advances over the last several decades in positron emission tomography (PET) and single photon emission computed tomography (SPECT) allow for targeted imaging of molecular and cellular events in the living systems. Angiogenesis, a multistep process regulated by the network of different angiogenic factors, has attracted world-wide interests, due to its pivotal role in the formation and progression of different diseases including cancer, cardiovascular diseases (CVD), and inflammation. In this review article, we will summarize the recent progress in PET or SPECT imaging of a wide variety of vascular targets in three major angiogenesis-related diseases: cancer, cardiovascular diseases, and inflammation. Faster drug development and patient stratification for a specific therapy will become possible with the facilitation of PET or SPECT imaging and it will be critical for the maximum benefit of patients. PMID:25086372

  11. Toward the Optimization of Bombesin-Based Radiotracers for Tumor Targeting.

    PubMed

    Valverde, Ibai E; Vomstein, Sandra; Mindt, Thomas L

    2016-04-28

    The peptide bombesin (BBN) is a peptide with high affinity for the gastrin-releasing peptide receptor (GRPr), a receptor that is overexpressed by, for example, breast and prostate cancers. Thus, GRPr agonists can be used as cancer-targeting vectors to shuttle diagnostic and therapeutic agents into tumor cells. With the aim of optimizing the tumor targeting properties of a radiolabeled [Nle(14)]BBN(7-14) moiety, novel BBN(7-14)- and BBN(6-14)-based radioconjugates were synthesized, labeled with Lu-177, and fully evaluated in vitro and in vivo. The effect of residue and backbone modification on several parameters such as the internalization of the radiolabeled peptides into PC3 and AR42J tumor cells, their affinity toward the human GRPr, metabolic stability in blood plasma, and biodistribution in mice bearing GRPr-expressing PC3 xenografts was studied. As a result of our investigations, a novel radiolabeled GRPr agonist with a high tumor uptake and a high tumor-to-kidney ratio was identified. PMID:27054526

  12. Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease

    PubMed Central

    Khare, Shilpi; Roach, Steven L.; Barnes, S. Whitney; Hoepfner, Dominic; Walker, John R.; Chatterjee, Arnab K.; Neitz, R. Jeffrey; Arkin, Michelle R.; McNamara, Case W.; Ballard, Jaime; Lai, Yin; Fu, Yue; Molteni, Valentina; Yeh, Vince; McKerrow, James H.; Glynne, Richard J.; Supek, Frantisek

    2015-01-01

    Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease. PMID:26186534

  13. A philosophy for CNS radiotracer design.

    PubMed

    Van de Bittner, Genevieve C; Ricq, Emily L; Hooker, Jacob M

    2014-10-21

    Decades after its discovery, positron emission tomography (PET) remains the premier tool for imaging neurochemistry in living humans. Technological improvements in radiolabeling methods, camera design, and image analysis have kept PET in the forefront. In addition, the use of PET imaging has expanded because researchers have developed new radiotracers that visualize receptors, transporters, enzymes, and other molecular targets within the human brain. However, of the thousands of proteins in the central nervous system (CNS), researchers have successfully imaged fewer than 40 human proteins. To address the critical need for new radiotracers, this Account expounds on the decisions, strategies, and pitfalls of CNS radiotracer development based on our current experience in this area. We discuss the five key components of radiotracer development for human imaging: choosing a biomedical question, selection of a biological target, design of the radiotracer chemical structure, evaluation of candidate radiotracers, and analysis of preclinical imaging. It is particularly important to analyze the market of scientists or companies who might use a new radiotracer and carefully select a relevant biomedical question(s) for that audience. In the selection of a specific biological target, we emphasize how target localization and identity can constrain this process and discuss the optimal target density and affinity ratios needed for binding-based radiotracers. In addition, we discuss various PET test-retest variability requirements for monitoring changes in density, occupancy, or functionality for new radiotracers. In the synthesis of new radiotracer structures, high-throughput, modular syntheses have proved valuable, and these processes provide compounds with sites for late-stage radioisotope installation. As a result, researchers can manage the time constraints associated with the limited half-lives of isotopes. In order to evaluate brain uptake, a number of methods are available

  14. A mitochondria-targeted antioxidant can inhibit peroxidase activity of cytochrome c by detachment of the protein from liposomes.

    PubMed

    Firsov, Alexander M; Kotova, Elena A; Orlov, Viktor N; Antonenko, Yuri N; Skulachev, Vladimir P

    2016-09-01

    Interaction of cytochrome c with cardiolipin converts this respiratory chain electron-transfer protein into a peroxidase, supposedly involved in mitochondria-mediated apoptosis initiation. Liposome membrane permeabilization provoked by peroxidase activity of the cytochrome c/cardiolipin complex has been previously shown to be suppressed by conventional antioxidants. Here, the mitochondria-targeted antioxidant SkQ1 (plastoquinonyl-decyl-triphenylphosphonium) was found to strongly inhibit both cytochrome c/cardiolipin peroxidase activity and the permeabilization of liposomes composed of phosphatidylcholine and cardiolipin. A number of binding assays revealed a significant inhibiting effect of SkQ1 on cytochrome c binding to liposomes, thus suggesting that SkQ1-mediated protection of liposomes from the cytochrome c/H2 O2 -induced permeabilization involved distortion of the cytochrome c-membrane binding. It is suggested that antioxidant and antiapoptotic effects of alkyltriphenylphosphonium cations can be related to the prevention of cytochrome c/cardiolipin interaction.

  15. Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs.

    PubMed

    Ortiz, Diana; Forquer, Isaac; Boitz, Jan; Soysa, Radika; Elya, Carolyn; Fulwiler, Audrey; Nilsen, Aaron; Polley, Tamsen; Riscoe, Michael K; Ullman, Buddy; Landfear, Scott M

    2016-08-01

    Endochin-like quinolones (ELQs) are potent and specific inhibitors of cytochrome bc1 from Plasmodium falciparum and Toxoplasma gondii and show promise for novel antiparasitic drug development. To determine whether the mitochondrial electron transport chain of Leishmania parasites could be targeted similarly for drug development, we investigated the activity of 134 structurally diverse ELQs. A cohort of ELQs was selectively toxic to amastigotes of Leishmania mexicana and L. donovani, with 50% inhibitory concentrations (IC50s) in the low micromolar range, but the structurally similar hydroxynaphthoquinone buparvaquone was by far the most potent inhibitor of electron transport, ATP production, and intracellular amastigote growth. Cytochrome bc1 is thus a promising target for novel antileishmanial drugs, and further improvements on the buparvaquone scaffold are warranted for development of enhanced therapeutics. PMID:27297476

  16. Structural and Functional Analysis of Novel Human Cytochrome c Targets in Apoptosis*

    PubMed Central

    Martínez-Fábregas, Jonathan; Díaz-Moreno, Irene; González-Arzola, Katiuska; Janocha, Simon; Navarro, José A.; Hervás, Manuel; Bernhardt, Rita; Velázquez-Campoy, Adrián; Díaz-Quintana, Antonio; De la Rosa, Miguel A.

    2014-01-01

    Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals. PMID:24643968

  17. Structural and functional analysis of novel human cytochrome C targets in apoptosis.

    PubMed

    Martínez-Fábregas, Jonathan; Díaz-Moreno, Irene; González-Arzola, Katiuska; Janocha, Simon; Navarro, José A; Hervás, Manuel; Bernhardt, Rita; Velázquez-Campoy, Adrián; Díaz-Quintana, Antonio; De la Rosa, Miguel A

    2014-06-01

    Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.

  18. Targeting Cytochrome P450 Enzymes: A New Approach in Anti-cancer Drug Development

    PubMed Central

    Bruno, Robert D.; Njar, Vincent C.O.

    2007-01-01

    Cytochrome P450s (CYPs) represent a large class of heme-containing enzymes that catalyze the metabolism of multitudes of substrates both endogenous and exogenous. Until recently, however, CYPs have been largely overlooked in cancer drug development, acknowledged only for their role in Phase I metabolism of chemotherapeutics. The first successful strategy targeting CYP enzymes in cancer therapy was the development of potent inhibitors of CYP19 (aromatase) for the treatment of breast cancer. Aromatase inhibitors ushered in a new era in hormone ablation therapy for estrogen dependent cancers, and have paved the way for similar strategies (i.e. inhibition of CYP17) that combat androgen dependent prostate cancer. Identification of CYPs involved in the inactivation of anti-cancer metabolites of Vitamin D3 and Vitamin A has triggered development of agents that target these enzymes as well. The discovery of the over-expression of exogenous metabolizing CYPs, such as CYP1B1, in cancer cells has roused interest in the development of inhibitors for chemoprevention and of prodrugs designed to be activated by CYPs only in cancer cells. Finally, the expression of CYPs within tumors has been utilized in the development of bioreductive molecules that are activated by CYPs only under hypoxic conditions. This review offers the first comprehensive analysis of strategies in drug development that either inhibit or exploit CYP enzymes for the treatment of cancer. PMID:17544277

  19. Bimodal targeting of microsomal cytochrome P450s to mitochondria: implications in drug metabolism and toxicity

    PubMed Central

    Sangar, Michelle C; Bansal, Seema

    2010-01-01

    Importance of the field Microsomal cytochrome P450s are critical for drug metabolism and toxicity. Recent studies show that these CYPs are also present in the mitochondrial compartment of human and rodent tissues. Mitochondrial CYP1A1 and 2E1 show both overlapping and distinct metabolic activities compared to microsomal forms. Mitochondrial CYP2E1 also induces oxidative stress. The mechanisms of mitochondria targeting of CYPs and their role in drug metabolism and toxicity are important factors to consider while determining the drug dose and in drug development. Areas covered in this review This review highlights the mechanisms of bimodal targeting of CYP1A1, 2B1, 2E1 and 2D6 to mitochondria and microsomes. The review also discusses differences in structure and function of mitochondrial CYPs. What the readers will gain A comprehensive review of the literature on drug metabolism in the mitochondrial compartment, and their potential for inducing mitochondrial dysfunction. Take home message Studies on the biochemistry, pharmacology and pharmacogenetic analysis of CYPs are mostly focused on the molecular forms associated with the microsomal membrane. However, the mitochondrial CYPs in some individuals can represent a substantial part of the tissue pool and contribute in a significant way to drug metabolism, clearance and toxicity. PMID:20629582

  20. Production of gaseous radiotracers for industrial applications.

    PubMed

    Sharma, V K; Pant, H J; Goswami, Sunil; Jagadeesan, K C; Anand, S; Chitra, S; Rana, Y S; Sharma, Archana; Singh, Tej; Gujar, H G; Dash, Ashutosh

    2016-10-01

    This paper describes prerequisite tests, analysis and the procedure for irradiation of gaseous targets and production of gaseous radioisotopes i.e. argon-41 ((41)Ar) and krypton-79 ((79)Kr) in a 100MWTh DHRUVA reactor located at Bhabha Atomic Research Center (BARC), Trombay, Mumbai, India. The produced radioisotopes will be used as radiotracers for tracing gas phase in industrial process systems. Various details and prequalification tests required for irradiation of gaseous targets are discussed. The procedure for regular production of (41)Ar and (79)Kr, and assay of their activity were standardized. Theoretically estimated and experimentally produced amounts of activities of the two radioisotopes, irradiated at identical conditions, were compared and found to be in good agreement. Based on the various tests, radiological safety analysis and standardization of the irradiation procedure, necessary approval was obtained from the competent reactor operating and safety authorities for regular production of gaseous radiotracers in DHRUVA reactor. PMID:27518216

  1. Molecular Imaging of Prostate Cancer: PET Radiotracers

    PubMed Central

    Jadvar, Hossein

    2012-01-01

    OBJECTIVE Recent advances in the fundamental understanding of the complex biology of prostate cancer have provided an increasing number of potential targets for imaging and treatment. The imaging evaluation of prostate cancer needs to be tailored to the various phases of this remarkably heterogeneous disease. CONCLUSION In this article, I review the current state of affairs on a range of PET radiotracers for potential use in the imaging evaluation of men with prostate cancer. PMID:22826388

  2. PET Radiotracers: crossing the blood-brain barrier and surviving metabolism

    PubMed Central

    Pike, Victor W.

    2009-01-01

    Radiotracers for imaging protein targets in living human brain with positron emission tomography (PET) are increasingly useful in clinical research and in drug development. Such radiotracers must fulfill many criteria, among which an ability to enter brain adequately and reversibly without contamination by troublesome radiometabolites is desirable for accurate measurement of the density of a target protein (e.g., neuroreceptor, transporter, enzyme or plaque). Candidate radiotracers may fail as a result of poor passive brain entry, rejection from brain by efflux transporters or undesirable metabolism. These issues are reviewed. Emerging PET radiotracers for measuring efflux transporter function, and new strategies for ameliorating radiotracer metabolism are discussed. A growing understanding of the molecular features affecting the brain penetration, metabolism and efflux transporter sensitivity of prospective radiotracers should ultimately lead to their more rational and efficient design, and also to their greater efficacy. PMID:19616318

  3. Diversity-Oriented Synthesis Probe Targets Plasmodium falciparum Cytochrome b Ubiquinone Reduction Site and Synergizes With Oxidation Site Inhibitors

    PubMed Central

    Lukens, Amanda K.; Heidebrecht, Richard W.; Mulrooney, Carol; Beaudoin, Jennifer A.; Comer, Eamon; Duvall, Jeremy R.; Fitzgerald, Mark E.; Masi, Daniela; Galinsky, Kevin; Scherer, Christina A.; Palmer, Michelle; Munoz, Benito; Foley, Michael; Schreiber, Stuart L.; Wiegand, Roger C.; Wirth, Dyann F.

    2015-01-01

    Background. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. Methods. We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. Results. We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. Conclusions. The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria. PMID:25336726

  4. A targeted proteomics approach for profiling murine cytochrome P450 expression.

    PubMed

    Hersman, Elisabeth M; Bumpus, Namandjé N

    2014-05-01

    The cytochrome P450 (P450) superfamily of enzymes plays a prominent role in drug metabolism. Although mice are a widely used preclinical model in pharmacology, the expression of murine P450 enzymes at the protein level has yet to be fully defined. Twenty-seven proteins belonging to P450 subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 2F, 2J, 2U, 3A, 4A, 4B, 4F, and 4V were readily detectable in Balb/c mouse tissue using a global mass spectrometry-based proteomics approach. Subsequently, a targeted mass spectrometry-based assay was developed to simultaneously quantify these enzymes in ranges of femtomoles of P450 per microgram of total protein concentration range. This screen was applied to mouse liver microsomes and tissue lysates of kidney, lung, intestine, heart, and brain isolated from mixed-sex fetuses; male and female mice that were 3-4 weeks, 9-10 weeks, and 8-10 months of age; and pregnant mice. CYP1A2 was consistently more abundant in male mouse liver microsomes compared with age-matched females. Hepatic expression of CYP2B9 was more abundant in 3- to 4-week-old male and female mice than in mice of other ages; in addition, CYP2B9 was the only enzyme that was detectable at higher levels in pregnant mouse liver microsomes compared with age-matched females. Interestingly, sexually dimorphic expression of CYP2B9, 2D26, 2E1, and 4B1 was observed in kidney only. The targeted proteomics assay described here can be broadly used as a tool for investigating the expression patterns of P450 enzymes in mice.

  5. Additive effects of mitochondrion-targeted cytochrome CYP2E1 and alcohol toxicity on cytochrome c oxidase function and stability of respirosome complexes.

    PubMed

    Bansal, Seema; Srinivasan, Satish; Anandasadagopan, Sureshkumar; Chowdhury, Anindya Roy; Selvaraj, Venkatesh; Kalyanaraman, Balaraman; Joseph, Joy; Avadhani, Narayan G

    2012-05-01

    Alcohol treatment induces oxidative stress by a combination of increased production of partially reduced oxygen species and decreased cellular antioxidant pool, including GSH. Recently, we showed that mitochondrion-targeted CYP2E1 augments alcohol-mediated toxicity, causing an increase in reactive oxygen species production and oxidative stress. Here, we show that cytochrome c oxidase (CcO), the terminal oxidase of the mitochondrial respiratory chain, is a critical target of CYP2E1-mediated alcohol toxicity. COS-7 and Hep G2 cell lines expressing predominantly mitochondrion-targeted (Mt(++)) CYP2E1 and livers from alcohol-treated rats showed loss of CcO activity and increased protein carbonylation, which was accompanied by a decline in the steady state levels of subunits I, IVI1, and Vb of the CcO complex. This was also accompanied by reduced mitochondrial DNA content and reduced mitochondrial mRNA. These changes were more prominent in Mt(++) cells in comparison with wild type (WT) CYP2E1-expressing or ER(+) (mostly microsome-targeted) cells. In addition, mitochondrion-specific antioxidants, ubiquinol conjugated to triphenyl phosphonium, triphenylphosphonium conjugated carboxyl proxyl, and the CYP2E1 inhibitor diallyl sulfide prevented the loss of CcO activity and the CcO subunits, most likely through reduced oxidative damage to the enzyme complex. Our results suggest that damage to CcO and dissociation of respirosome complexes are critical factors in alcohol-induced toxicity, which is augmented by mitochondrion-targeted CYP2E1. We propose that CcO is one of the direct and immediate targets of alcohol-induced toxicity causing respiratory dysfunction.

  6. 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

  7. MiR-34a regulates blood-brain barrier permeability and mitochondrial function by targeting cytochrome c.

    PubMed

    Bukeirat, Mimi; Sarkar, Saumyendra N; Hu, Heng; Quintana, Dominic D; Simpkins, James W; Ren, Xuefang

    2016-02-01

    The blood-brain barrier is composed of cerebrovascular endothelial cells and tight junctions, and maintaining its integrity is crucial for the homeostasis of the neuronal environment. Recently, we discovered that mitochondria play a critical role in maintaining blood-brain barrier integrity. We report for the first time a novel mechanism underlying blood-brain barrier integrity: miR-34a mediated regulation of blood-brain barrier through a mitochondrial mechanism. Bioinformatics analysis suggests miR-34a targets several mitochondria-associated gene candidates. We demonstrated that miR-34a triggers the breakdown of blood-brain barrier in cerebrovascular endothelial cell monolayer in vitro, paralleled by reduction of mitochondrial oxidative phosphorylation and adenosine triphosphate production, and decreased cytochrome c levels. PMID:26661155

  8. 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.

  9. 21-(/sup 18/F)fluoro-16 alpha-ethyl-19-norprogesterone: synthesis and target tissue selective uptake of a progestin receptor based radiotracer for positron emission tomography

    SciTech Connect

    Pomper, M.G.; Katzenellenbogen, J.A.; Welch, M.J.; Brodack, J.W.; Mathias, C.J.

    1988-07-01

    We have synthesized 21-(/sup 18/F)fluoro-16 alpha-ethyl-19-norprogesterone (FENP), a high affinity ligand for the progesterone receptor, labeled with the positron-emitting radionuclide fluorine-18 (t1/2 = 110 min). The synthesis proceeds in two steps from 21-hydroxy-16 alpha-ethyl-19-norprogesterone and involves (/sup 18/F)fluoride ion displacement of the 21-trifluoromethanesulfonate (21-triflate). This material is purified by HPLC and is obtained in 4-30% overall yield (decay corrected) within 40 min after the end of bombardment to produce (/sup 18/F)fluoride ion. The effective specific activity, determined by competitive radioreceptor binding assays, is 700-1400 Ci/mmol. In vivo, (/sup 18/F)FENP demonstrates highly selective, receptor-mediated uptake by the uterus of estrogen-primed rats; the uterus to blood and uterus to muscle ratios were respectively 26 and 16 at 1 h and 71 and 41 at 3 h after injection. The high target tissue selectivity of this uptake suggests that this compound may be useful for the in vivo imaging of progestin target tissues and receptor-rich tumors (such as human breast tumors) by positron emission tomography.

  10. The Heritage of Radiotracers for PET

    DOE R&D Accomplishments Database

    Fowler, J. S.; Wolf, A. P.

    1988-05-01

    The history of PET research clearly demonstrates that it is advances in chemistry coupled with a detailed examination of the biochemistry of new radiotracers which has allowed the PET method to be applied to new areas of biology and medicine. Radiotracers whose regional distribution reflects glucose metabolism, neutrotransmitter activity and enzyme activity have all required the development of rapid synthetic methods for the radiotracers themselves and the characterization of their biochemical behavior. This article traces some of the advances in the production of labeled precursors and in radiotracer synthesis and evaluation which have shaped the rapidly expanding application of PET to problems in the neurosciences, in cardiology and in oncology.

  11. The heritage of radiotracers for PET

    SciTech Connect

    Fowler, J.S.; Wolf, A.P.

    1988-05-01

    The history of PET research clearly demonstrates that it is advances in chemistry coupled with a detailed examination of the biochemistry of new radiotracers which has allowed the PET method to be applied to new areas of biology and medicine. Radiotracers whose regional distribution reflects glucose metabolism, neutrotransmitter activity and enzyme activity have all required the development of rapid synthetic methods for the radiotracers themselves and the characterization of their biochemical behavior. This article traces some of the advances in the production of labeled precursors and in radiotracer synthesis and evaluation which have shaped the rapidly expanding application of PET to problems in the neurosciences, in cardiology and in oncology. 54 refs.

  12. Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy.

    PubMed

    Mast, Natalia; Lin, Joseph B; Pikuleva, Irina A

    2015-09-01

    Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification.

  13. Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy

    PubMed Central

    Mast, Natalia; Lin, Joseph B.

    2015-01-01

    Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification. PMID:26082378

  14. Let-7b Inhibits Human Cancer Phenotype by Targeting Cytochrome P450 Epoxygenase 2J2

    PubMed Central

    Yang, Shenglan; Gong, Wei; Wang, Yan; Cianflone, Katherine; Tang, Jiarong; Wang, Dao Wen

    2012-01-01

    Background MicroRNAs (miRNAs) are small, noncoding RNA molecules of 20 to 22 nucleotides that regulate gene expression by binding to their 3′ untranslated region (3′UTR). Increasing data implicate altered miRNA participation in the progress of cancer. We previously reported that CYP2J2 epoxygenase promotes human cancer phenotypes. But whether and how CYP2J2 is regulated by miRNA is not understood. Methods and Results Using bioinformatics analysis, we found potential target sites for miRNA let-7b in 3′UTR of human CYP2J2. Luciferase and western blot assays revealed that CYP2J2 was regulated by let-7b. In addition, let-7b decreased the enzymatic activity of endogenous CYP2J2. Furthermore, let-7b may diminish cell proliferation and promote cell apoptosis of tumor cells via posttranscriptional repression of CYP2J2. Tumor xenografts were induced in nude mice by subcutaneous injection of MDA-MB-435 cells. The let-7b expression vector, pSilencer-let-7b, was injected through tail vein every 3 weeks. Let-7b significantly inhibited the tumor phenotype by targeting CYP2J2. Moreover, quantitative real-time polymerase chain reaction and western blotting were used to determine the expression levels of let-7b and CYP2J2 protein from 18 matched lung squamous cell cancer and adjacent normal lung tissues; the expression level of CYP2J2 was inversely proportional to that of let-7b. Conclusions Our results demonstrated that the decreased expression of let-7b could lead to the high expression of CYP2J2 protein in cancerous tissues. These findings suggest that miRNA let-7b reduces CYP2J2 expression, which may contribute to inhibiting tumor phenotypes. PMID:22761738

  15. Designing inhibitors of cytochrome c/cardiolipin peroxidase complexes: mitochondria-targeted imidazole-substituted fatty acids.

    PubMed

    Jiang, Jianfei; Bakan, Ahmet; Kapralov, Alexandr A; Silva, K Ishara; Huang, Zhentai; Amoscato, Andrew A; Peterson, James; Garapati, Venkata Krishna; Saxena, Sunil; Bayir, Hülya; Atkinson, Jeffrey; Bahar, Ivet; Kagan, Valerian E

    2014-06-01

    Mitochondria have emerged as the major regulatory platform responsible for the coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (Cyt) c. As this oxidation occurs within the peroxidase complex of Cyt c with CL, the latter represents a promising target for the discovery and design of drugs with antiapoptotic mechanisms of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogs of stearic acid TPP-n-ISAs with various positions of the attached imidazole group on the fatty acid (n = 6, 8, 10, 13, and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance and electron spin echo envelope modulation) we demonstrated that TPP-n-ISAs indeed were able to potently suppress CL-induced structural rearrangements in Cyt c, paving the way to its peroxidase competence. TPP-n-ISA analogs preserved the low-spin hexa-coordinated heme-iron state in Cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of Cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of Cyt c/CL complexes with a significant antiapoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all-atom molecular dynamics simulations. Based on the experimental data and computation predictions, we identified TPP-6-ISA as a candidate drug with optimized antiapoptotic potency.

  16. Designing Inhibitors of Cytochrome c/Cardiolipin Peroxidase Complexes: Mitochondria-Targeted Imidazole-Substituted Fatty Acids

    PubMed Central

    Jiang, Jianfei; Bakan, Ahmet; Kapralov, Alexandr A.; Silva, K. Ishara; Huang, Zhentai; Amoscato, Andrew A.; Peterson, James; Garapati, Venkata Krishna; Saxena, Sunil; Bayir, Hülya; Atkinson, Jeffrey; Bahar, Ivet; Kagan, Valerian E.

    2014-01-01

    Mitochondria have emerged as the major regulatory platform responsible for coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (cyt) c. As this oxidation occurs within the peroxidase complex of cyt c with CL, the latter represents a promising target for the discovery and design of drugs with anti-apoptotic mechanism of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogues of stearic acid TPP-n-ISA with different positions of the attached imidazole group on the fatty acid (n=6, 8, 10, 13 and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance, and electron spin echo envelope modulation) we demonstrated that TPP-n-ISA indeed were able to potently suppress CL induced structural re-arrangements in cyt c paving the way to its peroxidase competence. TPP-n-ISA analogues preserved the low spin hexa-coordinated heme iron state in cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of cyt c/CL complexes with a significant anti-apoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all atom molecular dynamics simulations. Based on the experimental data and computations predictions, we identified TPP-6-ISA as a candidate drug with optimized anti-apoptotic potency. PMID:24631490

  17. Human Cytochrome P450 2E1 Mutations That Alter Mitochondrial Targeting Efficiency and Susceptibility to Ethanol-induced Toxicity in Cellular Models*

    PubMed Central

    Bansal, Seema; Anandatheerthavarada, Hindupur K.; Prabu, Govindaswamy K.; Milne, Ginger L.; Martin, Martha V.; Guengerich, F. Peter; Avadhani, Narayan G.

    2013-01-01

    Human polymorphisms in the 5′-upstream regulatory regions and also protein coding regions of cytochrome P450 2E1 (CYP2E1) are known to be associated with several diseases, including cancer and alcohol liver toxicity. In this study, we report novel mutations in the N-terminal protein targeting regions of CYP2E1 that markedly affect subcellular localization of the protein. Variant W23R/W30R protein (termed W23/30R) is preferentially targeted to mitochondria but very poorly to the endoplasmic reticulum, whereas the L32N protein is preferentially targeted to the endoplasmic reticulum and poorly to mitochondria. These results explain the physiological significance of bimodal CYP targeting to the endoplasmic reticulum and mitochondria previously described. COS-7 cells and HepG2 cells stably expressing W23/30R mutations showed markedly increased alcohol toxicity in terms of increased production of reactive oxygen species, respiratory dysfunction, and loss of cytochrome c oxidase subunits and activity. Stable cells expressing the L32N variant, on the other hand, were relatively less responsive to alcohol-induced toxicity and mitochondrial dysfunction. These results further support our previous data, based on mutational studies involving altered targeting, indicating that mitochondria-targeted CYP2E1 plays an important role in alcohol liver toxicity. The results also provide an interesting new link to genetic variations affecting subcellular distribution of CYP2E1 with alcohol-induced toxicity. PMID:23471973

  18. Monoamine oxidase: radiotracer chemistry and human studies.

    PubMed

    Fowler, Joanna S; Logan, Jean; Shumay, Elena; Alia-Klein, Nelly; Wang, Gene-Jack; Volkow, Nora D

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serotonin, norepinephrine, and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson's disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 years since the first radiotracers were developed and the first positron emission tomography (PET) images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variables that have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe the following: (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological, and psychiatric disorders; and (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers that are currently used and possible new applications.

  19. Monoamine oxidase: Radiotracer chemistry and human studies

    DOE PAGES

    Fowler, Joanna S.; Logan, Jean; Shumay, Elena; Alia-Klein, Nelly; Wang, Gene-Jack; Volkow, Nora D.

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serot onin, norepinephrine and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson’s disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 since the first radiotracers were developed and the first PET images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variablesmore » which have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological and psychiatric disorders; (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers which are currently used and possible new applications.« less

  20. Monoamine oxidase: Radiotracer chemistry and human studies

    SciTech Connect

    Fowler, Joanna S.; Logan, Jean; Shumay, Elena; Alia-Klein, Nelly; Wang, Gene-Jack; Volkow, Nora D.

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serot onin, norepinephrine and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson’s disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 since the first radiotracers were developed and the first PET images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variables which have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological and psychiatric disorders; (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers which are currently used and possible new applications.

  1. Radiotracer investigation in a glass production unit.

    PubMed

    Pant, H J; Goswami, Sunil; Biswal, Jayashree; Samantaray, J S; Sharma, V K; Singhal, Sorabh

    2016-10-01

    A radiotracer investigation was carried out in a glass production unit in a glass industry. Lanthanum-140 as lanthanium oxide mixed with silica was used as a radiotracer to trace the molten glass in various sections of the unit. Residence time distributions of molten glass were measured and analyzed to identify the flow abnormities. The flow parameters such as breakthrough time, mean residence time, homogenization time, dead volume and flow patterns in different sections of the unit were obtained from the measured RTD data. The results of the investigation were used to improve and optimize the operation of the glass production unit. PMID:27474905

  2. 64Cu-labeled phosphonium cations as PET radiotracers for tumor imaging.

    PubMed

    Zhou, Yang; Liu, Shuang

    2011-08-17

    Alteration in mitochondrial transmembrane potential (ΔΨ(m)) is an important characteristic of cancer. The observation that the enhanced negative mitochondrial potential is prevalent in tumor cell phenotype provides a conceptual basis for development of mitochondrion-targeting therapeutic drugs and molecular imaging probes. Since plasma and mitochondrial potentials are negative, many delocalized organic cations, such as rhodamine-123 and (3)H-tetraphenylphosphonium, are electrophoretically driven through these membranes, and able to localize in the energized mitochondria of tumor cells. Cationic radiotracers, such as (99m)Tc-Sestamibi and (99m)Tc-Tetrofosmin, have been clinically used for diagnosis of cancer by single photon emission computed tomography (SPECT) and noninvasive monitoring of the multidrug resistance (MDR) transport function in tumors of different origin. However, their diagnostic and prognostic values are often limited due to their insufficient tumor localization (low radiotracer tumor uptake) and high radioactivity accumulation in the chest and abdominal regions (low tumor selectivity). In contrast, the (64)Cu-labeled phosphonium cations represent a new class of PET (positron emission tomography) radiotracers with good tumor uptake and high tumor selectivity. This review article will focus on our recent experiences in evaluation of (64)Cu-labeled phosphonium cations as potential PET radiotracers. The main objective is to illustrate the impact of radiometal chelate on physical, chemical, and biological properties of (64)Cu radiotracers. It will also discuss some important issues related to their tumor selectivity and possible tumor localization mechanism.

  3. Cyclotron production of ``very high specific activity'' platinum radiotracers in No Carrier Added form

    NASA Astrophysics Data System (ADS)

    Birattari, C.; Bonardi, M.; Groppi, F.; Gini, L.; Gallorini, M.; Sabbioni, E.; Stroosnijder, M. F.

    2001-12-01

    At the "Radiochemistry Laboratory" of Accelerators and Applied Superconductivity Laboratory, LASA, several production and quality assurance methods for short-lived and high specific activity radionuclides, have been developed. Presently, the irradiations are carried out at the Scanditronix MC40 cyclotron (K=38; p, d, He-4 and He-3) of JRC-Ispra, Italy, of the European Community, while both chemical purity and specific activity determination are carried out at the TRIGA MARK II research reactor of University of Pavia and at LASA itself. In order to optimize the irradiation conditions for platinum radiotracer production, both thin- and thick-target excitation function of natOs(α,xn) nuclear reactions were measured. A very selective radiochemical separation to obtain Pt radiotracers in No Carrier Added form, has been developed. Both real specific activity and chemical purity of radiotracer, have been determined by neutron activation analysis and atomic absorption spectrometry. An Isotopic Dilution Factor (IDF) of the order of 50 is achieved.

  4. Tomato allene oxide synthase and fatty acid hydroperoxide lyase, two cytochrome P450s involved in oxylipin metabolism, are targeted to different membranes of chloroplast envelope.

    PubMed

    Froehlich, J E; Itoh, A; Howe, G A

    2001-01-01

    Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) are related cytochrome P450s that metabolize a common fatty acid hydroperoxide substrate to different classes of bioactive oxylipins within chloroplasts. Here, we report the use of in vitro import assays to investigate the targeting of tomato (Lycopersicon esculentum) AOS (LeAOS) and HPL (LeHPL) to isolated chloroplasts. LeAOS, which contains a typical N-terminal transit peptide, was targeted to the inner envelope membrane by a route that requires both ATP and proteinase-sensitive components on the surface of chloroplasts. Imported LeAOS was peripherally associated with the inner envelope; the bulk of the protein facing the stroma. LeHPL, which lacks a typical chloroplast-targeting sequence, was targeted to the outer envelope by an ATP-independent and protease-insensitive pathway. Imported LeHPL was integrated into the outer envelope with most of the protein exposed to the inter-membrane space. We conclude that LeAOS and LeHPL are routed to different envelope membranes by distinct targeting pathways. Partitioning of AOS and HPL to different envelope membranes suggests differences in the spatial organization of these two branches of oxylipin metabolism.

  5. Design, evaluation, and comparison of ghrelin receptor agonists and inverse agonists as suitable radiotracers for PET imaging.

    PubMed

    Chollet, Constance; Bergmann, Ralf; Pietzsch, Jens; Beck-Sickinger, Annette G

    2012-04-18

    Ghrelin agonist and inverse agonist radiotracers, suitable for positron emission tomography (PET), were developed to study the behavior of ghrelin receptor ligands in vivo and for further design of druggable peptides. The target peptides were synthesized on solid support and conjugated to the bifunctional chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), which is known to form a stable complex with Ga(3+). Complexation with (68)Ga could be achieved under mild conditions and led to radiotracers with high radiochemical purity and specific activity. The biological activity of the radiotracers was evaluated in vitro by an inositol phosphate turnover assay. Pharmacokinetic profile and metabolic stability of the (68)Ga-NODAGA-radiotracers were investigated by small animal PET in rodent. Ghrelin derived agonists presented very high kidney accumulation, negligible tissue distribution, fast blood clearance, and poor stability in blood. Contrarily, the inverse agonist radiotracer exhibited very high stability in blood, large diffusion in tissues, reasonable kidney and liver metabolism, and slow blood clearance. This pharmacokinetic profile makes the ghrelin inverse agonist motif KwFwLL-CONH(2) suitable for further development of radiotracers and a promising lead to design peptide-based therapeutics against obesity. PMID:22372770

  6. An explosive-degrading cytochrome P450 activity and its targeted application for the phytoremediation of RDX.

    PubMed

    Rylott, Elizabeth L; Jackson, Rosamond G; Edwards, James; Womack, Grant L; Seth-Smith, Helena M B; Rathbone, Deborah A; Strand, Stuart E; Bruce, Neil C

    2006-02-01

    The widespread presence in the environment of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), one of the most widely used military explosives, has raised concern owing to its toxicity and recalcitrance to degradation. To investigate the potential of plants to remove RDX from contaminated soil and water, we engineered Arabidopsis thaliana to express a bacterial gene xplA encoding an RDX-degrading cytochrome P450 (ref. 1). We demonstrate that the P450 domain of XplA is fused to a flavodoxin redox partner and catalyzes the degradation of RDX in the absence of oxygen. Transgenic A. thaliana expressing xplA removed and detoxified RDX from liquid media. As a model system for RDX phytoremediation, A. thaliana expressing xplA was grown in RDX-contaminated soil and found to be resistant to RDX phytotoxicity, producing shoot and root biomasses greater than those of wild-type plants. Our work suggests that expression of xplA in landscape plants may provide a suitable remediation strategy for sites contaminated by this class of explosives. PMID:16429147

  7. Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

    PubMed

    Hafeez, Mian A; Shivaramaiah, Srichaitanya; Dorsey, Kristi Moore; Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Cobean, Julie; Barta, John R

    2015-05-01

    Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

  8. The revised human liver cytochrome P450 "Pie": absolute protein quantification of CYP4F and CYP3A enzymes using targeted quantitative proteomics.

    PubMed

    Michaels, Scott; Wang, Michael Zhuo

    2014-08-01

    The CYP4F subfamily of enzymes has been identified recently to be involved in the metabolism of endogenous compounds (arachidonic acid and leukotriene B4), nutrients (vitamins K1 and E), and xenobiotics (pafuramidine and fingolimod). CYP4F2 and CYP4F3B are reported to be expressed in the human liver. However, absolute concentrations of these enzymes in human liver microsomes (HLMs) and their interindividual variability have yet to be determined because of the lack of specific antibodies. Here, an liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based targeted quantitative proteomic approach was employed to determine the absolute protein concentrations of CYP4F2 and CYP4F3B compared with CYP3A in two panels of HLMs (n = 31). As a result, the human hepatic cytochrome P450 (P450) "pie" has been revised to include the contribution of CYP4F enzymes, which amounts to 15% of the total hepatic cytochrome P450 enzymes. CYP4F3B displayed low interindividual variability (3.3-fold) in the HLM panels whereas CYP4F2 displayed large variability (21-fold). However, CYP4F2 variability decreased to 3.4-fold if the two donors with the lowest expression were excluded. In contrast, CYP3A exhibited 29-fold interindividual variability in the same HLM panels. The proposed marker reaction for CYP4F enzymes pafuramidine/DB289 M1 formation did not correlate with CYP4F protein content, suggesting alternate metabolic pathways for DB289 M1 formation in HLMs. In conclusion, CYP4F enzymes are highly expressed in the human liver and their physiologic and pharmacologic roles warrant further investigation.

  9. Long-Circulating and pH-Sensitive Liposome Preparation Trapping a Radiotracer for Inflammation Site Detection.

    PubMed

    Mota, Luciene Das Graças; de Barros, André Luís Branco; Fuscaldi, Leonardo Lima; de Oliveira, Mônica Cristina; Cardoso, Valbert Nascimento

    2015-06-01

    Inflammatory and infectious diseases are one of the most common causes of mortality and morbidity. This paper aimed to prepare and to evaluate the ability of long-circulating and pH-sensitive liposomes, trapping a radiotracer, to identify inflamed focus. The physicochemical characterization of freeze-dried liposomes, using glucose as cryoprotectant, showed 80% of the vesicles with adequate mean diameter and good vesicle size homogeneity. Radiotracer encapsulation percentage in liposomes was 10.65%, of which 4.88% was adsorbed on the surface of the vesicles. Furthermore, liposomes presented positive zeta potential. Freeze-dried liposomes, stored for 180 days at 4 degrees C, did not show significant changes in the mean diameter, indicating good stability. Free radiotracer and radiolabeled liposomes were injected into inflammation focus-bearing rats, and ex-vivo biodistribution studies and scintigraphic images were performed. Results showed that radiopharmaceutical, free and encapsulated into liposomes, were able to identify the inflamed site. Target/non-target ratios, obtained by scintigraphic images, were greater than 1.5 at all investigated times. Data did not show significant differences between the free radiotracer and radiolabeled liposomes. Results suggest that this liposomal preparation could be employed as an alternative procedure for inflamed site detection by means of scintigraphic images. However, as the radiotracer is adsorbed onto the liposome surface by electrostatic forces, it is suggested that a neutral radiopharmaceutical be used to confirm the potential of this formulation as a scintigraphic probe for inflammation/infection detection. PMID:26369024

  10. Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection.

    PubMed

    Kawakami, Hirotaka; Ohtsuki, Sumio; Kamiie, Junichi; Suzuki, Takashi; Abe, Takaaki; Terasaki, Tetsuya

    2011-01-01

    Cytochrome P450 (CYP) proteins are involved in the biological oxidation and reduction of xenobiotics, affecting the pharmacological efficiency of drugs. This study aimed to establish a method to simultaneously quantify 11 CYP isoforms by multiplexed-multiple reaction monitoring analysis with liquid chromatography tandem mass spectrometry and in silico peptide selection to clarify CYP isoform expression profiles in human liver tissue. CYP1A2, 2A6, and 2D6 target peptides were identified by shot-gun proteomic analysis, and those of other isoforms were selected by in silico peptide selection criteria. The established quantification method detected target peptides at 10  fmol, and the dynamic range of calibration curves was at least 500-fold. The quantification value of CYP1A2 in Supersomes was not significantly different between the established method and quantitative immunoblot analysis. The absolute protein expression levels of 11 CYP isoforms were determined from one pooled and 10 individual human liver microsomes. In the individual microsomes, CYP2C9 showed the highest protein expression level, and CYP1A2, 2A6, 2C19, and 3A4 protein expression exhibited more than a 20-fold difference among individuals. This highly sensitive and selective quantification method is a useful tool for the analysis of highly homologous CYP isoforms and the contribution made by each CYP isoform to drug metabolism. PMID:20564338

  11. Metabolism of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine by Mitochondrion-targeted Cytochrome P450 2D6

    PubMed Central

    Bajpai, Prachi; Sangar, Michelle C.; Singh, Shilpee; Tang, Weigang; Bansal, Seema; Chowdhury, Goutam; Cheng, Qian; Fang, Ji-Kang; Martin, Martha V.; Guengerich, F. Peter; Avadhani, Narayan G.

    2013-01-01

    1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxic side product formed in the chemical synthesis of desmethylprodine opioid analgesic, which induces Parkinson disease. Monoamine oxidase B, present in the mitochondrial outer membrane of glial cells, catalyzes the oxidation of MPTP to the toxic 1-methyl-4-phenylpyridinium ion (MPP+), which then targets the dopaminergic neurons causing neuronal death. Here, we demonstrate that mitochondrion-targeted human cytochrome P450 2D6 (CYP2D6), supported by mitochondrial adrenodoxin and adrenodoxin reductase, can efficiently catalyze the metabolism of MPTP to MPP+, as shown with purified enzymes and also in cells expressing mitochondrial CYP2D6. Neuro-2A cells stably expressing predominantly mitochondrion-targeted CYP2D6 were more sensitive to MPTP-mediated mitochondrial respiratory dysfunction and complex I inhibition than cells expressing predominantly endoplasmic reticulum-targeted CYP2D6. Mitochondrial CYP2D6 expressing Neuro-2A cells produced higher levels of reactive oxygen species and showed abnormal mitochondrial structures. MPTP treatment also induced mitochondrial translocation of an autophagic marker, Parkin, and a mitochondrial fission marker, Drp1, in differentiated neurons expressing mitochondrial CYP2D6. MPTP-mediated toxicity in primary dopaminergic neurons was attenuated by CYP2D6 inhibitor, quinidine, and also partly by monoamine oxidase B inhibitors deprenyl and pargyline. These studies show for the first time that dopaminergic neurons expressing mitochondrial CYP2D6 are fully capable of activating the pro-neurotoxin MPTP and inducing neuronal damage, which is effectively prevented by the CYP2D6 inhibitor quinidine. PMID:23258538

  12. Radiotracers for PETT: new developments and perspectives

    SciTech Connect

    Fowler, J.S.; Wolf, A.P.

    1983-01-01

    Radiotracer development with positron emitters has its major focus on problems in the neurosciences. Progress is reviewed for high-level isotope production and labelled precurser synthesis with the medical cyclotron. The study of regional brain glucose metabolism represented the first extension of one of the methods of neurochemical autoradiography to humans and the study of brain protein synthesis and neurotransmitter receptors followed. In a more general sense, one PETT instrumentation will provide resolution in the 5 mm range is already emerging. Research status is reviewed. 103 references. (PSB)

  13. Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii.

    PubMed

    Gangl, Doris; Zedler, Julie A Z; Włodarczyk, Artur; Jensen, Poul Erik; Purton, Saul; Robinson, Colin

    2015-02-01

    The unicellular green alga Chlamydomonas reinhardtii has potential as a cell factory for the production of recombinant proteins and other compounds, but mainstream adoption has been hindered by a scarcity of genetic tools and a need to identify products that can be generated in a cost-effective manner. A promising strategy is to use algal chloroplasts as a site for synthesis of high value bioactive compounds such as diterpenoids since these are derived from metabolic building blocks that occur naturally within the organelle. However, synthesis of these complex plant metabolites requires the introduction of membrane-associated enzymes including cytochrome P450 enzymes (P450s). Here, we show that a gene (CYP79A1) encoding a model P450 can be introduced into the C. reinhardtii chloroplast genome using a simple transformation system. The gene is stably expressed and the P450 is efficiently targeted into chloroplast membranes by means of its endogenous N-terminal anchor domain, where it is active and accounts for 0.4% of total cell protein. These results provide proof of concept for the introduction of diterpenoid synthesis pathways into the chloroplast of C. reinhardtii. PMID:25556316

  14. Redox-Sensitive and Intrinsically Fluorescent Photoclick Hyaluronic Acid Nanogels for Traceable and Targeted Delivery of Cytochrome c to Breast Tumor in Mice.

    PubMed

    Li, Shuai; Zhang, Jian; Deng, Chao; Meng, Fenghua; Yu, Lin; Zhong, Zhiyuan

    2016-08-24

    In spite of their high specificity and potency, few protein therapeutics are applied in clinical cancer therapy owing to a lack of safe and efficacious delivery systems. Here, we report that redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels (HA-NGs) show highly efficient loading and breast tumor-targeted delivery of cytochrome c (CC). HA-NGs were obtained from hyaluronic acid-graft-oligo(ethylene glycol)-tetrazole (HA-OEG-Tet) via inverse nanoprecipitation and catalyst-free photoclick cross-linking with l-cystine dimethacrylamide (MA-Cys-MA). HA-NGs exhibited a superb CC loading content of up to 40.6 wt %, intrinsic fluorescence (λem = 510 nm), and a small size of ca. 170 nm. Notably, CC-loaded nanogels (CC-NGs) showed a fast glutathione-responsive protein release behavior. Importantly, released CC maintained its bioactivity. MTT assays revealed that CC-NGs were highly potent with a low IC50 of 3.07 μM to CD44+ MCF-7 human breast tumor cells. Confocal microscopy observed efficient and selective internalization of fluorescent HA-NGs into MCF-7 cells. Interestingly, HA-NGs exhibited also effective breast tumor penetration. The therapeutic results demonstrated that CC-NGs effectively inhibited the growth of MCF-7 breast tumor xenografts at a particularly low dose of 80 or 160 nmol CC equiv./kg. Moreover, CC-NGs did not cause any change in mice body weight, corroborating their low systemic side effects. Redox-sensitive and intrinsically fluorescent photoclick hyaluronic acid nanogels have appeared as a "smart" protein delivery nanoplatform enabling safe, efficacious, traceable, and targeted cancer protein therapy in vivo. PMID:27509045

  15. Pulmonary clearance of radiotracers after positive end-expiratory pressure or acute lung injury

    SciTech Connect

    Barrowcliffe, M.P.; Zanelli, G.D.; Jones, J.G.

    1989-01-01

    In anesthetized rabbits we measured clearance from lung to blood of eight aerosolized technetium-99m-labeled compounds: diethylenetriaminepentaacetate (99mTc-DTPA); cytochrome c; myoglobin; a myoglobin polymer; albumin; and anionic, cationic, and neutral dextrans of equivalent molecular size. We investigated the effect of applying positive end-expiratory pressure (PEEP) and, on a subsequent occasion, of injecting oleic acid intravenously to produce acute lung injury on the pulmonary clearance rate. Base-line clearance rates were monoexponential and varied with the molecular weights of the radiotracers. For each tracer the rate of clearance was increased a similar degree by either PEEP or oleic acid. However, with PEEP, clearance remained monoexponential, whereas after oleic acid, smaller molecular-weight radiotracers had multiexponential clearance curves. This suggests that after oleic acid the alveolar epithelium breaks down in a nonuniform fashion. We conclude that differentiation of the effect of PEEP from that of severe lung injury caused by oleic acid is not readily accomplished by either increasing the size of the tracer molecule or by varying the molecular charge.

  16. Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

    PubMed Central

    Jawallapersand, Poojah; Mashele, Samson Sitheni; Kovačič, Lidija; Stojan, Jure; Komel, Radovan; Pakala, Suresh Babu; Kraševec, Nada; Syed, Khajamohiddin

    2014-01-01

    Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative

  17. Cytochrome P450 monooxygenase CYP53 family in fungi: comparative structural and evolutionary analysis and its role as a common alternative anti-fungal drug target.

    PubMed

    Jawallapersand, Poojah; Mashele, Samson Sitheni; Kovačič, Lidija; Stojan, Jure; Komel, Radovan; Pakala, Suresh Babu; Kraševec, Nada; Syed, Khajamohiddin

    2014-01-01

    Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative

  18. SYBR Green Real-Time PCR-RFLP Assay Targeting the Plasmodium Cytochrome B Gene – A Highly Sensitive Molecular Tool for Malaria Parasite Detection and Species Determination

    PubMed Central

    Xu, Weiping; Morris, Ulrika; Aydin-Schmidt, Berit; Msellem, Mwinyi I.; Shakely, Delér; Petzold, Max; Björkman, Anders; Mårtensson, Andreas

    2015-01-01

    A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5–10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as ‘final positive’ if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5–100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7–100%) when compared against ‘final positive’ samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings. PMID:25774805

  19. SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene--a highly sensitive molecular tool for malaria parasite detection and species determination.

    PubMed

    Xu, Weiping; Morris, Ulrika; Aydin-Schmidt, Berit; Msellem, Mwinyi I; Shakely, Delér; Petzold, Max; Björkman, Anders; Mårtensson, Andreas

    2015-01-01

    A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5-10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5-100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7-100%) when compared against 'final positive' samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings.

  20. Diallyl Sulfide: Potential Use in Novel Therapeutic Interventions in Alcohol, Drugs, and Disease Mediated Cellular Toxicity by Targeting Cytochrome P450 2E1

    PubMed Central

    Rao, PSS; Midde, Narasimha M; Miller, Duane D.; Chauhan, Subhash; Kumar, Anil; Kumar, Santosh

    2015-01-01

    Diallyl sulfide (DAS) and other organosulfur compounds are chief constituents of garlic. These compounds have many health benefits, as they are very efficient in detoxifying natural agents. Therefore, these compounds may be useful for prevention/treatment of cancers. However, DAS has shown appreciable allergic reactions and toxicity, as they can also affect normal cells. Thus their use as in the prevention and treatment of cancer is limited. DAS is a selective inhibitor of cytochrome P450 2E1 (CYP2E1), which is known to metabolize many xenobiotics including alcohol and analgesic drugs in the liver. CYP2E1-mediated alcohol/drug metabolism produce reactive oxygen species and reactive metabolites, which damage DNA, protein, and lipid membranes, subsequently causing liver damage. Several groups have shown that DAS is not only capable of inhibiting alcohol- and drug-mediated cellular toxicities, but also HIV protein- and diabetes-mediated toxicities by selectively inhibiting CYP2E1 in various cell types. However, due to known DAS toxicities, its use as a treatment modality for alcohol/drug- and HIV/diabetes-mediated toxicity have only limited clinical relevance. Therefore, effort is being made to generate DAS analogs, which are potent and selective inhibitor of CYP2E1 and poor substrate of CYP2E1. This review summarizes current advances in the field of DAS, its anticancer properties, role as a CYP2E1 inhibitor, preventing agent of cellular toxicities from alcohol, analgesic drugs, xenobiotics, as well as, from diseases like HIV and diabetes. Finally, this review also provides insights toward developing novel DAS analogues for chemical intervention of many disease conditions by targeting CYP2E1 enzyme. PMID:26264202

  1. Peptide targeted copper-64 radiopharmaceuticals.

    PubMed

    Ma, Michelle T; Donnelly, Paul S

    2011-01-01

    Peptide targeted ⁶⁴Cu-labelled diagnostic agents for positron emission tomography are viable candidates for molecular imaging of cancer. In a clinical setting, optimal image quality relies on selective tumor uptake of the ⁶⁴Cu-labelled radiotracer. The three components of the radiotracer construct--the chelate group, linker and targeting peptide--all influence the biodistribution of the ⁶⁴Cu-labelled radiotracer in vivo. Low or moderate Cu complex stability in vivo results in transmetallation of ⁶⁴Cu to endogenous proteins, giving rise to high background activity. The length and the nature of the linker group affect the affinity of the ⁶⁴Cu-labelled radiotracer for the target receptor. Variations in the peptide sequence can impact on the metabolic stability and therefore the bioavailability and tumor retention of the ⁶⁴Cu-labelled radiotracer in vivo. Lastly, the hydrophilicity of the construct can influence radiotracer metabolism and clearance pathways. Recent advances in the field of peptide targeted ⁶⁴Cu-labelled radiopharmaceuticals involve GRPR-targeted and αvβ3 integrin receptor-targeted constructs. These constructs are based on the bombesin peptide sequence and the RGD recognition motif respectively. These examples are reviewed as case studies in the optimisation of ⁶⁴Cu radiotracer design.

  2. Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT2

    SciTech Connect

    Xiong, Yijia; Chen, Baowei; Shi, Liang; Fredrickson, Jim K.; Bigelow, Diana J.; Squier, Thomas C.

    2011-10-14

    Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) at its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore, carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC which is dependent on the presence of a functional type-2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 hr-1 that is insensitive to O2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 hr-1) that are consistent

  3. Radiotracer Technology in Mixing Processes for Industrial Applications

    PubMed Central

    Othman, N.; Kamarudin, S. K.

    2014-01-01

    Many problems associated with the mixing process remain unsolved and result in poor mixing performance. The residence time distribution (RTD) and the mixing time are the most important parameters that determine the homogenisation that is achieved in the mixing vessel and are discussed in detail in this paper. In addition, this paper reviews the current problems associated with conventional tracers, mathematical models, and computational fluid dynamics simulations involved in radiotracer experiments and hybrid of radiotracer. PMID:24616642

  4. A molecular model for the enzyme cytochrome P450(17 alpha), a major target for the chemotherapy of prostatic cancer.

    PubMed

    Laughton, C A; Neidle, S; Zvelebil, M J; Sternberg, M J

    1990-09-28

    The enzyme cytochrome P450(17 alpha) catalyses two key steps in the biosynthesis of the androgens from pregnanes: the 17 alpha hydroxylation step and the subsequent 17-20 lyase reaction. Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P450(17 alpha) based on that of P450cam, the only cytochrome P450 enzyme for which the crystal structure is known. The model suggests the possibility of two modes of binding of steroid substrates at the active site, perhaps reflecting the dual functionality of the enzyme.

  5. Exploring the transport of plant metabolites using positron emitting radiotracers

    PubMed Central

    Kiser, Matthew R.; Reid, Chantal D.; Crowell, Alexander S.; Phillips, Richard P.; Howell, Calvin R.

    2008-01-01

    Short-lived positron-emitting radiotracer techniques provide time-dependent data that are critical for developing models of metabolite transport and resource distribution in plants and their microenvironments. Until recently these techniques were applied to measure radiotracer accumulation in coarse regions along transport pathways. The recent application of positron emission tomography (PET) techniques to plant research allows for detailed quantification of real-time metabolite dynamics on previously unexplored spatial scales. PET provides dynamic information with millimeter-scale resolution on labeled carbon, nitrogen, and water transport over a small plant-size field of view. Because details at the millimeter scale may not be required for all regions of interest, hybrid detection systems that combine high-resolution imaging with other radiotracer counting technologies offer the versatility needed to pursue wide-ranging plant physiological and ecological research. In this perspective we describe a recently developed hybrid detection system at Duke University that provides researchers with the flexibility required to carry out measurements of the dynamic responses of whole plants to environmental change using short-lived radiotracers. Following a brief historical development of radiotracer applications to plant research, the role of radiotracers is presented in the context of various applications at the leaf to the whole-plant level that integrates cellular and subcellular signals and∕or controls. PMID:19404430

  6. Radiotracers Used for the Scintigraphic Detection of Infection and Inflammation

    PubMed Central

    Tsopelas, Chris

    2015-01-01

    Over the last forty years, a small group of commercial radiopharmaceuticals have found their way into routine medical use, for the diagnostic imaging of patients with infection or inflammation. These molecular radiotracers usually participate in the immune response to an antigen, by tagging leukocytes or other molecules/cells that are endogenous to the process. Currently there is an advancing effort by researchers in the preclinical domain to design and develop new agents for this application. This review discusses radiopharmaceuticals used in the nuclear medicine clinic today, as well as those potential radiotracers that exploit an organism's defence mechanisms to an infectious or inflammatory event. PMID:25741532

  7. Applications of Beta Particle Detection for Synthesis and Usage of Radiotracers Developed for Positron Emission Tomography

    NASA Astrophysics Data System (ADS)

    Dooraghi, Alex Abreu

    Positron Emission Tomography (PET) is a noninvasive molecular imaging tool that requires the use of a radioactive compound or radiotracer which targets a molecular pathway of interest. We have developed and employed three beta particle radiation detection systems to advance PET. Specifically, the goals of these systems are to: 1. Automate dispensing of solutions containing a positron emitting isotope. 2. Monitor radioactivity on-chip during synthesis of a positron emitting radiotracer. 3. Assay cellular uptake on-chip of a positron emitting radiotracer. Automated protocols for measuring and dispensing solutions containing radioisotopes are essential not only for providing an optimum environment for radiation workers, but also to ensure a quantitatively accurate workflow. For the first project, we describe the development and performance of a system for automated radioactivity distribution of beta particle emitting radioisotopes such as fluorine-18 (F-18). Key to the system is a radiation detector in-line with a peristaltic pump. The system demonstrates volume accuracy within 5 % for volumes of 20 muL or greater. When considering volumes of 20 muL or greater, delivered radioactivity is in agreement with the requested radioactivity as measured with the dose calibrator. The integration of the detector and pump leads to a flexible system that can accurately dispense solutions containing F-18 in radioactivity concentrations directly produced from a cyclotron (~ 0.1-1 mCi/muL), to low activity concentrations intended for preclinical mouse scans (~ 1-10 muCi/muL), and anywhere in between. Electrowetting on dielectric (EWOD) is an attractive microfluidic platform for batch synthesis of PET radiotracers. Visualization of radioisotopes on-chip is critical for synthesis optimization and technological development. For the second project, we describe the development and performance of a Cerenkov/real-time imaging system for PET radiotracer synthesis on EWOD. We also investigate

  8. Cross-coupling reactions as valuable tool for the preparation of PET radiotracers.

    PubMed

    Pretze, Marc; Grosse-Gehling, Philipp; Mamat, Constantin

    2011-01-26

    The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of new radiotracers and novel radiolabeling procedures with the most prominent short-lived positron emitters carbon-11 and fluorine-18. Radiolabeling with these radionuclides represents a remarkable challenge. Special attention has to be paid to synthesis time and specific labeling techniques due to the short physical half life of the respective radionuclides ¹¹C (t(½) = 20.4 min) and ¹⁸F (t½) = 109.8 min). In the past, numerous transition metal-catalyzed reactions were employed in organic chemistry, even though only a handful of these coupling reactions were adopted in radiochemical practice. Thus, the implementation of modern synthesis methods like cross-coupling reactions offers the possibility to develop a wide variety of novel radiotracers. The introduction of catalysts based on transition metal complexes bears a high potential for rapid, efficient, highly selective and functional group-tolerating incorporation of carbon-11 and fluorine-18 into target molecules. This review deals with design, application and improvement of transition metal-mediated carbon-carbon as well as carbon-heteroatom cross-coupling reactions as a labeling feature with the focus on the preparation of radiolabeled compounds for molecular imaging.

  9. Development of gamma-emitting, receptor binding radiotracers for imaging the brain and pancreas

    SciTech Connect

    Reba, R.C.

    1990-01-01

    This progress report covers period from Nov. 1, 1989 to Aug. 31, 1990. The long term objective was to develop receptor-binding radiotracers for SPECT or PET imaging of CNS or peripheral nervous system. The specific chemistry aims, as understood on the basis of past findings, were: to synthesize and develop a more polar analogs of 4IQNB, possessing similar binding characteristics but eliminated more rapidly from the surrounding tissues and the target organ, to design a method of introducing a technetium chelating group onto a molecule or cholinergic agent without drastic lowering of its apparent affinity, to synthesize and develop radiotracers based on m-AChR antagonists selective for one of the subtypes of the receptor. The chemistry service aims were to prepare and characterize (R,R)- and (R,S)-4IQNB and derivatives, to provide the triazene intermediate to other investigators, and to provide ({sup 123}I)4IQNB for in vivo imaging. The biochemistry aims were to characterize the vitro and in vivo properties of novel compounds and to perform the pharmacokinetic studies. 3 refs., 5 tabs.

  10. Cytochromes P450 in Nanodiscs

    PubMed Central

    Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators. PMID:20685623

  11. Improved detection of malaria cases in island settings of Vanuatu and Kenya by PCR that targets the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene.

    PubMed

    Isozumi, Rie; Fukui, Mayumi; Kaneko, Akira; Chan, Chim W; Kawamoto, Fumihiko; Kimura, Masatsugu

    2015-06-01

    Detection of sub-microscopic parasitemia is crucial for all malaria elimination programs. PCR-based methods have proven to be sensitive, but two rounds of amplification (nested PCR) are often needed to detect the presence of Plasmodium DNA. To simplify the detection process, we designed a nested PCR method whereby only the primary PCR is required for the detection of the four major human Plasmodium species. Primers designed for the detection of the fifth species, Plasmodium knowlesi, were not included in this study due to the absence of appropriate field samples. Compared to the standard 18S rDNA PCR method, our cytochrome c oxidase III (cox3) method detected 10-50% more cases while maintaining high sensitivities (1.00) for all four Plasmodium species in our samples from Vanuatu (n=77) and Kenya (n=76). Improvement in detection efficiency was more substantial for samples with sub-microscopic parasitemia (54%) than those with observable parasitemia (10-16%). Our method will contribute to improved malaria surveillance in low endemicity settings.

  12. 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.

  13. Radiotracer investigation in an industrial-scale oxidizer.

    PubMed

    Pant, H J; Sharma, V K

    2015-05-01

    A radiotracer investigation was carried out in an industrial-scale oxidizer. The main objectives of the investigation were to measure residence time distribution (RTD) of organic process fluid, determine the mean residence time (MRT) and investigate the degree of axial mixing. Bromine-82 as p-dibromo biphenyl was used as a radiotracer for measuring RTD of the organic process fluid. The MRT of the fluid in the oxidizer was determined to be 390min. An ideal stirred tank model with a plug flow reactor in recirculation stream was used to simulate the measured RTD data and was found suitable for describing flow in the system. Based on the model simulation the mean residence times in oxidizer and recycle stream were estimated. The results of the investigation showed that the oxidizer behaved as a well-mixed reactor whereas the recycle stream behaved as a plug flow reactor. PMID:25766114

  14. Optimization of Integrated Impeller Mixer via Radiotracer Experiments

    PubMed Central

    Othman, N.; Kamarudin, S. K.; Takriff, M. S.; Rosli, M. I.; Engku Chik, E. M. F.; Adnan, M. A. K.

    2014-01-01

    Radiotracer experiments are carried out in order to determine the mean residence time (MRT) as well as percentage of dead zone, Vdead (%), in an integrated mixer consisting of Rushton and pitched blade turbine (PBT). Conventionally, optimization was performed by varying one parameter and others were held constant (OFAT) which lead to enormous number of experiments. Thus, in this study, a 4-factor 3-level Taguchi L9 orthogonal array was introduced to obtain an accurate optimization of mixing efficiency with minimal number of experiments. This paper describes the optimal conditions of four process parameters, namely, impeller speed, impeller clearance, type of impeller, and sampling time, in obtaining MRT and Vdead (%) using radiotracer experiments. The optimum conditions for the experiments were 100 rpm impeller speed, 50 mm impeller clearance, Type A mixer, and 900 s sampling time to reach optimization. PMID:24741344

  15. Microbially mediated cobalt oxidation in seawater revealed by radiotracer experiments

    SciTech Connect

    Lee, B.G.; Fisher, N.S. )

    1993-12-01

    The influence of microbial activity on Co and Mn oxidation in decomposing diatom cultures was determined with radiotracer techniques. Adding a consortium of microorganisms collected from coastal seawater (0.2-3-[mu]m size fraction) to the cultures increased particulate Co formation rates at 18[degrees]C by an order of magnitude (to 3.8% d[sup [minus]1]) and particulate Mn formation rates 3-fold (to 7.9% d[sup [minus

  16. Radiotracer Dilution Method for Mercury Inventory Study in Electrolytic Cells

    SciTech Connect

    Sugiharto; Su'ud, Zaki; Kurniadi, Rizal; Waris, Abdul; Santoso, Sigit Budi; Abidin, Zainal; Santoso, Gatot Budi

    2010-06-22

    Purpose of the experiment is to demonstrate feasibility the use of radiotracer to measure weight of mercury in electrolytic cells of soda industry. The weight of mercury in each cell of the plant is designed approximately 1700 kg. Radiotracer is prepared by mixing {sup 203}Hg radioactive mercury with 2400 g of inactive mercury in a bath. The respective precisely weighted mercury aliquots to be injected into the cells are prepared by pouring approximately 130 g of radioactive mercury taken from the bath into 13 standard vials, in accordance with the number of the cells tested. Four standard references prepared by further dilution of {+-}2 g active mercury taken from the bath to obtain the dilution factors range of 12,000 to 20,000 from which the calibration graph is constructed. The injection process is conducting by pouring the radioactive mercury from aliquots into the flowing mercury at the inlet side of the cell and allows them to mix thoroughly. It is assumed that the mass of the radiotracer injected into a closed system remains constant, at least during the period of the test. From this experiment it was observed that the mixing time is two days after injection of radioactive mercury. The inactive mercury in each electrolytic cell calculated by the radiotracer method is of the range 1351.529 kg to 1966.354 kg with maximum error (95% confidence) is 1.52 %. The accuracy of measurement of the present method is better than gravimetric one which accounts 4 % of error on average.

  17. Radionuclides, radiotracers and radiopharmaceuticals for in vivo diagnosis

    NASA Astrophysics Data System (ADS)

    Wiebe, Leonard I.

    Radioactive tracers for in vivo clinical diagnosis fall within a narrow, strictly-defined set of specifications in respect of their physical properties, chemical and biochemical characteristics, and (approved) medical applications. The type of radioactive decay and physical half-life of the radionuclide are immutable properties which, along with the demands of production and supply, limit the choice of radionuclides used in medicine to only a small fraction of those known to exist. In use, the biochemical and physiological properties of a radiotracer are dictated by the chemical form of the radionuclide. This chemical form may range from elemental, molecular or ionic, to complex compounds formed by coordinate or covalent bonding of the radionuclide to either simple organic or inorganic molecules, or complex macromolecules. Few of the radiotracers which are tested in model systems ever become radiopharmaceuticals in the strictest sense. Radionuclides, radiotracers and radiopharmaceuticals in use are reviewed. Drug legislation and regulations concerning drug manufacture, as well as hospital ethical constraints and legislation concerning unsealed sources of radiation must all be satisfied in order to translate a radiopharmaceutical from the laboratory to clinical use.

  18. Severe liver cirrhosis markedly reduces AhR-mediated induction of cytochrome P450 in rats by decreasing the transcription of target genes.

    PubMed

    Floreani, Maura; De Martin, Sara; Gabbia, Daniela; Barbierato, Massimo; Nassi, Alberto; Mescoli, Claudia; Orlando, Rocco; Bova, Sergio; Angeli, Paolo; Gola, Elisabetta; Sticca, Antonietta; Palatini, Pietro

    2013-01-01

    Although the induction of cytochrome P450 (CYP) has long been investigated in patients with cirrhosis, the question whether liver dysfunction impairs the response to CYP inducers still remains unresolved. Moreover, the mechanism underlying the possible effect of cirrhosis on induction has not been investigated. Since ethical constraints do not permit methodologically rigorous studies in humans, this question was addressed by investigating the effect of the prototypical inducer benzo[a]pyrene (BP) on CYP1A1 and CYP1A2 in cirrhotic rats stratified according to the severity of liver dysfunction. We simultaneously assessed mRNA level, protein expression and enzymatic activity of the CYP1A enzymes, as well as mRNA and protein expressions of the aryl hydrocarbon receptor (AhR), which mediates the BP effect. Basal mRNA and protein expressions of CYP1A1 were virtually absent in both healthy and cirrhotic rats. On the contrary, CYP1A2 mRNA, protein and enzyme activity were constitutively present in healthy rats and decreased significantly as liver function worsened. BP treatment markedly increased the concentrations of mRNA and immunodetectable protein, and the enzymatic activities of both CYP1A enzymes to similar levels in healthy and non-ascitic cirrhotic rats. Induced mRNA levels, protein expressions and enzymatic activities of both CYPs were much lower in ascitic rats and were proportionally reduced. Both constitutive and induced protein expressions of AhR were significantly lower in ascitic than in healthy rats. These results indicate that the inducibility of CYP1A enzymes is well preserved in compensated cirrhosis, whereas it is markedly reduced when liver dysfunction becomes severe. Induction appears to be impaired at the transcriptional level, due to the reduced expression of AhR, which controls the transcription of CYP1A genes.

  19. Liver-specific cytochrome P450 CYP2C22 is a direct target of retinoic acid and a retinoic acid-metabolizing enzyme in rat liver.

    PubMed

    Qian, Linxi; Zolfaghari, Reza; Ross, A Catharine

    2010-07-01

    Several cytochrome P450 (CYP) enzymes catalyze the C4-hydroxylation of retinoic acid (RA), a potent inducer of cell differentiation and an agent in the treatment of several diseases. Here, we have characterized CYP2C22, a member of the rat CYP2C family with homology to human CYP2C8 and CYP2C9. CYP2C22 was expressed nearly exclusively in hepatocytes, where it was one of the more abundant mRNAs transcripts. In H-4-II-E rat hepatoma cells, CYP2C22 mRNA was upregulated by all-trans (at)-RA, and Am580, a nonmetabolizable analog of at-RA. In comparison, in primary human hepatocytes, at-RA increased CYP2C9 but not CYP2C8 mRNA. Analysis of the CYP2C22 promoter region revealed a RA response element (5'-GGTTCA-(n)5-AGGTCA-3') in the distal flanking region, which bound the nuclear hormone receptors RAR and RXR and which was required for transcriptional activation response of this promoter to RA in CYP2C22-luciferase-transfected RA-treated HepG2 cells. The cDNA-expressed CYP2C22 protein metabolized [3H]at-RA to more polar metabolites. While long-chain polyunsaturated fatty acids competed, 9-cis-RA was a stronger competitor. Our studies demonstrate that CYP2C22 is a high-abundance, retinoid-inducible, hepatic P450 with the potential to metabolize at-RA, providing additional insight into the role of the CYP2C gene family in retinoid homeostasis.

  20. 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.

  1. Simulation of multihaem cytochromes.

    PubMed

    Soares, Cláudio M; Baptista, António M

    2012-03-01

    This article presents an overview of the simulation studies of the behaviour of multihaem cytochromes using theoretical/computational methodologies, with an emphasis on cytochrome c(3). It starts with the first studies using rigid molecules and continuum electrostatic models, where protonation and redox events were treated as independent. The gradual addition of physical details is then described, from the inclusion of proton isomerism, to the proper treatment of the thermodynamics of electron-proton coupling, to the explicit inclusion of the solvent and protein structural reorganization into the models, culminating with the method for molecular dynamics simulations at constant pH and reduction potential, where the solvation, conformational, protonation and redox features are all simulated in a fully integrated and coupled way. We end with a discussion of the strategies used to study the interaction between multihaem cytochromes, taking into account the further coupling effect introduced by the molecular association.

  2. Synthesis, radioiodination and in vivo screening of novel potent iodinated and fluorinated radiotracers as melanoma imaging and therapeutic probes.

    PubMed

    Maisonial, Aurélie; Billaud, Emilie M F; Besse, Sophie; Rbah-Vidal, Latifa; Papon, Janine; Audin, Laurent; Bayle, Martine; Galmier, Marie-Josèphe; Tarrit, Sébastien; Borel, Michèle; Askienazy, Serge; Madelmont, Jean-Claude; Moins, Nicole; Auzeloux, Philippe; Miot-Noirault, Elisabeth; Chezal, Jean-Michel

    2013-05-01

    In order to develop new iodinated and fluorinated matched-pair radiotracers for Single-Photon Emission Computed Tomography (SPECT)/Positron Emission Tomography (PET) imaging and targeted radionuclide therapy of melanoma, we successfully synthesized and radiolabelled with iodine-125 seven new derivatives, starting from our previously described lead structure 3. The relevance of these radiotracers for gamma scintigraphic imaging of melanoma in rodent was assessed. The tumoural radioactivity uptake was most often high and specific even at early time points (12.1-18.3% ID/g at 3 h p.i. for [(125)I]39-42) and a fast clearance from the non-target organs was observed. Also, calculated effective doses that could be delivered to tumours when using corresponding [(131)I]-labelled analogues were generally higher than 100 cGy/MBq injected (98.9-150.5 cGy/MBq for [(131)I]39-42). These results make compounds 39-42 suitable candidates for (i) PET imaging of melanoma after labelling with fluorine-18 and (ii) targeted radionuclide therapy of disseminated melanoma after labelling with iodine-131.

  3. Functional investigation of bone implant viability using radiotracers in a new model of osteonecrosis

    PubMed Central

    Schiper, Luis; Faintuch, Bluma Linkowski; da Silva Badaró, Roberto José; de Oliveira, Erica Aparecida; Chavez, Victor E. Arana; Chinen, Elisangela; Faintuch, Joel

    2016-01-01

    OBJECTIVES: Conventional imaging methods are excellent for the morphological characterization of the consequences of osteonecrosis; however, only specialized techniques have been considered useful for obtaining functional information. To explore the affinity of radiotracers for severely devascularized bone, a new mouse model of isolated femur implanted in a subcutaneous abdominal pocket was devised. To maintain animal mobility and longevity, the femur was harvested from syngeneic donors. Two technetium-99m-labeled tracers targeting angiogenesis and bone matrix were selected. METHODS: Medronic acid and a homodimer peptide conjugated with RGDfK were radiolabeled with technetium-99m, and biodistribution was evaluated in Swiss mice. The grafted and control femurs were evaluated after 15, 30 and 60 days, including computed tomography (CT) and histological analysis. RESULTS: Radiolabeling achieved high (>95%) radiochemical purity. The biodistribution confirmed good blood clearance 1 hour after administration. For 99mTc-hydrazinonicotinic acid (HYNIC)-E-[c(RGDfK)2, remarkable renal excretion was observed compared to 99mTc-methylene diphosphonate (MDP), but the latter, as expected, revealed higher bone uptake. The results obtained in the control femur were equal at all time points. In the implanted femur, 99mTc-HYNIC-E-[c(RGDfK)2 uptake was highest after 15 days, consistent with early angiogenesis. Regarding 99mTc-MDP in the implant, similar uptake was documented at all time points, consistent with sustained bone viability; however, the uptake was lower than that detected in the control femur, as confirmed by histology. CONCLUSIONS: 1) Graft viability was successfully diagnosed using radiotracers in severely ischemic bone at all time points. 2) Analogously, indirect information about angiogenesis could be gathered using 999mTc-HYNIC-E-[c(RGDfK)2. 3) These techniques appear promising and warrant further studies to determine their potential clinical applications. PMID

  4. A one-step microwave-assisted synthetic method for an O/S-chemoselective route to derivatives of the first adenosine A3 PET radiotracer.

    PubMed

    Shanab, Karem; Neudorfer, Catharina; Holzer, Wolfgang; Mitterhauser, Markus; Wadsak, Wolfgang; Spreitzer, Helmut

    2014-01-01

    The synthesis of reference standards and expected in vivo metabolites of the first adenosine A3 PET radiotracer [18F]FE@SUPPY ([18F]fluoroethyl 4,6-diethyl-5-[(ethyl-sulfanyl)carbonyl]-2-phenylpyridine-3-carboxylate) was achieved by using a straightforward microwave assisted alkylation method, which allowed O/S-chemoselective alkylation of the starting material 1 to give each target compound 2-8 in a single step. PMID:24699149

  5. Discharge rate measurements in a canal using radiotracer methods.

    PubMed

    Pant, H J; Goswami, Sunil; Biswal, Jayashree; Samantray, J S; Sharma, V K

    2016-06-01

    Discharge rates of water were measured in a canal using radiotracer methods with an objective to validate the efficacy of Concrete Volute Pumps (CVPs) installed at various pumping stations along the canal. Pulse velocity and dilution methods were applied to measure the discharge rates using Iodine-131 as a radiotracer. The discharge rate measured in one of the sections of the canal using the pulse velocity method was found to be 22.5m(3)/s, whereas the discharge rates measured using the dilution method in four different sections of the canal varied from 20.27 to 20.62m(3)/s with single CVP in operation. The standard error in discharge rate measurements using dilution method ranged from ±1.1 to ±1.8%. The experimentally measured values of the discharge rate were in good agreement with the design value of the discharge rate (20m(3)/s) thus validating the performance of the CVPs used in the canal. PMID:27016711

  6. Discharge rate measurements in a canal using radiotracer methods.

    PubMed

    Pant, H J; Goswami, Sunil; Biswal, Jayashree; Samantray, J S; Sharma, V K

    2016-06-01

    Discharge rates of water were measured in a canal using radiotracer methods with an objective to validate the efficacy of Concrete Volute Pumps (CVPs) installed at various pumping stations along the canal. Pulse velocity and dilution methods were applied to measure the discharge rates using Iodine-131 as a radiotracer. The discharge rate measured in one of the sections of the canal using the pulse velocity method was found to be 22.5m(3)/s, whereas the discharge rates measured using the dilution method in four different sections of the canal varied from 20.27 to 20.62m(3)/s with single CVP in operation. The standard error in discharge rate measurements using dilution method ranged from ±1.1 to ±1.8%. The experimentally measured values of the discharge rate were in good agreement with the design value of the discharge rate (20m(3)/s) thus validating the performance of the CVPs used in the canal.

  7. PET-Specific Parameters and Radiotracers in Theoretical Tumour Modelling

    PubMed Central

    Marcu, Loredana G.; Bezak, Eva

    2015-01-01

    The innovation of computational techniques serves as an important step toward optimized, patient-specific management of cancer. In particular, in silico simulation of tumour growth and treatment response may eventually yield accurate information on disease progression, enhance the quality of cancer treatment, and explain why certain therapies are effective where others are not. In silico modelling is demonstrated to considerably benefit from information obtainable with PET and PET/CT. In particular, models have successfully integrated tumour glucose metabolism, cell proliferation, and cell oxygenation from multiple tracers in order to simulate tumour behaviour. With the development of novel radiotracers to image additional tumour phenomena, such as pH and gene expression, the value of PET and PET/CT data for use in tumour models will continue to grow. In this work, the use of PET and PET/CT information in in silico tumour models is reviewed. The various parameters that can be obtained using PET and PET/CT are detailed, as well as the radiotracers that may be used for this purpose, their utility, and limitations. The biophysical measures used to quantify PET and PET/CT data are also described. Finally, a list of in silico models that incorporate PET and/or PET/CT data is provided and reviewed. PMID:25788973

  8. Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3*

    PubMed Central

    Kim, Mi-Sun; Jang, Jichan; AB Rahman, Nurlilah Binte; Pethe, Kevin; Berry, Edward A.; Huang, Li-Shar

    2015-01-01

    Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2-a]pyridine derivatives, is believed to target the pathogen's homolog of the mitochondrial bc1 complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a “supercomplex” with a cytochrome aa3-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa3 supercomplex using a strain of M. smegmatis that has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding and provide structural insight into a third class of the bc complex superfamily. PMID:25861988

  9. Therapeutic doses of SkQ1 do not induce cytochromes P450 in rat liver.

    PubMed

    Myasoedova, K N; Silachev, D N

    2014-10-01

    The effect of SkQ1 (a mitochondria-targeted antioxidant) on the level of cytochromes P450 in rat liver was studied. It was found that administration of therapeutic dose of SkQ1 with drinking water for 5 days (250 nmol/kg of body weight per day) did not alter the level of cytochromes P450. Under the same conditions, the standard dose of phenobarbital used for the induction of cytochromes P450 caused the 2.7-fold increase in the content of these cytochromes. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in rats.

  10. Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain

    PubMed Central

    Bonomi, Robin; Mukhopadhyay, Uday; Shavrin, Aleksandr; Yeh, Hsien-Hsien; Majhi, Anjoy; Dewage, Sajeewa W.; Najjar, Amer; Lu, Xin; Cisneros, G. Andrés; Tong, William P.; Alauddin, Mian M.; Liu, Ren-Shuan; Mangner, Thomas J.; Turkman, Nashaat; Gelovani, Juri G.

    2015-01-01

    Histone deacetylases (HDAC’s) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa–specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [18F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [18F]DFAHA and previously reported [18F]FAHA. PET imaging with [18F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [18F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases. PMID:26244761

  11. Biogenesis of respiratory cytochromes in bacteria.

    PubMed Central

    Thöny-Meyer, L

    1997-01-01

    Biogenesis of respiratory cytochromes is defined as consisting of the posttranslational processes that are necessary to assemble apoprotein, heme, and sometimes additional cofactors into mature enzyme complexes with electron transfer functions. Different biochemical reactions take place during maturation: (i) targeting of the apoprotein to or through the cytoplasmic membrane to its subcellular destination; (ii) proteolytic processing of precursor forms; (iii) assembly of subunits in the membrane and oligomerization; (iv) translocation and/or modification of heme and covalent or noncovalent binding to the protein moiety; (v) transport, processing, and incorporation of other cofactors; and (vi) folding and stabilization of the protein. These steps are discussed for the maturation of different oxidoreductase complexes, and they are arranged in a linear pathway to best account for experimental findings from studies concerning cytochrome biogenesis. The example of the best-studied case, i.e., maturation of cytochrome c, appears to consist of a pathway that requires at least nine specific genes and more general cellular functions such as protein secretion or the control of the redox state in the periplasm. Covalent attachment of heme appears to be enzyme catalyzed and takes place in the periplasm after translocation of the precursor through the membrane. The genetic characterization and the putative biochemical functions of cytochrome c-specific maturation proteins suggest that they may be organized in a membrane-bound maturase complex. Formation of the multisubunit cytochrome bc, complex and several terminal oxidases of the bo3, bd, aa3, and cbb3 types is discussed in detail, and models for linear maturation pathways are proposed wherever possible. PMID:9293186

  12. The uses of radiotracers in the life sciences

    NASA Astrophysics Data System (ADS)

    Ruth, Thomas J.

    2009-01-01

    Radionuclides have been used to follow physical, chemical and biological processes almost from the time of their discovery. Probably the application with the biggest impact has been in the medical field where radionuclides have been incorporated into biologically active molecules and used to diagnose a wide variety of diseases and to treat many disorders. Other uses in the life sciences, in general, are related to using a radioactive isotope as marker for an existing species such as nitrogen-13 in plant studies or copper-67 to track copper catalysts in phytoplankton. This review describes in general terms these uses as well as providing the reader with the background related to the physical properties of radioactive decay, the concepts associated with the production of radionuclides using reactors or accelerators and the fundamentals of imaging radioactivity. The advances in imaging technology in recent years has had a profound impact on the use of radionuclides in positron emission tomography and the coupling of other imaging modalities to provide very precise insights into human disease. The variety of uses for radiotracers in science is almost boundless dependent only upon ones imagination.

  13. OpenPET: A Flexible Electronics System for Radiotracer Imaging.

    PubMed

    Moses, W W; Buckley, S; Vu, C; Peng, Q; Pavlov, N; Choong, W-S; Wu, J; Jackson, C

    2009-10-24

    We present the design for OpenPET, an electronics readout system designed for prototype radiotracer imaging instruments. The critical requirements are that it has sufficient performance, channel count, channel density, and power consumption to service a complete camera, and yet be simple, flexible, and customizable enough to be used with almost any detector or camera design. An important feature of this system is that each analog input is processed independently. Each input can be configured to accept signals of either polarity as well as either differential or ground referenced signals. Each signal is digitized by a continuously sampled ADC, which is processed by an FPGA to extract pulse height information. A leading edge discriminator creates a timing edge that is "time stamped" by a TDC implemented inside the FPGA. This digital information from each channel is sent to an FPGA that services 16 analog channels, and information from multiple channels is processed by this FPGA to perform logic for crystal lookup, DOI calculation, calibration, etc. As all of this processing is controlled by firmware and software, it can be modified / customized easily. The system is open source, meaning that all technical data (specifications, schematics and board layout files, source code, and instructions) will be publicly available.

  14. OpenPET: A Flexible Electronics System for Radiotracer Imaging

    PubMed Central

    Moses, W. W.; Buckley, S.; Vu, C.; Peng, Q.; Pavlov, N.; Choong, W.-S.; Wu, J.; Jackson, C.

    2011-01-01

    We present the design for OpenPET, an electronics readout system designed for prototype radiotracer imaging instruments. The critical requirements are that it has sufficient performance, channel count, channel density, and power consumption to service a complete camera, and yet be simple, flexible, and customizable enough to be used with almost any detector or camera design. An important feature of this system is that each analog input is processed independently. Each input can be configured to accept signals of either polarity as well as either differential or ground referenced signals. Each signal is digitized by a continuously sampled ADC, which is processed by an FPGA to extract pulse height information. A leading edge discriminator creates a timing edge that is “time stamped” by a TDC implemented inside the FPGA. This digital information from each channel is sent to an FPGA that services 16 analog channels, and information from multiple channels is processed by this FPGA to perform logic for crystal lookup, DOI calculation, calibration, etc. As all of this processing is controlled by firmware and software, it can be modified / customized easily. The system is open source, meaning that all technical data (specifications, schematics and board layout files, source code, and instructions) will be publicly available. PMID:21297894

  15. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  16. Isatin sulfonamides: potent caspases-3 and -7 inhibitors, and promising PET and SPECT radiotracers for apoptosis imaging.

    PubMed

    Limpachayaporn, Panupun; Schäfers, Michael; Haufe, Günter

    2015-01-01

    Caspases-3 and -7 play an essential role in apoptosis. Isatin sulfonamides have been identified as potent inhibitors of these executing caspases. Besides pharmacological application, these compounds can also serve as recognition units to target caspases using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) when labeled with a positron or a gamma emitter. Fluorinated, alkylated, arylated isatin derivatives, in addition to derivatives modified with heterocycles, have been prepared in order to improve their binding potency, selectivity and metabolic stability. Structural optimization has led to stable, highly active inhibitors, which after labeling have been applied in PET studies in tumor mouse models and for first preclinical and clinical investigations with healthy human volunteers. The results support further development of such radiotracers for clinical apoptosis imaging. PMID:26132525

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

    PubMed Central

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

    2015-01-01

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

  18. Development of a system for real-time measurements of metabolite transport in plants using short-lived positron-emitting radiotracers

    NASA Astrophysics Data System (ADS)

    Kiser, Matthew R.

    be introduced to plants as biologically active molecules such as 11CO2, N13O-3, 18F--[H2O], and H152O . Plants for these studies are grown in controlled-environment chambers at the Phytotron. The chambers offer an array of control for temperature, humidity, atmospheric CO2 concentration, and light intensity. Additionally, the Phytotron houses one large reach-in growth chamber that is dedicated to this project for radioisotope labeling measurements. There are several important properties of short-lived positron-emitting radio-tracers that make them well suited for use in investigating metabolite transport in plants. First, because the molecular mass of a radioisotope-tagged compound is only minutely different from the corresponding stable compound, radiotracer substances should be metabolized and transported in plants the same as their non-radioactive counterparts. Second, because the relatively high energy gamma rays emitted from electron-positron annihilation are attenuated very little by plant tissue, the real-time distribution of a radiotracer can be measured in vivo in plants. Finally, the short radioactive half-lives of these isotopes allow for repeat measurements on the same plant in a short period of time. For example, in studies of short-term environmental changes on plant metabolite dynamics, a single plant can be labeled multiple times to measure its responses to different, environmental conditions. Also, different short-lived radiotracers can be applied to the same plant over a short period of time to investigate the transport and allocation of various metabolites. This newly developed system provides the capabilities for production of 11CO2 at TUNL, transfer of the 11CO 2 gas from the target area at TUNL to a radiation-shielded cryogenic trap at the Phytotron, labeling of photoassimilates with 11C, and in vivo gamma-ray detection for real-time measurements of the radiotracer distribution in small plants. The experimental techniques and instrumentation

  19. New Arabidopsis thaliana Cytochrome c Partners: A Look Into the Elusive Role of Cytochrome c in Programmed Cell Death in Plants*

    PubMed Central

    Martínez-Fábregas, Jonathan; Díaz-Moreno, Irene; González-Arzola, Katiuska; Janocha, Simon; Navarro, José A.; Hervás, Manuel; Bernhardt, Rita; Díaz-Quintana, Antonio; De la Rosa, Miguel Á.

    2013-01-01

    Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280. PMID:24019145

  20. 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.

  1. Cyclotron production and potential clinical application of Iodine-124 labeled radiotracers

    NASA Astrophysics Data System (ADS)

    Finn, R.; Balatoni, J.; Kothari, P.; Pentlow, K.; Sheh, Y.; Lom, C.; Dahl, J.; Eckelman, W.; Plascjak, P.; Adams, H. R.; Larson, S. M.

    2001-07-01

    Positron emission tomography (PET) is a dynamic molecular imaging technique applicable to clinical research, drug development as well as clinical diagnoses. The potential for PET is derived from specificity of the radiotracers and radioligands that are synthesized to monitor the biochemical or physiological processes. Further developments will depend on an increasing availability of unique radiotracers. Iodine-124, a radionuclide that has potential for both diagnostic and therapeutic applications, possesses a half-life of 4.18 days and decays by positron emission (23.3%) and electron capture (76.7%). The preparation of this radionuclide via the 124Te(p,n)124I nuclear reaction is described as well as chemistry associated with the preparation of specific radiotracers and radiopharmaceuticals incorporating iodine-124 at Memorial Sloan-Kettering Cancer Center.

  2. Distributed microprocessor automation network for synthesizing radiotracers used in positron emission tomography

    SciTech Connect

    Russell, J.A.G.; Alexoff, D.L.; Wolf, A.P.

    1984-09-01

    This presentation describes an evolving distributed microprocessor network for automating the routine production synthesis of radiotracers used in Positron Emission Tomography. We first present a brief overview of the PET method for measuring biological function, and then outline the general procedure for producing a radiotracer. The paper identifies several reasons for our automating the syntheses of these compounds. There is a description of the distributed microprocessor network architecture chosen and the rationale for that choice. Finally, we speculate about how this network may be exploited to extend the power of the PET method from the large university or National Laboratory to the biomedical research and clinical community at large. 20 refs. (DT)

  3. Distributed Microprocessor Automation Network for Synthesizing Radiotracers Used in Positron Emission Tomography [PET

    DOE R&D Accomplishments Database

    Russell, J. A. G.; Alexoff, D. L.; Wolf, A. P.

    1984-09-01

    This presentation describes an evolving distributed microprocessor network for automating the routine production synthesis of radiotracers used in Positron Emission Tomography. We first present a brief overview of the PET method for measuring biological function, and then outline the general procedure for producing a radiotracer. The paper identifies several reasons for our automating the syntheses of these compounds. There is a description of the distributed microprocessor network architecture chosen and the rationale for that choice. Finally, we speculate about how this network may be exploited to extend the power of the PET method from the large university or National Laboratory to the biomedical research and clinical community at large. (DT)

  4. Measurement of leaching from simulated nuclear-waste glass using radiotracers

    SciTech Connect

    Bates, J.K.; Jardine, L.J.; Steindler, M.J.

    1982-09-01

    The use of radiotracer spiking as a method of measuring the leaching from simulated nuclear-waste glass is shown to give results comparable with other analytical detection methods. The leaching behavior of /sup 85/Sr, /sup 106/Ru, /sup 133/Ba, /sup 137/Cs, /sup 141/Ce, /sup 152/Eu, and other isotopes is measured for several defense waste glasses. These tests show that radiotracer spiking is a sensitive, multielement technique that can provide leaching data, for actual waste elements, that are difficult to obtain by other methods. Additionally, a detailed procedure is described that allows spiked glass to be prepared with a suitable distribution of radionuclides.

  5. Batch-reactor microfluidic device: first human use of a microfluidically produced PET radiotracer.

    PubMed

    Lebedev, Artem; Miraghaie, Reza; Kotta, Kishore; Ball, Carroll E; Zhang, Jianzhong; Buchsbaum, Monte S; Kolb, Hartmuth C; Elizarov, Arkadij

    2013-01-01

    The very first microfluidic device used for the production of (18)F-labeled tracers for clinical research is reported along with the first human Positron Emission Tomography scan obtained with a microfluidically produced radiotracer. The system integrates all operations necessary for the transformation of [(18)F]fluoride in irradiated cyclotron target water to a dose of radiopharmaceutical suitable for use in clinical research. The key microfluidic technologies developed for the device are a fluoride concentration system and a microfluidic batch reactor assembly. Concentration of fluoride was achieved by means of absorption of the fluoride anion on a micro ion-exchange column (5 μL of resin) followed by release of the radioactivity with 45 μL of the release solution (95 ± 3% overall efficiency). The reactor assembly includes an injection-molded reactor chip and a transparent machined lid press-fitted together. The resulting 50 μL cavity has a unique shape designed to minimize losses of liquid during reactor filling and liquid evaporation. The cavity has 8 ports for gases and liquids, each equipped with a 2-way on-chip mechanical valve rated for pressure up to 20.68 bar (300 psi). The temperature is controlled by a thermoelectric heater capable of heating the reactor up to 180 °C from RT in 150 s. A camera captures live video of the processes in the reactor. HPLC-based purification and reformulation units are also integrated in the device. The system is based on "split-box architecture", with reagents loaded from outside of the radiation shielding. It can be installed either in a standard hot cell, or as a self-shielded unit. Along with a high level of integration and automation, split-box architecture allowed for multiple production runs without the user being exposed to radiation fields. The system was used to support clinical trials of [(18)F]fallypride, a neuroimaging radiopharmaceutical under IND Application #109,880.

  6. Pre-clinical characterization of [11C]R05013853 as a novel radiotracer for imaging of the glycine transporter type 1 by positron emission tomography.

    PubMed

    Borroni, Edilio; Zhou, Yun; Ostrowitzki, Susanne; Alberati, Daniela; Kumar, Anil; Hainzl, Dominik; Hartung, Thomas; Hilton, John; Dannals, Robert F; Wong, Dean F

    2013-07-15

    A specific positron emission tomography (PET) radiotracer for the glycine transporter type 1 (GlyT1) would constitute an imaging biomarker to investigate the distribution of GlyT1 in normal individuals and those with neuropsychiatric disorders. In addition it could demonstrate the ability of a novel drug to reach its target in the brain and enable receptor occupancy studies, thus facilitating drug development. In this article we describe the evaluation in non-human primates of two candidate PET radiotracers ([(11)C]RO5013852 and [(11)C]RO5013853) previously characterized in the rat. Both radiotracers showed acceptable uptake in the baboon brain and heterogeneous distribution consistent with that reported for GlyT1. In vivo blockade studies with two specific glycine reuptake inhibitors (GRIs), RO5013853 and bitopertin (RG1678, reduced uptake of both tracers to homogenous levels across brain regions and demonstrated specificity of the signal. [(11)C]RO5013853 showed a larger specific signal and slightly higher brain uptake and was therefore selected for further characterization. Quantitative compartmental analysis of PET data showed that the 2-tissue compartment model with 5 parameters was the most appropriate to describe the kinetics of [(11)C]RO5013853. Two additional methods were used: a) the Logan graphical analysis using plasma input and, b) a linear parametric imaging approach with the 2-tissue compartmental model. These produced VT estimates of comparable magnitude, namely, pons, thalamus and cerebellum>caudate, putamen and cortical regions. High resolution autoradiography with tritiated RO5013853 was used to confirm the binding pattern observed by PET. In vivo metabolism studies in the baboon demonstrated the formation of a single, radiolabeled metabolite more polar than the parent compound. Finally, [(11)C]RO5013853 was used to quantify the degree of cerebral GlyT1 occupancy observed in the baboon following oral administration of bitopertin, a selective GRI

  7. "High specific activity" radiotracers for metallo-toxicological studies: cyclotron and nuclear reactor production, radiochemical separation and "quality control": platinum, iridium, gold, copper and gallium.

    PubMed

    Bonardi, Mauro; Groppi, Flavia; Birattari, Claudio; Arginelli, Dolores

    2002-09-01

    Very High Specific Activity RadioNuclides, HSARN, are a powerful tool to label a wide variety of chemical elements and compounds present in the biosphere in ultra-trace amounts. Medium and high Z radionuclides, can be produced by irradiation in light-ions accelerator and sometimes nuclear reactor. If the nuclear reaction product has atomic number different from irradiated target, it is possible separating the radioactive nuclide from irradiated target, without addition of isotopic carrier. These kinds of radionuclides are named No Carrier Added, NCA, and their specific activity is very high and can reach values close to the theoretical Carrier Free one. The true specific activity must be determined by use of very sensitive radioanalytical techniques. If a low isotopic dilution factor is obtained, these radiotracers are used to label inorganic species and complexes of elements, which are presently introduced into the echo-systems by human activities. New production methods for NCA Pt, Ir, Au, Cu and Ga radiotracers are presented, with some details on radiochemistry and quality controls.

  8. [(18)F]-Group 13 fluoride derivatives as radiotracers for positron emission tomography.

    PubMed

    Chansaenpak, Kantapat; Vabre, Boris; Gabbaï, François P

    2016-02-21

    The field of (18)F chemistry is rapidly expanding because of the use of this radionuclide in radiotracers for positron emission tomography (PET). Until recently, most [(18)F]-radiotracers were generated by the direct attachment of (18)F to a carbon in the organic backbone of the radiotracer. The past decade has witnessed the emergence of a new strategy based on the formation of an (18)F-group 13 element bond. This approach, which is rooted in the field of fluoride anion complexation/coordination chemistry, has led to the development of a remarkable family of boron, aluminium and gallium [(18)F]-fluoride anion complexing agents which can be conjugated with peptides and small molecules to generate disease specific PET radiotracers. This review is dedicated to the chemistry of these group 13 [(18)F]-fluorides anion complexing agents and their use in PET. Some of the key fluoride-binding motifs covered in this review include the trifluoroborate unit bound to neutral or cationic electron deficient backbones, the BF2 unit of BODIPY dyes, and AlF or GaF3 units coordinated to multidentate Lewis basic ligands. In addition to describing how these moieties can be converted into their [(18)F]-analogs, this review also dicusses their incorporation into bioconjugates for application in PET. PMID:26548467

  9. Increasing the accuracy of radiotracer monitoring in one-dimensional flow using polynomial deconvolution correction.

    PubMed

    Peyvandi, Reza Gholipour; Taheri, Ali

    2016-01-01

    Factors such as type of fluid movement and gamma-ray scattering may decrease the precision of the radiotracer monitoring as the response to a short tracer injection. Practical experiences using polynomial deconvolution techniques are presented. These techniques were successfully applied for correcting the obtained experimental results and increasing the time resolution of the method. PMID:26609682

  10. Methods to Increase the Metabolic Stability of (18)F-Radiotracers.

    PubMed

    Kuchar, Manuela; Mamat, Constantin

    2015-01-01

    The majority of pharmaceuticals and other organic compounds incorporating radiotracers that are considered foreign to the body undergo metabolic changes in vivo. Metabolic degradation of these drugs is commonly caused by a system of enzymes of low substrate specificity requirement, which is present mainly in the liver, but drug metabolism may also take place in the kidneys or other organs. Thus, radiotracers and all other pharmaceuticals are faced with enormous challenges to maintain their stability in vivo highlighting the importance of their structure. Often in practice, such biologically active molecules exhibit these properties in vitro, but fail during in vivo studies due to obtaining an increased metabolism within minutes. Many pharmacologically and biologically interesting compounds never see application due to their lack of stability. One of the most important issues of radiotracers development based on fluorine-18 is the stability in vitro and in vivo. Sometimes, the metabolism of (18)F-radiotracers goes along with the cleavage of the C-F bond and with the rejection of [(18)F]fluoride mostly combined with high background and accumulation in the skeleton. This review deals with the impact of radiodefluorination and with approaches to stabilize the C-F bond to avoid the cleavage between fluorine and carbon. PMID:26404227

  11. Use of supercritical carbon dioxide fluid as a solvent for the purification of pet radiotracers

    SciTech Connect

    Ferrieri, R.A.; Fowler, J.S.; Wolf, A.P.

    1993-12-31

    We have identified superfluid chromatography (SFC) as a promising method which could offer advantages in radiotracer purification through rapid separation, as well as, improved recovery and purity of labeled product. Using SF CO{sub 2} as the mobile phase for chromatographic separation of labeled product would eliminate the need for solvent removal from product prior to delivery.

  12. 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

  13. Mutational and biochemical analysis of cytochrome c', a nitric oxide-binding lipoprotein important for adaptation of Neisseria gonorrhoeae to oxygen-limited growth.

    PubMed

    Turner, Susan M; Moir, James W B; Griffiths, Lesley; Overton, Timothy W; Smith, Harry; Cole, Jeff A

    2005-06-01

    Neisseria gonorrhoeae is a prolific source of c-type cytochromes. Five of the constitutively expressed cytochromes are predicted, based on in silico analysis of the N. gonorrhoeae genome, to be components of the cytochrome bc1 complex, cytochrome c oxidase cbb3 or periplasmic cytochromes involved in electron transfer reactions typical of a bacterium with a microaerobic physiology. Cytochrome c peroxidase was previously shown to be a lipoprotein expressed only during oxygen-limited growth. The final c-type cytochrome, cytochrome c', similar to cytochrome c peroxidase, includes a lipobox required for targeting to the outer membrane. Maturation of cytochrome c' was partially inhibited by globomycin, an antibiotic that specifically inhibits signal peptidase II, resulting in the accumulation of the prolipoprotein in the cytoplasmic membrane. Disruption of the gonococcal cycP gene resulted in an extended lag phase during microaerobic growth in the presence but not in the absence of nitrite, suggesting that cytochrome c' protects the bacteria from NO generated by nitrite reduction during adaptation to oxygen-limited growth. The cytochrome c' gene was overexpressed in Escherichia coli and recombinant cytochrome c' was shown to be targeted to the outer membrane. Spectroscopic evidence is presented showing that gonococcal cytochrome c' is similar to previously characterized cytochrome c' proteins and that it binds NO in vitro. The demonstration that two of the seven gonococcal c-type cytochromes fulfil specialized functions and are outer membrane lipoproteins suggests that the localization of these lipoproteins close to the bacterial surface provides effective protection against external assaults from reactive oxygen and reactive nitrogen species.

  14. Role of Cytochrome P450s in Inflammation.

    PubMed

    Christmas, Peter

    2015-01-01

    Cytochrome P450 epoxygenases and hydroxylases play a regulatory role in the activation and suppression of inflammation by generating or metabolizing bioactive mediators. CYP2C and CYP2J epoxygenases convert arachidonic acid to anti-inflammatory epoxyeicosatrienoic acids, which have protective effects in a variety of disorders including cardiovascular disease and metabolic syndrome. CYP4A and CYP4F hydroxylases have the ability to metabolize multiple substrates related to the regulation of inflammation and lipid homeostasis, and it is a challenge to determine which substrates are physiologically relevant for each enzyme; the best-characterized activities include generation of 20-hydroxyeicosatetraenoic acid and inactivation of leukotriene B4. The expression of hepatic drug-metabolizing cytochrome P450s is modulated by cytokines during inflammation, resulting in changes to the pharmacokinetics of prescribed medications. Cytochrome P450s are therefore the focus of intersecting challenges in the pharmacology of inflammation: not only do they represent targets for development of new anti-inflammatory drugs but they also contribute to variability in drug efficacy or toxicity in inflammatory disease. Animal models and primary hepatocytes have been used extensively to study the effects of cytokines on cytochrome P450 expression and activity. However, it is difficult to predict changes in drug exposure in patients because the response to inflammation varies depending on the disease state, its time course, and the cytochrome P450 involved. In these circumstances, the development of endogenous markers of cytochrome P450 metabolism might provide a useful tool to reevaluate drug dosage and choice of therapy.

  15. ALB3 Insertase Mediates Cytochrome b6 Co-translational Import into the Thylakoid Membrane

    PubMed Central

    Króliczewski, Jarosław; Piskozub, Małgorzata; Bartoszewski, Rafał; Króliczewska, Bożena

    2016-01-01

    The cytochrome b6 f complex occupies an electrochemically central position in the electron-transport chain bridging the photosynthetic reaction center of PS I and PS II. In plants, the subunits of these thylakoid membrane protein complexes are both chloroplast and nuclear encoded. How the chloroplast-encoded subunits of multi-spanning cytochrome b6 are targeted and inserted into the thylakoid membrane is not fully understood. Experimental approaches to evaluate the cytochrome b6 import mechanism in vivo have been limited to bacterial membranes and were not a part of the chloroplast environment. To evaluate the mechanism governing cytochrome b6 integration in vivo, we performed a comparative analysis of both native and synthetic cytochrome b6 insertion into purified thylakoids. Using biophysical and biochemical methods, we show that cytochrome b6 insertion into the thylakoid membrane is a non-spontaneous co-translational process that involves ALB3 insertase. Furthermore, we provided evidence that CSP41 (chloroplast stem–loop-binding protein of 41 kDa) interacts with RNC-cytochrome b6 complexes, and may be involved in cytochrome b6 (petB) transcript stabilization or processing. PMID:27698412

  16. Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450.

    PubMed

    Stevenson, P M; Ruettinger, R T; Fulco, A J

    1983-05-16

    Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450. PMID:6405752

  17. Novel methodology for the study of mercury methylation and reduction in sediments and water using 197Hg radiotracer.

    PubMed

    Ribeiro Guevara, Sergio; Zizek, Suzana; Repinc, Urska; Pérez Catán, Soledad; Jaćimović, Radojko; Horvat, Milena

    2007-03-01

    Mercury tracers are powerful tools that can be used to study mercury transformations in environmental systems, particularly mercury methylation, demethylation and reduction in sediments and water. However, mercury transformation studies using tracers can be subject to error, especially when used to assess methylation potential. The organic mercury extracted can be as low as 0.01% of the endogenous labeled mercury, and artefacts and contamination present during methylmercury (MeHg) extraction processes can cause interference. Solvent extraction methods based on the use of either KBr/H2SO4 or HCl were evaluated in freshwater sediments using 197Hg radiotracer. Values obtained for the 197Hg tracer in the organic phase were up to 25-fold higher when HCl was used, which is due to the coextraction of 197Hg2+ into the organic phase during MeHg extraction. Evaluations of the production of MeHg gave similar results with both MeHg extraction procedures, but due to the higher Hg2+ contamination of the controls, the uncertainty in the determination was higher when HCl was used. The Hg2+ contamination of controls in the HCl extraction method showed a nonlinear correlation with the humic acid content of sediment pore water. Therefore, use of the KBr/H2SO4 method is recommended, since it is free from these interferences. 197Hg radiotracer (T1/2=2.673 d) has a production rate that is about 50 times higher than that of 203Hg (T1/2=46.595 d), the most frequently used mercury radiotracer. Hence it is possible to obtain a similar level of performance to 203Hg when it is used it in short-term experiments and produced by the irradiation of 196Hg with thermal neutrons, using mercury targets with the natural isotopic composition. However, if the 0.15% natural abundance of the 196Hg isotope is increased, the specific activity of the 197Hg tracer can be significantly improved. In the present work, 197Hg tracer was produced from mercury 51.58% enriched in the 196Hg isotope, and a 340-fold

  18. [(64)Cu]diacetyl-bis(N(4)-methyl-thiosemicarbazone) - a radiotracer for tumor hypoxia.

    PubMed

    Wood, Katie A; Wong, Wai Lup; Saunders, Michele I

    2008-05-01

    Positron emission tomography scanning using the radiotracer-labeled copper (II)-diacetyl-bis(N(4)-methylthiosemicarbazone) has been proposed as a noninvasive method for evaluating tumor hypoxia. Tumor hypoxia results in a more aggressive tumor phenotype together with resistance to both radiotherapy and chemotherapy. A noninvasive technique for evaluation of tumor hypoxia is not currently available. Validation of this technique would provide clinicians with a tool for determining the most appropriate cancer therapy, prognostic information, and subvolume delineation for the radiotherapy dose escalation to the radioresistant hypoxic regions within a tumor. This review article describes the background to the development of this tracer, its proposed retention mechanism, biodistribution dosimetry and the preclinical and clinical studies to date. It outlines the potential use of this radiotracer for imaging in the field of oncology.

  19. Synthesis of carbon-11, fluorine-18, and nitrogen-13 labeled radiotracers for biomedical applications

    SciTech Connect

    Fowler, J.S.; Wolf, A.P.

    1981-01-01

    A number of reviews, many of them recent, have appeared on various aspects of /sup 11/C, /sup 18/F and /sup 13/N-labeled radiotracers. This monograph treats the topic principally from the standpoint of synthetic organic chemistry while keeping in perspective the necessity of integrating the organic chemistry with the design and ultimate application of the radiotracer. Where possible, recent examples from the literature of organic synthesis are introduced to suggest potentially new routes which may be applied to problems in labeling organic molecules with the short-lived positron emitters, carbon-11, fluorine-18, and nitrogen-13. The literature survey of carbon-11, fluorine-18 and nitrogen-13 labeled compounds presented are of particular value to scientists working in this field. Two appendices are also included to provide supplementary general references. A subject index concludes this volume.

  20. Positron detection in silica monoliths for miniaturised quality control of PET radiotracers.

    PubMed

    Tarn, Mark D; Maneuski, Dzmitry; Alexander, Richard; Brown, Nathaniel J; O'Shea, Val; Pimlott, Sally L; Pamme, Nicole; Archibald, Stephen J

    2016-06-01

    We demonstrate the use of the miniaturised Medipix positron sensor for detection of the clinical PET radiotracer, [(68)Ga]gallium-citrate, on a silica-based monolith, towards microfluidic quality control. The system achieved a far superior signal-to-noise ratio compared to conventional sodium iodide-based radio-HPLC detection and allowed real-time visualisation of positrons in the monolith. PMID:27029282

  1. Residence time distribution measurements in a pilot-scale poison tank using radiotracer technique.

    PubMed

    Pant, H J; Goswami, Sunil; Samantray, J S; Sharma, V K; Maheshwari, N K

    2015-09-01

    Various types of systems are used to control the reactivity and shutting down of a nuclear reactor during emergency and routine shutdown operations. Injection of boron solution (borated water) into the core of a reactor is one of the commonly used methods during emergency operation. A pilot-scale poison tank was designed and fabricated to simulate injection of boron poison into the core of a reactor along with coolant water. In order to design a full-scale poison tank, it was desired to characterize flow of liquid from the tank. Residence time distribution (RTD) measurement and analysis was adopted to characterize the flow dynamics. Radiotracer technique was applied to measure RTD of aqueous phase in the tank using Bromine-82 as a radiotracer. RTD measurements were carried out with two different modes of operation of the tank and at different flow rates. In Mode-1, the radiotracer was instantaneously injected at the inlet and monitored at the outlet, whereas in Mode-2, the tank was filled with radiotracer and its concentration was measured at the outlet. From the measured RTD curves, mean residence times (MRTs), dead volume and fraction of liquid pumped in with time were determined. The treated RTD curves were modeled using suitable mathematical models. An axial dispersion model with high degree of backmixing was found suitable to describe flow when operated in Mode-1, whereas a tanks-in-series model with backmixing was found suitable to describe flow of the poison in the tank when operated in Mode-2. The results were utilized to scale-up and design a full-scale poison tank for a nuclear reactor.

  2. Cerenkov Luminescence Endoscopy: Improved Molecular Sensitivity with β−-Emitting Radiotracers

    PubMed Central

    Carpenter, Colin M.; Ma, Xiaowei; Liu, Hongguang; Sun, Conroy; Pratx, Guillem; Wang, Jing; Gambhir, Sanjiv S.; Xing, Lei; Cheng, Zhen

    2015-01-01

    Cerenkov luminescence endoscopy (CLE) is an optical technique that captures the Cerenkov photons emitted from highly energetic moving charged particles (β+ or β−) and can be used to monitor the distribution of many clinically available radioactive probes. A main limitation of CLE is its limited sensitivity to small concentrations of radiotracer, especially when used with a light guide. We investigated the improvement in the sensitivity of CLE brought about by using a β− radiotracer that improved Cerenkov signal due to both higher β-particle energy and lower γ noise in the imaging optics because of the lack of positron annihilation. Methods The signal-to-noise ratio (SNR) of 90Y was compared with that of 18F in both phantoms and small-animal tumor models. Sensitivity and noise characteristics were demonstrated using vials of activity both at the surface and beneath 1 cm of tissue. Rodent U87MG glioma xenograft models were imaged with radiotracers bound to arginine-glycine-aspartate (RGD) peptides to determine the SNR. Results γ noise from 18F was demonstrated by both an observed blurring across the field of view and a more pronounced fall-off with distance. A decreased γ background and increased energy of the β particles resulted in a 207-fold improvement in the sensitivity of 90Y compared with 18F in phantoms. 90Y-bound RGD peptide produced a higher tumor-to-background SNR than 18F in a mouse model. Conclusion The use of 90Y for Cerenkov endoscopic imaging enabled superior results compared with an 18F radiotracer. PMID:25300598

  3. Improving the In Vivo Profile of Minigastrin Radiotracers: A Comparative Study Involving the Neutral Endopeptidase Inhibitor Phosphoramidon.

    PubMed

    Kaloudi, Aikaterini; Nock, Berthold A; Lymperis, Emmanouil; Krenning, Eric P; de Jong, Marion; Maina, Theodosia

    2016-02-01

    Minigastrin radiotracers, such as [(111)In-DOTA]MG0 ([(111)In-DOTA-DGlu(1)]minigastrin), have been considered for diagnostic imaging and radionuclide therapy of CCK2R-positive human tumors, such as medullary thyroid carcinoma. However, the high kidney retention assigned to the pentaGlu(2-6) repeat in the peptide sequence has compromised their clinical applicability. On the other hand, truncated des(Glu)(2-6)-analogs, such as [(111)In-DOTA]MG11 ([(111)In-DOTA-DGlu(10),desGlu(2-6)]minigastrin), despite their low renal uptake, show poor bioavailability and tumor targeting. [(111)In]CP04 ([(111)In-DOTA-DGlu(1-6)]minigastrin) acquired by Glu(2-6)/DGlu(2-6) substitution showed promising tumor-to-kidney ratios in rodents. In the present study, we compare the biological profiles of [(111)In]CP04, [(111)In-DOTA]MG11, and [(111)In-DOTA]MG0 during in situ neutral endopeptidase (NEP) inhibition, recently shown to improve the bioavailability of several peptide radiotracers. After coinjection of the NEP inhibitor, phosphoramidon (PA), the stability of [(111)In]CP04 and [(111)In-DOTA]MG0 in peripheral mouse blood increased, with an exceptional >14-fold improvement monitored for [(111)In-DOTA]MG11. In line with these findings, PA treatment increased the uptake of [(111)In]CP04 (8.5 ± 0.4%ID/g to 16.0 ± 2.3%ID/g) and [(111)In-DOTA]MG0 (11.9 ± 2.2%ID/g to 17.2 ± 0.9%ID/g) in A431-CCK2R(+) tumors at 4 hours postinjection, whereas the respective increase for [(111)In-DOTA]MG11 was >6-fold (2.5 ± 0.9%ID/g to 15.1 ± 1.7%ID/g). Interestingly, kidney uptake remained lowest for [(111)In-DOTA]MG11, but unfavorably increased by PA treatment for [(111)In-DOTA]MG0. Thus, overall, the most favorable in vivo profile was displayed by [(111)In-DOTA]MG11 during NEP inhibition, highlighting the need to validate this promising concept in the clinic. PMID:26844849

  4. Carbon-11 labeling of CP-126,998*: A radiotracer for in vivo studies of acetylcholinesterase

    SciTech Connect

    Musachio, J.L.; Flesher, J.E.; Scheffel, U.

    1996-05-01

    The study of acetylcholinesterase (AChE) via PET is of interest as reduced activity of this enzyme has been observed in Alzheimer`s disease. Our efforts to develop a radiotracer for mapping of AChE have focused on the N-benzylpiperidine benzisoxazole, CP-126,998, a highly potent (IC{sub 50}=0.48 nm) and selective inhibitor of AChE. High specific activity [C-11] CP-126,998 was synthesized (14 - 24% radiochemical yield, non-decay corrected) by treatment of the desmethyl precursor, CP-118,954, with [C-11] methyl iodide and tetrabutylammonium hydroxide in DMF. In vivo studies with [C-11] CP-126,998 in mice show that this radiotracer displays highest uptake in striatum (6.2 %ID/g), a brain region known to be rich in AChE. The (striatum-cerebellum)/cerebellar radioactivity ratio reached a maximum of 4.3 at 30 min postinjection, and this ratio decreased to 2.4 at 120 min. .Radiotracer binding was saturable in vivo by pretreatment with CP-118,954. Pretreatment of mice with diisopropylfluorophosphate (4 mg/kg i.p.), a known AChE inhibitor, significantly inhibited binding in striatum in a dose-dependent manner. Initial results suggest that [C-11] CP-126,998 may prove useful as a marker for the study of AChE in humans via PET.

  5. In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase-A and -B Enzymatic Activity.

    PubMed

    Brooks, Allen F; Shao, Xia; Quesada, Carole A; Sherman, Phillip; Scott, Peter J H; Kilbourn, Michael R

    2015-12-16

    The isozymes of monoamine oxidase (MAO-A and MAO-B) are important enzymes involved in the metabolism of numerous biogenic amines, including the neurotransmitters serotonin, dopamine, and norepinephrine. Recently, changes in concentrations of MAO-B have been proposed to be an in vivo marker of neuroinflammation associated with Alzheimer's disease. Previous developments of in vivo radiotracers for imaging changes in MAO enzyme expression or activity have utilized the irreversible propargylamine-based suicide inhibitors or high-affinity reversibly binding inhibitors. As an alternative approach, we have investigated 1-[(11)C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as metabolic trapping agents for the monoamine oxidases. MAO-mediated oxidation and spontaneous hydrolysis yield 1-[(11)C]methyl-2,3-dihydro-4-pyridinone as a hydrophilic metabolite that is trapped within brain tissues. Radiotracers with phenyl, biphenyl, and 7-coumarinyl ethers were evaluated using microPET imaging in rat and primate brains. No isozyme selectivity for radiotracer trapping was observed in the rat brain for any compound, but in the monkey brain, the phenyl ether demonstrated MAO-A selectivity and the coumarinyl ether showed MAO-B selectivity. These are lead compounds for further development of 1-[(11)C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines with optimized brain pharmacokinetics and isozyme selectivity.

  6. SPECT-OPT multimodal imaging enables accurate evaluation of radiotracers for β-cell mass assessments

    PubMed Central

    Eter, Wael A.; Parween, Saba; Joosten, Lieke; Frielink, Cathelijne; Eriksson, Maria; Brom, Maarten; Ahlgren, Ulf; Gotthardt, Martin

    2016-01-01

    Single Photon Emission Computed Tomography (SPECT) has become a promising experimental approach to monitor changes in β-cell mass (BCM) during diabetes progression. SPECT imaging of pancreatic islets is most commonly cross-validated by stereological analysis of histological pancreatic sections after insulin staining. Typically, stereological methods do not accurately determine the total β-cell volume, which is inconvenient when correlating total pancreatic tracer uptake with BCM. Alternative methods are therefore warranted to cross-validate β-cell imaging using radiotracers. In this study, we introduce multimodal SPECT - optical projection tomography (OPT) imaging as an accurate approach to cross-validate radionuclide-based imaging of β-cells. Uptake of a promising radiotracer for β-cell imaging by SPECT, 111In-exendin-3, was measured by ex vivo-SPECT and cross evaluated by 3D quantitative OPT imaging as well as with histology within healthy and alloxan-treated Brown Norway rat pancreata. SPECT signal was in excellent linear correlation with OPT data as compared to histology. While histological determination of islet spatial distribution was challenging, SPECT and OPT revealed similar distribution patterns of 111In-exendin-3 and insulin positive β-cell volumes between different pancreatic lobes, both visually and quantitatively. We propose ex vivo SPECT-OPT multimodal imaging as a highly accurate strategy for validating the performance of β-cell radiotracers. PMID:27080529

  7. Potential therapeutic radiotracers: preparation, biodistribution and metabolic characteristics of 177Lu-labeled cyclic RGDfK dimer.

    PubMed

    Shi, Jiyun; Liu, Zhaofei; Jia, Bing; Yu, Zilin; Zhao, Huiyun; Wang, Fan

    2010-06-01

    In this study, we reported the preparation and evaluation of (177)Lu-DOTA-RGD2, (177)Lu-DOTA-Bz-RGD2 and (177)Lu-DTPA-Bz-RGD2 (RGD2 = E[c(RGDfK)](2)) as a potential therapeutic radiotracers for the treatment of integrin alpha(v)beta(3)-positive tumors. The BALB/c nude mice bearing the U87MG human glioma xenografts were used to evaluate the biodistribution characteristics and excretion kinetics of (177)Lu-DOTA-RGD2, (177)Lu-DOTA-Bz-RGD2 and (177)Lu-DTPA-Bz-RGD2. It was found that there were no major differences in their lipophilicity and biodistribution characteristics, particularly at latter time points. A major advantage of using DTPA-Bz as the bifunctional chelator (BFC) was its high radiolabeling efficiency (fast and high yield radiolabeling) at room temperature. Using DOTA and DOTA-Bz as BFCs, the radiolabeling kinetics was slow, and heating at 100 degrees C and higher DOTA-conjugate concentration were needed for successful (177)Lu-labeling. Therefore, DTPA-Bz is an optimal BFC for routine preparation of (177)Lu-labeled cyclic RGDfK peptides, and (177)Lu-DTPA-Bz-RGD2 is worthy of further investigation for targeted radiotherapy of integrin alpha(v)beta(3)-positive tumors.

  8. Synthesis and In Vitro Evaluation of Novel Nortropane Derivatives as Potential Radiotracers for Muscarinic M2 Receptors

    PubMed Central

    Knol, Remco J. J.; van den Bos, Jan C.; Janssen, Anton G. M.; de Bruin, Kora; van Eck-Smit, Berthe L. F.; Booij, Jan

    2011-01-01

    Disturbances of the cerebral cholinergic neurotransmitter system are present in neurodegenerative disorders. SPECT or PET imaging, using radiotracers that selectively target muscarinic receptor subtypes, may be of value for in vivo evaluation of such conditions. 6β-acetoxynortropane, a potent muscarinic M2 receptor agonist, has previously demonstrated nanomolar affinity and high selectivity for this receptor. Based on this compound we synthesized four nortropane derivatives that are potentially suitable for SPECT imaging of the M2 receptor. 6β-acetoxynortropane and the novel derivatives were tested in vitro for affinity to the muscarinic M1−3 receptors. The original 6β-acetoxynortropane displayed high affinity (Ki = 70–90 nM) to M2 receptors and showed good selectivity ratios to the M1 (65-fold ratio) and the M3 (70-fold ratio) receptors. All new derivatives showed reduced affinity to the M2 subtype and loss of subtype selectivity. It is therefore concluded that the newly synthesized derivatives are not suitable for human SPECT imaging of M2 receptors. PMID:21755053

  9. Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex.

    PubMed Central

    Edwards, A M; Blasie, J K; Bean, J C

    1998-01-01

    Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase. PMID:9512031

  10. Development of a system for real-time measurements of metabolite transport in plants using short-lived positron-emitting radiotracers

    NASA Astrophysics Data System (ADS)

    Kiser, Matthew R.

    be introduced to plants as biologically active molecules such as 11CO2, N13O-3, 18F--[H2O], and H152O . Plants for these studies are grown in controlled-environment chambers at the Phytotron. The chambers offer an array of control for temperature, humidity, atmospheric CO2 concentration, and light intensity. Additionally, the Phytotron houses one large reach-in growth chamber that is dedicated to this project for radioisotope labeling measurements. There are several important properties of short-lived positron-emitting radio-tracers that make them well suited for use in investigating metabolite transport in plants. First, because the molecular mass of a radioisotope-tagged compound is only minutely different from the corresponding stable compound, radiotracer substances should be metabolized and transported in plants the same as their non-radioactive counterparts. Second, because the relatively high energy gamma rays emitted from electron-positron annihilation are attenuated very little by plant tissue, the real-time distribution of a radiotracer can be measured in vivo in plants. Finally, the short radioactive half-lives of these isotopes allow for repeat measurements on the same plant in a short period of time. For example, in studies of short-term environmental changes on plant metabolite dynamics, a single plant can be labeled multiple times to measure its responses to different, environmental conditions. Also, different short-lived radiotracers can be applied to the same plant over a short period of time to investigate the transport and allocation of various metabolites. This newly developed system provides the capabilities for production of 11CO2 at TUNL, transfer of the 11CO 2 gas from the target area at TUNL to a radiation-shielded cryogenic trap at the Phytotron, labeling of photoassimilates with 11C, and in vivo gamma-ray detection for real-time measurements of the radiotracer distribution in small plants. The experimental techniques and instrumentation

  11. Canine cytochrome P-450 pharmacogenetics.

    PubMed

    Court, Michael H

    2013-09-01

    The cytochrome P-450 (CYP) drug metabolizing enzymes are essential for the efficient elimination of many clinically used drugs. These enzymes typically display high interindividual variability in expression and function resulting from enzyme induction, inhibition, and genetic polymorphism thereby predisposing patients to adverse drug reactions or therapeutic failure. There are also substantial species differences in CYP substrate specificity and expression that complicate direct extrapolation of information from humans to veterinary species. This article reviews the available published data regarding the presence and impact of genetic polymorphisms on CYP-dependent drug metabolism in dogs in the context of known human-dog CYP differences.

  12. The roles of c-type cytochromes in algal photosynthesis. Extraction from algae of a cytochrome similar to higher plant cytochrome f.

    PubMed

    Wood, P M

    1977-02-01

    A membrane-bound cytochrome resembling higher plant cytochrome f in many respects has been extracted from the algae Chlamydomonas. Euglena and Anacystis, and partially purified. The spectra of the cytochromes from Chlamydomonas and Euglena are virtually identical to that of parsley cytochrome f, with alpha-band maxima near 554 nm, very asymmetrical beta-bands, and gamma-band maxima at 421 nm. The cytochrome from Anacystis had alpha and gamma-bands both shifted to slightly longer wavelengths. The redox potential of the cytochrome from Chlamydomonas was determined as +350 mV, and its minimum molecular weight in sodium dodecyl sulphate as 31 000. The cytochrome from Euglena showed a rate of reaction with higher plant plastocyanin at least 100 times that of the soluble Euglena cytochrome c-552, and was unaffected by Euglena cytochrome c-552 antiserum. A very fast rate of electron transfer occurred between this cytochrome purified from Euglena and cytochrome c-552. The roles of the membrane-bound and soluble c-type cytochromes in algal photosynthesis are discussed, and it is recommended that the name cytochrome f should be reserved for the membrane-bound cytochrome (to emphasize its affinity with higher plant cytochrome f), while the soluble one should be named by its alpha-band (c-552, c-553, etc.) to make clear its distinctness from higher plant cytochrome f and homology with mitochondrial cytochrome c.

  13. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species.

    PubMed

    Gray, Joshua P; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  14. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    SciTech Connect

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and {beta}-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from {beta}-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  15. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species.

    PubMed

    Gray, Joshua P; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury. PMID:20561902

  16. Computer-aided design of aptamers for cytochrome p450.

    PubMed

    Shcherbinin, Dmitrii S; Gnedenko, Oksana V; Khmeleva, Svetlana A; Usanov, Sergey A; Gilep, Andrei A; Yantsevich, Aliaksei V; Shkel, Tatsiana V; Yushkevich, Ivan V; Radko, Sergey P; Ivanov, Alexis S; Veselovsky, Alexander V; Archakov, Alexander I

    2015-08-01

    Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M. PMID:26166326

  17. Metabolite identification of a radiotracer by electrochemistry coupled to liquid chromatography with mass spectrometric and radioactivity detection.

    PubMed

    Baumann, Anne; Faust, Andreas; Law, Marylin P; Kuhlmann, Michael T; Kopka, Klaus; Schäfers, Michael; Karst, Uwe

    2011-07-01

    Radioligands, which specifically bind to a receptor or enzyme (target), enable molecular imaging of the target expression by positron emission tomography (PET). One very promising PET tracer is (S)-1-(4-(2-[(18)F]-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (isatin), a caspase-3 inhibitor, which has been developed at the University Hospital of Münster to image cell death (apoptosis). The translation of this novel tracer from preclinical evaluation to clinical examinations requires biodistribution studies, which characterize the pharmakodynamics and metabolic fate of the compound. This information is used to further optimize the radioligands and to interpret radioactive signals from tissues upon injection of the radioligand in vivo with respect to their specificity. The analysis of the metabolism of radioligands is hampered by the low amount of the compound being typically injected (nano/picomolar amount per injection). In the present study, electrochemistry (EC) is applied to elucidate the oxidative metabolism pathway of the radiotracer. Previous studies have demonstrated that EC can be utilized as a complementary tool to conventional in vitro approaches in drug metabolism studies. Thereby, potential oxidative metabolites of the isatin are determined by EC coupled to electrospray ionization mass spectrometry (EC/ESI-MS). Moreover, using EC/liquid chromatography (LC) and ESI-ion trap MS(n), structural elucidation of the oxidation products is performed. Comparatively to EC, in vitro metabolism studies with rat liver microsomes are conducted. Finally, the developed LC/ESI-MS method is applied to determine metabolites in body fluids and cell extracts from in vivo studies with the nonradioactive ((19)F) and radioactive isatin ((18)F). On the basis of the electrochemically generated oxidation products of the radioligand, the major radioactive metabolite occurring in vivo was successfully identified.

  18. Crystallization of Mitochondrial Cytochrome Oxidase

    NASA Astrophysics Data System (ADS)

    Ozawa, Takayuki; Tanaka, Masashi; Wakabayashi, Takashi

    1982-12-01

    Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria. By washing the oxidase with detergent on a hydrophobic interaction column, phospholipids were depleted to the level of 1 mol of cardiolipin per mol of heme a. Hydrophobic impurities and partially denatured oxidase were separated from the intact oxidase on an affinity column with cytochrome c as the specific ligand. The final preparation of the oxidase contained seven distinct polypeptides. The molecular weight of the oxidase was estimated to be 130,000 from its specific heme a and copper content and from the subunit composition. Crystals of the oxidase were obtained by slow removal of the detergent from the buffer in which the oxidase was dissolved. The needle-shaped crystals were 100 μ m in average length and 5 μ m in width, and they strongly polarized visible light. Electron diffraction patterns were obtained with an unstained glutaraldehyde-fixed single crystal by electron microscopy using 1,000-kV electrons. From electron micrographs and the diffraction patterns of the crystal, it was concluded that the crystal is monoclinic in the space group P21, with unit cell dimensions a = 92 angstrom, b = 84 angstrom, and c = 103 angstrom, and α =β 90 degrees, γ = 126 degrees.

  19. Mitochondrial Cytochrome c Oxidase Deficiency

    PubMed Central

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-01-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance to study different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy. PMID:26846578

  20. Oxidative titrations of reduced cytochrome aa3: influence of cytochrome c and carbon monoxide on the midpoint potential values.

    PubMed

    Schroedl, N A; Hartzell, C R

    1977-11-15

    Oxidative titrations were performed on the electrostatic complex formed between cytochrome c and cytochrome aa3 at low ionic strength. Midpoint potentials of the redox centers in the proteins in 1:1 and 2:1 complexes were compared with those in mixtures of the cytochromes at high ionic strength. Computer simulations of all titrations yielded midpoint potentials for the components of cytochrome aa3 which were consistent with literature values for isolated cytochrome aa3 or mixture of cytochromes c and aa3. However, the unequal heme extinction coefficients observed previously (Schroedl, N.A., and Hartzell, C.R. (1977), Biochemistry 16, 1327) during oxidative titrations of cytochrome aa3 became equal in magnitude under these experimental conditions. The binding of cytochrome c to cytochrome aa3 changed the midpoint potentials of cytochrome aa3 by 15-20 mV, while the midpoint potentials for cytochrome c were altered by 50-60 mV. Careful analysis of these titrations including computer simulation revealed that cytochrome c was able to bind to cytochrome aa3 only after cytochrome aL2+ had become oxidized. When bound to cytochrome aa3, the midpoint potential of cytochrome c was 210 7V. Titrations performed under a carbon monoxide atmosphere revealed cytochrome aa3 midpoint potentials unchanged from reported values. Cytochrome c again exhibited a midpoint potential of 210 mV after binding to cytochrome aa3.

  1. Evaluation of [11C]metergoline as a PET radiotracer for 5HTR in nonhuman primates

    SciTech Connect

    Hooker, J.M.; Hooker, J.M.; Kim, S.W.; Reibel, A.T.; Alexoff, D.; Xu, Y.; Shea, C.

    2010-04-20

    Metergoline, a serotonin receptor antagonist, was labeled with carbon-11 in order to evaluate its pharmacokinetics and distribution in non-human primates using positron emission tomography. [{sup 11}C]Metergoline had moderate brain uptake and exhibited heterogeneous specific binding, which was blocked by pretreatment with metergoline and altanserin throughout the cortex. Non-specific binding and insensitivity to changes in synaptic serotonin limit its potential as a PET radiotracer. However, the characterization of [{sup 11}C]metergoline pharmacokinetics and binding in the brain and peripheral organs using PET improves our understanding of metergoline drug pharmacology.

  2. Radiotracer studies on ion-selective membranes based on poly(vinyl chloride) matrices.

    PubMed

    Jaber, A M; Moody, G J; Thomas, J D; Willcox, A

    1977-10-01

    Radiotracer studies with (45)Ca, (89)Sr and (133)Ba have provided evidence that the permeation of magnesium, strontium and barium ions through PVC membranes containing Orion 92-20-02 liquid ion-exchanger is inhibited by their low affinity for the liquid ion-exchanger sites. Experiments with (7)Be indicate a strong affinity of the membrane for beryllium ions with corresponding inhibition of permeation. When acid is present in the solution on one side of the membrane, preferential permeation by protons may lead to transport of ions against their concentration gradient in order to maintain the balance of charge.

  3. Cytochrome a1 of acetobacter aceti is a cytochrome ba functioning as ubiquinol oxidase.

    PubMed

    Matsushita, K; Shinagawa, E; Adachi, O; Ameyama, M

    1990-12-01

    Cytochrome a1 is a classic cytochrome that in the 1930s had already been detected in Acetobacter strains and in the 1950s was identified as a terminal oxidase. However, recent studies did not substantiate the previous observations. We have detected a cytochrome a1-like chromophore in Acetobacter aceti, which was purified and characterized in this study. The cytochrome was solubilized from membranes of the strain with octyl beta-D-glucopyranoside and was purified by single column chromatography. The purified cytochrome exhibited a broad alpha peak around 600-610 nm, which turned to a sharp peak at 589 nm in the presence of cyanide. Carbon monoxide difference spectra of the cytochrome indicated the presence of an alpha-type cytochrome. The cytochrome contained 1 mol each of hemes b and a and probably one copper ion. These results suggest that the cytochrome purified from A. aceti is the so-called cytochrome a1, and thus the existence of the classic cytochrome has been reconfirmed. The purified enzyme consisted of four polypeptides of 55, 35, 22, and 18 kDa, and it showed a sedimentation coefficient of 6.3 S in the native form. The enzyme had a high ubiquinol oxidase activity (140-160 mumol of ubiquinol-2 oxidized per min per mg of protein). When reconstituted into proteoliposomes, the cytochrome could generate an electrochemical proton gradient during oxidation of ubiquinol. Thus, cytochrome a1 of A. aceti has been shown to be a cytochrome ba terminal oxidase capable of generating an electrochemical proton gradient concomitant with ubiquinol oxidation.

  4. [(18)F]6-fluoro-3,4-dihydroxy-L-phenylalanine--recent modern syntheses for an elusive radiotracer.

    PubMed

    Edwards, Richard; Wirth, Thomas

    2015-05-15

    [(18)F]6-fluoro-3,4-dihydroxy-L-phenylalanine ([(18)F]F-DOPA) has been known to be a useful radiotracer for over 30 years. Its widespread clinical use has been hampered by the lack of a robust, high yielding synthesis. This review summarises new developments in radiochemistry that are providing solutions to long standing problems involved in the synthesis of this important but elusive radiotracer. Considerable advances in nucleophilic synthesis have been achieved by optimising multistep strategies and using both hypervalent iodine chemistry and transition metal-mediated fluorinations allowing for the production of high specific activity [(18)F]F-DOPA. PMID:25882075

  5. [(18)F]6-fluoro-3,4-dihydroxy-L-phenylalanine--recent modern syntheses for an elusive radiotracer.

    PubMed

    Edwards, Richard; Wirth, Thomas

    2015-05-15

    [(18)F]6-fluoro-3,4-dihydroxy-L-phenylalanine ([(18)F]F-DOPA) has been known to be a useful radiotracer for over 30 years. Its widespread clinical use has been hampered by the lack of a robust, high yielding synthesis. This review summarises new developments in radiochemistry that are providing solutions to long standing problems involved in the synthesis of this important but elusive radiotracer. Considerable advances in nucleophilic synthesis have been achieved by optimising multistep strategies and using both hypervalent iodine chemistry and transition metal-mediated fluorinations allowing for the production of high specific activity [(18)F]F-DOPA.

  6. Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation

    PubMed Central

    Kumar, Santosh

    2009-01-01

    Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts. PMID:20064075

  7. Anaerobic degradation of naphthalene in a fluvial aquifer: a radiotracer study.

    PubMed

    Bianchin, Mario; Smith, Leslie; Barker, James F; Beckie, Roger

    2006-03-20

    A radiotracer study was conducted in a creosote-contaminated aquifer beneath the Fraser River, British Columbia Canada to investigate the in situ degradation of naphthalene. The groundwater is anaerobic, with abundant methane, ferrous iron and carbon dioxide. This study followed earlier work at the site where the contaminant distribution could only be explained by invoking a mass loss through degradation, even though extensive field and laboratory microcosm studies closer to the source zone onshore could not confirm degradation. Accordingly, 14C-naphthalene was injected into the aquifer offshore, further from the source zone where modeling suggested degradation was occurring. During the 230-day monitoring period, 14CO2 was detected, confirming the degradation of the radio-labeled naphthalene tracer. A zero-order degradation rate of naphthalene of 5 microg/L-day was estimated based on the decrease in 14C-naphthalene concentration with time. While the degradation pathway could not be determined from the radiotracer study alone, the geochemistry of the site suggests that either iron reduction or methanogenesis is the terminal electron accepting processes responsible for naphthalene oxidation. PMID:16487624

  8. Effect of Cyclosporin A on the Uptake of D3-Selective PET Radiotracers in Rat Brain

    PubMed Central

    Tu, Zhude; Li, Shihong; Xu, Jinbin; Chu, Wenhua; Jones, Lynne A.; Luedtke, Robert R.; Mach, Robert H.

    2011-01-01

    Introduction Four benzamide analogs having a high affinity and selectivity for D3 versus D2 receptors were radiolabeled with 11C or 18F for in vivo evaluation. Methods Precursors were synthesized and the four D3 selective benzamide analogs were radiolabeled. The tissue distribution and brain uptake of the four compounds were evaluated in control rats and rats pretreated with cyclosporin A, a modulator of P-glycoprotein and an inhibitor of other ABC efflux transporters that contribute to the blood brain barrier. MicroPET imaging was carried out for [11C]6 in a control and a cyclosporin A pre-treated rat. Results All four compounds showed low brain uptake in control rats at 5 and 30 min post-injection; despite recently reported rat behavioral studies conducted on analogs 6 (WC-10) and 7 (WC-44). Following administration of cyclosporin A, increased brain uptake was observed with all four PET radiotracers at both 5 and 30 min post-i.v. injection. An increase in brain uptake following modulation/inhibition of the ABC transporters was also observed in the microPET study. Conclusions These data suggest that D3 selective conformationally-flexible benzamide analogs which contain a N-2-methoxyphenylpiperazine moiety are substrates for P-glycoprotein or other ABC transporters expressed at the blood-brain barrier, and that PET radiotracers containing this pharmacophore may display low brain uptake in rodents due to the action of these efflux transporters. PMID:21718948

  9. Optical reaction cell and light source for [18F] fluoride radiotracer synthesis

    DOEpatents

    Ferrieri, R.A.; Schlyer, D.; Becker, R.J.

    1998-09-15

    An apparatus is disclosed for performing organic synthetic reactions, particularly no-carrier-added nucleophilic radiofluorination reactions for PET radiotracer production. The apparatus includes an optical reaction cell and a source of broadband infrared radiant energy, which permits direct coupling of the emitted radiant energy with the reaction medium to heat the reaction medium. Preferably, the apparatus includes means for focusing the emitted radiant energy into the reaction cell, and the reaction cell itself is preferably configured to reflect transmitted radiant energy back into the reaction medium to further improve the efficiency of the apparatus. The apparatus is well suited to the production of high-yield syntheses of 2-[{sup 18}F]fluoro-2-deoxy-Dglucose. Also provided is a method for performing organic synthetic reactions, including the manufacture of [{sup 18}F]-labeled compounds useful as PET radiotracers, and particularly for the preparation of 2-[{sup 18}F]fluoro-2-deoxy-D-glucose in higher yields than previously possible. 4 figs.

  10. Optical reaction cell and light source for ›18F! fluoride radiotracer synthesis

    DOEpatents

    Ferrieri, Richard A.; Schlyer, David; Becker, Richard J.

    1998-09-15

    Apparatus for performing organic synthetic reactions, particularly no-carrier-added nucleophilic radiofluorination reactions for PET radiotracer production. The apparatus includes an optical reaction cell and a source of broadband infrared radiant energy, which permits direct coupling of the emitted radiant energy with the reaction medium to heat the reaction medium. Preferably, the apparatus includes means for focusing the emitted radiant energy into the reaction cell, and the reaction cell itself is preferably configured to reflect transmitted radiant energy back into the reaction medium to further improve the efficiency of the apparatus. The apparatus is well suited to the production of high-yield syntheses of 2-›.sup.18 F!fluoro-2-deoxy-D-glucose. Also provided is a method for performing organic synthetic reactions, including the manufacture of ›.sup.18 F!-labeled compounds useful as PET radiotracers, and particularly for the preparation of 2-›.sup.18 F!fluoro-2-deoxy-D-glucose in higher yields than previously possible.

  11. Designing steriod receptor-based radiotracers to image breast and prostate tumors

    SciTech Connect

    Katzenellenbogen, J.A.

    1995-06-01

    Imaging of breast or prostate cancers based on their content of steroid receptors poses a major challenge in the design of radiotracers. Receptors for steroid hormones are proteins that interact at specific sites in chromatin. Several analogs of estrogens, progestins and androgens have been radiolabeled and evaluated both in vitro and in vivo for receptor binding affinity and selectivity. Breast tumors in patients have been imaged with [{sup 18}F]fluoroestradiol. Scintigraphic images with radiolabeled progestin analogs may be useful for monitoring the efficacy of tamoxifen treatment in breast cancer patients. Tissue distribution and imaging studies in animals with fluorine-substituted androgens indicate that it may be possible to develop a steroid receptor-based radiotracer for staging prostate cancer. Radiochemists are reporting some progress in labeling steroid receptor ligands with {sup 99m}Tc. By using the techniques of molecular nuclear medicine, new imaging procedures could be developed that might provide more precise information to help characterize disease and effect treatment decisions in patients with breast or prostate cancers. 55 refs., 4 figs., 4 tabs.

  12. A world of cytochrome P450s.

    PubMed

    Nelson, David R

    2013-02-19

    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.

  13. 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

  14. Evaluation of the anti-HER2 C6.5 diabody as a PET radiotracer to monitor HER2 status and predict response to trastuzumab treatment

    PubMed Central

    Reddy, Smitha; Shaller, Calvin C.; Doss, Mohan; Shchaveleva, Irina; Marks, James D.; Yu, Jian Q.; Robinson, Matthew K.

    2011-01-01

    Purpose The rapid tumor targeting and pharmacokinetic properties of engineered antibodies make them potentially suitable for use in imaging strategies to predict and monitor response to targeted therapies. This study aims to evaluate C6.5 diabody (C6.5db), a non-covalent anti-HER2 single chain-Fv dimer, as a radiotracer for predicting response to HER2-targeted therapies such as trastuzumab. Experimental Design Immunodeficient mice bearing established HER2-positive tumor xenografts were injected with radioiodinated C6.5db and imaged using PET/CT. Radiotracer biodistribution was quantified using biopsied tumor and normal tissues. Potential competition between trastuzumab and C6.5db was examined in vitro by flow cytometry and co-immunoprecipitations. Results Biodistribution analysis of mice bearing xenografts with varying HER2 density revealed that the tumor uptake of 125I-C6.5db correlates with HER2 tumor density. In vitro competition experiments suggest that the C6.5db targets an epitope on HER2 that is distinct from that bound by trastuzumab. Treatment of SK-OV-3-tumored mice with trastuzumab for 3 d caused a 42% (P=0.002) decrease in tumor uptake of 125I-C6.5db. This is consistent with a dramatic decrease in the tumor PET signal of 124I-C6.5db after trastuzumab treatment. Furthermore, BT-474-tumored mice showed a ∼60% decrease (P=0.0026) in C6.5db uptake after 6 d of trastuzumab treatment. Immunohistochemistry of excised xenograft sections and in vitro flow cytometry revealed that the decreased C6.5db uptake upon trastuzumab treatment is not associated with HER2 downregulation. Conclusions These studies suggest that 124I-C6.5db-based imaging can be used to evaluate HER2 levels as a predictor of respone to HER2-directed therapies. PMID:21177408

  15. Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c oxidase.

    PubMed

    Pan, L P; Hibdon, S; Liu, R Q; Durham, B; Millett, F

    1993-08-24

    The reactions of bovine cytochrome c oxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis(bipyridine)ruthenium (II) were studied by laser flash photolysis. All of the derivatives form complexes with cytochrome c oxidase at low ionic strength (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in rapid electron transfer to the ferric heme group of cytochrome c, followed by electron transfer to cytochrome c oxidase. The photoreduced heme Fe(II) in the cytochrome c derivative modified at lysine 25 on the periphery of the heme crevice domain transferred an electron to CuA with a rate constant of 1.1 x 10(4) s-1. CuA then transferred an electron to cytochrome a with a rate constant of 2.3 x 10(4) s-1. The derivatives modified at lysines 7, 39, 55, and 60 remote from the heme crevice domain of cytochrome c have nearly the same kinetics. The rate constant for electron transfer from the cytochrome c heme to CuA is greater than 10(5) s-1, and the rate constant for electron transfer from CuA to cytochrome a is 2 x 10(4) s-1. The cytochrome c derivatives modified at lysines 13 and 27 in the heme crevice domain react much more slowly than the other derivatives, with intracomplex rate constants for oxidation of cytochrome c ranging from 1000 to 6000 s-1. The bulky ruthenium group at the heme crevice domain of these derivatives apparently alters the binding orientation, leading to smaller electron-transfer rates.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Resonance Raman fingerprinting of multiheme cytochromes from the cytochrome c3 family.

    PubMed

    Di Paolo, Roberto E; Pereira, Patrícia M; Gomes, Inês; Valente, Filipa M A; Pereira, Inês A C; Franco, Ricardo

    2006-03-01

    Resonance Raman (RR) spectroscopy was used to investigate conformational characteristics of the hemes of several ferricytochromes of the cytochrome c3 family, electron transfer proteins isolated from the periplasm and membranes of sulfate-reducing bacteria. Our analysis concentrated on the low-frequency region of the RR spectra, a fingerprint region that includes vibrations for heme-protein C-S bonds [nu(C(a)S)]. It has been proposed that these bonds are directly involved in the electron transfer process. The three groups of tetraheme cytochrome c3 analyzed, namely Type I cytochrome c (3) (TpIc (3)s), Type II cytochrome c (3) (TpIIc (3)s) and Desulfomicrobium cytochromes c3, display different frequency separations for the two nu(C(a)S) lines that are similar among members of each group. These spectral differences correlate with differences in protein structure observed among the three groups of cytochromes c3. Two larger cytochromes of the cytochrome c3 family display RR spectral characteristics for the nu(C(a)S) lines that are closer to TpIIc3 than to TpIc3. Two other multiheme cytochromes from Desulfovibrio that do not belong to the cytochrome c3 family display nu(C(a)S) lines with reverse relative areas in comparison with the latter family. This RR study shows that the small differences in protein structure observed among these cytochrome c3 correlate to differences on the heme-protein bonds, which are likely to have an impact upon the protein function, making RR spectroscopy a sensitive and useful tool for characterizing these cytochromes.

  17. Mammalian cytochromes P-450: Volume I and Volume II

    SciTech Connect

    Guengerich, F.P.

    1987-01-01

    This two volume set summarizes the current knowledge of mammalian cytochromes. Ten chapters cover the current understanding of the enzymology of rat, rabbit, and human liver cytochromes P-450, extrahepatic cytochromes P-450, the diversity of substrates for the individual cytochromes P0-450 proteins, the metabolism of pro-toxicants and -carcinogens by cytochrome P-450, the degradation of cytochrome P-450 proteins, and the regulation of cytochrome P-450 activities in vitro and in vivo. The individual chapters outline the historical development of each area, the approaches which are applied, the current state of knowledge, and future directions towards unresolved questions; and index.

  18. The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

    PubMed

    Díaz-Moreno, Irene; Hulsker, Rinske; Skubak, Pavol; Foerster, Johannes M; Cavazzini, Davide; Finiguerra, Michelina G; Díaz-Quintana, Antonio; Moreno-Beltrán, Blas; Rossi, Gian-Luigi; Ullmann, G Matthias; Pannu, Navraj S; De la Rosa, Miguel A; Ubbink, Marcellus

    2014-08-01

    The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

  19. Cytochromes P460 and c'-beta; a new family of high-spin cytochromes c.

    PubMed

    Elmore, Bradley O; Bergmann, David J; Klotz, Martin G; Hooper, Alan B

    2007-03-01

    Cytochromes-P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c' of M. capsulatus, believed to be involved in binding or transformation of N-oxides, are shown to represent an evolutionarily related new family of monoheme, approximately 17kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta-sheets in contrast to the predominantly alpha-helical cytochromes c' found in photoheterotrophic and denitrifying Proteobacteria.

  20. The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

    PubMed

    Díaz-Moreno, Irene; Hulsker, Rinske; Skubak, Pavol; Foerster, Johannes M; Cavazzini, Davide; Finiguerra, Michelina G; Díaz-Quintana, Antonio; Moreno-Beltrán, Blas; Rossi, Gian-Luigi; Ullmann, G Matthias; Pannu, Navraj S; De la Rosa, Miguel A; Ubbink, Marcellus

    2014-08-01

    The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process. PMID:24685428

  1. 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. PMID:16859410

  2. Islet secretory granules contain cytochrome b561.

    PubMed

    Mackin, R B; Jones, D P; Noe, B D

    1986-08-01

    A cytochrome has been detected in secretory granules prepared from anglerfish islets of Langerhans. The heme moiety was determined to be of the b type, and the dithionite-reduced cytochrome exhibited an alpha-band maximum at 561 nm with an extinction coefficient of 13.8 mM-1 X cm-1. The protein was present at a concentration of 40 +/- 4 pmol/mg of secretory granule protein. The cytochrome was found to be an integral membrane protein and to be reduced by ascorbic acid but not by NADH, NADPH, reduced glutathione (GSH), or succinate. Because of the similarity to previously characterized secretory granule cytochrome b561's from neuroendocrine tissues, this cytochrome is also referred to as cytochrome b561. Although its function has not yet been elucidated, the apparent specificity for ascorbate suggests that it may be a component of the ascorbate-dependent peptidyl-glycine alpha-amidating monooxygenase system that functions in the amidation of islet hormones. PMID:3525285

  3. Cytochrome bd Displays Significant Quinol Peroxidase Activity

    PubMed Central

    Al-Attar, Sinan; Yu, Yuanjie; Pinkse, Martijn; Hoeser, Jo; Friedrich, Thorsten; Bald, Dirk; de Vries, Simon

    2016-01-01

    Cytochrome bd is a prokaryotic terminal oxidase that catalyses the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Cytochrome bd is a tri-haem integral membrane enzyme carrying a low-spin haem b558, and two high-spin haems: b595 and d. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase (QPO) that reduces hydrogen peroxide to water. The highly active and pure enzyme preparation used in this study did not display the catalase activity recently reported for E. coli cytochrome bd. To our knowledge, cytochrome bd is the first membrane-bound quinol peroxidase detected in E. coli. The observation that cytochrome bd is a quinol peroxidase, can provide a biochemical basis for its role in detoxification of hydrogen peroxide and may explain the frequent findings reported in the literature that indicate increased sensitivity to hydrogen peroxide and decreased virulence in mutants that lack the enzyme. PMID:27279363

  4. Development of Water Target for Radioisotope Production

    NASA Astrophysics Data System (ADS)

    Tripp, Nathan

    2011-10-01

    Ongoing studies of plant physiology at TUNL require a supply of nitrogen-13 for use as a radiotracer. Production of nitrogen-13 using a water target and a proton beam follows the nuclear reaction 16-O(p,a)13-N. Unfortunately the irradiation of trace amounts of oxygen-18 within a natural water target produces fluorine-18 by the reaction 18-O(p, n)18-F. The presence of this second radioisotope reduces the efficacy of nitrogen-13 as a radiotracer. Designing a natural water target for nitrogen-13 production at TUNL required the design of several new systems to address the problems inherent in nitrogen-13 production. A heat exchanger cools the target water after irradiation within the target cell. The resulting improved thermal regulation of the target water prevents the system from overheating and minimizes the effect of the cavitations occurring within the target. Alumina pellets within a scrubbing unit remove the fluorine-18 contamination from the irradiated water. The modular design of the water target apparatus makes the system highly adaptable, allowing for easy reuse and adaptation of the different components into future projects. The newly designed and constructed water target should meet the current and future needs of TUNL researchers in the production of nitrogen-13. This TUNL REU project was funded in part by a grant from the National Science Foundation (NSF) NSF-PHY-08-51813.

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

    PubMed

    Arinç, E; Cakir, D

    1999-02-01

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

  6. Experimental observation of silver and gold penetration into dental ceramic by means of a radiotracer technique

    SciTech Connect

    Moya, F.; Payan, J.; Bernardini, J.; Moya, E.G.

    1987-12-01

    A radiotracer technique was used to study silver and gold diffusion into dental porcelain under experimental conditions close to the real conditions in prosthetic laboratories for porcelain bakes. It was clearly shown that these non-oxidizable elements were able to diffuse into the ceramic as well as oxidizable ones. The penetration depth varied widely according to the element. The ratio DAg/DAu was about 10(3) around 850 degrees C. In contrast to gold, the silver diffusion rate was high enough to allow silver, from the metallic alloy, to be present at the external ceramic surface after diffusion into the ceramic. Hence, the greening of dental porcelains baked on silver-rich alloys could be explained mainly by a solid-state diffusion mechanism.

  7. Distribution of nickel hydroxide in sintered nickel plaques measured by radiotracer method during electroimpregnation

    SciTech Connect

    Ng, P.K.; Schneider, E.W.

    1986-01-01

    Sintered nickel positive electrodes were prepared by electroimpregnating nickel hydroxide inside a porous nickel plaque in a nickel nitrate solution. The distribution of nickel hydroxide inside the plaque was measured using a radio-tracer method with /sup 63/Ni as the radioactivity source. Autoradiography and ..beta.. counting were used to follow qualitative and quantitative distributions, respectively, of the pore filling process. Relatively uniform distribution was observed at low current density, and the precipitation of Ni(OH)/sub 2/ extends to the center of the plaque. At high current density, most of the Ni(OH)/sub 2/ aggregated in the region just underneath the plaque surface, causing a somewhat nonuniform distribution. Nickel hydroxide also precipitates heavily on the surface of the plaque at high current density, reducing the penetration of electrolyte to the inside of the plaque.

  8. Working against time: Rapid radiotracer synthesis and imaging the human brain

    SciTech Connect

    Fowler, J.S.; Wolf, A.P.

    1997-04-01

    In this Account, the authors describe some advances in radiotracer chemistry which have made it possible to probe the chemical anatomy of the human brain while working within a very restricted time scale. Though we highlight research from our laboratory, it is important to emphasize that advances in PET brain imaging have come from many laboratories throughout the world. Thus, for a more comprehensive treatment of PET technology the reader is referred to textbooks and review articles cited in this Account. Since many of the milestones in delineating biochemical transformations and the movement of drugs in the human brain have involved radiosynthesis with carbon-11 and fluorine-18, we focus on these two isotopes. 50 refs., 6 figs., 1 tab.

  9. Radiotracer studies on calcium ion-selective electrode membranes based on poly(vinyl chloride) matrices.

    PubMed

    Craggs, A; Moody, G J; Thomas, J D; Willcox, A

    Radiotracer studies with (45)Ca and (36)Cl demonstrate that PVC matrix membranes containing Orion 92-20-02 liquid calcium ion-exchanger are permselective to counter-cations. Diffusion coefficients are quoted for the migration of (45)Ca between pairs of calcium solutions and are discussed in terms of solution concentration, membrane thickness and concentration level of sensor in the membrane. Migration of calcium ions from calcium chloride solution to a Group (II) metal chloride solution through a PVC membrane containing calcium liquid ion-exchanger is discussed in terms of solvent extraction and electrode selectivity coefficient parameters. Thus, magnesium, strontium and barium ions appear to inhibit migration through the membrane by their low affinity for the membrane liquid ion-exchanger sites, while the inhibition by beryllium ions is attributed to site blockage by the strong affinity of dialkylphosphate sites for beryllium.

  10. Radiotracer method for residence time distribution study in multiphase flow system.

    PubMed

    Sugiharto, S; Su'ud, Z; Kurniadi, R; Wibisono, W; Abidin, Z

    2009-01-01

    [(131)I] isotope in different chemical compounds have been injected into 24in hydrocarbon transmission pipeline containing approximately 95% water, 3% crude oil, 2% gas and negligible solid material, respectively. The system is operated at the temperature around 70 degrees C enabling fluids flow is easier in the pipeline. The segment of measurement was chosen far from the junction point of the pipeline, therefore, it was reasonably to assume that the fluids in such multiphase system were separated distinctively. Expandable tubing of injector was used to ensure that the isotopes were injected at the proper place in the sense that [(131)I]Na isotope was injected into water layer and iodo-benzene, ([131])IC(6)H(5,) was injected into crude oil regime. The radiotracer selection was based on the compatibility of radiotracer with each of fluids under investigation. [(131)I]Na was used for measuring flow of water while iodo-benzene, ([131])IC(6)H(5,) was used for measuring flow of crude oil. Two scintillation detectors were used and they are put at the distances 80 and 100m, respectively, from injection point. The residence time distribution data were utilized for calculation water and crude oil flows. Several injections were conducted in the experiments. Although the crude oil density is lighter than the density of water, the result of measurement shows that the water flow is faster than the crude oil flow. As the system is water-dominated, water may act as carrier and the movement of crude oil is slowed due to friction between crude oil with water and crude oil with gas at top layer. Above of all, this result was able to give answer on the question why crude oil always arrives behind water as it is checked at gathering station. In addition, the flow patterns of the water in the pipeline calculated by Reynolds number and predicted by simple tank-in-series model is turbulence in character.

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

    PubMed

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

    2014-05-01

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

  12. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    PubMed

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome.

  13. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    PubMed

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome. PMID:25091281

  14. The cytochrome bd respiratory oxygen reductases

    PubMed Central

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

    2011-01-01

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

  15. The cytochrome bd respiratory oxygen reductases.

    PubMed

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

    2011-11-01

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

  16. Thermal stability of the polyheme cytochrome c3 superfamily.

    PubMed

    Florens, L; Bianco, P; Haladjian, J; Bruschi, M; Protasevich, I; Makarov, A

    1995-10-16

    The cytochrome c3 superfamily includes Desulfovibrio polyheme cytochromes c. We report the characteristic thermal stability parameters of the Desulfovibrio desulfuricans Norway (D.d.N.) cytochromes c3 (M(r) 13,000 and M(r) 26,000) and the Desulfovibrio vulgaris Hildenborough (D.v.H.) cytochrome c3 (M(r) 13,000) and high molecular mass cytochrome c (Hmc), as obtained with the help of electronic spectroscopy, voltammetric techniques and differential scanning calorimetry. The polyheme cytochromes are denatured over a wide range of temperatures: the D.v.H. cytochrome c3 is highly thermostable (Td = 121 degrees C) contrary to the D.d.N. protein (Td = 73 degrees C). The thermostability of the polyheme cytochromes is redox state dependent. The results are discussed in the light of the structural and functional relationships within the cytochrome c3 superfamily. PMID:7589483

  17. Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c1.

    PubMed

    Heacock, D H; Liu, R Q; Yu, C A; Yu, L; Durham, B; Millett, F

    1993-12-25

    The reactions of a beef heart cytochrome c1 preparation containing the hinge protein with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) (Ru(II)) were studied by flash photolysis. All of the ruthenium-cytochrome c derivatives formed complexes with cytochrome c1 in low ionic strength buffer (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a 0.4-microseconds laser flash resulted in rapid electron transfer to the ferric heme group in cytochrome c, followed by electron transfer from the ferrous heme group of cytochrome c to the ferric heme group of cytochrome c1. The kinetic difference spectra displayed maxima at 546 nm and minima at 554 nm characteristic of electron transfer between the two cytochromes. The rate constants were independent of concentration at low ionic strength, indicating intracomplex electron transfer. The rate constants were 4,800, 6,800, 22,000, and 22,000 s-1 for cytochrome c derivatives modified at lysines 13, 27, 25, and 72, respectively. The observed rate constants were independent of ionic strength up to about 50 nM and then decreased progressively with further increases in ionic strength indicating dissociation of the complex. Second-order kinetics were observed at 310 mM ionic strength, with rate constants of 1.0 x 10(6), 1.6 x 10(7), 1.2 x 10(8), and 3.0 x 10(7) M-1 s-1 for the derivatives modified at lysines 13, 27, 25, and 72, respectively. The ionic strength dependence of the second-order rate constants is comparable to that involving native horse cytochrome c and is consistent with electron transfer reactions between oppositely charged proteins.

  18. Evidence for dimer structure of proton-pumping cytochrome c oxidase, an analysis by radiation inactivation.

    PubMed Central

    Sone, N; Kosako, T

    1986-01-01

    Cytochrome c oxidases, purified from bovine heart and the thermophilic bacterium PS3, were irradiated with a high-energy electron beam. The proton transport activities of both preparations and their electron transfer activities decreased as single exponential functions of the radiation dosage. Applying the target theory with alkaline phosphatase as an internal standard, the following functional molecular weights were obtained for cytochrome c oxidation and H+ pumping: 63-73 kd and 160-220 kd, respectively, for the bovine enzyme, and 80-100 kd and 190-230 kd for the PS3 enzyme. The results suggest that a dimer structure is necessary for H+ pumping, while a core part of monomer (presumably the largest two subunits, i.e. subunits I and II) is sufficient for cytochrome c oxidation. PMID:3017697

  19. Respiratory Flexibility in Response to Inhibition of Cytochrome c Oxidase in Mycobacterium tuberculosis

    PubMed Central

    Arora, Kriti; Ochoa-Montaño, Bernardo; Tsang, Patricia S.; Blundell, Tom L.; Dawes, Stephanie S.; Mizrahi, Valerie; Bayliss, Tracy; Mackenzie, Claire J.; Cleghorn, Laura A. T.; Ray, Peter C.; Wyatt, Paul G.; Uh, Eugene; Lee, Jinwoo; Barry, Clifton E.

    2014-01-01

    We report here a series of five chemically diverse scaffolds that have in vitro activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory bc1 complex in Mycobacterium tuberculosis in a strain-dependent manner. Deletion of the cytochrome bd oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in qcrB. These results highlight the promiscuity of the bc1 complex and the risk of targeting energy metabolism with new drugs. PMID:25155596

  20. Amyloid-β peptide binds to cytochrome C oxidase subunit 1.

    PubMed

    Hernandez-Zimbron, Luis Fernando; Luna-Muñoz, Jose; Mena, Raul; Vazquez-Ramirez, Ricardo; Kubli-Garfias, Carlos; Cribbs, David H; Manoutcharian, Karen; Gevorkian, Goar

    2012-01-01

    Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1-42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.

  1. Amyloid-β Peptide Binds to Cytochrome C Oxidase Subunit 1

    PubMed Central

    Hernandez-Zimbron, Luis Fernando; Luna-Muñoz, Jose; Mena, Raul; Vazquez-Ramirez, Ricardo; Kubli-Garfias, Carlos; Cribbs, David H.; Manoutcharian, Karen; Gevorkian, Goar

    2012-01-01

    Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1–42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1–42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1–42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1–42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1–42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD. PMID:22927926

  2. Label-free photoacoustic microscopy of cytochromes.

    PubMed

    Zhang, Chi; Zhang, Yu Shrike; Yao, Da-Kang; Xia, Younan; Wang, Lihong V

    2013-02-01

    Photoacoustic microscopy (PAM) has achieved submicron lateral resolution in showing subcellular structures; however, relatively few endogenous subcellular contrasts have so far been imaged. Given that the hemeprotein, mostly cytochromes in general cells, is optically absorbing around the Soret peak (~420 nm), we implemented label-free PAM of cytochromes in cytoplasm for the first time. By measuring the photoacoustic spectra of the oxidized and reduced states of fibroblast lysate and fitting the difference spectrum with three types of cytochromes, we found that the three cytochromes account for more than half the optical absorption in the cell lysate at 420 nm wavelength. Fixed fibroblasts on slides were imaged by PAM at 422 and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard staining histology. PAM was also applied to label-free histology of mouse ear sections by showing cytoplasms and nuclei of various cells. PAM of cytochromes in cytoplasm is expected to be a high-throughput, label-free technique for studying live cell functions, which cannot be accomplished by conventional histology.

  3. Yeast mutants overproducing iso-cytochromes c

    SciTech Connect

    Sherman, F.; Cardillo, T.S.; Errede, B.; Friedman, L.; McKnight, G.; Stiles, J.I.

    1980-01-01

    For over 15 years, the iso-cytochrome c system in the yeast Saccharomyces cerevisiae has been used to investigate a multitude of problems in genetics and molecular biology. More recently, attention has been focused on using mutants for examining translation and transcriptional processes and for probing regulatory regions governing gene expression. In an effort to explore regulatory mechanisms and to investigate mutational alterations that lead to increased levels of gene products, we have isolated and characterized mutants that overproduce cytochrome c. In this paper we have briefly summarized background information of some essential features of the iso-cytochrome c system and we have described the types of mutants that overproduce iso-1-cytochrome c or iso-2-cytochrome c. Genetic procedures and recombinant DNA procedures were used to demonstrate that abnormally high amounts of gene products occur in mutants as result of duplications of gene copies or of extended alteration of regulatory regions. The results summarized in this paper point out the requirements of gross mutational changes or rearrangements of chromosomal segments for augmenting gene products.

  4. Cytochrome P450 monooxygenase system in echinoderms.

    PubMed

    den Besten, P J

    1998-11-01

    The results of a limited number of studies on echinoderms provide evidence for the presence of a cytochrome P450 monooxygenase system in representatives of three classes of the phylum Echinodermata: the asteroids (sea stars), holothuroids (sea cucumbers) and echinoids (sea urchins). The monooxygenase system has been demonstrated to be involved in the metabolism of xenobiotic compounds, but is assumed to have its primary function in the metabolism of endogenous substrates, such as steroids. Available data on P450 cofactor requirement, P450-dependent metabolism of benzo[a]pyrene, studies with classical inhibitors of P450, specificity of P450 induction by planar compounds, and the changes in the benzo[a]pyrene metabolite profile in induced animals suggest similarities with the MO system present in vertebrates. However, the relatively high capacity of the monooxygenase system in sea stars to catalyse reactions with organic hydroperoxide as donor for activated oxygen, and the low induceability during exposure to xenobiotics indicate also important differences between the echinoderm cytochrome P450 monooxygenase system and that of vertebrates. Some evidence was found for the existence of different forms of cytochrome P450 in sea stars. Catalytic functions of the cytochrome P450 monooxygenase system of sea stars in the metabolism of steroids may be suppressed as a result of the induction of cytochrome P450 by xenobiotics. PMID:9972455

  5. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  6. Identification of ABC Transporter Interaction of a Novel Cyanoquinoline Radiotracer and Implications for Tumour Imaging by Positron Emission Tomography

    PubMed Central

    Slade, Rozanna L.; Pisaneschi, Federica; Nguyen, Quang-De; Smith, Graham; Carroll, Laurence; Beckley, Alice; Kaliszczak, Maciej A.; Aboagye, Eric O.

    2016-01-01

    Background The epidermal growth factor receptor (EGFR) is overexpressed in many cancers including lung, ovarian, breast, head and neck and brain. Mutation of this receptor has been shown to play a crucial role in the response of non-small cell lung carcinoma (NSCLC) to EGFR-targeted therapies. It is envisaged that imaging of EGFR using positron emission tomography (PET) could aid in selection of patients for treatment with novel inhibitors. We recognised multi-drug resistant phenotype as a threat to development of successful imaging agents. In this report, we describe discovery of a novel cyanoquinoline radiotracer that lacks ABC transporter activity. Methods Cellular retention of the prototype cyanoquinoline [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-({[1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-but-2-enamide ([18F]FED6) and [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-[({1-[(2R,5S)-3-fluoro-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-1H-1,2,3-triazol-4-yl}methyl)amino]but-2-enamide ([18F]FED20) were evaluated to establish potential for imaging specificity. The substrate specificity of a number of cyanoquinolines towards ABC transporters was investigated in cell lines proficient or deficient in ABCB1 or ABCG2. Results FED6 demonstrated substrate specificity for both ABCG2 and ABCB1, a property that was not observed for all cyanoquinolines tested, suggesting scope for designing novel probes. ABC transporter activity was confirmed by attenuating the activity of transporters with drug inhibitors or siRNA. We synthesized a more hydrophilic compound [18F]FED20 to overcome ABC transporter activity. FED20 lacked substrate specificity for both ABCB1 and ABCG2, and maintained a strong affinity for EGFR. Furthermore, FED20 showed higher inhibitory affinity for active mutant EGFR versus wild-type or resistant mutant EGFR; this property resulted in higher [18F]FED20 cellular retention in active

  7. Development of gamma emitting receptor binding radiotracers for imaging the brain and pancreas. Final technical progress report, March 1, 1988--May 31, 1993

    SciTech Connect

    1996-01-01

    This document give paragraph synopses of results in research on brain and pancreas imaging, using radiotracers. General catagories of research included chemistry, pharmacology, imaging physics, and kinetic modeling. A list of publications is included

  8. Two-dimensional crystallization of monomeric bovine cytochrome c oxidase with bound cytochrome c in reconstituted lipid membranes

    PubMed Central

    Osuda, Yukiho; Shinzawa-Itoh, Kyoko; Tani, Kazutoshi; Maeda, Shintaro; Yoshikawa, Shinya; Tsukihara, Tomitake; Gerle, Christoph

    2016-01-01

    Mitochondrial cytochrome c oxidase utilizes electrons provided by cytochrome c for the active vectorial transport of protons across the inner mitochondrial membrane through the reduction of molecular oxygen to water. Direct structural evidence on the transient cytochrome c oxidase–cytochrome c complex thus far, however, remains elusive and its physiological relevant oligomeric form is unclear. Here, we report on the 2D crystallization of monomeric bovine cytochrome c oxidase with tightly bound cytochrome c at a molar ratio of 1:1 in reconstituted lipid membranes at the basic pH of 8.5 and low ionic strength. PMID:26754561

  9. Reactive Intermediates in Cytochrome P450 Catalysis*

    PubMed Central

    Krest, Courtney M.; Onderko, Elizabeth L.; Yosca, Timothy H.; Calixto, Julio C.; Karp, Richard F.; Livada, Jovan; Rittle, Jonathan; Green, Michael T.

    2013-01-01

    Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis. PMID:23632017

  10. Genetic characterization of Bagarius species using cytochrome c oxidase I and cytochrome b genes.

    PubMed

    Nagarajan, Muniyandi; Raja, Manikam; Vikram, Potnuru

    2016-09-01

    In this study, we first inferred the genetic variability of two Bagarius bagarius populations collected from Ganges and Brahmaputra rivers of India using two mtDNA markers. Sequence analysis of COI gene did not show significant differences between two populations whereas cytochrome b gene showed significant differences between two populations. Followed by, genetic relationship of B. bagarius and B. yarrielli was analyzed using COI and cytochrome b gene and the results showed a higher level genetic variation between two species. The present study provides support for the suitability of COI and cytochrome b genes for the identification of B. bagarius and B. yarrielli.

  11. Semi-automated lab-on-a-chip for dispensing GA-68 radiotracers

    SciTech Connect

    Weinberg, Irving

    2014-03-12

    We solved a technical problem that is hindering American progress in molecular medicine, and restricting US citizens from receiving optimal diagnostic care. Specifically, the project deals with a mother/daughter generator of positron-emitting radiotracers (Ge-68/Ga-68). These generator systems are approved in Europe but cannot be used in the USA, because of safety issues related to possible breakthrough of long-lived Ge-68 (mother) atoms. Europeans have demonstrated abilities of Ga-68-labeled radiotracers to image cancer foci with high sensitivity and specificity, and to use such methods to effectively plan therapy.The USA Food and Drug Administration (FDA) and Nuclear Regulatory Commission (NRC) have taken the position that every patient administration of Ga-68 should be preceded by an assay demonstrated that Ge-68 breakthrough is within acceptable limits. Breakthrough of parent elements is a sensitive subject at the FDA, as evidenced by the recent recall of Rb-82 generators due to inadvertent administrations of Sr-82. Commercially, there is no acceptable rapid method for assaying breakthrough of Ge-68 prior to each human administration. The gamma emissions of daughter Ga-68 have higher energies than the parent Ge-68, so that the shielding assays typically employed for Mo-99/Tc-99m generators cannot be applied to Ga-68 generators. The half-life of Ga-68 is 68 minutes, so that the standard 10-half-life delay (used to assess breakthrough in Sr-82/Rb-82 generators) cannot be applied to Ga-68 generators. As a result of the aforementioned regulatory requirements, Ga-68 generators are sold in the USA for animal use only.The American clinical community’s inability to utilize Ga-68 generators impairs abilities to treat patients domestically, and puts the USA at a disadvantage in developing exportable products. The proposed DOE project aimed to take advantage of recent technological advances developed for lab-on-a-chip (LOC) applications. Based on our experiences

  12. Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes.

    PubMed

    Meyer, Terry E; Tsapin, Alexandre I; Vandenberghe, Isabel; de Smet, Lina; Frishman, Dmitrij; Nealson, Kenneth H; Cusanovich, Michael A; van Beeumen, Jozef J

    2004-01-01

    Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.

  13. Discovery of a fluorinated 4-oxo-quinoline derivative as a potential positron emission tomography radiotracer for imaging cannabinoid receptor type 2.

    PubMed

    Slavik, Roger; Müller Herde, Adrienne; Haider, Ahmed; Krämer, Stefanie D; Weber, Markus; Schibli, Roger; Ametamey, Simon M; Mu, Linjing

    2016-09-01

    The cannabinoid receptor type 2 (CB2) is part of the endocannabinoid system and has gained growing attention in recent years because of its important role in neuroinflammatory/neurodegenerative diseases. Recently, we reported on a carbon-11 labeled 4-oxo-quinoline derivative, designated RS-016, as a promising radiotracer for imaging CB2 using PET. In this study, three novel fluorinated analogs of RS-016 were designed, synthesized, and pharmacologically evaluated. The results of our efforts led to the identification of N-(1-adamantyl)-1-(2-(2-fluoroethoxy)ethyl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxamide (RS-126) as the most potent candidate for evaluation as a CB2 PET ligand. [(18) F]RS-126 was obtained in ≥ 99% radiochemical purity with an average specific radioactivity of 98 GBq/μmol at the end of the radiosynthesis. [(18) F]RS-126 showed a logD7.4 value of 1.99 and is stable in vitro in rat and human plasma over 120 min, whereas 55% intact parent compound was found in vivo in rat blood plasma at 10 min post injection. In vitro autoradiographic studies with CB2-positive rat spleen tissue revealed high and blockable binding which was confirmed in in vivo displacement experiments with rats by dynamic PET imaging. Ex vivo biodistribution studies confirmed accumulation of [(18) F]RS-126 in rat spleen with a specificity of 79% under blocking conditions. The moderate elevated CB2 levels in LPS-treated mice brain did not permit the detection of CB2 by [(18) F]RS-126 using PET imaging. In summary, [(18) F]RS-126 demonstrated high specificity toward CB2 receptor in vitro and in vivo and is a promising radioligand for imaging CB2 receptor expression. Cannabinoid receptor type 2 (CB2) is an interesting target for PET imaging. Specific binding of [(18) F]RS-126 in CB2-positive spleen tissue (white arrow head) was confirmed in in vivo displacement experiments with rats. Time activity curve of [(18) F]RS-126 in the spleen after the addition of GW405833 (CB2

  14. Synthesis of Diverse (11)C-Labeled PET Radiotracers via Direct Incorporation of [(11)C]CO2.

    PubMed

    Mossine, Andrew V; Brooks, Allen F; Jackson, Isaac M; Quesada, Carole A; Sherman, Phillip; Cole, Erin L; Donnelly, David J; Scott, Peter J H; Shao, Xia

    2016-05-18

    Three new positron emission tomography (PET) radiotracers of interest to our functional neuroimaging and translational oncology programs have been prepared through new developments in [(11)C]CO2 fixation chemistry. [(11)C]QZ (glutaminyl cyclase) was prepared via a tandem trapping of [(11)C]CO2/intramolecular cyclization; [(11)C]tideglusib (glycogen synthase kinase-3) was synthesized through a tandem trapping of [(11)C]CO2 followed by an intermolecular cycloaddition between a [(11)C]isocyanate and an isothiocyanate to form the 1,2,4-thiadiazolidine-3,5-dione core; [(11)C]ibrutinib (Bruton's tyrosine kinase) was synthesized through a HATU peptide coupling of an amino precursor with [(11)C]acrylic acid (generated from [(11)C]CO2 fixation with vinylmagnesium bromide). All radiochemical syntheses are fully automated on commercial radiochemical synthesis modules and provide radiotracers in 1-5% radiochemical yield (noncorrected, based upon [(11)C]CO2). All three radiotracers have advanced to rodent imaging studies and preliminary PET imaging results are also reported. PMID:27043721

  15. Synthesis of Diverse (11)C-Labeled PET Radiotracers via Direct Incorporation of [(11)C]CO2.

    PubMed

    Mossine, Andrew V; Brooks, Allen F; Jackson, Isaac M; Quesada, Carole A; Sherman, Phillip; Cole, Erin L; Donnelly, David J; Scott, Peter J H; Shao, Xia

    2016-05-18

    Three new positron emission tomography (PET) radiotracers of interest to our functional neuroimaging and translational oncology programs have been prepared through new developments in [(11)C]CO2 fixation chemistry. [(11)C]QZ (glutaminyl cyclase) was prepared via a tandem trapping of [(11)C]CO2/intramolecular cyclization; [(11)C]tideglusib (glycogen synthase kinase-3) was synthesized through a tandem trapping of [(11)C]CO2 followed by an intermolecular cycloaddition between a [(11)C]isocyanate and an isothiocyanate to form the 1,2,4-thiadiazolidine-3,5-dione core; [(11)C]ibrutinib (Bruton's tyrosine kinase) was synthesized through a HATU peptide coupling of an amino precursor with [(11)C]acrylic acid (generated from [(11)C]CO2 fixation with vinylmagnesium bromide). All radiochemical syntheses are fully automated on commercial radiochemical synthesis modules and provide radiotracers in 1-5% radiochemical yield (noncorrected, based upon [(11)C]CO2). All three radiotracers have advanced to rodent imaging studies and preliminary PET imaging results are also reported.

  16. Development of indazolylpyrimidine derivatives as high-affine EphB4 receptor ligands and potential PET radiotracers.

    PubMed

    Ebert, Kristin; Wiemer, Jens; Caballero, Julio; Köckerling, Martin; Steinbach, Jörg; Pietzsch, Jens; Mamat, Constantin

    2015-09-01

    Due to their essential role in the pathogenesis of cancer, members of the Eph (erythropoietin-producing hepatoma cell line-A2) receptor tyrosine kinase family represent promising candidates for molecular imaging. Thus, the development and preparation of novel radiotracers for the noninvasive imaging of the EphB4 receptor via positron emission tomography (PET) is described. First in silico investigations with the indazolylpyrimidine lead compound which is known to be highly affine to EphB4 were executed to identify favorable labeling positions for an introduction of fluorine-18 to retain the affinity. Based on this, reference compounds as well as precursors were developed and labeled with carbon-11 and fluorine-18, respectively. For this purpose, a protecting group strategy essentially had to be generated to prevent unwanted methylation and to enable the introduction of fluorine-18. Further, a convenient radiolabeling strategy using [(11)C]methyl iodide was established which afforded the isotopically labeled radiotracer in 30-35% RCY (d.c.) which is identical with the original inhibitor molecule. A spiro ammonium precursor was prepared for radiolabeling with fluorine-18. Unfortunately, the labeling did not lead to the desired (18)F-radiotracer under the chosen conditions. PMID:26189032

  17. Cytochrome C: A Biochemistry Laboratory Course

    ERIC Educational Resources Information Center

    Vincent, John B.; Woski, Stephen A.

    2005-01-01

    A laboratory course called cytochrome c that focuses on the theme of biochemical research is presented. The students follow this course by incorporating team-investigation and self-directed experimentation that provides them an opportunity to experience the excitement of research.

  18. Production of 191Pt radiotracer with high specific activity for the development of preconcentration procedures

    NASA Astrophysics Data System (ADS)

    Parent, M.; Strijckmans, K.; Cornelis, R.; Dewaele, J.; Dams, R.

    1994-04-01

    A radiotracer of Pt with suitable nuclear characteristics and high specific activity (i.e. activity to mass ratio) is a powerful tool when developing preconcentration methods for the determination of base-line levels of Pt in e.g. environmental and biological samples. Two methods were developed for the production of 191Pt with high specific activity and radionuclidic purity: (1) via the 190Pt(n, γ) 191Pt reaction by neutron irradiation of enriched Pt in a nuclear reactor at high neutron fluence rate and (2) via the 191Ir(p, n) 191Pt reaction by proton irradiation of natural Ir with a cyclotron, at an experimentally optimized proton energy. For the latter method it was necessary to separate Pt from the Ir matrix. For that reason either liquid-liquid extraction with dithizone or adsorption chromatography were used. The yields, the specific activities and the radionuclidic purities were experimentally determined as a function of the proton energy and compared to the former method. The half-life of 191Pt was accurately determined to be 2.802 ± 0.025 d.

  19. Biochemical quantification of sympathetic nervous activity in humans using radiotracer methodology: fallibility of plasma noradrenaline measurements

    SciTech Connect

    Esler, M.; Leonard, P.; O'Dea, K.; Jackman, G.; Jennings, G.; Korner, P.

    1982-01-01

    We have developed radiotracer techniques for studying noradrenaline kinetics, to assess better sympathetic nervous system function in humans. Tritiated l-noradrenaline was infused intravenously (0.35 microCi/m2/min) to plateau plasma concentration. Noradrenaline plasma clearance was calculated from plasma tritiated noradrenaline concentration at steady state, and the rate of spillover of noradrenaline to plasma derived from plasma noradrenaline specific radioactivity. Mean noradrenaline spillover at rest in 34 normal subjects was 0.33 micrograms/m2/min (range 0.17-0.61 micrograms/m2/min). Predictably, noradrenaline spillover was reduced in patients with subnormal sympathetic nervous system activity, 0.16 +/- 0.09 micrograms/m2/min in eight patients with idiopathic peripheral autonomic insufficiency, and 0.11 +/- 0.07 micrograms/m2/min (mean +/- SD) in six patients with essential hypertension treated with clonidine (0.45 mg daily). Noradrenaline line plasma clearance in normal subjects was 1.32 +/- 0.28 L/m2/min. Clearance fell with age, causing the previously described rise in plasma noradrenaline concentration with aging. Unexpected effects of drugs were encountered, for example chronic beta-adrenergic blockade in patients with essential hypertension reduced noradrenaline clearance. Plasma noradrenaline concentration measurements were not in agreement with noradrenaline release rate values, and do not reliably indicate sympathetic nervous system activity, in instances such as these where noradrenaline clearance is abnormal.

  20. Bioaccumulation of (63)Ni in the scleractinian coral Stylophora pistillata and isolated Symbiodinium using radiotracer techniques.

    PubMed

    Hédouin, Laetitia; Metian, Marc; Teyssié, Jean-Louis; Oberhänsli, François; Ferrier-Pagès, Christine; Warnau, Michel

    2016-08-01

    Development of nickel mining activities along the New Caledonia coasts threatens the biodiversity of coral reefs. Although the validation of tropical marine organisms as bioindicators of metal mining contamination has received much attention in the literature over the last decade, few studies have examined the potential of corals, the fundamental organisms of coral reefs, to monitor nickel (Ni) contamination in tropical marine ecosystems. In an effort to bridge this gap, the present work investigated the bioaccumulation of (63)Ni in the scleractinian coral Stylophora pistillata and in its isolated zooxanthellae Symbiodinium, using radiotracer techniques. Results highlight the high capacities of coral tissues (zooxanthellae and host tissues) to efficiently bioconcentrate (63)Ni compared to skeleton (Concentration Factors CF at 14 days of exposure are 3 orders of magnitude higher in tissues than in skeleton). When non-contaminated conditions were restored, (63)Ni was more efficiently retained in skeleton than in coral tissues, with biological half-lives (Tb½) of 44.3 and 6.5 days, respectively. In addition, our work showed that Symbiodinium bioconcentrated (63)Ni exponentially, with a vol/vol concentration factor at steady state (VCFSS) reaching 14,056. However, compilation of our results highlighted that despite efficient bioconcentration of (63)Ni in Symbiodinium, their contribution to the whole (63)Ni accumulation in coral nubbins represents less than 7%, suggesting that other biologically controlled processes occur in coral host allowing such efficient bioconcentration in coral tissues. PMID:27192479

  1. Indepth diagnosis of a secondary clarifier by the application of radiotracer technique and numerical modeling.

    PubMed

    Kim, H S; Shin, M S; Jang, D S; Jung, S H

    2006-01-01

    To make an indepth diagnosis of a full-scale rectangular secondary clarifier, an experimental and numerical study has been performed in a wastewater treatment facility. Calculation results by the numerical model with the adoption of the SIMPLE algorithm of Patankar are validated with radiotracer experiments. Emphasis is given to the prediction of residence time distribution (RTD) curves. The predicted RTD profiles are in good agreement with the experimental RTD curves at the upstream and center sections except for the withdrawal zone of the complex effluent weir structure. The simulation results predict successfully the well-known flow characteristics of each stage such as the waterfall phenomenon at the front of the clarifier, the bottom density current and the surface return flow in the settling zone, and the upward flow in the exit zone. The detailed effects of density current are thoroughly investigated in terms of high SS loading and temperature difference between influent and ambient fluid. The program developed in this study shows the high potential to assist in the design and determination of optimal operating conditions to improve effluent quality in a full-scale secondary clarifier.

  2. PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters in Vivo

    PubMed Central

    Mairinger, Severin; Erker, Thomas; Müller, Markus; Langer, Oliver

    2013-01-01

    Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer’s and Parkinson’s disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications. PMID:21434859

  3. Whole lake addition of cadmium-109: radiotracer accumulation in the mussel population in the first season.

    PubMed

    Malley, D F; Chang, P S; Hesslein, R H

    1989-11-01

    Cadmium with the radiotracer 109Cd was added to the epilimnion of Precambrian Shield Lake 382 in the Experimental Lakes Area, northwestern Ontario to monitor pathways of Cd from water to abiotic and biotic components, to quantify Cd accumulation and distribution in biota and to evaluate adverse biological and ecological effects. This experiment will permit evaluation of the adequacy of the water quality guidelines of 0.2 microgram Cd l-1 in protecting aquatic life in softwater lakes. As part of the whole-lake experiment, we monitored the activities of 109Cd in various body parts of the floater mussel Anodonta grandis grandis to determine accumulation and distribution of Cd. Additions of Cd from 23 June to the end of October 1987 (a total of 900 g Cd and 89 mCi of 109Cd) increased the total [Cd] in the water from 1.6 to about 85 ng l-1. Cadmium-109 was accumulated in body parts of the mussels, in increasing concentration: mantle less than foot less than gill less than visceral mass less than kidney. After 4 months exposure to the increased water [Cd], the mussels had increased body burden of Cd by an estimated 5-9 times. At the latter increase, the population of 7330 +/- 2100 mussels in the lake contained an estimated 0.011-0.020% of the added 109Cd.

  4. Investigating phosphorus uptake in anoxic and sulfidic surface sediments with 33P radiotracer experiments

    NASA Astrophysics Data System (ADS)

    Dijkstra, Nikki; Kraal, Peter; Gonzalez, Santiago; Slomp, Caroline

    2016-04-01

    Phosphorus (P) is a key nutrient for marine organisms. Enhanced P availability in the water column can fuel algal blooms and the development of bottom water anoxia. Recently, it was suggested that micro-organisms in sediments overlain by anoxic and sulfidic bottom waters might take up dissolved P and form Fe(II)-P minerals, thereby enhancing P removal. In this study, we investigated the uptake of P in surface sediments with 33P radiotracer experiments. The sediments were recovered from the anoxic and sulfidic deep basin of the Black Sea and, for comparison, from the adjacent oxic shelf. Results suggest a very fast sedimentary uptake of 33P at all sites but in particular for sediments from the oxic shelf. At all sites, most 33P was sequestered in the citrate-dithionite-bicarbonate-(CDB)-extractable sediment P fraction. No significant differences with abiotic controls were observed, implying that micro-organisms were not directly involved in the P uptake. Whereas 33P uptake by the oxic shelf sediment was likely controlled by sorption of 33P to iron(Fe)-(oxyhydr)oxides, the nature of the CDB-extractable P fraction in the deep basin sediments remains unclear. We discuss whether authigenic formation of Fe(II)-P minerals or fast adsorption of P to calcites may explain our findings.

  5. Food chains and biogeochemical pathways: contributions of fallout and other radiotracers.

    PubMed

    Whicker, F Ward; Pinder, John E

    2002-05-01

    This paper reviews examples of how measurements of global fallout in the environment and related tracer radionuclides have been used to enhance our basic knowledge of biogeochemical processes and food-chain pathways. Because it is these fundamental, natural processes that control the transport and accumulation of such trace substances in the environment, direct measurements of trace substances over time and space reveal strong insights into these processes. The necessity to monitor global fallout transport, although largely motivated by human health concerns, gave rise to a plethora of new information about plants, animals, and natural and agricultural ecosystems and how they function. This review provides a small selection of examples in the areas of plant and animal physiology, productivity and energy transfer in food chains, biogeochemical cycles of certain elements and their analogues, feeding relationships and movements of organisms, and the agriculture-based human food chain. It is concluded that if society is to cope successfully with continued growth of the human population and resource consumption, more knowledge is still required about these fundamental processes. The use of radiotracers can contribute greatly to this need, but current funding priorities, societal attitudes, and onerous regulations on the use of radioactivity may continue to limit such applications.

  6. Design of ruthenium-cytochrome c derivatives to measure electron transfer to cytochrome c peroxidase.

    PubMed

    Liu, R Q; Geren, L; Anderson, P; Fairris, J L; Peffer, N; McKee, A; Durham, B; Millet, F

    1995-01-01

    A new technique has been introduced to measure interprotein electron transfer which involves photoexcitation of a tris(bipyridine)ruthenium (Ru) complex covalently attached to one of the proteins. Four different strategies have been developed to specifically attach Ru to protein lysine amino groups, histidine imidazole groups, and cysteine sulhydryl groups. These strategies have been used to prepare more than 20 different singly-labeled Ru-cytochrome c derivatives. The new ruthenium photoexcitation technique has been used to study the mechanism for electron transfer between cytochrome c and cytochrome c peroxidase. Laser excitation of a complex between Ru-cytochrome c and cytochrome c peroxidase compound I results in formation of Ru(II*) which is a strong reducing agent, and rapidly transfers an electron to heme c Fe(III) to form Fe(II). The heme c Fe(II) then rapidly transfers an electron to the Trp-191 radical cation in CMPI. The rate constant for this reaction is 6 x 10(4) s-1 for a horse Ru-cytochrome c derivative labeled at lysine 27, and greater than 10(6) s-1 for yeast Ru-cytochrome c derivatives. A second laser flash results in electron transfer from heme c to the oxyferryl heme in cytochrome c peroxidase compound II with a rate constant of 350 s-1. The ruthenium photoreduction technique has been used to study the interaction domain between the two proteins, the pathway for electron transfer to the radical cation and the oxyferryl heme, and the specific residues in the heme crevice which control the electron transfer properties of the Trp-191 radical cation and the oxyferryl heme.

  7. Phosphorylation of mammalian cytochrome c and cytochrome c oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease.

    PubMed

    Hüttemann, Maik; Lee, Icksoo; Grossman, Lawrence I; Doan, Jeffrey W; Sanderson, Thomas H

    2012-01-01

    The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.

  8. Role of active oxygen species in the photodestruction of microsomal cytochrome P-450 and associated monooxygenases by hematoporphyrin derivative in rats

    SciTech Connect

    Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R.

    1985-02-01

    The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H/sub 2/O/sub 2/ such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization.

  9. Recent Structural Insights into Cytochrome P450 Function.

    PubMed

    Guengerich, F Peter; Waterman, Michael R; Egli, Martin

    2016-08-01

    Cytochrome P450 (P450) enzymes are important in the metabolism of drugs, steroids, fat-soluble vitamins, carcinogens, pesticides, and many other types of chemicals. Their catalytic activities are important issues in areas such as drug-drug interactions and endocrine function. During the past 30 years, structures of P450s have been very helpful in understanding function, particularly the mammalian P450 structures available in the past 15 years. We review recent activity in this area, focusing on the past 2 years (2014-2015). Structural work with microbial P450s includes studies related to the biosynthesis of natural products and the use of parasitic and fungal P450 structures as targets for drug discovery. Studies on mammalian P450s include the utilization of information about 'drug-metabolizing' P450s to improve drug development and also to understand the molecular bases of endocrine dysfunction. PMID:27267697

  10. Cytochrome P450 as dimerization catalyst in diketopiperazine alkaloid biosynthesis.

    PubMed

    Saruwatari, Takayoshi; Yagishita, Fumitoshi; Mino, Takashi; Noguchi, Hiroshi; Hotta, Kinya; Watanabe, Kenji

    2014-03-21

    As dimeric natural products frequently exhibit useful biological activities, identifying and understanding their mechanisms of dimerization is of great interest. One such compound is (−)-ditryptophenaline, isolated from Aspergillus flavus, which inhibits substance P receptor for potential analgesic and anti-inflammatory activity. Through targeted gene knockout in A. flavus and heterologous yeast gene expression, we determined for the first time the gene cluster and pathway for the biosynthesis of a dimeric diketopiperazine alkaloid. We also determined that a single cytochrome P450, DtpC, is responsible not only for pyrroloindole ring formation but also for concurrent dimerization of N-methylphenylalanyltryptophanyl diketopiperazine monomers into a homodimeric product. Furthermore, DtpC exhibits relaxed substrate specificity, allowing the formation of two new dimeric compounds from a non-native monomeric precursor, brevianamide F. A radical-mediated mechanism of dimerization is proposed.

  11. Interaction with membranes of cytochrome c554 from Nitrosomonas europaea.

    PubMed

    McTavish, H; Arciero, D M; Hooper, A B

    1995-12-01

    Two c-cytochromes extrinsically bound to the membranes of Nitrosomonas europaea have been identified. One is the tetraheme cytochrome c554, a protein previously described as soluble and periplasmic. Depending on the concentration of Fe and Cu in the growth medium, from 50 to 100% of the total cellular cytochrome c554 is membrane-associated. The cytochromes c554 found in the soluble or membrane fractions are identical in the spectroscopic, chromatographic, or primary structural properties examined. The interaction of cytochrome c554 with membranes is ionic in nature; it is disrupted by high concentrations of salt. Both membrane-derived and periplasmic forms of cytochrome c554 rebind tightly to membranes which have been washed free of the cytochrome. Cytochrome c554 binds to phospholipid vesicles, suggesting that phospholipids may play a role in the interaction of this cytochrome with the membrane. During the oxidation of NH2OH, the ability of the soluble hydroxylamine oxidoreductase (HAO) to transfer electrons to its natural electron acceptor, cytochrome c554, is substantially impaired when the latter is bound to phospholipid vesicles. The second c-cytochrome associated with membranes in N. europaea is identified as HAO based on its catalytic activity and the presence of a 464-nm ferrous absorption band. A small fraction of HAO is found to be membrane-bound and only in cells grown under low Fe/low Cu. This subpopulation of HAO can be released from the membranes without detergents. PMID:7503559

  12. Synthesis and in vivo Evaluation of Fluorine-18 and Iodine-123 Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine Derivatives as PET and SPECT Radiotracers for Mapping A2A Receptors.

    PubMed

    Vala, Christine; Morley, Thomas J; Zhang, Xuechun; Papin, Caroline; Tavares, Adriana Alexandre S; Lee, H Sharon; Constantinescu, Cristian; Barret, Olivier; Carroll, Vincent M; Baldwin, Ronald M; Tamagnan, Gilles D; Alagille, David

    2016-09-01

    Imaging agents that target adenosine type 2A (A2A ) receptors play an important role in evaluating new pharmaceuticals targeting these receptors, such as those currently being developed for the treatment of movement disorders like Parkinson's disease. They are also useful for monitoring progression and treatment efficacy by providing a noninvasive tool to map changes in A2A receptor density and function in neurodegenerative diseases. We previously described the successful evaluation of two A2A -specific radiotracers in both nonhuman primates and in subsequent human clinical trials: [(123) I]MNI-420 and [(18) F]MNI-444. Herein we describe the development of both of these radiotracers by selection from a series of A2A ligands, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core of preladenant. Each of this series of 16 ligands was found to bind to recombinant human A2A receptor in the low nanomolar range, and of these 16, six were radiolabeled with either fluorine-18 or iodine-123 and evaluated in nonhuman primates. These initial in vivo results resulted in the identification of 7-(2-(4-(4-(2-[(18) F]fluoroethoxy)phenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine ([(18) F]MNI-444) and 7-(2-(4-(2-fluoro-4-[(123) I]iodophenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-amine ([(123) I]MNI-420) as PET and SPECT radiopharmaceuticals for mapping A2A receptors in brain.

  13. Synthesis and in vivo Evaluation of Fluorine-18 and Iodine-123 Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine Derivatives as PET and SPECT Radiotracers for Mapping A2A Receptors.

    PubMed

    Vala, Christine; Morley, Thomas J; Zhang, Xuechun; Papin, Caroline; Tavares, Adriana Alexandre S; Lee, H Sharon; Constantinescu, Cristian; Barret, Olivier; Carroll, Vincent M; Baldwin, Ronald M; Tamagnan, Gilles D; Alagille, David

    2016-09-01

    Imaging agents that target adenosine type 2A (A2A ) receptors play an important role in evaluating new pharmaceuticals targeting these receptors, such as those currently being developed for the treatment of movement disorders like Parkinson's disease. They are also useful for monitoring progression and treatment efficacy by providing a noninvasive tool to map changes in A2A receptor density and function in neurodegenerative diseases. We previously described the successful evaluation of two A2A -specific radiotracers in both nonhuman primates and in subsequent human clinical trials: [(123) I]MNI-420 and [(18) F]MNI-444. Herein we describe the development of both of these radiotracers by selection from a series of A2A ligands, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core of preladenant. Each of this series of 16 ligands was found to bind to recombinant human A2A receptor in the low nanomolar range, and of these 16, six were radiolabeled with either fluorine-18 or iodine-123 and evaluated in nonhuman primates. These initial in vivo results resulted in the identification of 7-(2-(4-(4-(2-[(18) F]fluoroethoxy)phenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine ([(18) F]MNI-444) and 7-(2-(4-(2-fluoro-4-[(123) I]iodophenyl)piperazin-1-yl)ethyl)-2-(furan-2-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-amine ([(123) I]MNI-420) as PET and SPECT radiopharmaceuticals for mapping A2A receptors in brain. PMID:27407017

  14. Identification and validation of tetracyclic benzothiazepines as Plasmodium falciparum cytochrome bc1 inhibitors

    PubMed Central

    Dong, Carolyn; Urgaonkar, Sameer; Cortese, Joseph F.; Gamo, F. Javier; Garcia-Bustos, Jose F.; Lafuente, Maria J.; Patel, Vishal; Ross, Leila; Coleman, Bradley I.; Derbyshire, Emily R.; Clish, Clary B.; Serrano, Adelfa E.; Cromwell, Mandy; Barker, Robert H.; Dvorin, Jeffrey D.; Duraisingh, Manoj T.; Wirth, Dyann F.; Clardy, Jon; Mazitschek, Ralph

    2012-01-01

    Summary Here we report the discovery of tetracyclic benzothiazepines (BTZ) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc1 complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc1 activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy. PMID:22195562

  15. Cytochrome c maturation and the physiological role of c-type cytochromes in Vibrio cholerae.

    PubMed

    Braun, Martin; Thöny-Meyer, Linda

    2005-09-01

    Vibrio cholerae lives in different habitats, varying from aquatic ecosystems to the human intestinal tract. The organism has acquired a set of electron transport pathways for aerobic and anaerobic respiration that enable adaptation to the various environmental conditions. We have inactivated the V. cholerae ccmE gene, which is required for cytochrome c biogenesis. The resulting strain is deficient of all c-type cytochromes and allows us to characterize the physiological role of these proteins. Under aerobic conditions in rich medium, V. cholerae produces at least six c-type cytochromes, none of which is required for growth. Wild-type V. cholerae produces active fumarate reductase, trimethylamine N-oxide reductase, cbb3 oxidase, and nitrate reductase, of which only the fumarate reductase does not require maturation of c-type cytochromes. The reduction of nitrate in the medium resulted in the accumulation of nitrite, which is toxic for the cells. This suggests that V. cholerae is able to scavenge nitrate from the environment only in the presence of other nitrite-reducing organisms. The phenotypes of cytochrome c-deficient V. cholerae were used in a transposon mutagenesis screening to search for additional genes required for cytochrome c maturation. Over 55,000 mutants were analyzed for nitrate reductase and cbb3 oxidase activity. No transposon insertions other than those within the ccm genes for cytochrome c maturation and the dsbD gene, which encodes a disulphide bond reductase, were found. In addition, the role of a novel CcdA-like protein in cbb3 oxidase assembly is discussed.

  16. A radiotracer study of cerium and manganese uptake onto suspended particles in Chesapeake Bay

    SciTech Connect

    Moffett, J.W. )

    1994-01-01

    The oxidation kinetics of Ce(III) and Mn(II) were studied in Chesapeake Bay in March and July 1990 to establish the role of water column redox processes in contributing to Ce anomalies observed in this estuary (SHOLKOVITZ and ELDERFIELD, 1988; SHOLKOVITZ et al., 1992). Oxidation was measured by adding Mn(II) and Ce(III) to freshly collected water samples as radiotracers and measuring their uptake onto the ambient suspended particle assemblage. Mn(II) oxidation was measured by following the uptake of [sup 54]Mn(II) onto suspended particles and utilizing protocols established by other workers to distinguish oxidation from Mn(II) adsorption. The same protocols were applicable to Ce(III), using [sup 139]Ce(III), and were supported by the use of [sup 152]Eu(III) as a nonredox reactive control. Specific rates of Ce(III) and MN(II) oxidation measured at a station in the North Bay (depth = 4 m) in July were 2016% per day and 4032% per day, respectively. In March, at the same station, the specific rate of Mn(II) of oxidation was only 1-% per day, and Ce(III) oxidation was undetectable. Both Ce(III) and Mn(II) oxidation processes were inhibited by azide, indicating that they were microbially mediated. The seasonal differences probably reflect strong seasonal variation in the abundance of Mn oxidizing bacteria. No Ce(III) oxidation occured in samples collected below the oxic/anoxic interface in July. The specific rates of oxidation for both elements were over 1000 times higher than those measured in the Sargasso Sea. However, the specific rates for Ce(III) and Mn(II) were very similar to each other. This fact, coupled with similar spatial and temporal trends for specific oxidation rates, suggests a common mechanism of oxidation of both elements which may be significant in a wide range of marine environments.

  17. Improving AMS Detection of the Biomedical Radiotracer 41Ca with Segmented Radio-Frequency Quadrupoles

    NASA Astrophysics Data System (ADS)

    Alary, Jean-Francois; Javahery, Gholamreza; Kieser, William E.; Litherland, Albert E.; Cousins, Lisa M.

    41Ca is an important biomedical radiotracer finding many applications in biological, nutritional and medical studies. The detection of 41Ca by AMS is however limited by an important background signal of 41K originating from biological samples and from contaminated cesium in the source. An approach consisting of using PbF2-assisted in-source fluorination in combination with an Isobar Separator for Anions (ISA), a device incorporating a low energy radio frequency quadrupole (RFQ) gas cell, promises to push down the limit of detection of 41Ca attainable on small (<3 MV) accelerator mass spectrometry (AMS) systems by several orders of magnitude. Such on-line reduction of 41K should also result in a simplification of biological sample preparation and less concern about variable 41K contamination of the cesium beam. The selective collision-induced fragmentation of KF3- versus CaF3-, occurring in the gas cell of an ISA equipped with a double segment RFQ, have been reported earlier1), leading to K being suppressed by a factor of 1e4 over Ca. We present here the future configuration of the ISA, redesigned using multi-segmented RFQ to enhance further this effect and improve transmission through the gas cell. A segmented RFQ is an appropriate tool to finely control ion energy down to the few eV's separating the fragmentation energies of the two fluoride species. This pre-commercial ISA destined to be used at the newly established A. E. Lalonde AMS laboratory at University of Ottawa (Canada) will be presented. Some practicalities of integrating a low energy RFQ-based device in a high energy AMS system will also be discussed.

  18. SU-E-I-80: Beta-Minus Emitting Radiotracers Improves Molecular Endoscopy

    SciTech Connect

    Carpenter, C; Ma, X; Sun, C; Pratx, G; Cheng, Z; Xing, L

    2014-06-01

    Purpose: Molecular Endoscopy using Cerenkov Luminescence can be used to monitor the distribution of many clinically-available PET and SPECT probes for endoscopic applications. A main limitation of Cerenkov is its limited sensitivity to small concentrations of radiotracer when using light guides s. Herein we demonstrate that the use of a high energy beta emitting radioisotope, exemplified here with 90Y provides superior sensitivity to 18F because of its higher light output and its lack of corresponding gamma emission. Methods: A series of phantom experiments were performed to compare the sensitivity and noise of the CLE system for imaging 90Y and 18F. Three vials of known concentrations of 90Y (0.008 μCi, 0.08 μCi, 1 μCi) were placed in centrifuge tubes and isolated from each other. One vial of 18F (100 μCi) was placed in the imaging chamber and imaged over the course of decay (19 hours, 43 minutes, or ∼10 half-lives). Image time-points were formed from 5-minute integrations. Results: Using an SNR of 10 to define the noise-floor, the 90Y minimum detectable activity was 0.056 μCi. To the contrast, the minimum detectable activity for 18F was 11.63 μCi. These data demonstrate a 207-fold improvement in SNR of 90Y compared to 18F, when controlled for activity. Conclusion: This study demonstrated that a pure β- radionuclide such as 90Y be used is superior to 18F for Cerenkov Endoscopy. Further study is needed to demonstrate its utility in preclinical studies, endoscopic applications, intraoperative, and radiotherapy applications.

  19. Biological evaluation of new [(18) F]F-labeled synthetic amino acid derivatives as oncologic radiotracers.

    PubMed

    Kim, Yeseulmi; Lee, Sang Ju; Yook, Cheol-Min; Oh, Seung Jun; Ryu, Jin-Sook; Lee, Jong Jin

    2016-08-01

    The present study evaluated the tumoral uptake of the novel synthetic amino acid positron emission tomography (PET) tracers (S)-2-amino-3-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)propanoic acid (AMC-101), (S)-2-amino-4-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)butanoic acid (AMC-102), and (S)-2-amino-5-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)pentanoic acid (AMC-103), all of which are (S)-2-amino-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)alkyl acids. In vitro cellular uptake was investigated using the rat glioma cell lines 9L and C6. In vitro competitive inhibition tests were performed to identify the involvement of specific amino acid transporters. In vivo dynamic PET images of 9L xenograft tumor-bearing model mice were acquired over 2 h after AMC administration. [(18) F]FDOPA PET studies were performed with and without S-carbidopa pretreatment for comparison. All three AMCs exhibited good in vitro cell uptake through the L and alanine-serine-cysteine transporters and enabled clear tumor visualization on PET, leaving the brain devoid of the tracer. Thirty minutes after injection, the mean tumor standardized uptake values were 1.59 ± 0.05, 1.89 ± 0.27, and 1.74 ± 0.13 for AMC-101, AMC-102, and AMC-103, respectively. Although the tumor uptake values of AMCs were lower than that of [(18) F]FDOPA with S-carbidopa pretreatment, AMCs enabled higher contrast images with lower background activity compared with [(18) F]FDOPA with S-carbidopa pretreatment. Our results indicate the potential uses of these new synthetic amino acids as oncologic radiotracers.

  20. Addressing Shewanella oneidensis cytochromome : a first step towards high-throughput expression of cytochromes c.

    SciTech Connect

    Londer, Y. Y.; Giuliani, S.; Peppler, T.; Collart, F.; Biosciences Division

    2008-11-01

    Integrated studies that address proteins structure and function in the new era of systems biology and genomics often require the application of high-throughput approaches for parallel production of many different purified proteins from the same organism. Cytochromes c-electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain--are essential in most organisms. However, they are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. We have addressed this challenge by designing two families of vectors (total of 6 vectors) suitable for ligation-independent cloning and developing a pipeline for expression and solubility analysis of cytochromes c. This system has been validated by expression analysis of thirty genes from Shewanella oneidensis coding for cytochromes c or cytochromes c-type domains predicted to have 1-4 hemes. Out of 30 targets, 26 (87%) were obtained in soluble form in one or more vectors. This work establishes a methodology for high-throughput expression of this class of proteins and provides a clone resource for the microbiological and functional genomics research communities.

  1. Inhibition of the synthesis of a cytochrome-c-oxidase subunit isoform by antisense RNA.

    PubMed

    Sandonà, D; Bisson, R

    1994-02-01

    To investigate the role of subunit VIIe, an oxygen-regulated subunit isoform of Dictyostelium discoideum cytochrome-c oxidase, the full-length cDNA was inserted into an expression vector under the control of an actin promoter in the sense and antisense orientation. The DNA constructs were used for stable transformation of the slime mold amoebae. In most of the 28 antisense clones tested, the concentration of cytochrome-c oxidase was lowered compared to the wild type, while no significant changes were found in the sense mutants. Antisense RNA was abundantly expressed, leading to a drastic reduction of the steady-state level of the endogenous subunit VIIe mRNA, which was decreased up to 20-30% the level observed in parent cells. In these transformants, the amount of the target polypeptide and cytochrome c oxidase was 40-50% and 60-70% of control, respectively. A similar decrease was found in the level of the remaining nuclear and mitochondrial subunits. Unexpectedly, these changes affected neither basal nor uncoupled cell respiration suggesting an increase of the enzyme specific activity. Hypoxia completely relieved the cytochrome-c-oxidase deficit. These results indicate that subunit VII is needed for an efficient assembly of the protein complex and provide evidence for its involvement in the modulation of the enzyme activity. PMID:8112318

  2. Probing the cytochrome c' folding landscape.

    PubMed

    Pletneva, Ekaterina V; Zhao, Ziqing; Kimura, Tetsunari; Petrova, Krastina V; Gray, Harry B; Winkler, Jay R

    2007-11-01

    The folding kinetics of R. palustris cytochrome c' (cyt c') have been monitored by heme absorption and native Trp72 fluorescence at pH 5. The Trp72 fluorescence burst signal suggests early compaction of the polypeptide ensemble. Analysis of heme transient absorption spectra reveals deviations from two-state behavior, including a prominent slow phase that is accelerated by the prolyl isomerase cyclophilin. A nonnative proline configuration (Pro21) likely interferes with the formation of the helical bundle surrounding the heme.

  3. PET studies of binding competition between endogenous dopamine and the D1 radiotracer [11C]NNC 756.

    PubMed

    Abi-Dargham, A; Simpson, N; Kegeles, L; Parsey, R; Hwang, D R; Anjilvel, S; Zea-Ponce, Y; Lombardo, I; Van Heertum, R; Mann, J J; Foged, C; Halldin, C; Laruelle, M

    1999-05-01

    NNC 756 ((+)-8-chloro-5-(2,3-dihydrobenzofuran-7-yl)-7-hydroxy-3-methyl-2,3,4,5- tetrahydro-1H-3-benzazepine) is a new high affinity dopamine (DA) D1 receptor antagonist. Labeled with C-11, it has been used as a PET radiotracer to visualize D1 receptors both in striatal and extrastriatal areas, such as the prefrontal cortex. The goal of this study was to evaluate several methods for derivation of D1 receptor binding potential (BP) with [11C]NNC 756 in baboons, and to use these methods to assess the vulnerability of [11C]NNC 756 binding to competition by endogenous DA. A three-compartment model provided a good fit to PET data acquired following a single bolus injection. BP values obtained with this analysis were in good agreement with values derived from in vitro studies. BP values measured following injection of the potent DA releaser amphetamine (1 mg/kg, n=2) were similar to values measured under control conditions. Kinetic parameters derived from single bolus experiments were used to design a bolus plus continuous infusion administration protocol aimed at achieving a state of sustained binding equilibrium. Injection of amphetamine during sustained equilibrium did not affect [11C]NNC 756 binding. Similar results were observed with another D1 radiotracer, [11C]SCH 23390. Doses of amphetamine used in this study are known to reduce by 20-40% the binding potential of several D2 receptors radiotracers. Therefore, the absence of displacement of [11C]NNC 756 by an endogenous DA surge may indicate important differences between D1 and D2 receptors in vivo, such as differences in proportion of high affinity states not occupied by DA at baseline. These findings may also imply that a simple binding competition model is inadequate to account for the effects of manipulation of endogenous DA levels on the in vivo binding of radiolabeled antagonists.

  4. Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase

    PubMed Central

    Muntyan, Maria S.; Cherepanov, Dmitry A.; Malinen, Anssi M.; Bloch, Dmitry A.; Sorokin, Dimitry Y.; Severina, Inna I.; Ivashina, Tatiana V.; Lahti, Reijo; Muyzer, Gerard; Skulachev, Vladimir P.

    2015-01-01

    Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb3 cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ion-pumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme. PMID:26056262

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

    PubMed Central

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

    2001-01-01

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

  6. Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase.

    PubMed

    Hill, B C; Greenwood, C

    1984-01-30

    The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.

  7. Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo′' to inhibition by the carbon monoxide-releasing molecule, CORM-3☆☆☆

    PubMed Central

    Jesse, Helen E.; Nye, Tacita L.; McLean, Samantha; Green, Jeffrey; Mann, Brian E.; Poole, Robert K.

    2013-01-01

    Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24 μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a

  8. Biogenesis of cytochrome b6 in photosynthetic membranes

    PubMed Central

    Saint-Marcoux, Denis; Wollman, Francis-André

    2009-01-01

    In chloroplasts, binding of a c′-heme to cytochrome b6 on the stromal side of the thylakoid membranes requires a specific mechanism distinct from the one at work for c-heme binding to cytochromes f and c6 on the lumenal side of membranes. Here, we show that the major protein components of this pathway, the CCBs, are bona fide transmembrane proteins. We demonstrate their association in a series of hetero-oligomeric complexes, some of which interact transiently with cytochrome b6 in the process of heme delivery to the apoprotein. In addition, we provide preliminary evidence for functional assembly of cytochrome b6f complexes even in the absence of c′-heme binding to cytochrome b6. Finally, we present a sequential model for apo- to holo-cytochrome b6 maturation integrated within the assembly pathway of b6f complexes in the thylakoid membranes. PMID:19564403

  9. Purification and characterization of cytochrome c' from Neisseria meningitidis.

    PubMed

    Huston, W M; Lowe, E C; Butler, C S; Moir, J W B

    2005-02-01

    Cytochrome c', a c-type cytochrome with unique spectroscopic and magnetic properties, has been characterized in a variety of denitrifying and photosynthetic bacteria. Cytochrome c' has a role in defence and/or removal of NO but the mechanism of action is not clear. To examine the function of cytochrome c' from Neisseria meningitidis, the protein was purified after heterologous overexpression in Escherichia coli. The electronic spectra of the oxidized c' demonstrated a pH-dependent transition (over the pH range of 6-10) typical of known c'-type cytochromes. Interestingly, the form in which NO is supplied determines the redox state of the resultant haem-nitrosyl complex. Fe(III)-NO complexes were formed when Fe(II) or Fe(III) cytochrome c' was sparged with NO gas, whereas an Fe(II)-NO complex was generated when NO was supplied using DEA NONOate (diazeniumdiolate).

  10. Synthesis and Biological Evaluation of Thiophene-Based Cannabinoid Receptor Type 2 Radiotracers for PET Imaging

    PubMed Central

    Haider, Ahmed; Müller Herde, Adrienne; Slavik, Roger; Weber, Markus; Mugnaini, Claudia; Ligresti, Alessia; Schibli, Roger; Mu, Linjing; Mensah Ametamey, Simon

    2016-01-01

    . Evaluation of the CB2-positive spleen, however, showed no accumulation of the radiotracer. Despite the promising in vitro binding affinities, specific binding of [11C]AAT-015, and [11C]AAT-778 could not be demonstrated. PMID:27512365

  11. Preclinical radiation dosimetry for the novel SV2A radiotracer [18F]UCB-H

    PubMed Central

    2013-01-01

    Background [18F]UCB-H was developed as a novel radiotracer with a high affinity for synaptic vesicle protein 2A, the binding site for the antiepileptic levetiracetam. The objectives of this study were to evaluate the radiation dosimetry of [18F]UCB-H in a preclinical trial and to determine the maximum injectable dose according to guidelines for human biomedical research. The radiation dosimetry was derived by organ harvesting and dynamic micro positron emission tomography (PET) imaging in mice, and the results of both methods were compared. Methods Twenty-four male C57BL-6 mice were injected with 6.96 ± 0.81 MBq of [18F]UCB-H, and the biodistribution was determined by organ harvesting at 2, 5, 10, 30, 60, and 120 min (n = 4 for each time point). Dynamic microPET imaging was performed on five male C57BL-6 mice after the injection of 9.19 ± 3.40 MBq of [18F]UCB-H. A theoretical dynamic bladder model was applied to simulate urinary excretion. Human radiation dose estimates were derived from animal data using the International Commission on Radiological Protection 103 tissue weighting factors. Results Based on organ harvesting, the urinary bladder wall, liver and brain received the highest radiation dose with a resulting effective dose of 1.88E-02 mSv/MBq. Based on dynamic imaging an effective dose of 1.86E-02 mSv/MBq was calculated, with the urinary bladder wall and liver (brain was not in the imaging field of view) receiving the highest radiation. Conclusions This first preclinical dosimetry study of [18F]UCB-H showed that the tracer meets the standard criteria for radiation exposure in clinical studies. The dose-limiting organ based on US Food and Drug Administration (FDA) and European guidelines was the urinary bladder wall for FDA and the effective dose for Europe with a maximum injectable single dose of approximately 325 MBq was calculated. Although microPET imaging showed significant deviations from organ harvesting, the Pearson’s correlation coefficient

  12. Deeply branching c6-like cytochromes of cyanobacteria.

    PubMed

    Bialek, Wojciech; Nelson, Matthew; Tamiola, Kamil; Kallas, Toivo; Szczepaniak, Andrzej

    2008-05-20

    The cyanobacterium Synechococcus sp. PCC 7002 carries two genes, petJ1 and petJ2, for proteins related to soluble, cytochrome c6 electron transfer proteins. PetJ1 was purified from the cyanobacterium, and both cytochromes were expressed with heme incorporation in Escherichia coli. The expressed PetJ1 displayed spectral and biochemical properties virtually identical to those of PetJ1 from Synechococcus. PetJ1 is a typical cytochrome c6 but contains an unusual KDGSKSL insertion. PetJ2 isolated from E. coli exhibited absorbance spectra characteristic of cytochromes, although the alpha, beta, and gamma bands were red-shifted relative to those of PetJ1. Moreover, the surface electrostatic properties and redox midpoint potential of PetJ2 (pI 9.7; E(m,7) = 148 +/- 1.7 mV) differed substantially from those of PetJ1 (pI 3.8; E(m,7) = 319 +/- 1.6 mV). These data indicate that the PetJ2 cytochrome could not effectively replace PetJ1 as an electron acceptor for the cytochrome bf complex in photosynthesis. Phylogenetic comparisons against plant, algal, bacterial, and cyanobacterial genomes revealed two novel and widely distributed clusters of previously uncharacterized, cyanobacterial c 6-like cytochromes. PetJ2 belongs to a group that is distinct from both c6 cytochromes and the enigmatic chloroplast c 6A cytochromes. We tentatively designate the PetJ2 group as c6C cytochromes and the other new group as c6B cytochromes. Possible functions of these cytochromes are discussed.

  13. Structure of the Schizosaccharomyces pombe cytochrome c gene

    SciTech Connect

    Russell, P.R.; Hall, B.D.

    1982-02-01

    The cytochrome c gene of the fission yeast Schizosaccharomyces pombe has been cloned by using the Saccharomyces cerevisiae iso-1-cytochrome c gene as a molecular hybridization probe. The DNA sequence and the 5' termini of the mRNA transcripts of the gene have been determined. The DNA sequence has confirmed, with two exceptions, the previously determined protein sequence. The nonrandom distribution of silent third base differences which was observed between the two cytochrome c genes of S. cerevisiae does not extend to the S. pombe cytochrome c gene, suggesting that there are no constraints other than protein function and codon usage which have acted to conserve the cytochrome c DNA sequences of the two yeasts. Introduction of the S. pombe cytochrome c gene on a yeast plasmid into a S. cerevisiae mutant which lacked functional cytochrome c transformed that recipient strain for the ability to grow on a nonfermentable carbon source. This implies that the S. pombe cytochrome c gene has all the regulatory signals which are required for its expression in S. cerevisiae, and that none of the amino acid differences between the cytochrome c proteins of the two yeasts has a drastic effect on the function of the protein in vivo.

  14. 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.

  15. Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris.

    PubMed

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

    1993-11-01

    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.

  16. Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration.

    PubMed

    Costa, N L; Carlson, H K; Coates, J D; Louro, R O; Paquete, C M

    2015-07-01

    Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled.

  17. Measurements of liquid phase residence time distributions in a pilot-scale continuous leaching reactor using radiotracer technique.

    PubMed

    Pant, H J; Sharma, V K; Shenoy, K T; Sreenivas, T

    2015-03-01

    An alkaline based continuous leaching process is commonly used for extraction of uranium from uranium ore. The reactor in which the leaching process is carried out is called a continuous leaching reactor (CLR) and is expected to behave as a continuously stirred tank reactor (CSTR) for the liquid phase. A pilot-scale CLR used in a Technology Demonstration Pilot Plant (TDPP) was designed, installed and operated; and thus needed to be tested for its hydrodynamic behavior. A radiotracer investigation was carried out in the CLR for measurement of residence time distribution (RTD) of liquid phase with specific objectives to characterize the flow behavior of the reactor and validate its design. Bromine-82 as ammonium bromide was used as a radiotracer and about 40-60MBq activity was used in each run. The measured RTD curves were treated and mean residence times were determined and simulated using a tanks-in-series model. The result of simulation indicated no flow abnormality and the reactor behaved as an ideal CSTR for the range of the operating conditions used in the investigation.

  18. Aldehyde Reduction by Cytochrome P450

    PubMed Central

    Amunom, Immaculate; Srivastava, Sanjay; Prough, Russell A.

    2011-01-01

    This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model α,β-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions. PMID:21553396

  19. 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

  20. Structural basis of resistance to anti-cytochrome bc₁ complex inhibitors: implication for drug improvement.

    PubMed

    Esser, Lothar; Yu, Chang-An; Xia, Di

    2014-01-01

    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 reductionsite 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.

  1. Antimalarial 4(1H)-pyridones bind to the Qi site of cytochrome bc1

    PubMed Central

    Capper, Michael J.; O’Neill, Paul M.; Fisher, Nicholas; Strange, Richard W.; Moss, Darren; Ward, Stephen A.; Berry, Neil G.; Lawrenson, Alexandre S.; Hasnain, S. Samar; Biagini, Giancarlo A.; Antonyuk, Svetlana V.

    2015-01-01

    Cytochrome bc1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Qo site (one of two potential binding sites within cytochrome bc1) using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Qo site but bind at the Qi site. The discovery that these compounds bind at the Qi site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Qi also explains the ability of this class to overcome parasite Qo-based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles. PMID:25564664

  2. Pharmacogenetic biomarkers: cytochrome P450 3A5.

    PubMed

    MacPhee, Iain A M

    2012-09-01

    The immunosuppressive drugs used for solid organ transplantation all have a narrow therapeutic index with wide variation between individuals in the blood concentration achieved by a given dose. Therapeutic drug monitoring is employed routinely but may not allow optimisation of drug exposure during the critical period two to three days following transplantation. A key factor in the inter-individual variability for tacrolimus, and probably sirolimus, is whether an individual is genetically predicted to express the drug metabolising enzyme cytochrome P450 3A5 (CYP3A5). Individuals predicted to express CYP3A5 by possession of at least one wild-type CYP3A5*1 allele require 1.5-2 times higher doses of tacrolimus to achieve target blood concentrations than individuals homozygous for the CYP3A5*3 allele who are functional non-expressers of CYP3A5. Planning the initial tacrolimus dose based on the CYP3A5 genotype has been shown to allow more rapid achievement of target blood concentrations after transplantation than a standard dose given to all patients. However, it remains to be demonstrated that use of this approach as an adjunct to therapeutic drug monitoring can reduce either efficacy failure (transplant rejection) or toxicity. Use of a pharmacogenetic approach to dosing sirolimus awaits testing and it is unlikely to be useful for ciclosporin or everolimus.

  3. 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

  4. Structural re-arrangement and peroxidase activation of cytochrome c by anionic analogues of vitamin E, tocopherol succinate and tocopherol phosphate.

    PubMed

    Yanamala, Naveena; Kapralov, Alexander A; Djukic, Mirjana; Peterson, Jim; Mao, Gaowei; Klein-Seetharaman, Judith; Stoyanovsky, Detcho A; Stursa, Jan; Neuzil, Jiri; Kagan, Valerian E

    2014-11-21

    Cytochrome c is a multifunctional hemoprotein in the mitochondrial intermembrane space whereby its participation in electron shuttling between respiratory complexes III and IV is alternative to its role in apoptosis as a peroxidase activated by interaction with cardiolipin (CL), and resulting in selective CL peroxidation. The switch from electron transfer to peroxidase function requires partial unfolding of the protein upon binding of CL, whose specific features combine negative charges of the two phosphate groups with four hydrophobic fatty acid residues. Assuming that other endogenous small molecule ligands with a hydrophobic chain and a negatively charged functionality may activate cytochrome c into a peroxidase, we investigated two hydrophobic anionic analogues of vitamin E, α-tocopherol succinate (α-TOS) and α-tocopherol phosphate (α-TOP), as potential inducers of peroxidase activity of cytochrome c. NMR studies and computational modeling indicate that they interact with cytochrome c at similar sites previously proposed for CL. Absorption spectroscopy showed that both analogues effectively disrupt the Fe-S(Met(80)) bond associated with unfolding of cytochrome c. We found that α-TOS and α-TOP stimulate peroxidase activity of cytochrome c. Enhanced peroxidase activity was also observed in isolated rat liver mitochondria incubated with α-TOS and tBOOH. A mitochondria-targeted derivative of TOS, triphenylphosphonium-TOS (mito-VES), was more efficient in inducing H2O2-dependent apoptosis in mouse embryonic cytochrome c(+/+) cells than in cytochrome c(-/-) cells. Essential for execution of the apoptotic program peroxidase activation of cytochrome c by α-TOS may contribute to its known anti-cancer pharmacological activity. PMID:25278024

  5. Structural Re-arrangement and Peroxidase Activation of Cytochrome c by Anionic Analogues of Vitamin E, Tocopherol Succinate and Tocopherol Phosphate*

    PubMed Central

    Yanamala, Naveena; Kapralov, Alexander A.; Djukic, Mirjana; Peterson, Jim; Mao, Gaowei; Klein-Seetharaman, Judith; Stoyanovsky, Detcho A.; Stursa, Jan; Neuzil, Jiri; Kagan, Valerian E.

    2014-01-01

    Cytochrome c is a multifunctional hemoprotein in the mitochondrial intermembrane space whereby its participation in electron shuttling between respiratory complexes III and IV is alternative to its role in apoptosis as a peroxidase activated by interaction with cardiolipin (CL), and resulting in selective CL peroxidation. The switch from electron transfer to peroxidase function requires partial unfolding of the protein upon binding of CL, whose specific features combine negative charges of the two phosphate groups with four hydrophobic fatty acid residues. Assuming that other endogenous small molecule ligands with a hydrophobic chain and a negatively charged functionality may activate cytochrome c into a peroxidase, we investigated two hydrophobic anionic analogues of vitamin E, α-tocopherol succinate (α-TOS) and α-tocopherol phosphate (α-TOP), as potential inducers of peroxidase activity of cytochrome c. NMR studies and computational modeling indicate that they interact with cytochrome c at similar sites previously proposed for CL. Absorption spectroscopy showed that both analogues effectively disrupt the Fe-S(Met80) bond associated with unfolding of cytochrome c. We found that α-TOS and α-TOP stimulate peroxidase activity of cytochrome c. Enhanced peroxidase activity was also observed in isolated rat liver mitochondria incubated with α-TOS and tBOOH. A mitochondria-targeted derivative of TOS, triphenylphosphonium-TOS (mito-VES), was more efficient in inducing H2O2-dependent apoptosis in mouse embryonic cytochrome c+/+ cells than in cytochrome c−/− cells. Essential for execution of the apoptotic program peroxidase activation of cytochrome c by α-TOS may contribute to its known anti-cancer pharmacological activity. PMID:25278024

  6. Production of recombinant multiheme cytochromes c in Wolinella succinogenes.

    PubMed

    Kern, Melanie; Simon, Jörg

    2011-01-01

    Respiratory nitrogen cycle processes like nitrification, nitrate reduction, denitrification, nitrite ammonification, or anammox involve a variety of dissimilatory enzymes and redox-active cofactors. In this context, an intriguing protein class are cytochromes c, that is, enzymes containing one or more covalently bound heme groups that are attached to heme c binding motifs (HBMs) of apo-cytochromes. The key enzyme of the corresponding maturation process is cytochrome c heme lyase (CCHL), an enzyme that catalyzes the formation of two thioether linkages between two vinyl side chains of a heme and two cysteine residues arranged in the HBM. In recent years, many multiheme cytochromes c involved in nitrogen cycle processes, such as hydroxylamine oxidoreductase and cytochrome c nitrite reductase, have attracted particular interest. Structurally, these enzymes exhibit conserved heme packing motifs despite displaying very different enzymic properties and largely unrelated primary structures. The functional and structural characterization of cytochromes c demands their purification in sufficient amounts as well as the feasibility to generate site-directed enzyme variants. For many interesting organisms, however, such systems are not available, mainly hampered by genetic inaccessibility, slow growth rates, insufficient cell yields, and/or a low capacity of cytochrome c formation. Efficient heterologous cytochrome c overproduction systems have been established using the unrelated proteobacterial species Escherichia coli and Wolinella succinogenes. In contrast to E. coli, W. succinogenes uses the cytochrome c biogenesis system II and contains a unique set of three specific CCHL isoenzymes that belong to the unusual CcsBA-type. Here, W. succinogenes is presented as host for cytochrome c overproduction focusing on a recently established gene expression system designed for large-scale production of multiheme cytochromes c.

  7. Production of recombinant multiheme cytochromes c in Wolinella succinogenes.

    PubMed

    Kern, Melanie; Simon, Jörg

    2011-01-01

    Respiratory nitrogen cycle processes like nitrification, nitrate reduction, denitrification, nitrite ammonification, or anammox involve a variety of dissimilatory enzymes and redox-active cofactors. In this context, an intriguing protein class are cytochromes c, that is, enzymes containing one or more covalently bound heme groups that are attached to heme c binding motifs (HBMs) of apo-cytochromes. The key enzyme of the corresponding maturation process is cytochrome c heme lyase (CCHL), an enzyme that catalyzes the formation of two thioether linkages between two vinyl side chains of a heme and two cysteine residues arranged in the HBM. In recent years, many multiheme cytochromes c involved in nitrogen cycle processes, such as hydroxylamine oxidoreductase and cytochrome c nitrite reductase, have attracted particular interest. Structurally, these enzymes exhibit conserved heme packing motifs despite displaying very different enzymic properties and largely unrelated primary structures. The functional and structural characterization of cytochromes c demands their purification in sufficient amounts as well as the feasibility to generate site-directed enzyme variants. For many interesting organisms, however, such systems are not available, mainly hampered by genetic inaccessibility, slow growth rates, insufficient cell yields, and/or a low capacity of cytochrome c formation. Efficient heterologous cytochrome c overproduction systems have been established using the unrelated proteobacterial species Escherichia coli and Wolinella succinogenes. In contrast to E. coli, W. succinogenes uses the cytochrome c biogenesis system II and contains a unique set of three specific CCHL isoenzymes that belong to the unusual CcsBA-type. Here, W. succinogenes is presented as host for cytochrome c overproduction focusing on a recently established gene expression system designed for large-scale production of multiheme cytochromes c. PMID:21185447

  8. Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)

    ClinicalTrials.gov

    2016-10-24

    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

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

    PubMed

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

    1986-02-01

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

  10. Function of copper in cytochrome production in yeast

    SciTech Connect

    Connelly, J.L.; Mattoon, J.R.; Ortegon, V.

    1986-05-01

    Investigations which are attempting to elucidate the nature of the involvement of copper (Cu) in hemoprotein production in yeast have disclosed that the syntheses of cytochromes aa/sub 3/, b and c are influenced differentially by the absence of Cu. Cells are grown in minimal media (29/sup 0/C, 200 rpm, 48-72 hrs) +/- bathocuproine disulfonate (BC), a specific Cu chelator; spectra of standardized samples are compared for evaluation of porphyrin and hemoprotein production. The use of mutants helped localize the effect of BC (or Cu) along the biosynthesis pathway. Mutants included the parent (D28), which provided a general reference point, D28/F8, deficient in ferrochelatase, which accumulated protoporphyrin, B231/C1, deficient in uroporphyrinogen decarboxylase, which accumulated uroporphyrins and GT38/7A, deficient in amino levulinic acid (ALA) synthetase, which synthesized cytochromes in proportion to the ALA added. In each case BC lowered porphyrin and cytochrome production. Cytochrome production in GT 38/7A and D28 was differentially affected by BC. Cytochrome aa/sub 3/ was eliminated by low levels as expected. Cytochrome b was eliminated by 50X and Cytochrome c was decreased markedly but not eliminated by 100X BC. These observations suggest (1) that porphyrin biosynthesis may be influenced but not blocked (2) a function of Cu in the mitochondrial production (assembly) of Cytochrome b and (3) a possible function of Cu in the transport, across mitochondrial membranes, of hemoprotein or porphyrin intermediates.

  11. Homology in the structure and the prosthetic groups between two different terminal ubiquinol oxidases, cytochrome a1 and cytochrome o, of Acetobacter aceti.

    PubMed

    Matsushita, K; Ebisuya, H; Adachi, O

    1992-12-01

    Acetobacter aceti produces two different terminal oxidases dependent on the culture conditions, shaking and static cultures. Cells grown on shaking culture contain cytochrome a1, while cytochrome o is present in cells grown on static culture. Cytochrome a1 and cytochrome o of A. aceti were compared especially with respect to the protein structure and the prosthetic groups. Cytochrome a1 exhibited lower CN sensitivity and higher affinity for O2 than cytochrome o. Both terminal oxidases consisted of four nonidentical polypeptides of which the molecular sizes were identical between both enzymes. Cytochrome a1 cross-reacted with an antibody raised against cytochrome o at the same level as cytochrome o did, and an antibody elicited against cytochrome a1 cross-reacted with both cytochrome o and cytochrome a1 at the same intensity, which indicates that both oxidases are indistinguishable immunochemically. Furthermore, almost the same peptide mapping pattern with chymotrypsin was observed in subunit I and in subunit II between both terminal oxidases, and the amino-terminal sequences in the subunit II of both oxidases were identical at least in their 10 amino acids. As for the prosthetic groups, both oxidases were shown to contain two heme-irons and one copper atom. Further, high performance liquid chromatography analysis of the heme moieties extracted from both the purified enzymes indicated that cytochrome a1 contains hemes b and a at a ratio of 1 to 1, whereas cytochrome o contains the same amounts of hemes b and o. Thus, data indicate that cytochrome a1 and cytochrome o of A. aceti are cytochrome ba and cytochrome bo ubiquinol oxidases, respectively, and that both oxidases have a closely similar protein structure and prosthetic groups, in which only heme a in the heme/copper binuclear center of cytochrome a1 is replaced by heme o in that of cytochrome o.

  12. A Radiotracer Strategy to Quantify PARP-1 Expression In Vivo Provides a Biomarker That Can Enable Patient Selection for PARP Inhibitor Therapy.

    PubMed

    Makvandi, Mehran; Xu, Kuiying; Lieberman, Brian P; Anderson, Redmond-Craig; Effron, Samuel Sander; Winters, Harrison D; Zeng, Chenbo; McDonald, Elizabeth S; Pryma, Daniel A; Greenberg, Roger A; Mach, Robert H

    2016-08-01

    Despite the availability of PARP inhibitors for cancer therapy, a biomarker to clearly stratify patients for selection of this treatment remains lacking. Here we describe a radiotracer-based method that addresses this issue, using the novel compound [(125)I] KX1: as a PARP-1-selective radiotracer that can accurately measure PARP-1 expression in vitro and in vivo The pharmacologic properties of the PARP radiotracer [(125)I] KX1: was characterized in multiple cell lines where single-agent sensitivity was correlated with [(125)I] KX1: binding to PARP-1. In vivo evaluation of [(125)I] KX1: verified in vitro results, validating PARP radiotracers to define PARP-1 enzyme expression as an in vivo biomarker. Notably, PARP-1 expression as quantified by [(125)I] KX1: correlated positively with the cytotoxic sensitivity of cell lines evaluated with PARP inhibitors. Overall, our results defined a novel technology with the potential to serve as a companion diagnostic to identify patients most likely to respond therapeutically to a PARP inhibitor. Cancer Res; 76(15); 4516-24. ©2016 AACR. PMID:27261505

  13. Thermal stability of cytochrome c' from mesophilic Shewanella amazonensis.

    PubMed

    Kato, Yuki; Fujii, Sotaro; Kuribayashi, Taka-aki; Masanari, Misa; Sambongi, Yoshihiro

    2015-01-01

    Cytochrome c' (SACP) from mesophilic Shewanella amazonensis, growing optimally at 37 °C, was thermally more stable than cytochrome c' (AVCP) from mesophilic Allochromatium vinosum, growing optimally at 25 °C. In contrast, SACP was less stable than cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus, growing optimally at 52 °C. Although only 28% of the SACP amino acid sequence was identical to those of AVCP and PHCP, the latter two being 55% identical, the overall main chain structures of the three cytochromes c' were similar, and SACP exhibited thermal stability intermediate between those of AVCP and PHCP. For these three proteins, the higher the stability is, the lesser the number of Gly residues in the putative α-helical regions is. Cytochromes c' including the present three are suitable for examining the protein stabilization mechanisms, because they are structurally similar and available from environments with a wide range of temperatures.

  14. Mitochondrial cytochrome c biogenesis: no longer an enigma

    PubMed Central

    Babbitt, Shalon E.; Sutherland, Molly C.; Francisco, Brian San; Mendez, Deanna L.; Kranz, Robert G.

    2015-01-01

    Cytochromes c and c1are heme proteins that are essential for aerobic respiration. Release of cytochrome c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for import of apocytochrome c. Thus, HCCS affects cellular levels of cytochrome c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles played by heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways. PMID:26073510

  15. Targeted molecular imaging in oncology.

    PubMed

    Yang, David J; Kim, E Edmund; Inoue, Tomio

    2006-01-01

    Improvement of scintigraphic tumor imaging is extensively determined by the development of more tumor specific radiopharmaceuticals. Thus, to improve the differential diagnosis, prognosis, planning and monitoring of cancer treatment, several functional pharmaceuticals have been developed. Application of molecular targets for cancer imaging, therapy and prevention using generator-produced isotopes is the major focus of ongoing research projects. Radionuclide imaging modalities (positron emission tomography, PET; single photon emission computed tomography, SPECT) are diagnostic cross-sectional imaging techniques that map the location and concentration of radionuclide-labeled radiotracers. 99mTc- and 68Ga-labeled agents using ethylenedicysteine (EC) as a chelator were synthesized and their potential uses to assess tumor targets were evaluated. 99mTc (t1/2 = 6 hr, 140 keV) is used for SPECT and 68Ga (t1/2 = 68 min, 511 keV) for PET. Molecular targets labeled with Tc-99m and Ga-68 can be utilized for prediction of therapeutic response, monitoring tumor response to treatment and differential diagnosis. Molecular targets for oncological research in (1) cell apoptosis, (2) gene and nucleic acid-based approach, (3) angiogenesis (4) tumor hypoxia, and (5) metabolic imaging are discussed. Numerous imaging ligands in these categories have been developed and evaluated in animals and humans. Molecular targets were imaged and their potential to redirect optimal cancer diagnosis and therapeutics were demonstrated. PMID:16485568

  16. Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

    PubMed

    Ponnamperuma, K; Croteau, R

    1996-05-01

    Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

  17. Cytochrome oxidase heme-protein dynamics: a transient Raman study of carbon monoxide photolysis from cytochrome a

    SciTech Connect

    Findsen, E.W.; Ondrias, M.R.

    1984-09-19

    Data are reported on results of initial efforts to probe the mechanism of cytochrome oxidase function by utilizing time-resolved resonance Raman spectroscopy. Preparation of the reduced beef-heart cytochrome oxidase sample and cytochrome oxidase-CO sample is described. At the laser powers and concentrations employed, the reduced cytochrome oxidase-CO sample underwent almost complete photolysis during the laser pulse. Principal conclusions drawn from spectral analysis are that time-resolved resonance Raman investigation of the transient heme species generated by ligand photolysis is a viable technique for the study of heme-ligand dynamics in proteins other than hemoglobin. A transient proximal geometry leading to a strengthened iron-histidine bond is present in these. The interplay of porphyrin core size, pi electron density, and Fe-His bonding as modulated by heme-protein dynamics is different for the ligand binding sites of hemoglobin and cytochrome oxidase. 17 references, 1 figure.

  18. Diffusion Experiment in Lithium Ionic Conductors with the Radiotracer of {sup 8}Li: from Micro- to Nano-diffusion

    SciTech Connect

    Jeong, Sun-Chan; Katayama, Ichiro; Kawakami, Hirokane; Watanabe, Yutaka; Ishiyama, Hironobu; Imai, Nobuaki; Hirayama, Yoshikazu; Miyatake, Hiroari; Sataka, Masao; Sugai, Hiroyuki; Okayasu, Satoru; Ichikawa, Shin-Ichi; Nishio, Katsuhisa; Mitsuoka, Shinichi; Nakanoya, Takamitsu; Hashimoto, Takashi; Hashimoto, Takanori; Yahagi, Masahito

    2009-05-04

    We have developed a radiotracer method for diffusion studies in lithium ionic conductors, by using, as the tracer, the short-lived {alpha}-emitting radioisotope of {sup 8}Li from TRIAC (Tokai Radioactive Ion Accelerator Complex). In the method, we measured {alpha}-particles coming out of the sample of interest and have found that the time-dependent yields of {alpha}-particle from the diffusing {sup 8}Li primarily implanted is a good measure of the Li diffusion in the sample. The method has been successfully applied to measure the lithium diffusion coefficients in a typical defect-mediated lithium ionic conductor of LiGa, well demonstrating that the method is very efficient to measure the diffusion in the micro-meter regime per second. Further development, as an extension of the present method, was proposed to measure the diffusion on the nanoscale in lithium ionic conductors.

  19. UV-photochemical vapor generation of selenium for atomic absorption spectrometry: Optimization and 75Se radiotracer efficiency study

    NASA Astrophysics Data System (ADS)

    Rybínová, Marcela; Musil, Stanislav; Červený, Václav; Vobecký, Miloslav; Rychlovský, Petr

    2016-09-01

    Volatile selenium compounds were generated UV-photochemically in the continuous flow mode using four UV-photoreactors differing in the material of the reaction coil; Teflon tubing and quartz tubes with various inner diameters and wall thicknesses were tested. Atomic absorption spectrometry with an externally heated quartz furnace atomizer was employed as the detector. The relevant experimental generation parameters were optimized and the basic analytical characteristics were determined. Using formic acid as the photochemical agent, limits of detection achieved for selenium were in the range 46-102 ng L- 1 in dependence on the type of UV-photoreactor employed. When nitric acid was also added to the photochemical agent, the limits of detection were reduced to 27-44 ng L- 1. The repeatability did not exceed 2.4% (5 μg L- 1 Se(IV), n = 10). Experiments with 75Se radiotracer have been performed for the first time to quantify the efficiency of UV-photochemical vapor generation (UV-PVG) of selenium. The highest efficiency of 67 ± 1% was obtained for a UV-photoreactor containing a quartz reaction coil (2.0 mm i.d., 4.0 mm o.d.). The generation efficiency of 61 ± 1% was obtained for a Teflon reaction coil (1.0 mm i.d., 1.4 mm o.d.). Mapping of the radiotracer distribution in the individual parts of the apparatus did not reveal substantial transport losses of the analyte in the UV-PVG system.

  20. Sodium-22-radiolabeled silica nanoparticles as new radiotracer for biomedical applications: in vivo positron emission tomography imaging, biodistribution, and biocompatibility

    PubMed Central

    Al Faraj, Achraf; Alotaibi, Basem; Shaik, Abjal Pasha; Shamma, Khaled Z; Al Jammaz, Ibrahim; Gerl, Jürgen

    2015-01-01

    Despite their advantageous chemical properties for nuclear imaging, radioactive sodium-22 (22Na) tracers have been excluded for biomedical applications because of their extremely long lifetime. In the current study, we proposed, for the first time, the use of 22Na radiotracers for pre-clinical applications by efficiently loading with silica nanoparticles (SiNPs) and thus offering a new life for this radiotracer. Crown-ether-conjugated SiNPs (300 nm; −0.18±0.1 mV) were successfully loaded with 22Na with a loading efficacy of 98.1%±1.4%. Noninvasive positron emission tomography imaging revealed a transient accumulation of 22Na-loaded SiNPs in the liver and to a lower extent in the spleen, kidneys, and lung. However, the signal gradually decreased in a time-dependent manner to become not detectable starting from 2 weeks postinjection. These observations were confirmed ex vivo by quantifying 22Na radioactivity using γ-counter and silicon content using inductively coupled plasma-mass spectrometry in the blood and the different organs of interest. Quantification of Si content in the urine and feces revealed that SiNPs accumulated in the organs were cleared from the body within a period of 2 weeks and completely in 1 month. Biocompatibility evaluations performed during the 1-month follow-up study to assess the possibility of synthesized nanocarriers to induce oxidative stress or DNA damage confirmed their safety for pre-clinical applications. 22Na-loaded nanocarriers can thus provide an innovative diagnostic agent allowing ultra-sensitive positron emission tomography imaging. On the other hand, with its long lifetime, onsite generators or cyclotrons will not be required as 22Na can be easily stored in the nuclear medicine department and be used on-demand. PMID:26504381

  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. Characterization of Cytochrome 579, an Unusual Cytochrome Isolated from an Iron-Oxidizing Microbial Community

    SciTech Connect

    Singer, Steven; Chan, Clara S; Zemla, Adam; Verberkmoes, Nathan C; Hwang, Mona; Hettich, Robert {Bob} L; Banfield, Jillian F.; Thelen, Michael P.

    2008-01-01

    Proteogenomic studies of Fe(II)-oxidizing microbial biofilms collected from an extremely acidic environment have identified a novel, soluble cytochrome as one of the most abundant proteins produced by these communities. This red cytochrome, extracted from biofilms with dilute sulfuric acid and purified by cation exchange chromatography, has an unusual visible spectral signature at 579 nm. Fe(II)-dependent reduction of Cyt579 was thermodynamically favorable at pH>3, raising the possibility that Cyt579 acts as an accessory protein for electron transfer. Transmission electron microscopy of immuno-gold labeled biofilm indicated that the Cyt579 is localized near the bacterial cell surface, consistent with periplasmic localization. Further protein analysis of Cyt579, using preparative chromatofocusing and SDS-PAGE, revealed three forms of the protein that correspond to different N-terminal truncations of the amino acid sequence. Intact protein analysis corroborated the post-translational modifications of these forms and identified a genomically uncharacterized Cyt579 variant. Homology modeling was used to predict the overall cytochrome structure and heme binding site; positions of nine amino acid substitutions found in 3 Cyt579 variants all map to the surface of the protein and away from the heme group. Based on this detailed characterization of Cyt579, we propose that Cyt579 acts an electron transfer protein shuttling electrons derived from Fe(II) oxidation to support critical metabolic functions in the acidophilic microbial community.

  3. Evolution of the couple cytochrome c and cytochrome c oxidase in Primates

    PubMed Central

    Pierron, Denis; Wildman, Derek E.; Hüttemann, Maik; Letellier, Thierry; Grossman, Lawrence I.

    2013-01-01

    Mitochondrial energy metabolism has been affected by a broad set of ancient and recent evolutionary events. The oldest example is the endosymbiosis theory that led to mitochondria and a recently proposed example is adaptation to cold climate by anatomically modern human lineages. Mitochondrial energy metabolism has also been associated with an important area in anthropology and evolutionary biology, brain enlargement in human evolution. Indeed, several studies have pointed to the need for a major metabolic rearrangement to supply a sufficient amount of energy for brain development in primates. The gene encoding for the coupled cytochrome c (cyt c) / cytochrome c oxidase (COX, complex IV, EC 1.9.3.1) seems to have an exceptional pattern of evolution in the anthropoid lineage. It has been proposed that this evolution was linked to the rearrangement of energy metabolism needed for brain enlargement. This hypothesis is reinforced by the fact that the COX enzyme was proposed to have a large role in control of the respiratory chain and thereby global energy production. After summarizing major events that occurred during the evolution of COX and cytochrome c on the primate lineage, we review the different evolutionary forces that could have influenced primate COX evolution and discuss the probable causes and consequence of this evolution. Finally, we discuss and review the co-occurring primate phenotypic evolution. PMID:22729859

  4. Adaptive evolution of cytochrome c oxidase: Infrastructure for a carnivorous plant radiation.

    PubMed

    Jobson, Richard W; Nielsen, Rasmus; Laakkonen, Liisa; Wikström, Mårten; Albert, Victor A

    2004-12-28

    Much recent attention in the study of adaptation of organismal form has centered on developmental regulation. As such, the highly conserved respiratory machinery of eukaryotic cells might seem an unlikely target for selection supporting novel morphologies. We demonstrate that a dramatic molecular evolutionary rate increase in subunit I of cytochrome c oxidase (COX) from an active-trapping lineage of carnivorous plants is caused by positive Darwinian selection. Bladderworts (Utricularia) trap plankton when water-immersed, negatively pressured suction bladders are triggered. The resetting of traps involves active ion transport, requiring considerable energy expenditure. As judged from the quaternary structure of bovine COX, the most profound adaptive substitutions are two contiguous cysteines absent in approximately 99.9% of databased COX I sequences from Eukaryota, Archaea, and Bacteria. This motif lies directly at the docking point of COX I helix 3 and cytochrome c, and modeling of bovine COX I suggests the possibility of an unprecedented helix-terminating disulfide bridge that could alter COX/cytochrome c dissociation kinetics. Thus, the key adaptation in Utricularia likely lies in molecular energetic changes that buttressed the mechanisms responsible for the bladderworts' radical morphological evolution. Along with evidence for COX evolution underlying expansion of the anthropoid neocortex, our findings underscore that important morphological and physiological innovations must often be accompanied by specific adaptations in proteins with basic cellular functions. PMID:15596720

  5. Adaptive evolution of cytochrome c oxidase: Infrastructure for a carnivorous plant radiation.

    PubMed

    Jobson, Richard W; Nielsen, Rasmus; Laakkonen, Liisa; Wikström, Mårten; Albert, Victor A

    2004-12-28

    Much recent attention in the study of adaptation of organismal form has centered on developmental regulation. As such, the highly conserved respiratory machinery of eukaryotic cells might seem an unlikely target for selection supporting novel morphologies. We demonstrate that a dramatic molecular evolutionary rate increase in subunit I of cytochrome c oxidase (COX) from an active-trapping lineage of carnivorous plants is caused by positive Darwinian selection. Bladderworts (Utricularia) trap plankton when water-immersed, negatively pressured suction bladders are triggered. The resetting of traps involves active ion transport, requiring considerable energy expenditure. As judged from the quaternary structure of bovine COX, the most profound adaptive substitutions are two contiguous cysteines absent in approximately 99.9% of databased COX I sequences from Eukaryota, Archaea, and Bacteria. This motif lies directly at the docking point of COX I helix 3 and cytochrome c, and modeling of bovine COX I suggests the possibility of an unprecedented helix-terminating disulfide bridge that could alter COX/cytochrome c dissociation kinetics. Thus, the key adaptation in Utricularia likely lies in molecular energetic changes that buttressed the mechanisms responsible for the bladderworts' radical morphological evolution. Along with evidence for COX evolution underlying expansion of the anthropoid neocortex, our findings underscore that important morphological and physiological innovations must often be accompanied by specific adaptations in proteins with basic cellular functions.

  6. Upregulation of Mitochondrial Content in Cytochrome c Oxidase Deficient Fibroblasts

    PubMed Central

    Kogot-Levin, Aviram; Saada, Ann; Leibowitz, Gil; Soiferman, Devorah; Douiev, Liza; Raz, Itamar; Weksler-Zangen, Sarah

    2016-01-01

    Cytochrome-c-oxidase (COX) deficiency is a frequent cause of mitochondrial disease and is associated with a wide spectrum of clinical phenotypes. We studied mitochondrial function and biogenesis in fibroblasts derived from the Cohen (CDs) rat, an animal model of COX deficiency. COX activity in CDs-fibroblasts was 50% reduced compared to control rat fibroblasts (P<0.01). ROS-production in CDs fibroblasts increased, along with marked mitochondrial fragmentation and decreased mitochondrial membrane-potential, indicating mitochondrial dysfunction. Surprisingly, cellular ATP content, oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were unchanged. To clarify the discrepancy between mitochondrial dysfunction and ATP production, we studied mitochondrial biogenesis and turnover. The content of mitochondria was higher in CDs-fibroblasts. Consistently, AMPK activity and the expression of NRF1-target genes, NRF2 and PGC1-α that mediate mitochondrial biogenesis were increased (P<0.01 vs control fibroblast). In CDs-fibrobalsts, the number of autophagosomes (LC3+ puncta) containing mitochondria in CDs fibroblasts was similar to that in control fibroblasts, suggesting that mitophagy was intact. Altogether, our findings demonstrate that mitochondrial dysfunction and oxidative stress are associated with an increase in mitochondrial biogenesis, resulting in preservation of ATP generation. PMID:27780242

  7. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

    PubMed Central

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes. PMID:26129850

  8. 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

  9. Heterologous synthesis of cytochrome c' by Escherichia coli is not dependent on the System I cytochrome c biogenesis machinery.

    PubMed

    Inoue, Hiroki; Wakai, Satoshi; Nishihara, Hirofumi; Sambongi, Yoshihiro

    2011-07-01

    Hydrogenophilus thermoluteolus cytochrome c' (PHCP) has typical spectral properties previously observed for other cytochromes c', which comprise Ambler's class II cytochromes c. The PHCP protein sequence (135 amino acids) deduced from the cloned gene is the most homologous (55% identity) to that of cytochrome c' from Allochromatium vinosum (AVCP). These findings indicate that PHCP forms a four-helix bundle structure, similar to AVCP. Strikingly, PHCP with a covalently bound heme was heterologously synthesized in the periplasm of Escherichia coli strains deficient in the DsbD protein, a component of the System I cytochrome c biogenesis machinery. The heterologous synthesis of PHCP by aerobically growing E. coli also occurred without a plasmid carrying the genes for Ccm proteins, other components of the System I machinery. Unlike Ambler's class I general cytochromes c, the synthesis of PHCP is not dependent on the System I machinery and exhibits similarity to that of E. coli periplasmic cytochrome b(562), a 106-residue four-helix bundle.

  10. Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

    PubMed Central

    Lynch, S. R.; Copeland, R. A.

    1992-01-01

    The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues. PMID:1338946

  11. 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

  12. Activation of Oxygen by Cytochrome P-450 and Other Haemoproteins

    NASA Astrophysics Data System (ADS)

    Metelitsa, D. I.

    1982-11-01

    Data on the activation of molecular oxygen by the full microsomal hydroxylating system containing cytochrome P-450 as the terminal oxygenase are examined. The nature of the hydroxylating agent, which is the oxenoid Fe3+O, is analysed. The autoxidation reactions of cytochrome P-450 from various sources, haemoglobin, myoglobin, and peroxidases are compared and the role of the axial ligands of the haem iron and the structure of the active centres of the haemoproteins in this process is demonstrated. The possible mechanisms of the oxidation of organic compounds by peroxides with participation of cytochrome P-450, cytochrome c, haemoglobin, and catalase are examined critically. Haemoproteins have been divided into three groups in terms of the type of peroxide oxidation reactions. The relative contributions of the radical and two-electron reactions in the oxidation of compounds by peroxides with participation of different haemoproteins are analysed. The bibliography includes 184 references.

  13. Genetics Home Reference: cytochrome P450 oxidoreductase deficiency

    MedlinePlus

    ... P450 oxidoreductase deficiency is a disorder of hormone production. This condition specifically affects steroid hormones, which are ... activity of cytochrome P450 oxidoreductase, which disrupts the production of steroid hormones. Changes in sex hormones such ...

  14. Differential action on cancer and normal tissue by adrenochrome monoaminoguanidine methanesulfonate and cytochrome C combined with radiotherapy

    SciTech Connect

    Nakatsugawa, S. ); Sugahara, T. )

    1994-06-15

    The possibility that radioprotective effects on potent natural killer (NK) cells by adrenochrome monoaminoguanidine methanesulfonate (AMM) + cytochrome C during radiotherapy (RT) for lung cancer might result in the radiosensitization of human lung cancer cells in vivo is examined. Human lung cancer xenografts in the right hind legs of KSN mice (10 weeks old) were locally irradiated with 20 Gy of X ray. AMM (10 mg/kg/day) and/or cytochrome C (CCC) (5 mg/kg/day) were given intraperitoneally immediately before or after RT, followed by daily administration for 4 days. Natural killer activities of host splenocytes were also tested with the standard [sup 51]Cr releasing assay with YAC-1 cells as target cells. In a clinical study, 65 patients with lung cancer were treated with more than 50 Gy of RT with or without combination with AMM + CCC, OK-432 or AMM + CCC + OK-432. Before and after RT, lymphocyte subsets in the peripheral blood were examined with dichromatic analysis using an Ortho Spectrum IIIFCM system and fluorescent MABs. In this study, the change in the absolute number of each subset was investigated. AMM + cytochrome C augumented NK activity in KSN nude mice, protected potent NK cells in patients with lung cancer against RT and sensitized the human lung cancer xenografts to RT. AMM + cytochrome C may have potential as a differential modulator of radiosensitivity of normal tissues and of tumors. 8 refs., 2 figs., 1 tab.

  15. Role of lysines in cytochrome c-cardiolipin interaction.

    PubMed

    Sinibaldi, Federica; Howes, Barry D; Droghetti, Enrica; Polticelli, Fabio; Piro, Maria Cristina; Di Pierro, Donato; Fiorucci, Laura; Coletta, Massimo; Smulevich, Giulietta; Santucci, Roberto

    2013-07-01

    Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c-cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c-cardiolipin interaction, as these side chains appear to be critical for cytochrome c-cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet-visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c-cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.

  16. The reactivity of cytochrome c with soft ligands.

    PubMed

    Schejter, A; Plotkin, B; Vig, I

    1991-03-25

    The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron-methionine sulfur bond of this heme protein is enhanced by delocalization of the metal t2g electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.

  17. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele).

  18. Geometrical analysis of cytochrome c unfolding

    NASA Astrophysics Data System (ADS)

    Urie, Kristopher G.; Pletneva, Ekaterina; Gray, Harry B.; Winkler, Jay R.; Kozak, John J.

    2011-01-01

    A geometrical model has been developed to study the unfolding of iso-1 cytochrome c. The model draws on the crystallographic data reported for this protein. These data were used to calculate the distance between specific residues in the folded state, and in a sequence of extended states defined by n = 3, 5, 7, 9, 11, 13, and 15 residue units. Exact calculations carried out for each of the 103 residues in the polypeptide chain demonstrate that different regions of the chain have different unfolding histories. Regions where there is a persistence of compact structures can be identified, and this geometrical characterization is fully consistent with analyses of time-resolved fluorescence energy-transfer (TrFET) data using dansyl-derivatized cysteine side-chain probes at positions 39, 50, 66, 85, and 99. The calculations were carried out assuming that different regions of the polypeptide chain unfold synchronously. To test this assumption, lattice Monte Carlo simulations were performed to study systematically the possible importance of asynchronicity. Calculations show that small departures from synchronous dynamics can arise if displacements of residues in the main body of the chain are much more sluggish than near-terminal residues.

  19. Cytochrome P450 expression in oesophageal cancer.

    PubMed Central

    Murray, G I; Shaw, D; Weaver, R J; McKay, J A; Ewen, S W; Melvin, W T; Burke, M D

    1994-01-01

    The cytochrome P450 superfamily of enzymes play a central part in the metabolism of carcinogens and anti-cancer drugs. The expression, cellular localisation, and distribution of different forms of P450 and the functionally associated enzymes epoxide hydrolase and glutathione S-transferases have been investigated in oesophageal cancer and non-neoplastic oesophageal tissue using immunohistochemistry. Expression of the different enzymes was confined to epithelial cells in both non-neoplastic samples and tumour samples except the CYP3A was also identified in mast cells and glutathione S-transferase pi was present in chronic inflammatory cells. CYP1A was present in a small percentage of non-neoplastic samples but both CYP2C and CYP3A were absent. Epoxide hydrolase was present in half of the non-neoplastic samples and the different classes of glutathione S-transferase were present in a low number of samples. In carcinomas CYP1A, CYP3A, epoxide hydrolase, and glutathione S-transferase pi were expressed in at least 60% of samples. The expression of glutathione S-transferases alpha and mu were significantly less in adenocarcinoma compared with squamous carcinoma. Images Figure 1 Figure 2 Figure 3 PMID:8200549

  20. Novel extrahepatic cytochrome P450s

    SciTech Connect

    Karlgren, Maria . E-mail: Maria.Karlgren@imm.ki.se; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

    2005-09-01

    The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis.

  1. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele). PMID:16719392

  2. Crystallization of beef heart cytochrome c oxidase

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Shinya; Shinzawa, Kyoko; Tsukihara, Tomitake; Abe, Toshio; Caughey, Winslow S.

    1991-03-01

    The three-dimensional structure of cytochrome c oxidase, a complex (multimetal, multisubunit) membrane protein is critical to elucidation of the mechanism of the enzymic reactions and their control. Our recent developments in the crystallization of the enzyme isolated from beef hearts are presented. The crystals appeared more readily at higher protein concentration, lower ionic strength, higher detergent concentration (Brij-35) and lower temperature. Large crystals were obtained by changing one of these parameters to the crystallization point as slowly as possible, keeping the other parameters constant. Increasing the detergent concentration was the most successful method, producing green crystals of the resting oxidized form as hexagonal bipyramids with typical dimensions of 0.6 mm. The usual procedures for crystallization of water soluble proteins, such as increasing ionic strength by vapor diffusion, were not applicable for this enzyme. Crystals of the resting oxidized enzyme belong to a space group of P6 2 or P6 4 with cell dimensions, a = b = 208.7 Å and c = 282.3 Å. The Patterson function shows that the crystal exhibited a non-crystallographic two-fold axis parallel to the c-axis in the asymmetric unit.

  3. Heterogeneity of amino acid sequence in hippopotamus cytochrome c.

    PubMed

    Thompson, R B; Borden, D; Tarr, G E; Margoliash, E

    1978-12-25

    The amino acid sequences of chymotryptic and tryptic peptides of Hippopotamus amphibius cytochrome c were determined by a recent modification of the manual Edman sequential degradation procedure. They were ordered by comparison with the structure of the hog protein. The hippopotamus protein differs in three positions: serine, alanine, and glutamine replace alanine, glutamic acid, and lysine in positions 43, 92, and 100, respectively. Since the artiodactyl suborders diverged in the mid-Eocene some 50 million years ago, the fact that representatives of some of them show no differences in their cytochromes c (cow, sheep, and hog), while another exhibits as many as three such differences, verifies that even in relatively closely related lines of descent the rate at which cytochrome c changes in the course of evolution is not constant. Furthermore, 10.6% of the hippopotamus cytochrome c preparation was shown to contain isoleucine instead of valine at position 3, indicating that one of the four animals from which the protein was obtained was heterozygous in the cytochrome c gene. Such heterogeneity is a necessary condition of evolutionary variation and has not been previously observed in the cytochrome c of a wild mammalian population.

  4. Methionine ligand lability of homologous monoheme cytochromes c.

    PubMed

    Levin, Benjamin D; Walsh, Kelly A; Sullivan, Kristal K; Bren, Kara L; Elliott, Sean J

    2015-01-01

    Direct electrochemical analysis of adsorbed bacterial monoheme cytochromes c has revealed a phenomenological loss of the axial methionine when examined using pyrolytic "edge-plane" graphite (EPG) electrodes. While prior findings have reported that the Met-loss state may be quantitatively understood using the cytochrome c from Hydrogenobacter thermophilus as a model system, here we demonstrate that the formation of the Met-loss state upon EPG electrodes can be observed for a range of cytochrome orthologs. Through an electrochemical comparison of the wild-type proteins from organisms of varying growth temperature optima, we establish that Met-ligand losses at graphite surfaces have similar energetics to the "foldons" for known protein folding pathways. Furthermore, a downward shift in reduction potential to approximately -100 mV vs standard hydrogen electrode was observed, similar to that of the alkaline transition found in mitochondrial cytochromes c. Pourbaix diagrams for the Met-loss forms of each cytochrome, considered here in comparison to mutants where the Met-ligand has been substituted to His or Ala, suggest that the nature of the Met-loss state is distinct from either a His-/aquo- or a bis-His-ligated heme center, yet more closely matches the pKa values found for bis-His-ligated hemes., We find the propensity for adoption of the Met-loss state in bacterial monoheme cytochromes c scales with their overall thermal stability, though not with the specific stability of the Fe-Met bond.

  5. Effect of Long-Term Treatment with Antioxidant SkQ1 Added to Drinking Water on Cytochromes P450 Level in Rat Liver.

    PubMed

    Myasoedova, K N; Silachev, D N

    2015-12-01

    Mitochondria-targeted cationic antioxidant plastoquinonyl decyltriphenylphosphonium (SkQ1) added to drinking water in therapeutic doses (250 nmol/kg per day) for a long time (up to 24 months) does not induce cytochromes P450 in rat liver.

  6. Detection of cytochrome P4501A in several species using antibody against a synthetic peptide derived from rainbow trout cytochrome P4501A1

    SciTech Connect

    Lin, S.; Bandiera, S.M.; Bullock, P.L.; Addison, R.F.

    1998-03-01

    Induction of cytochrome P4501A (CYP1A) is being used increasingly as a biomarker to indicate exposure of organisms to environmental contaminants such as some polycyclic and polychlorinated aromatic hydrocarbons. Measurement of CYPIA protein in wildlife would be facilitated by the use of a specific antibody that recognized the isozyme in several species. In the present study, a polyclonal antibody targeted to CYP1A1 was generated using a synthetic peptide corresponding to amino acids 277--294 of the trout enzyme as the antigen of immunization. Specificity of the resulting antibody was assessed by noncompetitive enzyme-linked immunosorbent assay with several purified rat CYP isozymes and by immunoblot analysis with liver microsomes from diverse species. The antibody reacted strongly with the immunizing peptide and with purified rat cytochrome P4501A1 but did not react with rat CYP1A2, a closely related isozyme, or with six other purified rat CYP proteins in enzyme-linked immunosorbent assay. On immunoblots, the antibody recognized a single protein band in hepatic microsomes from the various mammal and fish species tested. Two protein bands were detected in liver microsomes from 3-methylcholanthrene-treated chickens. The results suggest that the antigenic determinant to which the antibody binds is unique to CYP1A and is conserved in different species. Because of its specificity, this anti-peptide antibody should be suitable as a probe to measure CYP1A protein levels in wildlife.

  7. Chapter X. Phosphorylation of mammalian cytochrome c and cytochrome c oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease

    PubMed Central

    Hüttemann, Maik; Lee, Icksoo; Grossman, Lawrence I.; Doan, Jeffrey W.; Sanderson, Thomas H.

    2013-01-01

    The mitochondrial oxidative phosphorylation (OxPhos) system generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke. PMID:22729861

  8. The [18F]2-Fluoro-1,3-thiazolyl Moiety - an Easily-Accessible Structural Motif for Prospective Molecular Imaging Radiotracers

    PubMed Central

    Siméon, Fabrice G.; Wendahl, Matthew T.; Pike, Victor W.

    2010-01-01

    2-Fluoro-1,3-thiazoles were rapidly and efficiently labeled with no-carrier-added fluorine-18 (t1/2 = 109.7 min) by treatment of readily prepared 2-halo precursors with cyclotron-produced [18F]fluoride ion. The [18F]2-fluoro-1,3-thiazolyl moiety constitutes a new and easily-labeled structural motif for prospective molecular imaging radiotracers. PMID:21057601

  9. Estimating endogenous dopamine levels at D2 and D3 receptors in humans using the agonist radiotracer [(11)C]-(+)-PHNO.

    PubMed

    Caravaggio, Fernando; Nakajima, Shinichiro; Borlido, Carol; Remington, Gary; Gerretsen, Philip; Wilson, Alan; Houle, Sylvain; Menon, Mahesh; Mamo, David; Graff-Guerrero, Ariel

    2014-11-01

    Using positron emission tomography (PET) and an acute dopamine depletion challenge it is possible to estimate endogenous dopamine levels occupying dopamine D2/3 receptors (D2/3R) in humans in vivo. Our group has developed [(11)C]-(+)-PHNO, the first agonist radiotracer with preferential in vivo affinity for D3R. Thus, the use of [(11)C]-(+)-PHNO offers the novel possibility of (i) estimating in vivo endogenous dopamine levels at D2/3R using an agonist radiotracer, and (ii) estimating endogenous dopamine levels at D3R in extrastriatal regions such as the substantia nigra, hypothalamus, and ventral pallidum. Ten healthy participants underwent a [(11)C]-(+)-PHNO PET scan under baseline conditions and another under acute endogenous dopamine depletion achieved via oral administration of alpha-methyl-para-tyrosine (64 mg/kg). [(11)C]-(+)-PHNO binding was sensitive to acute dopamine depletion, allowing in vivo estimates of endogenous dopamine in D2R-rich regions (caudate and putamen), mixed D2/3R-rich regions (ventral striatum and globus pallidus), and extrastriatal D3R-rich regions (hypothalamus and ventral pallidum). Dopamine depletion decreased self-reported vigor, which was correlated with the reduction in dopamine levels in the globus pallidus. [(11)C]-(+)-PHNO is a suitable radiotracer for use in estimating endogenous dopamine levels at D2R and D3R in neuropsychiatric populations.

  10. Estimating Endogenous Dopamine Levels at D2 and D3 Receptors in Humans using the Agonist Radiotracer [11C]-(+)-PHNO

    PubMed Central

    Caravaggio, Fernando; Nakajima, Shinichiro; Borlido, Carol; Remington, Gary; Gerretsen, Philip; Wilson, Alan; Houle, Sylvain; Menon, Mahesh; Mamo, David; Graff-Guerrero, Ariel

    2014-01-01

    Using positron emission tomography (PET) and an acute dopamine depletion challenge it is possible to estimate endogenous dopamine levels occupying dopamine D2/3 receptors (D2/3R) in humans in vivo. Our group has developed [11C]-(+)-PHNO, the first agonist radiotracer with preferential in vivo affinity for D3R. Thus, the use of [11C]-(+)-PHNO offers the novel possibility of (i) estimating in vivo endogenous dopamine levels at D2/3R using an agonist radiotracer, and (ii) estimating endogenous dopamine levels at D3R in extrastriatal regions such as the substantia nigra, hypothalamus, and ventral pallidum. Ten healthy participants underwent a [11C]-(+)-PHNO PET scan under baseline conditions and another under acute endogenous dopamine depletion achieved via oral administration of alpha-methyl-para-tyrosine (64 mg/kg). [11C]-(+)-PHNO binding was sensitive to acute dopamine depletion, allowing in vivo estimates of endogenous dopamine in D2R-rich regions (caudate and putamen), mixed D2/3R-rich regions (ventral striatum and globus pallidus), and extrastriatal D3R-rich regions (hypothalamus and ventral pallidum). Dopamine depletion decreased self-reported vigor, which was correlated with the reduction in dopamine levels in the globus pallidus. [11C]-(+)-PHNO is a suitable radiotracer for use in estimating endogenous dopamine levels at D2R and D3R in neuropsychiatric populations. PMID:24874713

  11. Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1

    NASA Technical Reports Server (NTRS)

    Tsapin, A. I.; Vandenberghe, I.; Nealson, K. H.; Scott, J. H.; Meyer, T. E.; Cusanovich, M. A.; Harada, E.; Kaizu, T.; Akutsu, H.; Leys, D.; Van Beeumen, J. J.

    2001-01-01

    Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

  12. Arsenic stimulates release of cytochrome c from isolated mitochondria via induction of mitochondrial permeability transition

    SciTech Connect

    Bustamante, Juanita; Nutt, Leta; Orrenius, Sten; Gogvadze, Vladimir . E-mail: Vladimir.Gogvadze@imm.ki.se

    2005-09-01

    Arsenic trioxide, As(III), is a known environmental toxicant, co-carcinogen, and potent chemotherapeutic agent. In model experiments with isolated rat liver mitochondria, As(III) stimulated a dose-dependent, cyclosporin A-sensitive release of cytochrome c via induction of mitochondrial permeability transition and subsequent swelling of mitochondria. Mitochondrial GSH does not seem to be a target for As(III) which, however, appears to cause oxidative modification of thiol groups of pore forming proteins, notably adenine nucleotide translocase. In mouse embryonic fibroblasts, 10 {mu}M As(III) stimulated cytochrome c release and apoptosis via a Bax/Bak-dependent mechanism. At high concentrations (125 {mu}M and higher), cells died by Bax/Bak-independent necrosis; at this concentration range As(III) targets mitochondria directly, particularly complex I of the mitochondrial respiratory chain. Since pyruvate, a substrate of complex I, is a predominant mitochondrial substrate in the cell, inhibition of complex I will cause mitochondrial instability and a decrease of {delta}{psi} that facilitates permeability transition and necrotic cell death.

  13. A 1-methyl-4-piperidinyl cytectrene carboxylate labeled by the technetium 99m, a radiotracer for rat brain acetylcholinesterase activity.

    PubMed

    Mejri, Najoua; Barhoumi, Chokri; Trabelsi, Moez; Mekni, Abdelkader; Said, Nadia Malek; Saidi, Mouldi

    2010-02-01

    Alzheimer's disease (AD) is a degenerative neurological disorder that causes progressive and irreversible loss of connections between brain cells and loss of mental functions. Clinical and postmortem studies show that the biochemical changes in brains of AD patients include decrease in acetylcholinesterase (AChE) activity. Our aim was to study AChE activity using piperidinyl ester labelled with technetium-99m. In vivo and in vitro studies demonstrated that labelled piperidinyl ester was a substrate for AChE. The hydrolytic rate of this substrate was measured and the specificity was evaluated using the inhibitor BW284c51. The rhenium analogues of the technetium-labelled substrate were used to determine the affinity constant (K(m)) and the maximum reaction velocity (V(max)) because of the high specific activity of technetium. The high hydrolytic rate and high specificity of the substrate for AChE make it suitable as an in vivo radiotracer for studying AChE activity in the brain.

  14. Investigation of flow dynamics of liquid phase in a pilot-scale trickle bed reactor using radiotracer technique.

    PubMed

    Pant, H J; Sharma, V K

    2016-10-01

    A radiotracer investigation was carried out to measure residence time distribution (RTD) of liquid phase in a trickle bed reactor (TBR). The main objectives of the investigation were to investigate radial and axial mixing of the liquid phase, and evaluate performance of the liquid distributor/redistributor at different operating conditions. Mean residence times (MRTs), holdups (H) and fraction of flow flowing along different quadrants were estimated. The analysis of the measured RTD curves indicated radial non-uniform distribution of liquid phase across the beds. The overall RTD of the liquid phase, measured at the exit of the reactor was simulated using a multi-parameter axial dispersion with exchange model (ADEM), and model parameters were obtained. The results of model simulations indicated that the TBR behaved as a plug flow reactor at most of the operating conditions used in the investigation. The results of the investigation helped to improve the existing design as well as to design a full-scale industrial TBR for petroleum refining applications. PMID:27544314

  15. Grain boundary diffusion and segregation of Bi in Cu: radiotracer measurements in B and C diffusion regimes

    SciTech Connect

    Divinski, Sergiy; Lohmann, Maik; Herzig, Christian

    2004-08-02

    Grain boundary (GB) radiotracer diffusion of {sup 207}Bi in polycrystalline Cu was measured in Harrison's B (1198-843 K) and C (659-536 K) kinetic regimes. In the B regime the triple product P=s{center_dot}{delta}{center_dot}D{sub gb} (where s is the segregation factor and {delta} the GB width) was found to obey the Arrhenius law P=6.6x10{sup -12}xexp (-102.8 kJ mol{sup -1}/RT) m{sup 3} s{sup -1}. The measurements in the C regime yielded the temperature dependence of the Bi GB diffusion coefficient D{sub gb} in Cu, D{sub gb}=0.24xexp (-156.2 kJ mol{sup -1}/RT) m{sup 2} s{sup -1}. Assuming that {delta} congruent with 5x10{sup -10} m the segregation factor s of Bi in Cu was determined in the limit of very dilute solute concentration to follow an Arrhenius dependence s=s{sub 0}exp(-{delta}H{sub s}/RT) with the pre-exponential factor s{sub 0}=0.054 and the segregation enthalpy {delta}H{sub s}=-53.4 kJ mol{sup -1}.

  16. Investigation of flow dynamics of liquid phase in a pilot-scale trickle bed reactor using radiotracer technique.

    PubMed

    Pant, H J; Sharma, V K

    2016-10-01

    A radiotracer investigation was carried out to measure residence time distribution (RTD) of liquid phase in a trickle bed reactor (TBR). The main objectives of the investigation were to investigate radial and axial mixing of the liquid phase, and evaluate performance of the liquid distributor/redistributor at different operating conditions. Mean residence times (MRTs), holdups (H) and fraction of flow flowing along different quadrants were estimated. The analysis of the measured RTD curves indicated radial non-uniform distribution of liquid phase across the beds. The overall RTD of the liquid phase, measured at the exit of the reactor was simulated using a multi-parameter axial dispersion with exchange model (ADEM), and model parameters were obtained. The results of model simulations indicated that the TBR behaved as a plug flow reactor at most of the operating conditions used in the investigation. The results of the investigation helped to improve the existing design as well as to design a full-scale industrial TBR for petroleum refining applications.

  17. Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation.

    PubMed

    Sigman, J A; Pond, A E; Dawson, J H; Lu, Y

    1999-08-24

    In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation. PMID:10460168

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

    SciTech Connect

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

    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 proteomic 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 more

  19. Enantiopure bifunctional chelators for copper radiopharmaceuticals--does chirality matter in radiotracer design?

    PubMed

    Singh, Ajay N; Dakanali, Marianna; Hao, Guiyang; Ramezani, Saleh; Kumar, Amit; Sun, Xiankai

    2014-06-10

    It is well recognized that carbon chirality plays a critical role in the design of drug molecules. However, very little information is available regarding the effect of stereoisomerism of macrocyclic bifunctional chelators (BFC) on biological behaviors of the corresponding radiopharmaceuticals. To evaluate such effects, three enantiopure stereoisomers of a copper radiopharmaceutical BFC bearing two chiral carbon atoms were synthesized in forms of R,R-, S,S-, and R,S-. Their corresponding peptide conjugates were prepared by coupling with a model peptide sequence, c(RGDyK), which targets the αvβ3 integrin for in vitro and in vivo evaluation of their biological behaviors as compared to the racemic conjugate. Despite the chirality differences, all the conjugates showed a similar in vitro binding affinity profile to the αvβ3 integrin (106, 108, 85 and 100 nM for rac-H2-1, RR-H2-1, SS-H2-1, and RS-H2-1 respectively with all p values > 0.05) and a similar level of in vivo tumor uptake (2.72 ± 0.45, 2.60 ± 0.52, 2.45 ± 0.48 and 2.88 ± 0.59 for rac-(64)Cu-1, RR-(64)Cu-1, SS-(64)Cu-1, and RS-(64)Cu-1 at 1 h p.i. respectively). Furthermore, they demonstrated a nearly identical biodistribution pattern in major organs (e.g. 2.07 ± 0.21, 2.13 ± 0.58, 1.70 ± 0.20 and 1.90 ± 0.46 %ID/g at 24 h p.i. in liver for rac-(64)Cu-1, RR-(64)Cu-1, SS-(64)Cu-1, and RS-(64)Cu-1 respectively; 1.80 ± 0.46, 2.30 ± 1.49, 1.73 ± 0.31 and 2.23 ± 0.71 at 24 h p.i. in kidneys for rac-(64)Cu-1, RR-(64)Cu-1, SS-(64)Cu-1, and RS-(64)Cu-1 respectively). Therefore we conclude that the chirality of BFC plays a negligible role in αvβ3-targeted copper radiopharmaceuticals. However, we believe it is still worthwhile to consider the chirality effects of BFCs on other targeted imaging or therapeutic agents.

  20. Cytochrome p450nor, a novel class of mitochondrial cytochrome P450 involved in nitrate respiration in the fungus Fusarium oxysporum.

    PubMed

    Takaya, N; Suzuki, S; Kuwazaki, S; Shoun, H; Maruo, F; Yamaguchi, M; Takeo, K

    1999-12-15

    Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen. In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide. We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol. Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F. oxysporum and acts as nitric oxide reductase. Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria.

  1. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G.

    2010-11-16

    Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

  2. Energy-Dependent Reversal of the Cytochrome Oxidase Reaction

    NASA Astrophysics Data System (ADS)

    Wikstrom, Marten

    1981-07-01

    Energization of isolated rat liver mitochondria with ATP under conditions in which cytochrome c is poised in a highly oxidized state shifts the state of cytochrome oxidase (cytochrome c oxidase; ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) from fully oxidized to two new spectroscopically distinguishable states depending on the applied phosphorylation potential and redox potential at cytochrome c. Both new states are spectrally similar or identical to two previously described intermediates in the reaction between reduced enzyme and O2. The data suggest that the energy-dependent transitions are due to reversed electron transfer from water to ferricytochrome c linked to accumulation of intermediates of O2 reduction at the catalytic heme a3/copper center. Titrations with redox potential indicate that each transition is a one-electron step, a finding that would identify the second observed compound as enzyme-bound peroxide or its equivalent. This is consistent with this compound being spectrally identical to ``Compound C,'' previously described as the reaction product between half-reduced oxidase (two electrons) and O2. On the basis of these data a catalytic scheme of O2 reduction is proposed for the heme a3/copper center of cytochrome oxidase.

  3. Electrochemical investigations on the oxygen activation by cytochrome P-450.

    PubMed

    Scheller, F; Renneberg, R; Schwarze, W; Strnad, G; Pommerening, K; Prümke, H J; Mohr, P

    1979-01-01

    The application of cytochrome P-450 in substrate conversion is complicated both due to the limited stability and the cofactor regeneration problems. To overcome the disadvantages of NADPH consumption the transfer of the reduction equivalents from an electrode into the cytochrome P-450-system was studied: 1. NADPH was cathodically reduced at a mercury pool electrode. By immobilization of NADP on dialdehyde Sephadex the reductive recycling was possible. 2. Different forms of reduced oxygen were produced by the cathode: a) The reaction of O2- with deoxycorticosterone yields a carboxylic acid derivative. In contrast the cytochrome P-450 catalyzed NADPH-dependent reaction with the same substrate gives corticosterone, O2- represents only an intermediate in the activation of oxygen and is not the "activated oxygen" species. b) Molecular oxygen was reduced to HO2- and H2O2, respectively. The interaction of adsorbed cytochrome P-450 on the electrode surface with the reduced oxygen species in the absence of NADPH was studied. The electrochemically generated peroxide seems to be more active than added H2O2. 3. In a model of electro-enzyme-reactor several substrates were hydroxylated by microsomal cytochrome P-450 with cathodically reduced oxygen which substitutes NADPH.

  4. Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

    SciTech Connect

    Ascenzi, Paolo; Ciaccio, Chiara; Sinibaldi, Federica; Santucci, Roberto; Coletta, Massimo

    2011-01-07

    Research highlights: {yields} Cardiolipin binding to cytochrome c. {yields} Cardiolipin-dependent peroxynitrite isomerization by cytochrome c. {yields} Cardiolipin-cytochrome c complex plays pro-apoptotic effects. {yields} Cardiolipin-cytochrome c complex plays anti-apoptotic effects. -- Abstract: Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k{sub on}) is (3.2 {+-} 0.4) x 10{sup 5} M{sup -1} s{sup -1}. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 {+-} 0.8) x 10{sup -5} M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.

  5. Reduction of Heavy Metals by Cytochrome c(3)

    SciTech Connect

    ABDELOUAS,A.; GONG,W.L.; LUTZE,W.; NUTTALL,E.H.; SPRAGUE,F.; SHELNUTT,JOHN A.; STRIETELMEIER,B.A.; FRANCO,R.; MOURA,I.; MOURA,J.J.G.

    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 without 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.

  6. Radical formation in cytochrome c oxidase☆

    PubMed Central

    Yu, Michelle A.; Egawa, Tsuyoshi; Shinzawa-Itoh, Kyoko; Yoshikawa, Shinya; Yeh, Syun-Ru; Rousseau, Denis L.; Gerfen, Gary J.

    2015-01-01

    The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 µs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO2•− ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H2O2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50 µs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (R – O – O•) species was formed, presumably by the reaction between O2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  8. A cytochrome cbb3 (cytochrome c) terminal oxidase in Azospirillum brasilense Sp7 supports microaerobic growth.

    PubMed

    Marchal, K; Sun, J; Keijers, V; Haaker, H; Vanderleyden, J

    1998-11-01

    Spectral analysis indicated the presence of a cytochrome cbb3 oxidase under microaerobic conditions in Azospirillum brasilense Sp7 cells. The corresponding genes (cytNOQP) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an A. brasilense cytN mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (mu(e)) of the A. brasilense cytN mutant was comparable to the wild-type specific growth rate (m(e) of approximately 0.2 h-1). In microaerobic NH4+-supplemented conditions, the low respiration of the A. brasilense cytN mutant affected its specific growth rate (mu(e) of approximately 0.02 h-1) compared to the wild-type specific growth rate (mu(e) of approximately 0.2 h-1). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH4+-supplemented conditions. Differences in growth rates and respiration between the wild type and the A. brasilense cytN mutant were less pronounced under these nitrogen-fixing conditions (mu(e) of approximately 0.03 h-1 for the wild type and 0.02 h-1 for the A. brasilense cytN mutant). The nitrogen-fixing capacity of the A. brasilense cytN mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the A. brasilense cytochrome cbb3 oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.

  9. The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s.

    PubMed

    Nelson, David R; Goldstone, Jared V; Stegeman, John J

    2013-02-19

    The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the 'cytochrome P450 genesis locus', where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.

  10. Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine.

    PubMed

    McCue, Jeffrey M; Driscoll, William J; Mueller, Gregory P

    2008-01-11

    Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo.

  11. Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine

    SciTech Connect

    McCue, Jeffrey M.; Driscoll, William J.; Mueller, Gregory P.

    2008-01-11

    Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo.

  12. The mechanism of cytochrome C oxidase inhibition by nitric oxide.

    PubMed

    Antunes, Fernando; Cadenas, Enrique

    2007-01-01

    The basic biochemistry of the inhibition of cytochrome oxidase by NO is reviewed. Three possible mechanisms that include the binding of NO to the fully reduced Fe(a3)-Cu(B) site, to the semi-reduced Fe(a3)-Cu(B) site, and to the fully oxidized Fe(a3)-Cu(B) site are confronted with the experimental data. Mathematical models are used to facilitate the analysis and to solve puzzling observations concerning the NO inhibition of cytochrome oxidase. It is concluded that the inhibition of cytochrome oxidase by NO is mixed, having both competitive and uncompetitive components, but under physiological electron flows the competitive component is largely predominant. The physiological and pathological relevance of this inhibition is briefly discussed.

  13. In vitro synthesis of arachidonoyl amino acids by cytochrome c.

    PubMed

    McCue, Jeffrey M; Driscoll, William J; Mueller, Gregory P

    2009-11-01

    Arachidonoyl amino acids are a class of endogenous lipid messengers that are expressed in the mammalian central nervous system and peripherally. While several of their prominent pharmacologic effects have been documented, the mechanism by which arachidonoyl amino acids are biosynthesized has not been defined. We have previously observed that the mitochondrial protein, cytochrome c, is capable of catalyzing the formation of the prototypic arachidonoyl amino acid, arachidonoyl glycine, utilizing arachidonoyl CoA and glycine as substrates, in the presence of hydrogen peroxide. Here we report that cytochrome c is similarly able to catalyze the formation of N-arachidonoyl serine, N-arachidonoyl alanine, and N-arachidonoyl gamma aminobutyric acid from arachidonoyl CoA and the respective amino acids. The identities of the arachidonoyl amino acid products were verified by mass spectral fragmentation pattern analysis. The synthetic reactions exhibited Michaelis-Menten kinetics and continued favorably at physiologic temperature and pH. Spectral data indicate that both cytochrome c protein structure and a +3 heme iron oxidation state are required for the reaction mechanism to proceed optimally. Reactions designed to catalyze the formation of N-arachidonoyl dopamine were not efficient due to the rapid oxidation of dopamine substrate by hydrogen peroxide, consuming both reactants. Finally, under standard assay conditions, arachidonoyl CoA and ethanolamine were found to react spontaneously to form anandamide, independent of cytochrome c and hydrogen peroxide. Accordingly, it was not possible to demonstrate a potential role for cytochrome c in the biosynthetic mechanism for either arachidonoyl dopamine or anandamide. However, the ability of cytochrome c to effectively catalyze the formation of N-arachidonoyl serine, N-arachidonoyl alanine, and N-arachidonoyl gamma aminobutyric acid in vitro highlights its potential role for the generation of these lipid messengers in vivo.

  14. Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition.

    PubMed

    Borisov, Vitaliy B; Forte, Elena; Siletsky, Sergey A; Sarti, Paolo; Giuffrè, Alessandro

    2015-02-01

    Cytochrome bd is a prokaryotic respiratory quinol oxidase phylogenetically unrelated to heme-copper oxidases, that was found to promote virulence in some bacterial pathogens. Cytochrome bd from Escherichia coli was previously reported to contribute not only to proton motive force generation, but also to bacterial resistance to nitric oxide (NO) and hydrogen peroxide (H2O2). Here, we investigated the interaction of the purified enzyme with peroxynitrite (ONOO(-)), another harmful reactive species produced by the host to kill invading microorganisms. We found that addition of ONOO(-) to cytochrome bd in turnover with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) causes the irreversible inhibition of a small (≤15%) protein fraction, due to the NO generated from ONOO(-) and not to ONOO(-) itself. Consistently, addition of ONOO(-) to cells of the E. coli strain GO105/pTK1, expressing cytochrome bd as the only terminal oxidase, caused only a minor (≤5%) irreversible inhibition of O2 consumption, without measurable release of NO. Furthermore, by directly monitoring the kinetics of ONOO(-) decomposition by stopped-flow absorption spectroscopy, it was found that the purified E. coli cytochrome bd in turnover with O2 is able to metabolize ONOO(-) with an apparent turnover rate as high as ~10 mol ONOO(-) (mol enzyme)(-1) s(-1) at 25°C. To the best of our knowledge, this is the first time that the kinetics of ONOO(-) decomposition by a terminal oxidase has been investigated. These results strongly suggest a protective role of cytochrome bd against ONOO(-) damage.

  15. Investigating the pharmacokinetics and biological distribution of silver-loaded polyphosphoester-based nanoparticles using 111Ag as a radiotracer

    PubMed Central

    Aweda, Tolulope A.; Zhang, Shiyi; Mupanomunda, Chiedza; Burkemper, Jennifer; Heo, Gyu Seong; Bandara, Nilantha; Lin, Mai; Cutler, Cathy S.; Cannon, Carolyn L.; Youngs, Wiley; Wooley, Karen L.; Lapi, Suzanne E.

    2015-01-01

    Purified 111Ag was used as a radiotracer to investigate silver loading and release, pharmacokinetics and biodistribution of polyphosphoester-based degradable shell crosslinked knedel-like (SCK) nanoparticles as a comparison to the previously reported small molecule, N-heterocyclic silver carbene complex analogue (SCC1) for the delivery of therapeutic silver ions in mouse models. Biodistribution studies were conducted by aerosol administration of 111Ag acetate, [111Ag]SCC1 and [111Ag]SCK doses directly into the lungs of C57BL/6 mice. Nebulization of the 111Ag antimicrobials resulted in an average uptake of 1.07 ± 0.12% of the total aerosolized dose given per mouse. The average dose taken into the lungs of mice was estimated to be 2.6 ± 0.3% of the dose inhaled per mouse for [111Ag]SCC1 and twice as much dose was observed for the [111Ag]SCKs (5.0 ± 0.3% and 5.9 ± 0.8% for [111Ag]aSCK and [111Ag]zSCK, respectively) at 1 h post administration (p.a.). [111Ag]SCKs also exhibited higher dose retention in the lungs; 62 – 68% for [111Ag]SCKs and 43% for [111Ag]SCC1 of the initial 1 h dose was observed in the lungs at 24 h post administration (p.a.). This study demonstrates the utility of 111Ag as a useful tool for monitoring the pharmacokinetics of silver loaded antimicrobials in vivo. PMID:25952472

  16. 2'[(18)F]-fluoroethylrhodamine B is a promising radiotracer to measure P-glycoprotein function.

    PubMed

    Trencsényi, György; Kertész, István; Krasznai, Zoárd T; Máté, Gábor; Szalóki, Gábor; Szabó Judit, P; Kárpáti, Levente; Krasznai, Zoltán; Márián, Teréz; Goda, Katalin

    2015-07-10

    In vivo detection of the emergence of P-glycoprotein (Pgp) mediated multidrug resistance in tumors could be beneficial for patients treated with anticancer drugs. PET technique in combination with appropriate radiotracers could be the most convenient method for detection of Pgp function. Rhodamine derivatives are validated fluorescent probes for measurement of mitochondrial membrane potential and also Pgp function. The aim of this study was to investigate whether 2'[(18)F]-fluoroethylrhodamine B ((18)FRB) a halogenated rhodamine derivative previously synthesized for PET assessment of myocardial perfusion preserved its Pgp substrate character. ATPase assay as well as accumulation experiments carried out using Pgp(+) and Pgp(-) human gynecologic (A2780/A2780(AD) and KB-3-1/KB-V1) and a mouse fibroblast cell pairs (NIH 3T3 and NIH 3T3 MDR1) were applied to study the interaction of (18)FRB with Pgp. ATPase assay proved that (18)FRB is a high affinity substrate of Pgp. Pgp(-) cells accumulated the (18)FRB rapidly in accordance with its lipophilic character. Dissipation of the mitochondrial proton gradient by a proton ionophore CCCP decreased the accumulation of rhodamine 123 (R123) and (18)FRB into Pgp(-) cells. Pgp(+) cells exhibited very low R123 and (18)FRB accumulation (around 1-8% of the Pgp(-) cell lines) which was not sensitive to the mitochondrial proton gradient; rather it was increased by the Pgp inhibitor cyclosporine A (CsA). Based on the above data we conclude that (18)FRB is a high affinity Pgp substrate and consequently a potential PET tracer to detect multidrug resistant tumors as well as the function of physiological barriers expressing Pgp.

  17. Investigating the pharmacokinetics and biological distribution of silver-loaded polyphosphoester-based nanoparticles using (111) Ag as a radiotracer.

    PubMed

    Aweda, Tolulope A; Zhang, Shiyi; Mupanomunda, Chiedza; Burkemper, Jennifer; Heo, Gyu Seong; Bandara, Nilantha; Lin, Mai; Cutler, Cathy S; Cannon, Carolyn L; Youngs, Wiley J; Wooley, Karen L; Lapi, Suzanne E

    2015-05-30

    Purified (111) Ag was used as a radiotracer to investigate silver loading and release, pharmacokinetics, and biodistribution of polyphosphoester-based degradable shell crosslinked knedel-like (SCK) nanoparticles as a comparison to the previously reported small molecule, N-heterocyclic silver carbene complex analog (SCC1) for the delivery of therapeutic silver ions in mouse models. Biodistribution studies were conducted by aerosol administration of (111) Ag acetate, [(111) Ag]SCC1, and [(111) Ag]SCK doses directly into the lungs of C57BL/6 mice. Nebulization of the (111) Ag antimicrobials resulted in an average uptake of 1.07 ± 0.12% of the total aerosolized dose given per mouse. The average dose taken into the lungs of mice was estimated to be 2.6 ± 0.3% of the dose inhaled per mouse for [(111) Ag]SCC1 and twice as much dose was observed for the [(111) Ag]SCKs (5.0 ± 0.3% and 5.9 ± 0.8% for [(111) Ag]aSCK and [(111) Ag]zSCK, respectively) at 1 h post administration (p.a.). [(111) Ag]SCKs also exhibited higher dose retention in the lungs; 62-68% for [(111) Ag]SCKs and 43% for [(111) Ag]SCC1 of the initial 1 h dose were observed in the lungs at 24 h p.a.. This study demonstrates the utility of (111) Ag as a useful tool for monitoring the pharmacokinetics of silver-loaded antimicrobials in vivo.

  18. Investigating the pharmacokinetics and biological distribution of silver-loaded polyphosphoester-based nanoparticles using (111) Ag as a radiotracer.

    PubMed

    Aweda, Tolulope A; Zhang, Shiyi; Mupanomunda, Chiedza; Burkemper, Jennifer; Heo, Gyu Seong; Bandara, Nilantha; Lin, Mai; Cutler, Cathy S; Cannon, Carolyn L; Youngs, Wiley J; Wooley, Karen L; Lapi, Suzanne E

    2015-05-30

    Purified (111) Ag was used as a radiotracer to investigate silver loading and release, pharmacokinetics, and biodistribution of polyphosphoester-based degradable shell crosslinked knedel-like (SCK) nanoparticles as a comparison to the previously reported small molecule, N-heterocyclic silver carbene complex analog (SCC1) for the delivery of therapeutic silver ions in mouse models. Biodistribution studies were conducted by aerosol administration of (111) Ag acetate, [(111) Ag]SCC1, and [(111) Ag]SCK doses directly into the lungs of C57BL/6 mice. Nebulization of the (111) Ag antimicrobials resulted in an average uptake of 1.07 ± 0.12% of the total aerosolized dose given per mouse. The average dose taken into the lungs of mice was estimated to be 2.6 ± 0.3% of the dose inhaled per mouse for [(111) Ag]SCC1 and twice as much dose was observed for the [(111) Ag]SCKs (5.0 ± 0.3% and 5.9 ± 0.8% for [(111) Ag]aSCK and [(111) Ag]zSCK, respectively) at 1 h post administration (p.a.). [(111) Ag]SCKs also exhibited higher dose retention in the lungs; 62-68% for [(111) Ag]SCKs and 43% for [(111) Ag]SCC1 of the initial 1 h dose were observed in the lungs at 24 h p.a.. This study demonstrates the utility of (111) Ag as a useful tool for monitoring the pharmacokinetics of silver-loaded antimicrobials in vivo. PMID:25952472

  19. 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

    to proteins, including c-type cytochromes that have not been characterized. The distinctive expression of c-type cytochromes in response to growth with different terminal electron acceptors offers opportunities for functional (i.e., activity) in situ monitoring using metaproteomics or transcript-targeted approaches.

  20. Musk xylene is a novel specific inducer of cytochrome P-450IA2.

    PubMed

    Iwata, N; Minegishi, K; Suzuki, K; Ohno, Y; Kawanishi, T; Takahashi, A

    1992-04-15

    The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.

  1. Sensor sensationalism? Alternative views on the nature and role of 'cytochrome a1' in bacteria.

    PubMed

    Poole, R K; Baines, B S; Williams, H D

    1985-01-01

    Replying to a recent proposal that 'cytochrome a1' functions as an oxygen sensor, we argue that this speculation is flawed by the failure to appreciate that cytochrome a1-like haemoproteins are a diverse group of haemoproteins. PMID:3939981

  2. An Engineered Cytochrome b6c1 Complex with a Split Cytochrome b Is Able To Support Photosynthetic Growth of Rhodobacter capsulatus

    PubMed Central

    Saribas, A. Sami; Mandaci, Sevnur; Daldal, Fevzi

    1999-01-01

    The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280–16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into

  3. Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus.

    PubMed

    Allen, James W A; Sawyer, Elizabeth B; Ginger, Michael L; Barker, Paul D; Ferguson, Stuart J

    2009-04-01

    c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys-Xaa-Xaa-Cys-His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b(562) containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b(562) to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CX(n)CH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.

  4. Cytochrome c4 is required for siderophore expression by Legionella pneumophila, whereas cytochromes c1 and c5 promote intracellular infection.

    PubMed

    Yip, Emily S; Burnside, Denise M; Cianciotto, Nicholas P

    2011-03-01

    A panel of cytochrome c maturation (ccm) mutants of Legionella pneumophila displayed a loss of siderophore (legiobactin) expression, as measured by both the chrome azurol S assay and a Legionella-specific bioassay. These data, coupled with the finding that ccm transcripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes siderophore expression by L. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individual c-type cytochromes. Whereas ubiquinol-cytochrome c reductase (petC) mutants specifically lacking cytochrome c(1) and cycB mutants lacking cytochrome c(5) had normal siderophore expression, cyc4 mutants defective for cytochrome c(4) completely lacked legiobactin. These data, along with the expression pattern of cyc4 mRNA, indicate that cytochrome c(4) in particular promotes siderophore expression. In intracellular infection assays, petC mutants and cycB mutants, but not cyc4 mutants, had a reduced ability to infect both amoebae and macrophage hosts. Like ccm mutants, the cycB mutants were completely unable to grow in amoebae, highlighting a major role for cytochrome c(5) in intracellular infection. To our knowledge, these data represent both the first direct documentation of the importance of a c-type cytochrome in expression of a biologically active siderophore and the first insight into the relative importance of c-type cytochromes in intracellular infection events.

  5. Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization.

    PubMed Central

    Rodrigues, C. M.; Solá, S.; Silva, R.; Brites, D.

    2000-01-01

    activate the apoptotic machinery in neural cells. Toxicity occurs through a mitochondrial-dependent pathway, which in part involves opening of the permeability transition pore. Furthermore, membrane permeabilization is required for cytochrome c release from mitochondria and can be prevented by UDC or TUDC. These data suggest that the mitochondria is a pharmacological target for cytoprotection during unconjugated hyperbilirubinemia and neurodegenerative disorders, and that UDC or TUDC may be potential therapeutic agents. PMID:11147571

  6. Silencing NADPH-cytochrome P450 reductase results in reduced acaricide resistance in Tetranychus cinnabarinus (Boisduval)

    PubMed Central

    Shi, Li; Zhang, Jiao; Shen, Guangmao; Xu, Zhifeng; Wei, Peng; Zhang, Yichao; Xu, Qiang; He, Lin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are involved in metabolic resistance to insecticides and require NADPH cytochrome P450 reductase (CPR) to transfer electrons when they catalyze oxidation reactions. The carmine spider mite, Tetranychus cinnabarinus is an important pest mite of crop and vegetable plants worldwide, and its resistance to acaricides has quickly developed. However, the role of CPR on the formation of acaricide-resistance in T. cinnabarinus is still unclear. In this study, a full-length cDNA encoding CPR was cloned and characterized from T. cinnabarinus (designated TcCPR). TcCPR expression was detectable in all developmental stages of T. cinnabarinus, but it’s much lower in eggs. TcCPR was up-regulated and more inducible with fenpropathrin treatment in the fenpropathrin-resistant (FeR) strain compared with the susceptible SS strain. Feeding of double-strand RNA was effective in silencing the transcription of TcCPR in T. cinnabarinus, which resulted in decreasing the activity of P450s and increasing the susceptibility to fenpropathrin in the FeR strain but not in the susceptible strain. The current results provide first evidence that the down-regulation of TcCPR contributed to an increase of the susceptibility to fenpropathrin in resistant mites. TcCPR could be considered as a novel target for the development of new pesticides. PMID:26493678

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

    PubMed

    Billen, M J; Squires, E J

    2009-01-01

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

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

    PubMed

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

    2006-10-01

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

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

    PubMed Central

    Sacco, James C.; Trepanier, Lauren A.

    2010-01-01

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

  10. Environmental biochemistry of current environmental levels of heavy metals: preparation of radiotracers with very high specific radioactivity for metallobiochemical experiments on laboratory animals.

    PubMed

    Sabbioni, E; Goetz, L; Birattari, C; Bonardi, M

    1981-03-01

    Environmental toxicology research on dose-response relationships of heavy metals requires experiments on laboratory animals exposed to "low doses" of trace elements which should reflect "present or actual environmental levels" characteristic of polluted environments. Unfortunately no criteria exist to establish the "low doses" to which laboratory animals must be exposed, in practice the choice of the level used is made in an almost arbitrary manner. In order to define the "present environmental levels" of heavy metals which should be administered to laboratory animals an approach is suggested, based upon knowledge of the concentrations of trace elements in the diet, air and food as well as the fractions absorbed. Today daily intakes of trace elements by man are of the order of few micrograms or nanograms thus requiring the use of extremely sensitive analytical techniques to determine the very low amounts of heavy metals in tissues and cellular components. In these fields of research the use of radiotracers with very high specific radioactivity appears particularly advantageous but requires considerable care during their preparation and use. The first part of this paper deals with a definition of the ranges of concentrations of trace elements which should be used for metabolic studies on laboratory animals when they are exposed via different routes such as ingestion, inhalation in injection; the second part describes the production of radiotracers with very high specific radioactivity by proton activation in the cyclotron and by neutron irradiation in the nuclear reactor. Their use to label present levels of heavy metals under conditions adapted for biochemical purposes, as well as the preparation of different metal-labelled chemical species is also reported. Particular attention is directed to quality control of the radiotracer solutions which are administered to the animals including those of radioactivity concentrations, radioisotopic purity, radiochemical purity

  11. Magnetic Circular Dichroism Studies XXV. A Preliminary Investigation of Microsomal Cytochromes*

    PubMed Central

    Dolinger, Peter M.; Kielczewski, Michael; Trudell, James R.; Barth, Günter; Linder, Robert E.; Bunnenberg, Edward; Djerassi, Carl

    1974-01-01

    The application of magnetic circular dichroism as an optical probe for simultaneous identification and determination of at least two microsomal cytochromes is demonstrated. The assignments of the bands in the spectra of microsomal suspensions are made from the spectra of soluble preparations of cytochrome P-450 obtained from Pseudomonas putida and of cytochrome b5 obtained from rat livers. PMID:4521811

  12. Concentration and function of membrane-bound cytochromes in cyanobacterial heterocysts

    SciTech Connect

    Houchins, J.P.; Hind, G.

    1984-10-01

    Membranes isolated from heterocysts and vegetative cells of Anabaena 7120 were assayed for content of cytochrome f, cytochrome b-563, cytochrome b-559/sub HP/, cytochrome b-559/sub LP/, and cytochrome aa/sub 3/ by use of reduced-minus-oxidized difference spectra. The level of cytochrome aa/sub 3/ in heterocyst membranes was 4 to 100 times higher than that in vegetative cells of Anabaena 7120 or other species of cyanobacteria. Heterocyst membranes lack cytochrome b-559/sub HP/ but contain cytochrome b-559/sub LP/ (E/sub m7.5/ = +77 millivolts, n = 1) at approximately the same concentration as cytochrome f. The role of cytochrome b-559/sub LP/ in the hydrogenase-dependent electron transfer pathway was investigated with the inhibitor 2-(n-heptyl)-4-hydroxyquinoline N-oxide which blocks electron flow from hydrogenase to acceptors reacting with the plastoquinone pool. Addition of inhibitor elicited no change in the reduction level of cytochrome b-559/sub LP/ indicating that this cytochrome is not directly involved in this pathway. 30 references, 6 figures, 1 table.

  13. Molecular dynamics in cytochrome c oxidase Moessbauer spectra deconvolution

    SciTech Connect

    Bossis, Fabrizio; Palese, Luigi L.

    2011-01-07

    Research highlights: {yields} Cytochrome c oxidase molecular dynamics serve to predict Moessbauer lineshape widths. {yields} Half height widths are used in modeling of Lorentzian doublets. {yields} Such spectral deconvolutions are useful in detecting the enzyme intermediates. -- Abstract: In this work low temperature molecular dynamics simulations of cytochrome c oxidase are used to predict an experimentally observable, namely Moessbauer spectra width. Predicted lineshapes are used to model Lorentzian doublets, with which published cytochrome c oxidase Moessbauer spectra were simulated. Molecular dynamics imposed constraints to spectral lineshapes permit to obtain useful information, like the presence of multiple chemical species in the binuclear center of cytochrome c oxidase. Moreover, a benchmark of quality for molecular dynamic simulations can be obtained. Despite the overwhelming importance of dynamics in electron-proton transfer systems, limited work has been devoted to unravel how much realistic are molecular dynamics simulations results. In this work, molecular dynamics based predictions are found to be in good agreement with published experimental spectra, showing that we can confidently rely on actual simulations. Molecular dynamics based deconvolution of Moessbauer spectra will lead to a renewed interest for application of this approach in bioenergetics.

  14. Cytochrome allelic variants and clopidogrel metabolism in cardiovascular diseases therapy.

    PubMed

    Jarrar, Mohammed; Behl, Shalini; Manyam, Ganiraju; Ganah, Hany; Nazir, Mohammed; Nasab, Reem; Moustafa, Khaled

    2016-06-01

    Clopidogrel and aspirin are among the most prescribed dual antiplatelet therapies to treat the acute coronary syndrome and heart attacks. However, their potential clinical impacts are a subject of intense debates. The therapeutic efficiency of clopidogrel is controlled by the actions of hepatic cytochrome P450 (CYPs) enzymes and impacted by individual genetic variations. Inter-individual polymorphisms in CYPs enzymes affect the metabolism of clopidogrel into its active metabolites and, therefore, modify its turnover and clinical outcome. So far, clinical trials fail to confirm higher or lower adverse cardiovascular effects in patients treated with combinations of clopidogrel and proton pump inhibitors, compared with clopidogrel alone. Such inconclusive findings may be due to genetic variations in the cytochromes CYP2C19 and CYP3A4/5. To investigate potential interactions/effects of these cytochromes and their allele variants on the treatment of acute coronary syndrome with clopidogrel alone or in combination with proton pump inhibitors, we analyze recent literature and discuss the potential impact of the cytochrome allelic variants on cardiovascular events and stent thrombosis treated with clopidogrel. The diversity of CYP2C19 polymorphisms and prevalence span within various ethnic groups, subpopulations and demographic areas are also debated. PMID:27072373

  15. Isolation of a cytochrome aa3 gene from Bradyrhizobium japonicum

    PubMed Central

    O'Brian, Mark R.; Maier, Robert J.

    1987-01-01

    Bradyhizobium japonicum strain LO501 is a Tn5-induced mutant that does not express the terminal oxidase cytochrome aa3 (cytochrome-c oxidase, EC 1.9.3.1). Two and one-half kilobase pairs of LO501 genomic DNA that flanks the transposon was isolated and used as a hybridization probe to obtain the wild-type gene from a cosmid library. Two subcloned fragments from two of the isolated cosmids were ligated into broad host range vectors, and restriction maps of these fragments were generated. The resultant plasmids, pCA1 and pBL33, each contained DNA homologous to that mutated in strain LO501. The two plasmids were each introduced into strain LO501 by conjugal transfer, and it was found that pCA1, but not pBL33, complemented the oxidase mutant. The transconjugant strain LO501[pCA1] expressed wild-type levels of cytochrome aa3, as discerned spectrophotometrically, and had restored N,N,N′,N′-tetramethyl-p-phenylenediamine oxidase activity. Furthermore, the frequency of complementation of LO501 cells that received pCA1 by conjugation was 1.0, demonstrating that pCA1 complemented the mutant in trans. The results show that pCA1 contains the entire wild-type gene that was mutated in strain LO501, and this gene is required for cytochrome aa3 expression. Images PMID:16593835

  16. Relationship between horn fly infestation and polymorphisms in cytochrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Individual animal variation occurs regarding external parasite infestation in beef cattle. Our objective was to determine if horn flies infestations present on beef cattle are associated with the single nucleotide polymorphism (SNP; T-318C) in the cytochrome P450 gene (CYP3A28) and the prolactin (PR...

  17. Low cytochrome b variation in bream Abramis brama.

    PubMed

    Hayden, B; Coscia, I; Mariani, S

    2011-05-01

    Variability in cytochrome b (cytb) in European populations of bream Abramis brama was assessed. The cytb gene was found to be strongly conserved in A. brama relative to other cyprinid taxa. This limits the usefulness of this marker in examining geographical genetic structure in this species and raises interesting questions as to the recent evolutionary history of the species.

  18. Direct simulation of plastocyanin and cytochrome f interactions in solution

    NASA Astrophysics Data System (ADS)

    Kovalenko, I. B.; Abaturova, A. M.; Gromov, P. A.; Ustinin, D. M.; Grachev, E. A.; Riznichenko, G. Yu; Rubin, A. B.

    2006-06-01

    Most biological functions, including photosynthetic activity, are mediated by protein interactions. The proteins plastocyanin and cytochrome f are reaction partners in a photosynthetic electron transport chain. We designed a 3D computer simulation model of diffusion and interaction of spinach plastocyanin and turnip cytochrome f in solution. It is the first step in simulating the electron transfer from cytochrome f to photosystem 1 in the lumen of thylakoid. The model is multiparticle and it can describe the interaction of several hundreds of proteins. In our model the interacting proteins are represented as rigid bodies with spatial fixed charges. Translational and rotational motion of proteins is the result of the effect of stochastic Brownian force and electrostatic force. The Poisson-Boltzmann formalism is used to determine the electrostatic potential field generated around the proteins. Using this model we studied the kinetic characteristics of plastocyanin-cytochrome f complex formation for plastocyanin mutants at pH 7 and a variety of ionic strength values.

  19. In vitro import and assembly of the nucleus-encoded mitochondrial subunit III of cytochrome c oxidase (Cox3).

    PubMed

    Vázquez-Acevedo, Miriam; Rubalcava-Gracia, Diana; González-Halphen, Diego

    2014-11-01

    The cox3 gene, encoding subunit III of cytochrome c oxidase (Cox3) is in mitochondrial genomes except in chlorophycean algae, where it is localized in the nucleus. Therefore, algae like Chlamydomonas reinhardtii, Polytomella sp. and Volvox carteri, synthesize the Cox3 polypeptide in the cytosol, import it into mitochondria, and integrate it into the cytochrome c oxidase complex. In this work, we followed the in vitro internalization of the Cox3 precursor by isolated, import-competent mitochondria of Polytomella sp. In this colorless alga, the precursor Cox3 protein is synthesized with a long, cleavable, N-terminal mitochondrial targeting sequence (MTS) of 98 residues. In an import time course, a transient Cox3 intermediate was identified, suggesting that the long MTS is processed more than once. The first processing step is sensitive to the metalo-protease inhibitor 1,10-ortophenantroline, suggesting that it is probably carried out by the matrix-located Mitochondrial Processing Protease. Cox3 is readily imported through an energy-dependent import pathway and integrated into the inner mitochondrial membrane, becoming resistant to carbonate extraction. Furthermore, the imported Cox3 protein was assembled into cytochrome c oxidase, as judged by the presence of a labeled band co-migrating with complex IV in Blue Native Electrophoresis. A model for the biogenesis of Cox3 in chlorophycean algae is proposed. This is the first time that the in vitro mitochondrial import of a cytosol-synthesized Cox3 subunit is described. PMID:24561572

  20. Deletion of the Leader Peptide of the Mitochondrially Encoded Precursor of Saccharomyces Cerevisiae Cytochrome C Oxidase Subunit II

    PubMed Central

    Torello, A. T.; Overholtzer, M. H.; Cameron, V. L.; Bonnefoy, N.; Fox, T. D.

    1997-01-01

    Cytochrome c oxidase subunit II (Cox2p) of Saccharomyces cerevisiae is synthesized within mitochondria as a precursor, pre-Cox2p. The 15-amino acid leader peptide is processed after export to the intermembrane space. Leader peptides are relatively unusual in mitochondrially coded proteins: indeed mammalian Cox2p lacks a leader peptide. We generated two deletions in the S. cerevisiae COX2 gene, removing either the leader peptide (cox2-20) or the leader peptide and processing site (cox2-21) without altering either the promoter or the mRNA-specific translational activation site. When inserted into mtDNA, both deletions substantially reduced the steady-state levels of Cox2p and caused a tight nonrespiratory phenotype. A respiring pseudorevertant of the cox2-20 mutant was heteroplasmic for the original mutant mtDNA and a ρ(-) mtDNA whose deletion fused the first 251 codons of the mitochondrial gene encoding cytochrome b to the cox2-20 sequence. The resulting fusion protein was processed to yield functional Cox2p. Thus, the presence of amino-terminal cytochrome b sequence bypassed the need for the pre-Cox2p leader peptide. We propose that the pre-Cox2p leader peptide contains a targeting signal necessary for membrane insertion, without which it remains in the matrix and is rapidly degraded. PMID:9093845

  1. Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

    PubMed Central

    Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

    2013-01-01

    There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

  2. Gallium arsenide differentially affects processing of phagolysosomal targeted antigen by macrophages.

    PubMed

    Lewis, T A; Hartmann, C B; McCoy, K L

    1998-03-01

    Gallium arsenide, a semiconductor utilized in the electronics industry, causes immunosuppression in animals. The chemical's effect on macrophages to process antigen for activating pigeon cytochrome-specific helper T cell hybridoma was investigated. Mice were administered 200 mg/kg gallium arsenide or vehicle intraperitoneally. Five-day exposure suppressed processing by splenic macrophages but augmented processing by thioglycollate-elicited and resident peritoneal macrophages. Cytochrome coupled to latex beads was targeted to phagolysosomes to examine processing in lysosomes. Cytochrome beads required phagocytosis for processing and were located in phagolysosomes. Gallium arsenide did not alter the phagocytic ability of macrophages. Peritoneal macrophages normally processed the targeted antigen, indicating that gallium arsenide influenced compartment(s) preceding lysosomes. However, the processing efficiency of exposed splenic macrophages depended on the size of particulate cytochrome, suggesting that processing varied in phagolysosomes of different sizes. Gallium arsenide impacted different intracellular compartments in these macrophages, perhaps contributing to systemic immunotoxicity and local inflammation caused by exposure.

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

    PubMed

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

    2015-02-01

    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.

  4. Cytochrome P450 polymorphism--molecular, metabolic and pharmacogenetic aspects. I. Mechanisms of activity of cytochrome P450 monooxygenases.

    PubMed

    Pachecka, Jan; Tomaszewski, Piotr; Kubiak-Tomaszewska, Grazyna

    2008-01-01

    Cytochrome P450, initially perceived as a type of cell pigment, was soon identified as a hemoprotein with an enzymatic activity characteristic for monooxygenases with an affinity for differentiated endo- or exogenous substrates, including drugs. So far in the human organism 58 CYP isoenzymes belonging to 18 families have been described. Most from the CYP monooxygenases superfamily turned out to be integral elements of hepatocytic reticular monooxygenase complexes which also contain NADPH-dependent cytochrome P450 reductase (CPR). Later investigations indicated the possibility of the participation in electron transport for reticular CYP isoenzymes, alternative NADH-dependent reticular system composed of cytochrome b5 reductase (CBR) and cytochrome b5. The demonstration of the activity of some CYP superfamily isoenzymes not only in hepatocytes but also in many other cells of the human organism, numerous plant and animal tissues and even in cells of fungi, protists and prokaryotes has contributed to the significantly increased understanding of the role of CYP in biological systems. In addition, some CYP isoenzymes were found to be characteristic for the inner mitochondrial membrane monooxygenase complexes which contain NADPH-dependent adrenodoxin reductase (AR) and adrenodoxin (Ad), which is identical with ferredoxin-1 (Fd-1) and hepatoredoxin (Hd).

  5. Cytochrome c551 and the cytochrome c maturation pathway affect virulence gene expression in Bacillus cereus ATCC 14579.

    PubMed

    Han, Hesong; Sullivan, Thomas; Wilson, Adam C

    2015-02-01

    Loss of the cytochrome c maturation system in Bacillus cereus results in increased transcription of the major enterotoxin genes nhe, hbl, and cytK and the virulence regulator plcR. Increased virulence factor production occurs at 37°C under aerobic conditions, similar to previous findings in Bacillus anthracis. Unlike B. anthracis, much of the increased virulence gene expression can be attributed to loss of only c551, one of the two small c-type cytochromes. Additional virulence factor expression occurs with loss of resBC, encoding cytochrome c maturation proteins, independently of the presence of the c-type cytochrome genes. Hemolytic activity of strains missing either cccB or resBC is increased relative to that in the parental strain, while sporulation efficiency is unaffected in the mutants. Increased virulence gene expression in the ΔcccB and ΔresBC mutants occurs only in the presence of an intact plcR gene, indicating that this process is PlcR dependent. These findings suggest a new mode of regulation of B. cereus virulence and reveal intriguing similarities and differences in virulence regulation between B. cereus and B. anthracis.

  6. Binding of ferulic acid to cytochrome c enhances stability of the protein at physiological pH and inhibits cytochrome c-induced apoptosis.

    PubMed

    Yang, Fang; Zhou, Bing-Rui; Zhang, Peng; Zhao, Yan-Feng; Chen, Jie; Liang, Yi

    2007-12-15

    Ferulic acid (FA) is one of the most effective components of a traditional Chinese medicine, angelica, and cytochrome c plays a vital role in apoptosis. Here we report the application of fluorescence spectroscopy, isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and circular dichroism (CD) to investigate the mechanism for the interaction of bovine heart cytochrome c with FA and the effect of the binding on native state stability of the protein at physiological pH. Fluorescence spectroscopic studies together with ITC measurements indicate that FA binds to cytochrome c with moderate affinity and quenches the intrinsic fluorescence of the protein in a static way. ITC experiments show that the interaction of cytochrome c with FA is driven by a moderately favorable entropy increase in combination with a less favorable enthalpy decrease for the first binding site of the protein. The melting temperature of cytochrome c in the presence of FA measured by DSC and CD increases 4.0 and 5.0 degrees C, respectively, compared with that in the absence of FA. Taken together, these results indicate that FA binds to and stabilizes cytochrome c at physiological pH. Furthermore, binding of FA to cytochrome c inhibits cytochrome c-induce apoptosis of human hepatoma cell line SMMC-7721. Our data provide insight into the mechanism of drug-protein interactions, and will be helpful to the understanding of the mechanism for FA-inhibited and cytochrome c-induced apoptosis.

  7. Synthesis and Evaluation of [11C]LY2795050 as a Novel Kappa Opioid Receptor Antagonist Radiotracer for PET Imaging

    PubMed Central

    Zheng, Ming-Qiang; Nabulsi, Nabeel; Kim, Su Jin; Tomasi, Giampaolo; Lin, Shu-fei; Mitch, Charles; Quimby, Steven; Barth, Vanessa; Rash, Karen; Masters, John; Navarro, Antonio; Seest, Eric; Morris, Evan E.; Carson, Richard E.; Huang, Yiyun

    2013-01-01

    is a suitable ligand for imaging the KOR in primates. This newly developed KOR antagonist tracer has since been advanced to PET imaging of KOR in humans and constitutes the first successful KOR antagonist radiotracer. PMID:23353688

  8. [{sup 11}C]d-threo-Methylphenidate, a new radiotracer for the dopamine transporter. Characterization in baboon and human brain

    SciTech Connect

    Ding, Y.S.; Volkow, N.D.; Fowler, J.S.

    1995-05-01

    dl-threo Methylphenidate (MP, Ritalin) is a psychostimulant drug which binds to the dopamine transporter (DAT). We evaluated [{sup 11}C]d-threo-methylphenidate ([{sup 11}C]d-MP), the more active enantiomer, as a radiotracer for the DAT in baboons and human brain. Stereoselectivity, saturability and pharmacological specificity and reproducibility were examined. Stereoselectivity was examined in baboons by comparing [{sup 11C}]d-MP,[{sup 11}C]l-MP and [{sup 11}C]dl-MP. Unlabeled MP was used to assess the reversibility and saturability of the binding. GBR 12909,{beta}-(4-iodophenyl)tropane-2-carboxylic acid methyl ester ({beta}-CIT), tomoxetine and citalopram were used to assess the specificity of the binding. The ratios between the radioactivity in the striatum to that in cerebellum (ST/CB) were 3.3,2.2 and 1.1 for [{sup 11}C]d-MP,[{sup 11}C]dl-MP and [{sup 11}C]l-MP respectively. Most of the striatal binding of [{sup 11}C]d-threo-MP was displaced by injection of nonradioactive MP demonstrating reversibility. Pretreatment with MP (0.5 mg/kg), GBR12909 (1.5 mg/kg) or {beta}-CIT (0.3 mg/kg) reduced ST/CB by about 60% and the ratios of distribution volumes at the steady-state for the triatum to cerebellum (DV{sub st/}DV{sub cb}) by about 50%. Pretreatment with tomoxetine (3.0 mg/kg) or citalopram (2.0 mg/kg), inhibitors of the norepinephrine and serotonin transporter, had no effect. Studies of [{sup 11}C]d-MP in the human brain showed highest uptake in basal ganglia with a half clearance time of about 60 minutes. Repeated studies in 6 normal human subjects showed differences in DV{sub st/}DV{sub cb} between -7% and 8%. MP pretreatment decreased BG but no cortical or cerebellar binding and reduced Bmax/Kd by 91%.

  9. 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

  10. Cytochrome c/cardiolipin relations in mitochondria: a kiss of death

    PubMed Central

    Kagan, Valerian E.; Bayir, Hülya A.; Belikova, Natalia A.; Kapralov, Olexandr; Tyurina, Yulia Y.; Tyurin, Vladimir A.; Jiang, Jianfei; Stoyanovsky, Detcho A.; Wipf, Peter; Kochanek, Patrick M.; Greenberger, Joel S.; Pitt, Bruce; Shvedova, Anna A.; Borisenko, Grigory

    2009-01-01

    Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt c's conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria – its asymmetric trans-membrane distribution and mechanisms of collapse, regulation of its synthesis, remodeling and fatty acid composition – are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with anti-apoptotic effects are presented. PMID:19285551

  11. Production, isolation and characterization of monoclonal antibodies to cytochromes c of beef heart and Paracoccus denitrificans.

    PubMed

    Kuo, L M; Davies, H C

    1983-08-01

    Hybridoma cell lines secreting monoclonal antibodies which bind beef heart cytochrome c or Paracoccus denitrificans cytochrome c have been produced using spleen cells from BALB/c mice immunized with cytochrome c. Immunization was performed with either the native cytochrome c, succinylated hemocyanin-conjugated cytochrome c, or beef heart cytochrome c polymerized with glutaraldehyde. Of 10 such fusions, the hybridization frequency ranged from 0 to 42%. The cell fusion efficiency, the possible factors involved in the cell fusion efficiency and the frequency of antibody producing hybridomas are described. The percentage of hybridomas positive for anti-cytochrome c antibody production as screened for by radioimmunoassay or ELISA was 2%. Of the antibodies from 12 hybridoma cell lines which resulted from 10 fusions, three were specific to beef heart cytochrome c, another three were specific to P. denitrificans cytochrome c, and the remainder reacted with both cytochromes c. These groups of monoclonal antibodies react to different sets of sites on these two cytochromes c. The monoclonal antibodies from ten representative clones have been isolated and characterized by different methods.

  12. Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation

    PubMed Central

    Hildenbeutel, Markus; Hegg, Eric L.; Stephan, Katharina; Gruschke, Steffi; Meunier, Brigitte

    2014-01-01

    Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3–Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b. PMID:24841564

  13. 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

  14. Effect of the hinge protein on the heme iron site of cytochrome c sub 1

    SciTech Connect

    Kim, C.H.; Yencha, A.J.; Bunker, G.; Zhang, G.; Chance, B.; King, T.E. )

    1989-02-21

    X-ray absorption spectroscopic (XAS) studies on cytochrome c{sub 1} from beef heart mitochondria were conducted to identify the effect of the hinge protein on the structure of the heme site in cytochrome c{sub 1}. A comparison of XAS data of highly purified one-band and two-band cytochrome c{sub 1} demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c{sub 1}: a conformational change occurs within a radius of approximately 5 {angstrom} from the heme iron in cytochrome c{sub 1} when the hinge protein is bound to cytochrome c{sub 1}. This result may be correlated with the previous observations that the structure and reactivity of cytochrome c{sub 1} are affected by the hinge protein.

  15. Design, synthesis and evaluation of [(3)H]PF-7191, a highly specific nociceptin opioid peptide (NOP) receptor radiotracer for in vivo receptor occupancy (RO) studies.

    PubMed

    Zhang, Lei; Drummond, Elena; Brodney, Michael A; Cianfrogna, Julie; Drozda, Susan E; Grimwood, Sarah; Vanase-Frawley, Michelle A; Villalobos, Anabella

    2014-11-15

    Herein we report the identification of (+)-N-(2-((1H-pyrazol-1-yl)methyl)-3-((1R,3r,5S)-6'-fluoro-8-azaspiro[bicyclo[3.2.1]octane-3,1'-isochroman]-8-yl)propyl)-N-[(3)H]-methylacetamide {[(3)H]PF-7191 [(+)-11]} as a promising radiotracer for the nociceptin opioid peptide (NOP) receptor. (+)-11 demonstrated high NOP binding affinity (Ki = 0.1 nM), excellent selectivity over other opioid receptors (>1000×) and good brain permeability in rats (C(b,u)/C(p,u) = 0.29). Subsequent characterization of [(3)H](+)-11 showed a high level of specific binding and a brain bio-distribution pattern consistent with known NOP receptor expression. Furthermore, the in vivo brain binding of [(3)H](+)-11 in rats was inhibited by a selective NOP receptor antagonist in a dose-responsive manner. This overall favorable profile indicated that [(3)H](+)-11 is a robust radiotracer for pre-clinical in vivo receptor occupancy (RO) measurements and a possible substrate for carbon-11 labeling for positron emission tomography (PET) imaging in higher species.

  16. Synthesis of Radioisotope Mn-56@SiO2, Sm-153@SiO2, and Dy-165@SiO2 Hybrid Nanoparticles for Use as Radiotracer.

    PubMed

    Seo, Sang-Ei; Kang, Yun Ok; Jung, Sung-Hee; Choi, Seong-Ho

    2015-09-01

    Radioisotope hybrid nanoparticles (NPs) of Mn-56@SiO2, Sm-153@SiO2, and Dy-165@SiO2 were synthesized by neutron irradiation of Mn-55@SiO2, Sm-150@SiO2, and Dy-163@SiO2 NPs respectively using the HANARO research reactor. The Mn-55@SiO2, Sm-150@SiO2, and Dy-163@SiO2 NPs were synthesized by calcination in air flow at 500 degrees C for 8 h of the hybrid NPs that has been prepared by the sol-gel reaction of tetraethyl silicate in the presence of the complex precursors. Mn-55, Sm-150, and Dy-163 were selected for use as radiotracers were selected because these elements can be easily gamma-activated by neutrons (activation limits: 1 picogram (Dy), 1-10 picogram (Mn), 10-100 picogram (Sm)). The successful synthesis of the radioisotope hybrid NPs was confirmed by Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Spectrometry (EDS), Scanning Electron Microscopy (SEM), and Gamma Spectroscopy analysis. The synthesized the radioisotope hybrid NPs could be used as radiotracers in the scientific, environmental, engineering, and industrial fields.

  17. Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli.

    PubMed

    Gao, Tao; O'Brian, Mark R

    2007-09-01

    c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.

  18. 18F-FP-PEG2-β-Glu-RGD2: A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging

    PubMed Central

    Tang, Ganghua; Yao, Shaobo; Yao, Baoguo; Wang, Hongliang; Nie, Dahong; Liang, Xiang; Tang, Caihua; He, Shanzhen

    2015-01-01

    Radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive determination of integrin αvβ3 expression in tumors. In this study, we performed radiosynthesis and biological evaluation of a new 18F-labeled RGD homodimeric peptide with one 8-amino-3,6-dioxaoctanoic acid (PEG2) linker on the glutamate β-amino group (18F-FP-PEG2-β-Glu-RGD2) as a symmetric PET tracer for tumor imaging. Biodistribution studies showed that radioactivity of 18F-FP-PEG2-β-Glu-RGD2 was rapidly cleared from blood by predominately renal excretion. MicroPET-CT imaging with 18F-FP-PEG2-β-Glu-RGD2 revealed high tumor contrast and low background in A549 human lung adenocarcinoma-bearing mouse models, PC-3 prostate cancer-bearing mouse models, and orthotopic transplanted C6 brain glioma models. 18F-FP-PEG2-β-Glu-RGD2 exhibited good stability in vitro and in vivo. The results suggest that this tracer is a potential PET tracer for tumor imaging. PMID:26397833

  19. Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b/sub 5/ and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

    SciTech Connect

    Klasing, S.A.; Mora, M.I.; Wilson, W.C.; Fahey, G.C. Jr.; Garst, J.E.

    1985-09-01

    Experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b/sub 5/, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose, or 6.0% arenaceous flour. Chicks fed 12.0% wood molasses had a higher cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic, had a higher cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.

  20. Protein camouflage in cytochrome c-calixarene complexes

    NASA Astrophysics Data System (ADS)

    McGovern, Róise E.; Fernandes, Humberto; Khan, Amir R.; Power, Nicholas P.; Crowley, Peter B.

    2012-07-01

    Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.

  1. Redox processes controlling the biogenesis of c-type cytochromes.

    PubMed

    Bonnard, Géraldine; Corvest, Vincent; Meyer, Etienne H; Hamel, Patrice P

    2010-11-01

    In mitochondria, two mono heme c-type cytochromes are essential electron shuttles of the respiratory chain. They are characterized by the covalent attachment of their heme C to a CXXCH motif in the apoproteins. This post-translational modification occurs in the intermembrane space compartment. Dedicated assembly pathways have evolved to achieve this chemical reaction that requires a strict reducing environment. In mitochondria, two unrelated machineries operate, the rather simple System III in yeast and animals and System I in plants and some protozoans. System I is also found in bacteria and shares some common features with System II that operates in bacteria and plastids. This review aims at presenting how different systems control the chemical requirements for the heme ligation in the compartments where cytochrome c maturation takes place. A special emphasis will be given on the redox processes that are required for the heme attachment reaction onto apocytochromes c.

  2. Structural Changes and Proton Transfer in Cytochrome c Oxidase.

    PubMed

    Vilhjálmsdóttir, Jóhanna; Johansson, Ann-Louise; Brzezinski, Peter

    2015-08-27

    In cytochrome c oxidase electron transfer from cytochrome c to O2 is linked to transmembrane proton pumping, which contributes to maintaining a proton electrochemical gradient across the membrane. The mechanism by which cytochrome c oxidase couples the exergonic electron transfer to the endergonic proton translocation is not known, but it presumably involves local structural changes that control the alternating proton access to the two sides of the membrane. Such redox-induced structural changes have been observed in X-ray crystallographic studies at residues 423-425 (in the R. sphaeroides oxidase), located near heme a. The aim of the present study is to investigate the functional effects of these structural changes on reaction steps associated with proton pumping. Residue Ser425 was modified using site-directed mutagenesis and time-resolved spectroscopy was used to investigate coupled electron-proton transfer upon reaction of the oxidase with O2. The data indicate that the structural change at position 425 propagates to the D proton pathway, which suggests a link between redox changes at heme a and modulation of intramolecular proton-transfer rates.

  3. Cytochrome C Biosensor—A Model for Gas Sensing

    PubMed Central

    Hulko, Michael; Hospach, Ingeborg; Krasteva, Nadejda; Nelles, Gabriele

    2011-01-01

    This work is about gas biosensing with a cytochrome c biosensor. Emphasis is put on the analysis of the sensing process and a mathematical model to make predictions about the biosensor response. Reliable predictions about biosensor responses can provide valuable information and facilitate biosensor development, particularly at an early development stage. The sensing process comprises several individual steps, such as phase partition equilibrium, intermediate reactions, mass-transport, and reaction kinetics, which take place in and between the gas and liquid phases. A quantitative description of each step was worked out and finally combined into a mathematical model. The applicability of the model was demonstrated for a particular example of methanethiol gas detection by a cytochrome c biosensor. The model allowed us to predict the optical readout response of the biosensor from tabulated data and data obtained in simple liquid phase experiments. The prediction was experimentally verified with a planar three-electrode electro-optical cytochrome c biosensor in contact with methanethiol gas in a gas tight spectroelectrochemical measurement cell. PMID:22163937

  4. Sulfide inhibition of and metabolism by cytochrome c oxidase.

    PubMed

    Nicholls, Peter; Marshall, Doug C; Cooper, Chris E; Wilson, Mike T

    2013-10-01

    Hydrogen sulfide (H2S), a classic cytochrome c oxidase inhibitor, is also an in vitro oxidase substrate and an in vivo candidate hormonal ('gasotransmitter') species affecting sleep and hibernation. H2S, nitric oxide (NO) and carbon monoxide (CO) share some common features. All are low-molecular-mass physiological effectors and also oxidase inhibitors, capable of binding more than one enzyme site, and each is an oxidizable 'substrate'. The oxidase oxidizes CO to CO2, NO to nitrite and sulfide to probable persulfide species. Mitochondrial cytochrome c oxidase in an aerobic steady state with ascorbate and cytochrome c is rapidly inhibited by sulfide in a biphasic manner. At least two successive inhibited species are involved, probably partially reduced. The oxidized enzyme, in the absence of turnover, occurs in at least two forms: the 'pulsed' and 'resting' states. The pulsed form reacts aerobically with sulfide to form two intermediates, 'P' and 'F', otherwise involved in the reaction of oxygen with reduced enzyme. Sulfide can directly reduce the oxygen-reactive a3CuB binuclear centre in the pulsed state. The resting enzyme does not undergo such a step, but only a very slow one-electron reduction of the electron-transferring haem a. In final reactivation phases, both the steady-state inhibition of catalysis and the accumulation of P and F states are reversed by slow sulfide oxidation. A model for this complex reaction pattern is presented. PMID:24059525

  5. Using Cytochrome c{sub 3} to Make Selenium Nanowires

    SciTech Connect

    ABDELOUAS,A.; FRANCO,R.; GONG,W.L.; LUTZE,W.; MOURA,I.; SHELNUTT,JOHN A.

    1999-11-24

    We report on a new method to make nanostructures, in this case selenium nanowires, in aqueous solution at room temperature. We used the protein cytochrome c{sub 3} to reduce selenate (SeO{sub 4}{sup 2{minus}}) to selenium (Se{sup 0}). Cytochrome c{sub 3} is known for its ability to catalyze reduction of metals including U{sup VI} {yields} U{sup IV}, Cr{sup VI} {yields} Cr{sup III}, Mo{sup VI} {yields} Mo{sup IV}, Cu{sup II} {yields} Cu{sup 0}, Pb{sup II} {yields} Pb{sup 0}, Hg{sup II} {yields} Hg{sup 0}. Nanoparticles of Se{sup 0} precipitated from an aqueous solution at room temperature, followed by spontaneous self-assembling into nanowires. Cytochrome c{sub 3} was extracted from the sulfate-reducing bacteria Desulfovibrio vulgaris (strain Holdenborough) and isolated by the procedure of DerVartanian and Legall.

  6. Enhanced expression of cytochrome P450 in stomach cancer.

    PubMed Central

    Murray, G. I.; Taylor, M. C.; Burke, M. D.; Melvin, W. T.

    1998-01-01

    The cytochromes P450 have a central role in the oxidative activation and detoxification of a wide range of xenobiotics, including many carcinogens and several anti-cancer drugs. Thus the cytochrome P450 enzyme system has important roles in both tumour development and influencing the response of tumours to chemotherapy. Stomach cancer is one of the commonest tumours of the alimentary tract and environmental factors, including dietary factors, have been implicated in the development of this tumour. This type of tumour has a poor prognosis and responds poorly to current therapies. In this study, the presence and cellular localization of several major forms of P450, CYP1A, CYP2E1 and CYP3A have been investigated in stomach cancer and compared with their expression in normal stomach. There was enhanced expression of CYP1A and CYP3A in stomach cancer with CYP1A present in 51% and CYP3A present in 28% of cases. In contrast, no P450 was identified in normal stomach. The presence of CYP1A and CYP3A in stomach cancer provides further evidence for the enhanced expression of specific forms of cytochrome P450 in tumours and may be important therapeutically for the development of anti-cancer drugs that are activated by these forms of P450. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9569036

  7. Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

    SciTech Connect

    Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

    1986-04-01

    In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with (/sup 14/C)-5-aminolevulinic acid (/sup 14/C)-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H/sub 2/O/sub 2/ stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with (/sup 14/C)-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed.

  8. Evolution of the primate cytochrome c oxidase subunit II gene.

    PubMed

    Adkins, R M; Honeycutt, R L

    1994-03-01

    We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. PMID:8006990

  9. Rational redesign of the biodegradative enzyme cytochrome P450 cam:

    SciTech Connect

    Ornstein, R.; Paulsen, M.; Bass, M.; Arnold, G.

    1991-03-01

    Cytochromes P450, a superfamily of monooxygenase enzymes present in all kingdoms of living organisms, are very versatile with respect to substrate range and catalytic functionality. Many recalcitrant halogenated hydrocarbons, on DOE sites and throughout the nation, result in serious environmental impact. Cytochromes P450 have been shown to be catalytically capable of, at least partial, dehalogenation of some such compounds. Clearly, however, their active site stereochemistry and related functional components are not well suited for this role because the rates of dehalogenation are generally rather modest. The evolution of modified active site and access channel structures may proceed very slowly if multiple genetic changes are simultaneously required for enzyme adaptation. Since each mutational event is by itself a rare event, a basic premise of our research is that designing multiple changes into an enzyme may be more timely than waiting for them to occur biologically either via natural selection or under laboratory-controlled conditions. Starting with available high-resolution x-ray crystal structures, molecular modeling and molecular dynamics simulations have been used to probe the basic structure/function principles and conformational fluctuations of the biodegradative enzyme, cytochrome P450cam (camphor hydroxylase from Pseudomonas putida) and active site mutants, to provide the fundamental understanding necessary for rational engineering of the enzyme for modified substrate specificity. In the present paper, we review our progress to data, in the area of molecular dynamics simulations and active site redesign of P450cam. 36 refs., 2 figs.

  10. Cytochrome c(2) Exit Strategy: Dissociation Studies and Evolutionary Implications.

    PubMed

    Pogorelov, Taras V; Autenrieth, Felix; Roberts, Elijah; Luthey-Schulten, Zaida A

    2007-01-25

    Small, water-soluble, type c cytochromes form a transient network connecting major bioenergetic membrane protein complexes in both photosynthesis and respiration. In the photosynthesis cycle of Rhodobacter sphaeroides, cytochrome c2 (cyt c2) docks to the reaction center (RC), undergoes electron transfer, and exits for the cytochrome bc1 complex. Translations of cyt c2 about the RC-cyt c2 docking interface and surrounding membrane reveal possible exit pathways. A pathway at a minimal elevation allowed by the architecture of the RC is analyzed using both an all-atom steered molecular dynamics simulation of the RC-cyt c2 complex and a bioinformatic analysis of the structures and sequences of cyt c. The structure-based phylogenetic analysis allows for the identification of structural elements that have evolved to satisfy the requirements of having multiple functional partners. The patterns of evolutionary variation obtained from the phylogenetic analysis of both docking partners of cyt c2 reveal conservation of key residues involved in the interaction interfaces that would be candidates for further experimental studies. Additionally, using the molecular mechanics Poisson-Boltzmann surface area method we calculate that the binding free energy of reduced cyt c2 to the RC is nearly 6 kcal/mol more favorable than with oxidized cyt c2. The redox-dependent variations lead to changes in structural flexibility, behavior of the interfacial water molecules, and eventually changes in the binding free energy of the complex. PMID:17228920

  11. Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts.

    PubMed Central

    Hampton, M B; Zhivotovsky, B; Slater, A F; Burgess, D H; Orrenius, S

    1998-01-01

    The export of cytochrome c from mitochondria to the cytoplasm has been detected during apoptosis. Addition of cytochrome c to cytosolic extracts can activate the caspases, suggesting that this export could be an important intracellular signal for initiating the apoptotic programme. We have investigated the mechanism of caspase activation by cytochrome c. Mitochondrial cytochrome c normally shuttles electrons between complexes III and IV of the electron transport chain. Interaction with these complexes is dependent on electrostatic interactions via a polylysine binding pocket. Cytosolic caspase activation was only observed with intact holocytochrome c, and increasing the ionic composition of the extracts prevented activation, suggesting that stringent allosteric interactions between cytochrome c and other cytoplasmic factors are necessary. Cytochrome c was fully reduced within 5 min of addition to the cytosolic extracts. Potassium ferricyanide could maintain cytochrome c in an oxidized state, but care was taken to use ferricyanide at concentrations where its polyanion effect did not cause interference. The oxidized form of cytochrome c was able to activate the caspases. We conclude that reduced cytochrome c will function in the cytoplasm; however, its reduction is not a critical step, and electron transfer from cytochrome c to its cytoplasmic-binding partner(s) is not necessary in the pathway leading to apoptosis. PMID:9405280

  12. Determining the IC50 Values for Vorozole and Letrozole, on a Series of Human Liver Cytochrome P450s, to Help Determine the Binding Site of Vorozole in the Liver

    PubMed Central

    Raymond, Lendelle; Rayani, Nikita; Polson, Grace; Sikorski, Kylie; Lian, Ailin; VanAlstine-Parris, Melissa A.

    2015-01-01

    Vorozole and letrozole are third-generation aromatase (cytochrome P450 19A1) inhibitors. [11C]-Vorozole can be used as a radiotracer for aromatase in living animals but when administered by IV, it collects in the liver. Pretreatment with letrozole does not affect the binding of vorozole in the liver. In search of finding the protein responsible for the accumulation of vorozole in the liver, fluorometric high-throughput screening assays were used to test the inhibitory capability of vorozole and letrozole on a series of liver cytochrome P450s (CYP1A1, CYP1A2, CYP2A6, and CYP3A4). It was determined that vorozole is a potent inhibitor of CYP1A1 (IC50 = 0.469 μM) and a moderate inhibitor of CYP2A6 and CYP3A4 (IC50 = 24.4 and 98.1 μM, resp.). Letrozole is only a moderate inhibitor of CYP1A1 and CYP2A6 (IC50 = 69.8 and 106 μM) and a very weak inhibitor of CYP3A4 (<10% inhibition at 1 mM). Since CYP3A4 makes up the majority of the CYP content found in the human liver, and vorozole inhibits it moderately well but letrozole does not, CYP3A4 is a good candidate for the protein that [11C]-vorozole is binding to in the liver. PMID:26635974

  13. Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis.

    PubMed

    Knaff, D B; Willie, A; Long, J E; Kriauciunas, A; Durham, B; Millett, F

    1991-02-01

    The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Downstream targets of WRKY33.

    PubMed

    Petersen, Klaus; Fiil, Berthe Katrine; Mundy, John; Petersen, Morten

    2008-11-01

    Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. In a recent publication we show that MPK4 and its substrate MKS1 interact with WRKY33 in vivo, and that WRKY33 is released from complexes with MPK4 upon infection. Transcriptome analysis of a wrky33 loss-of-function mutant identified a subset of defense-related genes as putative targets of WRKY33. These genes include PAD3 and CYP71A13, which encode cytochrome P450 monoxygenases required for synthesis of the antimicrobial phytoalexin camalexin. Chromatin immunoprecipitation confirmed that WRKY33 bound the promoter of PAD3 when plants were inoculated with pathogens. Here we further discuss the involvement of two other targets of WRKY33, NUDT6 and ROF2 in defense responses against invading pathogens.

  15. In-silico Assessment of Protein-Protein Electron Transfer. A Case Study: Cytochrome c Peroxidase – Cytochrome c

    PubMed Central

    Wallrapp, Frank H.; Voityuk, Alexander A.; Guallar, Victor

    2013-01-01

    The fast development of software and hardware is notably helping in closing the gap between macroscopic and microscopic data. Using a novel theoretical strategy combining molecular dynamics simulations, conformational clustering, ab-initio quantum mechanics and electronic coupling calculations, we show how computational methodologies are mature enough to provide accurate atomistic details into the mechanism of electron transfer (ET) processes in complex protein systems, known to be a significant challenge. We performed a quantitative study of the ET between Cytochrome c Peroxidase and its redox partner Cytochrome c. Our results confirm the ET mechanism as hole transfer (HT) through residues Ala194, Ala193, Gly192 and Trp191 of CcP. Furthermore, our findings indicate the fine evolution of the enzyme to approach an elevated turnover rate of 5.47×106 s−1 for the ET between Cytc and CcP through establishment of a localized bridge state in Trp191. PMID:23555224

  16. Targets and targeting.

    PubMed

    Will, E

    2001-08-01

    Using the vocabulary of ballistics in medicine for emphasis can result in misleading exaggeration and semantic confusion. The dual meaning of target as either aiming point (aim at) or outcome (aim to achieve) creates a muddle in the efforts to comply with quality assurance initiatives. Disentangling the two meanings allows new approaches to the clinical technology required in a modern health care environment. An example can be shown in new strategies for the management of renal anemia with iron and erythropoietin. The potential to shape outcome distributions through validated, preemptive intervention thresholds offers the predictable results required by patients and payers. Using the management of patient cohorts as a platform for outcomes creates no necessary conflict with individualized clinical care. Future guideline statements should include the likely characteristics of compliant outcome populations, as a prompt to clinical goals and as an indication of the necessary cost and effort of compliance with treatment standards. Overemphasis in language is no substitute for considered clinical methodology.

  17. Inducing effect of oxfendazole on cytochrome P450IA2 in rabbit liver. Consequences on cytochrome P450 dependent monooxygenases.

    PubMed

    Gleizes, C; Eeckhoutte, C; Pineau, T; Alvinerie, M; Galtier, P

    1991-06-15

    Male New Zealand rabbits were dosed with either 0.9, 4.5 or 22.5 mg/kg/day of oxfendazole by gastric intubation for 10 days. Oxfendazole administered at the therapeutic dose (4.5 mg/kg) and at the highest dose (22.5 mg/kg) increased 1.54- and 2.36-fold the total liver microsomal cytochrome P450 and more particularly the isoenzyme P450IA2 (95 and 184% increases) as demonstrated by western blotting. Increases in ethoxyresorufin O-deethylation and hydroxylations of benzopyrene and acetanilide occurred in livers of the same animals without any change in N-demethylation of aminopyrine, benzphetamine or erythromycin. Because of the unchanged level of mRNA specific to cytochrome P450IA2, as shown by northern blot analysis of poly mRNA, an enzyme stabilization rather than a transcriptional activation of IA2 genes should be involved in the P450IA2 regulation mechanisms. Oxfendazole bound strongly to cytochrome P450, giving rise to a type II spectrum, and inhibited noncompetitively the ethoxyresorufin O-deethylase and acetanilide hydroxylase activities, this confirmed that oxfendazole interacts only with the P450IA2 family. On the basis of a comparison of the enzymatic activities induced by various imidazole drugs, it was concluded that oxfendazole, like omeprazole and albendazole, behaved as a 3-methylcholanthrene-type inducer. These three benzimidazoles did not all belong to the same category of cytochrome P450 inducers as the antifungal drugs miconazole, clotrimazole and ketoconazole.

  18. Cytochrome c biogenesis in Campylobacter jejuni requires cytochrome c6 (CccA; Cj1153) to maintain apocytochrome cysteine thiols in a reduced state for haem attachment.

    PubMed

    Liu, Yang-Wei; Kelly, David J

    2015-06-01

    The microaerophilic food-borne pathogen Campylobacter jejuni uses complex cytochrome-rich respiratory chains for growth and host colonisation. Cytochrome c biogenesis requires haem ligation to reduced apocytochrome cysteines, catalysed by the cytochrome c synthase, CcsBA. While ccsBA could not be deleted, we showed that the thiol reductase DsbD and the CcsX homologue Cj1207 are involved in, but not essential for, cytochromes c biogenesis. Mutant phenotypic analyses and biochemical studies with purified proteins revealed that the mono-haem c-type cytochromes Cj1153 (CccA) and Cj1020 (CccB) and the di-haem Cj0037 (CccC) are electron donors to the cb-oxidase (CcoNOQP), with CccC being more efficient than CccA. Remarkably, cccA deletion or site-directed mutagenesis resulted in an almost complete loss of all other c-type cytochromes. Cytochrome c structural and biogenesis genes were still transcribed in the cccA deletion mutant and the quinol oxidase genes (cioAB) were up-regulated. Cytochrome c production could be rescued in this mutant by growth with exogenous dithiothreitol or L-cysteine, suggesting that in the absence of CccA, apocytochrome c haem binding motifs become oxidised, preventing haem attachment. Our results identify CccA, the most abundant periplasmic c-type cytochrome in C. jejuni, as a novel and unexpected protein required for cytochrome c biogenesis in this pathogen.

  19. Model studies using supercritical carbon dioxide fluid (SF CO{sub 2}) as a reaction medium for radiotracer synthesis and purification

    SciTech Connect

    Ferrieri, R.A.; Fowler, J.S.; Wolf, A.P.

    1994-05-01

    Supercritical fluids (SFs) have found widespread use in the analytical field as solvents for compound purification, and initial results on their use for radiotracer synthesis have been reported. SF`s possess the unique feature that their solvating strength can be altered drastically through small changes in pressure and temperature of the fluid within the supercritical regime. We have modified a SF chromatograph to allow us to investigate its use in radiotracer synthesis and purification. The solubility of several PET radiotracers was measured in SF CO{sub 2} at 5000 psi and 55{degrees}C and showed the following: raclopride, 68 {mu}g/mL{sup 2}; (L)-deprenyl, 85 {mu}g/mL; flumazenil, 61 {mu}g/mL; (-)cocaine, 108 {mu}g/mL; ritalin, 45 {mu}g/Ml; and cogentin, 250 {mu}g/mL. Analytical separations were achieved on 30 to 50 {mu}g amounts of (L)-deprenyl (3.9 min RT) and nor-deprenyl (4.7 min RT), as well as raclopride (10.8 min RT) and nor-raclopride (10.3 min RT) using 250 mm x 4.5 mm i.d. Ultracarb 5 ODS (30), and 75 mm x 4.5 mm i.d. silica columns, respectively, and pure SF CO{sub 2} as the mobile phase. Model studies on simple N-alkylation reactions were also carried out using pur SF CO{sub 2} as the reaction medium on a modified alumina support. (L)-Deprenyl was synthesized from only 100 {mu}g of the starting labelling substrate using 500 mg of alumina impregnated with triphenylphosphine diiodide (20% by wt.) and maintained at 170{degrees}C. The methylating agent, methyl iodide, was generated in situ from methanol, but was always present in excess of the substrate. Studies are in progress to reduce methanol amounts. Pressure studies of SF CO{sub 2} ranging from 3000 to 6000 psi showed an 80% increase in the methylation reaction relative to the amount of starting substrate suggesting an effect of the fluid density. Temperature was also a critical parameter here as the reaction did not proceed at 80{degrees}C for similiar pressures.

  20. Cox26 is a novel stoichiometric subunit of the yeast cytochrome c oxidase.

    PubMed

    Levchenko, Maria; Wuttke, Jan-Moritz; Römpler, Katharina; Schmidt, Bernhard; Neifer, Klaus; Juris, Lisa; Wissel, Mirjam; Rehling, Peter; Deckers, Markus

    2016-07-01

    The cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain. The complex accepts electrons from cytochrome c and passes them onto molecular oxygen. This process contributes to energy capture in the form of a membrane potential across the inner membrane. The enzyme complex assembles in a stepwise process from the three mitochondria-encoded core subunits Cox1, Cox2 and Cox3, which associate with nuclear-encoded subunits and cofactors. In the yeast Saccharomyces cerevisiae, the cytochrome c oxidase associates with the bc1-complex into supercomplexes, allowing efficient energy transduction. Here we report on Cox26 as a protein found in respiratory chain supercomplexes containing cytochrome c oxidase. Our analyses reveal Cox26 as a novel stoichiometric structural subunit of the cytochrome c oxidase. A loss of Cox26 affects cytochrome c oxidase activity and respirasome organization.

  1. The structure of an electron transfer complex containing a cytochrome c and a peroxidase.

    PubMed

    Pettigrew, G W; Prazeres, S; Costa, C; Palma, N; Krippahl, L; Moura, I; Moura, J J

    1999-04-16

    Efficient biological electron transfer may require a fluid association of redox partners. Two noncrystallographic methods (a new molecular docking program and 1H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) of Paracoccus denitrificans and cytochromes c. For the natural redox partner, cytochrome c550, the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase. In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase. These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility. PMID:10196231

  2. Participation of NADPH-cytochrome C reductase in thyroid hormone biosynthesis.

    PubMed

    Yamamoto, K; DeGroot, L J

    1975-04-01

    Purified rat liver NADPH-cytochrome c reductase supports iodination of tyrosine in a system including NADPH, cytochrome c and thyroid perioxidase. Catalase inhibits the iodination of tyrosine, while superoxide dismutase has no effect. Antibody developed in the rabbit against purified rat liver NADPH-cytochrome c reductase inhibits both reduction of cytochrome c and tyrosine iodination supported by the enzyme. The antibody forms a single precipitation line with thyroid extract, and inhibits NADPH cytochrome c reductase activity of the thyroid. The antibody partially inhibits iodination in a thyroid mitochondrial-microsomal fraction, but does not inhibit NADH-dependent iodination. The immunochemical studies indicate the participation of NADPH-cytochrome c reductase in thyroidal H2O generation, and the independent existence of NADPH-dependent and NADH-dependent H2O2 generation mechanisms in the thyroid. PMID:235416

  3. Inappropriate Intra-cervical Injection of Radiotracer for Sentinel Lymph Node Mapping in a Uterine Cervix Cancer Patient: Importance of Lymphoscintigraphy and Blue Dye Injection.

    PubMed

    Kadkhodayan, Sima; Farahabadi, Elham Hosseini; Yousefi, Zohreh; Hasanzadeh, Malihe; Sadeghi, Ramin

    2014-01-01

    Herein, we report a case of sentinel lymph node mapping in a uterine cervix cancer patient, referring to the nuclear medicine department of our institute. Lymphoscintigraphy images showed inappropriate intra-cervical injection of radiotracer. Blue dye technique was applied for sentinel lymph node mapping, using intra-cervical injection of methylene blue. Two blue/cold sentinel lymph nodes, with no pathological involvement, were intra-operatively identified, and the patient was spared pelvic lymph node dissection. The present case underscores the importance of lymphoscintigraphy imaging in sentinel lymph node mapping and demonstrates the added value of blue dye injection in selected patients. It is suggested that pre-operative lymphoscintigraphy imaging be considered as an integral part of sentinel lymph node mapping in surgical oncology. Detailed results of lymphoscintigraphy images should be provided for surgeons prior to surgery, and in case the sentinel lymph nodes are not visualized, use of blue dye for sentinel node mapping should be encouraged.

  4. Isolation of a Rhizobium phaseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation.

    PubMed Central

    Soberón, M; Williams, H D; Poole, R K; Escamilla, E

    1989-01-01

    Cultured cells of a Rhizobium phaseoli wild-type strain (CE2) possess b-type and c-type cytochromes and two terminal oxidases: cytochromes o and aa3. Cytochrome aa3 was partially expressed when CE2 cells were grown on minimal medium, during symbiosis, and in well-aerated liquid cultures in a complex medium (PY2). Two cytochrome mutants of R. phaseoli were obtained and characterized. A Tn5-mob-induced mutant, CFN4201, expressed diminished amounts of b-type and c-type cytochromes, showed an enhanced expression of cytochrome oxidases, and had reduced levels of N,N,N',N'-tetramethyl-p-phenylenediamine, succinate, and NADH oxidase activities. Nodules formed by this strain had no N2 fixation activity. The other mutant, CFN4205, which was isolated by nitrosoguanidine mutagenesis, had reduced levels of cytochrome o and higher succinate oxidase activity but similar NADH and N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activities when compared with the wild-type strain. Strain CFN4205 expressed a fourfold-higher cytochrome aa3 content when cultured on minimal and complex media and had twofold-higher cytochrome aa3 levels during symbiosis when compared with the wild-type strain. Nodules formed by strain CFN4205 fixed 33% more N2 than did nodules formed by the wild-type strain, as judged by the total nitrogen content found in plants nodulated by these strains. Finally, low-temperature photodissociation spectra of whole cells from strains CE2 and CFN4205 reveal cytochromes o and aa3. Both cytochromes react with O2 at -180 degrees C to give a light-insensitive compound. These experiments identify cytochromes o and aa3 as functional terminal oxidases in R. phaseoli. PMID:2644201

  5. Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

    PubMed Central

    Kelly, Steven L.; Kelly, Diane E.

    2013-01-01

    The first eukaryote genome revealed three yeast cytochromes P450 (CYPs), hence the subsequent realization that some microbial fungal genomes encode these proteins in 1 per cent or more of all genes (greater than 100) has been surprising. They are unique biocatalysts undertaking a wide array of stereo- and regio-specific reactions and so hold promise in many applications. Based on ancestral activities that included 14α-demethylation during sterol biosynthesis, it is now seen that CYPs are part of the genes and metabolism of most eukaryotes. In contrast, Archaea and Eubacteria often do not contain CYPs, while those that do are frequently interesting as producers of natural products undertaking their oxidative tailoring. Apart from roles in primary and secondary metabolism, microbial CYPs are actual/potential targets of drugs/agrochemicals and CYP51 in sterol biosynthesis is exhibiting evolution to resistance in the clinic and the field. Other CYP applications include the first industrial biotransformation for corticosteroid production in the 1950s, the diversion into penicillin synthesis in early mutations in fungal strain improvement and bioremediation using bacteria and fungi. The vast untapped resource of orphan CYPs in numerous genomes is being probed and new methods for discovering function and for discovering desired activities are being investigated. PMID:23297358

  6. Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization.

    PubMed Central

    Miles, J S; Munro, A W; Rospendowski, B N; Smith, W E; McKnight, J; Thomson, A J

    1992-01-01

    1. The gene CYP102 encoding cytochrome P-450 BM-3 and subgenes encoding the cytochrome P-450 and cytochrome P-450 reductase domains have been cloned in Escherichia coli. 2. The protein products of these genes have been overexpressed and purified to homogeneity. 3. The cytochrome P-450 domain is purified in the ferric low-spin state, but is readily converted into the high-spin state by addition of the substrate palmitate (Ks = 1 microM). The cytochrome P-450 reductase domain readily reduces cytochrome c. Mixing the two domains reconstitutes only about one-thousandth of the fatty acid hydroxylase activity associated with the intact cytochrome P-450 BM-3. 4. The X-band e.p.r. spectra of both the cytochrome P-450 domain and intact cytochrome P-450 BM-3 give g-values indicating low-spin ferric haem. The spectra are virtually identical with those of the equivalent form of cytochrome P-450 cam indicating that the haem ligation in cytochrome P-450 BM-3 is identical with that of cytochrome P-450 cam. 5. Resonance Raman spectra of the substrate-free and substrate-bound forms of the cytochrome P-450 domain are given. Spectral differences in comparison with cytochrome P-450 cam may reflect subtle electronic differences between the respective haem environments. Images Fig. 1. PMID:1334408

  7. Structural characterization of a monoclonal antibody immunopurified pulmonary cytochrome P-450 from 3-methylcholanthrenetreated rats

    SciTech Connect

    Robinson, R.C.; Cheng, K.C.; Park, S.S.; Gelboin, H.V.; Friedman, F.K.

    1986-05-01

    Extrahepatic cytochromes P-450 have not been as extensively studied as the hepatic forms, owing to the low concentrations of these enzymes in extrahepatic tissues. A cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to the major MC-inducible form of rat liver cytochrome P-450. The lung cytochrome P-450 is related to this liver form by at least two common epitopes, recognized by monoclonal antibodies 1-7-1 and 1-31-2. The isolated pulmonary cytochrome P-450 is MC-inducible and has an apparent molecular weight of 57 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight as well as the NH/sub 2/-terminal sequence of the pulmonary cytochrome P-450 is identical to that of the major MC-inducible form of rat liver cytochrome P-450. In addition, limited proteolytic digestion of both cytochromes P-450 generates the same peptide patterns on SDS-PAGE. By several criteria, treatment of rats with MC thus induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme.

  8. Cytochrome c release from mitochondria proceeds by a two-step process

    PubMed Central

    Ott, Martin; Robertson, John D.; Gogvadze, Vladimir; Zhivotovsky, Boris; Orrenius, Sten

    2002-01-01

    Cytochrome c is often released from mitochondria during the early stages of apoptosis, although the precise mechanisms regulating this event remain unclear. In this study, with isolated liver mitochondria, we demonstrate that cytochrome c release requires a two-step process. Because cytochrome c is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin, this interaction must first be disrupted to generate a soluble pool of this protein. Specifically, solubilization of cytochrome c involves a breaching of the electrostatic and/or hydrophobic affiliations that this protein usually maintains with cardiolipin. Once cytochrome c is solubilized, permeabilization of the outer mitochondrial membrane by Bax is sufficient to allow the extrusion of this protein into the extramitochondrial environment. Neither disrupting the interaction of cytochrome c with cardiolipin, nor permeabilizing the outer membrane with Bax, alone, is sufficient to trigger this protein's release. This mechanism also extends to conditions of mitochondrial permeability transition insofar as cytochrome c release is significantly depressed when the electrostatic interaction between cytochrome c and cardiolipin remains intact. Our results indicate that the release of cytochrome c involves a distinct two-step process that is undermined when either step is compromised. PMID:11818574

  9. Kinetics of the photoreduction of cytochrome b-559 by photosystem II in chloroplasts.

    PubMed

    Whitmarsh, J; Cramer, W A

    1977-05-11

    The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e, hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100 +/- 10 ms, compared to 6-10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a t 1/2 = 25-35 ms. The addition of uncouplers, which caused stimulatory effect on ferricyanide reduction under the same experimental conditions resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems.

  10. Reduction of U(VI) and Toxic Metals by Desulfovibrio Cytochrome c3

    SciTech Connect

    Wall, Judy D.

    2003-06-01

    The project, ''Reduction of U(VI) and toxic metals by Desulfovibrio cytochrome c3'', is designed to obtain spectroscopic information for or against a functional interaction of cytochrome c3 and uranium in the whole cells. That is, is the cytochrome c3 the uranium reductase? Our approach has been to start with purified cytochrome and determine any unique spectral disturbances during electron flow to U(VI). Then we will attempt to identify these signals emanating from cells actively reducing uranium. This project is being carried out in collaboration with Dr. William Woodruff at the Los Alamos National Laboratory where the spectral experiments are being carried out.

  11. Fusing two cytochromes b of Rhodobacter capsulatus cytochrome bc1 using various linkers defines a set of protein templates for asymmetric mutagenesis.

    PubMed

    Czapla, Monika; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur

    2012-01-01

    Cytochrome bc(1) (mitochondrial complex III), one of the key enzymes of biological energy conversion, is a functional homodimer in which each monomer contains three catalytic subunits: cytochrome c(1), the iron-sulfur subunit and cytochrome b. The latter is composed of eight transmembrane α-helices which, in duplicate, form a hydrophobic core of a dimer. We show that two cytochromes b can be fused into one 16-helical subunit using a number of different peptide linkers that vary in length but all connect the C-terminus of one cytochrome with the N-terminus of the other. The fusion proteins replace two cytochromes b in the dimer defining a set of available protein templates for introducing mutations that allow breaking symmetry of a dimer. A more detailed comparison of the form with the shortest, 3 amino acid, linker to the form with 12 amino acid linker established that both forms display similar level of structural plasticity to accommodate several, but not all, asymmetric patterns of mutations that knock out individual segments of cofactor chains. While the system based on a fused gene does not allow for the assessments of the functionality of electron-transfer paths in vivo, the family of proteins with fused cytochrome b offers attractive model for detailed investigations of molecular mechanism of catalysis at in vitro/reconstitution level.

  12. Folding of horse cytochrome c in the reduced state.

    PubMed

    Bhuyan, A K; Udgaonkar, J B

    2001-10-01

    Equilibrium and kinetic folding studies of horse cytochrome c in the reduced state have been carried out under strictly anaerobic conditions at neutral pH, 10 degrees C, in the entire range of aqueous solubility of guanidinium hydrochloride (GdnHCl). Equilibrium unfolding transitions observed by Soret heme absorbance, excitation energy transfer from the lone tryptophan residue to the ferrous heme, and far-UV circular dichroism (CD) are all biphasic and superimposable, implying no accumulation of structural intermediates. The thermodynamic parameters obtained by two-state analysis of these transitions yielded DeltaG(H2O)=18.8(+/-1.45) kcal mol(-1), and C(m)=5.1(+/-0.15) M GdnHCl, indicating unusual stability of reduced cytochrome c. These results have been used in conjunction with the redox potential of native cytochrome c and the known stability of oxidized cytochrome c to estimate a value of -164 mV as the redox potential of the unfolded protein. Stopped-flow kinetics of folding and unfolding have been recorded by Soret heme absorbance, and tryptophan fluorescence as observables. The refolding kinetics are monophasic in the transition region, but become biphasic as moderate to strongly native-like conditions are approached. There also is a burst folding reaction unobservable in the stopped-flow time window. Analyses of the two observable rates and their amplitudes indicate that the faster of the two rates corresponds to apparent two-state folding (U<-->N) of 80-90 % of unfolded molecules with a time constant in the range 190-550 micros estimated by linear extrapolation and model calculations. The remaining 10-20 % of the population folds to an off-pathway intermediate, I, which is required to unfold first to the initial unfolded state, U, in order to refold correctly to the native state, N (I<-->U<-->N). The slower of the two observable rates, which has a positive slope in the linear functional dependence on the denaturant concentration indicating that an

  13. Change of the terminal oxidase from cytochrome a1 in shaking cultures to cytochrome o in static cultures of Acetobacter aceti.

    PubMed

    Matsushita, K; Ebisuya, H; Ameyama, M; Adachi, O

    1992-01-01

    Acetobacter aceti has an ability to grow under two different culture conditions, on shaking submerged cultures and on static pellicle-forming cultures. The respiratory chains of A. aceti grown on shaking and static cultures were compared, especially with respect to the terminal oxidase. Little difference was detected in several oxidase activities and in cytochrome b and c contents between the respiratory chains of both types of cells. Furthermore, the results obtained here suggested that the respiratory chains consist of primary dehydrogenases, ubiquinone, and terminal ubiquinol oxidase, regardless of the culture conditions. There was a remarkable difference, however, in the terminal oxidase, which is cytochrome a1 in cells in shaking culture but cytochrome o in cells grown statically. Change of the culture condition from shaking to static caused a change in the terminal oxidase from cytochrome a1 to cytochrome o, which is concomitant with an increase of pellicle on the surface of the static culture. In contrast, reappearance of cytochrome a1 in A. aceti was attained only after serial successive shaking cultures of an original static culture; cytochrome a1 predominated after the culture was repeated five times. In the culture of A. aceti, two different types of cells were observed; one forms a rough-surfaced colony, and the other forms a smooth-surfaced colony. Cells of the former type predominated in the static culture, while the cells of the latter type predominated in the shaking culture. Thus, data suggest that a change of the culture conditions, from static to shaking or vice versa, results in a change of the cell type, which may be related to the change in the terminal oxidase from cytochrome a1 to cytochrome o in A. aceti.

  14. Evolution of the cytochrome b gene of mammals.

    PubMed

    Irwin, D M; Kocher, T D; Wilson, A C

    1991-02-01

    With the polymerase chain reaction (PCR) and versatile primers that amplify the whole cytochrome b gene (approximately 1140 bp), we obtained 17 complete gene sequences representing three orders of hoofed mammals (ungulates) and dolphins (cetaceans). The fossil record of some ungulate lineages allowed estimation of the evolutionary rates for various components of the cytochrome b DNA and amino acid sequences. The relative rates of substitution at first, second, and third positions within codons are in the ratio 10 to 1 to at least 33. For deep divergences (greater than 5 million years) it appears that both replacements and silent transversions in this mitochondrial gene can be used for phylogenetic inference. Phylogenetic findings include the association of (1) cetaceans, artiodactyls, and perissodactyls to the exclusion of elephants and humans, (2) pronghorn and fallow deer to the exclusion of bovids (i.e., cow, sheep, and goat), (3) sheep and goat to the exclusion of other pecorans (i.e., cow, giraffe, deer, and pronghorn), and (4) advanced ruminants to the exclusion of the chevrotain and other artiodactyls. Comparisons of these cytochrome b sequences support current structure-function models for this membrane-spanning protein. That part of the outer surface which includes the Qo redox center is more constrained than the remainder of the molecule, namely, the transmembrane segments and the surface that protrudes into the mitochondrial matrix. Many of the amino acid replacements within the transmembrane segments are exchanges between hydrophobic residues (especially leucine, isoleucine, and valine). Replacement changes at first and second positions of codons approximate a negative binomial distribution, similar to other protein-coding sequences. At four-fold degenerate positions of codons, the nucleotide substitutions approximate a Poisson distribution, implying that the underlying mutational spectrum is random with respect to position. PMID:1901092

  15. Expression of the Rhodobacter sphaeroides cytochrome c2 structural gene.

    PubMed Central

    Brandner, J P; McEwan, A G; Kaplan, S; Donohue, T J

    1989-01-01

    A Rhodobacter sphaeroides mutant (CYCA1) lacking cytochrome c2 (cyt c2) was previously constructed (T. J. Donohue, A. G. McEwan, S. Van Doren, A. R. Crofts, and S. Kaplan, Biochemistry, 27: 1918-1924, 1988) by a combination of in vivo and in vitro molecular genetic techniques. CYCA1 was incapable of photosynthetic growth (PS-); in this presentation, we show that chemoheterotrophically grown CYCA1 contained significant quantities of a high potential soluble c-type cytochrome(s) with an alpha band of approximately 554 nm which had previously gone undetected under these physiological conditions in wild-type cells. In addition, the PS- phenotype of CYCA1 can be complemented in trans with stable low-copy-number (approximately 5 to 9 per R. sphaeroides genome) broad-host-range plasmids containing the wild-type cyt c2 structural gene (cycA) and upstream regulatory sequences. cyt c2 and cycA-specific mRNA levels were elevated in both the wild type and CYCA1 derivatives harboring intact cycA genes in trans, presumably as a result of increased gene dosage. Although photosynthetically grown wild-type cells contained approximately twofold more cycA-specific transcripts than chemoheterotrophically grown cells, there was an approximately four- to sevenfold increase in cyt c2 levels under photosynthetic conditions. Similarly, complemented CYCA1 strains contained between 1.3- and 2.3-fold more cycA mRNA under photosynthetic conditions than under chemoheterotrophic conditions and had 6- to 12-fold higher steady-state levels of cyt c2 under the same physiological conditions. These data are discussed in terms of possible posttranscriptional control over cyt c2. Images PMID:2536660

  16. Regulation of cytochrome P-450Ia1 gene expression

    SciTech Connect

    Kamps, C.A.

    1989-01-01

    The mechanism by which cytochrome P-450IA1 gene expression is induced by polycyclic aromatic hydrocarbons and various polychlorinated dibenzo-p-dioxins involves an intracellular protein known as the Ah receptor. Within the past few years, a second protein has been identified which binds to certain polycyclic aromatic hydrocarbons (PAHs) but not to the receptor ligand, 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD). The protein, named the 4S PAH binding protein, has been reported to bind to a site on the DNA in the 5{prime} regulatory region for the cytochrome P-450IA1 gene. This finding led to the hypothesis that the 4S PAH binding protein may be involved in the trans-regulation of this gene. The work presented in this manuscript addressed this hypothesis by (1) screening animals and cell lines for the presence or absence of the Ah receptor and 4S PAH binding protein, (2) screening polycyclic aromatic hydrocarbons (PAHs) to identify ligands which specifically bind only the 4S protein, (3) determining dose-response curves for TCDD and 4S protein specific ligands in mammalian cell lines, (4) co-administering a 4S binding protein ligand and TCDD in mammalian cell lines to determine the effects of the 4S protein-ligand complex on TCDD-induced cytochrome P-450IA1 expression, and (5) co-administering TCDD and 6-methyl 1,3,8-trichlorodibenzofuran (MCDF), a compound reported to be an antagonist of TCDD-induced benzo(a)pyrene-3-hydroxylase (AHH) activity, to determine whether antagonism occurs at the transcriptional level. The results of gradient assays show that the Ah receptor and the 4S binding protein were expressed in the rat strains which were studied. In the cell lines, H4IIE cells (rat hepatoma expressed only the receptor whereas Hepa1c1c7 cells mouse hepatoma) expressed both proteins.

  17. Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants.

    PubMed

    Wang, K; Zhen, Y; Sadoski, R; Grinnell, S; Geren, L; Ferguson-Miller, S; Durham, B; Millett, F

    1999-12-31

    The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).

  18. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    SciTech Connect

    Qin, Ling; Mills, Denise A.; Buhrow, Leann; Hiser, Carrie; Ferguson-Miller, Shelagh

    2010-09-02

    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

  19. Functions of the hydrophilic channels in protonmotive cytochrome c oxidase

    PubMed Central

    Rich, Peter R.; Maréchal, Amandine

    2013-01-01

    The structures and functions of hydrophilic channels in electron-transferring membrane proteins are discussed. A distinction is made between proton channels that can conduct protons and dielectric channels that are non-conducting but can dielectrically polarize in response to the introduction of charge changes in buried functional centres. Functions of the K, D and H channels found in A1-type cytochrome c oxidases are reviewed in relation to these ideas. Possible control of function by dielectric channels and their evolutionary relation to proton channels is explored. PMID:23864498

  20. Cytochrome P450: taming a wild type enzyme

    PubMed Central

    Jung, Sang Taek; Lauchli, Ryan; Arnold, Frances H

    2011-01-01

    Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly 'evolvable'--they are particularly robust to mutation in their active sites and readily accept new substrates and exhibit new selectivities. Their ability to adapt to new challenges upon mutation may reflect the nonpolar nature of their active sites as well as their high degree of conformational variability. PMID:21411308

  1. Higd1a is a positive regulator of cytochrome c oxidase

    PubMed Central

    Hayashi, Takaharu; Asano, Yoshihiro; Shintani, Yasunori; Aoyama, Hiroshi; Kioka, Hidetaka; Tsukamoto, Osamu; Hikita, Masahide; Shinzawa-Itoh, Kyoko; Takafuji, Kazuaki; Higo, Shuichiro; Kato, Hisakazu; Yamazaki, Satoru; Matsuoka, Ken; Nakano, Atsushi; Asanuma, Hiroshi; Asakura, Masanori; Minamino, Tetsuo; Goto, Yu-ichi; Ogura, Takashi; Kitakaze, Masafumi; Komuro, Issei; Sakata, Yasushi; Tsukihara, Tomitake; Yoshikawa, Shinya; Takashima, Seiji

    2015-01-01

    Cytochrome c oxidase (CcO) is the only enzyme that uses oxygen to produce a proton gradient for ATP production during mitochondrial oxidative phosphorylation. Although CcO activity increases in response to hypoxia, the underlying regulatory mechanism remains elusive. By screening for hypoxia-inducible genes in cardiomyocytes, we identified hypoxia inducible domain family, member 1A (Higd1a) as a positive regulator of CcO. Recombinant Higd1a directly integrated into highly purified CcO and increased its activity. Resonance Raman analysis revealed that Higd1a caused structural changes around heme a, the active center that drives the proton pump. Using a mitochondria-targeted ATP biosensor, we showed that knockdown of endogenous Higd1a reduced oxygen consumption and subsequent mitochondrial ATP synthesis, leading to increased cell death in response to hypoxia; all of these phenotypes were rescued by exogenous Higd1a. These results suggest that Higd1a is a previously unidentified regulatory component of CcO, and represents a therapeutic target for diseases associated with reduced CcO activity. PMID:25605899

  2. Sputter target

    DOEpatents

    Gates, Willard G.; Hale, Gerald J.

    1980-01-01

    The disclosure relates to an improved sputter target for use in the deposition of hard coatings. An exemplary target is given wherein titanium diboride is brazed to a tantalum backing plate using a gold-palladium-nickel braze alloy.

  3. Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774.

    PubMed

    da Silva, Sofia Marques; Pacheco, Isabel; Pereira, Inês A Cardoso

    2012-06-01

    Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.

  4. Spectroscopic studies on electron transfer between plastocyanin and cytochrome b6f complex.

    PubMed

    Sujak, A; Drepper, F; Haehnel, W

    2004-05-27

    This paper reports the results of the research on the interaction between the highly active cytochrome b(6)f complex and plastocyanin, both isolated from the same source - spinachia oleracea plants. An equilibrium constant K between the cytochrome f of the cytochrome b(6)f complex and plastocyanin has been estimated by two independent spectroscopic techniques: steady-state absorption spectroscopy and stopped-flow. The second-order rate constants k2 for forward and backward electron transfer between cytochrome f and plastocyanin have been found between 1.4-2 x 10(7) and 8-10 x 10(6) M(-1)s(-1), respectively, giving the value of an equilibrium constant of about 2+/-0.4 or a difference in redox potential between plastocyanin and cytochrome f of cytochrome b(6)f complex of ca. 17 mV. The value of K=1.7+/-0.3 has been estimated from steady-state experiments in which the initial and final concentrations of participating components after mixing have been estimated via differential spectra analysis or spectra deconvolution. We propose a method of evaluation of the final plastocyanin concentration after the electron transfer reaction between cytochrome bf complex and plastocyanin that overcomes the interference by the strong chlorophyll absorption in the spectral region where oxidised plastocyanin has its low extinction absorption band. The data from both experiments, in the system devoid of quinol being the electron donor to cytochrome b(6), suggest that in case of electron transfer from cytochrome f to plastocyanin electron transfer can either bypass cytochrome f or the Rieske iron-sulfur protein can be reduced prior to its movement to the quinol binding site of cytochrome b(6). The role of the Rieske protein in forward and backward electron transfer reactions is discussed.

  5. 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.

  6. Site-directed nanoparticle labeling of cytochrome c

    PubMed Central

    Aubin-Tam, Marie-Eve; Hwang, Wonmuk; Hamad-Schifferli, Kimberly

    2009-01-01

    Although nanoparticle-protein conjugates have been synthesized for numerous applications, bioconjugation remains a challenge, often resulting in denaturation or loss of protein function. This is partly because the protein–nanoparticle interface is poorly understood, which impedes the use of nanoparticles in nanomedicine. Although the effects of nanoparticle ligand and material on protein structure have been explored, the choice of the labeling site on the protein has not yet been systematically studied. To address this issue, we label cytochrome c site-specifically with a negatively charged Au nanoparticle via a covalent thiol–Au bond. The attachment site is controlled by cysteine mutations of surface residues. The effect of labeling on protein structure is probed by circular dichroism. Protein unfolding is the most severe when the nanoparticle is attached to the N- and C-terminal foldon, the core motif of cytochrome c. Also, when the nanoparticle is attached in the vicinity of charged residues, the amount of structural damage is greater because of salt-dependent electrostatic interactions with charged ligand bis(p-sulfonatophenyl) phenylphosphine on the nanoparticle. Molecular dynamics simulations also elucidate local to global structural perturbation depending on labeling site. These results suggest that the labeling site must be considered as one of the main design criteria for nanoparticle–protein conjugates. PMID:19251670

  7. Vaccine-Drug Interactions: Cytokines, Cytochromes, and Molecular Mechanisms.

    PubMed

    Pellegrino, Paolo; Perrotta, Cristiana; Clementi, Emilio; Radice, Sonia

    2015-09-01

    Vaccinations are recommended throughout life to reduce the risk of vaccine-preventable diseases and their sequelae. Vaccines are often administered in patients with chronic diseases who are likely to be treated with several drugs. A growing number of clinical observations have indicated the possibility of interactions between vaccines and drugs, leading to changes in drug metabolism after vaccination. These interactions represent a significant concern because of the increasing use of vaccines in older patients who are likely to be treated with several drugs. Because of the possible implications of adverse reactions in terms of public health, several studies were performed to verify the risk posed by these interactions and to clarify the biologic mechanisms that drive these events. Of the several mechanisms proposed to be at the basis of vaccine-drug interactions, the most convincing evidence suggests a role of inflammatory cytokines on the regulation of specific cytochrome P450 enzymes in the liver. Differences in the cytochrome P450 enzymes involved in the metabolism of these drugs could explain these contrasting results and provide important insights to fully understand the clinical importance of these events. Further studies are required to verify whether vaccine-drug interactions may occur in other clinical settings, especially the ones for which patients are required to be vaccinated against specific diseases.

  8. Spectroscopic features of cytochrome P450 reaction intermediates

    PubMed Central

    Luthra, Abhinav; Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Preface Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2-3]. Historically these enzymes received their name from ‘pigment 450’ due to the unusual position of the Soret band in UV-Vis absorption spectra of the reduced CO-saturated state [4-5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other ‘P450-like heme enzymes’ such as nitric oxide synthase and chloroperoxidase, the phenomenological term ‘cytochrome P450’ is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420 nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle. PMID:21167809

  9. The Cox3p assembly module of yeast cytochrome oxidase

    PubMed Central

    Su, Chen-Hsien; McStay, Gavin P.; Tzagoloff, Alexander

    2014-01-01

    Yeast cytochrome oxidase (COX) was previously inferred to assemble from three modules, each containing one of the three mitochondrially encoded subunits and a different subset of the eight nuclear gene products that make up this respiratory complex. Pull-down assays of pulse-labeled mitochondria enabled us to characterize Cox3p subassemblies that behave as COX precursors and contain Cox4p, Cox7p, and Cox13p. Surprisingly, Cox4p is a constituent of two other complexes, one of which was previously proposed to be an intermediate of Cox1p biogenesis. This suggests that Cox4p, which contacts Cox1p and Cox3p in the holoenzyme, can be incorporated into COX by two alternative pathways. In addition to subunits of COX, some Cox3p intermediates contain Rcf1p, a protein associated with the supercomplex that stabilizes the interaction of COX with the bc1 (ubiquinol-cytochrome c reductase) complex. Finally, our results indicate that although assembly of the Cox1p module is not contingent on the presence of Cox3p, the converse is not true, as none of the Cox3p subassemblies were detected in a mutant blocked in translation of Cox1p. These studies support our proposal that Cox3p and Cox1p are separate assembly modules with unique compositions of ancillary factors and subunits derived from the nuclear genome. PMID:24478450

  10. Ivory identification by DNA profiling of cytochrome b gene.

    PubMed

    Lee, James Chun-I; Hsieh, Hsing-Mei; Huang, Li-Hung; Kuo, Yi-Chen; Wu, Jane-Hong; Chin, Shih-Chien; Lee, An-Hsing; Linacre, Adrian; Tsai, Li-Chin

    2009-03-01

    Ivory can be visually identified in its native form as coming from an elephant species; however, determining from which of the three extant elephant species a section of ivory originates is more problematic. We report on a method that will identify and distinguish the protected and endangered elephant species, Elephas maximus or Loxodonta sp. To identify the species of elephant from ivory products, we developed three groups of nested PCR amplifications within the cytochrome b gene that generate amplification products using highly degraded DNA isolated from confiscated ivory samples dating from 1995. DNA from a total of 382 out of 453 ivory samples were successfully isolated and amplified leading to species identification. All sequences were searched against GenBank and found to match with E. maximus and Loxodonta sp. with at least 99% similarity. The samples that were tested came from eight Asian elephants, 14 African forest elephants (Loxodonta cyclotis), and 360 African savannah elephants (Loxodonta africana). This study demonstrates a high success rate in species identification of ivory by a nested PCR approach within the cytochrome b gene which provides the necessary information for the protection of endangered species conservation.

  11. Cytochrome b gene for species identification of the conservation animals.

    PubMed

    Hsieh, H M; Chiang, H L; Tsai, L C; Lai, S Y; Huang, N E; Linacre, A; Lee, J C

    2001-10-15

    A partial DNA sequence of cytochrome b gene was used to identify the remains of endangered animals and species endemic to Taiwan. The conservation of animals species included in this study were: the formosan gem-faced civets, leopard cats, tigers, clouded leopards, lion, formosan muntjacs, formosan sika deers, formosan sambars, formosan serows, water buffalo, formosan pangolins and formosan macaques. The control species used included domestic cats, domestic dogs, domestic sheeps, domestic cattles, domestic pigs and humans. Heteroplasmy was detected in the formosan macaque, domestic pig and domestic cats. The frequencies of heteroplasmy in these animals were about 0.25% (1 in 402bp). Sequences were aligned by Pileup program of GCG computer package, and the phylogenetic tree was constructed by the neighbor-joining method. The results of sequence comparison showed that the percentage range of sequence diversity in the same species was from 0.25 to 2.74%, and that between the different species was from 5.97 to 34.83%. The results of phylogenetic analysis showed that the genetic distance between the different species was from 6.33 to 40.59. Animals of the same species, both the endangered animal species and domestic animals, were clustered together in the neighbor-joining tree. Three unknown samples of animal remains were identified by this system. The partial sequence of cytochrome b gene adopted in this study proved to be usable for animal identification.

  12. Cytochrome P450-derived eicosanoids: the neglected pathway in cancer

    PubMed Central

    Kaipainen, Arja; Greene, Emily R.; Huang, Sui

    2010-01-01

    Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed. PMID:20941528

  13. Biological diversity of cytochrome P450 redox partner systems.

    PubMed

    McLean, Kirsty J; Luciakova, Dominika; Belcher, James; Tee, Kang Lan; Munro, Andrew W

    2015-01-01

    Cytochrome P450 enzymes (P450s or CYPs) catalyze an enormous variety of oxidative reactions in organisms from all major domains of life. Their monooxygenase activity relies on the reductive scission of molecular oxygen (O2) bound to P450 heme iron, and thus on the delivery of two electrons to the heme iron at discrete points in the catalytic cycle. Early studies suggested that P450 redox partner machinery fell into only two major classes: either the eukaryotic diflavin enzyme NADPH-cytochrome P450 oxidoreductase, or bacterial/mitochondrial NAD(P)H-ferredoxin reductase and ferredoxin partners. However, more recent studies, aided by genome sequence data, reveal a much more complex scenario. Several new types of P450 redox partner systems have now been characterized, including P450s naturally linked to their redox partners, or to a component protein of their P450 electron delivery system. Other P450s have evolved to bypass requirements for redox partners, and instead react directly with hydrogen peroxide or NAD(P)H to facilitate oxidative or reductive catalysis. Further P450s are fused to non-redox partner enzymes and can catalyse consecutive reactions in a common pathway. This chapter describes the biochemistry and the enormous natural diversity of P450 redox systems, including descriptions of novel P450s fused to non-redox partner proteins.

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

    PubMed

    Percy, Melanie J; Lappin, Terry R

    2008-05-01

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

  15. Utility and evolution of cytochrome b in insects.

    PubMed

    Simmons, R B; Weller, S J

    2001-08-01

    Cytochrome b (cyt-b) is widely used in molecular phylogenetic studies of vertebrate, but not invertebrate, taxa. To determine whether this situation is an historical accident or reflects the utility of cyt-b, we compared the abilities of cyt-b, COI, and one nuclear ribosomal gene region (D1 of 28S) to recover intergeneric relationships within the tiger moth tribes Ctenuchini and Euchromiini. Additionally, we compared the rate of sequence and amino acid evolution of cyt-b across insects. Cytochrome b had the same level of sequence variation and A/T bias as COI, but was less useful for recovering intergeneric relationships. The total evidence tree casts doubt on the traditional taxonomy of the group. For the class Insecta, we found that functional conservation of amino acids occurs for the same regions as those found in vertebrates with the exception of Mallophaga (lice). Lice have an accelerated rate of nonsynonymous substitutions. Accelerated rate of cyt-b nucleotide and amino acid evolution in Apidae (bees) may be correlated with increased metabolic rates associated with facultative endothermy (= heterothermy).

  16. Electrochemistry of cytochromes p450: analysis of current-voltage characteristics of electrodes with immobilized cytochromes p450 for the screening of substrates and inhibitors.

    PubMed

    Shumyantseva, V V; Bulko, T V; Kuznetsova, G P; Samenkova, N F; Archakov, A I

    2009-04-01

    In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14alpha-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450.

  17. Direct Measurement of Cyclic Current-Voltage Responses of Integral Membrane Proteins at a Self-Assembled Lipid-Bilayer-Modified Electrode: Cytochrome f and Cytochrome c Oxidase

    NASA Astrophysics Data System (ADS)

    Salamon, Z.; Hazzard, J. T.; Tollin, G.

    1993-07-01

    Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.

  18. Direct measurement of cyclic current-voltage responses of integral membrane proteins at a self-assembled lipid-bilayer-modified electrode: Cytochrome f and cytochrome c oxidase

    SciTech Connect

    Salamon, Z.; Hazzard, J.T.; Tollin, G. )

    1993-07-15

    Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic [open quotes]voltammograms,[close quotes] 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins. 63 refs., 2 figs., 1 tab.

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

    PubMed

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

    2012-07-26

    Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). 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 c. 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.

  20. Proton-assisted two-electron transfer in natural variants of tetraheme cytochromes from Desulfomicrobium Sp.

    PubMed

    Correia, Ilídio J; Paquete, Catarina M; Coelho, Ana; Almeida, Claudia C; Catarino, Teresa; Louro, Ricardo O; Frazão, Carlos; Saraiva, Lígia M; Carrondo, Maria Arménia; Turner, David L; Xavier, António V

    2004-12-10

    The tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 --> Ser, Val-45 --> Ala, and Phe-88 --> Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats.

  1. KINETICS OF BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P450 ISOENZYMES IN HUMAN LIVER MICROSOMES

    EPA Science Inventory

    Kinetics of Bromodichloromethane Metabolism by
    Cytochrome P450 Isoenzymes in Human Liver Microsomes

    Guangyu Zhao and John W. Allis

    ABSTRACT
    The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have ...

  2. Mitofilin regulates cytochrome c release during apoptosis by controlling mitochondrial cristae remodeling

    SciTech Connect

    Yang, Rui-feng; Zhao, Guo-wei; Liang, Shu-ting; Zhang, Yuan; Sun, Li-hong; Chen, Hou-zao; Liu, De-pei

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Mitofilin deficiency caused disruption of the cristae structures in HeLa cells. Black-Right-Pointing-Pointer Mitofilin deficiency reduced cell proliferation and increased cell sensitivity to apoptotic stimuli. Black-Right-Pointing-Pointer Mitofilin deficiency accelerated the release of cytochrome c from mitochondria. Black-Right-Pointing-Pointer Mitofilin deficiency accelerated STS-induced intrinsic apoptotic pathway without interfering with the activation of Bax. -- Abstract: Mitochondria amplify caspase-dependent apoptosis by releasing proapoptotic proteins, especially cytochrome c. This process is accompanied by mitochondrial cristae remodeling. Our studies demonstrated that mitofilin, a mitochondrial inner membrane protein, acted as a cristae controller to regulate cytochrome c release during apoptosis. Knockdown of mitofilin in HeLa cells with RNAi led to fragmentation of the mitochondrial network and disorganization of the cristae. Mitofilin-deficient cells showed cytochrome c redistribution between mitochondrial cristae and the intermembrane space (IMS) upon intrinsic apoptotic stimuli. In vitro cytochrome c release experiments further confirmed that, compared with the control group, tBid treatment led to an increase in cytochrome c release from mitofilin-deficient mitochondria. Furthermore, the cells with mitofilin knockdown were more prone to apoptosis by accelerating cytochrome c release upon the intrinsic apoptotic stimuli than controls. Moreover, mitofilin deficiency did not interfere with the activation of proapoptotic member Bax upon intrinsic apoptotic stimuli. Thus, mitofilin distinctly functions in cristae remodeling and controls cytochrome c release during apoptosis.

  3. Delivery of nitric oxide for analysis of the function of cytochrome c'.

    PubMed

    Cole, Lindsay J; Huston, Wilhelmina M; Moir, James W B

    2008-01-01

    On delivery of nitric oxide (NO) to protein samples (e.g., cytochrome c'), for spectroscopic experiments it is important to avoid exposure to oxygen and to remove contaminants from the NO gas. We describe a number of techniques for steady-state UV/Vis spectrophotometry and pre-steady-state stopped-flow spectrophotometry analysis of cytochrome c'.

  4. Liposome effect on the cytochrome c-catalyzed peroxidation of carbonyl substrates to triplet species.

    PubMed

    Nantes, I L; Faljoni-Alario, A; Vercesi, A E; Santos, K E; Bechara, E J

    1998-09-01

    Cytochrome c exhibits peroxidase activity on diphenylacetaldehyde (DPAA) and 3-methylacetoacetone (MAA), which is greatly affected by the presence and nature of charged liposome or micelle interfaces interacting with the enzyme. The ferricytochrome c reaction with DPAA is accelerated when the enzyme is attached to negatively charged interfaces. Whatever the medium, bulk solution or negatively charged dicetylphosphate (DCP), phosphatidylcholine/phosphatidylethanolamine/cardiolipin (PC/PE/CL) liposomes, this chemiluminescent reaction is accompanied by reduction of cytochrome c to its ferrous form. In turn, MAA is oxidized by cytochrome c exclusively when bound to DCP liposomes. Contrary to DPAA oxidation, the MAA reaction is followed by bleaching of cytochrome c, reflecting damage to the hemeprotein chromophore. The cytochrome-c-catalyzed oxidation of either DPAA or MAA leads to concomitant disappearance of the enzyme charge transfer absorption band at 695 nm. This suggests that the peroxidase activity of cytochrome c involves substrate-induced loss of the methionine ligand at the iron sixth coordination position, which is favored by interaction of cytochrome c with negatively charged interfaces. Accordingly, a decrease and blue shift of the charge transfer band could be observed in cytochrome-c-containing negatively charged DCP, PC/PE/CL liposomes or lysophosphatidylethanolamine micelles in the presence of DPAA or MAA. PMID:9741591

  5. Measurement of Cytochrome P450 Enzyme Induction and Inhibition in Human Hepatoma Cells.

    PubMed

    Rodrigues, Robim M; De Kock, Joery; Doktorova, Tatyana Y; Rogiers, Vera; Vanhaecke, Tamara

    2015-01-01

    Cytochrome P450 enzymes are a diverse group of catalytic enzymes in the liver that are mainly responsible for the biotransformation of organic substances. Cytochrome P450 activity as well as both its induction and inhibition are key factors in drug biotransformation and can be involved in deactivation, activation, detoxification and toxification processes. Thus, the modulation of cytochrome P450 activity is an important parameter when evaluating the potential toxicity of chemical compounds using an in vitro system. The cytochrome P450 3A subfamily proteins are among the most important drug-metabolizing enzymes in human liver and are responsible for about half of all cytochrome P450-dependent drug oxidations. In vitro, these enzymes are active not only in primary human hepatocyte cultures, but also in differentiated human hepatoma HepaRG cells. The present protocol describes the culture of cryopreserved differentiated HepaRG cells and the evaluation of its cytochrome P450 activity upon exposure to a chemical compound using a commercially available luminogenic cytochrome P450 assay. This in vitro model can be used to monitor the induction and inhibition of cytochrome P450 3A following exposure to a particular test compound.

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

    PubMed

    Brankova, Liliana; Ivanov, Sergei; Alexieva, Vera

    2007-09-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

  9. Molecular interactions between multihaem cytochromes: probing the protein-protein interactions between pentahaem cytochromes of a nitrite reductase complex.

    PubMed

    Lockwood, Colin; Butt, Julea N; Clarke, Thomas A; Richardson, David J

    2011-01-01

    The cytochrome c nitrite reductase NrfA is a 53 kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2- to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21 kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein-protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer-dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2- or HSO3-. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50 nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

  10. Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

    PubMed Central

    Pokkuluri, P. Raj; Londer, Yuri Y.; Duke, Norma E.C.; Erickson, Jill; Pessanha, Miguel; Salgueiro, Carlos A.; Schiffer, Marianne

    2004-01-01

    The structure of a novel c7-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 Å resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c7-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c7 molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, Eapp, of domain C is −105 mV, 50 mV higher than that of PpcA. PMID:15133162

  11. Hepatic metabolism of cyclodiene insecticides by constitutive forms of cytochrome P-450 from lower vertebrates.

    PubMed

    Ronis, M J; Walker, C H; Peakall, D

    1987-01-01

    1. Multiple forms of cytochrome P-450 were separated from the hepatic microsomes of untreated male rats, pigeons (Columbia livia), razorbills (Alca torda), puffins (Fratercula arctica), and rainbow trout (Salmo gairdnerii), using anion exchange chromatography and DEAE-cellulose. 2. In some cases cytochrome P-450 forms were further purified on hydroxylapatite and carboxymethyl-sephadex columns. 3. Considerable differences in the distribution of forms between these five species were evident from elution profiles on DEAE cellulose, and on analysis of the cytochrome P-450 containing pools by SDS-PAGE. 4. The metabolism of two organochlorine compounds, aldrin and the dieldrin analogue HCE, were studied in (a) intact microsomes and (b) reconstituted systems containing cytochrome P-450, from each of the five species. 5. In spite of their close structural similarity, significant differences were found between the two substrates in the distribution of catalytic activity between the cytochrome P-450 isozymes of each species. PMID:2888582

  12. Cytochromes c': Structure, Reactivity and Relevance to Haem-Based Gas Sensing.

    PubMed

    Hough, Michael A; Andrew, Colin R

    2015-01-01

    Cytochromes c' are a group of class IIa cytochromes with pentacoordinate haem centres and are found in photosynthetic, denitrifying and methanotrophic bacteria. Their function remains unclear, although roles in nitric oxide (NO) trafficking during denitrification or in cellular defence against nitrosoative stress have been proposed. Cytochromes c' are typically dimeric with each c-type haem-containing monomer folding as a four-α-helix bundle. Their hydrophobic and crowded distal sites impose severe restrictions on the binding of distal ligands, including diatomic gases. By contrast, NO binds to the proximal haem face in a similar manner to that of the eukaryotic NO sensor, soluble guanylate cyclase and bacterial analogues. In this review, we focus on how structural features of cytochromes c' influence haem spectroscopy and reactivity with NO, CO and O2. We also discuss the relevance of cytochrome c' to understanding the mechanisms of gas binding to haem-based sensor proteins.

  13. Rcf1 mediates cytochrome oxidase assembly and respirasome formation, revealing heterogeneity of the enzyme complex.

    PubMed

    Vukotic, Milena; Oeljeklaus, Silke; Wiese, Sebastian; Vögtle, F Nora; Meisinger, Chris; Meyer, Helmut E; Zieseniss, Anke; Katschinski, Doerthe M; Jans, Daniel C; Jakobs, Stefan; Warscheid, Bettina; Rehling, Peter; Deckers, Markus

    2012-03-01

    The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S. cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.

  14. Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase.

    PubMed

    Faletto, M B; Koser, P L; Battula, N; Townsend, G K; Maccubbin, A E; Gelboin, H V; Gurtoo, H L

    1988-09-01

    Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

  15. Integrin αvβ3-Targeted Imaging of Lung Cancer1

    PubMed Central

    Chen, Xiaoyuan; Sievers, Eric; Hou, Yingping; Park, Ryan; Tohme, Michel; Bart, Robert; Bremner, Ross; Bading, James R; Conti, Peter S

    2005-01-01

    Abstract A series of radiolabeled cyclic arginine-glycine-aspartic acid (RGD) peptide ligands for cell adhesion molecule integrin αvβ3-targeted tumor angiogenesis targeting are being developed in our laboratory. In this study, this effort continues by applying a positron emitter 64Cu-labeled PEGylated dimeric RGD peptide radiotracer 64Cu-DOTA-PEG-E[c(RGDyK)]2 for lung cancer imaging. The PEGylated RGD peptide indicated integrin αvβ3 avidity, but the PEGylation reduced the receptor binding affinity of this ligand compared to the unmodified RGD dimer. The radiotracer revealed rapid blood clearance and predominant renal clearance route. The minimum nonspecific activity accumulation in normal lung tissue and heart rendered high-quality orthotopic lung cancer tumor images, enabling clear demarcation of both the primary tumor at the upper lobe of the left lung, as well as metastases in the mediastinum, contralateral lung, and diaphragm. As a comparison, fluorodeoxyglucose (FDG) scans on the same mice were only able to identify the primary tumor, with the metastatic lesions masked by intense cardiac uptake and high lung background. 64Cu-DOTA-PEG-E[c(RGDyK)]2 is an excellent positron emission tomography (PET) tracer for integrin-positive tumor imaging. Further studies to improve the receptor binding affinity of the tracer and subsequently to increase the magnitude of tumor uptake without comprising the favorable in vivo kinetics are currently in progress. PMID:15799827

  16. Forced Cytochrome B gene mutation expression induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells

    PubMed Central

    Dasgupta, Santanu; Hoque, Mohammad Obaidul; Upadhyay, Sunil; Sidransky, David

    2010-01-01

    Mitochondria encoded Cytochrome B (CYTB) gene mutations were reported in tumors of different anatomic origin but the functional significance of these mutations are not well studied. Earlier, we found a 7-amino acid deletion mutation in the CYTB gene in a primary bladder cancer patient. In the present study, we overexpressed this 7-amino acid deletion mutation of CYTB gene in SV-40 transformed human uroepithelial HUC-1 cells. The nuclear transcribed mitochondrial CYTB (mtCYTB) was targeted into the mitochondria and generated increased copies of mitochondria and mitochondrial COX-I protein in the transfected HUC-1 cells. The pro-apoptotic protein Bax largely remained confined to the cytoplasm of the mtCYTB transfected HUC-1 cells without release of Cytochrome C. The downstream apoptotic proteins PARP also remained uncleaved along with increased Lamin B1 in the mtCYTB transfected cells. Our results demonstrate that forced overexpression of mtCYTB in transformed human uroepithelial HUC-1 cells triggered mitochondrial proliferation and induction of an anti-apoptotic signaling cascade favoring sustained cellular growth. Coding mitochondrial DNA mutations appear to have significant functional contribution in tumor progression. PMID:19569044

  17. Promising Tools in Prostate Cancer Research: Selective Non-Steroidal Cytochrome P450 17A1 Inhibitors

    PubMed Central

    Bonomo, Silvia; Hansen, Cecilie H.; Petrunak, Elyse M.; Scott, Emily E.; Styrishave, Bjarne; Jørgensen, Flemming Steen; Olsen, Lars

    2016-01-01

    Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using non-steroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC50 values in the nanomolar range, without affinity for the major drug-metabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells. PMID:27406023

  18. Promising Tools in Prostate Cancer Research: Selective Non-Steroidal Cytochrome P450 17A1 Inhibitors.

    PubMed

    Bonomo, Silvia; Hansen, Cecilie H; Petrunak, Elyse M; Scott, Emily E; Styrishave, Bjarne; Jørgensen, Flemming Steen; Olsen, Lars

    2016-01-01

    Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using non-steroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC50 values in the nanomolar range, without affinity for the major drug-metabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells. PMID:27406023

  19. 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.

  20. Interactions among Cytochromes P450 in Microsomal Membranes

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Sineva, Elena V.; Halpert, James R.

    2015-01-01

    The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1, and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the intersubunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov, D. R., Davydova, N. Y., Sineva, E. V., Kufareva, I., and Halpert, J. R. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone. Biochem. J. 453, 219–230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the luminescence resonance energy transfer measurements of interprobe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures. According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species. PMID:25533469

  1. Proton interactions in the resting form of cytochrome oxidase.

    PubMed

    Papadopoulos, P G; Walter, S A; Li, J W; Baker, G M

    1991-01-22

    The effect of pH on the near-UV absorption spectrum of cytochrome oxidase has been examined. Several lines of evidence implicate a proton binding site that can modulate the optical properties of cytochrome alpha 3 in the resting enzyme. Changing the pH within the range 6.5-10.5 was found to reversibly shift the position of the Soret band over an 11-nm range. The lower pH values caused a progressive blue shift in the Soret band, whereas the high-pH range promoted a gradual red shift. Limiting band positions were approximately 416 and 427 nm. The incubation time required to reach a stable band position varied somewhat as did the actual extent of the shift. In most cases, the shift was associated with an isosbestic point. A pH titration profile for the apparent equilibrium position of the Soret band was obtained. Nonlinear least-squares fitting to a scatter plot, assuming a single acid/base group, showed an apparent pKa of 7.8. Magnetic circular dichroism (MCD) spectra of the low-pH form at 416 nm, the high-pH form at 427 nm, and the cyanide derivative at 428 nm were compared. No evidence of a high-pH-dependent low-spin transition or a change in the redox state of cytochrome a3 was found, confirming earlier work [Baker, G. M., Noguchi, M., & Palmer, G. (1987) J. Biol. Chem. 262, 595-604]. Subtraction of ferricytochrome a [spectrum taken from Vanneste, W. H. (1966) Biochemistry 5, 838-848] from a series of blue-shifting spectra showed a band at 414 nm that progressively gained amplitude and a band at 430 nm that correspondingly lost amplitude. A series of red-shifting spectra showed the opposite behavior with a clear isosbestic point being evident in both cases. The difference extinction change at 414 and 430 nm depended linearly on the position of the Soret band, both showing a reversible dependence on pH. The 430-nm band is noted to be unusually red-shifted for high-spin ferric heme a. An additional, pH-insensitive band was observed at 408-410 nm which was eliminated

  2. Oxidation state-dependent conformational changes in cytochrome c.

    PubMed

    Berghuis, A M; Brayer, G D

    1992-02-20

    High-resolution three-dimensional structural analyses of yeast iso-1-cytochrome c have now been completed in both oxidation states using isomorphous crystalline material and similar structure determination methodologies. This approach has allowed a comprehensive comparison to be made between these structures and the elucidation of the subtle conformational changes occurring between oxidation states. The structure solution of reduced yeast iso-1-cytochrome c has been published and the determination of the oxidized protein and a comparison of these structures are reported herein. Our data show that oxidation state-dependent changes are expressed for the most part in terms of adjustments to heme structure, movement of internally bound water molecules and segmental thermal parameter changes along the polypeptide chain, rather than as explicit polypeptide chain positional shifts, which are found to be minimal. This result is emphasized by the retention of all main-chain to main-chain hydrogen bond interactions in both oxidation states. Observed thermal factor changes primarily affect four segments of polypeptide chain. Residues 37-39 show less mobility in the oxidized state, with Arg38 and its side-chain being most affected. In contrast, residues 47-59, 65-72 and 81-85 have significantly higher thermal factors, with maximal increases being observed for Asn52, Tyr67 and Phe82. The side-chains of two of these residues are hydrogen bonded to the internally bound water molecule, Wat166, which shows a large 1.7 A displacement towards the positively charged heme iron atom in the oxidized protein. Further analyses suggest that Wat166 is a major factor in stabilizing both oxidation states of the heme through differential orientation of dipole moment, shift in distance to the heme iron atom and alterations in the surrounding hydrogen bonding network. It also seems likely that Wat166 movement leads to the disruption of the hydrogen bond from the side-chain of Tyr67 to the Met80

  3. Synthesis and characterization of (68)Ga-labeled curcumin and curcuminoid complexes as potential radiotracers for imaging of cancer and Alzheimer's disease.

    PubMed

    Asti, Mattia; Ferrari, Erika; Croci, Stefania; Atti, Giulia; Rubagotti, Sara; Iori, Michele; Capponi, Pier C; Zerbini, Alessandro; Saladini, Monica; Versari, Annibale

    2014-05-19

    Curcumin (CUR) and curcuminoids complexes labeled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer's disease. Gallium-68 is a positron-emitting, generator-produced radionuclide, and its properties can be exploited in situ in medical facilities without a cyclotron. Moreover, CUR showed a higher uptake in tumor cells compared to normal cells, suggesting potential diagnostic applications in this field. In spite of this, no studies using labeled CUR have been performed in this direction, so far. Herein, (68)Ga-labeled complexes with CUR and two curcuminoids, namely diacetyl-curcumin (DAC) and bis(dehydroxy)curcumin (bDHC), were synthesized and characterized by means of experimental and theoretical approaches. Moreover, a first evaluation of their affinity to synthetic β-amyloid fibrils and uptake by A549 lung cancer cells was performed to show the potential application of these new labeled curcuminoids in these diagnostic fields. The radiotracers were prepared by reacting (68)Ga(3+) obtained from a (68)Ge/(68)Ga generator with 1 mg/mL curcuminoids solutions. Reaction parameters (precursor amount, reaction temperature, and pH) were optimized to obtain high and reproducible radiochemical yield and purity. Stoichiometry and formation of the curcuminoid complexes were investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR, ultraviolet-visible, and fluorescence spectroscopy on the equivalent (nat)Ga-curcuminoids (nat = natural) complexes, and their structure was computed by theoretical density functional theory calculations. The analyses evidenced that CUR, DAC, and bDHC were predominantly in the keto-enol form and attested to Ga(L)2(+) species formation. Identity of the (68)Ga(L)2(+) complexes was confirmed by coelution with the equivalent (nat)Ga(L)2(+) complexes in ultrahigh-performance liquid chromatography analyses.(68)Ga(CUR)2(+), (68)Ga(DAC)2(+), and (68)Ga(bDHC)2

  4. Outer-membrane cytochrome-c, OmcF from Geobacter sulfurreducens: high structural similarity to an algal cytochrome c6.

    SciTech Connect

    Pokkuluri, P. R.; Londer, Y. Y.; Wood, S. J.; Duke, N. E. C.; Morgado, L.; Salgueiro, C. A.; Schiffer, M.; Biosciences Division; Univ. Nova de Lisboa

    2009-01-01

    Putative outer membrane c-type cytochromes have been implicated in metal ion reducing properties of Geobacter sulfurreducens. OmcF (GSU2432), OmcB (GSU2731), and OmcC (GSU2737) are three such proteins that have predicted lipid anchors. OmcF is a monoheme cytochrome, whereas OmcB and OmcC are multiheme cytochromes. Deletion of OmcF was reported to affect the expression of OmcB and OmcC in G. sulfurreducens. The OmcF deficient strain was impaired in its ability to both reduce and grow on Fe(III) citrate probably because the expression of OmcB, which is crucial for iron reduction, is low in this strain. U(VI) reduction activity of this bacterium is also lower on deletion of OmcB or OmcF. The U(VI) reduction activity is affected more by the deletion of OmcF than by the deletion of OmcB. The soluble part of OmcF (residues 20-104, referred to as OmcF{sub S} hereafter) has sequence similarity to soluble cytochromes c{sub 6} of photosynthetic algae and cyanobacteria. The cytochrome c{sub 6} proteins in algae and cyanobacteria are electron transport proteins that mediate the transfer of electrons from cytochrome b{sub 6}f to photosystem I and have high reduction potentials of about +350 mV and low pI. The structures of seven cytochromes c{sub 6} have been previously determined. Further, a c{sub 6}-like cytochrome (PetJ2) of unknown function was recently identified in Synechoccus sp. PCC 7002 with a reduction potential of +148 mV and high pI. Here, we report the structure of OmcF{sub S} and its remarkable structural similarity to that of cytochrome c{sub 6} from the green alga, Monoraphidium braunii. To our knowledge, OmcF{sub S} is the first example of a cytochrome c{sub 6}-like structure from a nonphotosynthetic organism.

  5. Cytochrome b nucleotide sequence variation among the Atlantic Alcidae.

    PubMed

    Friesen, V L; Montevecchi, W A; Davidson, W S

    1993-01-01

    Analysis of cytochrome b nucleotide sequences of the six extant species of Atlantic alcids and a gull revealed an excess of adenines and cytosines and a deficit of guanines at silent sites on the coding strand. Phylogenetic analyses grouped the sequences of the common (Uria aalge) and Brünnich's (U. lomvia) guillemots, followed by the razorbill (Alca torda) and little auk (Alle alle). The black guillemot (Cepphus grylle) sequence formed a sister taxon, and the puffin (Fratercula arctica) fell outside the other alcids. Phylogenetic comparisons of substitutions indicated that mutabilities of bases did not differ, but that C was much more likely to be incorporated than was G. Imbalances in base composition appear to result from a strand bias in replication errors, which may result from selection on secondary RNA structure and/or the energetics of codon-anticodon interactions. PMID:7916741

  6. Porphyrin fluorescence dominates UV photoemission of folded cytochrome c.

    PubMed

    Löwenich, Dennis; Kleinermanns, Karl

    2007-01-01

    In this article we reinvestigate the bimodal fluorescence of cytochrome c (Cyt c) by using excitation-wavelength-dependent fluorescence spectroscopy. We show that its major contributions at pH 3-7 do not arise from tryptophan (Trp-59) fluorescence as hitherto assumed. Instead, different chromophores of Cyt c contribute at different pH values. At pH 3-7, the porphyrin system contributes about 80% and tryptophan about 20% to the total fluorescence upon excitation of Cyt c at 280 nm. At pH 2, the fluorescence originates nearly completely from the tryptophan residue. Porphyrin fluorescence is still present at pH 2 but its contribution is too small for quantitative deconvolution. Our results show that the UV fluorescence of Cyt c has to be deconvoluted before it can be used to perform time-resolved measurements of the folding of this small protein.

  7. Cytochrome c peroxidase activity of heme bound amyloid β peptides.

    PubMed

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh

    2016-09-01

    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP.

  8. Highly selective ligand binding by Methylophilus methylotrophus cytochrome c''.

    PubMed

    Quintas, Pedro O; Catarino, Teresa; Todorovic, Smilja; Turner, David L

    2011-06-28

    Cytochrome c'' (cyt c'') from Methylophilus methylotrophus is unusual insofar as the heme has two axial histidine ligands in the oxidized form but one is detached when the protein is reduced. Despite cyt c'' having an axial site available for binding small ligands, we show here that only NO binds readily to the ferrous cyt c''. Binding of CO, as well as CN(-), on the other hand requires considerable structural reorganization, or reduction of the disulfide bridge close to the heme. Standard free energies for the binding of NO and CO reveal high selectivity of the ferrous cyt c'' for NO, indicating its putative physiological role. In this work, we characterize in detail the kinetics of NO binding and the structural features of the Fe(2+)-NO adduct by stopped-flow and resonance Raman spectroscopy, respectively.

  9. Impacts of diversification of cytochrome P450 on plant metabolism.

    PubMed

    Mizutani, Masaharu

    2012-01-01

    Cytochrome P450 monooxygenases (P450s) catalyze a wide variety of monooxygenation reactions in primary and secondary metabolism in plants. The share of P450 genes in each plant genome is estimated to be up to 1%. This implies that the diversification of P450 has made a significant contribution to the ability to acquire the emergence of new metabolic pathways during land plant evolution. The P450 families conserved universally in land plants contribute to their chemical defense mechanisms. Several P450s are involved in the biosynthesis and catabolism of plant hormones. Species-specific P450 families are essential for the biosynthetic pathways of phytochemicals such as terpenoids and alkaloids. Genome wide analysis of the gene clusters including P450 genes will provide a clue to defining the metabolic roles of orphan P450s. Metabolic engineering with plant P450s is an important technology for large-scale production of valuable phytochemicals such as medicines.

  10. Novel Bioactivation Pathway of Benzbromarone Mediated by Cytochrome P450.

    PubMed

    Kitagawara, Yumina; Ohe, Tomoyuki; Tachibana, Kumiko; Takahashi, Kyoko; Nakamura, Shigeo; Mashino, Tadahiko

    2015-09-01

    Benzbromarone (BBR) is a hepatotoxic drug, but the detailed mechanism of its toxicity remains unknown. We identified 2,6-dibromohydroquinone (DBH) and mono-debrominated catechol (2-ethyl-3-(3-bromo-4,5-dihydroxybenzoyl)benzofuran; CAT) as novel metabolites of BBR in rat and human liver microsomal systems by comparison with chemically synthesized authentic compounds, and we also elucidated that DBH is formed by cytochrome P450 2C9 and that CAT is formed mainly by CYP1A1, 2D6, 2E1, and 3A4. Furthermore, CAT, DBH, and the oxidized form of DBH are highly cytotoxic in HepG2 compared with BBR. Taken together, our data demonstrate that DBH, a novel reactive metabolite, may be relevant to BBR-induced hepatotoxicity. PMID:26106235

  11. Biogenesis and Assembly of Eukaryotic Cytochrome c Oxidase Catalytic Core

    PubMed Central

    Soto, Ileana C.; Fontanesi, Flavia; Liu, Jingjing; Barrientos, Antoni

    2011-01-01

    Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin which assembly is intricate and highly regulated. The COX catalytic core is formed by three mitochondrial DNA encoded subunits, Cox1, Cox2 and Cox3, conserved in the bacterial enzyme. Their biogenesis requires the action of messenger-specific and subunit-specific factors which facilitate the synthesis, membrane insertion, maturation or assembly of the core subunits. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to identify these ancillary factors. Here we review the current state of knowledge of the biogenesis and assembly of the eukaryotic COX catalytic core and discuss the degree of conservation of the players and mechanisms operating from yeast to human. PMID:21958598

  12. [Cytochrome P450 enzymes and microbial drug development - A review].

    PubMed

    Li, Zhong; Zhang, Wei; Li, Shengying

    2016-03-01

    Cytochrome P450 enzymes broadly exist in animals, plants and microorganisms. This superfamily of monooxygenases holds the greatest diversity of substrate structures and catalytic reaction types among all enzymes. P450 enzymes play important roles in natural product biosynthesis. In particular, P450 enzymes are capable of catalyzing the regio- and stereospecific oxidation of non-activated C-H bonds in complex organic compounds under mild conditions, which overrides many chemical catalysts. This advantage thus warrants their great potential in microbial drug development. In this review, we introduce a variety of P450 enzymes involved in natural product biosynthesis; provide a brief overview on protein engineering, biotransformation and practical application of P450 enzymes; and discuss the limits, challenges and prospects of industrial application of P450 enzymes.

  13. Role of cytochrome P450 in drug interactions

    PubMed Central

    Bibi, Zakia

    2008-01-01

    Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events. PMID:18928560

  14. Cytochrome c peroxidase activity of heme bound amyloid β peptides.

    PubMed

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh

    2016-09-01

    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP. PMID:27270708

  15. Cytochrome P450 epoxygenase pathway of polyunsaturated fatty acid metabolism

    PubMed Central

    Spector, Arthur A.; Kim, Hee-Yong

    2014-01-01

    Polyunsaturated fatty acids (PUFA) are oxidized by cytochrome P450 epoxygenases to PUFA epoxides which function as potent lipid mediators. The major metabolic pathways of PUFA epoxides are incorporation into phospholipids and hydrolysis to the corresponding PUFA diols by soluble epoxide hydrolase. Inhibitors of soluble epoxide hydrolase stabilize PUFA epoxides and potentiate their functional effects. The epoxyeicosatrienoic acids (EETs) synthesized from arachidonic acid produce vasodilation, stimulate angiogenesis, have anti-inflammatory actions, and protect the heart against ischemia-reperfusion injury. EETs produce these functional effects by activating receptor-mediated signaling pathways and ion channels. The epoxyeicosatetraenoic acids synthesized from eicosapentaenoic acid and epoxydocosapentaenoic acids synthesized from docosahexaenoic acid are potent inhibitors of cardiac arrhythmias. Epoxydocosapentaenoic acids also inhibit angiogenesis, decrease inflammatory and neuropathic pain, and reduce tumor metastasis. These findings indicate that a number of the beneficial functions of PUFA may be due to their conversion to PUFA epoxides. PMID:25093613

  16. [Cytochrome P450 enzymes and microbial drug development - A review].

    PubMed

    Li, Zhong; Zhang, Wei; Li, Shengying

    2016-03-01

    Cytochrome P450 enzymes broadly exist in animals, plants and microorganisms. This superfamily of monooxygenases holds the greatest diversity of substrate structures and catalytic reaction types among all enzymes. P450 enzymes play important roles in natural product biosynthesis. In particular, P450 enzymes are capable of catalyzing the regio- and stereospecific oxidation of non-activated C-H bonds in complex organic compounds under mild conditions, which overrides many chemical catalysts. This advantage thus warrants their great potential in microbial drug development. In this review, we introduce a variety of P450 enzymes involved in natural product biosynthesis; provide a brief overview on protein engineering, biotransformation and practical application of P450 enzymes; and discuss the limits, challenges and prospects of industrial application of P450 enzymes. PMID:27382792

  17. The production of ammonia by multiheme cytochromes C.

    PubMed

    Simon, Jörg; Kroneck, Peter M H

    2014-01-01

    The global biogeochemical nitrogen cycle is essential for life on Earth. Many of the underlying biotic reactions are catalyzed by a multitude of prokaryotic and eukaryotic life forms whereas others are exclusively carried out by microorganisms. The last century has seen the rise of a dramatic imbalance in the global nitrogen cycle due to human behavior that was mainly caused by the invention of the Haber-Bosch process. Its main product, ammonia, is a chemically reactive and biotically favorable form of bound nitrogen. The anthropogenic supply of reduced nitrogen to the biosphere in the form of ammonia, for example during environmental fertilization, livestock farming, and industrial processes, is mandatory in feeding an increasing world population. In this chapter, environmental ammonia pollution is linked to the activity of microbial metalloenzymes involved in respiratory energy metabolism and bioenergetics. Ammonia-producing multiheme cytochromes c are discussed as paradigm enzymes. PMID:25416396

  18. Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

    PubMed Central

    Pachence, J M; Amador, S; Maniara, G; Vanderkooi, J; Dutton, P L; Blasie, J K

    1990-01-01

    We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c

  19. Genomic analyses of bacterial porin-cytochrome gene clusters

    DOE PAGES

    Shi, Liang; Fredrickson, James K.; Zachara, John M.

    2014-11-26

    In this study, the porin-cytochrome (Pcc) protein complex is responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III) by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens PCA. The identified and characterized Pcc complex of G. sulfurreducens PCA consists of a porin-like outer-membrane protein, a periplasmic 8-heme c type cytochrome (c-Cyt) and an outer-membrane 12-heme c-Cyt, and the genes encoding the Pcc proteins are clustered in the same regions of genome (i.e., the pcc gene clusters) of G. sulfurreducens PCA. A survey of additionally microbial genomes has identified the pcc gene clusters in all sequenced Geobacter spp. and other bacteriamore » from six different phyla, including Anaeromyxobacter dehalogenans 2CP-1, A. dehalogenans 2CP-C, Anaeromyxobacter sp. K, Candidatus Kuenenia stuttgartiensis, Denitrovibrio acetiphilus DSM 12809, Desulfurispirillum indicum S5, Desulfurivibrio alkaliphilus AHT2, Desulfurobacterium thermolithotrophum DSM 11699, Desulfuromonas acetoxidans DSM 684, Ignavibacterium album JCM 16511, and Thermovibrio ammonificans HB-1. The numbers of genes in the pcc gene clusters vary, ranging from two to nine. Similar to the metal-reducing (Mtr) gene clusters of other Fe(III)-reducing bacteria, such as Shewanella spp., additional genes that encode putative c-Cyts with predicted cellular localizations at the cytoplasmic membrane, periplasm and outer membrane often associate with the pcc gene clusters. This suggests that the Pcc-associated c-Cyts may be part of the pathways for extracellular electron transfer reactions. The presence of pcc gene clusters in the microorganisms that do not reduce solid-phase Fe(III) and Mn(IV) oxides, such as D. alkaliphilus AHT2 and I. album JCM 16511, also suggests that some of the pcc gene clusters may be involved in extracellular electron transfer reactions with the substrates other than Fe(III) and Mn(IV) oxides.« less

  20. Electrostatic properties of cytochrome f: implications for docking with plastocyanin.

    PubMed Central

    Pearson, D C; Gross, E L; David, E S

    1996-01-01

    The electrostatic properties of cytochrome f (cyt f), a member of the cytochrome b6f complex and reaction partner with plastocyanin (PC) in photosynthetic electron transport, are qualitatively studied with the goal of determining the mechanism of electron transfer between cyt f and PC. A crystal structure for cyt f was analyzed with the software package GRASP, revealing a large region of positive potential generated by a patch of positively charged residues (including K58, K65, K66, K122, K185, K187, and R209) and reinforced by the iron center of the heme. This positive field attracts the negative charges of the two acidic patches on the mobile electron carrier PC. Three docked complexes are obtained for the two proteins, based on electrostatic or hydrophobic interactions or both and on steric fits by manual docking methods. The first of these three complexes shows strong electrostatic interactions between K187 on cyt f and D44 on PC and between E59 on PC and K58 on cyt f. Two other manually docked complexes are proposed, implicating H87 on PC as the electron-accepting site from the iron center of cyt f through Y1. The second complex maintains the D44/K187 cross-link (but not the E59/K58 link) while increasing hydrophobic interactions between PC and cyt f. Hydrophobic interactions are increased still further in the third complex, whereas the link between K187 on cyt f and D44 on PC is broken. The proposed reaction mechanism, therefore, involves an initial electrostatic docking complex that gives rise to a nonpolar attraction between the regions surrounding H87 on PC and Y1 on cyt f, providing for an electron-transfer active complex. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:8804589

  1. The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling

    PubMed Central

    Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

    2013-01-01

    The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450–CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate–enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies. PMID:23844938

  2. Human bone marrow niche chemoprotection mediated by cytochrome p450 enzymes

    PubMed Central

    Alonso, Salvador; Su, Meng; Jones, Jace W.; Ganguly, Sudipto; Kane, Maureen A.; Jones, Richard J.; Ghiaur, Gabriel

    2015-01-01

    Substantial evidence now demonstrates that interactions between the tumor microenvironment and malignant cells are a critical component of clinical drug resistance. However, the mechanisms responsible for microenvironment-mediated chemoprotection remain unclear. We showed that bone marrow (BM) stromal cytochrome P450 (CYP)26 enzymes protect normal hematopoietic stem cells (HSCs) from the pro-differentiation effects of retinoic acid. Here, we investigated if stromal expression of CYPs is a general mechanism of chemoprotection. We found that similar to human hepatocytes, human BM-derived stromal cells expressed a variety of drug-metabolizing enzymes. CYP3A4, the liver's major drug-metabolizing enzyme, was at least partially responsible for BM stroma's ability to protect multiple myeloma (MM) and leukemia cells from bortezomib and etoposide, respectively, both in vitro and in vivo. Moreover, clarithromycin overcame stromal-mediated MM resistance to dexamethasone, suggesting that CYP3A4 inhibition plays a role in its ability to augment the activity of lenalidomide and dexamethasone as part of the BiRd regimen. We uncovered a novel mechanism of microenvironment-mediated drug resistance, whereby the BM niche creates a sanctuary site from drugs. Targeting these sanctuaries holds promise for eliminating minimal residual tumor and improving cancer outcomes. PMID:25915157

  3. The effects of flubendazole and mebendazole on cytochromes P4501A in pheasant hepatocytes.

    PubMed

    Savlík, M; Polásková, P; Szotáková, B; Lamka, J; Skálová, L

    2005-10-01

    Many benzimidazoles are known inducers of cytochromes P4501A (CYP1A) in laboratory animals and cell lines. As flubendazole and mebendazole are benzimidazole anthelmintics often used in a pheasant, in the present study an effect of these drugs in primary cultures of pheasant (Phasianus colchicus) hepatocytes was investigated. After 48 h incubation of the hepatocytes with the benzimidazoles (0.2-5 microM), CYP1A activities -- ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-demethylation (MROD) activities were measured and the CYP1A protein levels were determined by Western blotting. None of the tested benzimidazoles influenced the CYP1A protein content. No pharmacologically significant enhancement of CYP1A after exposure of the hepatocytes to flubendazole and mebendazole was found. Inhibition of the EROD/MROD activities caused by both tested substances was observed only at the highest concentration (5 microM). From a point of view of CYP1A induction or inhibition, the treatment of pheasants by both anthelmintics tested seems to be safe. Our study demonstrates the inter-species differences in CYP1A inducibility and the importance of induction/inhibition studies on target animals.

  4. Development of NanoART for HIV Treatment: Minding the Cytochrome P450 (CYP) Enzymes

    PubMed Central

    Midde, Narasimha M.; Kumar, Santosh

    2015-01-01

    Sustained suppression of HIV viral load is the primary objective for HIV treatment, which successfully achieved by the use of a wide array of antiretroviral therapies (ART). Despite this enormous success low level of virus persists in the anatomical and cellular reservoirs of the body causing a multitude of immunological and neurocognitive deficits. Towards this, nano-formulations are gaining attention to solve these problems by delivering ART to the targeted locations such as brain, lymphoid tissues, and monocytes/macrophages. As cytochrome P450 (CYP) enzymes play a critical role in the metabolism of drugs and other xenobiotics, it is expected that the interaction of nanoparticles with CYP enzymes may result in adverse drug reactions, cellular toxicity, and alterations in CYP-mediated metabolism of other drug molecules. Considering these potential adverse outcomes it is imperative to design the nano-carriers that will have minimal impact on CYP enzymes. Therefore, developing a long-acting nanoART regimen with minimal side effects is an essential step to improve patient’s adherence to the treatment paradigm, effective treatment strategy, and to combat the HIV infection & AIDS. PMID:26635972

  5. Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

    PubMed Central

    Daley, Daniel O.; Clifton, Rachel; Whelan, James

    2002-01-01

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2. PMID:12142462

  6. Inhibitory effect of Aristolochia fruit on Cytochrome P450 isozymes in vitro and in vivo.

    PubMed

    Zhu, Shuzhen; Gao, Jingwen; Shi, Zhan; QLi, George; Chen, Zuanguang; Yao, Meicun

    2015-05-01

    The mature fruits of Aristolochia debilis, known in China by the name, "Madouling" has been popularly prescribed in Asia, particularly in China, to treat a range of conditions including gynaecological problems, arthritis and wound healing. This study was aimed to evaluate the potential effect of Madouling on the cytochrome P450 (CYP) isozymes in vitro in microsomal fractions and in vivo in rats. The influence of Madouling on CYPs activity was first explored by an in vitro method of estimating levels of four respective metabolites in rat liver microsomes. The results were re-examined in vivo in rats by using a cocktail approach involving the probe drugs theophylline, tolbutamide, chlorzoxazone and dapsone. Pharmacokinetics of the four substrates was used to analyze the activities of the targeting isozymes. In vitro study revealed that Madouling decreased the activity of CYP1A2, 3A1 and 2E1. However, no significant influence on CYP2C6 was found. These results coincided with those of in vivo study to a great degree except that in vivo estimation the herb didn't inhibit CYP1A2 significantly. From the data obtained, Madouling is suggested as a candidate for clinically significant CYP interactions. Drug co-administrated with Madouling may need dose adjustment.

  7. Effect of ergot alkaloids associated with fescue toxicosis on hepatic cytochrome P450 and antioxidant proteins

    SciTech Connect

    Settivari, Raja S.; Evans, Tim J.; Rucker, Ed; Rottinghaus, George E.; Spiers, Donald E.

    2008-03-15

    Intake of ergot alkaloids found in endophyte-infected tall fescue grass is associated with decreased feed intake and reduction in body weight gain. The liver is one of the target organs of fescue toxicosis with upregulation of genes involved in xenobiotic metabolism and downregulation of genes associated with antioxidant pathways. It was hypothesized that short-term exposure of rats to ergot alkaloids would change hepatic cytochrome P450 (CYP) and antioxidant expression, as well as reduce antioxidant enzyme activity and hepatocellular proliferation rates. Hepatic gene expression of various CYPs, selected nuclear receptors associated with the CYP induction, and antioxidant enzymes were measured using real-time PCR. Hepatic expression of CYP, antioxidant and proliferating cell nuclear antigen (PCNA) proteins were measured using Western blots. The CYP3A1 protein expression was evaluated using primary rat hepatocellular cultures treated with ergovaline, one of the major ergot alkaloids produced by fescue endophyte, in order to assess the direct role of ergot alkaloids in CYP induction. The enzyme activities of selected antioxidants were assayed spectrophotometrically. While hepatic CYP and nuclear receptor expression were increased in ergot alkaloid-exposed rats, the expression and activity of antioxidant enzymes were reduced. This could potentially lead to increased oxidative stress, which might be responsible for the decrease in hepatocellular proliferation after ergot alkaloid exposure. This study demonstrated that even short-term exposure to ergot alkaloids can potentially induce hepatic oxidative stress which can contribute to the pathogenesis of fescue toxicosis.

  8. Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis

    PubMed Central

    2016-01-01

    Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products. PMID:27119279

  9. The Effect of Vinpocetine on Human Cytochrome P450 Isoenzymes by Using a Cocktail Method

    PubMed Central

    Kong, Lingti; Song, Chunli; Ye, Linhu; Guo, Daohua; Yu, Meiling; Xing, Rong

    2016-01-01

    Vinpocetine is a derivative of the alkaloid vincamine, which had been prescribed for chronic cerebral vascular ischemia and acute ischemic stroke or used as a dietary supplement for its several different mechanisms of biological activities. However, information on the cytochrome P450 (CYP) enzyme-mediated drug metabolism has not been previously studied. The present study was performed to investigate the effects of vinpocetine on CYPs activity, and cocktail method was used, respectively. To evaluate the effects of vinpocetine on the activity of human CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, human liver microsomes were utilized to incubate with the mixed CYPs probe substrates and the target components. The results indicate that vinpocetine exhibited weak inhibitory effect on the CYP2C9, where the IC50 value is 68.96 μM, whereas the IC50 values for CYP3A4, CYP2C19, CYP2D6, and CYP2E1 were all over range of 100 μM, which showed that vinpocetine had no apparent inhibitory effects on these CYPs. In conclusion, the results indicated that drugs metabolized by CYP2C9 coadministrated with vinpocetine may require attention or dose adjustment. PMID:27006677

  10. Regulation of Porcine Hepatic Cytochrome P450 — Implication for Boar Taint

    PubMed Central

    Rasmussen, Martin Krøyer; Zamaratskaia, Galia

    2014-01-01

    Cytochrome P450 (CYP450) is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole), one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint. PMID:25408844

  11. Regulation of porcine hepatic cytochrome p450 - implication for boar taint.

    PubMed

    Rasmussen, Martin Krøyer; Zamaratskaia, Galia

    2014-09-01

    Cytochrome P450 (CYP450) is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole), one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint. PMID:25408844

  12. Modeling of interactions between xenobiotics and cytochrome P450 (CYP) enzymes

    PubMed Central

    Raunio, Hannu; Kuusisto, Mira; Juvonen, Risto O.; Pentikäinen, Olli T.

    2015-01-01

    The adverse effects to humans and environment of only few chemicals are well known. Absorption, distribution, metabolism, and excretion (ADME) are the steps of pharmaco/toxicokinetics that determine the internal dose of chemicals to which the organism is exposed. Of all the xenobiotic-metabolizing enzymes, the cytochrome P450 (CYP) enzymes are the most important due to their abundance and versatility. Reactions catalyzed by CYPs usually turn xenobiotics to harmless and excretable metabolites, but sometimes an innocuous xenobiotic is transformed into a toxic metabolite. Data on ADME and toxicity properties of compounds are increasingly generated using in vitro and modeling (in silico) tools. Both physics-based and empirical modeling approaches are used. Numerous ligand-based and target-based as well as combined modeling methods have been employed to evaluate determinants of CYP ligand binding as well as predicting sites of metabolism and inhibition characteristics of test molecules. In silico prediction of CYP–ligand interactions have made crucial contributions in understanding (1) determinants of CYP ligand binding recognition and affinity; (2) prediction of likely metabolites from substrates; (3) prediction of inhibitors and their inhibition potency. Truly predictive models of toxic outcomes cannot be created without incorporating metabolic characteristics; in silico methods help producing such information and filling gaps in experimentally derived data. Currently modeling methods are not mature enough to replace standard in vitro and in vivo approaches, but they are already used as an important component in risk assessment of drugs and other chemicals. PMID:26124721

  13. Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

    PubMed

    Hrycay, E G; Bandiera, S M

    2009-12-01

    The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

  14. LIQUID TARGET

    DOEpatents

    Martin, M.D.; Salsig, W.W. Jr.

    1959-01-13

    A liquid handling apparatus is presented for a liquid material which is to be irradiated. The apparatus consists essentially of a reservoir for the liquid, a target element, a drain tank and a drain lock chamber. The target is in the form of a looped tube, the upper end of which is adapted to be disposed in a beam of atomic particles. The lower end of the target tube is in communication with the liquid in the reservoir and a means is provided to continuously circulate the liquid material to be irradiated through the target tube. Means to heat the reservoir tank is provided in the event that a metal is to be used as the target material. The apparatus is provided with suitable valves and shielding to provide maximum safety in operation.

  15. Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation.

    PubMed Central

    Bott, M; Thöny-Meyer, L; Loferer, H; Rossbach, S; Tully, R E; Keister, D; Appleby, C A; Hennecke, H

    1995-01-01

    Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor. PMID:7721713

  16. 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.

  17. Influence of copper on the interaction between cytochrome c and sulfite in vitro.

    PubMed

    Wang, Jinsheng; Guo, Dongsheng; Yuan, Xiaoying

    2006-01-01

    The quantitative influence of copper on the interaction between cytochrome c and sulfite, which is a derivate of sulfur dioxide in the human body, has been studied by fluorescence spectrometry and ultraviolet absorption spectrometry in vitro. The results indicate that copper may intensely combine with protein component and ferroporphyrin component in cytochrome c at the concentration of 0.1 mM, and the respective association constants (K(A)) are 3.77 x 10(4) L mol(-1) and 9.38 x 10(3) L mol(-1). Sulfite has little interaction with the protein component in cytochrome c (K(A) = 0.094 L mol(-1)), at either low concentrations or relatively high concentrations (<0.15 M). However, it can react with the ferroporphyrin component in cytochrome c (K(A) = 4.297 L mol(-1)). After copper is added to the sulfite-cytochrome c binary systems, the reaction between sulfite and the protein component in cytochrome c is obviously strengthened at a low concentration (K(A) = 7.289 L mol(-1)), while the addition of copper merely has a little effect on the interaction between sulfite and the ferroporphyrin component in cytochrome c. PMID:17009250

  18. Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.

    PubMed Central

    Menting, J G; Cornish, E; Scopes, R K

    1994-01-01

    NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4). PMID:7991686

  19. Comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis

    PubMed Central

    Dorner, Mariah E; McMunn, Ryan D; Bartholow, Thomas G; Calhoon, Brecken E; Conlon, Michelle R; Dulli, Jessica M; Fehling, Samuel C; Fisher, Cody R; Hodgson, Shane W; Keenan, Shawn W; Kruger, Alyssa N; Mabin, Justin W; Mazula, Daniel L; Monte, Christopher A; Olthafer, Augustus; Sexton, Ashley E; Soderholm, Beatrice R; Strom, Alexander M; Hati, Sanchita

    2015-01-01

    Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes. PMID:26130403

  20. 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. PMID:26806033

  1. Chlorella saccharophila cytochrome f and its involvement in the heat shock response

    PubMed Central

    Zuppini, Anna; Gerotto, Caterina; Moscatiello, Roberto; Bergantino, Elisabetta; Baldan, Barbara

    2009-01-01

    Cytochrome f is an essential component of the major redox complex of the thylakoid membrane. Cloning and characterization are presented here of a novel partial cDNA (ChspetA) encoding cytochrome f in the psychrophile unicellular green alga Chlorella saccharophila and its involvement in the heat shock (HS) response pathway has been analysed. Semi-quantitative reverse transcriptase PCR analysis showed that ChspetA expression is up-regulated in heat-shocked cells and the protein profile of cytochrome f highlighted a release of cytochrome f into the cytosol depending on the time lapse from the HS. Evans Blue assay, analysis of chromatin condensation, and chloroplast alterations showed the induction of cell death in cell suspensions treated with cytosolic extracts from heat-shocked cells. This study identifies cytochrome f in C. saccharophila that seems to be involved in the HS-induced programmed cell death process. The data suggest that cytochrome f fulfils its role through a modulation of its transcription and translation levels, together with its intracellular localization. This work focuses on a possible role of cytochrome f into the programmed cell death-like process in a unicellular chlorophyte and suggests the existence of chloroplast-mediated programmed cell death machinery in an organism belonging to one of the primary lineages of photosynthetic eukaryotes. PMID:19773387

  2. Bioconcentration of Ag, Cd, Co, Mn and Zn in the Mangrove Oyster (Crassostrea gasar) and Preliminary Human Health Risk Assessment: A Radiotracer Study.

    PubMed

    Kuranchie-Mensah, Harriet; Teyssié, Jean-Louis; Oberhänsli, François; Tumnoi, Yutthana; Pouil, Simon; Warnau, Michel; Metian, Marc

    2016-09-01

    Bioaccumulation kinetics of five dissolved metals were determined in the mangrove oyster Crassostrea gasar, using corresponding radiotracers ((54)Mn, (57)Co, (65)Zn, (109)Cd and (110m)Ag). Additionally, their bioaccessibility to human consumers was estimated. Results indicated that over a 14-day exposure (54)Mn and (57)Co were linearly concentrated in oysters whereas (109)Cd, (65)Zn and (110m)Ag were starting to saturate (steady-state not reached). Whole-body concentration factors at 14 days (CF14d in toto) ranged from 187 ± 65 to 629 ± 179 with the lowest bioconcentration capacity for Co and the highest for Ag. Depuration kinetics were best described by a double-exponential model with associated biological half-lives ranging from 26 days (Ag) to almost 8 months (Zn and Cd). Bioaccessible fraction of the studied elements was estimated using in vitro digestions, which suggested that oysters consumed seasoned with lemon enhanced the accessibility of Cd, Mn and Zn to human consumers, but not Ag and Co. PMID:27194421

  3. Affinity of (nat/68)Ga-Labelled Curcumin and Curcuminoid Complexes for β-Amyloid Plaques: Towards the Development of New Metal-Curcumin Based Radiotracers.

    PubMed

    Rubagotti, Sara; Croci, Stefania; Ferrari, Erika; Iori, Michele; Capponi, Pier C; Lorenzini, Luca; Calzà, Laura; Versari, Annibale; Asti, Mattia

    2016-01-01

    Curcumin derivatives labelled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer's disease. Nevertheless, no study by exploiting the labelling with gallium-68 has been performed so far, in spite of its suitable properties (positron emitter, generator produced radionuclide). Herein, an evaluation of the affinity for synthetic β-amyloid fibrils and for amyloid plaques of three (nat/68)Ga-labelled curcumin analogues, namely curcumin curcumin (CUR), bis-dehydroxy-curcumin (bDHC) and diacetyl-curcumin (DAC), was performed. Affinity and specificity were tested in vitro on amyloid synthetic fibrils by using gallium-68 labelled compounds. Post-mortem brain cryosections from Tg2576 mice were used for the ex vivo visualization of amyloid plaques. The affinity of (68)Ga(CUR)₂⁺, (68)Ga(DAC)₂⁺, and (68)Ga(bDHC)₂⁺ for synthetic β-amyloid fibrils was moderate and their uptake could be observed in vitro. On the other hand, amyloid plaques could not be visualized on brain sections of Tg2576 mice after injection, probably due to the low stability of the complexes in vivo and of a hampered passage through the blood-brain barrier. Like curcumin, all (nat/68)Ga-curcuminoid complexes maintain a high affinity for β-amyloid plaques. However, structural modifications are still needed to improve their applicability as radiotracers in vivo. PMID:27608011

  4. Radiotracers binding to estrogen receptors: I: tissue distribution of 17 alpha-ethynylestradiol and moxestrol in normal and tumor-bearing rats

    SciTech Connect

    Feenstra, A.; Nolten, G.M.; Vaalburg, W.; Reiffers, S.; Woldring, M.G.

    1982-07-01

    Ethynylestradiol and moxestrol can be labeled with /sup 11/C by introducing this positron emitter in the 17 alpha-ethynyl group. To investigate their potential as radiotracers binding to estrogen receptors, researchers studied the tissue distribution of tritiated ethynylestradiol and moxestrol, with specific activities of 57 Ci/mmol and 77-90 Ci/mmol, respectively, in the adult female rat. At 30 min after injection, both compounds showed specific uptake in the uterus (% dose/g): 2.52 for ethynylestradiol and of 2.43 for moxestrol. A decrease of the specific activity to 6-9 Ci/mmol resulted in uterine uptakes of 1.60 and 2.10 respectively, for ethynylestradiol and moxestrol, at 30 min. In the female rat bearing DMBA-induced mammary tumors, specific uptake was also measured in the tumors, although the values were only 25-30% of the uterine uptake. Moxestrol showed a greater uptake selectivity in the tumors compared with ethynylestradiol. From this study researchers conclude that ethynylestradiol and moxestrol have good potential as tracers binding to mammary tumors that contain estrogen receptors.

  5. Affinity of nat/68Ga-Labelled Curcumin and Curcuminoid Complexes for β-Amyloid Plaques: Towards the Development of New Metal-Curcumin Based Radiotracers

    PubMed Central

    Rubagotti, Sara; Croci, Stefania; Ferrari, Erika; Iori, Michele; Capponi, Pier C.; Lorenzini, Luca; Calzà, Laura; Versari, Annibale; Asti, Mattia

    2016-01-01

    Curcumin derivatives labelled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer’s disease. Nevertheless, no study by exploiting the labelling with gallium-68 has been performed so far, in spite of its suitable properties (positron emitter, generator produced radionuclide). Herein, an evaluation of the affinity for synthetic β-amyloid fibrils and for amyloid plaques of three nat/68Ga-labelled curcumin analogues, namely curcumin curcumin (CUR), bis-dehydroxy-curcumin (bDHC) and diacetyl-curcumin (DAC), was performed. Affinity and specificity were tested in vitro on amyloid synthetic fibrils by using gallium-68 labelled compounds. Post-mortem brain cryosections from Tg2576 mice were used for the ex vivo visualization of amyloid plaques. The affinity of 68Ga(CUR)2+, 68Ga(DAC)2+, and 68Ga(bDHC)2+ for synthetic β-amyloid fibrils was moderate and their uptake could be observed in vitro. On the other hand, amyloid plaques could not be visualized on brain sections of Tg2576 mice after injection, probably due to the low stability of the complexes in vivo and of a hampered passage through the blood–brain barrier. Like curcumin, all nat/68Ga-curcuminoid complexes maintain a high affinity for β-amyloid plaques. However, structural modifications are still needed to improve their applicability as radiotracers in vivo. PMID:27608011

  6. Depuration and uptake kinetics of I, Cs, Mn, Zn and Cd by the earthworm (Lumbricus terrestris) in radiotracer-spiked litter

    SciTech Connect

    Sheppard, S.C.; Evenden, W.G.; Cornwell, T.C.

    1997-10-01

    The relative depuration and uptake kinetics of contaminants should be known to interpret appropriately the use of organisms such as earthworms in environmental bioassays and monitoring. For example, 14-d earthworm bioassays should be interpreted with the knowledge that some contaminants will continue to accumulate in tissues for months. The radiotracers {sup 125}I, {sup 134}Cs, {sup 54}Mn, {sup 65}Zn, and {sup 109}Cd were applied to deciduous litter and specimens of Lumbricus terrestris were exposed, either to litter alone or to litter on the top of soil columns. Depuration was monitored for 120 d and uptake, in a separate experiment, for 20 d. Both depuration and uptake were described using two-phase, first-order statistical models. Cut clearance had a mean half-time of 1.4 d. The mean half-time for physiological depuration decreased from I (210 d) > Cd (150 d) > Zn (69 d) > Mn (40 d) > Cs (24 d). Both the depuration and the uptake experiments were necessary to resolve even partially the multiphase processes. Earthworm/soil dry weight concentration ratios decreased from Cd > Zn > I {ge} Cs {ge} Mn. The very slow kinetics indicate that tissue concentrations will increase continuously for a long time, with important implications for subsequent food-chain transfers.

  7. Metabolism, distribution, and excretion of deoxynivalenol with combined techniques of radiotracing, high-performance liquid chromatography ion trap time-of-flight mass spectrometry, and online radiometric detection.

    PubMed

    Wan, Dan; Huang, Lingli; Pan, Yuanhu; Wu, Qinghua; Chen, Dongmei; Tao, Yanfei; Wang, Xu; Liu, Zhenli; Li, Juan; Wang, Liye; Yuan, Zonghui

    2014-01-01

    Dispositions of deoxynivalenol (DON) in rats and chickens were investigated, using a radiotracer method coupled with a novel γ-accurate radioisotope counting (γ-ARC) radio-high-performance liquid chromatography ion trap time-of-flight tandem mass spectrometry (radio-HPLC-IT-TOF-MS/MS) system. 3β-(3)H-DON was chemically synthesized and orally administrated to both sexes of rats and chickens as single or multiple doses. The results showed that DON was widely distributed and quickly eliminated in all tissues. The highest concentration was found in the gastrointestinal tract at 6 h post-administration. Substantially lower levels were detected in the kidney, liver, heart, lung, spleen, and brain. Three new metabolites were identified tentatively as 10-deoxynivalenol-sulfonate, 10-deepoxy-deoxynivalenol (DOM-1)-sulfonate, and deoxynivalenol-3α-sulfate. Deoxynivalenol-3α-sulfate was a major metabolite in chickens, while the major forms in rats were DOM-1 and DON. Additionally, a higher excretion rate in urine was observed in female rats than in male rats. The differences in metabolite profiles and excretion rates, which suggested diverse ways to detoxify, may relate to the different tolerances in different genders or species.

  8. Methodological considerations regarding the use of inorganic 197Hg(II) radiotracer to assess mercury methylation potential rates in lake sediment.

    PubMed

    Pérez Catán, Soledad; Guevara, Sergio Ribeiro; Marvin-DiPasquale, Mark; Magnavacca, Cecilia; Cohen, Isaac Marcos; Arribere, María

    2007-09-01

    Methodological considerations on the determination of benthic methyl-mercury (CH(3)Hg) production potentials were investigated on lake sediment, using (197)Hg radiotracer. Three methods to arrest bacterial activity were compared: flash freezing, thermal sterilization, and gamma-irradiation. Flash freezing showed similar CH(3)Hg recoveries as thermal sterilization, which was both 50% higher than the recoveries obtained with gamma-ray irradiation. No additional radiolabel was recovered in kill-control samples after an additional 24 or 65 h of incubation, suggesting that all treatments were effective at arresting Hg(II)-methylating bacterial activity, and that the initial recoveries are likely due to non-methylated (197)Hg(II) carry-over in the organic extraction and/or [(197)Hg]CH(3)Hg produced via abiotic reactions. Two CH(3)Hg extraction methods from sediment were compared: (a) direct extraction into toluene after sediment leaching with CuSO(4) and HCl and (b) the same extraction with an additional back-extraction step to thiosulphate. Similar information was obtained with both methods, but the low efficiency observed and the extra work associated with the back-extraction procedure represent significant disadvantages, even tough the direct extraction involves higher Hg(II) carry over.

  9. Methodological considerations regarding the use of inorganic 197Hg(II) radiotracer to assess mercury methylation potential rates in lake sediment

    USGS Publications Warehouse

    Perez, Catan S.; Guevara, S.R.; Marvin-DiPasquale, M.; Magnavacca, C.; Cohen, I.M.; Arribere, M.

    2007-01-01

    Methodological considerations on the determination of benthic methyl-mercury (CH3Hg) production potentials were investigated on lake sediment, using 197Hg radiotracer. Three methods to arrest bacterial activity were compared: flash freezing, thermal sterilization, and ??-irradiation. Flash freezing showed similar CH3Hg recoveries as thermal sterilization, which was both 50% higher than the recoveries obtained with ??-ray irradiation. No additional radiolabel was recovered in kill-control samples after an additional 24 or 65 h of incubation, suggesting that all treatments were effective at arresting Hg(II)-methylating bacterial activity, and that the initial recoveries are likely due to non-methylated 197Hg(II) carry-over in the organic extraction and/or [197Hg]CH3Hg produced via abiotic reactions. Two CH3Hg extraction methods from sediment were compared: (a) direct extraction into toluene after sediment leaching with CuSO4 and HCl and (b) the same extraction with an additional back-extraction step to thiosulphate. Similar information was obtained with both methods, but the low efficiency observed and the extra work associated with the back-extraction procedure represent significant disadvantages, even tough the direct extraction involves higher Hg(II) carry over. ?? 2007 Elsevier Ltd. All rights reserved.

  10. Synthesis and evaluation of inhaled [11C]butane and intravenously injected [11C]acetone as potential radiotracers for studying inhalant abuse.

    PubMed

    Gerasimov, Madina R; Ferrieri, Richard A; Pareto, Deborah; Logan, Jean; Alexoff, David; Ding, Yu-Shin

    2005-02-01

    The phenomenon of inhalant abuse is a growing problem in the US and many countries around the world. Yet, relatively little is known about the pharmacokinetic properties of inhalants that underlie their abuse potential. While the synthesis of 11C-labeled toluene, acetone and butane has been proposed in the literature, none of these compounds has been developed as radiotracers for PET studies. In the present report we extend our previous studies with [11C]toluene to include [11C]acetone and [11C]butane with the goal of comparing the pharmacokinetic profiles of these three volatile abused substances. Both [11C]toluene and [11C]acetone were administered intravenously and [11C]butane was administered via inhalation to anesthesized baboons. Rapid and efficient uptake of radiolabeled toluene and acetone into the brain was followed by fast clearance in the case of toluene and slower kinetics in the case of acetone. [11C]Butane was detected in the blood and brain following inhalation, but the levels of radioactivity in both tissues dropped to half of the maximal values over the period of less than a minute. To our knowledge, this is the first reported study of the in vivo brain pharmacokinetics of labeled acetone and butane in nonhuman primates. These data provide insight into the pharmacokinetic features possibly associated with the abuse liability of toluene, acetone and butane.

  11. (54)Mn Radiotracers Demonstrate Continuous Dissolution and Reprecipitation of Vernadite (δ-MnO2) during Interaction with Aqueous Mn(II).

    PubMed

    Elzinga, Evert J

    2016-08-16

    (54)Mn radiotracers were used to assess Mn atom exchange between aqueous Mn(II) and vernadite (δ-MnO2) at pH 5.0. Continuous solid-liquid redistribution of (54)Mn atoms occurred, and systems are near isotopic equilibrium after reaction for 3 months. Despite this extensive exchange, X-ray diffraction and X-ray absorption spectroscopy data showed no major changes in vernadite bulk mineralogy. These results demonstrate that the vernadite-Mn(II) interface is dynamic, with the substrate undergoing continuous dissolution and reprecipitation mediated by aqueous Mn(II) without observable impacts on its mineralogy. Interfacial redox reactions between adsorbed Mn(II) and solid-phase Mn(IV) and Mn(III) are proposed as the main drivers of this process. Interaction between aqueous Mn(II) and structural Mn(III) likely involves interfacial electron transfer coupled with Mn atom exchange. The exchange of aqueous Mn(II) and solid-phase Mn(IV) is more complex and is proposed to result from coupled interfacial comproportionation-disproportionation reactions, where electron transfer from adsorbed Mn(II) to lattice Mn(IV) produces transient Mn(III) species that disproportionate to regenerate aqueous Mn(II) and structural Mn(IV). These findings provide further evidence of the importance of Mn(II)(aq)-MnO2(s) interactions and the attendant production of transient Mn(III) intermediates to the geochemical functioning of phyllomanganates in environments undergoing Mn redox cycling.

  12. A Mn-54 Radiotracer Study of Mn Isotope Solid-Liquid Exchange during Reductive Transformation of Vernadite (δ-MnO₂) by Aqueous Mn(II)

    SciTech Connect

    Elzinga, Evert J.; Kustka, Adam B.

    2015-04-09

    We employed Mn-54 radiotracers to characterize the extent and dynamics of Mn atom exchange between aqueous Mn(II) and vernadite (δ-Mn(IV)O2) at pH 7.5 under anoxic conditions. Exchange of Mn atoms between the solid and liquid phase is rapid, reaching dynamic equilibrium in 2–4 days. We propose that during the initial stages of reaction, Mn atom exchange occurs through consecutive comproportionation-disproportionation reactions where interfacial electron transfer from adsorbed Mn(II) to lattice Mn(IV) generates labile Mn(III) cations that rapidly disproportionate to reform aqueous Mn(II) and solid-phase Mn(IV). Following nucleation of Mn(III)OOH phases, additional exchange likely occurs through electron transfer from aqueous Mn(II) to solid-phase Mn(III). Our results provide evidence for the fast and extensive production of transient Mn(III) species at the vernadite surface upon contact of this substrate with dissolved Mn(II). We further show that HEPES buffer is a reductant of lattice Mn(IV) in the vernadite structure in our experiments. The methods and results presented here introduce application of Mn-54 tracers as a facile tool to further investigate the formation kinetics of labile Mn(III) surface species and their impacts on Mn-oxide structure and reactivity over a range of environmentally relevant geochemical conditions.

  13. A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect.

    PubMed Central

    Abdullaev, Ziedulla Kh; Bodrova, Marina E; Chernyak, Boris V; Dolgikh, Dmitry A; Kluck, Ruth M; Pereverzev, Mikhail O; Arseniev, Alexander S; Efremov, Roman G; Kirpichnikov, Mikhail P; Mokhova, Elena N; Newmeyer, Donald D; Roder, Heinrich; Skulachev, Vladimir P

    2002-01-01

    A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12. PMID:11879204

  14. Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver

    PubMed Central

    Zhang, Hai-Feng; Li, Zhi-Hui; Liu, Jia-Yu; Liu, Ting-Ting; Wang, Ping; Fang, Yan; Zhou, Jun; Cui, Ming-Zhu; Gao, Na; Tian, Xin; Gao, Jie; Wen, Qiang; Jia, Lin-Jing

    2016-01-01

    Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism. PMID:27271371

  15. A panel of cytochrome P450 BM3 variants to produce drug metabolites and diversify lead compounds.

    PubMed

    Sawayama, Andrew M; Chen, Michael M Y; Kulanthaivel, Palaniappan; Kuo, Ming-Shang; Hemmerle, Horst; Arnold, Frances H

    2009-11-01

    Herein we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant. PMID:19774562