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Sample records for radiotracer targeting cytochrome

  1. Radiolabeled multimeric cyclic RGD peptides as integrin alphavbeta3 targeted radiotracers for tumor imaging.

    PubMed

    Liu, Shuang

    2006-01-01

    Integrin alphavbeta3 plays a significant role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (RGD) tripeptide sequence. These include vitronectin, fibronectin, fibrinogen, lamin, collagen, Von Willibrand's factor, osteoponin, and adenovirus particles. Integrin alphavbeta3 is expressed at low levels on epithelial cells and mature endothelial cells, but it is overexpressed on the activated endothelial cells of tumor neovasculature and some tumor cells. The highly restricted expression of integrin alphavbeta3 during tumor growth, invasion, and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. In the past decade, many radiolabeled linear and cyclic RGD peptide antagonists have been evaluated as the integrin alphavbeta3 targeted radiotracers. Significant progress has been made on their use for imaging tumors of different origin by single photon emission computed tomography (SPECT) or positron emission tomography (PET) in several tumor-bearing animal models. [18F]Galacto-RGD is under clinical investigation as the first integrin alphavbeta3 targeted radiotracer for noninvasive visualization of the activated integrin alphavbeta3 in cancer patients. This review will focus on the radiolabeled multimeric cyclic RGD peptides (dimers and tetramers) useful as radiotracers to image the tumor integrin alphavbeta3 expression by SPECT and PET, and some fundamental aspects for the development of integrin alphavbeta3 targeted radiotracers. These include the choice of radionuclide and bifunctional chelators, selection of targeting biomolecules, and factors influencing the integrin alphavbeta3 binding affinity and tumor uptake, as well as different approaches for modification of radiotracer pharmacokinetics. PMID:17009846

  2. (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

  3. 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. PMID:26033907

  4. 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

  5. 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

  6. 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

  7. Optimizing tumor targeting of the lipophilic EGFR-binding radiotracer SKI 243 using a liposomal nanoparticle delivery system.

    PubMed

    Medina, Oula Penate; Pillarsetty, Nagavarakishore; Glekas, Athanasios; Punzalan, Blesida; Longo, Valerie; Gönen, Mithat; Zanzonico, Pat; Smith-Jones, Peter; Larson, Steven M

    2011-02-10

    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 [SKI 212243]), 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 SKI 243. SKI 243 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 SKI 212243 (SKI 243) with that of the same tracer embedded in liposomes. SKI 243 and liposomal SKI 243 are both taken up by tumor xenografts but liposomal SKI 243 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

  8. Evaluation of 64Cu-Labeled Acridinium Cation: A PET Radiotracer Targeting Tumor Mitochondria

    PubMed Central

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

    2011-01-01

    This report presents the synthesis and evaluations of 64Cu(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 64Cu(DO3A-xy-ACR) was excreted mainly through the renal system with >65% of injected radioactivity being recovered from urine samples at 1 h post-injection (p.i.). The tumor uptake of 64Cu(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. 64Cu(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 64Cu(DO3A-xy-ACR). The high liver radioactivity accumulation was also seen. 64Cu(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 64Cu(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 64Cu(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 64Cu-labeled acridinium cations are indeed able to localize in the energized mitochondria of tumor cells. PMID:21413736

  9. 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

  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. A Philosophy for CNS Radiotracer Design

    PubMed Central

    2015-01-01

    Conspectus 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

  13. 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

  14. A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation induced death

    PubMed Central

    Atkinson, Jeffrey; Kapralov, Alexandr A.; Yanamala, Naveena; Tyurina, Yulia Y.; Amoscato, Andrew A.; Pearce, Linda; Peterson, Jim; Huang, Zhentai; Jiang, Jianfei; Samhan-Arias, Alejandro K.; Maeda, Akihiro; Feng, Weihong; Wasserloos, Karla; Belikova, Natalia A.; Tyurin, Vladimir A.; Wang, Hong; Fletcher, Jackie; Wang, Yongsheng; Vlasova, Irina I.; Klein-Seetharaman, Judith; Stoyanovsky, Detcho A.; Bayîr, Hülya; Pitt, Bruce R.; Epperly, Michael W.; Greenberger, Joel S.; Kagan, Valerian E.

    2013-01-01

    The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of hemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids which blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its heme-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 hr before through 24 hrs after the irradiation. PMID:21988913

  15. A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death.

    PubMed

    Atkinson, Jeffrey; Kapralov, Alexandr A; Yanamala, Naveena; Tyurina, Yulia Y; Amoscato, Andrew A; Pearce, Linda; Peterson, Jim; Huang, Zhentai; Jiang, Jianfei; Samhan-Arias, Alejandro K; Maeda, Akihiro; Feng, Weihong; Wasserloos, Karla; Belikova, Natalia A; Tyurin, Vladimir A; Wang, Hong; Fletcher, Jackie; Wang, Yongsheng; Vlasova, Irina I; Klein-Seetharaman, Judith; Stoyanovsky, Detcho A; Bayîr, Hülya; Pitt, Bruce R; Epperly, Michael W; Greenberger, Joel S; Kagan, Valerian E

    2011-01-01

    The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 h before through 24 h after the irradiation. PMID:21988913

  16. Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis

    PubMed Central

    Birk, A V; Chao, W M; Bracken, C; Warren, J D; Szeto, H H

    2014-01-01

    BACKGROUND AND PURPOSE Cardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c. EXPERIMENTAL APPROACH Interactions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria. KEY RESULTS SS-31 interacted only with liposomes and bicelles containing cardiolipin in about 1:1 ratio. NMR studies demonstrated that the aromatic residues of SS-31 penetrated deep into cardiolipin-containing bilayers. SS-31 restored cytochrome c reduction and mitochondrial oxygen consumption in the presence of added cardiolipin. In fresh mitochondria, SS-31 increased state 3 respiration and efficiency of ATP synthesis. CONCLUSIONS AND IMPLICATIONS SS-31 selectively targeted cardiolipin and modulated its interaction with cytochrome c. SS-31 inhibited the cytochrome c/cardiolipin complex peroxidase activity while protecting its ability to serve as an electron carrier, thus optimizing mitochondrial electron transport and ATP synthesis. This novel class of cardiolipin therapeutics has the potential to restore mitochondrial bioenergetics for treatment of numerous age-related diseases. LINKED ARTICLES This article is

  17. Synthesis, radiolabeling and preliminary in vivo evaluation of multimodal radiotracers for PET imaging and targeted radionuclide therapy of pigmented melanoma.

    PubMed

    Billaud, Emilie M F; Maisonial-Besset, Aurélie; Rbah-Vidal, Latifa; Vidal, Aurélien; Besse, Sophie; Béquignat, Jean-Baptiste; Decombat, Caroline; Degoul, Françoise; Audin, Laurent; Deloye, Jean-Bernard; Dollé, Frédéric; Kuhnast, Bertrand; Madelmont, Jean-Claude; Tarrit, Sébastien; Galmier, Marie-Josèphe; Borel, Michèle; Auzeloux, Philippe; Miot-Noirault, Elisabeth; Chezal, Jean-Michel

    2015-03-01

    Melanin pigment represents an attractive target to address specific treatment to melanoma cells, such as cytotoxic radionuclides. However, less than half of the patients have pigmented metastases. Hence, specific marker is required to stratify this patient population before proceeding with melanin-targeted radionuclide therapy. In such a context, we developed fluorinated analogues of a previously studied melanin-targeting ligand, N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide (ICF01012). These latter can be labeled either with (18)F or (131)I/(125)I for positron emission tomography imaging (melanin-positive patient selection) and targeted radionuclide therapy purposes. Here we describe the syntheses, radiosyntheses and preclinical evaluations on melanoma-bearing mice model of several iodo- and fluoro(hetero)aromatic derivatives of the ICF01012 scaffold. After preliminary planar gamma scintigraphic and positron emission tomography imaging evaluations, [(125)I]- and [(18)F]-N-[2-(diethylamino)ethyl]-4-fluoro-3-iodobenzamides ([(125)I]4, [(18)F]4) were found to be chemically and biologically stable with quite similar tumor uptakes at 1 h p.i. (9.7 ± 2.6% ID/g and 6.8 ± 1.9% ID/g, respectively). PMID:25637883

  18. 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

  19. 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

  20. 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

  1. Cytochrome P450s in the development of target-based anticancer drugs.

    PubMed

    Purnapatre, Kedar; Khattar, Sunil K; Saini, Kulvinder Singh

    2008-01-18

    Enzymes of the cytochrome P450 (CYP) superfamily are the major determinants of half-life and execute pharmacological effects of many therapeutic drugs. In new drug discovery research, recombinant (human) CYPs are also used for identifying active or inactive metabolites that could lead to increased potency or toxicity of a molecule. In addition, CYP inhibition by anticancer drugs might lead to adverse drug reactions, multiple-drug resistance, and drug-drug interactions. During the discovery and pre-clinical evaluation of a New Chemical Entity (NCE), large amounts of purified recombinant CYPs are required for studying metabolism and pharmacokinetic parameters. Therefore, present research efforts are focused to over-express these human CYPs in bacteria, yeast, insect and mammalian cells, followed by their purification on an industrial scale to facilitate identification of novel anticancer drugs. This review summarizes the merits and limitations of these expression systems for an optimized production of individual CYP isoforms, and their usefulness in the discovery and development of target-based, safe and efficacious NCEs for the treatment of cancer. PMID:18053638

  2. 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

  3. Small-Animal SPECT/CT of the Progression and Recovery of Rat Liver Fibrosis by Using an Integrin αvβ3-targeting Radiotracer.

    PubMed

    Yu, Xinhe; Wu, Yue; Liu, Hao; Gao, Liquan; Sun, Xianlei; Zhang, Chenran; Shi, Jiyun; Zhao, Huiyun; Jia, Bing; Liu, Zhaofei; Wang, Fan

    2016-05-01

    Purpose To assess the potential utility of an integrin αvβ3-targeting radiotracer, technetium 99m-PEG4-E[PEG4-cyclo(arginine-glycine-aspartic acid-D-phenylalanine-lysine)]2 ((99m)Tc-3PRGD2), for single photon emission computed tomography (SPECT)/computed tomography (CT) for monitoring of the progression and prognosis of liver fibrosis in a rat model. Materials and Methods All animal experiments were performed by following the protocol approved by the institutional animal care and use committee. (99m)Tc-3PRGD2 was prepared and longitudinal SPECT/CT was performed to monitor the progression (n = 8) and recovery (n = 5) of liver fibrosis induced in a rat model by means of thioacetamide (TAA) administration. The mean liver-to-background radioactivity per unit volume ratio was analyzed for comparisons between the TAA and control (saline) groups at different stages of liver fibrosis. Data were compared by using Student t and Mann-Whitney tests. Results of SPECT/CT were compared with those of ex vivo biodistribution analysis (n = 5). Results Accumulation of (99m)Tc-3PRGD2 in the liver increased in proportion to the progression of fibrosis and TAA exposure time; accumulation levels were significantly different between the TAA and control groups as early as week 4 of TAA administration (liver-to-background ratio: 32.30 ± 3.39 vs 19.01 ± 3.31; P = .0002). Results of ex vivo immunofluorescence staining demonstrated the positive expression of integrin αvβ3 on the activated hepatic stellate cells, and the integrin αvβ3 levels in the liver corresponded to the results of SPECT/CT (R(2) = 0.75, P < .0001). (99m)Tc-3PRGD2 uptake in the fibrotic liver decreased after antifibrotic therapy with interferon α2b compared with that in the control group (relative liver-to-background ratio: 0.45 ± 0.05 vs 1.01 ± 0.05; P < .0001) or spontaneous recovery (relative liver-to-background ratio: 0.56 ± 0.06 vs 1.01 ± 0.05; P < .0001). Conclusion (99m)Tc-3PRGD2 SPECT/CT was successfully

  4. 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

  5. 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

  6. Targeting cytochrome C oxidase in mitochondria with Pt(II)-porphyrins for photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Börsch, Michael

    2010-02-01

    Mitochondria are the power house of living cells, where the synthesis of the chemical "energy currency" adenosine triphosphate (ATP) occurs. Oxidative phosphorylation by a series of membrane protein complexes I to IV, that is, the electron transport chain, is the source of the electrochemical potential difference or proton motive force (PMF) of protons across the inner mitochondrial membrane. The PMF is required for ATP production by complex V of the electron transport chain, i.e. by FoF1-ATP synthase. Destroying cytochrome C oxidase (COX; complex IV) in Photodynamic Therapy (PDT) is achieved by the cationic photosensitizer Pt(II)-TMPyP. Electron microscopy revealed the disruption of the mitochondrial christae as a primary step of PDT. Time resolved phosphorescence measurements identified COX as the binding site for Pt(II)-TMPyP in living HeLa cells. As this photosensitizer competed with cytochrome C in binding to COX, destruction of COX might not only disturb ATP synthesis but could expedite the release of cytochrome C to the cytosol inducing apoptosis.

  7. 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

  8. 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

  9. Synthesis and biological evaluation of positron emission tomography radiotracers targeting serotonin 4 receptors in brain: [18F]MNI-698 and [18F]MNI-699.

    PubMed

    Caillé, Fabien; Morley, Thomas J; Tavares, Adriana Alexandre S; Papin, Caroline; Twardy, Nicole M; Alagille, David; Lee, H Sharon; Baldwin, Ronald M; Seibyl, John P; Barret, Olivier; Tamagnan, Gilles D

    2013-12-01

    Two new benzodioxane derivatives were synthesized as candidates to image the serotonin 4 receptors by positron emission tomography (PET) and radiolabeled with fluorine-18 via a two-step procedure. Competition binding assays demonstrated that MNI-698 and MNI-699 had sub-nanomolar binding affinities against rat striatal 5-HT4 receptors (Ki of 0.20 and 0.07 nM, respectively). PET imaging in rhesus monkey showed that the regional brain distribution of [(18)F]MNI-698 and [(18)F]MNI-699 were consistent with the known densities of 5-HT4 in brain. [(18)F]MNI-698 and [(18)F]MNI-699 are among the first fluorine-18 radiotracers developed for imaging the 5-HT4 receptors in vivo and are currently under preclinical investigation in primates for future human use. PMID:24157369

  10. 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

  11. 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. PMID:26082378

  12. 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

  13. 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

  14. 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. PMID:24631490

  15. 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.

  16. 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

  17. t-Bu2SiF-derivatized D2-receptor ligands: the first SiFA-containing small molecule radiotracers for target-specific PET-imaging.

    PubMed

    Iovkova-Berends, Ljuba; Wängler, Carmen; Zöller, Thomas; Höfner, Georg; Wanner, Klaus Theodor; Rensch, Christian; Bartenstein, Peter; Kostikov, Alexey; Schirrmacher, Ralf; Jurkschat, Klaus; Wängler, Björn

    2011-01-01

    The synthesis, radiolabeling and in vitro evaluation of new silicon-fluoride acceptor (SiFA) derivatized D(2)-receptor ligands is reported. The SiFA-technology simplifies the introduction of fluorine-18 into target specific biomolecules for Positron-Emission-Tomography (PET). However, one of the remaining challenges, especially for small molecules such as receptor-ligands, is the bulkiness of the SiFA-moiety. We therefore synthesized four Fallypride SiFA-conjugates derivatized either directly at the benzoic acid ring system (SiFA-DMFP, SiFA-FP, SiFA-DDMFP) or at the butyl-side chain (SiFA-M-FP) and tested their receptor affinities. We found D(2)-receptor affinities for all compounds in the nanomolar range (K(i(SiFA-DMFP)) = 13.6 nM, K(i(SiFA-FP)) = 33.0 nM, K(i(SiFA-DDMFP)) = 62.7 nM and K(i(SiFA-M-FP)) = 4.21 nM). The radiofluorination showed highest yields when 10 nmol of the precursors were reacted with [(18)F]fluoride/TBAHCO(3) in acetonitrile. After a reversed phased cartridge purification the desired products could be isolated as an injectable solution after only 10 min synthesis time with radiochemical yields (RCY) of more than 40% in the case of SiFA-DMFP resulting in specific activities >41 GBq/µmol (>1,100 Ci/mmol). Furthermore, the radiolabeled products were shown to be stable in the injectable solutions, as well as in human plasma, for at least 90 min. PMID:21892125

  18. 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.

  19. 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.

  20. Principle component analysis for radiotracer signal separation.

    PubMed

    Kasban, H; Arafa, H; Elaraby, S M S

    2016-06-01

    Radiotracers can be used in several industrial applications by injecting the radiotracer into the industrial system and monitoring the radiation using radiation detectors for obtaining signals. These signals are analyzed to obtain indications about what is happening within the system or to determine the problems that may be present in the system. For multi-phase system analysis, more than one radiotracer is used and the result is a mixture of radiotracers signals. The problem is in such cases is how to separate these signals from each other. The paper presents a proposed method based on Principle Component Analysis (PCA) for separating mixed two radiotracer signals from each other. Two different radiotracers (Technetium-99m (Tc(99m)) and Barium-137m (Ba(137m))) were injected into a physical model for simulation of chemical reactor (PMSCR-MK2) for obtaining the radiotracer signals using radiation detectors and Data Acquisition System (DAS). The radiotracer signals are mixed and signal processing steps are performed include background correction and signal de-noising, then applying the signal separation algorithms. Three separation algorithms have been carried out; time domain based separation algorithm, Independent Component Analysis (ICA) based separation algorithm, and Principal Components Analysis (PCA) based separation algorithm. The results proved the superiority of the PCA based separation algorithm to the other based separation algorithm, and PCA based separation algorithm and the signal processing steps gives a considerable improvement of the separation process. PMID:26974488

  1. Cytochrome f

    SciTech Connect

    Soriano, G.M.; Smith, J.L.; Cramer, W.A.

    2001-07-17

    Cytochrome f (f, folium, leaf), a c-type cytochrome with a characteristic CysXXCysHis amino acid sequence for heme ligation, is the largest of the four major protein subunits of the membrane-embedded cytochrome b{sub 6}{sup f} complex of oxygenic photosynthesis. It contains 285-86 amino acids, consisting of a soluble 250-residue domain on the p-side (positive-side) or lumen-side of the membrane, a single trans-membrane 20-residue {alpha}-helix, and an n- or stromal-side segment consisting of 15 residues. These domains contain, respectively, the heme prosthetic group and intraprotein electron transfer pathway, the membrane anchor and a short segment that is important in the assembly of the b{sub 6}{sup f} complex. The function of the cytochrome f in oxygenic photosynthesis is to act as the terminal electron acceptor in the membrane-embedded cytochrome b{sub 6}{sup f} complex that provides the electron transport connection between the photosystem II and photosystem I reaction centers. Electron transfer through the complex is coupled to proton translocation and generation of a proton electrochemical potential that is utilized to drive the synthesis of ATP through the proton-motive ATP synthase. These functions of the cytochrome b{sub 6}{sup f} complex are analogous to those of the multisubunit cytochrome bc{sub 1} complex (ubiquinol:cytochrome c oxidoreductase) of the mitochondrial respiratory chain and photosynthetic bacteria. Both complexes contain four redox centers with very similar redox and structural properties: a covalently bound c-type heme in cytochrome f or c{sub 1}, the 2Fe-2S cluster of the Rieske ISP, and the two noncovalently bound hemes of cytochrome b. The structure properties have been defined in 3.0-3.1 {angstrom} structures of the b{sub 6}{sup f} complex from a thermophilic cyanobacterium and a green alga. These structures also defined a fifth redox prosthetic group, a novel covalently bound heme, tentatively called heme x. With the exception of

  2. Monoamine oxidase: Radiotracer chemistry and human studies

    DOE PAGESBeta

    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

  3. 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.

  4. 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. PMID:25678350

  5. 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.

  6. 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

  7. 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

  8. 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

  9. An unusual TOM20/TOM22 bypass mechanism for the mitochondrial targeting of cytochrome P450 proteins containing N-terminal chimeric signals.

    PubMed

    Anandatheerthavarada, Hindupur K; Sepuri, Naresh Babu V; Biswas, Gopa; Avadhani, Narayan G

    2008-07-11

    Previously we showed that xenobiotic-inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In the present study, we investigated the mechanism of delivery of chimeric signal-containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP+33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP+5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal-containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of chimeric signal-containing client proteins by Hsp90 required the cytosol-exposed N-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to bind CYP proteins. These results suggest that, compared with the unimodal mitochondria-targeting signals, the chimeric mitochondria-targeting signals are highly evolved and dynamic in nature. PMID:18480056

  10. Mitochondrial targeting of cytochrome P450 proteins containing NH2-terminal chimeric signals involves an unusual TOM20/TOM22 bypass mechanism.

    PubMed

    Anandatheerthavarada, Hindupur K; Sepuri, Naresh Babu V; Avadhani, Narayan G

    2009-06-19

    Previously we showed that xenobiotic inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In this study, we investigated the mechanism of delivery of chimeric signal containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP+33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP+5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27A1 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of a chimeric signal containing client protein by Hsp90 required the cytosol-exposed NH(2)-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to import CYP proteins. These results suggest that compared with the unimodal mitochondrial targeting signals, the chimeric mitochondrial targeting signals are highly evolved and dynamic in nature. PMID:19401463

  11. 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

  12. 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

  13. Impact of expression system on the function of the C6.5 diabody PET radiotracer.

    PubMed

    Miller, Joshua; Doss, Mohan; McQuillen, Ryan; Shaller, Calvin C; Tolner, Berend; Yu, Jian Q; Chester, Kerry; Robinson, Matthew K

    2012-06-01

    The ability of engineered antibodies to rapidly and selectively target tumors that express their target antigen makes them well suited for use as radioimaging tracers. The combination of molecular size and bivalent nature makes diabody molecules a particularly promising structure for use as radiotracers for diagnostic imaging. Previous data have demonstrated that the anti-HER2 C6.5 diabody (C6.5db) is an effective radiotracer in preclinical models of HER2-positive cancer. The aim of this study was to evaluate the impact on radiotracer performance, associated with expressing the C6.5db in the Pichia pastoris (P-C6.5db) system as compared to Escherichia coli (E. C6.5db). Glycosylation of P-C6.5db led to faster blood clearance and lower overall tumor uptake than seen with E. coli-produced C6.5db. However, P-C6.5db achieved high tumor/background ratios that are critical for effective imaging. Dosimetry measurements determined in this study for both (124)I-P-C6.5db and (124)I-E-C6.5db suggest that they are equivalent to other radiotracers currently being administered to patients. PMID:22383295

  14. 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

  15. 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

  16. MicroRNA-30c-1-3p is a silencer of the pregnane X receptor by targeting the 3'-untranslated region and alters the expression of its target gene cytochrome P450 3A4.

    PubMed

    Vachirayonstien, Thaveechai; Yan, Bingfang

    2016-09-01

    The pregnane X receptor (PXR) is a master regulator of genes involved in drug elimination. Recently, activation of PXR has also been linked to the development of many disease conditions such as metabolic disorders and malignancies. MicroRNAs (miRs) emerge as important molecular species involved in these conditions. This study was undertaken to test a large number of miRs for their ability to regulate PXR expression. As many as 58 miRs were tested and miR-30c-1-3p was identified to suppress PXR expression. The suppression was achieved by targeting the 3'-untranslated region, 438 nucleotides from the stop codon. The suppression was detected in multiple cell lines from different organ origins. In addition, miR-30c-1-3p altered basal and induced expression of cytochrome P450 3A4 (CYP3A4), a prototypical target gene of PXR. The alteration varied depending on the time and amounts of miR-30c-1-3p. CYP3A4 is responsible for the metabolism of more than 50% medicines. The interconnection between miR-30c-1-3p and PXR signifies a role of miRs in drug-drug interactions and chemosensitivity. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. PMID:27085140

  17. 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

  18. 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

  19. Targeted protein degradation of outer membrane decaheme cytochrome MtrC metal reductase in Shewanella oneidensis MR-1 measured using biarsenical probe CrAsH-EDT(2).

    PubMed

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

    2011-11-15

    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 (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 h(-1) that is insensitive to O(2) 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 h(-1)) that are

  20. Radiolabeled Cyclic RGD Peptides as Radiotracers for Imaging Tumors and Thrombosis by SPECT.

    PubMed

    Zhou, Yang; Chakraborty, Sudipta; Liu, Shuang

    2011-01-01

    The integrin family is a group of transmembrane glycoprotein comprised of 19 α- and 8 β-subunits that are expressed in 25 different α/β heterodimeric combinations on the cell surface. Integrins play critical roles in many physiological processes, including cell attachment, proliferation, bone remodeling, and wound healing. Integrins also contribute to pathological events such as thrombosis, atherosclerosis, tumor invasion, angiogenesis and metastasis, infection by pathogenic microorganisms, and immune dysfunction. Among 25 members of the integrin family, the α(v)β(3) is studied most extensively for its role of tumor growth, progression and angiogenesis. In contrast, the α(IIb)β(3 )is expressed exclusively on platelets, facilitates the intercellular bidirectional signaling ("inside-out" and "outside-in") and allows the aggregation of platelets during vascular injury. The α(IIb)β(3) plays an important role in thrombosis by its activation and binding to fibrinogen especially in arterial thrombosis due to the high blood flow rate. In the resting state, the α(IIb)β(3) on platelets does not bind to fibrinogen; on activation, the conformation of platelet is altered and the binding sites of α(IIb)β(3 )are exposed for fibrinogen to crosslink platelets. Over the last two decades, integrins have been proposed as the molecular targets for diagnosis and therapy of cancer, thrombosis and other diseases. Several excellent review articles have appeared recently to cover a broad range of topics related to the integrin-targeted radiotracers and their nuclear medicine applications in tumor imaging by single photon emission computed tomography (SPECT) or a positron-emitting radionuclide for positron emission tomography (PET). This review will focus on recent developments of α(v)β(3)-targeted radiotracers for imaging tumors and the use of α(IIb)β(3)-targeted radiotracers for thrombosis imaging, and discuss different approaches to maximize the targeting capability of

  1. Mechanistic role of cytochrome P450 (CYP)1B1 in oxygen-mediated toxicity in pulmonary cells: A novel target for prevention of hyperoxic lung injury.

    PubMed

    Dinu, Daniela; Chu, Chun; Veith, Alex; Lingappan, Krithika; Couroucli, Xanthi; Jefcoate, Colin R; Sheibani, Nader; Moorthy, Bhagavatula

    2016-08-01

    Supplemental oxygen, which is routinely administered to preterm infants with pulmonary insufficiency, contributes to bronchopulmonary dysplasia (BPD) in these infants. Hyperoxia also contributes to the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in adults. The mechanisms of oxygen-mediated pulmonary toxicity are not completely understood. Recent studies have suggested an important role for cytochrome P450 (CYP)1A1/1A2 in the protection against hyperoxic lung injury. The role of CYP1B1 in oxygen-mediated pulmonary toxicity has not been studied. In this investigation, we tested the hypothesis that CYP1B1 plays a mechanistic role in oxygen toxicity in pulmonary cells in vitro. In human bronchial epithelial cell line BEAS-2B, hyperoxic treatment for 1-3 days led to decreased cell viability by about 50-80%. Hyperoxic cytotoxicity was accompanied by an increase in levels of reactive oxygen species (ROS) by up to 110%, and an increase of TUNEL-positive cells by up to 4.8-fold. Western blot analysis showed hyperoxia to significantly down-regulate CYP1B1 protein level. Also, there was a decrease of CYP1B1 mRNA by up to 38% and Cyp1b1 promoter activity by up to 65%. On the other hand, CYP1B1 siRNA appeared to rescue the cell viability under hyperoxia stress, and overexpression of CYP1B1 significantly attenuated hyperoxic cytotoxicity after 48 h of incubation. In immortalized lung endothelial cells derived from Cyp1b1-null and wild-type mice, hyperoxia increased caspase 3/7 activities in a time-dependent manner, but endothelial cells lacking the Cyp1b1 gene showed significantly decreased caspase 3/7 activities after 48 and 72 h of incubation, implying that CYP1B1 might promote apoptosis in wild type lung endothelial cells under hyperoxic stress. In conclusion, our results support the hypothesis that CYP1B1 plays a mechanistic role in pulmonary oxygen toxicity, and CYP1B1-mediated apoptosis could be one of the mechanisms of oxygen

  2. Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics.

    PubMed

    Gröer, C; Busch, D; Patrzyk, M; Beyer, K; Busemann, A; Heidecke, C D; Drozdzik, M; Siegmund, W; Oswald, S

    2014-11-01

    Cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGT) are major determinants in the pharmacokinetics of most drugs on the market. To investigate their impact on intestinal and hepatic drug metabolism, we developed and validated quantification methods for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using in each case stable isotope labelled peptides as internal standard. The chromatography of the respective peptides was performed with gradient elution using a reversed phase (C18) column (Ascentis(®) Express Peptide ES-C18, 100mm×2.1mm, 2.7μm) and 0.1% formic acid (FA) as well as acetonitrile with 0.1% FA as mobile phases at a flow rate of 300μl/min. The MS/MS detection of all peptides was done simultaneously with a scheduled multiple reaction monitoring (MRM) method in the positive mode by monitoring in each case three mass transitions per proteospecific peptide and the internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (0.25-50nM), within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was successfully applied to determine the CYP and UGT protein amount in human liver and intestinal microsomes. The method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to quantify clinically relevant human CYP and UGT enzymes. PMID:25218440

  3. 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)

  4. 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.

  5. Improving the stability of peptidic radiotracers by the introduction of artificial scaffolds: which structure element is most useful?

    PubMed

    Bacher, Lisa; Fischer, Gabriel; Litau, Shanna; Schirrmacher, Ralf; Wängler, Björn; Baller, Marko; Wängler, Carmen

    2015-08-01

    Peptidic radiotracers are highly potent substances for the specific in vivo imaging of various biological targets with Single Photon Emission Computed Tomography and Positron Emission Tomography. However, some radiolabeled peptides such as bombesin analogs were shown to exhibit only a limited stability, hampering a successful target visualization. One option to positively influence the stability of radiolabeled peptides is the introduction of certain artificial molecular scaffolds. In order to comparatively assess the influence of different structure elements on the stability of radiolabeled peptides and to identify those structure elements being most useful for peptide radiotracer stabilization, several monomeric and dimeric bombesin derivatives were synthesized, exhibiting differing molecular designs and the chelator NODAGA for (68) Ga-labeling. The radiolabeled peptides were evaluated regarding their in vitro stability in human serum to determine the influence of the introduced molecular scaffolds on the peptides' serum stabilities. The results of the evaluations showed that the introduction of scaffold structures and the overall molecular design have a substantial impact on the stabilities of the resulting peptidic radiotracers. But besides some general trends found using certain scaffold structures, the obtained results point to the necessity to empirically assess their influence on stability for each susceptible peptidic radiotracer individually. PMID:26219022

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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. PMID:26138548

  12. Vivid tumor imaging utilizing liposome-carried bimodal radiotracer.

    PubMed

    Kim, Jonghee; Pandya, Darpan N; Lee, Woonghee; Park, Jang Woo; Kim, Youn Ji; Kwak, Wonjung; Ha, Yeong Su; Chang, Yongmin; An, Gwang Il; Yoo, Jeongsoo

    2014-04-10

    By developing a new bimodal radioactive tracer that emits both luminescence and nuclear signals, a trimodal liposome for optical, nuclear, and magnetic resonance imaging is efficiently prepared. Fast clearance of the radiotracer from reticuloendothelial systems enables vivid tumor imaging with minimum background. PMID:24900846

  13. 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

  14. Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

    PubMed Central

    O'Keefe, Daniel P.; Leto, Kenneth J.

    1989-01-01

    The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450. Images Figure 3 PMID:16666677

  15. 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

  16. Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction*

    PubMed Central

    Bajpai, Prachi; Srinivasan, Satish; Ghosh, Jyotirmoy; Nagy, Leslie D.; Wei, Shouzou; Guengerich, F. Peter; Avadhani, Narayan G.

    2014-01-01

    In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues. PMID:25160618

  17. Optimization of integrated impeller mixer via radiotracer experiments.

    PubMed

    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, V dead (%), 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 V dead (%) 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

  18. 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

  19. 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

  20. Radiotracers for PET and SPECT studies of neurotransmitter systems

    SciTech Connect

    Fowler, J.S.

    1991-01-01

    The study of neurotransmitter systems is one of the major thrusts in emission tomography today. The current generation of Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) radiotracers examines neurotransmitter properties from a number of different perspectives including their pre and post synaptic sites and the activity of the enzymes which regulate their concentration. Although the dopamine system has been the most extensively investigated, other neurotransmitter systems including the acetylcholine muscarine, serotonin, benzodiazepine, opiate, NMDA and others are also under intensive development. Enzymes involved in the synthesis and regulation of neurotransmitter concentration, for example monoamine oxidase and amino acid decarboxylase has also been probed in vivo. Medical applications range from the study of normal function and the characterization of neurotransmitter activity in neurological and psychiatric diseases and in heart disease and cancer to the study of the binding of therapeutic drugs and substances of abuse. This chapter will provide an overview of the current generation of radiotracers for PET and SPECT studies of neurotransmitter systems including radiotracer design, synthesis localization mechanisms and applications in emission tomography. 60 refs., 1 tab.

  1. Design of a serotonin 4 receptor radiotracer with decreased lipophilicity for single photon emission computed tomography.

    PubMed

    Fresneau, Nathalie; Dumas, Noé; Tournier, Benjamin B; Fossey, Christine; Ballandonne, Céline; Lesnard, Aurélien; Millet, Philippe; Charnay, Yves; Cailly, Thomas; Bouillon, Jean-Philippe; Fabis, Frédéric

    2015-04-13

    With the aim to develop a suitable radiotracer for the brain imaging of the serotonin 4 receptor subtype (5-HT4R) using single photon emission computed tomography (SPECT), we synthesized and evaluated a library of di- and triazaphenanthridines with lipophilicity values which were in the range expected to favour brain penetration, and which demonstrated specific binding to the target of interest. Adding additional nitrogen atoms to previously described phenanthridine ligands exhibiting a high unspecific binding, we were able to design a radioiodinated compound [(125)I]14. This compound exhibited a binding affinity value of 0.094 nM toward human 5-HT4R and a high selectivity over other serotonin receptor subtypes (5-HTR). In vivo SPECT imaging studies and competition experiments demonstrated that the decreased lipophilicity (in comparison with our previously reported compounds 4 and 5) allowed a more specific labelling of the 5-HT4R brain-containing regions. PMID:25778994

  2. 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

  3. The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase.

    PubMed

    Bickar, D; Turrens, J F; Lehninger, A L

    1986-11-01

    When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c. PMID:3021740

  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. 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

  7. Cytochromes p450.

    PubMed

    Werck-Reichhart, Danièle; Bak, Søren; Paquette, Suzanne

    2002-01-01

    There are 272 cytochrome P450 genes (including 26 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest families of proteins in higher plants. This explosion of the P450 family is thought to have occurred via gene duplication and conversion, and to result from the need of sessile plants to adapt to a harsh environment and to protect themselves from pathogens and predators. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions. Their biological functions range from the synthesis of structural macromolecules such as lignin, cutin or suberin, to the synthesis or catabolism of all types of hormone or signaling molecules, the synthesis of pigments and defense compounds, and to the metabolism of xenobiotics. In despite of a huge acceleration in our understanding of plant P450 functions in the recent years, the vast majority of these functions remain completely unknown. PMID:22303202

  8. 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

  9. Cytochromes P450

    PubMed Central

    Werck-Reichhart, Danièle; Bak, Søren; Paquette, Suzanne

    2002-01-01

    There are 272 cytochrome P450 genes (including 26 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest families of proteins in higher plants. This explosion of the P450 family is thought to have occurred via gene duplication and conversion, and to result from the need of sessile plants to adapt to a harsh environment and to protect themselves from pathogens and predators. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions. Their biological functions range from the synthesis of structural macromolecules such as lignin, cutin or suberin, to the synthesis or catabolism of all types of hormone or signaling molecules, the synthesis of pigments and defense compounds, and to the metabolism of xenobiotics. In despite of a huge acceleration in our understanding of plant P450 functions in the recent years, the vast majority of these functions remain completely unknown. PMID:22303202

  10. Cytochromes p450.

    PubMed

    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

  11. Cytochrome P450 database.

    PubMed

    Lisitsa, A V; Gusev, S A; Karuzina, I I; Archakov, A I; Koymans, L

    2001-01-01

    This paper describes a specialized database dedicated exclusively to the cytochrome P450 superfamily. The system provides the impression of superfamily's nomenclature and describes structure and function of different P450 enzymes. Information on P450-catalyzed reactions, substrate preferences, peculiarities of induction and inhibition is available through the database management system. Also the source genes and appropriate translated proteins can be retrieved together with corresponding literature references. Developed programming solution provides the flexible interface for browsing, searching, grouping and reporting the information. Local version of database manager and required data files are distributed on a compact disk. Besides, there is a network version of the software available on Internet. The network version implies the original mechanism, which is useful for the permanent online extension of the data scope. PMID:11769119

  12. 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

  13. Fluorinated quinazolinones as potential radiotracers for imaging kinesin spindle protein expression.

    PubMed

    Holland, Jason P; Jones, Michael W; Cohrs, Susan; Schibli, Roger; Fischer, Eliane

    2013-01-15

    Anti-mitotic anti-cancer drugs offer a potential platform for developing new radiotracers for imaging proliferation markers associated with the mitosis-phase of the cell-cycle. One interesting target is kinesin spindle protein (KSP)-an ATP-dependent motor protein that plays a vital role in bipolar spindle formation. In this work we synthesised a range of new fluorinated-quinazolinone compounds based on the structure of the clinical candidate KSP inhibitor, ispinesib, and investigated their properties in vitro as potential anti-mitotic agents targeting KSP expression. Anti-proliferation (MTT and BrdU) assays combined with additional studies including fluorescence-assisted cell sorting (FACS) analysis of cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Additional studies using confocal fluorescence microscopy showed that these compounds induce M-phase arrest via monoaster spindle formation. Structural studies revealed that compound 20-(R) is the most potent fluorinated-quinazolinone inhibitor of KSP and represents a suitable lead candidate for further studies on designing (18)F-radiolabelled agents for positron-emission tomography (PET). PMID:23245569

  14. Synthesis, characterization and application of Au-198 nanoparticles as radiotracer for industrial applications.

    PubMed

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

    2016-05-01

    This paper describes synthesis and characterization of radioactive gold nanoparticles ((198)Au-NPs), and explores their utility as a radiotracer for tracing an aqueous phase in a continuous laboratory-scale bubble column at ambient conditions. The performance of the (198)Au-NPs as a radiotracer was compared with the results obtained with a conventional radiotracer i.e. bromine-82 ((82)Br) as ammonium bromide and found to be identical. A tank-in-series with backmixing model (TISBM) was used to simulate the RTDs of the aqueous phase and characterize flow in the bubble column. PMID:26897465

  15. Coupling in cytochrome c oxidase

    PubMed Central

    Kessler, R. J.; Blondin, G. A.; Zande, H. Vande; Haworth, R. A.; Green, D. E.

    1977-01-01

    Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K+ when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome c and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid. Images PMID:198794

  16. 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

  17. Role of cytochrome bd oxidase from Corynebacterium glutamicum in growth and lysine production.

    PubMed

    Kabus, Armin; Niebisch, Axel; Bott, Michael

    2007-02-01

    Corynebacterium glutamicum possesses two terminal oxidases, cytochrome aa3 and cytochrome bd. Cytochrome aa3 forms a supercomplex with the cytochrome bc1 complex, which contains an unusual diheme cytochrome c1. Both the bc1 -aa3 supercomplex and cytochrome bd transfer reducing equivalents from menaquinol to oxygen; however, they differ in their proton translocation efficiency by a factor of three. Here, we analyzed the role of cytochrome bd for growth and lysine production. When cultivated in glucose minimal medium, a cydAB deletion mutant of C. glutamicum ATCC 13032 grew like the wild type in the exponential phase, but growth thereafter was inhibited, leading to a biomass formation 40% less than that of the wild type. Constitutive overproduction of functional cytochrome bd oxidase in ATCC 13032 led to a reduction of the growth rate by approximately 45% and of the maximal biomass by approximately 35%, presumably as a consequence of increased electron flow through the inefficient cytochrome bd oxidase. In the L-lysine-producing C. glutamicum strain MH20-22B, deletion of the cydAB genes had only minor effects on growth rate and biomass formation, but lysine production was increased by approximately 12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by C. glutamicum. PMID:17142369

  18. Cytochrome c' of Methylococcus capsulatus Bath.

    PubMed

    Zahn, J A; Arciero, D M; Hooper, A B; Dispirito, A A

    1996-09-15

    Cytochrome c' was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The native and subunit molecular masses of the cytochrome were 34.9 kDa and 16.2 kDa, respectively, with an isoelectric pH of 7.0. The amino acid composition and N-terminal amino acid sequence were consistent with identification of the protein as a cytochrome c'. The electron paramagnetic resonance spectrum of the monoheme cytochrome indicated the presence of a high spin, S = 5/2, heme center that is diagnostic of cytochromes c'. The optical absorption spectra of ferric or ferrous cytochrome c' were also characteristic of cytochromes c'. The ferrocytochrome bound carbon monoxide and nitric oxide, but not isocyanide, cyanide, or azide. Changes in physical properties due to binding of CO or NO to some other c'-type cytochromes have been interpreted as an indication of dimer dissociation. In the case of cytochrome c' from M. capsulatus Bath, analytical ultracentrifugation of the ferricytochrome, the ferrocytochrome, and the ferrocytochrome-CO complex indicate that the changes induced by binding of CO are conformational and are not consistent with dimer dissociation. EPR spectra show that cytochrome c' was reduced in the presence of hydroxylamine only when in a complex with cytochrome P-460. The value of the midpoint potential, Em 7.0, was -250 mV for cytochrome c' from M. capsulatus Bath, which is well below the range of values reported for other cytochromes c'. The values of midpoint potentials for cytochrome P-460 (Em 7.0 = -300 mV to -380 mV) and cytochrome C555 (Em 7.0 = +175 mV to +195 mV) are less than and greater than, respectively, the value for cytochrome c' and suggest the possibility that the latter may function as an electron shuttle between cytochrome P-460 and cytochrome C555. PMID:8856071

  19. The Mycobacterium tuberculosis Cytochrome P450 System

    PubMed Central

    Ouellet, Hugues; Johnston, Jonathan B.; Ortiz de Montellano, Paul R.

    2009-01-01

    Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis, the causative agent, that are resistant to the major frontline antitubercular drugs increases the urgency for the development of new therapeutic agents. Sequencing of the M. tuberculosis genome revealed the existence of twenty cytochrome P450 enzymes, some of which are potential candidates for drug targeting. The recent burst of studies reporting microarray-based gene essentiality and transcriptome analyses under in vitro, ex vivo and in vivo conditions highlight the importance of selected P450 isoforms for M. tuberculosis viability and pathogenicity. Current knowledge of the structural and biochemical properties of the M. tuberculosis P450 enzymes and their putative redox partners is reviewed, with an emphasis on findings related to their physiological function(s) as well as their potential as drug targets. PMID:19635450

  20. Activation of caspase-dependent apoptosis by intracellular delivery of cytochrome c-based nanoparticles

    PubMed Central

    2014-01-01

    Background Cytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery. Results Cytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4’,6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles. Conclusions Our results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing

  1. Diffusion of radiotracers in normal and ischemic brain slices.

    PubMed

    Patlak, C S; Hospod, F E; Trowbridge, S D; Newman, G C

    1998-07-01

    Diffusion in the extracellular space (ECS) is important in physiologic and pathologic brain processes but remains poorly understood. To learn more about factors influencing tissue diffusion and the role of diffusion in solute-tissue interactions, particularly during cerebral ischemia, we have studied the kinetics of several radiotracers in control and hypoxic 450-microm hippocampal slices and in 1,050-microm thick slices that model the ischemic penumbra. Kinetics were analyzed by nonlinear least squares methods using models that combine extracellular diffusion with tissue compartments in series or in parallel. Studies with 14C-polyethylene glycol confirmed prior measurements of extracellular volume and that ECS shrinks during ischemia. Separating diffusion from transport also revealed large amounts of 45Ca that bind to or enter brain as well as demonstrating a small, irreversibly bound compartment during ischemia. The rapidity of 3H2O entry into cells made it impossible for us to distinguish intracellular from extracellular diffusion. The diffusion-compartment analysis of 3-O-methylglucose data appears to indicate that 5 mmol/L glucose is inadequate to support glycolysis fully in thick slices. Unexpectedly, the diffusion coefficient for all four tracers rose in thick slices compared with thin slices, suggesting that ECS becomes less tortuous in the penumbra. PMID:9663508

  2. 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.

  3. 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. PMID:26233907

  4. 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

  5. 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)

  6. 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.

  7. Methylsulfonylmethane modulates apoptosis of LPS/IFN-γ-activated RAW 264.7 macrophage-like cells by targeting p53, Bax, Bcl-2, cytochrome c and PARP proteins.

    PubMed

    Karabay, Arzu Z; Aktan, Fugen; Sunguroğlu, Asuman; Buyukbingol, Zeliha

    2014-12-01

    Methylsulfonylmethane (MSM) is a non-toxic, natural organosulfur compound, which is known to possess antioxidant and anti-inflammatory activities. In recent years, MSM has been widely used as a dietary supplement for its beneficial effects against various diseases, especially arthritis. Despite being a popular supplement product, the mechanism of action of MSM is not well known. This study was designed to investigate the effects of MSM on cytotoxic signals induced by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in RAW 264.7 macrophage-like cells. The results showed that MSM reversed apoptosis of RAW 264.7 macrophage-like cells at non-cytotoxic concentrations probably through the modulation of apoptotic proteins. After pre-treatment of cells with non-toxic doses of MSM; caspase-3 activation, p53 accumulation, cytochrome c release and Bax/Bcl-2 ratio were significantly decreased and full length poly ADP-ribose polymerase (PARP) was significantly increased. In addition, the loss of mitochondrial membrane potential was decreased with MSM pretreatment in activated macrophages. Since excess nitric oxide production causes apoptosis of macrophages, anti-apoptotic effects of MSM are thought to be mediated by its inhibitor effects on inducible nitric oxide synthase (iNOS) protein and nitric oxide levels. More interestingly, higher doses of MSM exhibited biphasic effects, inhibited cell viability, induced apoptosis of macrophages, increased caspase-3 activity and PARP cleavage. Thus, our results reveal the molecular mechanism of of MSM indicating that MSM supplementation may be beneficial for complications related to nitric oxide-dependent apoptosis in inflammatory conditions. However, the optimum concentration of MSM must be chosen carefully to elicit the desired effect. PMID:25211405

  8. Radiation Dosimetry and Biodistribution of the Translocator Protein Radiotracer [11C]DAA1106 Determined with PET/CT in Healthy Human Volunteers

    PubMed Central

    Brody, Arthur L.; Okita, Kyoji; Shieh, Jennifer; Liang, Lidia; Hubert, Robert; Mamoun, Michael; Farahi, Judah; Mandelkern, Mark A.

    2014-01-01

    Introduction When microglia become activated (an integral part of neuroinflammation), cellular morphology changes and expression of translocator protein (TSPO) 18 kDa is increased. Over the past several years, [11C]DAA1106 has emerged as a reliable radiotracer for labeling TSPO with high affinity during positron emission tomography (PET) scanning. While [11C]DAA1106 PET scanning has been used in several research studies, a radiation dosimetry study of this radiotracer in humans has not yet been published. Methods Twelve healthy participants underwent full body dynamic [11C]DAA1106 PET scanning, with 8 sequential whole body scans (approximately 12 bed positions each), following a single injection. Regions of interest were drawn manually and time activity curves (TACs) were obtained for 15 organs. OLINDA/EXM 1.1 was used to compute radiation absorbed doses to the target organs, as well as effective dose (ED) and effective dose equivalent (EDE). Results The ED and EDE were 4.06 ± 0.58 μSv/MBq and 5.89 ± 0.83 μSv/MBq, respectively. The highest absorbed doses were to the heart wall, kidney, liver, pancreas, and spleen. TACs revealed that peak dose rates are during the first scan (at 6 min) for all organs other than the urinary bladder wall, which had its peak dose rate during the fourth scan (at 30 min). Conclusions The recently developed radiotracer [11C]DAA1106 has its EDE and target-organ absorbed dose such that, for a single administration, its radiation dosimetry is well within the U.S. FDA guidelines for basic research studies in adults. This dose level implies that the dosimetry for multiple [11C]DAA1106 scans within a given year also falls within FDA guidelines, and this favorable property makes this radiotracer suitable for examining microglial activation repeatedly over time, which may in the future be useful for longitudinal tracking of disease progression and monitoring of therapy response in conditions marked by neuroinflammation (e.g., head trauma and

  9. 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

  10. 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.

  11. Mutations in Cytochrome Assembly and Periplasmic Redox Pathways in Bordetella pertussis

    PubMed Central

    Feissner, Robert E.; Beckett, Caroline S.; Loughman, Jennifer A.; Kranz, Robert G.

    2005-01-01

    Transposon mutagenesis of Bordetella pertussis was used to discover mutations in the cytochrome c biogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochrome c oxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesize c-type cytochromes and possessed insertions in the genes for cytochrome c oxidase subunits (ctaC, -D, and -E), heme a biosynthesis (ctaB), assembly of cytochrome c oxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize all c-type cytochromes. Seven of these had transposons in dipZ (dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochrome c assembly by exogenous dithiothreitol, which was consistent with the cytochrome c cysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in the ccsBA operon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent in ccsA mutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbA or dsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochrome c biogenesis in B. pertussis, a targeted knockout was made in dsbB. The DsbB− mutant makes holocytochromes c like the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. A dipZ mutant is not corrected by a dsbB mutation. Alternative mechanisms to oxidize disulfides in B. pertussis are analyzed and discussed. PMID:15937156

  12. An Investigation of the Effectiveness of Radiotracer Techniques for Instruction in Microbiology.

    ERIC Educational Resources Information Center

    Hurlburt, Evelyn McClelland

    Students in a junior college microbiology course were randomly assigned to one of two laboratory treatments: one using radiotracer techniques to investigate aspects of microbial metabolism, and the other using conventional techniques to investigate the same metabolic features. An achievement test administered at the completion of the unit and six…

  13. [(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

  14. 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.

  15. 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

  16. Branching in the sequential folding pathway of cytochrome c.

    PubMed

    Krishna, Mallela M G; Maity, Haripada; Rumbley, Jon N; Englander, S Walter

    2007-09-01

    Previous results indicate that the folding pathways of cytochrome c and other proteins progressively build the target native protein in a predetermined stepwise manner by the sequential formation and association of native-like foldon units. The present work used native state hydrogen exchange methods to investigate a structural anomaly in cytochrome c results that suggested the concerted folding of two segments that have little structural relationship in the native protein. The results show that the two segments, an 18-residue omega loop and a 10-residue helix, are able to unfold and refold independently, which allows a branch point in the folding pathway. The pathway that emerges assembles native-like foldon units in a linear sequential manner when prior native-like structure can template a single subsequent foldon, and optional pathway branching is seen when prior structure is able to support the folding of two different foldons. PMID:17660254

  17. Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designed I-CreI homing endonuclease.

    PubMed

    Djukanovic, Vesna; Smith, Jeff; Lowe, Keith; Yang, Meizhu; Gao, Huirong; Jones, Spencer; Nicholson, Michael G; West, Ande; Lape, Janel; Bidney, Dennis; Carl Falco, Saverio; Jantz, Derek; Alexander Lyznik, Leszek

    2013-12-01

    The I-CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double-strand breaks and enhancing DNA recombination reactions in maize cells. The DNA-binding properties of this protein were re-designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single-chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium-mediated transformation, the cleavage resulted in mutations at a co-delivered extra-chromosomal ms26-site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26-site in 5.8% of transgenic T(0) plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double-strand break repair generated by non-homologous end joining. One of 21 mutagenized T(0) plants carried two mutated alleles of the MS26 gene. As expected, the bi-allelic mutant T(0) plant and the T(1) progeny homozygous for the ms26 mutant alleles were male-sterile. This paper described the second maize chromosomal locus (liguless-1 being the first one) mutagenized by a re-designed I-CreI-based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants. PMID:24112765

  18. 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

  19. 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.

  20. 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

  1. 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. PMID:23135409

  2. In vivo imaging of beta cells with radiotracers: state of the art, prospects and recommendations for development and use.

    PubMed

    Eriksson, Olof; Laughlin, Maren; Brom, Maarten; Nuutila, Pirjo; Roden, Michael; Hwa, Albert; Bonadonna, Riccardo; Gotthardt, Martin

    2016-07-01

    Radiotracer imaging is characterised by high in vivo sensitivity, with a detection limit in the lower picomolar range. Therefore, radiotracers represent a valuable tool for imaging pancreatic beta cells. High demands are made of radiotracers for in vivo imaging of beta cells. Beta cells represent only a small fraction of the volume of the pancreas (usually 1-3%) and are scattered in the tiny islets of Langerhans throughout the organ. In order to be able to measure a beta cell-specific signal, one has to rely on highly specific tracer molecules because current in vivo imaging technologies do not allow the resolution of single islets in humans non-invasively. Currently, a considerable amount of preclinical data are available for several radiotracers and three are under clinical evaluation. We summarise the current status of the evaluation of these tracer molecules and put forward recommendations for their further evaluation. PMID:27094935

  3. The cytochrome bd-type quinol oxidase is important for survival of Mycobacterium smegmatis under peroxide and antibiotic-induced stress.

    PubMed

    Lu, Ping; Heineke, Marieke H; Koul, Anil; Andries, Koen; Cook, Gregory M; Lill, Holger; van Spanning, Rob; Bald, Dirk

    2015-01-01

    Targeting respiration and ATP synthesis has received strong interest as a new strategy for combatting drug-resistant Mycobacterium tuberculosis. Mycobacteria employ a respiratory chain terminating with two branches. One of the branches includes a cytochrome bc1 complex and an aa3-type cytochrome c oxidase while the other branch terminates with a cytochrome bd-type quinol oxidase. In this communication we show that genetic inactivation of cytochrome bd, but not of cytochrome bc1, enhances the susceptibility of Mycobacterium smegmatis to hydrogen peroxide and antibiotic-induced stress. The type-II NADH dehydrogenase effector clofazimine and the ATP synthase inhibitor bedaquiline were bacteriostatic against wild-type M. smegmatis, but strongly bactericidal against a cytochrome bd mutant. We also demonstrated that the quinone-analog aurachin D inhibited mycobacterial cytochrome bd at sub-micromolar concentrations. Our results identify cytochrome bd as a key survival factor in M. smegmatis during antibiotic stress. Targeting the cytochrome bd respiratory branch therefore appears to be a promising strategy that may enhance the bactericidal activity of existing tuberculosis drugs. PMID:26015371

  4. 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.

  5. Development of a Fluorinated Class-I HDAC Radiotracer Reveals Key Chemical Determinants of Brain Penetrance.

    PubMed

    Strebl, Martin G; Wang, Changning; Schroeder, Frederick A; Placzek, Michael S; Wey, Hsiao-Ying; Van de Bittner, Genevieve C; Neelamegam, Ramesh; Hooker, Jacob M

    2016-05-18

    Despite major efforts, our knowledge about many brain diseases remains remarkably limited. Epigenetic dysregulation has been one of the few leads toward identifying the causes and potential treatments of psychiatric disease over the past decade. A new positron emission tomography radiotracer, [(11)C]Martinostat, has enabled the study of histone deacetylase in living human subjects. A unique property of [(11)C]Martinostat is its profound brain penetrance, a feature that is challenging to engineer intentionally. In order to understand determining factors for the high brain-uptake of Martinostat, a series of compounds was evaluated in rodents and nonhuman primates. The study revealed the major structural contributors to brain uptake, as well as a more clinically relevant fluorinated HDAC radiotracer with comparable behavior to Martinostat, yet longer half-life. PMID:26675505

  6. 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

  7. 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

  8. 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. PMID:26057343

  9. 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

  10. 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

  11. Regulation of cytochrome P450 (CYP) genes by nuclear receptors.

    PubMed Central

    Honkakoski, P; Negishi, M

    2000-01-01

    Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID:10749660

  12. Cytochrome P450-like substrate oxidation catalyzed by cytochrome c and immobilized cytochrome c.

    PubMed

    Akasaka, R; Mashino, T; Hirobe, M

    1993-03-01

    Cytochrome c (cyt.c) was shown to catalyze cytochrome P450 (P450)-like oxidative reactions, such as N-, and O-demethylation, S-oxidation, and epoxidation of olefins. A more detailed examination showed that (i) N-methylcarbazole and thioanisole oxidation with H2(18)O2 catalyzed by cyt.c resulted in introduction of 18O into the product, and (ii) during the epoxidation of cis-stilbene catalyzed by cyt.c, the stereochemistry of the substrate was retained and 18O was introduced when H2(18)O2 was used as an oxidant. These results show that cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as P450. To utilize these P450-like reactivities effectively, cyt.c was immobilized on poly-gamma-methyl-L-glutamate. Up to 99% of the cyt.c used was immobilized. This immobilized cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as both P450 and free cyt.c, and the activities of these reactions were increased by the immobilization. In N-demethylation of N,N-dimethylaniline with cumene hydroperoxide (CHP) catalyzed by cyt.c, the Vmax for CHP was increased by 4.4-fold by the immobilization of the enzyme, while the Km remained unchanged. Since P450 is involved in the metabolism of many xenobiotics, the above results suggest that immobilized cyt.c may be useful in drug metabolism research. PMID:7681661

  13. Investigation of liquid phase axial dispersion in Taylor bubble flow by radiotracer residence time distribution analysis

    NASA Astrophysics Data System (ADS)

    Chughtai, I. R.; Iqbal, W.; Din, G. U.; Mehdi, S.; Khan, I. H.; Inayat, M. H.; Jin, J. H.

    2013-05-01

    A gas-liquid Taylor bubble flow occurs in small diameter channels in which gas bubbles are separated by slugs of pure liquid. This type of flow regime is well suited for solid catalyzed gas-liquid reactors in which the reaction efficiency is a strong function of axial dispersion in the regions of pure liquid. This paper presents an experimental study of liquid phase axial dispersion in a Taylor bubble flow developed in a horizontal tube using high speed photography and radiotracer residence time distribution (RTD) analysis. A parametric dependence of axial dispersion on average volume fraction of gas phase was also investigated by varying the relative volumetric flow rates of the two phases. 137mBa produced from a 137Cs/137mBa radionuclide generator was used as radiotracer and measurements were made using the NaI(Tl) scintillation detectors. Validation of 137mBa in the form of barium chloride as aqueous phase radiotracer was also carried out. Axial Dispersion Model (ADM) was used to simulate the hydrodynamics of the system and the results of the experiment are presented. It was observed that the system is characterized by very high values of Peclet Number (Pe˜102) which reveals an approaching plug type flow. The experimental and model estimated values of mean residence times were observed in agreement with each other.

  14. 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.

  15. 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

  16. A system to obtain radiotracer uptake data simultaneously with NMR spectra in a high field magnet

    SciTech Connect

    Buchanan, M.; Marsden, P.K.; Garlick, P.B.; Mielke, C.H.

    1996-06-01

    Radiotracer techniques and nuclear magnetic resonance (NMR) spectroscopy are two complementary methods that are widely used to investigate cardiac metabolism. The authors have now developed a novel gamma photon detector system that will operate within a wide-bore, 9.4 T magnet. With this detector in position, it is possible to acquire radiotracer uptake data while simultaneously collecting NMR spectra. The advantages of this new system are firstly, that it enables correlations between radiotracer and NMR data to be made on individual rat hearts, and secondly that it allows the number of experiments required to obtain results of statistical significance to be greatly decreased. The extension of the system, to one in which positron emission tomography (PET) and magnetic resonance imaging (MRI) data are acquired simultaneously, clearly has enormous clinical potential. The detector consists of a NaI(Tl) scintillation crystal coupled to a magnetic field-insensitive photomultiplier tube by a 72.5 cm long, acrylic light pipe. This detector configuration satisfies the two, conflicting requirements of the crystal being near the sample, and thus in a high magnetic field, and the PMT being in a low magnetic field and thus far from the sample. In this paper the authors present the technical specifications of their new system together with what they believe are the first examples of simultaneously acquired NMR spectra and {sup 18}F-fluorodeoxyglucose ({sup 18}FDG) uptake data, obtained from isolated, perfused rat hearts.

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

    PubMed

    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, (111)In-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 (111)In-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

  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. PMID:24685428

  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. PMID:17292891

  20. Cytochrome c adducts with PCB quinoid metabolites.

    PubMed

    Li, Miao; Teesch, Lynn M; Murry, Daryl J; Pope, R Marshal; Li, Yalan; Robertson, Larry W; Ludewig, Gabriele

    2016-02-01

    Polychlorinated biphenyls (PCBs) are a group of 209 individual congeners widely used as industrial chemicals. PCBs are found as by-products in dye and paint manufacture and are legacy, ubiquitous, and persistent as human and environmental contaminants. PCBs with fewer chlorine atoms may be metabolized to hydroxy- and dihydroxy-metabolites and further oxidized to quinoid metabolites both in vitro and in vivo. Specifically, quinoid metabolites may form adducts on nucleophilic sites within cells. We hypothesized that the PCB-quinones covalently bind to cytochrome c and, thereby, cause defects in the function of cytochrome c. In this study, synthetic PCB quinones, 2-(4'-chlorophenyl)-1,4-benzoquinone (PCB3-pQ), 4-4'-chlorophenyl)-1,2-benzoquinone (PCB3-oQ), 2-(3', 5'-dichlorophenyl)-1,4-benzoquinone, 2-(3',4', 5'-trichlorophenyl)-1,4-benzoquinone, and 2-(4'-chlorophenyl)-3,6-dichloro-1,4-benzoquinone, were incubated with cytochrome c, and adducts were detected by liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to separate the adducted proteins, while trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to identify the amino acid binding sites on cytochrome c. Conformation change of cytochrome c after binding with PCB3-pQ was investigated by SYBYL-X simulation and cytochrome c function was examined. We found that more than one molecule of PCB-quinone may bind to one molecule of cytochrome c. Lysine and glutamic acid were identified as the predominant binding sites. Software simulation showed conformation changes of adducted cytochrome c. Additionally, cross-linking of cytochrome c was observed on the SDS-PAGE gel. Cytochrome c was found to lose its function as electron acceptor after incubation with PCB quinones. These data provide evidence that the covalent

  1. The Expanding Reach of Environmental Radiotracers - New Chronometers And More Sensitive Measurements

    NASA Astrophysics Data System (ADS)

    Aalseth, C.

    2015-12-01

    Radiotracers in the environment provide powerful tools for understanding environmental processes. Viewed as an age continuum, methods using shorter-lived radionuclides (<100 y) like 3H, 7Be, 85Kr, 134Cs, and 137Cs generally rely on measurements of radioactive decay in samples. Methods using longer-lived radionuclides (>1,000 y) like 10Be, 14C, 36Cl, and 81Kr generally rely on atom-counting measurements such as accelerator mass spectrometry. Significant challenges exist in the age range between 100 and 1,000 years where useful radiotracers are difficult to measure by either method and can have very low abundance. These challenges are being addressed with more sensitive measurements using both atom counting and radioactive decay, extending the reach of established radiotracers as well as adding new chronometers. Improvements in atom-counting methods will be reviewed; the practicality of using 81Kr (abundance ~5×10-13 in atmospheric krypton) for age-dating old aquifers has been established and current work focuses on improving sample utilization efficiency to allow smaller samples to be measured. Better efficiency also brings lower-abundance isotopes within reach, for example 39Ar. Improvements in radioactive decay counting will be reviewed; these take advantage of ultra-pure materials to achieve lower backgrounds and are adding new age-dating reach to the environmental science tool-set with intermediate half-life radionuclides, for example 32Si for sediment cores. These methods also improve sensitivity for established radiotracers like 3H and will allow smaller samples to be measured, allowing specific processes to be traced. For example, using 3H as an indicator of carbon cycling through organic compounds in soil systems. Progress in both atom counting and decay counting is expanding the use of 39Ar for age-dating aquifers, measuring ocean mixing, and age-dating younger glacial ice. Argon-39 is a challenging intermediate-age radiotracer (269-year half-life) with

  2. 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

  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. Cytochrome P450 humanised mice

    PubMed Central

    2004-01-01

    Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

  5. Cytochrome f function in photosynthetic electron transport.

    PubMed Central

    Whitmarsh, J; Cramer, W A

    1979-01-01

    The questions of whether the stoichiometry of the turnover of cytochrome f, and the time-course of its reduction subsequent to a light flash, are consistent with efficient function in noncyclic electron transport have been investigated. Measurements were made of the absorbance change at the 553-nm alpha-band maximum relative to a reference wavelength. In the dark cytochrome f is initially fully reduced, oxidized by a 0.3-s flash, and reduced again in the dark period after the flash. In the presence of gramicidin at 18 degrees C, the dark reduction was characterized by a half-time of 25-30 ms, stoichiometries of cytochrome f:chlorophyll and P700:chlorophyll of 1:670 and 1:640, respectively, and a short time delay. The time delay in the dark reduction of cytochrome f, which is expected for a component in an intermediate position in the chain, becomes more apparent in the presence of valinomycin and K+. Under these conditions the half-time for cytochrome f dark reduction is 130-150 ms, and the delay is approximately equal to 20 ms. The measured value for the activation energy of the dark reduction of cytochrome f (11 +/- 1 kcal/mol) is the same as that for noncyclic electron transport in steady-state light. A sigmoidal time-course for the reduction of cytochrome f has been calculated for a simple linear electron transport chain. The kinetics for reduction of cytochrome f predicted by the calculation, in the presence of valinomycin and K+, are in reasonably good agreement with the experimental data. There is an appreciable amount of data in the literature to document complex properties of cytochrome f after illumination with short flashes, and evidence for complexity in a light-minus-dark transition. The data presented here, obtained after a long flash that should establish steady-state conditions, either fulfill or are consistent with the basic criteria for efficient function of cytochrome f in noncyclic electron transport. PMID:262417

  6. 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

  7. The carboxyl-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of tung tree (Vernicia fordii) tail-anchored cytochrome b5 (Cb5) target specifically to endoplasmic reti...

  8. 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

  9. Cytochrome bc1 complexes of microorganisms.

    PubMed Central

    Trumpower, B L

    1990-01-01

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

  10. Induction of cytochrome P-450, cytochrome b-5, NADPH-cytochrome c reductase and change of cytochrome P-450 isozymes with long-term trichloroethylene treatment.

    PubMed

    Kawamoto, T; Hobara, T; Nakamura, K; Imamura, A; Ogino, K; Kobayashi, H; Iwamoto, S; Sakai, T

    1988-12-30

    Several reports have described the effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system (MFOS). These studies suggest that repeated TCE administration induces MFOS, especially cytochrome P-450 and NADPH-cytochrome c reductase. However, it is uncertain what isozymes are induced by TCE treatment, and it is not clear how microsomal enzymes or cytochrome P-450 isozymes are altered when TCE is administered for a duration longer than 28 days. We investigated the changes of MFOS by long-term TCE treatment. Male Wistar rats were injected with TCE, 1.0 g/kg body weight once a day for 5 continuous days or 2.0 g/kg body weight twice a week for 15 days. The mean body weight of the rats treated with TCE for 15 weeks was slightly, but not significantly, less than that of the control rats. Relative liver weights (liver wt/body wt) of the TCE-treated group were however significantly larger (21%) than those of the control group. The weights of the other organs were not changed by long-term TCE treatment. Trichloroethylene treatments for 5 days and 15 weeks caused significant increases in microsomal protein, cytochrome P-450, cytochrome b-5 and NADPH-cytochrome c reductase. TCE treatments produced an increase in a polypeptide band at 52,000 molecular weight range observed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This increase in similar to, but less pronounced than that induced by phenobarbital (PB) treatment. There were no remarkable changes at 56,000 molecular weight range where a band appeared after the treatment with 3-methylcholanthrene (MC). It is likely that the induction of cytochrome P-450 by TCE is relatively similar to that by PB. PMID:3145630

  11. 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

  12. Biosynthesis of artificial microperoxidases by exploiting the secretion and cytochrome c maturation apparatuses of Escherichia coli

    PubMed Central

    Braun, Martin; Thöny-Meyer, Linda

    2004-01-01

    Microperoxidases were initially isolated as peptide fragments containing covalently bound heme and are derived from naturally occurring c-type cytochromes. They are not only used as model compounds but also have potential applications as biosensors, electron carriers, photoreceptors, microzymes, and drugs. In a systematic attempt to define the minimal requirements for covalent attachment of hemes to c-type cytochromes, we have succeeded to produce artificial microperoxidases with peptide sequences that do not occur naturally and can be manipulated. The in vivo production of these microperoxidases requires targeting of the peptide to the bacterial periplasm, proteolytic processing of the signal peptide, and covalent attachment of heme to the signature motif CXXCH by the cytochrome c maturation proteins CcmA-H. The peptides that bind heme carry a C-terminal histidine tag, presumably to stabilize the heme peptide. We present a heme cassette that is the basis for the de novo design of functional hemoproteins. PMID:15328415

  13. Model studies in cytochrome P-450 mediated toxicity of halogenated compounds: radical processes involving iron porphyrins

    SciTech Connect

    Brault, D.

    1985-12-01

    Haloalkane toxicity originates from attack on biological targets by reactive intermediates derived from haloalkane metabolism by a hemoprotein, cytochrome P-450. Carbon-centered radicals and their peroxylderivatives are most likely involved. The reactions of iron porphyrin - a model for cytochrome P-450 - with various carbon-centered and peroxyl radicals generated by pulse radiolysis are examined. Competition between iron porphyrin and unsaturated fatty acids for attack by peroxyl radicals is pointed out. These kinetic data are used to derive a model for toxicity of haloalkanes with particular attention to carbon tetrachloride and halothane. The importance of local oxygen concentration and structural arrangement of fatty acids around cytochrome P-450 is emphasized. 56 references.

  14. 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

  15. Cytochrome P450-based cancer gene therapy: current status.

    PubMed

    Kan, On; Kingsman, Susan; Naylor, Stuart

    2002-12-01

    Results from a number of preclinical studies have demonstrated that a P450-based gene-directed enzyme prodrug therapy (GDEPT) strategy for the treatment of cancer is both safe and efficacious. This strategy has now moved forward into the clinic. At least two different approaches using different delivery methods (retroviral vector MetXia [Oxford BioMedica] and encapsulated P450 expressing cells), different cytochrome P450 isoforms (human CYP2B6 versus rat CYP2B1) and different prodrugs (cyclophosphamide [CPA] versus ifosfamide [IFA]) have concluded Phase I/II clinical trial with encouraging results. In the future, P450-based GDEPT can potentially be further enhanced by improved vectors for P450 gene delivery and disease-targeted promoters for focused gene expression at the target site. In addition, there is scope for developing synthetic P450s and their respective prodrugs to improve both enzyme kinetics and the profile of the active moiety. PMID:12517265

  16. 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.

  17. 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.

  18. 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.

  19. Prediction of water vapor transport rates across polyvinylchloride packaging systems using a novel radiotracer method

    SciTech Connect

    Wood, R.W.; Mulski, M.J.; Kuu, W.Y. )

    1990-09-01

    A radiotracer method is used to study the transport properties of water vapor in polyvinylchloride (PVC), a plastic commonly used in the packaging of parenteral solutions. Water vapor transport across a PVC film appears to be Fickian in nature. Using the steady-state solution of Fick's second law and the permeability coefficient of water vapor across the PVC film obtained using the described method, the predicted water vapor transport rate (WVTR) for a parenteral solution packaged in PVC is in reasonable agreement with actual WVTR as determined by weight loss under precisely controlled conditions.

  20. 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.

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

    PubMed

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

    2016-06-01

    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

  2. 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

  3. Cooperative properties of cytochromes P450

    PubMed Central

    Denisov, Ilia G.; Frank, Daniel J.; Sligar, Stephen G.

    2009-01-01

    Cytochromes P450 form a large and important class of heme monooxygenases with a broad spectrum of substrates and corresponding functions, from steroid hormone biosynthesis to the metabolism of xenobiotics. Despite decades of study, the molecular mechanisms responsible for the complex non-Michaelis behavior observed with many members of this super-family during metabolism, often termed ‘cooperativity,’ remain to be fully elucidated. Although there is evidence that oligomerization may play an important role in defining the observed cooperativity, some monomeric cytochromes P450, particularly those involved in xenobiotic metabolism, also display this behavior due to their ability to simultaneously bind several substrate molecules. As a result, formation of distinct enzyme-substrate complexes with different stoichiometry and functional properties can give rise to homotropic and heterotropic cooperative behavior. This review aims to summarize the current understanding of cooperativity in cytochromes P450, with a focus on the nature of cooperative effects in monomeric enzymes. PMID:19555717

  4. Cytochrome c biogenesis: the Ccm system.

    PubMed

    Sanders, Carsten; Turkarslan, Serdar; Lee, Dong-Woo; Daldal, Fevzi

    2010-06-01

    Cytochromes of c-type contain covalently attached hemes that are formed via thioether bonds between the vinyls of heme b and cysteines within C(1)XXC(2)H motifs of apocytochromes. In diverse organisms this post-translational modification relies on membrane-associated specific biogenesis proteins, referred to as cytochrome c maturation (Ccm) systems. A highly complex version of these systems, Ccm or System I, is found in Gram-negative bacteria, archaea and plant mitochondria. We describe emerging functional interactions between the Ccm components categorized into three conserved modules, and present a mechanistic view of the molecular basis of ubiquitous vinyl-2 approximately Cys(1) and vinyl-4 approximately Cys(2) heme b-apocytochrome thioether bonds in c-type cytochromes. PMID:20382024

  5. CYTOCHROME OXIDASE IN NORMAL AND REGENERATING NEURONS

    PubMed Central

    Howe, Howard A.; Mellors, Robert C.

    1945-01-01

    Manometric determinations of cytochrome oxidase activity were carried out on grey matter from the thalamus and anterior horn of cats and monkeys under various experimental conditions. The thalamus of the cat was studied following the degeneration of virtually all the thalamic neurons secondary to decortication. In comparing the deneuronated thalamus with the normal one, it was found that approximately 34 per cent of the cytochrome oxidase activity was contributed by the neurons and the balance by neuroglia and mesodermal tissues which on the operated side remained comparable to that of the normal side. Total activity of the normal thalamus averaged 5.52 units per mg. of dry weight where I unit is defined as the amount of cytochrome oxidase required to produce a net oxygen consumption of 10 c.mm. per hour under the specified conditions of the experiment. The grey matter of the anterior horns of the spinal cord was isolated by a special technique and its cytochrome oxidase activity was compared with anterior horns in which motoneurons had been stimulated to regenerative activity by section of peripheral nerves. Each animal was studied in relation to an anterior horn which was normal and one in which only the functional state of the motoneurons had been changed. Average normal levels of 2.23 units were found for cat anterior horn and 0.69 units for the monkey. Reductions of cytochrome oxidase activity in the range of 22 to 23 per cent were observed for both cat and monkey following nerve section. In the latter the time sequence was carefully studied in relation to the cytological cycle known as chromatolysis and a virus refractory state previously described by us. It was found that maximal reduction of cytochrome oxidase activity coincided with maximal refractoriness of the cells to poliomyelitis virus (30 to 70 days following nerve section). Neither of these states could be correlated in time with maximal chromatolysis (10 to 15 days). PMID:19871471

  6. 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

  7. 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. PMID:26369789

  8. Cytochrome c Oxidase Biogenesis: New levels of Regulation

    PubMed Central

    Fontanesi, Flavia; Soto, Ileana C.; Barrientos, Antoni

    2008-01-01

    Summary Eukaryotic cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a multimeric enzyme of dual genetic origin, whose assembly is a complicated and highly regulated process. COX displays a concerted accumulation of its constitutive subunits. Data obtained from studies performed with yeast mutants indicate that most catalytic core unassembled subunits are post-translationally degraded. Recent data obtained in the yeast Saccharomyces cerevisiae have revealed another contribution to the stoichiometric accumulation of subunits during COX biogenesis targeting subunit 1 or Cox1p. Cox1p is a mitochondrially encoded catalytic subunit of COX which acts as a seed around which the full complex is assembled. A regulatory mechanism exists by which Cox1p synthesis is controlled by the availability of its assembly partners. The unique properties of this regulatory mechanism offer a means to catalyze multiple-subunit assembly. New levels of COX biogenesis regulation have been recently proposed. For example, COX assembly and stability of the fully assembled enzyme depend on the presence in the mitochondrial compartments of two partners of the oxidative phosphorylation system, the mobile electron carrier cytochrome c and the mitochondrial ATPase. The different mechanisms of regulation of COX assembly are reviewed and discussed. PMID:18465791

  9. Combination of sealed source and radiotracer technique to understand malfunctioning in a chemical plant.

    PubMed

    Yelgaonkar, V N; Jayakumar, T K; Singh, Sudhir; Sharma, M K

    2009-01-01

    Pure terphthalic acid (PTA) is produced by the oxidation of paraxylene in an oxidation reactor of a PTA plant. Since the reaction is exothermic, the temperature rises above 210 degrees C. Vapours formed in the reactor are passed through a series of heat exchangers and the cooled liquid is fed back to the reactor, which flows to the reactor by gravity. In one of the heat exchangers, improper flow distribution in the inlet and outlet pipelines was suspected. Maldistribution of flow in the heat exchanger was also suspected. Gamma scanning of the pipelines and a radiotracer experiment were carried out in the heat exchanger to study the malfunctioning. A specially fabricated pipe scanner was used to scan both 24in diameter and 16in diameter pipelines. From gamma scanning of the pipelines mostly on the bends, absence of the full bore flow of the liquid was observed. Presence of vapours along with the liquid could be obstructing the liquid flow, thereby causing the malfunctioning. A radiotracer experiment was also carried out to study the flow pattern in the heat exchanger. From the experiment, mean residence time of the heat exchanger was estimated as 470s, which theoretically should be about 102s. It indicated that the flow is decelerated in the heat exchanger because of the presence of vapour lock in the tube side. PMID:19318264

  10. 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.

  11. 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.

  12. 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

  13. 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.

  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. Intronic polymorphisms of cytochromes P450

    PubMed Central

    2010-01-01

    The cytochrome P450 enzymes active in drug metabolism are highly polymorphic. Most allelic variants have been described for enzymes encoded by the cytochrome P450 family 2 (CYP2) gene family, which has 252 different alleles. The intronic polymorphisms in the cytochrome P450 genes account for only a small number of the important variant alleles; however, the most important ones are CYP2D6*4 and CYP2D6*41, which cause abolished and reduced CYP2D6 activity, respectively, and CYP3A5*3 and CYP3A5*5, common in Caucasian populations, which cause almost null activity. Their discoveries have been based on phenotypic alterations within individuals in a population, and their identification has, in several cases, been difficult and taken a long time. In light of the next-generation sequencing projects, it is anticipated that further alleles with intronic mutations will be identified that can explain the hitherto unidentified genetic basis of inter-individual differences in cytochrome P450-mediated drug and steroid metabolism. PMID:20846929

  16. 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.

  17. Cholesterol-metabolizing cytochromes P450: implications for cholesterol lowering

    PubMed Central

    Pikuleva, Irina A.

    2010-01-01

    Cardiovascular disease (CVD) continues to be a leading cause of death worldwide. Elevated serum cholesterol is one of the classical risk factors for CVD which also include age, hypertension, smoking, diabetes mellitus, obesity and family history. A number of therapeutic drug classes have been developed to treat hypercholesterolemia, yet, an important percentage of patients do not reach their treatment goals. Therefore, new cholesterol-lowering medications, having a site of action different from that of currently available drugs need to be developed. This review summarizes new information about cytochrome P450 enzymes 7A1, 27A1, and 46A1, that play key roles in cholesterol elimination and that have potential to serve as targets for cholesterol-lowering. PMID:18950282

  18. 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

  19. Personalized Cancer Therapy Considering Cytochrome P450 Variability.

    PubMed

    Preissner, Saskia; Simmaco, Maurizio; Gentile, Giovanna; Preissner, Robert

    2015-01-01

    The individual variability of pharmacokinetics is underestimated and few systematic studies exist in this field. In most cases, this leads to unwanted side effects or toxicity. In polychemotherapy, prodrugs (like ifosfamide), which have to be activated by cytochrome P450 enzymes (CYPs), play an important role. If patients are poor metabolizers for these drugs, the therapy will be ineffective. Furthermore, CYPs and transporters can be (over)expressed in target tissues, which is also not examined and considered in clinical routine. Here, we present a body map showing relevant enzymes in some organs and tissues. Finally, a typical case of a Caucasian chemotherapy patient with breast cancer is presented and discussed regarding a personalized cancer therapy considering the single nucleotide polymorphisms found via genotyping. PMID:26233905

  20. b-Type Cytochromes in Higher Plant Plasma Membranes 1

    PubMed Central

    Asard, Han; Venken, Mireille; Caubergs, Roland; Reijnders, Willem; Oltmann, Fred L.; De Greef, Jan A.

    1989-01-01

    The composition and characteristics of b-type cytochromes from higher plant plasma membranes, purified using aqueous two-phase partitioning, were investigated. At least three different cytochromes were identified by their wavelength maxima and redox midpoint potentials (E0′). Cytochrome b-560.7 (E0′ from + 110 to + 160 millivolts) was present in zucchini (Cucurbita pepo) hypocotyls and bean (Phaseolus vulgaris L.) hooks, although in different concentrations. The main component in cauliflower (Brassica oleracea L.) inflorescences (cytochrome b-558.8) is probably functionally similar to this cytochrome. The plasma membrane generally contains two to three cytochrome species. However, the occurrence and concentrations were species dependent. The high potential cytochrome can be reduced by ascorbate but not NADH, and may be involved in blue light perception. PMID:16666854

  1. 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

  2. Genetic analysis of the phenobarbital regulation of the cytochrome P-450 2b-9 and aldehyde dehydrogenase type 2 mRNAs in mouse liver.

    PubMed Central

    Damon, M; Fautrel, A; Guillouzo, A; Corcos, L

    1996-01-01

    The aim of this study was to investigate the effect of the genetic background on the phenobarbital inducibility of cytochrome P-450 2b-9, cytochrome P-450 2b-10 and aldehyde dehydrogenase type 2 mRNAs in mice. We analysed the basal expression and the phenobarbital inducibility of both cytochrome P-450 mRNAs by semi-quantitative specific reverse transcription-PCR analyses in five inbred mouse strains (A/J,BALB/cByJ,C57BL/6J, DBA/2J and SWR/J). Male mice constitutively expressed cytochrome P-450 2b-9 and cytochrome P-450 2b-10 mRNAs, but a number of differences in their response to phenobarbital were observed. In all these mouse strains, phenobarbital induced cytochrome P-450 2b-10 mRNA whereas it could have either a positive or a negative effect on cytochrome P-450 2b-9 expression, depending on the strain and the sex of the mice. Specifically, phenobarbital increased cytochrome P-450 2b-9 expression in C57BL/6J males while it decreased it in DBA/2J mice. Interestingly, dexamethasone was able to mimic the phenobarbital effect on both cytochromes P-450 in these two strains. Aldehyde dehydrogenase type 2 mRNA was always induced by phenobarbital, except in the C57BL/6J strain. Genetic analysis revealed that the phenobarbital-inducible phenotype was either a semi-dominant or a recessive trait in F1 animals from a C57BL/6J x DBA/2J cross for the cytochrome P-450 2b-9 and the aldehyde dehydrogenase type 2 genes, respectively. This study suggests that the genetic basis for phenobarbital induction in mice depends on the target gene, and that more than one regulatory step would by involved in this response pathway. PMID:8713075

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. In vivo distribution of liposome encapsulated hemoglobin studied with imaging radiotracers. Progress report

    SciTech Connect

    Phillips, W.T.

    1992-12-01

    This project has as its objective the development of radiotracer imaging technology to follow the in vivo circulation and organ deposition of liposome encapsulated hemoglobin (LEH). LEH will be labeled with technetium-99m or indium-111 and infused into small animals to monitor any in vivo differences between different LEH formulations. These studies will be correlated with any hematological and pathological changes associated with LEH treatment. Development of such non-invasive monitoring techniques may lead to significant cost effective manufacturing and formulation improvements, and ultimately a more efficacious LEH product. The development of this elegant labeling technique should make it possible to study the effect of various LEH modifications on biodistribution non-invasively in primates and humans.

  8. Radiotracers for Cardiac Sympathetic Innervation: Transport Kinetics and Binding Affinities for the Human Norepinephrine Transporter

    PubMed Central

    Raffel, David M.; Chen, Wei; Jung, Yong-Woon; Jang, Keun Sam; Gu, Guie; Cozzi, Nicholas V.

    2013-01-01

    Introduction Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [11C]-(−)-meta-hydroxyephedrine, [11C]-(−)-epinephrine, and a series of [11C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [3H]-labeled biogenic amines were also determined. Methods Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis-Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [3H]mazindol provided estimates of binding affinities (KI) for NET. Results Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for ‘tracer concentrations’ of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r2 = 0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics. Conclusion The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density. PMID:23306137

  9. Unusual Cytochrome P450 Enzymes and Reactions*

    PubMed Central

    Guengerich, F. Peter; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO3+), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. PMID:23632016

  10. Recombinant human erythrocyte cytochrome b5.

    PubMed

    Lloyd, E; Ferrer, J C; Funk, W D; Mauk, M R; Mauk, A G

    1994-09-27

    The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10). The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium. PMID:7918357

  11. Cytochrome c peroxidase from Methylococcus capsulatus Bath.

    PubMed

    Zahn, J A; Arciero, D M; Hooper, A B; Coats, J R; DiSpirito, A A

    1997-11-01

    A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively. PMID:9325424

  12. 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

  13. 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

  14. Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions

    PubMed Central

    Brusnichkin, Anton V.; Nedosekin, Dmitry A.; Galanzha, Ekaterina I.; Vladimirov, Yuri A.; Shevtsova, Elena F.; Proskurnin, Mikhail A.; Zharov, Vladimir P.

    2012-01-01

    Light-absorbing endogenous cellular proteins, in particular cytochrome c, are used as intrinsic biomarkers for studies of cell biology and environment impacts. To sense cytochrome c against real biological backgrounds, we combined photothermal (PT) thermal-lens single channel schematic in a back-synchronized measurement mode and a multiplex thermal-lens schematic in a transient high resolution (ca. 350 nm) imaging mode. These multifunctional PT techniques using continuous-wave (cw) Ar+ laser and a nanosecond pulsed optical parametric oscillator in the visible range demonstrated the capability for label-free spectral identification and quantification of trace amounts of cytochrome c in a single mitochondrion alone or within a single live cell. PT imaging data were verified in parallel by molecular targeting and fluorescent imaging of cellular cytochrome c. The detection limit of cytochrome c in a cw mode was 5 × 10−9 mol/L (80 attomols in the signal-generation zone); that is ca. 103 lower than conventional absorption spectroscopy. Pulsed fast PT microscopy provided the detection limit for cytochrome c at the level of 13 zmol (13 × 10−21 mol) in the ultra-small irradiated volumes limited by optical diffraction effects. For the first time, we demonstrate a combination of high resolution PT imaging with PT spectral identification and ultrasensitive quantitative PT characterization of cytochrome c within individual mitochondria in single live cells. A potential of far-field PT microscopy to sub-zeptomol detection thresholds, resolution beyond diffraction limit, PT Raman spectroscopy, and 3D imaging are further highlighted. PMID:20572284

  15. Direct evidence for the atovaquone action on the Plasmodium cytochrome bc1 complex.

    PubMed

    Siregar, Josephine E; Kurisu, Genji; Kobayashi, Tamaki; Matsuzaki, Motomichi; Sakamoto, Kimitoshi; Mi-ichi, Fumika; Watanabe, Yoh-ichi; Hirai, Makoto; Matsuoka, Hiroyuki; Syafruddin, Din; Marzuki, Sangkot; Kita, Kiyoshi

    2015-06-01

    Atovaquone, a coenzyme Q analogue has been indicated to specifically target the cytochrome bc1 complex of the mitochondrial respiratory chain in the malarial parasite and other protozoan. Various mutations in the quinone binding site of the cytochrome b gene of Plasmodium spp. such as M133I, L144S, L271V, K272R, Y268C, Y268S, Y268N, and V284F are suggesting to associate with resistance to atovaquone. There is no direct evidence of relation between the mutations and resistance to atovaquone in Plasmodium parasite that has been available. Technical difficulties in isolating active assayable mitochondria in the malarial parasite hinder us to obtain direct biochemical evidence to support the relation between the mutations and drug resistance. The establishment of a mitochondrial isolation method for the malaria parasite has allowed us to test the degree of resistance of Plasmodium berghei isolates to atovaquone directly. We have tested the activity of dihydroorotate (DHO)-cytochrome c reductase in various P. berghei atovaquone resistant clones in the presence of a wide concentration range of atovaquone. Our results show the IC(50) of P. berghei atovaquone resistant clones is much higher (1.5 up to 40 nM) in comparison to the atovaquone sensitive clones (0.132-0.465 nM). The highest IC(50) was revealed in clones carrying Y268C and Y268N mutations (which play an important role in atovaquone resistance in Plasmodium falciparum), with an approximately 100-fold increase. The findings indicate the importance of the mutation in the quinone binding site of the cytochrome b gene and that provide a direct evidence for the atovaquone inhibitory mechanism in the cytochrome bc1 complex of the parasite. PMID:25264100

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Measurement of residence time distribution of liquid phase in an industrial-scale continuous pulp digester using radiotracer technique.

    PubMed

    Sheoran, Meenakshi; Goswami, Sunil; Pant, Harish J; Biswal, Jayashree; Sharma, Vijay K; Chandra, Avinash; Bhunia, Haripada; Bajpai, Pramod K; Rao, S Madhukar; Dash, A

    2016-05-01

    A series of radiotracer experiments was carried out to measure residence time distribution (RTD) of liquid phase (alkali) in an industrial-scale continuous pulp digester in a paper industry in India. Bromine-82 as ammonium bromide was used as a radiotracer. Experiments were carried out at different biomass and white liquor flow rates. The measured RTD data were treated and mean residence times in individual digester tubes as well in the whole digester were determined. The RTD was also analyzed to identify flow abnormalities and investigate flow dynamics of the liquid phase in the pulp digester. Flow channeling was observed in the first section (tube 1) of the digester. Both axial dispersion and tanks-in-series with backmixing models preceded with a plug flow component were used to simulate the measured RTD and quantify the degree of axial mixing. Based on the study, optimum conditions for operating the digester were proposed. PMID:26896681

  4. Establishment of a trimodality analytical platform for tracing, imaging and quantification of gold nanoparticles in animals by radiotracer techniques.

    PubMed

    Chen, Chien-Hung; Lin, Fong-Sian; Liao, Wei-Neng; Liang, Sanching L; Chen, Min-Hua; Chen, Yo-Wen; Lin, Wan-Yu; Hsu, Ming-Hua; Wang, Mei-Ya; Peir, Jinn-Jer; Chou, Fong-In; Chen, Ching-Ya; Chen, Sih-Yu; Huang, Su-Chin; Yang, Mo-Hsiung; Hueng, Dueng-Yuan; Hwu, Yeukuang; Yang, Chung-Shi; Chen, Jen-Kun

    2015-01-01

    This study aims to establish a (198)Au-radiotracer technique for in vivo tracing, rapid quantification, and ex vivo visualization of PEGylated gold nanoparticles (GNPs) in animals, organs and tissue dissections. The advantages of GNPs lie in its superior optical property, biocompatibility and versatile conjugation chemistry, which are promising to develop diagnostic probes and drug delivery systems. (198)Au is used as a radiotracer because it simultaneously emits beta and gamma radiations with proper energy and half-life; therefore, (198)Au can be used for bioanalytical purposes. The (198)Au-tagged radioactive gold nanoparticles ((198)Au-GNPs) were prepared simply by irradiating the GNPs in a nuclear reactor through the (197)Au(n,γ)(198)Au reaction and subsequently the (198)Au-GNPs were subjected to surface modification with polyethylene glycol to form PEGylated (198)Au-GNPs. The (198)Au-GNPs retained physicochemical properties that were the same as those of GNP before neutron irradiation. Pharmacokinetic and biodisposition studies were performed by intravenously injecting three types of (198)Au-GNPs with or without PEGylation into mice; the γ radiation in blood specimens and dissected organs was then measured. The (198)Au-radiotracer technique enables rapid quantification freed from tedious sample preparation and shows more than 95% recovery of injected GNPs. Clinical gamma scintigraphy was proved feasible to explore spatial- and temporal-resolved biodisposition of (198)Au-GNPs in living animals. Moreover, autoradiography, which recorded beta particles from (198)Au, enabled visualizing the heterogeneous biodisposition of (198)Au-GNPs in different microenvironments and tissues. In this study, the (198)Au-radiotracer technique facilitated creating a trimodality analytical platform for tracing, quantifying and imaging GNPs in animals. PMID:25424326

  5. Measurement of Bmax and Kd with the glycine transporter 1 radiotracer ¹⁸F-MK6577 using a novel multi-infusion paradigm.

    PubMed

    Xia, Yan; Zheng, Ming-Qiang; Holden, Daniel; Lin, Shu-fei; Kapinos, Michael; Ropchan, Jim; Gallezot, Jean-Dominique; Huang, Yiyun; Carson, Richard E

    2015-12-01

    Glycine is a co-agonist of glutamate at the NMDA receptor. Glycine transporter 1 (GlyT1) inhibitors are reported to be potential therapeutic agents for schizophrenia. (18)F-MK6577 is a new positron emission tomography (PET) radiotracer useful for imaging brain GlyT1 and its occupancy in humans. We devised a novel multi-infusion paradigm of radiolabeled and unlabeled compound and an iterative linear/nonlinear alternating fitting method to allow for the determination of in vivo affinity (Kd) and target concentration (Bmax) images, constraining Kd to be uniform across the brain. This paradigm was tested with (18)F-MK6577 in baboons. Voxel-based analysis produced high quality Bmax images and reliable Kd estimates, and also suggested that the nondisplaceable distribution volume (VND) is not uniform throughout the brain. In vivo GlyT1 Kd was estimated to be 1.87 nmol/L for (18)F-MK6577, and the rank order of GlyT1 distribution measured in the baboon brain was: high in the brainstem (133 nmol/L), medium in the cerebellum (83 nmol/L), and low in the cortex (30 nmol/L). These in vivo Kd and Bmax values agreed well with those determined in vitro, thus validating our novel multi-infusion approach. PMID:26198176

  6. 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

  7. 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

  8. Target foil rupture scenario and provision for handling different models of medical cyclotrons used in India

    PubMed Central

    Shaiju, V. S.; Sharma, S. D.; Kumar, Rajesh; Sarin, B.

    2009-01-01

    Medical cyclotron is a particle accelerator used in producing short lived radiotracers such as 18F, 11C, 15O, 13N etc. These radiotracers are labeled with suitable pharmaceuticals for use to gather information related to metabolic activity of the cell using Positron Emission Tomography (PET) scan. Target foil rupture is considered one of the major emergency situations during medical cyclotron operations because there is a potential of over exposure to the working personnel. Radiation protection survey of a self-shielded medical cyclotron installation was carried out during normal and emergency conditions. It is found that the induced activity in the target foil increases with its successive usages. As a case study, we have evaluated the emergency handling procedures of GE PETtrace-6 medical cyclotron. Recommendations have also been made to reduce personal exposure while handling the target foil rupture condition such as the use of L-Bench near the target area and participation of experienced personnel. PMID:20098564

  9. Cytochromes P450: Roles in Diseases*

    PubMed Central

    Pikuleva, Irina A.; Waterman, Michael R.

    2013-01-01

    The cytochrome P450 superfamily consists of a large number of heme-containing monooxygenases. Many human P450s metabolize drugs used to treat human diseases. Others are necessary for synthesis of endogenous compounds essential for human physiology. In some instances, alterations in specific P450s affect the biological processes that they mediate and lead to a disease. In this minireview, we describe medically significant human P450s (from families 2, 4, 7, 11, 17, 19, 21, 24, 27, 46, and 51) and the diseases associated with these P450s. PMID:23632021

  10. 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

  11. 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

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

    ClinicalTrials.gov

    2015-12-09

    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

  13. A simple system for remote processing and delivery of H{sub 2}[{sup 15}O] produced from a N{sub 2}/H{sub 2} target

    SciTech Connect

    Ferrieri, R.A.; Alexoff, D.L.; Schlyer, D.L.; Wolf, A.P.

    1993-12-31

    The N{sub 2} + H{sub 2} target can produce more than enough H{sub 2}[{sup 15}O] to meet the demands of any PET imaging facility. We have shown that the radiotracer can be transported across 300 feet of tubing without significant loss of activity, using fast tows of target gas. Thus, there is no need to maintain an extraneous chemical Processing station at the PET imaging facility. A simple remotely operated system is reported that performs the three step operation for H{sub 2}[{sup 15}O] delivery at the PET imaging facility: collection of the radiotracer in water; removal of ammonia from the preparation and delivery of the radiotracer to the injection syringe. The system can process and make available for injection 100 mCi of H{sub 2}[{sup 15}O]. The machine is easily prepped for subsequent deliveries. So that additional doses of radiotracer can be made available within 12 minutes. A general syringe loading device with remote hydraulic injector is that is compatible for use with any H{sub 2}[{sup 15}O] radiotracer processing station. The device allows for direct measurement of syringe dose while filling, and for easy, as well as safe transfer of the injection syringe assembly to a delivery cart that houses the remote hydraulic injector. The injection syringe is never handled directly during transport nor during injection except, to connect it to the intravenous line, thus minimizing radiation exposure to personnel.

  14. 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.

  15. Calcium carbonate scaling kinetics determined from radiotracer experiments with calcium-47

    SciTech Connect

    Turner, C.W.; Smith, D.W.

    1998-02-01

    The deposition of calcium carbonate is one of the principal modes of fouling of the heat-transfer surface of a fresh-water-cooled heat exchanger. The deposition rate of calcium carbonate on a heat-transfer surface has been measured using a calcium-47 radiotracer and compared to the measured rate of thermal fouling. The crystalline phase of calcium carbonate that precipitates depends on the degree of supersaturation at the heat-transfer surface, with aragonite precipitating at higher supersaturations and calcite precipitating at lower supersaturations. Whereas the mass deposition rates were constant with time, the thermal fouling rates decreased throughout the course of each experiment as a result of densification of the deposit. It is proposed that the densification was driven by the temperature gradient across the deposit together with the retrograde solubility of calcium carbonate. The temperature dependence of the deposition rate yielded an activation energy of 79 {+-} 4 kJ/mol for the precipitation of calcium carbonate on a heat-transfer surface.

  16. 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.

  17. Design and evaluation of radiotracers for determination of regional cerebral blood flow with PET

    SciTech Connect

    Lambrecht, R.M.; Duncan, C.C.; Shiue, C.Y.

    1982-01-01

    The tracer kinetics of 4-Fluoro(/sup 18/F)-, 4-Bromo(/sup 82/Br)- and 4-Iodo(/sup 125/I)-antipyrine and /sup 15/O-water were compared in a cat or baboon animal model. First-pass cerebral extraction and clearance with alterations in PaCO/sub 2/ were measured for whole brain. The Renkin/Crone model was used to evaluate brain capillary permeability-surface area product for 4-/sup 18/FAP in cats. Positron-emission-tomographic measurements required development of an instrument and technique for control of the arterial concentration of the radiotracer as a ramp function, so that tracer concentration changes due to radioactive decay or altered physiological processes could be accurately described with PET. Pharmacokinetic and tissue-distribution studies in cats were used to determine dosimetry for 4-/sup 18/FAP. 4-Bromoantipyrine labeled with /sup 78/Br (t = 6.5 m) is suggested as a tracer for determination of rCBF with PET.

  18. 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

  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. Characterization of fast-decaying PET radiotracers solely through LC-MS/MS of constituent radioactive and carrier isotopologues

    PubMed Central

    2013-01-01

    Background The characterization of fast-decaying radiotracers that are labeled with carbon-11 (t1/2 = 20.38 min), including critical measurement of specific radioactivity (activity per mole at a specific time) before release for use in positron-emission tomography (PET), has relied heavily on chromatographic plus radiometric measurements, each of which may be vulnerable to significant errors. Thus, we aimed to develop a mass-specific detection method using sensitive liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) for identifying 11C-labeled tracers and for verifying their specific radioactivities. Methods The LC-MS/MS was tuned and set up with methods to generate and measure the product ions specific for carbon-11 species and M + 1 carrier (predominantly the carbon-13 isotopologue) in four 11C-labeled tracers. These radiotracers were synthesized and then analyzed before extensive carbon-11 decay. The peak areas of carbon-11 species and M + 1 carrier from the LC-MS/MS measurement and the calculated abundances of carbon-12 carrier and M + 1 radioactive species gave the mole fraction of carbon-11 species in each sample. This value upon multiplication with the theoretical specific radioactivity of carbon-11 gave the specific radioactivity of the radiotracer. Results LC-MS/MS of each 11C-labeled tracer generated the product ion peaks for carbon-11 species and M + 1 carrier at the expected LC retention time. The intensity of the radioactive peak diminished as time elapsed and was undetectable after six half-lives of carbon-11. Measurements of radiotracer-specific radioactivity determined solely by LC-MS/MS at timed intervals gave a half-life for carbon-11 (20.43 min) in excellent agreement with the value obtained radiometrically. Additionally, the LC-MS/MS measurement gave specific radioactivity values (83 to 505 GBq/μmol) in good agreement with those from conventional radiometric methods. Conclusions 11C-Labeled tracers were

  1. 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

  2. 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

  3. Docking of cytochrome c6 and plastocyanin to the aa3-type cytochrome c oxidase in the cyanobacterium Phormidium laminosum.

    PubMed

    Hart, Sarah E; Howe, Christopher J; Mizuguchi, Kenji; Fernandez-Recio, Juan

    2008-12-01

    The interactions between redox proteins are transient in nature. Therefore, very few crystal structures are available for the complexes formed between these proteins. Computational docking simulations thus provide a useful alternative method for studying the interactions between electron transfer proteins. In this paper, we have studied the interactions between the aa(3)-type cytochrome c oxidase of the cyanobacterium Phormidium laminosum and its redox partners plastocyanin and cytochrome c(6) using a combination of comparative modelling techniques and docking simulations. Rigid-body docking orientations were scored with a combined energy function that accounts for electrostatics and desolvation. These simulations have identified two plausible docking sites, one of which appears to be unique to the binding of plastocyanin to the oxidase. This unique binding site may be due to the presence of a long loop region in the subunit II of cyanobacterial oxidases. Control simulations were performed with the ba(3)-type cytochrome c oxidase and its redox partner cytochrome c(552) from Thermus thermophilus. The docking between cytochrome c oxidase and its redox partners plastocyanin and cytochrome c(6) is dominated by hydrophobic residues, a feature already observed from kinetic and structural studies in other complexes of P. laminosum (e.g. plastocyanin or cytochrome c(6) with cytochrome f and photosystem I). PMID:18824464

  4. Interaction of yeast iso-1-cytochrome c with cytochrome c peroxidase investigated by [15N, 1H] heteronuclear NMR spectroscopy.

    PubMed

    Worrall, J A; Kolczak, U; Canters, G W; Ubbink, M

    2001-06-19

    The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed. For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex. In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs. In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase. This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding. PMID:11401551

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

    PubMed Central

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

    2004-01-01

    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 ≈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

  6. Immunoquantitation of cytochrome. beta. /sub 5/ and methylcholanthrene-induced cytochromes P-450

    SciTech Connect

    Shires, T.K.; Krieter, P.A.; Shawver, L.K.; Seidel, S.L.

    1987-06-01

    The enzyme-linked immunosorbent assay (ELISA) has been investigated for its ability to quantitate hydrophobic proteins like cytochromes ..beta../sub 5/ and P-450 at the subnanogram level. Issues encountered that have broad significance not only for ELISA, but for other qualitative and quantitative immunoassays as well, include the effects of detergent, the discriminatory capacity of ELISA, and the method for determining an assay's selectivity.

  7. Spectroelectrochemistry of cytochrome P450cam.

    PubMed

    Bistolas, Nikitas; Christenson, Andreas; Ruzgas, Tautgirdas; Jung, Christiane; Scheller, Frieder W; Wollenberger, Ulla

    2004-02-13

    The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis. PMID:14741708

  8. Dihydrolipoic acid reduces cytochrome b561 proteins.

    PubMed

    Bérczi, Alajos; Zimányi, László; Asard, Han

    2013-03-01

    Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins. PMID:22526465

  9. 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

  10. 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

  11. Polymorphism of human cytochrome P-450.

    PubMed

    Guengerich, F P; Umbenhauer, D R; Churchill, P F; Beaune, P H; Böcker, R; Knodell, R G; Martin, M V; Lloyd, R S

    1987-03-01

    The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3577206

  12. 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.

  13. 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.

  14. 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.

  15. Mapping of four discontiguous antigenic determinants on horse cytochrome c.

    PubMed

    Oertle, M; Immergluck, K; Paterson, Y; Bosshard, H R

    1989-07-01

    The epitopes (antigenic determinants) recognized by four different monoclonal antibodies on horse cytochrome c have been partially characterized by differential acetylation of lysine residues of free and antibody-bound cytochrome c. The degree of acetylation in the bound and free antigen molecule was assessed by a double-labeling procedure with [3H]acetic anhydride and [14C]acetic anhydride. Out of the 19 lysine residues of cytochrome c only very few were less reactive in the antigen-antibody complex, i.e. presumably located at the epitope for the antibody under study. The protection varied from 1.5-fold to over 20-fold lower reactivity in antibody-bound cytochrome c. The present results are complemented by previous data obtained by cross-reactivity analysis with cytochromes c from different species, with chemically modified cytochrome c derivatives, and by inhibition of proteolysis of cytochrome c in the presence of the antibodies. From the combined data we conclude that each of the four epitopes depends on the precise spatial folding of the antigen and contains residues which are brought together by the folding of the polypeptide chain. This work exemplifies that mapping of conformation-dependent epitopes can be achieved by applying a combination of mapping procedures of which each by itself provides partial information. PMID:2473902

  16. [18F]Fluoromethyl-[1,2-2H4]-choline: A novel radiotracer for imaging choline metabolism in tumors by positron emission tomography

    PubMed Central

    Leyton, Julius; Smith, Graham; Zhao, Yongjun; Perumal, Meg; Nguyen, Quang-De; Robins, Edward; Årstad, Erik; Aboagye, Eric O.

    2009-01-01

    Current radiotracers for positron emission tomography (PET) imaging of choline metabolism have poor systemic metabolic stability in vivo. We describe a novel radiotracer, [18F]fluoromethyl-[1,2-2H4]-choline (D4-FCH), that employs deuterium isotope effect to improve metabolic stability. D4-FCH proved more resistant to oxidation than its non-deuterated analog, [18F]fluoromethylcholine (FCH), in plasma, kidneys, liver and tumor, while retaining phosphorylation potential. Tumor radiotracer levels, a determinant of sensitivity in imaging studies, was improved by deuterium substitution; tumor uptake values expressed as %injected dose/voxel at 60 min were 7.43 ± 0.47 and 5.50 ± 0.49 for D4-FCH and FCH, respectively, (P = 0.04). D4-FCH was also found to be a useful response biomarker. Treatment with the mitogenic extracellular kinase inhibitor, PD0325901, resulted in a reduction in tumor radiotracer uptake that occurred in parallel with reductions in choline kinase A expression. In conclusion, D4-FCH is a very promising metabolically stable radiotracer for imaging choline metabolism in tumors. PMID:19773436

  17. 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

  18. 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

  19. Evidence that two forms of bovine erythrocyte cytochrome b5 are identical to segments of microsomal cytochrome b5.

    PubMed Central

    Douglas, R H; Hultquist, D E

    1978-01-01

    Homogeneous preparations of two forms of soluble cytochrome b5 have been obtained from bovine erythrocytes by successive chromatography on DEAE-cellulose, Bio-Gel P-60, and DEAE-Sephadex. Although the two forms could be separated on disc gel electrophoresis, they appeared to have similar molecular weights of approximately 12,000 and identical visible absorbance spectra. The tryptic hemepeptides derived from the two forms of bovine erythrocyte cytochrome b5 are electrophoretically indistinguishable from each other and from the tryptic core hemepeptide derived from liver microsomal cytochrome b5. The bovine erythrocyte tryptic hemepeptide was purified to homogeneity; its amino acid composition was shown to be identical to that of tryptic hemepeptide from liver microsomal cytochrome b5. The amino acid compositions of the two isolatable forms of erythrocyte cytochrome b5 correspond well to the compositions of the 97- and 95-residue segments of native liver microsomal cytochrome b5 that begin at the NH2 terminus. These results agree with the hypothesis that soluble erythrocyte cytochrome b5 is derived from microsomal protein by proteolysis during erythroid maturation. PMID:277914

  20. Mitochondrial gene cytochrome b developmental and environmental expression in Aedes aegypti.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome b, coded by mitochondrial DNA, is one of the cytochromes involved in electron transport in the respiratory chain of mitochondria. Cytochrome b is a critical intermediate in a mitochondrial death pathway. To reveal whether cytochrome b of the mosquito Aedes aegypti L. (AeaCytB) is developm...

  1. Diffuse pulmonary uptake of bone-seeking radiotracer in bone scintigraphy of a rare case of pulmonary alveolar microlithiasis

    PubMed Central

    Fallahi, Babak; Ghafary, Bahar Moasses; Fard-Esfahani, Armaghan; Eftekhari, Mohammad

    2015-01-01

    Pulmonary alveolar microlithiasis (PAM) is a rare diffuse pulmonary disease representing microliths formed by deposition of calcium phosphonate in the alveolar airspaces. PAM is often diagnosed incidentally during chest X-ray imaging. Most of them are asymptomatic. We present a 39-year-old man referring for a bone scan due to a complaint of right leg pain. Bone scan showed diffuse uptake of bone-seeking radiotracer on both lung fields predominantly in basal regions. The bronchoalveolar lavage test confirmed the diagnosis of PAM. PMID:26170578

  2. 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

  3. 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.

  4. MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association.

    PubMed

    Brown, K; Nurizzo, D; Besson, S; Shepard, W; Moura, J; Moura, I; Tegoni, M; Cambillau, C

    1999-06-18

    The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely. The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit. The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552

  5. Purification and preliminary characterization of three c-type cytochromes from Pseudomonas nautica strain 617.

    PubMed

    Saraiva, L M; Besson, S; Moura, I; Fauque, G

    1995-07-26

    Three c-type cytochromes, namely cytochrome c553, cytochrome c553(548) and cytochrome c', were purified from the marine denitrifying bacterium Pseudomonas nautica strain 617. These three monohemic cytochromes present in small amounts were preliminarily characterized by physiochemical and spectroscopic techniques. The visible and the 1H-NMR spectra show that cytochrome c553 and cytochrome c553(548) have histidine-methionine as iron axial ligands. Cytochrome c553 and cytochrome c553(548) have mid-point redox potentials of +269 mV and +223 mV, at pH 7.6, and their molecular masses are 14 kDa and 17 kDa, respectively. Cytochrome c' has a molecular mass of 21 kDa and its visible spectrum is typical of a high spin heme. PMID:7626097

  6. Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids

    PubMed Central

    Hallahan, David L.; Nugent, Jonathan H. A.; Hallahan, Beverly J.; Dawson, Glenn W.; Smiley, Diane W.; West, Jevon M.; Wallsgrove, Roger M.

    1992-01-01

    The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity. PMID:16668790

  7. TERATOGEN METABOLISM: THALIDOMIDE ACTIVATION IS MEDIATED BY CYTOCHROME P-450

    EPA Science Inventory

    A metabolite of thalidomide generated by hepatic microsomes inhibited the attachment of tumor cells to concanavalin A-coated polyethylene. Evidence that metabolite formation is mediated by microsomal cytochrome P-450 is presented. Microsomes incubated with thalidomide underwent a...

  8. 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 ...

  9. Rearrangement Reactions Catalyzed by Cytochrome P450s

    PubMed Central

    Ortiz de Montellano, Paul R.; Nelson, Sidney D.

    2010-01-01

    Cytochrome P450s promote a variety of rearrangement reactions both as a consequence of the nature of the radical and other intermediates generated during catalysis, and of the neighboring structures in the substrate that can interact either with the initial radical intermediates or with further downstream products of the reactions. This article will review several kinds of previously published cytochrome P450-catalyzed rearrangement reactions, including changes in stereochemistry, radical clock reactions, allylic rearrangements, “NIH” and related shifts, ring contractions and expansions, and cyclizations that result from neighboring group interactions. Although most of these reactions can be carried out by many members of the cytochrome P450 superfamily, some have only been observed with select P450s, including some reactions that are catalyzed by specific endoperoxidases and cytochrome P450s found in plants. PMID:20971058

  10. 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