Sample records for c-type cytochrome cyma

  1. Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes


    Shi, Liang; Squier, Thomas C.; Zachara, John M.; Fredrickson, James K.


    Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer ...

  2. Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes. (United States)

    Shi, Liang; Squier, Thomas C; Zachara, John M; Fredrickson, James K


    Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope.

  3. Identification and Characterization of MtoA: a Decaheme c-Type Cytochrome of the Neutrophilic Fe(II-oxidizing Bacterium Sideroxydans lithotrophicus ES-1

    Directory of Open Access Journals (Sweden)

    Juan eLiu


    Full Text Available The Gram-negative bacterium Sideroxydans lithotrophicus ES-1 (ES-1 grows on FeCO3 or FeS at oxic-anoxic interfaces at circumneutral pH, and the ES-1-mediated Fe(II oxidation occurs extracellularly. However, the molecular mechanisms underlying ES-1’s ability to oxidize Fe(II remain unknown. Survey of the ES-1 genome for candidate genes for microbial extracellular Fe(II oxidation revealed that it contained a three-gene cluster encoding homologues of MtrA, MtrB and CymA of Shewanella oneidensis MR-1 (MR-1 that are involved in extracellular Fe(III reduction. Homologues of MtrA and MtrB were also involved in extracellular Fe(II oxidation by Rhodopseudomonas palustris TIE-1. To distinguish them from those found in MR-1, the identified homologues were named MtoAB and CymAES-1. Cloned mtoA partially complemented an MR-1 mutant without MtrA with regards to ferrihydrite reduction. Characterization of purified MtoA showed that it was a decaheme c-type cytochrome and oxidized soluble Fe(II. Oxidation of Fe(II by MtoA was pH- and Fe(II-complexing ligand-dependent. Under conditions tested, MtoA oxidized Fe(II from pH 7-9 with the optimal rate at pH 9. MtoA oxidized Fe(II complexed with different ligands at different rates. The reaction rates followed the order Fe(IICl2 > Fe(II-citrate > Fe(II-NTA > Fe(II-EDTA with the second-order rate constants ranging from 6.3 × 10-3 μM-1s-1 for oxidation of Fe(IICl2 to 1.0 × 10-3 μM-1s-1 for oxidation of Fe(II-EDTA. Thermodynamic modeling shows that redox reaction rates for the different Fe(II-complexes correlated with their respective estimated reaction-free energies. Collectively, these results demonstrate that MtoA is a functional Fe(II-oxidizing protein and, by working in concert with MtoB and CymAES 1, may oxidize Fe(II at the bacterial surface and transfer released electrons across the bacterial cell envelope to the quinone pool in the inner membrane during extracellular Fe(II oxidation by ES-1.

  4. Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation. (United States)

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


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

  5. Importance of c-Type cytochromes for U(VI reduction by Geobacter sulfurreducens

    Directory of Open Access Journals (Sweden)

    Leang Ching


    Full Text Available Abstract Background In order to study the mechanism of U(VI reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI with acetate serving as the electron donor was investigated. Results The ability of several c-type cytochrome deficient mutants to reduce U(VI was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60% the ability of G. sulfurreducens to reduce U(VI. Involvement in U(VI reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI reduction. A subpopulation of both wild type and U(VI reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III revealed no correlation between the impact of cytochrome deletion on U(VI reduction and reduction of Fe(III hydroxide and chelated Fe(III. Conclusion This study indicates that c-type cytochromes are involved in U(VI reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium

  6. U(VI) reduction by diverse outer surface c-type cytochromes of Geobacter sulfurreducens. (United States)

    Orellana, Roberto; Leavitt, Janet J; Comolli, Luis R; Csencsits, Roseann; Janot, Noemie; Flanagan, Kelly A; Gray, Arianna S; Leang, Ching; Izallalen, Mounir; Mester, Tünde; Lovley, Derek R


    Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.

  7. c-Type cytochrome-dependent formation of U(IV nanoparticles by Shewanella oneidensis.

    Directory of Open Access Journals (Sweden)

    Matthew J Marshall


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

  8. OmcF, a Putative c-Type Monoheme Outer Membrane Cytochrome Required for the Expression of Other Outer Membrane Cytochromes in Geobacter sulfurreducens



    Outer membrane cytochromes are often proposed as likely agents for electron transfer to extracellular electron acceptors, such as Fe(III). The omcF gene in the dissimilatory Fe(III)-reducing microorganism Geobacter sulfurreducens is predicted to code for a small outer membrane monoheme c-type cytochrome. An OmcF-deficient strain was constructed, and its ability to reduce and grow on Fe(III) citrate was found to be impaired. Following a prolonged lag phase (150 h), the OmcF-deficient strain de...

  9. A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase. (United States)

    Ray, A; Williams, H D


    Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c-type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway's terminal oxidase is a quinol oxidase.

  10. C-type cytochromes in the photosynthetic electron transfer pathways in green sulfur bacteria and heliobacteria. (United States)

    Azai, Chihiro; Tsukatani, Yusuke; Itoh, Shigeru; Oh-oka, Hirozo


    Green sulfur bacteria and heliobacteria are strictly anaerobic phototrophs that have homodimeric type 1 reaction center complexes. Within these complexes, highly reducing substances are produced through an initial charge separation followed by electron transfer reactions driven by light energy absorption. In order to attain efficient energy conversion, it is important for the photooxidized reaction center to be rapidly rereduced. Green sulfur bacteria utilize reduced inorganic sulfur compounds (sulfide, thiosulfate, and/or sulfur) as electron sources for their anoxygenic photosynthetic growth. Membrane-bound and soluble cytochromes c play essential roles in the supply of electrons from sulfur oxidation pathways to the P840 reaction center. In the case of gram-positive heliobacteria, the photooxidized P800 reaction center is rereduced by cytochrome c-553 (PetJ) whose N-terminal cysteine residue is modified with fatty acid chains anchored to the cytoplasmic membrane.

  11. The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus. (United States)

    Allen, James W A; Leach, Nicholas; Ferguson, Stuart J


    c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

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

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


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

  13. Surface multiheme c-type cytochromes from Thermincola potens: Implications for dissimilatory metal reduction by Gram-positive bacteria (United States)

    Carlson, H. K.; Iavarone, A. T.; Gorur, A.; Yeo, B. S.; Tran, R.; Melnyk, R. A.; Mathies, R. A.; Auer, M.; Coates, J. D.


    Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they have been shown to be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or the humic substances analog, anthraquinone-2,6-disulfonate (AQDS). The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS and that several MHCs are localized to the cell wall or cell surface of T. potens. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results are the first direct evidence for cell-wall associated cytochromes and MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

  14. Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria. (United States)

    Carlson, Hans K; Iavarone, Anthony T; Gorur, Amita; Yeo, Boon Siang; Tran, Rosalie; Melnyk, Ryan A; Mathies, Richard A; Auer, Manfred; Coates, John D


    Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

  15. Real-time measurements of the redox states of c-type cytochromes in electroactive biofilms: a confocal resonance Raman Microscopy study.

    Directory of Open Access Journals (Sweden)

    Bernardino Virdis

    Full Text Available Confocal Resonance Raman Microscopy (CRRM was used to probe variations of redox state of c-type cytochromes embedded in living mixed-culture electroactive biofilms exposed to different electrode polarizations, under potentiostatic and potentiodynamic conditions. In the absence of the metabolic substrate acetate, the redox state of cytochromes followed the application of reducing and oxidizing electrode potentials. Real-time monitoring of the redox state of cytochromes during cyclic voltammetry (CV in a potential window where cytochromes reduction occurs, evidenced a measurable time delay between the oxidation of redox cofactors probed by CV at the electrode interface, and oxidation of distal cytochromes probed by CRRM. This delay was used to tentatively estimate the diffusivity of electrons through the biofilm. In the presence of acetate, the resonance Raman spectra of young (10 days, j = 208 ± 49 µA cm(-2 and mature (57 days, j = 267 ± 73 µA cm(-2 biofilms show that cytochromes remained oxidized homogeneously even at layers as far as 70 µm from the electrode, implying the existence of slow metabolic kinetics that do not result in the formation of a redox gradient inside the biofilm during anode respiration. However, old biofilms (80 days, j = 190 ± 37 µA cm(-2 with thickness above 100 µm were characterized by reduced catalytic activity compared to the previous developing stages. The cytochromes in these biofilm were mainly in the reduced redox state, showing that only aged mixed-culture biofilms accumulate electrons during anode respiration. These results differ substantially from recent observations in pure Geobacter sulfurreducens electroactive biofilms, in which accumulation of reduced cytochromes is already observed in thinner biofilms, thus suggesting different bottlenecks in current production for mixed-culture and G. sulfurreducens biofilms.

  16. Functional roles of CymA and NapC in reduction of nitrate and nitrite by Shewanella putrefaciens W3-18-1

    Energy Technology Data Exchange (ETDEWEB)

    Beliav, Alex; Qiu, Dongru; Fredrickson, James K.; Wei, Hehong; Nealson, Kenneth H.; Xia, Ming; Zhou, Jizhong; Dai, Jingcheng; Shi, Liang; Tiedje, James M.; Romine, Margaret F.


    Shewanella putrefaciens W3-18-1 harbours two periplasmic nitrate reductase (Nap) gene clusters, NapC-associated nap-alpha (napEDABC) and CymA-dependent nap-beta (napDAGHB), for dissimilatory nitrate respiration. CymA is a member of the NapC/NirT quinol dehydrogenase family and acts as a hub to support different respiratory pathways, including those on iron [Fe(III)] and manganese [Mn(III, IV)] (hydr)oxide, nitrate, nitrite, fumarate and arsenate in Shewanella strains. However, in our analysis it was shown that another NapC/NirT family protein, NapC, was only involved in nitrate reduction, although both CymA and NapC can transfer quinol-derived electrons to a periplasmic terminal reductase or an electron acceptor. Furthermore, our results showed that NapC could only interact specifically with the Nap-alpha nitrate reductase while CymA could interact promiscuously with Nap-alpha, Nap-beta and the NrfA nitrite reductase for nitrate and nitrite reduction. To further explore the difference in specificity, site-directed mutagenesis on both CymA and NapC was conducted and the phenotypic changes in nitrate and nitrite reduction were tested. Our analyses demonstrated that the Lys-91 residue played a key role in nitrate reduction for quinol oxidation and the Asp-166 residue might influence the maturation of CymA. The Asp-97 residue might be one of the key factors that influence the interaction of CymA with the cytochromes NapB and NrfA.

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

    Thrall, Elizabeth S.

    This thesis describes spectroscopic studies of three different systems: silver nanoparticles, the dye molecule rhodamine 6G adsorbed on graphene, and the type IV pili and c-type cytochromes produced by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens. Although these systems are quite different in some ways, they can all be considered examples of nanomaterials. A nanomaterial is generally defined as having at least one dimension below 100 nm in size. Silver nanoparticles, with sub-100 nm size in all dimensions, are examples of zero-dimensional nanomaterials. Graphene, a single atomic layer of carbon atoms, is the paradigmatic two-dimensional nanomaterial. And although bacterial cells are on the order of 1 μm in size, the type IV pili and multiheme c-type cytochromes produced by G. sulfurreducens can be considered to be one- and zero-dimensional nanomaterials respectively. A further connection between these systems is their strong interaction with visible light, allowing us to study them using similar spectroscopic tools. The first chapter of this thesis describes research on the plasmon-mediated photochemistry of silver nanoparticles. Silver nanoparticles support coherent electron oscillations, known as localized surface plasmons, at resonance frequencies that depend on the particle size and shape and the local dielectric environment. Nanoparticle absorption and scattering cross-sections are maximized at surface plasmon resonance frequencies, and the electromagnetic field is amplified near the particle surface. Plasmonic effects can enhance the photochemistry of silver particles alone or in conjunction with semiconductors according to several mechanisms. We study the photooxidation of citrate by silver nanoparticles in a photoelectrochemical cell, focusing on the wavelength-dependence of the reaction rate and the role of the semiconductor substrate. We find that the citrate photooxidation rate does not track the plasmon resonance of the silver

  18. In Situ Spectral Kinetics of Cr(VI) Reduction by c-Type Cytochromes in A Suspension of Living Shewanella putrefaciens 200 (United States)

    Liu, Tongxu; Li, Xiaomin; Li, Fangbai; Han, Rui; Wu, Yundang; Yuan, Xiu; Wang, Ying


    Although c-type cytochromes (c-Cyts) mediating metal reduction have been mainly investigated with in vitro purified proteins of dissimilatory metal reducing bacteria, the in vivo behavior of c-Cyts is still unclear given the difficulty in measuring the proteins of intact cells. Here, c-Cyts in living Shewanella putrefaciens 200 (SP200) was successfully quantified using diffuse-transmission UV/Vis spectroscopy due to the strong absorbance of hemes, and the in situ spectral kinetics of Cr(VI) reduction by c-Cyts were examined over time. The reduced product Cr(III) observed on the cell surface may play a role in inhibiting the Cr(VI) reduction and reducing the cell numbers with high concentrations (>200 μM) of Cr(VI) evidenced by the 16S rRNA analysis. A brief kinetic model was established with two predominant reactions, redox transformation of c-Cyts and Cr(VI) reduction by reduced c-Cyts, but the fitting curves were not well-matched with c-Cyts data. The Cr(III)-induced inhibitory effect to the cellular function of redox transformation of c-Cyts was then added to the model, resulting in substantially improved the model fitting. This study provides a case of directly examining the reaction properties of outer-membrane enzyme during microbial metal reduction processes under physiological conditions.

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

    Directory of Open Access Journals (Sweden)

    Camille S Stephen

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

  20. Purification of a cytochrome bc1-aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum


    Niebisch, A.; Bott, M.


    The aerobic respiratory chain of the Gram-positive Corynebacterium glutamicum involves a bc(1) complex with a diheme cytochrome c(1) and a cytochrome aa(3) oxidase but no additional c-type cytochromes. Here we show that the two enzymes form a supercomplex, because affinity chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochrome aa(3) always resulted in the copurification of the subunits of the bc(1) complex (QcrA, QcrB, QcrC) and the aa(3) comp...

  1. A severe reduction in the cytochrome C content of Geobacter sulfurreducens eliminates its capacity for extracellular electron transfer. (United States)

    Estevez-Canales, Marta; Kuzume, Akiyoshi; Borjas, Zulema; Füeg, Michael; Lovley, Derek; Wandlowski, Thomas; Esteve-Núñez, Abraham


    The ability of Geobacter species to transfer electrons outside the cell enables them to play an important role in a number of biogeochemical and bioenergy processes. Gene deletion studies have implicated periplasmic and outer-surface c-type cytochromes in this extracellular electron transfer. However, even when as many as five c-type cytochrome genes have been deleted, some capacity for extracellular electron transfer remains. In order to evaluate the role of c-type cytochromes in extracellular electron transfer, Geobacter sulfurreducens was grown in a low-iron medium that included the iron chelator (2,2'-bipyridine) to further sequester iron. Haem-staining revealed that the cytochrome content of cells grown in this manner was 15-fold lower than in cells exposed to a standard iron-containing medium. The low cytochrome abundance was confirmed by in situ nanoparticle-enhanced Raman spectroscopy (NERS). The cytochrome-depleted cells reduced fumarate to succinate as well as the cytochrome-replete cells do, but were unable to reduce Fe(III) citrate or to exchange electrons with a graphite electrode. These results demonstrate that c-type cytochromes are essential for extracellular electron transfer by G. sulfurreducens. The strategy for growing cytochrome-depleted G. sulfurreducens will also greatly aid future physiological studies of Geobacter species and other microorganisms capable of extracellular electron transfer.

  2. Identification of c-Type Heme-Containing Peptides Using Non-Activated Immobilized Metal Affinity Cchromatography Resin Enrichment and Higher-Energy Collisional Dissociation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Haizhen; Yang, Feng; Qian, Weijun; Brown, Roslyn N.; Wang, Yuexi; Merkley, Eric D.; Park, Jea H.; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Shi, Liang; Fredrickson, Jim K.; Pasa-Tolic, Ljiljana; Smith, Richard D.; Lipton, Mary S.


    c-type cytochromes play essential roles in many biological activities of both prokaryotic and eukaryotic cells, including electron transfer, enzyme catalysis and induction of apoptosis. We report a novel enrichment strategy for identifying c-type heme-containing peptides that uses non-activated IMAC resin. The strategy demonstrated at least seven-fold enrichment for heme-containing peptides digested from a cytochrome c protein standard, and quantitative linear performance was also assessed for heme-containing peptide enrichment. Heme-containing peptides extracted from the periplasmic fraction of Shewanella oneidensis MR-1 were further identified using higher-energy collisional dissociation tandem mass spectrometry. The results demonstrated the applicability of this enrichment strategy to identify c-type heme-containing peptides from a highly complex biological sample, and at the same time, confirmed the periplasmic localization of heme-containing proteins during suboxic respiration activities of S. oneidensis MR-1.

  3. Reduction of nitrate in Shewanella oneidensis depends on atypical NAP and NRF systems with NapB as a preferred electron transport protein from CymA to NapA. (United States)

    Gao, Haichun; Yang, Zamin K; Barua, Soumitra; Reed, Samantha B; Romine, Margaret F; Nealson, Kenneth H; Fredrickson, James K; Tiedje, James M; Zhou, Jizhong


    In the genome of Shewanella oneidensis, a napDAGHB gene cluster encoding periplasmic nitrate reductase (NapA) and accessory proteins and an nrfA gene encoding periplasmic nitrite reductase (NrfA) have been identified. These two systems seem to be atypical because the genome lacks genes encoding cytoplasmic membrane electron transport proteins, NapC for NAP and NrfBCD/NrfH for NRF, respectively. Here, we present evidence that reduction of nitrate to ammonium in S. oneidensis is carried out by these atypical systems in a two-step manner. Transcriptional and mutational analyses suggest that CymA, a cytoplasmic membrane electron transport protein, is likely to be the functional replacement of both NapC and NrfH in S. oneidensis. Surprisingly, a strain devoid of napB encoding the small subunit of nitrate reductase exhibited the maximum cell density sooner than the wild type. Further characterization of this strain showed that nitrite was not detected as a free intermediate in its culture and NapB provides a fitness gain for S. oneidensis to compete for nitrate in the environments. On the basis results from mutational analyses of napA, napB, nrfA and napBnrfA in-frame deletion mutants, we propose that NapB is able to favor nitrate reduction by routing electrons to NapA exclusively.

  4. Unique organizational and functional features of the cytochrome c maturation system in Shewanella oneidensis.

    Directory of Open Access Journals (Sweden)

    Miao Jin

    Full Text Available Shewanella are renowned for their ability to respire on a wide range of electron acceptors, which has been partially accredited to the presence of a large number of the c-type cytochromes. In the model species S. oneidensis MR-1, at least 41 genes encode c-type cytochromes that are predicted to be intact, thereby likely functional. Previously, in-frame deletion mutants for 36 of these genes were obtained and characterized. In this study, first we completed the construction of an entire set of c-type cytochrome mutants utilizing a newly developed att-based mutagenesis approach, which is more effective and efficient than the approach used previously by circumventing the conventional cloning. Second, we investigated the cytochrome c maturation (Ccm system in S. oneidensis. There are two loci predicted to encode components of the Ccm system, SO0259-SO0269 and SO0476-SO0478. The former is proven essential for cytochrome c maturation whereas the latter is dispensable. Unlike the single operon organization observed in other γ-proteobacteria, genes at the SO0259-SO0269 locus are uniquely organized into four operons, ccmABCDE, scyA, SO0265, and ccmFGH-SO0269. Functional analysis revealed that the SO0265 gene rather than the scyA and SO0269 genes are relevant to cytochrome c maturation.

  5. Purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon Aeropyrum pernix


    Kabashima Yoshiki; Sakamoto Junshi


    Abstract Background The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from Archaea. In particular, c-type cytochromes have been reported only for a limited number of species. Results Here, we isolated a c-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, Aeropyrum pernix, grown aerobically. The redox spectr...

  6. Cytochromes c': Structure, Reactivity and Relevance to Haem-Based Gas Sensing. (United States)

    Hough, Michael A; Andrew, Colin R


    Cytochromes c' are a group of class IIa cytochromes with pentacoordinate haem centres and are found in photosynthetic, denitrifying and methanotrophic bacteria. Their function remains unclear, although roles in nitric oxide (NO) trafficking during denitrification or in cellular defence against nitrosoative stress have been proposed. Cytochromes c' are typically dimeric with each c-type haem-containing monomer folding as a four-α-helix bundle. Their hydrophobic and crowded distal sites impose severe restrictions on the binding of distal ligands, including diatomic gases. By contrast, NO binds to the proximal haem face in a similar manner to that of the eukaryotic NO sensor, soluble guanylate cyclase and bacterial analogues. In this review, we focus on how structural features of cytochromes c' influence haem spectroscopy and reactivity with NO, CO and O2. We also discuss the relevance of cytochrome c' to understanding the mechanisms of gas binding to haem-based sensor proteins.

  7. C-type lectins%C型凝集素

    Institute of Scientific and Technical Information of China (English)

    谢建辉; 顾建新


    C型凝集素(C-type lectin)代表一个识别碳水化合物配体依赖于钙离子(Ca2+)参与的糖原结合蛋白家族,含有一个或多个一级结构和二级结构同源的碳水化合物识别结构域.随着研究的深入,越来越多的C型凝集素能够识别体内的非糖类的配体,包括蛋白质和脂类等.这些C型凝集素在维持机体稳态、免疫防御以及免疫监视等重要生理病理过程中发挥着重要作用.就C型凝集素的结构、分类和在免疫系统中的功能作一介绍.%C-type lectins are Ca2+-dependent glycan-binding proteins and share primary and secondary structural homology in their carbohydrate-recognition domains (CRDs). However, many members of this family are recently identified not to bind carbohydrates and have evolved to recognize non-sugar ligands such as proteins and lipids. The large family of C-type lectins has an important role in the physiological functions and pathological processes including immune homeostasis, immune defenses, and immune surveillance and so on. In this short review, we summarize the structure of C-type lectin domain, the classification of C-type lectins and their role in the immune system.

  8. Magnetite Compensates for the Lack of a Pilin-Associated c-Type Cytochrome in Extracellular Electron Exchange

    DEFF Research Database (Denmark)

    Liu, Fanghua; Rotaru, Amelia-Elena; Shrestha, Pravin;


    Nano-scale magnetite can facilitate microbial extracellular electron transfer that plays an important role in biogeochemical cycles, bioremediation, and several bioenergy strategies, but the mechanisms for the stimulation of extracellular electron transfer are poorly understood. Further investiga......Nano-scale magnetite can facilitate microbial extracellular electron transfer that plays an important role in biogeochemical cycles, bioremediation, and several bioenergy strategies, but the mechanisms for the stimulation of extracellular electron transfer are poorly understood. Further......S-deficient strain unable to participate in interspecies electron transfer or Fe(III) oxide reduction. In the presence of magnetite wild-type cells repressed expression of the OmcS gene, suggesting that cells might need to produce less OmcS when magnetite was available. The finding that magnetite can compensate...

  9. OmcB, a c-Type Polyheme Cytochrome, Involved in Fe(III) Reduction in Geobacter sulfurreducens



    Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative o...

  10. The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase. (United States)

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


    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.

  11. Purification and characterization of cytochrome f-556.5 from the blue-green alga Spirulina platensis. (United States)

    Böhme, H; Pelzer, B; Böger, P


    The membrane-bound cytochrome f-556.5 from the blue-green alga Spirulina platensis was purified to apparent homogeneity. Most of its properties are comparable to cytochrome f isolated from higher plants and green algae. It is clearly distinguishable from soluble cytochrome c-554, also present in Spirulina, which probably replaces the function of plastocyanin in photosynthetic electron transport. 1. The reduced form of cytochrome f exhibits an asymmetrical alpha-band with a maximum at 556.5 nm, and a pronounced shoulder at 550 nm. The beta-, gamma and delta-bands coincide with those described for Scenedesmus cytochrome f-553, with maxima at 524 (532), 422, 331 and a protein peak at 276 nm. The maximum of ferricytochrome f is at 410.5 nm; there is no indication of a weak 695 nm band, described for soluble c-type cytochromes. The purest preparations had a delta/protein-peak ratio of 0.8; the gamma/alpha ratio was 7.3. Formation of a pyridine hemochromogen with a maximum at 550 nm indicated a c-type cytochrome. The molar extinction coefficient at 556.5 nm is 30200, the differential extinction coefficient 21 500. 2. The molecular weight determined by gel filtration or SDS-polyacrylamide gel electrophoresis is 33 000 and 34 000, respectively. 3. The redox properties differ from those described for other cytochromes f isolated from green algae and higher plants: the midpoint redox potential is significantly more negative (+318 mV, pH 7.0) and from pH 6 to 10 no pH dependence is observed. 4. The isoelectric point was determined at pH 3.95, which is more acidic as compared to other cytochromes f. 5. Comparison of the amino acid composition indicated a distant relationship to higher plant cytochrome f and a closer relationship to cytochrome f from green algae.

  12. C-type natriuretic peptide in prostate cancer

    DEFF Research Database (Denmark)

    Nielsen, Soeren Junge; Iversen, Peter; Rehfeld, Jens F.;


    C-type natriuretic peptide (CNP) is expressed in the male reproductive organs in pigs. To examine whether the human prostate also expresses the CNP gene, we measured CNP and N-terminal proCNP in prostate cancer tissue extracts and performed immunohistochemical biopsy staining. Additionally, pro......CNP-derived peptides were quantitated in plasma from patients with prostate cancer. Blood was collected from healthy controls and patients before surgery for localized prostate cancer. Tissue extracts were prepared from tissue biopsies obtained from radical prostatectomy surgery. N-terminal proCNP, proCNP (1...... demonstrated the presence of the peptides in prostatic epithelial cells. The N-terminal proCNP concentrations in plasma were marginally lower in patients with localized prostate cancer compared with control subjects [13.8 pmol/l (11.0-17.2) vs. 15.1 pmol/l (10.4-23.2), p=0.002] but not enough to justify...

  13. C-type natriuretic peptide and its precursor

    DEFF Research Database (Denmark)

    Lippert, Solvej; Iversen, Peter; Brasso, Klaus;


    AIM: Seminal plasma offer a more organ-specific matrix for markers in prostatic disease. We hypothesized that C-type natriuretic peptide (CNP) expression may constitute such a new target. METHODS: Patients with benign prostatic hyperplasia, clinically localized and metastatic prostate cancer were...... examined for CNP and CNP precursor (proCNP) concentrations in blood and seminal plasma. Furthermore, CNP and the CNP receptor (NPR-B) mRNA contents in tissue from prostate and seminal vesicles were analyzed by qPCR. RESULTS: CNP and NPR-B concentrations decreased with increasing tumor burden (p = 0.......0027 and p = 0.0096, respectively). In contrast, seminal plasma CNP and proCNP concentrations were markedly increased with increased tumor burden (p prostate cancer....

  14. Production, purification, and capsid stability of rhinovirus C types. (United States)

    Griggs, Theodor F; Bochkov, Yury A; Nakagome, Kazuyuki; Palmenberg, Ann C; Gern, James E


    The rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification.

  15. Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III. (United States)

    Richard-Fogal, Cynthia L; San Francisco, Brian; Frawley, Elaine R; Kranz, Robert G


    The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E. coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E. coli periplasmic DsbC/DsbD thiol-reduction pathway. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

  16. C-type Lectin Receptors for Tumor Eradication: Future Directions

    Energy Technology Data Exchange (ETDEWEB)

    Streng-Ouwehand, Ingeborg; Unger, Wendy W. J.; Kooyk, Yvette van, E-mail: [Department of Molecular Cell Biology and Immunology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam (Netherlands)


    Dendritic cells are key regulators in directing immune responses and therefore are under extensive research for the induction of anti-tumor responses. DCs express a large array of receptors by which they scan their surroundings for recognition and uptake of pathogens. One of the receptor-families is the C-type lectins (CLR), which bind carbohydrate structures and internalize antigens upon recognition. Intracellular routing of antigen through CLR enhances loading and presentation of antigen through MHC class I and II, inducing antigen-specific CD4{sup +} and CD8{sup +} T-cell proliferation and skewing T-helper cells. These characteristics make CLRs very interesting targets for DC-based immunotherapy. Profound research has been done on targeting specific tumor antigens to CLR using either antibodies or the natural ligands such as glycan structures. In this review we will focus on the current data showing the potency of CLR-targeting and discuss improvements that can be achieved to enhance anti-tumor activity in the near future.

  17. C-type natriuretic peptide in prostate cancer

    DEFF Research Database (Denmark)

    Nielsen, Soeren Junge; Iversen, Peter; Rehfeld, Jens F.;


    C-type natriuretic peptide (CNP) is expressed in the male reproductive organs in pigs. To examine whether the human prostate also expresses the CNP gene, we measured CNP and N-terminal proCNP in prostate cancer tissue extracts and performed immunohistochemical biopsy staining. Additionally, pro......CNP-derived peptides were quantitated in plasma from patients with prostate cancer. Blood was collected from healthy controls and patients before surgery for localized prostate cancer. Tissue extracts were prepared from tissue biopsies obtained from radical prostatectomy surgery. N-terminal proCNP, proCNP (1......-50) and CNP were measured in plasma and tissue extracts. Biopsies were stained for CNP-22 and N-terminal proCNP. Tissue extracts from human prostate cancer contained mostly N-terminal proCNP [median 5.3 pmol/g tissue (range 1.0-12.9)] and less CNP [0.14 pmol/g tissue (0.01-1.34)]. Immunohistochemistry...

  18. Targeting C-type Lectin Receptors for Cancer Immunity

    Directory of Open Access Journals (Sweden)

    Huimin eYan


    Full Text Available C-type lectin receptors (CLRs are a large family of soluble and trans-membrane pattern recognition receptors that are widely and primarily expressed on myeloid cells. CLRs are important for cell-cell communication and host defense against pathogens through the recognition of specific carbohydrate structures. Similar to a family of Toll-like receptors (TLRs, CLRs signaling are involved in the various steps for initiation of innate immune responses and promote secretion of soluble factors such as cytokines and interferons, Moreover, CLRs contribute to endocytosis and antigen-presentation, thereby fine-tune adaptive immune responses. In addition, there may also be a direct activation of acquired immunity. On the other hand, glycans, such as mannose structures, Lewis-type antigens or GalNAc are components of tumor antigens and ligate CLRs, leading to immunoregulation. Therefore agonists or antagonists of CLRs signaling are potential therapeutic reagents for cancer immunotherapy. We aim to overview the current knowledge of CLRs signaling and the application of their ligands on tumor-associating immune response.

  19. Mathematical model of various statements of C-type Language

    Directory of Open Access Journals (Sweden)

    Manoj Kumar Srivastav


    Full Text Available Some of the important components of high level languages are statements, keywords, variable declarations, arrays, user defined functions etc. In case of object oriented programming language we use class, object, inheritance, operator overloading, function overloading, polymorphism etc. There are some common category of statements such as control statement, loop statements etc. Pointers are also one important concept in C-language. User defined functions, function subprograms or subroutines are also important concepts in different programming languages. The language like ALGOL was developed using Chomsky context free grammar. The similar concept used in C-type languages. The high level languages are now based on mathematical derivations and logic. Most of the components of any high level language can be obtained from simple mathematical logic and derivations. In the present study the authors have tried to give some unified mathematical model of few statements, arrays, user defined functions of C-language. However, the present method may further be extended to any other high level language.

  20. Cloning and characterization of a putative human holocytochrome c-type synthetase gene (HCCS) isolated from the critical region for microphthalmia with linear skin defects (MLS)

    Energy Technology Data Exchange (ETDEWEB)

    Schaefer, L.; Ballabio, A.; Zoghbi, H.Y. [Baylor College of Medicine, Houston, TX (United States)


    Microphthalmia with linear skin defects syndrome (MLS) is an X-linked male-lethal disorder associated with X chromosomal rearrangements resulting in monosomy from Xpter to Xp22. Features include microphthalmia, sclerocornea, linear skin defects, and agenesis of the corpus callosum. Using a cross-species conservation strategy, an expressed sequence from the 450- to the 550-kb MLS critical region on Xp22 was identified by screening a human embryo cDNA library. Northern analysis revealed a transcript of {approx}2.6 kb in all tissues examined, with weaker expression of {approx}1.2- and {approx}5.2-kb transcripts. The strongest expression was observed in heart and skeletal muscle. Sequence analysis of a 3-kb cDNA contig revealed an 807-bp open reading frame encoding a putative 268-amino-acid-protein. Comparison of the sequence with sequences in the databases revealed homology with holocytochrome c-type synthetases, which catalyze the covalent addition of a heme group onto c-type cytochromes in the mitochondria. The c-type cytochromes are required for proper functioning of the electron transport pathway. The human gene (HGMW-approved symbol HCCS) and the corresponding murine gene characterized in this paper are the first mammalian holocytochrome c-type synthetases to be described in the literature. Because of the lack of a neuromuscular phenotype in MLS, it is uncertain whether the deletion of a mitochondrial holocytochrome synthetase would contribute to the phenotype seen in MLS. The expression pattern of this gene and knowledge about the function of holocytochrome synthetases, however, suggest that it is a good candidate for X-linked encephalomyopathies typically associated with mitochondrial dysfunction. 25 refs., 4 figs.

  1. The cytochrome p450 homepage. (United States)

    Nelson, David R


    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  2. DMPD: C-type lectin receptors in antifungal immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18160296 C-type lectin receptors in antifungal immunity. Willment JA, Brown GD. Tre...nds Microbiol. 2008 Jan;16(1):27-32. Epub 2007 Dec 21. (.png) (.svg) (.html) (.csml) Show C-type lectin receptors in anti...fungal immunity. PubmedID 18160296 Title C-type lectin receptors in antifungal immunity. Author

  3. Diving into the redox properties of Geobacter sulfurreducens cytochromes: a model for extracellular electron transfer. (United States)

    Santos, Telma C; Silva, Marta A; Morgado, Leonor; Dantas, Joana M; Salgueiro, Carlos A


    Geobacter bacteria have a remarkable respiratory versatility that includes the dissimilatory reduction of insoluble metal oxides in natural habitats and electron transfer to electrode surfaces from which electricity can be harvested. In both cases, electrons need to be exported from the cell interior to the exterior via a mechanism designated as extracellular electron transfer (EET). Several c-type cytochromes from G. sulfurreducens (Gs) were identified as key players in this process. Biochemical and biophysical data have been obtained for ten Gs cytochromes, including inner-membrane associated (MacA), periplasmic (PpcA, PpcB, PpcC, PpcD, PpcE and GSU1996) and outer membrane-associated (OmcF, OmcS and OmcZ). The redox properties of these cytochromes have been determined, except for PpcC and GSU1996. In this perspective, the reduction potentials of these two cytochromes were determined by potentiometric redox titrations followed by visible spectroscopy. The data obtained are taken together with those available for other key cytochromes to present a thorough overview of the current knowledge of Gs EET mechanisms and provide a possible rationalization for the existence of several multiheme cytochromes involved in the same respiratory pathways.

  4. C-type lectins do not act as functional receptors for filovirus entry into cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuno, Keita; Nakayama, Eri; Noyori, Osamu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan); Marzi, Andrea; Ebihara, Hideki [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Irimura, Tatsuro [Graduate School of Pharmaceutical Science, University of Tokyo, Tokyo (Japan); Feldmann, Heinz [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Takada, Ayato, E-mail: [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan)


    Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

  5. C-type natriuretic-derived peptides as biomarkers in human disease

    DEFF Research Database (Denmark)

    Lippert, Solvej Kølvraa; Goetze, Jens Peter


    and extracellular fluid volume. Atrial natriuretic peptide and B-type natriuretic peptide have gained considerable diagnostic interest as biomarkers in cardiovascular disease. By contrast, C-type natriuretic peptide has not yet been ascribed a role in human diagnostics. This perspective aims at recapitulating...... the present biochemical and clinical issues concerning C-type natriuretic peptide measurement in plasma as a potential biomarker....

  6. A novel pathway of cytochrome c biogenesis is involved in the assembly of the cytochrome b6f complex in arabidopsis chloroplasts. (United States)

    Lezhneva, Lina; Kuras, Richard; Ephritikhine, Geneviève; de Vitry, Catherine


    We recently characterized a novel heme biogenesis pathway required for heme c(i)' covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6. These mutants were functionally complemented with the corresponding wild type cDNAs. Using fluorescent protein reporters, we demonstrated that the CCB1, CCB2, CCB3, and CCB4 proteins are targeted to the chloroplast compartment of Arabidopsis. We have extended our study to the YGGT family, to which CCB3 belongs, by studying insertion mutants of two additional members of this family for which no mutants were previously characterized, and we showed that they are not functionally involved in the CCB system. Thus, we demonstrate the ubiquity of the CCB proteins in chloroplast heme c(i)' binding.

  7. A Novel Pathway of Cytochrome c Biogenesis Is Involved in the Assembly of the Cytochrome b6f Complex in Arabidopsis Chloroplasts*S⃞ (United States)

    Lezhneva, Lina; Kuras, Richard; Ephritikhine, Geneviève; de Vitry, Catherine


    We recently characterized a novel heme biogenesis pathway required for heme ci′ covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6. These mutants were functionally complemented with the corresponding wild type cDNAs. Using fluorescent protein reporters, we demonstrated that the CCB1, CCB2, CCB3, and CCB4 proteins are targeted to the chloroplast compartment of Arabidopsis. We have extended our study to the YGGT family, to which CCB3 belongs, by studying insertion mutants of two additional members of this family for which no mutants were previously characterized, and we showed that they are not functionally involved in the CCB system. Thus, we demonstrate the ubiquity of the CCB proteins in chloroplast heme ci′ binding. PMID:18593701

  8. Cytochrome c and insect cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Kai-Yu Liu; Hong Yang; Jian-Xin Peng; Hua-Zhu Hong


    The role ofcytochrome c in insect cell apoptosis has drawn considerable attention and has been subject to considerable controversy.In Drosophila,the majority of studies have demonstrated that cytochrome c may not be involved in apoptosis,although there are conflicting reports.Cytochrome c is not released from mitochondria into the cytosol and activation of the initiator caspase Dronc or effector caspase Drice is not associated with cytochrome c during apoptosis in Drosophila SL2 cells or BG2 cells.Cytochrome c failed to induce caspase activation and promote caspase activation in Drosophila cell lysates,but remarkably caused caspase activation in extracts from human cells.Knockdown of cytochrome c does not protect cells from apoptosis and over-expression of cytochrome c also does not promote apoptosis.Structural analysis has revealed that cytochrome c is not required for Dapaf-1 complex assembly.In Lepidoptera,the involvement of cytochrome c in apoptosis has been demonstrated by the accumulating evidence.Cytochrome c release from mitochondria into cytosol has been observed in different cell lines such as Spodoptera frugiperda Sf9,Spodoptera litura S1-1 and Lymantria dispar LdFB.Silencing of cytochrome c expression significantly affected apoptosis and activation of caspase and the addition of cytochrome c to cell-free extracts results in caspase activation,suggesting the activation of caspase is dependent on cytochrome c.Although Apaf- 1 has not been identified in Lepidoptera,the inhibitor of apoptosome formation can inhibit apoptosis and caspase activation.Cytochrome c may be exclusively required for Lepidoptera apoptosis.

  9. Cytochrome c2-independent respiratory growth of Rhodobacter capsulatus.


    Daldal, F


    To assess the role of cytochrome c2 as a respiratory electron carrier, we obtained a double mutant of Rhodobacter capsulatus defective in cytochrome c2 and in the quinol oxidase260. This mutant was able to grow chemoheterotrophically, indicating that an electron pathway independent of cytochrome c2 was functional between the ubiquinol:cytochrome c2 oxidoreductase and the cytochrome oxidase410.

  10. The role of multihaem cytochromes in the respiration of nitrite in Escherichia coli and Fe(III) in Shewanella oneidensis. (United States)

    Clarke, Thomas A; Holley, Tracey; Hartshorne, Robert S; Fredrickson, Jim K; Zachara, John M; Shi, Liang; Richardson, David J


    The periplasmic nitrite reductase system from Escherichia coli and the extracellular Fe(III) reductase system from Shewanella oneidensis contain multihaem c-type cytochromes as electron carriers and terminal reductases. The position and orientation of the haem cofactors in multihaem cytochromes from different bacteria often show significant conservation despite different arrangements of the polypeptide chain. We propose that the decahaem cytochromes of the iron reductase system MtrA, MtrC and OmcA comprise pentahaem 'modules' similar to the electron donor protein, NrfB, from E. coli. To demonstrate this, we have isolated and characterized the N-terminal pentahaem module of MtrA by preparing a truncated form containing five covalently attached haems. UV-visible spectroscopy indicated that all five haems were low-spin, consistent with the presence of bis-His ligand co-ordination as found in full-length MtrA.

  11. Mitochondrial cytochrome c oxidase deficiency. (United States)

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


    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 of studying 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.

  12. A C-Type Cytochrome and a Transcriptional Regulator Responsible for Enhanced Extracellular Electron Transfer in Geobacter Sulfurreducens Revealed by Adaptive Evolution (United States)


    ducens fuel cells. Appl Environ Microbiol 72: 7345–7348. Sambrook, J., Fritsch, E.F., and Maniatis , T. (1989) Molecular Cloning : A Laboratory Manual... Molecular Science and Engineering, Naval Research Laboratory, Washington, DC, USA. 3Department of Bioengineering, University of California, San Diego, La...ORGANIZATION NAME(S) AND ADDRESS(ES) Naval Research Laboratory,Center for Bio/ Molecular Science and Engineering,Washington,DC,20375 8. PERFORMING

  13. Macrophage galactose-type C-type lectin receptor for DC targeting of antitumor glycopeptide vaccines

    DEFF Research Database (Denmark)

    Nuti, M; Zizzari, I; Napoletano, C;


    e13528 Background: Dendritic cells (DCs) are the most potent antigen presenting cells and are employed in cancer vaccination. Several receptors are being studied in order to identif strategies to increase DCs activating capacity. The C-type lectin macrophage galactose type C-type lectin (MGL...... of IFNg and IL-2 secretion by both CD8 and CD4 T cells. CONCLUSIONS: These results demonstrate that MGL engagement profoundly affects DC plasticity inducing and directing a Th1 immune response. Moreover, MGL receptor expressed on human DC can be targeted by glycopeptide based vaccines with adjuvant...

  14. A hypothesis on the origin of C-type asteroids and carbonaceous chondrites


    Busarev, V. V.


    A hypothesis based on observational and theoretical results on the origin of C-type asteroids and carbonaceous chondrites is proposed. Asteroids of C-type and close BGF-types could form from hydrated silicate-organic matter accumulated in the cores of water-differentiated (due to 26Al and other short-lived isotopes decay) bodies existed in the growth zones of Jupiter. Gravitational scattering of such bodies by Jupiter at its final stage of formation to the main asteroid belt might have led to...

  15. Genomic Analyses of Bacterial Porin-Cytochrome Gene Clusters

    Directory of Open Access Journals (Sweden)

    Liang eShi


    Full Text Available The porin-cytochrome (Pcc protein complex is responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens PCA. The identified and characterized Pcc complex of G. sulfurreducens PCA consists of a porin-like outer-membrane protein, a periplasmic 8-heme c-type cytochrome (c-Cyt and an outer-membrane 12-heme c-Cyt, and the genes encoding the Pcc proteins are clustered in the same regions of genome (i.e., the pcc gene clusters of G. sulfurreducens PCA. A survey of additionally microbial genomes has identified the pcc gene clusters in all sequenced Geobacter spp. and other bacteria from six different phyla, including Anaeromyxobacter dehalogenans 2CP-1, A. dehalogenans 2CP-C, Anaeromyxobacter sp. K, Candidatus Kuenenia stuttgartiensis, Denitrovibrio acetiphilus DSM 12809, Desulfurispirillum indicum S5, Desulfurivibrio alkaliphilus AHT2, Desulfurobacterium thermolithotrophum DSM 11699, Desulfuromonas acetoxidans DSM 684, Ignavibacterium album JCM 16511, and Thermovibrio ammonificans HB-1. The numbers of genes in the pcc gene clusters vary, ranging from two to nine. Similar to the metal-reducing (Mtr gene clusters of other Fe(III-reducing bacteria, such as Shewanella spp., additional genes that encode putative c-Cyts with predicted cellular localizations at the cytoplasmic membrane, periplasm and outer membrane often associate with the pcc gene clusters. This suggests that the Pcc-associated c-Cyts may be part of the pathways for extracellular electron transfer reactions. The presence of pcc gene clusters in the microorganisms that do not reduce solid-phase Fe(III and Mn(IV oxides, such as D. alkaliphilus AHT2 and I. album JCM 16511, also suggests that some of the pcc gene clusters may be involved in extracellular electron transfer reactions with the substrates other than Fe(III and Mn(IV oxides.

  16. Targeting of macrophage galactose-type C-type lectin (MGL) induces DC signaling and activation

    DEFF Research Database (Denmark)

    Napoletano, Chiara; Zizzari, Ilaria G; Rughetti, Aurelia;


    Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen-associated molecular patterns. In particular, C-type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca(2+) -dependent manner. Targe...

  17. Levels of complexity in pathogen recognition by C-type lectins.

    NARCIS (Netherlands)

    Cambi, A.; Figdor, C.G.


    In pathogen recognition by C-type lectins, several levels of complexity can be distinguished; these might modulate the immune response in different ways. Firstly, the pathogen-associated molecular pattern repertoire expressed at the microbial surface determines the interactions with specific recepto

  18. Transmission-blocking antibodies against mosquito C-type lectins for dengue prevention. (United States)

    Liu, Yang; Zhang, Fuchun; Liu, Jianying; Xiao, Xiaoping; Zhang, Siyin; Qin, Chengfeng; Xiang, Ye; Wang, Penghua; Cheng, Gong


    C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1), facilitating the attachment of West Nile virus (WNV) on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E) protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden.

  19. Processing-independent analysis for pro-C-type natriuretic peptide

    DEFF Research Database (Denmark)

    Lippert, Solvej Kølvraa; Rehfeld, Jens F.; Gøtze, Jens Peter


    C-type natriuretic peptide (CNP) is expressed in several human tissues. We designed a specific processing-independent assay for proCNP-derived products and quantitated the concentrations in human seminal plasma from normal and vasectomized men. Antibodies were raised against the N-terminus of human...

  20. Intestinally secreted C-type lectin Reg3b attenuates salmonellosis but not listeriosis in mice

    NARCIS (Netherlands)

    Ampting, van M.T.J.; Loonen, L.M.P.; Schonewille, A.J.; Konings, I.; Vink, C.; Iovanna, J.; Chamaillard, M.; Dekker, J.; Meer, van der R.; Wells, J.; Bovee-Oudenhoven, I.M.J.


    The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal i

  1. Transmission-blocking antibodies against mosquito C-type lectins for dengue prevention.

    Directory of Open Access Journals (Sweden)

    Yang Liu


    Full Text Available C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1, facilitating the attachment of West Nile virus (WNV on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden.

  2. C-type lectin interactions with Schistosoma mansoni SEA : Molecular basis and function

    NARCIS (Netherlands)

    Liempt, van P.A.G.


    Outline of this thesis The studies described in this thesis have been performed to gain more insight in the recognition of Schistosoma mansoni glycans by C-type lectins and the consequences for dendritic cell mediated immune responses. As a first approach to understand the molecular interactions o

  3. Computational and experimental prediction of human C-type lectin receptor druggability

    Directory of Open Access Journals (Sweden)

    Jonas eAretz


    Full Text Available Mammalian C-type lectin receptors are involved in many aspects of immune cell regulation such as pathogen recognition, clearance of apoptotic bodies and lymphocyte homing. Despite a great interest in modulating C-type lectin receptor recognition of carbohydrates, the number of specific molecular probes is limited. To this end, we predicted the druggability of a panel of 22 C-type lectin receptors using DoGSiteScorer. The computed druggability scores of most structures were low, characterizing this family as either challenging or even undruggable. To further explore these findings, we employed a fluorine-based NMR screening of fragment mixtures against DC-SIGN, a receptor of pharmacological interest. To our surprise, we found many fragment hits associated with the carbohydrate recognition site (hit rate = 13.5%. An SPR-based follow-up assay confirmed 18 of these fragments (47% and equilibrium dissociation constants were determined. Encouraged by these findings we expanded our experimental druggability prediction to Langerin and MCL and found medium to high hit rates as well, being 15.7% and 10.0%, respectively. Our results highlight limitations of current in silico approaches to druggability assessment, in particular with regard to carbohydrate-binding proteins. In sum, our data indicate that small molecule ligands for a larger panel of C-type lectin receptors can be developed.

  4. A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans.


    Ludwig, B.; Schatz, G


    Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC was purified from the cytoplasmic membrane of the bacterium Paracoccus denitrificans. The enzyme contains two heme groups (a and a3) and two copper atoms per minimal unit, oxidizes mammalian cytochrome c at a high rate, and, when incorporated into liposomes, generates an electrochemical proton gradient during cytochrome c oxidation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis reveals only two subunits of...

  5. Cytochromes P460 and c'-beta; a new family of high-spin cytochromes c. (United States)

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


    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.

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


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


    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 chan...

  7. Transient evolution of C-type shocks in dusty regions of varying density

    CERN Document Server

    Ashmore, I; Caselli, P; Falle, S A E G; Hartquist, T W


    Outflows of young stars drive shocks into dusty, molecular regions. Most models of such shocks assume that they are steady and propagating perpendicular to the magnetic field. Real shocks often violate both of these assumptions and the media through which they propagate are inhomogeneous. We use the code employed previously to produce the first time-dependent simulations of fast-mode, oblique C-type shocks interacting with density perturbations. We include a self-consistent calculation of the thermal and ionisation balances and a fluid treatment of grains. We identify features that develop when a multifluid shock encounters a density inhomogeneity to investigate whether any part of the precursor region ever behaves in a quasi-steady fashion. If it does the shock may be modelled approximately without solving the time-dependent hydromagnetic equations. Simulations were made for initially steady oblique C-type shocks encountering density inhomogeneities. For a semi-finite inhomogeneity with a density larger than...

  8. Cytochrome c1 exhibits two binding sites for cytochrome c in plants. (United States)

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


    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.

  9. Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

    Directory of Open Access Journals (Sweden)

    PSF Barbosa


    Full Text Available Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC. BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15%. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 µg/mL increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.

  10. Crotacetin, a novel snake venom C-type lectin, is homolog of convulxin

    Directory of Open Access Journals (Sweden)

    G. Rádis-Baptista


    Full Text Available Snake venom (sv C-type lectins encompass a group of hemorrhagic toxins, which are able to interfere with hemostasis. They share significant similarity in their primary structures with C-type lectins of other animals, and also present a conserved carbohydrate recognition domain (CRD. A very well studied sv C-type lectin is the heterodimeric toxin, convulxin (CVX, from the venoms of South American rattlesnakes, Crotalus durissus terrificus and C. d. cascavella. It consists of two subunits, alfa (CVXalpha , 13.9 kDa and beta (CVXbeta , 12.6 kDa, joined by inter and intra-chain disulfide bounds, and is arranged in a tetrameric alpha4beta4 conformation. Convulxin is able to activate platelet and induce their aggregation by acting via p62/GPVI collagen receptor. Several cDNA precursors, homolog of CVX subunits, were cloned by PCR homology screening. As determined by computational analysis, one of them, named crotacetin beta subunit, was predicted as a polypeptide with a tridimensional conformation very similar to other subunits of convulxin-like snake toxins. Crotacetin was purified from C. durissus venoms by gel permeation and reverse phase high performance liquid chromatography. The heterodimeric crotacetin is expressed in the venoms of several C. durissus subspecies, but it is prevalent in the venom of C. durissus cascavella. As inferred from homology modeling, crotacetin induces platelet aggregation but noticeably exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria.

  11. Cytochrome b5 from Giardia lamblia. (United States)

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


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

  12. Mechanisms of Electron Transfer in Two Decaheme Cytochromes from a Metal-Reducing Bacterium

    Energy Technology Data Exchange (ETDEWEB)

    Wigginton, Nicholas S.; Rosso, Kevin M.; Hochella, Michael F.


    Single-molecule current-voltage (I–V) spectra were collected using a scanning tunneling microscope for two decaheme c-type cytochromes, OmcA and MtrC, which are outer-membrane proteins from the dissimilatory metal-reducing bacterium Shewanella oneidensis. Although the two cytochromes are similar in heme count, charge-carrying amino-acid content, and molecular mass, their I–V spectra are significantly different. The I–V spectra for OmcA show smoothly varying symmetric exponential behavior. These spectra are well fit by a coherent tunneling model that is based on a simple square barrier description of the tunneling junction. In contrast, the I–V spectra for MtrC have pronounced breaks in slope in the positive tip bias range. Two large peaks in the normalized differential conductance spectra of MtrC were fit to a tunneling model that accounts for the possibility of transient population of empty states stabilized by vibrational relaxation. Reorganization energies deduced for the two features are similar to those normally assigned to metal centers in other metalloproteins. Work function measurements of the cytochrome films were used to convert the energies of these two spectral features to the normal hydrogen electrode scale for comparison with the midpoint potential measured using protein film voltammetry, which showed good correspondence. We conclude that MtrC mediates tunneling current by heme orbital participation. The difference in tunneling behavior between OmcA and MtrC suggests distinct physiological functions for the two cytochromes; in contrast to OmcA, MtrC appears to be tuned to a specific operating potential.

  13. Functional characterization of PccH, a key cytochrome for electron transfer from electrodes to the bacterium Geobacter sulfurreducens. (United States)

    Dantas, Joana M; Tomaz, Diogo M; Morgado, Leonor; Salgueiro, Carlos A


    The cytochrome PccH from Geobacter sulfurreducens (Gs) plays a crucial role in current-consuming fumarate-reducing biofilms. Deletion of pccH gene inhibited completely electron transfer from electrodes toward Gs cells. The pccH gene was cloned and the protein heterologously expressed in Escherichia coli. Complementary biophysical techniques including CD, UV-visible and NMR spectroscopy were used to characterize PccH. This cytochrome contains one low-spin c-type heme with His-Met axial coordination and unusual low-reduction potential. This reduction potential is pH-dependent, within the Gs physiological pH range, and is discussed within the context of the electron transfer mechanisms from electrodes to Gs cells.

  14. Two-dimensional crystallization of monomeric bovine cytochrome c oxidase with bound cytochrome c in reconstituted lipid membranes. (United States)

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


    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.

  15. Different Origins or Different Evolutions? Decoding the Spectral Diversity Among C-type Asteroids (United States)

    Vernazza, P.; Castillo-Rogez, J.; Beck, P.; Emery, J.; Brunetto, R.; Delbo, M.; Marsset, M.; Marchis, F.; Groussin, O.; Zanda, B.; Lamy, P.; Jorda, L.; Mousis, O.; Delsanti, A.; Djouadi, Z.; Dionnet, Z.; Borondics, F.; Carry, B.


    Anhydrous pyroxene-rich interplanetary dust particles (IDPs) have been proposed as surface analogs for about two-thirds of all C-complex asteroids. However, this suggestion appears to be inconsistent with the presence of hydrated silicates on the surfaces of some of these asteroids, including Ceres. Here, we report the presence of enstatite (pyroxene) on the surface of two C-type asteroids (Ceres and Eugenia) based on their spectral properties in the mid-infrared range. The presence of this component is particularly unexpected in the case of Ceres, because most thermal evolution models predict a surface consisting of hydrated compounds only. The most plausible scenario is that Ceres’ surface has been partially contaminated by exogenous enstatite-rich material, possibly coming from the Beagle asteroid family. This scenario questions a similar origin for Ceres and the remaining C-types, and it possibly supports recent results obtained by the Dawn mission (NASA) that Ceres may have formed in the very outer solar system. Concerning the smaller D ∼ 200 km C-types such as Eugenia, both their derived surface composition (enstatite and amorphous silicates) and low density (ice), and that a significant volume fraction of these bodies has remained unaffected by hydrothermal activity likely implying a late accretion. In addition, their current heliocentric distance may best explain the presence or absence of water ice at their surfaces. Finally, we raise the possibility that CI chondrites, Tagish-Lake-like material, or hydrated IDPs may be representative samples of the cores of these bodies.

  16. Nerval influences on liver cytochrome P450. (United States)

    Klinger, W; Karge, E; Danz, M; Krug, M


    In male young adult Wistar rats the influences of nucleus raphe electrocoagulation, spinal cord dissection (cordotomy between C7 and Th1), vagotomy and denervation of liver hilus by phenol on liver cytochrome P450-system (cytochrome P450 concentration, ethylmorphine N-demethylation and ethoxycoumarin O-deethylation activities, hexobarbitone sleeping time) were investigated. In general the influences were small or negligible when compared with sham operated controls, only after vagotomy the depressing effect of sham operation was abolished. In all cases sham operation had a depressing effect until up to five weeks after operation.

  17. Flower colour and cytochromes P450†


    Tanaka, Yoshikazu; Brugliera, Filippa


    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 ...

  18. Genetic characterization of Bagarius species using cytochrome c oxidase I and cytochrome b genes. (United States)

    Nagarajan, Muniyandi; Raja, Manikam; Vikram, Potnuru


    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.

  19. Unbalanced fermentation of glycerol in Escherichia coli via heterologous production of an electron transport chain and electrode interaction in microbial electrochemical cells. (United States)

    Sturm-Richter, Katrin; Golitsch, Frederik; Sturm, Gunnar; Kipf, Elena; Dittrich, André; Beblawy, Sebastian; Kerzenmacher, Sven; Gescher, Johannes


    Microbial electrochemical cells are an emerging technology for achieving unbalanced fermentations. However, organisms that can serve as potential biocatalysts for this application are limited by their narrow substrate spectrum. This study describes the reprogramming of Escherichia coli for the efficient use of anodes as electron acceptors. Electron transfer into the periplasm was accelerated by 183% via heterologous expression of the c-type cytochromes CymA, MtrA and STC from Shewanella oneidensis. STC was identified as a target for heterologous expression via a two-stage screening approach. First, mass spectroscopic analysis revealed natively expressed cytochromes in S. oneidensis. Thereafter, the corresponding genes were cloned and expressed in E. coli to quantify periplasmic electron transfer activity using methylene blue. This redox dye was further used to expand electron transfer to carbon electrode surfaces. The results demonstrate that E. coli can be reprogrammed from glycerol fermentation to respiration upon production of the new electron transport chain.

  20. Affinity chromatography purification of cytochrome c binding enzymes.


    Azzi, A; Bill, K; Broger, C


    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 co...

  1. Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco. (United States)

    Ke, F; Zhang, H B; Wang, Y; Hou, L F; Dong, H J; Wang, Z F; Pan, G W; Cao, X Y


    This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR).

  2. Effect of granule size on the properties of lotus rhizome C-type starch. (United States)

    Lin, Lingshang; Huang, Jun; Zhao, Lingxiao; Wang, Juan; Wang, Zhifeng; Wei, Cunxu


    Lotus rhizome C-type starch was separated into different size fractions. Starch morphologies changed from irregular to elongated, ellipsoid, oval, and spherical with decreasing granule size. The small- and very-small-sized fractions had a centric hilum, and the other size fractions had an eccentric hilum. The different size fractions all showed C-type crystallinity, pseudoplasticity and shear-thinning rheological properties. The range of amylose content was 25.6 to 26.6%, that of relative crystallinity was 23.9 to 25.8%, that of swelling power was 29.0 to 31.4 g/g, and that of gelatinization enthalpy was 12.4 to 14.2J/g. The very-small-sized fraction had a significantly lower short-range ordered degree and flow behavior index and higher scattering peak intensity, water solubility, gelatinization peak temperature, gelatinization conclusion temperature, consistency coefficient, hydrolysis degrees, and digestion rate than the large-sized fraction. Granule size significantly positively influenced short-range ordered structure and swelling power and negatively influenced scattering peak intensity, water solubility, hydrolysis and digestion of starch (p<0.01).

  3. Crystalline and structural properties of acid-modified lotus rhizome C-type starch. (United States)

    Cai, Jinwen; Cai, Canhui; Man, Jianmin; Yang, Yang; Zhang, Fengmin; Wei, Cunxu


    The crystalline and structural properties of acid-modified C-type starch from lotus rhizomes were investigated using a combination of techniques. The degradation of granule during hydrolysis began from the end distant from the hilum and then propagated into the center of granule, accompanied by loss of birefringence. The crystallinity changed from C-type to A-type via CA-type during hydrolysis. At the early stage of hydrolysis, the amylose content substantially reduced, the peak and conclusion gelatinization temperatures increased, and the enthalpy decreased. During hydrolysis, the double helix content gradually increased and the amorphous component decreased, the lamellar peak intensity firstly increased and then decreased accompanied by hydrolysis of amorphous and crystalline regions. This study elucidated that B-type allomorph was mainly arranged in the distal region of eccentric hilum, A-type allomorph was mainly located in the periphery of hilum end, and the center of granule was a mixed distribution of A- and B-type allomorphs.

  4. A hypothesis on the origin of C-type asteroids and carbonaceous chondrites

    CERN Document Server

    Busarev, V V


    A hypothesis based on observational and theoretical results on the origin of C-type asteroids and carbonaceous chondrites is proposed. Asteroids of C-type and close BGF-types could form from hydrated silicate-organic matter accumulated in the cores of water-differentiated (due to 26Al and other short-lived isotopes decay) bodies existed in the growth zones of Jupiter. Gravitational scattering of such bodies by Jupiter at its final stage of formation to the main asteroid belt might have led to fragmentation and re-accretion of their primitive materials on the surfaces of many asteroids and/or asteroid parent bodies. The hypothesis makes clear a row of long-standing puzzling facts, the main of which are as follows. The low-albedo and carbonaceous-chondritic surface properties of (1) Ceres contradict to its probable differentiated structure and icy crust (e. g., Thomas et al., 2005, Nature 437: 224-226; Castillo-Rogez et al., 2010, Icarus 205, 443-459), but it could be explained by the process of primitive matte...

  5. Effects of Glutamic Acid on C-type Inactivation of Kvl.4△N Channel

    Institute of Scientific and Technical Information of China (English)

    Cheng Ye; Xiaoyan Li; Zhouwu Shu; Xuejun Jiang


    Objectives Acidosis has an inhibitory effect on the inactivation of Kvl.4 AN channel through the position H508.So in order to show the effects of glutamic acid on the mutant Kv 1.4 channel that lacks N-type inactivation(Kvl.4A2-146),we studied in the expression system of the Xenopus oocytes.Methods The two-electrode voltage-clamp technique(TEV)was used to record the currents.Results Acidosis increased fKvl.4 △2-146 C-type inactivation.After application of glutamic acid(1 mmol/L) to Kvl.4 △2-146 increased C-type inactivation further,changed inactivation time constants from (2.02±0.39s)to(1.71±0.23 s)(P<0.05)at +50my,and shifted the steadystate inactivation curves of Kvl.4 △N to positive potential,which was from(-44.30±0.59 mV) to(-39.88±0.29 mV)(P<0.05 ).and slowed the rate of recovery from inactivation,which was from(1.64±0.19s) to (1.91±0.23 s)(P<0.05).Conclusions Together,these results suggest that 1 mmol/L glutamic acid accelerates the Ctype inactivation of Kvl.4 AN in pH 6.8.

  6. Isolation and characterization of a c-type lysozyme from the nurse shark. (United States)

    Hinds Vaughan, Nichole; Smith, Sylvia L


    Lysozyme is a ubiquitous antibacterial enzyme that occurs in numerous invertebrate and vertebrate species. Three forms have been described c-type, g-type and i-type which differ in primary structure. Shark lysozyme has not been characterized; here we report on the isolation and characterization of lysozyme from unstimulated shark (Ginglymostoma cirratum) leukocytes and provide amino acid sequence data across the highly conserved active site of the molecule identifying it to be a c-type lysozyme. A leukocyte lysate was applied either (a) to the first of two sequential DE-52 cellulose columns or alternatively, (b) to a DEAE-Sepharose column. Lysozyme activity in lysate and active fractions was identified by zones of lysis of Micrococcus lysodeikticus cell walls on lysoplates and zones of growth inhibition in agar diffusion assays using Planococcus citreus as the target organism. SDS-PAGE analysis revealed a 14 kDa protein which was identified as lysozyme by mass spectroscopic analysis of peptides, reactivity against anti-HEWL antibodies on a Western blot, hydrolysis of M. lysodeikticus cell walls, and inhibition of growth of P. citreus on AU-gel blots in which the area of growth inhibition correlated to a 14 kDa protein.

  7. Light-driven cytochrome P450 hydroxylations

    DEFF Research Database (Denmark)

    Jensen, Kenneth; Jensen, Poul Erik; Møller, Birger Lindberg


    Plants are light-driven "green" factories able to synthesize more than 200,000 different bioactive natural products, many of which are high-value products used as drugs (e.g., artemisinin, taxol, and thapsigargin). In the formation of natural products, cytochrome P450 (P450) monooxygenases play...

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


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


    Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cys...

  9. Purification of the Cytochrome c Reductase/Cytochrome c Oxidase Super Complex of Yeast Mitochondria


    Braun, Hans-Peter; Sunderhaus, Stephanie; Boekema, Egbert J.; Kouřil, Roman


    The protein complexes of the respiratory chain interact by forming large protein particles called respiratory supercomplexes or ‘‘respirasomes’’. Biochemical characterization of these particles proved to be difficult because of their instability. Here we describe a strategy to isolate and characterize the cytochrome c reductase/cytochrome c oxidase supercomplex of yeast, also termed the III + IV supercomplex, which is based on lactate cultivation of yeast, gentle isolation of mitochondria, me...

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


    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; Rosa, MIguel A. de la; Díaz-Moreno, Irene


    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-dri...

  11. The structure of PccH from Geobactersulfurreducens-a novel low reduction potential monoheme cytochrome essential for accepting electrons from an electrode

    Energy Technology Data Exchange (ETDEWEB)

    Dantas, Joana; Campelo, Luisa M.; Duke, Norma E. C.; Salgueiro, Carlos A.; Pokkuluri, P. Raj


    The structure of cytochrome c (GSU3274) designated as PccH from Geobactersulfurreducens was determined at a resolution of 2.0 angstrom. PccH is a small (15kDa) cytochrome containing one c-type heme, found to be essential for the growth of G.sulfurreducens with respect to accepting electrons from graphite electrodes poised at -300mV versus standard hydrogen electrode. with fumarate as the terminal electron acceptor. The structure of PccH is unique among the monoheme cytochromes described to date. The structural fold of PccH can be described as forming two lobes with the heme sandwiched in a cleft between the two lobes. In addition, PccH has a low reduction potential of -24mV at pH7, which is unusual for monoheme cytochromes. Based on difference in structure, together with sequence phylogenetic analysis, we propose that PccH can be regarded as a first characterized example of a new subclass of class I monoheme cytochromes. The low reduction potential of PccH may enable the protein to be redox active at the typically negative potential ranges encountered by G. sulfurreducens. Because PccH is predicted to be located in the periplasm of this bacterium, it could not be involved in the first step of accepting electrons from the electrode but is very likely involved in the downstream electron transport events in the periplasm.

  12. The structure of PccH from Geobacter sulfurreducens - a novel low reduction potential monoheme cytochrome essential for accepting electrons from an electrode. (United States)

    Dantas, Joana M; Campelo, Luísa M; Duke, Norma E C; Salgueiro, Carlos A; Pokkuluri, P Raj


    The structure of cytochrome c (GSU3274) designated as PccH from Geobacter sulfurreducens was determined at a resolution of 2.0 Å. PccH is a small (15 kDa) cytochrome containing one c-type heme, found to be essential for the growth of G. sulfurreducens with respect to accepting electrons from graphite electrodes poised at -300 mV versus standard hydrogen electrode. with fumarate as the terminal electron acceptor. The structure of PccH is unique among the monoheme cytochromes described to date. The structural fold of PccH can be described as forming two lobes with the heme sandwiched in a cleft between the two lobes. In addition, PccH has a low reduction potential of -24 mV at pH 7, which is unusual for monoheme cytochromes. Based on difference in structure, together with sequence phylogenetic analysis, we propose that PccH can be regarded as a first characterized example of a new subclass of class I monoheme cytochromes. The low reduction potential of PccH may enable the protein to be redox active at the typically negative potential ranges encountered by G. sulfurreducens. Because PccH is predicted to be located in the periplasm of this bacterium, it could not be involved in the first step of accepting electrons from the electrode but is very likely involved in the downstream electron transport events in the periplasm.

  13. Processing-independent analysis for pro-C-type natriuretic peptide

    DEFF Research Database (Denmark)

    Lippert, Solvej Kølvraa; Rehfeld, Jens F.; Gøtze, Jens Peter


    proCNP concentrations similar to non-vasectomized men (range 107-705 pmol/L, age 34-44 years). Taken together, our new proCNP assay shows that proCNP is abundantly present in human seminal plasma and that seminal proCNP is secreted from the prostate gland and/or the seminal vesicles.......C-type natriuretic peptide (CNP) is expressed in several human tissues. We designed a specific processing-independent assay for proCNP-derived products and quantitated the concentrations in human seminal plasma from normal and vasectomized men. Antibodies were raised against the N-terminus of human...... proCNP 11-27. Samples were incubated with trypsin prior to immunoassay, which allows for the measurement of "total" proCNP irrespective of the degree of post-translational processing. Seminal plasma from normal young men and vasectomized men were collected and quantitated; the molecular heterogeneity...

  14. Identification of a novel human rhinovirus C type by antibody capture VIDISCA-454. (United States)

    Jazaeri Farsani, Seyed Mohammad; Oude Munnink, Bas B; Canuti, Marta; Deijs, Martin; Cotten, Matthew; Jebbink, Maarten F; Verhoeven, Joost; Kellam, Paul; Loens, Katherine; Goossens, Herman; Ieven, Margareta; van der Hoek, Lia


    Causative agents for more than 30 percent of respiratory infections remain unidentified, suggesting that unknown respiratory pathogens might be involved. In this study, antibody capture VIDISCA-454 (virus discovery cDNA-AFLP combined with Roche 454 high-throughput sequencing) resulted in the discovery of a novel type of rhinovirus C (RV-C). The virus has an RNA genome of at least 7054 nt and carries the characteristics of rhinovirus C species. The gene encoding viral protein 1, which is used for typing, has only 81% nucleotide sequence identity with the closest known RV-C type, and, therefore, the virus represents the first member of a novel type, named RV-C54.

  15. Outbreak of infection caused by Neisseria meningitidis group C type 2 in a nursery. (United States)

    Sáez-Nieto, J A; Perucha, M; Casamayor, H; Marcen, J J; Llacer, A; Garcia-Barreno, B; Casal, J


    An outbreak of meningococcal infection which took place in a nursery in Rioja, Spain, is reported. Between November 1981 and February 1982, 11 patients had meningitis with or without septicaemia. Two died. Three meningococcal strains from the patients isolated were studied. All three were group C type 2 and were resistant to sulphadiazine (MIC 50 mg/l) but susceptible to penicillin, ampicillin, chloramphenicol, rifampicin and spiramycin. This outbreak took place during an epidemic in which serogroup B was the most prevalent in Spain. Two surveys before and after chemoprophylaxis were made to determine the carrier rate in the nursery population. The strain causing the outbreak was found in 2.5 and 4 per cent of persons respectively. Rifampicin was administered to all carriers after the first survey and to carriers of the virulent strain after the second survey. The remaining children were given polysaccharide C vaccine. No more cases arose after this last prophylactic measure.

  16. Characterization of cytochrome mutants for pellicle formation in Shewanella onedensis MR-1

    Institute of Scientific and Technical Information of China (English)

    LIANG Yi-li; HE Zhi-li; GAO Hai-chun; QIU Guan-zhou; ZHOU Ji-zhong; LIU Xue-duan


    Biofilm systems are effective for biosorption of metal ions. Shewanella oneidensis MR-1, a Gram-negative facultative anaerobe, is a natural pellicle-like biofilm former. The mechanisms of pellicle formation by S. oneidensis MR-1 have not yet been understood. 17 S. oneidensis MR-1 deletion mutants, including 12 c-type cytochromes were generated and tested if they were involved in pellicle formation. The results show that △SO4666, △SO1777, △SO1782, △SO2361 and △SO2363 have varying deficiency in pellicle formation. The deletion mutant △SO4666 cannot form a pellicle under non-shake conditions, suggesting that it may play an important role in pellicle formation by S. oneidensis MR-1. Overall, these data suggest a very complex picture of aerobic respiration by S. oneidensis MR-1.

  17. A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity. (United States)

    Zhao, Zhi-Ying; Yin, Zhi-Xin; Xu, Xiao-Peng; Weng, Shao-Ping; Rao, Xia-Yu; Dai, Zong-Xian; Luo, Yong-Wen; Yang, Gan; Li, Zong-Sheng; Guan, Hao-Ji; Li, Se-Dong; Chan, Siu-Ming; Yu, Xiao-Qiang; He, Jian-Guo


    C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type carbohydrate recognition domain with an EPN (Glu(99)-Pro(100)-Asn(101)) motif that has a predicted ligand binding specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically expressed in the hepatopancreas of L. vannamei. Recombinant LvCTL1 (rLvCTL1) had hemagglutinating activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1 to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that has direct anti-WSSV activity.

  18. Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens. (United States)

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


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

  19. Electrostatic effect on electron transfer between cytochrome b5 and cytochrome c

    Institute of Scientific and Technical Information of China (English)


    The binding and electron transfer between wild type, E44A, E56A, E44/56A, E44/48/56A/D60Aand F35Y variants of cytochrome b5 and cytochrome c were studied. When mixed with cytochrome c, the cytochrome b5E44/48/56A/D60A did not show the typical UV-vis difference spectrum of absorption, indicating that the alteration ofthe surface electrostatic potential obviously influenced the spectrum. The electron transfer rates of wild type cytochromeb5, its variants and cytochrome e at different temperature and ionic strength exhibited an order of F35Y > wild type >E56A > E44A > E44/48/56A/D60A. The enthalpy and entropy of the reaction did not change obviously, suggestingthat the mutation did not significantly disturb the electron transfer conformation. The investigation of electron transfer rateconstants at different ionic strength demonstrated that electrostatic interaction obviously affected the electron transfer pro-cess. The significant difference of Cyt b5 F35Y and E44/48/56A/D60A from the wild type protein further confirmed thegreat importance of the electrostatic interaction in the protein electron transfer.

  20. Functional coadaptation between cytochrome c and cytochrome c oxidase within allopatric populations of a marine copepod. (United States)

    Rawson, Paul D; Burton, Ronald S


    Geographically isolated populations may accumulate alleles that function well on their own genetic backgrounds but poorly on the genetic backgrounds of other populations. Consequently, interpopulation hybridization may produce offspring of low fitness as a result of incompatibilities arising in allopatry. Genes participating in these epistatic incompatibility systems remain largely unknown. In fact, despite the widely recognized importance of epistatic interactions among gene products, few data directly address the functional consequences of such interactions among natural genetic variants. In the marine copepod, Tigriopus californicus, we found that the cytochrome c variants isolated from two different populations each had significantly higher activity with the cytochrome c oxidase derived from their respective source population. Three amino acid substitutions in the cytochrome c protein appear to be sufficient to confer population specificity. These results suggest that electron transport system (ETS) proteins form coadapted sets of alleles within populations and that disruption of the coadapted ETS gene complex leads to functional incompatibilities that may lower hybrid fitness.

  1. Macrophage-inducible C-type lectin underlies obesity-induced adipose tissue fibrosis. (United States)

    Tanaka, Miyako; Ikeda, Kenji; Suganami, Takayoshi; Komiya, Chikara; Ochi, Kozue; Shirakawa, Ibuki; Hamaguchi, Miho; Nishimura, Satoshi; Manabe, Ichiro; Matsuda, Takahisa; Kimura, Kumi; Inoue, Hiroshi; Inagaki, Yutaka; Aoe, Seiichiro; Yamasaki, Sho; Ogawa, Yoshihiro


    In obesity, a paracrine loop between adipocytes and macrophages augments chronic inflammation of adipose tissue, thereby inducing systemic insulin resistance and ectopic lipid accumulation. Obese adipose tissue contains a unique histological structure termed crown-like structure (CLS), where adipocyte-macrophage crosstalk is known to occur in close proximity. Here we show that Macrophage-inducible C-type lectin (Mincle), a pathogen sensor for Mycobacterium tuberculosis, is localized to macrophages in CLS, the number of which correlates with the extent of interstitial fibrosis. Mincle induces obesity-induced adipose tissue fibrosis, thereby leading to steatosis and insulin resistance in liver. We further show that Mincle in macrophages is crucial for CLS formation, expression of fibrosis-related genes and myofibroblast activation. This study indicates that Mincle, when activated by an endogenous ligand released from dying adipocytes, is involved in adipose tissue remodelling, thereby suggesting that sustained interactions between adipocytes and macrophages within CLS could be a therapeutic target for obesity-induced ectopic lipid accumulation.

  2. Urinary C-type natriuretic peptide: an emerging biomarker for heart failure and renal remodeling. (United States)

    Zakeri, Rosita; Burnett, John C; Sangaralingham, S Jeson


    The public health and economic burden of heart failure (HF) is staggering and the need for relevant pathophysiologic and clinical biomarkers to advance the field and improve HF therapy remains high. Renal dysfunction is common among HF patients and is associated with increased HF hospitalization and mortality. It is widely recognized that mechanisms contributing to HF pathogenesis include a complex bidirectional interaction between the kidney and heart, encompassed by the term cardiorenal syndrome (CRS). Among a new wave of urinary biomarkers germane to CRS, C-type natriuretic peptide (CNP) has emerged as an innovative biomarker of renal structural and functional impairment in HF and chronic renal disease states. CNP is a hormone, synthesized in the kidney, and is an important regulator of cell proliferation and organ fibrosis. Hypoxia, cytokines and fibrotic growth factors, which are inherent to both cardiac and renal remodeling processes, are among the recognized stimuli for CNP production and release. In this review we aim to highlight current knowledge regarding the biology and pathophysiological correlates of urinary CNP, and its potential clinical utility as a diagnostic and prognostic biomarker in HF and renal disease states.

  3. SiO line emission from C-type shock waves : interstellar jets and outflows

    CERN Document Server

    Gusdorf, A; Flower, D R; Forets, G Pineau des


    We study the production of SiO in the gas phase of molecular outflows, through the sputtering of Si--bearing material in refractory grain cores, which are taken to be olivine; we calculate also the rotational line spectrum of the SiO. The sputtering is driven by neutral particle impact on charged grains, in steady--state C-type shock waves, at the speed of ambipolar diffusion. The emission of the SiO molecule is calculated by means of an LVG code. A grid of models has been generated. We compare our results with those of an earlier study (Schilke et al. 1997). Improvements in the treatment of the coupling between the charged grains and the neutral fluid lead to narrower shock waves and lower fractions of Si being released into the gas phase. More realistic assumptions concerning the initial fractional abundance of O2 lead to SiO formation being delayed, so that it occurs in the cool, dense postshock flow. Good agreement is obtained with recent observations of SiO line intensities in the L1157 and L1448 molecul...

  4. The Macrophage Galactose-Type C-Type Lectin (MGL Modulates Regulatory T Cell Functions.

    Directory of Open Access Journals (Sweden)

    Ilaria Grazia Zizzari

    Full Text Available Regulatory T cells (Tregs are physiologically designed to prevent autoimmune disease and maintain self-tolerance. In tumour microenvironments, their presence is related to a poor prognosis, and they influence the therapeutic outcome due to their capacity to suppress the immune response by cell-cell contact and to release immunosuppressive cytokines. In this study, we demonstrate that Treg immunosuppressive activity can be modulated by the cross-linking between the CD45RA expressed by Tregs and the C-type lectin MGL. This specific interaction strongly decreases the immunosuppressive activity of Tregs, restoring the proliferative capacity of co-cultured T lymphocytes. This effect can be attributed to changes in CD45RA and TCR signalling through the inhibition of Lck and inactivation of Zap-70, an increase in the Foxp3 methylation status and, ultimately, the reduced production of suppressive cytokines. These results indicate a role of MGL as an immunomodulator within the tumour microenvironment interfering with Treg functions, suggesting its possible use in the design of anticancer vaccines.

  5. Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c{sub 552} and its b-type variant

    Energy Technology Data Exchange (ETDEWEB)

    Tozawa, Kaeko; Ferguson, Stuart J.; Redfield, Christina, E-mail: [University of Oxford, Department of Biochemistry (United Kingdom); Smith, Lorna J., E-mail: [University of Oxford, Department of Chemistry (United Kingdom)


    Cytochrome c{sub 552} from the thermophilic bacterium Hydrogenobacter thermophilus is a typical c-type cytochrome which binds heme covalently via two thioether bonds between the two heme vinyl groups and two cysteine thiol groups in a CXXCH sequence motif. This protein was converted to a b-type cytochrome by substitution of the two cysteine residues by alanines (Tomlinson and Ferguson in Proc Natl Acad Sci USA 97:5156–5160, 2000a). To probe the significance of the covalent attachment of the heme in the c-type protein, {sup 15}N relaxation and hydrogen exchange studies have been performed for the wild-type and b-type proteins. The two variants share very similar backbone dynamic properties, both proteins showing high {sup 15}N order parameters in the four main helices, with reduced values in an exposed loop region (residues 18–21), and at the C-terminal residue Lys80. Some subtle changes in chemical shift and hydrogen exchange protection are seen between the wild-type and b-type variant proteins, not only for residues at and neighbouring the mutation sites, but also for some residues in the heme binding pocket. Overall, the results suggest that the main role of the covalent linkages between the heme group and the protein chain must be to increase the stability of the protein.

  6. A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA. (United States)

    Liu, Yimo; Wang, Zheming; Liu, Juan; Levar, Caleb; Edwards, Marcus J; Babauta, Jerome T; Kennedy, David W; Shi, Zhi; Beyenal, Haluk; Bond, Daniel R; Clarke, Thomas A; Butt, Julea N; Richardson, David J; Rosso, Kevin M; Zachara, John M; Fredrickson, James K; Shi, Liang


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

  7. The reaction of neuroglobin with potential redox protein partners cytochrome b5  and cytochrome c

    DEFF Research Database (Denmark)

    Fago, Angela; Mathews, A.J.; Moens, L.


    Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b5 is relatively slow (k=6×102M-1s-1 at pH 7.0) and thus is unlikely to be of physiological...... significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2×107M-1s-1) with an apparent overall equilibrium constant of 1μM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis...

  8. Biogenesis of cytochrome b6 in photosynthetic membranes. (United States)

    Saint-Marcoux, Denis; Wollman, Francis-André; de Vitry, Catherine


    In chloroplasts, binding of a c'-heme to cytochrome b(6) on the stromal side of the thylakoid membranes requires a specific mechanism distinct from the one at work for c-heme binding to cytochromes f and c(6) on the lumenal side of membranes. Here, we show that the major protein components of this pathway, the CCBs, are bona fide transmembrane proteins. We demonstrate their association in a series of hetero-oligomeric complexes, some of which interact transiently with cytochrome b(6) in the process of heme delivery to the apoprotein. In addition, we provide preliminary evidence for functional assembly of cytochrome b(6)f complexes even in the absence of c'-heme binding to cytochrome b(6). Finally, we present a sequential model for apo- to holo-cytochrome b(6) maturation integrated within the assembly pathway of b(6)f complexes in the thylakoid membranes.

  9. The nature of CuA in cytochrome c oxidase


    Stevens, Tom H.; Martin, Craig T.; Wang, Hsin; Brudvig, Gary W.; Scholes, Charles P.; Chan, Sunney I.


    The isolation and purification of yeast cytochrome c oxidase is described. Characterization of the purified protein indicates that it is spectroscopically identical with cytochrome c oxidase isolated from beef heart. Preparations of isotopically substituted yeast cytochrome c oxidase are obtained incorporating [1,3-15N2]histidine or [beta,beta- 2H2]cysteine. Electron paramagnetic resonance and electron nuclear double resonance spectra of the isotopically substituted proteins identify unambigu...

  10. Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes

    Directory of Open Access Journals (Sweden)

    Beier Frank


    Full Text Available Abstract Background Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation – such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias – generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components. Methods Primary mouse chondrocytes in monolayer where incubated with the synthetic glucocorticoid Dexamethasone (DEX for 12 to 72 hours. Cell numbers were determined by counting, and real-time PCR was performed to examine regulation of genes in the CNP signaling pathway by DEX. Results We show that DEX does influence expression of key genes in the CNP pathway. Most importantly, DEX significantly increases RNA expression of the gene encoding CNP itself (Nppc. In addition, DEX stimulates expression of Prkg2 (encoding cGMP-dependent protein kinase II and Npr3 (natriuretic peptide decoy receptor genes. Conversely, DEX was found to down-regulate the expression of the gene encoding its receptor, Nr3c1 (glucocorticoid receptor, as well as the Npr2 gene (encoding the CNP receptor. Conclusion Our data suggest that the growth-suppressive activities of DEX are not due to blockade of CNP signaling. This study reveals a novel, unanticipated relationship between glucocorticoid and CNP signaling and provides the first evidence that CNP expression in chondrocytes is regulated by endocrine

  11. Bradykinin-potentiating peptides and C-type natriuretic peptides from snake venom. (United States)

    Higuchi, S; Murayama, N; Saguchi, K; Ohi, H; Fujita, Y; Camargo, A C; Ogawa, T; Deshimaru, M; Ohno, M


    Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (approximately 25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (approximately 144 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily.

  12. Thermal Infrared Imaging Experiments of C-Type Asteroid 162173 Ryugu on Hayabusa2 (United States)

    Okada, Tatsuaki; Fukuhara, Tetsuya; Tanaka, Satoshi; Taguchi, Makoto; Imamura, Takeshi; Arai, Takehiko; Senshu, Hiroki; Ogawa, Yoshiko; Demura, Hirohide; Kitazato, Kohei; Nakamura, Ryosuke; Kouyama, Toru; Sekiguchi, Tomohiko; Hasegawa, Sunao; Matsunaga, Tsuneo; Wada, Takehiko; Takita, Jun; Sakatani, Naoya; Horikawa, Yamato; Endo, Ken; Helbert, Jörn; Müller, Thomas G.; Hagermann, Axel


    The thermal infrared imager TIR onboard Hayabusa2 has been developed to investigate thermo-physical properties of C-type, near-Earth asteroid 162173 Ryugu. TIR is one of the remote science instruments on Hayabusa2 designed to understand the nature of a volatile-rich solar system small body, but it also has significant mission objectives to provide information on surface physical properties and conditions for sampling site selection as well as the assessment of safe landing operations. TIR is based on a two-dimensional uncooled micro-bolometer array inherited from the Longwave Infrared Camera LIR on Akatsuki (Fukuhara et al., 2011). TIR takes images of thermal infrared emission in 8 to 12 μm with a field of view of 16 × 12° and a spatial resolution of 0.05° per pixel. TIR covers the temperature range from 150 to 460 K, including the well calibrated range from 230 to 420 K. Temperature accuracy is within 2 K or better for summed images, and the relative accuracy or noise equivalent temperature difference (NETD) at each of pixels is 0.4 K or lower for the well-calibrated temperature range. TIR takes a couple of images with shutter open and closed, the corresponding dark frame, and provides a true thermal image by dark frame subtraction. Data processing involves summation of multiple images, image processing including the StarPixel compression (Hihara et al., 2014), and transfer to the data recorder in the spacecraft digital electronics (DE). We report the scientific and mission objectives of TIR, the requirements and constraints for the instrument specifications, the designed instrumentation and the pre-flight and in-flight performances of TIR, as well as its observation plan during the Hayabusa2 mission.

  13. Respiratory cytochrome c oxidase can be efficiently reduced by the photosynthetic redox proteins cytochrome c6 and plastocyanin in cyanobacteria. (United States)

    Navarro, José A; Durán, Raúl V; De la Rosa, Miguel A; Hervás, Manuel


    Plastocyanin and cytochrome c6 are two small soluble electron carriers located in the intrathylacoidal space of cyanobacteria. Although their role as electron shuttle between the cytochrome b6f and photosystem I complexes in the photosynthetic pathway is well established, their participation in the respiratory electron transport chain as donors to the terminal oxidase is still under debate. Here, we present the first time-resolved analysis showing that both cytochrome c6 and plastocyanin can be efficiently oxidized by the aa3 type cytochrome c oxidase in Nostoc sp. PCC 7119. The apparent electron transfer rate constants are ca. 250 and 300 s(-1) for cytochrome c6 and plastocyanin, respectively. These constants are 10 times higher than those obtained for the oxidation of horse cytochrome c by the oxidase, in spite of being a reaction thermodynamically more favourable.

  14. Absence of C-type virus production in human leukemic B cell, T cell and null cell lines.

    Directory of Open Access Journals (Sweden)



    Full Text Available Electron microscope observation of cultured human leukemic B cell, T cell and null cell lines and reverse transcriptase assay of the culture supernatants were all negative for the presence of C-type virus. Bat cell line, which propagates primate C-type viruses well, was cocultivated with the human leukemic cell lines, in the hope of amplification of virus if present. Three weeks after mixed culture, the culture supernatants were again examined for reverse transcriptase activity and the cells were tested for syncytia formation by cocultivation with rat XC, human KC and RSb cell lines. All these tests, except for the positive control using a simian sarcoma virus, were negative, suggesting that no C-type was produced from these human leukemic cell lines.

  15. The ccoNOQP gene cluster codes for a cb-type cytochrome oxidase that functions in aerobic respiration of Rhodobacter capsulatus. (United States)

    Thöny-Meyer, L; Beck, C; Preisig, O; Hennecke, H


    The genes for a new type of a haem-copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe. Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb-type oxidase. CcoN is a b-type cytochrome, CcoO and CcoP are membrane-bound mono- and dihaem c-type cytochromes and CcoQ is a small membrane protein of unknown function. Genes for a similar oxidase are also present in other non-rhizobial bacterial species such as Azotobacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis. A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R. capsulatus and which is probably identical to the cbb3-type oxidase that was recently purified from a different strain of the same species. Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen-fixation activity.

  16. A-current modifies the spike of C-type neurones in the rabbit nodose ganglion. (United States)

    Ducreux, C; Puizillout, J J


    1. In the rabbit nodose ganglion, C-type fibre neurones (C neurones) can be divided into two subtypes according to their after-hyperpolarizing potential (AHP) i.e. those with a fast AHP only and those with a fast AHP and a subsequent slow AHP produced by a slow calcium-dependent potassium current. In addition we have shown that some C neurones can be divided into two groups according to the effect of membrane hyperpolarization on their spikes i.e. type 1 in which duration and amplitude do not change and type 2 in which duration and amplitude decrease significantly. 2. In the present report we studied the effect of A-current (IA) on spike duration, amplitude and slow AHP using intracellular recording techniques. 3. To detect the presence of IA, we first applied a series of increasing rectangular hyperpolarizing pulses to remove IA inactivation and then a short depolarizing pulse to trigger a spike. In type 1 C neurones the lag time of the spike in relation to hyperpolarization remains constant whereas in type 2 C neurones the spike only appears after IA inactivation and lag time in relation to hyperpolarization is lengthened. Thus, type 2 C neurones have an IA while type 1 C neurones do not. The fact that addition of cadmium did not change the lag time in type 2 C neurones shows that the IA is not calcium dependent. 4. Nodose neurones can be orthodromically activated by stimulation of the vagal peripheral process. In this way, after a hyperpolarizing pulse, IA can be fully activated by the orthodromic spike itself. Under these conditions it is possible to analyse the effects of IA on the spike. This was done by increasing either the hyperpolarizing potential, pulse duration, or the delay of the spike after the end of the pulse. We observed that maximum IA inactivation removal was always associated with the lowest duration and amplitude of the spike. 5. When IA inhibitors, 4-aminopyridine (4-AP) or catechol, were applied to type 2 C neurones, the delay of the spike

  17. Cytochrome c as a peroxidase : tuning of heme reactivity

    NARCIS (Netherlands)

    Diederix, Rutger Ernest Michiel


    This thesis describes the peroxidase activity of the electron-transfer protein cytochrome c, and how it is controlled by the protein matrix. It is shown that unfolding cytochrome c has the effect to significantly enhance its peroxidase activity of (up to several thousand-fold). This can be achieved

  18. C-type lectin receptors and RIG-I-like receptors: new points on the oncogenomics map

    Directory of Open Access Journals (Sweden)

    Yuzhalin AE


    Full Text Available Anton G Kutikhin, Arseniy E YuzhalinDepartment of Epidemiology, Kemerovo State Medical Academy, Kemerovo, Russian FederationAbstract: The group of pattern recognition receptors includes families of Toll-like receptors, NOD-like receptors, C-type lectin receptors, and RIG-I-like receptors. They are key sensors for a number of infectious agents, some of which are oncogenic, and they launch an immune response against them, normally promoting their eradication. Inherited variations in genes encoding these receptors and proteins and their signaling pathways may affect their function, possibly modulating cancer risk and features of cancer progression. There are numerous studies investigating the association of single nucleotide polymorphisms within or near genes encoding Toll-like receptors and NOD-like receptors, cancer risk, and features of cancer progression. However, there is an almost total absence of articles analyzing the correlation between polymorphisms of genes encoding C-type lectin receptors and RIG-I-like receptors and cancer risk or progression. Nevertheless, there is some evidence supporting the hypothesis that inherited C-type lectin receptor and RIG-I-like receptor variants can be associated with increased cancer risk. Certain C-type lectin receptors and RIG-I-like receptors recognize pathogen-associated molecular patterns of potentially oncogenic infectious agents, and certain polymorphisms of genes encoding C-type lectin receptors and RIG-I-like receptors may have functional consequences at the molecular level that can lead to association of such single nucleotide polymorphisms with risk or progression of some diseases that may modulate cancer risk, so these gene polymorphisms may affect cancer risk indirectly. Polymorphisms of genes encoding C-type lectin receptors and RIG-I-like receptors thereby may be correlated with a risk of lung, oral, esophageal, gastric, colorectal, and liver cancer, as well as nasopharyngeal carcinoma

  19. Interspecies Complementation of Escherichia coli ccm Mutants: CcmE (CycJ) from Bradyrhizobium japonicum Acts as a Heme Chaperone during Cytochrome c Maturation


    Schulz, Henk; Thöny-Meyer, Linda


    Biogenesis of c-type cytochromes in α- and γ-proteobacteria requires the function of a set of orthologous genes (ccm genes) that encode specific maturation factors. The Escherichia coli CcmE protein is a periplasmic heme chaperone. The membrane protein CcmC is required for loading CcmE with heme. By expressing CcmE (CycJ) from Bradyrhizobium japonicum in E. coli we demonstrated that heme is bound covalently to this protein at a strictly conserved histidine residue. The B. japonicum homologue ...

  20. Borrelia burgdorferi RST1 (OspC type A) genotype is associated with greater inflammation and more severe Lyme disease. (United States)

    Strle, Klemen; Jones, Kathryn L; Drouin, Elise E; Li, Xin; Steere, Allen C


    Evidence is emerging for differential pathogenicity among Borrelia burgdorferi genotypes in the United States. By using two linked genotyping systems, ribosomal RNA intergenic spacer type (RST) and outer surface protein C (OspC), we studied the inflammatory potential of B. burgdorferi genotypes in cells and patients with erythema migrans or Lyme arthritis. When macrophages were stimulated with 10 isolates of each RST1, RST2, or RST3 strain, RST1 (OspC type A)-stimulated cells expressed significantly higher levels of IL-6, IL-8, chemokine ligand (CCL) 3, CCL4, tumor necrosis factor, and IL-1β, factors associated with innate immune responses. In peripheral blood mononuclear cells, RST1 strains again stimulated significantly higher levels of these mediators. Moreover, compared with RST2, RST1 isolates induced significantly more interferon (IFN)-α, IFN-γ, and CXCL10, which are needed for adaptive immune responses; however, OspC type I (RST3) approached RST1 (OspC type A) in stimulating these adaptive immune mediators. Similarly, serum samples from patients with erythema migrans who were infected with the RST1 genotype had significantly higher levels of almost all of these mediators, including exceptionally high levels of IFN-γ-inducible chemokines, CCL2, CXCL9, and CXCL10; and this pronounced inflammatory response was associated with more symptomatic infection. Differences among genotypes were not as great in patients with Lyme arthritis, but those infected with RST1 strains more often had antibiotic-refractory arthritis. Thus, the B. burgdorferi RST1 (OspC type A) genotype, followed by the RST3 (OspC type I) genotype, causes greater inflammation and more severe disease, establishing a link between spirochetal virulence and host inflammation.

  1. Structures of Cytochrome b 5 Mutated at the Charged Surface-Residues and Their Interactions with Cytochrome c

    Institute of Scientific and Technical Information of China (English)

    WU,Jian(邬键); WANG,Yun-Hua(王韵华); GAN,Jian-Hua(甘建华); WANG,Wen-Hu(王文虎); SUN,Bing-Yun(孙炳耘); HUANG,Zhong-Xian(黄仲贤); XIA,Zong-Xiang(夏宗芗)


    Glu44, Glu48, Glu56 and Asp60 are the negatively charged residues located at the molecular surface of cytochrome b5@Two mutants of cytochrome b5 were prepared, in which two or all of these four residues were mutated to alanines. The mutations give rise to slightly positive shifts of the redox potentials of cytochrome b5 and obvious decrease of the cytochrome b5-cytochrome c binding constants and electron transfer rates. The crystal structures of the two mutants were determined at 0.18 nm resolution, showing no alteration in overall structures and exhibiting slight chages in the local conformations around the mutation sites as compared with the wild-type protein. Based on the crystal structure of the quadruple-site mutant, a model for the binding of this mutant with cytochrome c is proposed, which involves the salt bridges from Glu37, Glu38 and heme propionate of cytochrome b5 to three lysines of cytochrome c and can well account for the properties and behaviors of this mutant.

  2. Prediction of cytochrome P450 mediated metabolism

    DEFF Research Database (Denmark)

    Olsen, Lars; Oostenbrink, Chris; Jørgensen, Flemming Steen


    Cytochrome P450 enzymes (CYPs) form one of the most important enzyme families involved in the metabolism of xenobiotics. CYPs comprise many isoforms, which catalyze a wide variety of reactions, and potentially, a large number of different metabolites can be formed. However, it is often hard...... to rationalize what metabolites these enzymes generate. In recent years, many different in silico approaches have been developed to predict binding or regioselective product formation for the different CYP isoforms. These comprise ligand-based methods that are trained on experimental CYP data and structure-based...... methods that consider how the substrate is oriented in the active site or/and how reactive the part of the substrate that is accessible to the heme group is. We will review key aspects for various approaches that are available to predict binding and site of metabolism (SOM), what outcome can be expected...

  3. Flower colour and cytochromes P450. (United States)

    Tanaka, Yoshikazu; Brugliera, Filippa


    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.

  4. Calcium transport in vesicles energized by cytochrome oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Rosier, Randy N.


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

  5. Interaction of apo cytochrome c with sulfonated polystyrene nanoparticles. (United States)

    Liang, Li; Yao, Ping; Gong, Jie; Jiang, Ming


    Stable nanoparticle dispersion in aqueous solutions was obtained with partially sulfonated polystyrene. The hydrophobic association of the backbone chains and phenyl groups is balanced by the electrostatic repulsion of the sulfonate groups on the particle surface. The size distribution of the sulfonated polystyrene particles in relation to concentration, degree of sulfonation and chain length, and pH was characterized by dynamic laser light-scattering. The structure and morphology of the particles were characterized with fluorescence and atom force microscopy. Highly sulfonated polystyrene particles can form large complex particles with positively charged protein, apo cytochrome c. Dynamic laser light-scattering and atom force microscopy studies show that the size and distribution of the complex particles depend on the relative amount of apo cytochrome c and sulfonated polystyrene. When sulfonated polystyrene is in excess, apo cytochrome c interacts with sulfonated polystyrene particles forming stable complexes and excessive sulfonated polystyrene particles bind to the periphery of the complexes preventing them from further aggregation. When apo cytochrome c is in excess, apo cytochrome c links the complexes forming much larger particles. Fluorescence study demonstrates that the hydrophobicity/hydrophility of the complex particles is relative to the ratio of apo cytochrome c and sulfonated polystyrene, degree of sulfonation, and pH. Apo cytochrome c not only can neutralize the negative charges on the surface of sulfonated polystyrene particles, but may also insert into the cores disrupting the original structure of sulfonated polystyrene particles.

  6. Intracerebroventricular Administration of C-Type Natriuretic Peptide Suppresses Food Intake via Activation of the Melanocortin System in Mice


    Yamada-Goto, Nobuko; Katsuura, Goro; Ebihara, Ken; Inuzuka, Megumi; Ochi, Yukari; Yamashita, Yui; Kusakabe, Toru; Yasoda, Akihiro; Satoh-Asahara, Noriko; Ariyasu, Hiroyuki; Hosoda, Kiminori; Nakao, Kazuwa


    C-type natriuretic peptide (CNP) and its receptor are abundantly distributed in the brain, especially in the arcuate nucleus (ARC) of the hypothalamus associated with regulating energy homeostasis. To elucidate the possible involvement of CNP in energy regulation, we examined the effects of intracerebroventricular administration of CNP on food intake in mice. The intracerebroventricular administration of CNP-22 and CNP-53 significantly suppressed food intake on 4-h refeeding after 48-h fastin...

  7. Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea

    Directory of Open Access Journals (Sweden)

    Jingqun Ao


    Full Text Available The C-type lectin-like receptors (CTLRs play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD, an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1–C6, a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp and WYD (Trp-Tyr-Asp. Ca2+ binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus and guppy (Poecilia retitculata CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca2+-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.

  8. Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase. (United States)

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


    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.

  9. Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase.

    Directory of Open Access Journals (Sweden)

    Laura Pastorino

    Full Text Available 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.

  10. Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3. (United States)

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


    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.

  11. Heterologous synthesis of cytochrome c' by Escherichia coli is not dependent on the System I cytochrome c biogenesis machinery. (United States)

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


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

  12. Modular assembly of yeast cytochrome oxidase. (United States)

    McStay, Gavin P; Su, Chen Hsien; Tzagoloff, Alexander


    Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high-molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.

  13. Cytochrome C stabilization and immobilization in aerogels. (United States)

    Harper-Leatherman, Amanda S; Wallace, Jean Marie; Rolison, Debra R


    Sol-gel-derived aerogels are three-dimensional, nanoscale materials that combine large surface areas and high porosities. These traits make them useful for any rate-critical chemical process, particularly sensing or electrochemical applications, once physical or chemical moieties are incorporated into the gels to add their functionality into the ultraporous scaffold. Incorporating biomolecules into aerogels has been challenging due to the inability of most biomolecules to remain structurally intact within the gels during the necessary supercritical fluid processing. However, the heme protein cytochrome c (cyt. c) forms self-organized superstructures around gold (or silver) nanoparticles in buffer that can be encapsulated within silica and processed to form aerogels in which cyt. c retains its characteristic visible absorption. The gold (or silver) nanoparticle-nucleated superstructures protect the majority of the protein from the harsh physicochemical conditions necessary to form an aerogel. The Au∼cyt. c superstructures exhibit rapid gas-phase recognition of nitric oxide (NO) within the aerogel matrix, as facilitated by the high-quality pore structure of the aerogel, and remain viable for weeks at room temperature.

  14. Cytochrome 572 is a conspicuous membrane protein with iron oxidation activity purified directly from a natural acidophilic microbial community. (United States)

    Jeans, Chris; Singer, Steven W; Chan, Clara S; Verberkmoes, Nathan C; Shah, Manesh; Hettich, Robert L; Banfield, Jillian F; Thelen, Michael P


    Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5-1.2 and laden with toxic metals. To approach the mechanism of Fe(II) oxidation as a means of cellular energy acquisition, we isolated proteins from natural samples and found a conspicuous and novel cytochrome, Cyt(572), which is unlike any known cytochrome. Both the character of its covalently bound prosthetic heme group and protein sequence are unusual. Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. The purified protein has a cytochrome c-type heme binding motif, CxxCH, but a unique spectral signature at 572 nm, and thus is called Cyt(572). It readily oxidizes Fe(2+) in the physiologically relevant acidic regime, from pH 0.95-3.4. Other physical characteristics are indicative of a membrane-bound multimeric protein. Circular dichroism spectroscopy indicates that the protein is largely beta-stranded, and 2D Blue-Native polyacrylamide gel electrophoresis and chemical crosslinking independently point to a multi-subunit structure for Cyt(572). By analyzing environmental genomic information from biofilms in several distinctly different mine locations, we found multiple genetic variants of Cyt(572). MS proteomics of extracts from these biofilms substantiated the prevalence of these variants in the ecosystem. Due to its abundance, cellular location and Fe(2+) oxidation activity at very low pH, we propose that Cyt(572) provides a critical function for fitness within the ecological niche of these acidophilic microbial communities.

  15. The Putative Assembly Factor CcoH Is Stably Associated with the cbb3-Type Cytochrome Oxidase ▿ (United States)

    Pawlik, Grzegorz; Kulajta, Carmen; Sachelaru, Ilie; Schröder, Sebastian; Waidner, Barbara; Hellwig, Petra; Daldal, Fevzi; Koch, Hans-Georg


    Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for cofactor incorporation adds an additional level of complexity to their assembly. cbb3-type cytochrome c oxidases (cbb3-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for cbb3-Cox stability. Biogenesis of cbb3-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active cbb3-Cox. Most bacteria expressing cbb3-Cox also contain the ccoGHIS genes, which encode putative cbb3-Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in cbb3-Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out-C-terminus-in (Nout-Cin) topology. In its absence, neither the fully assembled cbb3-Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembrane domain to the CcoP subunit of cbb3-Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the active cbb3-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active cbb3-Cox. Surprisingly, CcoH does not seem to interact only transiently with the cbb3-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona fide subunit of the cbb3-Cox than an assembly factor per se. PMID:20952576

  16. Molecular Organization of Cytochrome c 2 near the Binding Domain of Cytochrome bc 1 Studied by Electron Spin–Lattice Relaxation Enhancement


    Pietras, Rafał; Sarewicz, Marcin; Osyczka, Artur


    Measurements of specific interactions between proteins are challenging. In redox systems, interactions involve surfaces near the attachment sites of cofactors engaged in interprotein electron transfer (ET). Here we analyzed binding of cytochrome c 2 to cytochrome bc 1 by measuring paramagnetic relaxation enhancement (PRE) of spin label (SL) attached to cytochrome c 2. PRE was exclusively induced by the iron atom of heme c 1 of cytochrome bc 1, which guaranteed that only the configurations wit...

  17. Molecular characterization and expression analysis of a novel dual-CRD C-type lectin in kuruma shrimp (Marsupenaeus japonicus)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Man; MAO Yong; WANG Jun; FENG Wenrong; SONG Xiaohong; SU Yongquan


    C-type lectins are among the most significant pattern recognition receptors (PRRs) found in invertebrate. They are a class of carbohydrate-binding proteins that can recognize specific sugar moieties on the surface of pathogens. In the present study, a novel C-type lecitn (termed MjLectin) from kuruma shrimp Marsupenaeus japonicus was identified. The full-length cDNA of MjLectin was 1 245 bp with a 1 011 bp open reading frame (ORF) that encoded a polypeptide of 336 amino acid residues. MjLectin consisted of two tandemly arrayed carbohydrate-recognition domains (CRDs), unlike other reported M. japonicus C-type lectins with only one CRD. It showed a high similarity to other shrimp dual-CRD lectins. Among the Ca2+-binding Site 2, the tripeptide motif dictating the carbohydrate binding specificity was exhibited as a rare mutant LPN (Leu134-Pro135-Asn136) in CRD1 and a traditional EPN (Glu299-Pro300-Asn301) in CRD2, respectively. MjLectin showed a specific expression pattern in both tissue and cellular levels, for its mRNA transcript was mainly expressed in the F-cells of the hepatopancreas. After white spot syndrome virus (WSSV) challenge (3.6×108 virions/μL), the expression of MjLectin in the hepatopancreas was up-regulated significantly at 48 h (P<0.01) compared with the control group. These results suggested that MjLectin might be involved in the innate immune defense against WSSV infection.

  18. The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement


    Caminschi, Irina; Proietto, Anna I.; Ahmet, Fatma; Kitsoulis, Susie; Shin Teh, Joo; Lo, Jennifer C. Y.; Rizzitelli, Alexandra; Wu, Li; Vremec, David; van Dommelen, Serani L.H.; Campbell, Ian K.; Maraskovsky, Eugene; Braley, Hal; Davey, Gayle M.; Mottram, Patricia


    A novel dendritic cell (DC)–restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the...

  19. Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster

    DEFF Research Database (Denmark)

    Williamson, M; Lenz, C; Winther, A M


    The insect allatostatins are a diverse group of neuropeptides that obtained their names by their inhibitory actions on the corpora allata (two endocrine glands near the insect brain), where they block the biosynthesis of juvenile hormone (a terpenoid important for development and reproduction...... neurons of the brain and abdominal ganglia and in endocrine cells of the midgut. This is the first publication on the structure of a C-type allatostatin from insects other than moths and the first report on the presence of all three types of allatostatins in a representative of the insect order Diptera...

  20. Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release

    Institute of Scientific and Technical Information of China (English)

    Yushan Zhu; Min Li; Xiaohui Wang; Haijing Jin; Shusen Liu; Jianxin Xu; Quan Chen


    Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions.The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood.Here we addressed the underlying mechanism,explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome e (cyto.c) release.We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3).We found that cyto.c1 was cleaved at the site of D106,which is critical for binding with cyto.c,following apoptotic stresses or targeted expression of casp.3 into tbe mitochondrial intermembrane space.We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe.These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk.Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.

  1. Molecular organization of cytochrome c2 near the binding domain of cytochrome bc1 studied by electron spin-lattice relaxation enhancement. (United States)

    Pietras, Rafał; Sarewicz, Marcin; Osyczka, Artur


    Measurements of specific interactions between proteins are challenging. In redox systems, interactions involve surfaces near the attachment sites of cofactors engaged in interprotein electron transfer (ET). Here we analyzed binding of cytochrome c2 to cytochrome bc1 by measuring paramagnetic relaxation enhancement (PRE) of spin label (SL) attached to cytochrome c2. PRE was exclusively induced by the iron atom of heme c1 of cytochrome bc1, which guaranteed that only the configurations with SL to heme c1 distances up to ∼30 Å were detected. Changes in PRE were used to qualitatively and quantitatively characterize the binding. Our data suggest that at low ionic strength and under an excess of cytochrome c2 over cytochrome bc1, several cytochrome c2 molecules gather near the binding domain forming a "cloud" of molecules. When the cytochrome bc1 concentration increases, the cloud disperses to populate additional available binding domains. An increase in ionic strength weakens the attractive forces and the average distance between cytochrome c2 and cytochrome bc1 increases. The spatial arrangement of the protein complex at various ionic strengths is different. Above 150 mM NaCl the lifetime of the complexes becomes so short that they are undetectable. All together the results indicate that cytochrome c2 molecules, over the range of salt concentration encompassing physiological ionic strength, do not form stable, long-lived complexes but rather constantly collide with the surface of cytochrome bc1 and ET takes place coincidentally with one of these collisions.

  2. Cytochrome P450 gene polymorphism and cancer. (United States)

    Agundez, Jose A G


    Human cytochrome P450 (CYP) enzymes play a key role in the metabolism of drugs and environmental chemicals. Several CYP enzymes metabolically activate procarcinogens to genotoxic intermediates. Phenotyping analyses revealed an association between CYP enzyme activity and the risk to develop several forms of cancer. Research carried out in the last decade demonstrated that several CYP enzymes are polymorphic due to single nucleotide polymorphisms, gene duplications and deletions. As genotyping procedures became available for most human CYP, an impressive number of association studies on CYP polymorphisms and cancer risk were conducted. Here we review the findings obtained in these studies regarding CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP8A1 and CYP21 gene polymorphisms. Consistent evidences for association between CYP polymorphisms and lung, head and neck, and liver cancer were reported. Controversial findings suggest that colorectal and prostate cancers may be associated to CYP polymorphisms, whereas no evidences for a relevant association with breast or bladder cancers were reported. We summarize the available information related to the association of CYP polymorphisms with leukaemia, lymphomas and diverse types of cancer that were investigated only for some CYP genes, including brain, esophagus, stomach, pancreas, pituitary, cervical epithelium, melanoma, ovarian, kidney, anal and vulvar cancers. This review discusses on causes of heterogeneity in the proposed associations, controversial findings on cancer risk, and identifies topics that require further investigation. In addition, some recommendations on study design, in order to obtain more conclusive findings in further studies, are provided.

  3. Cytochromes P450 and insecticide resistance. (United States)

    Scott, J G


    The cytochrome P450-dependent monooxygenases (monooxygenases) are an extremely important metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. Monooxygenase-mediated metabolism is a common mechanism by which insects become resistant to insecticides as evidenced by the numerous insect species and insecticides affected. This review begins by presenting background information about P450s, the role of monooxygenases in insects, and the different techniques that have been used to isolate individual insect P450s. Next, insecticide resistance is briefly described, and then historical information about monooxygenase-mediated insecticide resistance is reviewed. For any case of monooxygenase-mediated resistance, identification of the P450(s) involved, out of the dozens that are present in an insect, has proven very challenging. Therefore, the next section of the review focuses on the minimal criteria for establishing that a P450 is involved in resistance. This is followed by a comprehensive examination of the literature concerning the individual P450s that have been isolated from insecticide resistant strains. In each case, the history of the strain and the evidence for monooxygenase-mediated resistance are reviewed. The isolation and characterization of the P450(s) from the strain are then described, and the evidence of whether or not the isolated P450(s) is involved in resistance is summarized. The remainder of the review summarizes our current knowledge of the molecular basis of monooxygenase-mediated resistance and the implications for the future. The importance of these studies for development of effective insecticide resistance management strategies is discussed.

  4. Direct regulation of cytochrome c oxidase by calcium ions.

    Directory of Open Access Journals (Sweden)

    Tatiana Vygodina

    Full Text Available Cytochrome c oxidase from bovine heart binds Ca(2+ reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+ shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca(2+ on the functional characteristics of cytochrome oxidase was revealed earlier. Here we report that Ca(2+ inhibits cytochrome oxidase activity of isolated bovine heart enzyme by 50-60% with Ki of ∼1 µM, close to Kd of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (∼10 s(-1 or less. No inhibitory effect of Ca(2+ is observed under conventional conditions of cytochrome c oxidase activity assays (turnover number >100 s(-1 at pH 8, which may explain why the effect was not noticed earlier. The inhibition is specific for Ca(2+ and is reversed by EGTA. Na(+ ions that compete with Ca(2+ for binding with the Cation Binding Site, do not affect significantly activity of the enzyme but counteract the inhibitory effect of Ca(2+. The Ca(2+-induced inhibition of cytochrome c oxidase is observed also with the uncoupled mitochondria from several rat tissues. At the same time, calcium ions do not inhibit activity of the homologous bacterial cytochrome oxidases. Possible mechanisms of the inhibition are discussed as well as potential physiological role of Ca(2+ binding with cytochrome oxidase. Ca(2+- binding at the Cation Binding Site is proposed to inhibit proton-transfer through the exit part of the proton conducting pathway H in the mammalian oxidases.

  5. Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity. (United States)

    Huang, Xin; Huang, Ying; Shi, Yan-Ru; Ren, Qian; Wang, Wen


    C-type lectins play crucial roles in innate immunity. In the present study, a novel C-type lectin gene, designated as MrCTL, was identified from Macrobrachium rosenbergii. MrCTL contains 2 carbohydrate-recognition domains (CRDs), namely MrCRD1 and MrCRD2. The MrCRD1 contains a QEP motif and MrCRD2 contains a motif of EPD. MrCTL was mainly expressed in the hepatopancreas. The expression level of MrCTL in hepatopancreas was significantly upregulated after a challenge with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). The recombinant MrCTL, MrCRD1 and MrCRD2 have an ability to agglutinate both Gram-negative (V. parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) in a calcium dependent manner. The recombinant MrCTL, MrCRD1 and MrCRD2 bind directly to all tested microorganisms. All these results suggested that MrCTL may have important roles in immune defense against invading pathogens in prawns.

  6. Molecular cloning and expression of a C-type lectin-like protein from orange-spotted grouper Epinephelus coioides. (United States)

    Ji, H; Wei, J; Wei, S; Yan, Y; Huang, Y; Huang, X; Zhou, S; Zhou, Y; Qin, Q


    A C-type lectin-like protein (Ec-CTLP) was cloned from the grouper Epinephelus coioides. The full-length cDNA of Ec-CTLP was composed of 905 bp with a 522 bp open reading frame that encodes a 174-residue protein. The putative amino acid sequence of Ec-CTLP contains a signal peptide of 19 residues at the N-terminus and a CLECT domain from Cys43 to Arg169 and a conserved imperfect WND (Trp-Asn-Asp) motif. The homologous identity of deduced amino acid sequences is from 32 to 42% with other fishes. The expression of Ec-CTLP was differently upregulated in E. coioides spleen (germline stem) cells after being challenged at 16 and 4° C. Intracellular localization revealed that Ec-CTLP was distributed only in the cytoplasm. Recombinant Ec-CTLP (rEc-CTLP) was expressed in Escherichia coli BL21 (DE3) and purified for mouse Mus musculus anti-Ec-CTLP serum preparation. The rEc-CTLP fusion protein does not possess haemagglutinating activity, but improves survival from frozen bacteria. The survival of bacteria (including gram-negative E. coli and gram-positive Staphylococcus aureus) was positively correlated with the concentration of the rEc-CTLP. These findings can provide clues to help understand the probable C-type lectin in marine fish innate immunity.

  7. A shrimp C-type lectin inhibits proliferation of the hemolymph microbiota by maintaining the expression of antimicrobial peptides. (United States)

    Wang, Xian-Wei; Xu, Ji-Dong; Zhao, Xiao-Fan; Vasta, Gerardo Raul; Wang, Jin-Xing


    Some aquatic invertebrates such as shrimp contain low albeit stable numbers of bacteria in the circulating hemolymph. The proliferation of this hemolymph microbiota in such a nutrient-rich environment is tightly controlled in healthy animals, but the mechanisms responsible had remained elusive. In the present study, we report a C-type lectin (MjHeCL) from the kuruma shrimp (Marsupenaeus japonicus) that participates in restraining the hemolymph microbiota. Although the expression of MjHeCL did not seem to be modulated by bacterial challenge, the down-regulation of its expression by RNA interference led to proliferation of the hemolymph microbiota, ultimately resulting in shrimp death. This phenotype was rescued by the injection of recombinant MjHeCL, which restored the healthy status of the knockdown shrimp. A mechanistic analysis revealed that MjHeCL inhibited bacterial proliferation by modulating the expression of antimicrobial peptides. The key function of MjHeCL in the shrimp immune homeostasis might be related to its broader recognition spectrum of the hemolymph microbiota components than other lectins. Our study demonstrates the role of MjHeCL in maintaining the healthy status of shrimp and provides new insight into the biological significance of C-type lectins, a diversified and abundant lectin family in invertebrate species.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  9. Pulse-radiolysis study of cytochrome c/sub 3/

    Energy Technology Data Exchange (ETDEWEB)

    van Leeuwen, W.; van Dijk, C.; Grande, H.J.; Veeger, C.


    Pulse-radiolysis experiments were performed in the presence of methyl viologen and cytochrome c/sub 3/. After the pulse, methyl viologen radicals are formed and the kinetics of these radicals with cytochrome c/sub 3/ are studied. The reaction between cytochrome c/sub 3/ and methyl viologen radicals (MV/sup +/) is diffusion controlled. The ionic strength dependence and the pH-dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome c/sub 3/ molecule was Z = +4.7 +- 0.7. After the pulse an equilibrium is reached for the reaction of MV/sup +/ with cytochrome c/sub 3/. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, ..cap alpha... With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: E/sub 0/ = -0.250-0.088 x (in V). An estimation is made of the energy of interaction between the haems due to electrostatic interactions ( < 32 mV) and due to ionic strength effects (-12 mV < < 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome c/sub 3/ molecule are dependent on the degree of reduction of the other haems in the molecule. The reaction of cytochrome c/sub 3/ with MV/sup +/ or with ethanol radicals (EtOHsup(.)) has been compared with the reactions of horse-heart cytochrome c and of metmyoglobin with the same radicals. The reaction of MV/sup +/ or EtOHsup(.) with horse-heart cytochrome c is found to be diffusion controlled; the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy.

  10. Proline-40 is Essential to Maintaining Cytochrome b5's Stability and Its Electron Transfer with Cytochrome c

    Institute of Scientific and Technical Information of China (English)

    WANG,Zhi-Qiang(王志强); WU,Jian(邬建); WANG,Yun-Hua(王韵华); QIAN,Wen(钱雯); XIE,Yi(谢毅); XIA,Zong-Xiang(夏宗芗); HUANG,Zhong-Xian(黄仲贤)


    In order to illustrate the roles played by Pro40 in the sturcture,properties and functions of Cytochrome b5, three mutated genes, P40V, P40Y, P40G were constructed in this work. Only the P40V gene was successfully expressed into holoprotein in E. coli JM83. According to the results of X-ray crystallographic analysis and various kinds of spectrostoscopy, it is evident that substituting valine for Pro40 does not result in significant alterations in the protein' soverall structure; however,local coformational perturbations in the proximity of the heme do occur. The redox potential of the P40V mutant is 40 mV lower than that of the wild type protein. Its stability towards heat, urea, acid and ethanol were significantly decreased. The mutation leads to a decrease in the hydrophobicity of the heme pocket, which is probably the major factor contributing to the above changes. Binding constants and electron transfer rates between cytochrome b5 and cytochrome c were determined using UV-visible spectroscopy and stopped-flow techniques for both the wild type and the mutant. The results showed that the substitution of Pro40 by valine does not influence the binding constant of cytochrome b5 to cytochrome c ; however, the electron transfer rate between them decreased significantly. This indicates that proline-40 is essential to maintaining cytochrome b5's stability and its electron transfer with cytochrome c.These studies also provided a good example that property and functional changes of a protein do not necessarily require large overall structural alterations; in most cases, only perturbations on the local conformations are suffcient to induce significant changes in protein′s properties and functions.

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

    Energy Technology Data Exchange (ETDEWEB)



    We report on reduction and precipitation of Se(VI), Pb(II), CU(II), U(VI), Mo(VI), and Cr(VI) in water by cytochrome c{sub 3} isolated from Desulfomicrobium baczdatum [strain 9974]. The tetraheme protein cytochrome c{sub 3} was reduced by sodium dithionite. Redox reactions were monitored by UV-visible spectroscopy of cytochrome c{sub 3}. Analytical electron microscopy work showed that Se(VI), Pb(II), and CU(II) were reduced to the metallic state, U(W) and Mo(W) to U(IV) and Mo(IV), respectively, and Cr(VI) probably to Cr(III). U(IV) and Mo(W) precipitated as oxides and Cr(III) as an amorphous hydroxide. Cytochrome c{sub 3} was used repeatedly in the same solution without loosing its effectiveness. The results suggest usage of cytochrome c{sub 3} to develop innovative and environmentally benign methods to remove heavy metals from waste- and groundwater.

  12. Homotropic cooperativity of monomeric cytochrome P450 3A4

    Energy Technology Data Exchange (ETDEWEB)

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G. (UIUC)


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

  13. The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s. (United States)

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


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

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

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


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

  15. Salsolinol, a catechol neurotoxin, induces oxidative modification of cytochrome c

    Directory of Open Access Journals (Sweden)

    Jung Hoon Kang


    Full Text Available Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol,an endogenous neurotoxin, is known to perform a role in thepathogenesis of Parkinson’s disease (PD. In this study, weevaluated oxidative modification of cytochrome c occurring afterincubation with salsolinol. When cytochrome c was incubatedwith salsolinol, protein aggregation increased in a dosedependentmanner. The formation of carbonyl compounds andthe release of iron were obtained in salsolinol- treated cytochromec. Salsolinol also led to the release of iron fromcytochrome c. Reactive oxygen species (ROS scavengers andiron specific chelator inhibited the salsolinol-mediated cytochromec modification and carbonyl compound formation. It issuggested that oxidative damage of cytochrome c by salsolinolmight induce the increase of iron content in cells, subsequentlyleading to the deleterious condition which was observed. Thismechanism may, in part, provide an explanation for thedeterioration of organs under neurodegenerative disorders suchas PD. [BMB Reports 2013; 46(2: 119-123

  16. Mechanisms of Cytochrome C Extraction by Reverse Micelles

    Institute of Scientific and Technical Information of China (English)


    The extraction of cytochrome C was carried out by means of phase transfer technique with three different reverse micellar systems, i.e., a CTAB micellar solution in n-butyl alcohol-chloroform(volume ratio 4∶1), an AOT micellar solution in isooctane and a SDSS-D2EHPA micellar solution in isooctane. The extraction mechanisms were studied. The results show that the extraction mechanisms for the same proteins with different types of reverse micellar systems can be distinct. The extraction of cytochrome C with CTAB and SDSS-D2EHPA reverse micellar systems are carried out according to the mechanism of electrostatic interaction. However, in the extraction of cytochrome C with the AOT reverse micellar system, the electrostatic interaction between the protein and the surfactant is not important.

  17. Activation of endogenous C-type retroviral genomes by internal alpha-irradiation of mice with /sup 224/Radium

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, J.; Erfle, V.; Mueller, W.A.


    Sensitive co-cultivation techniques were applied to study the radiation-induced activation of endogenous retroviral genomes in different mouse strains by the alpha-emitting radionuclide /sup 224/Radium. Activated infectious C-type retroviruses were detected in spleen, bone marrow and bone tissues of C57BL/6-, BALB/c- and NMRI mice. The titres of high-dose-irradiated animals were higher than those found in low-dose-irradiated animals. Infectious retrovirus could be detected with a dose of 13.2 rad (maximum dose rate 0.9 rad/day) in the skeleton, and a dose of 4.2 rad (maximum dose rate 0.3 rad/day) in the spleen. The virus activation pattern was different in the three mouse strains. These data indicate that activation of endogenous retroviral genomes by alpha-irradiation shows a dose-effect relationship and a dependence on the genetic background of the mouse.

  18. Lactobacillus reuteri Surface Mucus Adhesins Upregulate Inflammatory Responses Through Interactions With Innate C-Type Lectin Receptors. (United States)

    Bene, Krisztián P; Kavanaugh, Devon W; Leclaire, Charlotte; Gunning, Allan P; MacKenzie, Donald A; Wittmann, Alexandra; Young, Ian D; Kawasaki, Norihito; Rajnavolgyi, Eva; Juge, Nathalie


    The vertebrate gut symbiont Lactobacillus reuteri exhibits strain-specific adhesion and health-promoting properties. Here, we investigated the role of the mucus adhesins, CmbA and MUB, upon interaction of L. reuteri ATCC PTA 6475 and ATCC 53608 strains with human monocyte-derived dendritic cells (moDCs). We showed that mucus adhesins increased the capacity of L. reuteri strains to interact with moDCs and promoted phagocytosis. Our data also indicated that mucus adhesins mediate anti- and pro-inflammatory effects by the induction of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), IL-1β, IL-6, and IL-12 cytokines. L. reuteri ATCC PTA 6475 and ATCC 53608 were exclusively able to induce moDC-mediated Th1 and Th17 immune responses. We further showed that purified MUB activates moDCs and induces Th1 polarized immune responses associated with increased IFNγ production. MUB appeared to mediate these effects via binding to C-type lectin receptors (CLRs), as shown using cell reporter assays. Blocking moDCs with antibodies against DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) or Dectin-2 did not affect the uptake of the MUB-expressing strain, but reduced the production of TNF-α and IL-6 by moDCs significantly, in line with the Th1 polarizing capacity of moDCs. The direct interaction between MUB and CLRs was further confirmed by atomic force spectroscopy. Taken together these data suggest that mucus adhesins expressed at the cell surface of L. reuteri strains may exert immunoregulatory effects in the gut through modulating the Th1-promoting capacity of DCs upon interaction with C-type lectins.

  19. Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides. (United States)

    Wei, Shina; Huang, Youhua; Cai, Jia; Huang, Xiaohong; Fu, Jing; Qin, Qiwei


    Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and "GSTDYGIFQINS" motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.

  20. A hepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity. (United States)

    Sun, Yun-Dong; Fu, Li-Dong; Jia, Yu-Ping; Du, Xin-Jun; Wang, Qian; Wang, Yu-Hang; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing


    Lectins play important roles in animal innate immune responses by serving as pattern recognition receptors, opsonins, or effector molecules. Here, we report a novel hepatopancreas-specific C-type lectin, designated Fc-hsL, from the hepatopancreas of the Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-hsL is 571 bp long with a 480 bp open reading frame that encodes a 159-residue protein. Fc-hsL contains a signal peptide and a single C-type lectin-like domain (CTLD) or carbohydrate recognition domain (CRD). It has an EPN(Glu-Pro-Asn) motif with a predicted ligand-binding site specific for mannose. Fc-hsL was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated following challenge of shrimp with bacteria or virus. Fc-hsL was not detected in other tissues but was induced in the stomach of immune-challenged shrimp. Fc-hsL protein was detected in both hemolymph and the hepatopancreas of bacteria- and virus-challenged shrimp. Recombinant mature Fc-hsL has no hemagglutinating activity, but calcium-dependent agglutinating activity against some Gram-positive and Gram-negative bacteria was detected. The rFc-hsL also has binding activity to some Gram-positive and Gram-negative bacteria and high antimicrobial activity against some bacteria and fungi. These in vitro functions of recombinant Fc-hsL were calcium-independent. Fc-hsL may act as a pattern recognition receptor in antibacterial defense and as an effector in innate immunity of Chinese shrimp.

  1. Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Míriam Cristina Sakuragui Matuo


    Full Text Available Saccharomyces cerevisiae has been widely used in mutagenicity tests due to the presence of a cytochrome P-450 system, capable of metabolizing promutagens to active mutagens. There are a large number of S. cerevisiae strains with varying abilities to produce cytochrome P-450. However, strain selection and ideal cultivation conditions are not well defined. We compared cytochrome P-450 levels in four different S. cerevisiae strains and evaluated the cultivation conditions necessary to obtain the highest levels. The amount of cytochrome P-450 produced by each strain varied, as did the incubation time needed to reach the maximum level. The highest cytochrome P-450 concentrations were found in media containing fermentable sugars. The NCYC 240 strain produced the highest level of cytochrome P-450 when grown in the presence of 20 % (w/v glucose. The addition of ethanol to the media also increased cytochrome P-450 synthesis in this strain. These results indicate cultivation conditions must be specific and well-established for the strain selected in order to assure high cytochrome P-450 levels and reliable mutagenicity results.Linhagens de Saccharomyces cerevisiae tem sido amplamente empregadas em testes de mutagenicidade devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias pró-mutagênicas à sua forma ativa. Devido à grande variedade de linhagens de S. cerevisiae com diferentes capacidades de produção de citocromo P-450, torna-se necessária a seleção de cepas, bem como a definição das condições ideais de cultivo. Neste trabalho, foram comparados os níveis de citocromo P-450 em quatro diferentes linhagens de S. cerevisiae e avaliadas as condições de cultivo necessárias para obtenção de altas concentrações deste sistema enzimático. O maior nível enzimático foi encontrado na linhagem NCYC 240 em presença de 20 % de glicose (p/v. A adição de etanol ao meio de cultura também produziu um aumento na s

  2. The role of protein dynamics and thermal fluctuations in regulating cytochrome c/cytochrome c oxidase electron transfer. (United States)

    Alvarez-Paggi, Damian; Zitare, Ulises; Murgida, Daniel H


    In this overview we present recent combined electrochemical, spectroelectrochemical, spectroscopic and computational studies from our group on the electron transfer reactions of cytochrome c and of the primary electron acceptor of cytochrome c oxidase, the CuA site, in biomimetic complexes. Based on these results, we discuss how protein dynamics and thermal fluctuations may impact on protein ET reactions, comment on the possible physiological relevance of these results, and finally propose a regulatory mechanism that may operate in the Cyt/CcO electron transfer reaction in vivo. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  3. Single catalytic site model for the oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase.


    Speck, S.H.; Dye, D.; Margoliash, E


    A single catalytic site model is proposed to account for the multiphasic kinetics of oxidation of ferrocytochrome c by cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC This model involves nonproductive binding of substrate to sites near the catalytic site on cytochrome c oxidase for cytochrome c, decreasing the binding constant for cytochrome c at the catalytic site. This substrate inhibition results in an increase in the first-order rate constant for the dissociati...

  4. How important is intestinal cytochrome P450 3A metabolism?

    NARCIS (Netherlands)

    Herwaarden, A.E. van; Waterschoot, R.A. van; Schinkel, A.H.


    Cytochrome P450 3A (CYP3A) enzymes metabolize a wide variety of xenobiotics including many drugs. Because CYP3A is localized in both the liver and intestine, it can make a major contribution to the presystemic elimination of substrate drugs after oral administration ('first-pass metabolism'). Howeve

  5. Trends in predicted chemoselectivity of cytochrome P450 oxidation

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Lonsdale, Richard; Harvey, Jeremy N;


    Prediction of epoxide formation in drug metabolism is a difficult but important task, as epoxide formation is linked to drug toxicity. A comparison of the energy barriers for cytochrome P450 mediated epoxidation of alkenes to the barriers for the hydroxylation of an aliphatic carbon atom next to ...

  6. Molecular dynamics in cytochrome c oxidase Moessbauer spectra deconvolution

    Energy Technology Data Exchange (ETDEWEB)

    Bossis, Fabrizio [Department of Medical Biochemistry, Medical Biology and Medical Physics (DIBIFIM), University of Bari ' Aldo Moro' , Bari (Italy); Palese, Luigi L., E-mail: [Department of Medical Biochemistry, Medical Biology and Medical Physics (DIBIFIM), University of Bari ' Aldo Moro' , Bari (Italy)


    Research highlights: {yields} Cytochrome c oxidase molecular dynamics serve to predict Moessbauer lineshape widths. {yields} Half height widths are used in modeling of Lorentzian doublets. {yields} Such spectral deconvolutions are useful in detecting the enzyme intermediates. -- Abstract: In this work low temperature molecular dynamics simulations of cytochrome c oxidase are used to predict an experimentally observable, namely Moessbauer spectra width. Predicted lineshapes are used to model Lorentzian doublets, with which published cytochrome c oxidase Moessbauer spectra were simulated. Molecular dynamics imposed constraints to spectral lineshapes permit to obtain useful information, like the presence of multiple chemical species in the binuclear center of cytochrome c oxidase. Moreover, a benchmark of quality for molecular dynamic simulations can be obtained. Despite the overwhelming importance of dynamics in electron-proton transfer systems, limited work has been devoted to unravel how much realistic are molecular dynamics simulations results. In this work, molecular dynamics based predictions are found to be in good agreement with published experimental spectra, showing that we can confidently rely on actual simulations. Molecular dynamics based deconvolution of Moessbauer spectra will lead to a renewed interest for application of this approach in bioenergetics.

  7. Cation binding site of cytochrome c oxidase: progress report. (United States)

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A


    Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  8. Cytochrome allelic variants and clopidogrel metabolism in cardiovascular diseases therapy. (United States)

    Jarrar, Mohammed; Behl, Shalini; Manyam, Ganiraju; Ganah, Hany; Nazir, Mohammed; Nasab, Reem; Moustafa, Khaled


    Clopidogrel and aspirin are among the most prescribed dual antiplatelet therapies to treat the acute coronary syndrome and heart attacks. However, their potential clinical impacts are a subject of intense debates. The therapeutic efficiency of clopidogrel is controlled by the actions of hepatic cytochrome P450 (CYPs) enzymes and impacted by individual genetic variations. Inter-individual polymorphisms in CYPs enzymes affect the metabolism of clopidogrel into its active metabolites and, therefore, modify its turnover and clinical outcome. So far, clinical trials fail to confirm higher or lower adverse cardiovascular effects in patients treated with combinations of clopidogrel and proton pump inhibitors, compared with clopidogrel alone. Such inconclusive findings may be due to genetic variations in the cytochromes CYP2C19 and CYP3A4/5. To investigate potential interactions/effects of these cytochromes and their allele variants on the treatment of acute coronary syndrome with clopidogrel alone or in combination with proton pump inhibitors, we analyze recent literature and discuss the potential impact of the cytochrome allelic variants on cardiovascular events and stent thrombosis treated with clopidogrel. The diversity of CYP2C19 polymorphisms and prevalence span within various ethnic groups, subpopulations and demographic areas are also debated.

  9. Electron-paramagnetic-resonance studies of structure and function of the two-haem enzymes Pseudomonas cytochrome c peroxidase and beef heart cytochrome c oxidase. (United States)

    Vänngård, T


    Beef heart cytochrome c oxidase contains two cytochromes, a and a3, and Pseudomonas aeruginosa cytochrome c peroxidase has one high- and one low-potential c haem, cHP and cLP. The parallelism in co-ordination and spin states between cytochrome a and haem cHP on the one hand and between cytochrome a3 and haem cLP on the other is illustrated. The two latter haems become accessible to cyanide, when the former are reduced. Such reduction also leads to an activation of the enzymes. Mechanisms are presented in which ferryl forms of cytochromes a3 and haem cLP take part. The enzymes reach an oxidation state, formally the same as resting enzyme, but with different properties.

  10. The C-Type Lectin Receptor MCL Mediates Vaccine-Induced Immunity against Infection with Blastomyces dermatitidis. (United States)

    Wang, Huafeng; Li, Mengyi; Lerksuthirat, Tassanee; Klein, Bruce; Wüthrich, Marcel


    C-type lectin receptors (CLRs) are essential in shaping the immune response to fungal pathogens. Vaccine-induced resistance requires Dectin-2 to promote differentiation of antifungal Th1 and Th17 cells. Since Dectin-2 and MCL heterodimerize and both CLRs use FcRγ as the signaling adaptor, we investigated the role of MCL in vaccine immunity to the fungal pathogen Blastomyces dermatitidis. MCL(-/-) mice showed impaired vaccine resistance against B. dermatitidis infection compared to that of wild-type animals. The lack of resistance correlated with the reduced recruitment of Th17 cells to the lung upon recall following experimental challenge and impaired interleukin-17 (IL-17) production by vaccine antigen-stimulated splenocytes in vitro. Soluble MCL fusion protein recognized and bound a water-soluble ligand from the cell wall of vaccine yeast, but the addition of soluble Dectin-2 fusion protein did not augment ligand recognition by MCL. Taken together, our data indicate that MCL regulates the development of vaccine-induced Th17 cells and protective immunity against lethal experimental infection with B. dermatitidis.

  11. Physiological levels of A-, B- and C-type natriuretic peptide shed the endothelial glycocalyx and enhance vascular permeability. (United States)

    Jacob, Matthias; Saller, Thomas; Chappell, Daniel; Rehm, Markus; Welsch, Ulrich; Becker, Bernhard F


    Atrial natriuretic peptide (ANP) is a peptide hormone released from the cardiac atria during hypervolemia. Though named for its well-known renal effect, ANP has been demonstrated to acutely increase vascular permeability in vivo. Experimentally, this phenomenon was associated with a marked shedding of the endothelial glycocalyx, at least for supraphysiological intravascular concentrations. This study investigates the impact and mechanism of action of physiological doses of ANP and related peptides on the vascular barrier. In isolated guinea pig hearts, prepared and perfused in a modified Langendorff mode with and without the intravascular presence of the colloid hydroxyethyl starch (HES), we measured functional changes in vascular permeability and glycocalyx shedding related to intracoronary infusion of physiological concentrations of A-, B- and C-type natriuretic peptide (ANP, BNP and CNP). Significant coronary venous washout of glycocalyx constituents (syndecan-1 and heparan sulfate) was observed. As tested for ANP, this effect was positively related to the intracoronary concentration. Intravascular shedding of the glycocalyx was morphologically confirmed by electron microscopy. Also, functional vascular barrier competence decreased, as indicated by significant increases in transudate formation and HES extravasation. Ortho-phenanthroline, a non-specific inhibitor of matrix metalloproteases, was able to reduce ANP-induced glycocalyx shedding. These findings suggest participation of natriuretic peptides in pathophysiological processes like heart failure, inflammation or sepsis. Inhibition of metalloproteases might serve as a basis for future therapeutical options.

  12. Intracerebroventricular administration of C-type natriuretic peptide suppresses food intake via activation of the melanocortin system in mice. (United States)

    Yamada-Goto, Nobuko; Katsuura, Goro; Ebihara, Ken; Inuzuka, Megumi; Ochi, Yukari; Yamashita, Yui; Kusakabe, Toru; Yasoda, Akihiro; Satoh-Asahara, Noriko; Ariyasu, Hiroyuki; Hosoda, Kiminori; Nakao, Kazuwa


    C-type natriuretic peptide (CNP) and its receptor are abundantly distributed in the brain, especially in the arcuate nucleus (ARC) of the hypothalamus associated with regulating energy homeostasis. To elucidate the possible involvement of CNP in energy regulation, we examined the effects of intracerebroventricular administration of CNP on food intake in mice. The intracerebroventricular administration of CNP-22 and CNP-53 significantly suppressed food intake on 4-h refeeding after 48-h fasting. Next, intracerebroventricular administration of CNP-22 and CNP-53 significantly decreased nocturnal food intake. The increment of food intake induced by neuropeptide Y and ghrelin was markedly suppressed by intracerebroventricular administration of CNP-22 and CNP-53. When SHU9119, an antagonist for melanocortin-3 and melanocortin-4 receptors, was coadministered with CNP-53, the suppressive effect of CNP-53 on refeeding after 48-h fasting was significantly attenuated by SHU9119. Immunohistochemical analysis revealed that intracerebroventricular administration of CNP-53 markedly increased the number of c-Fos-positive cells in the ARC, paraventricular nucleus, dorsomedial hypothalamus, ventromedial hypothalamic nucleus, and lateral hypothalamus. In particular, c-Fos-positive cells in the ARC after intracerebroventricular administration of CNP-53 were coexpressed with α-melanocyte-stimulating hormone immunoreactivity. These results indicated that intracerebroventricular administration of CNP induces an anorexigenic action, in part, via activation of the melanocortin system.

  13. Mechanistic insights into the role of C-type lectin receptor/CARD9 signaling in human antifungal immunity

    Directory of Open Access Journals (Sweden)

    Rebecca A. Drummond


    Full Text Available Human CARD9 deficiency is an autosomal recessive primary immunodeficiency disorder caused by biallelic mutations in the gene CARD9, which encodes a signaling protein that is found downstream of many C-type lectin receptors (CLRs. CLRs encompass a large family of innate recognition receptors, expressed predominantly by myeloid and epithelial cells, which bind fungal carbohydrates and initiate antifungal immune responses. Accordingly, human CARD9 deficiency is associated with the spontaneous development of persistent and severe fungal infections that primarily localize to the skin and subcutaneous tissue, mucosal surfaces and/or central nervous system (CNS. In the last few years, more than 15 missense and nonsense CARD9 mutations have been reported which associate with the development of a wide spectrum of fungal infections caused by a variety of fungal organisms. The mechanisms by which CARD9 provides organ-specific protection against these fungal infections are now emerging. In this review, we summarize recent immunological and clinical advances that have provided significant mechanistic insights into the pathogenesis of human CARD9 deficiency. We also discuss how genetic mutations in CARD9-coupled receptors (Dectin-1, Dectin-2 and CARD9-binding partners (MALT1, BCL10 affect human antifungal immunity relative to CARD9 deficiency, and we highlight major understudied research questions which merit future investigation.

  14. Plasma C-type natriuretic peptide as a predictor for therapeutic response to metoprolol in children with postural tachycardia syndrome.

    Directory of Open Access Journals (Sweden)

    Jing Lin

    Full Text Available POTS is a global public-health disease, but predictor for therapeutic response to metoprolol in children with POTS is lacking. This study was designed to investigate predictive value of plasma C-type natriuretic peptide (CNP in the therapeutic efficacy of metoprolol on postural tachycardia syndrome (POTS in children. Totally 34 children with POTS and 27 healthy children were included in the study. The head-up test or head-up tilt test was used to check heart rate and blood pressure from supine to upright in subjects. A double antibody (competitive sandwich immunoluminometric assay was used to detect plasma CNP. Metoprolol was used to treat children with POTS. The difference in plasma concentrations of CNP between responders and non-responders was compared. An ROC curve was used to analyze plasma CNP to predict efficacy of metoprolol on POTS in children. Plasma CNP in children with POTS was significantly higher than that of healthy children [(51.9 ± 31.4 vs. (25.1 ± 19.1 pg/ml, P 32.55 pg/ml yielded a sensitivity of 95.8% and specificity of 70% in predicting therapeutic efficacy of metoprolol on POTS children. Plasma CNP might serve as a useful predictor for the therapeutic efficacy of metoprolol on POTS in children.

  15. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.; Garboczi, David N. (NIH)


    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

  16. C-type lectin like receptor 2 (CLEC-2) signals independently of lipid raft microdomains in platelets. (United States)

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol A; Kunapuli, Satya P


    C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling.

  17. Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development.

    Directory of Open Access Journals (Sweden)

    Shigeto Yoshida


    Full Text Available The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50 of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.

  18. Epidermal Langerhans cells rapidly capture and present antigens from C-type lectin-targeting antibodies deposited in the dermis. (United States)

    Flacher, Vincent; Tripp, Christoph H; Stoitzner, Patrizia; Haid, Bernhard; Ebner, Susanne; Del Frari, Barbara; Koch, Franz; Park, Chae Gyu; Steinman, Ralph M; Idoyaga, Juliana; Romani, Nikolaus


    Antigen-presenting cells can capture antigens that are deposited in the skin, including vaccines given subcutaneously. These include different dendritic cells (DCs) such as epidermal Langerhans cells (LCs), dermal DCs, and dermal langerin+ DCs. To evaluate access of dermal antigens to skin DCs, we used mAb to two C-type lectin endocytic receptors, DEC-205/CD205 and langerin/CD207. When applied to murine and human skin explant cultures, these mAbs were efficiently taken up by epidermal LCs. In addition, anti-DEC-205 targeted langerin+ CD103+ and langerin- CD103- mouse dermal DCs. Unexpectedly, intradermal injection of either mAb, but not isotype control, resulted in strong and rapid labeling of LCs in situ, implying that large molecules can diffuse through the basement membrane into the epidermis. Epidermal LCs targeted in vivo by ovalbumin-coupled anti-DEC-205 potently presented antigen to CD4+ and CD8+ T cells in vitro. However, to our surprise, LCs targeted through langerin were unable to trigger T-cell proliferation. Thus, epidermal LCs have a major function in uptake of lectin-binding antibodies under standard vaccination conditions.

  19. Differential expression of skin mucus C-type lectin in two freshwater eel species, Anguilla marmorata and Anguilla japonica. (United States)

    Tsutsui, Shigeyuki; Yoshinaga, Tatsuki; Komiya, Kaoru; Yamashita, Hiroka; Nakamura, Osamu


    Two types of lactose-specific lectins, galectin (AJL-1) and C-type lectin (AJL-2), were previously identified in the mucus of adult Anguilla japonica. Here, we compared the expression profiles of these two homologous lectins at the adult and juvenile stages between the tropical eel Anguilla marmorata and the temperate eel A. japonica. Only one lectin, predicted to be an orthologue of AJL-1 by LC-MS/MS, was detected in the mucus of adult A. marmorata. We also found that an orthologous gene to AJL-2 was expressed at very low levels, or not at all, in the skin of adult A. marmorata. However, we detected the gene expression of an AJL-2-orthologue in the skin of juvenile A. marmorata, and a specific antibody also detected the lectin in the juvenile fish epidermis. These findings suggest that expression profiles of mucosal lectins vary during development as well as between species in the Anguilla genus.

  20. C-type Lectin Mincle Recognizes Glucosyl-diacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia. (United States)

    Behler-Janbeck, Friederike; Takano, Tomotsugu; Maus, Regina; Stolper, Jennifer; Jonigk, Danny; Tort Tarrés, Meritxell; Fuehner, Thomas; Prasse, Antje; Welte, Tobias; Timmer, Mattie S M; Stocker, Bridget L; Nakanishi, Yoichi; Miyamoto, Tomofumi; Yamasaki, Sho; Maus, Ulrich A


    Among various innate immune receptor families, the role of C-type lectin receptors (CLRs) in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae) is not fully defined. We here show that Mincle gene expression was induced in alveolar macrophages and neutrophils in bronchoalveolar lavage fluids of mice and patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG), which was identified as the ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae.

  1. Effect of urea on synchronous fluorescence spectra and electrochemical behaviour of cytochrome

    Institute of Scientific and Technical Information of China (English)

    侴菊; 陆天虹; 吴越


    The changes of the synchronous fluorescence spectra and the electrochemical behaviour of cytochrome c with the urea concentration are studied. It has been found that with the increase of urea concentration, there occur sequentially the deaggregation of cytochrome c molecules, the increase of exposure extent of the heme group to the solvent, the disruption of Fe-S bond of the heme group and the change in the electrochemical behaviour of cytochrome c. It is suggested that the reason why the electrochemical reaction of cytochrome c is irreversible is that cytochrome c molecules exist in the concentrated solution as oligomers which are electrochemically inactive.

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

    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.


    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

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

    Directory of Open Access Journals (Sweden)

    Alexandr N Simonov

    Full Text Available 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.

  4. Multiple redox states of multiheme cytochromes may enable bacterial response to changing redox environments (United States)

    Arbour, T.; Wrighton, K. C.; Mullin, S. W.; Castelle, C.; Luef, B.; Gilbert, B.; Banfield, J. F.


    Multiheme c-type cytochromes (MHCs) are key components in electron-transport pathways that enable some microorganisms to transfer electron byproducts of metabolism to a variety of minerals. As a response to changes in mineral redox potential, microbial communities may shift their membership, or individual organisms may adjust protein expression. Alternatively, the ability to respond may be conferred by the innate characteristics of certain electron-transport-chain components. Here, we used potentiostat-controlled microbial fuel cells (MFCs) to measure the timescale of response to imposed changes in redox conditions, thus placing constraints on the importance of these different mechanisms. In the experiments, a solid electrode acts as an electron-accepting mineral whose redox potential can be precisely controlled. We inoculated duplicate MFCs with a sediment/groundwater mixture from an aquifer at Rifle, Colorado, supplied acetate as an electron donor, and obtained stable, mixed-species biofilms dominated by Geobacter and a novel Geobacter-related family. We poised the anode at potentials spanning the range of natural Fe(III)-reduction, then performed cyclic voltammetry (CV) to characterize the overall biofilm redox signature. The apparent biofilm midpoint potential shifted directly with anode set potential when the latter was changed within the range from about -250 to -50 mV vs. SHE. Following a jump in set potential by 200 mV, the CV-midpoint shift by ~100 mV over a timescale of ~30 minutes to a few hours, depending on the direction of the potential change. The extracellular electron transfer molecules, whose overall CV signature is very similar to those of purified MHCs, appear to span a broad redox range (~200 mV), supporting the hypothesis that MHCs confer substantial redox flexibility. This flexibility may be a principle reason for the abundance of MHCs expressed by microorganisms capable of extracellular electron transfer to minerals.

  5. Electron Flow in Multiheme Bacterial Cytochromes is a Balancing Act Between Heme Electronic Interaction and Redox Potentials

    Energy Technology Data Exchange (ETDEWEB)

    Breuer, Marian; Rosso, Kevin M.; Blumberger, Jochen


    The naturally widespread process of electron transfer from metal reducing bacteria to extracellular solid metal oxides entails unique biomolecular machinery optimized for long-range electron transport. To perform this function efficiently microorganisms have adapted multi-heme c-type cytochromes to arrange heme cofactors into wires that cooperatively span the cellular envelope, transmitting electrons along distances greater than 100 Angstroms. Implications and opportunities for bionanotechnological device design are self-evident. However, at the molecular level how these proteins shuttle electrons along their heme wires, navigating intraprotein intersections and interprotein interfaces effciently, remains a mystery so far inaccessible to experiment. To shed light on this critical topic, we carried out extensive computer simulations to calculate Marcus theory quantities for electron transfer along the ten heme cofactors in the recently crystallized outer membrane cytochrome MtrF. The combination of electronic coupling matrix elements with free energy calculations of heme redox potentials and reorganization energies for heme-to-heme electron transfer allows the step-wise and overall electron transfer rate to be estimated and understood in terms of structural and dynamical characteristics of the protein. By solving a master equation for electron hopping, we estimate an intrinsic, maximum possible electron flux through solvated MtrF of 104-105 s-1, consistent with recently measured rates for the related MtrCAB protein complex. Intriguingly, this flux must navigate thermodynamically uphill steps past low potential hemes. Our calculations show that the rapid electron transport through MtrF is the result of a clear correlation between heme redox potential and the strength of electronic coupling along the wire: Thermodynamically uphill steps occur only between electronically well connected stacked heme pairs. This suggests that the protein evolved to harbor low potential

  6. Structural transformation of cytochrome c and apo cytochrome c induced by sulfonated polystyrene. (United States)

    Gong, Jie; Yao, Ping; Duan, Hongwei; Jiang, Ming; Gu, Shaohua; Chunyu, Lijuan


    The structural transformation of cytochrome c (cyt c) and its heme-free precursor, apo cyt c, induced by negatively charged sulfonated polystyrene (SPS) with different charge density (degree of sulfonation) and chain length was studied to understand the factors that influence the folding and unfolding of the protein. SPS forms stable transparent nanoparticles in aqueous solution. The hydrophobic association of the backbone chain and phenyl groups is balanced by the electrostatic repulsion of the sulfonate groups on the particle surface. The binding of cyt c to negatively charged SPS particles causes an extensive disruption of the native compact structure of cyt c: the cleavage of Fe-Met80 ligand, about 40% loss of the helical structure, and the disruption of the asymmetry environment of Trp59. On the other hand, SPS particle-bound apo cyt c undergoes a conformational change from the random coil to alpha-helical structure. The folding of apo cyt c in SPS particles was influenced by pH and ionic strength of the solution, SPS concentration, and the degree of sulfonation and chain length of SPS. The folding can reach more than 90% of the alpha-helix content of native cyt c in solution. Poly(sodium 4-styrenesulfonate) (PSS), which is 100% sulfonated polystyrene and cannot form hydrophobic cores in the solution, induces only two-thirds of the alpha-helix content compared with SPS. It appears that the electrostatic interaction between PSS/SPS and apo cyt c induces an early partially folded state of apo cyt c. The hydrophobic interaction between nonpolar residues in apo cyt c and the hydrophobic cores in SPS particles extends the alpha-helical structure of apo cyt c.

  7. Polarimetric survey of main-belt asteroids. II. Results for 58 B- and C-type objects (United States)

    Gil-Hutton, R.; Cañada-Assandri, M.


    Aims: We present results of a polarimetric survey of main-belt asteroids at Complejo Astronómico el Leoncito (CASLEO), San Juan, Argentina. The aims of this survey are to increase the database of asteroid polarimetry, to estimate diversity in polarimetric properties of asteroids that belong to different taxonomic classes, and to search for objects that exhibit anomalous polarimetric properties. Methods: The data were obtained with the Torino and CASPROF polarimeters at the 2.15m telescope. The Torino polarimeter is an instrument that allows simultaneous measurement of polarization in five different bands, and the CASPROF polarimeter is a two-hole aperture polarimeter with rapid modulation. Results: The survey began in 2003, and up to 2009 data on a sample of more than 170 asteroids were obtained. In this paper the results for 58 B- and C-type objects are presented, most of them polarimetrically observed for the first time. Using these data we find phase-polarization curves and polarimetric parameters for these taxonomic classes. Based on observations carried out at the Complejo Astronómico El Leoncito, operated under agreement between the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and the National Universities of La Plata, Córdoba, and San Juan.Tables 1 and 2 are available in electronic form at CDS via anonymous ftp to ( or via

  8. Thermal Inertia Determination of C-type Asteroid Ryugu from in-situ Surface Brightness Temperature Measurements (United States)

    Hamm, Maximilian; Grott, Matthias; Knollenberg, Jörg; Kührt, Ekkehard; Pelivan, Ivanka


    The Japanese Hayabusa-2 mission is a sample-return mission currently on its way to the C-type asteroid Ryugu. Hayabusa-2 carries the small lander MASCOT (Mobile Asteroid Surface Scout), whose scientific payload includes the infrared radiometer MARA. The primary science goal of MARA is to determine Ryugu's surface brightness temperatures at the landing site for a full asteroid rotation, which will be measured using a long-pass filter, an 8 to 12 µm bandpass, as well as four narrow bandpasses centered at wavelengths between 5 and 15 µm. From these measurements, surface thermal inertia will be derived, but because MARA performs single pixel measurements, heterogeneity in the field of view cannot be resolved. Yet, the surface will likely exhibit different surface textures, and thermal inertia in the field of view could vary from 600 (small rocks) to 50 Jm-2s-0.5K-1 (fine regolith grains). Sub-pixel heterogeneity is a common problem when interpreting radiometer data, since the associated ambiguities cannot be resolved without additional information on surface texture. For MARA, this information will be provided by the MASCOT camera, and in the present paper we have investigated to what extent different thermal inertias can be retrieved from MARA data. To test the applied approach, we generated synthetic MARA data using a thermal model of Ryugu, assuming different thermal inertias for sections of the field of view. We find that sub-pixel heterogeneity systematically deforms the diurnal temperature curve so that it is not possible to fit the data using a single thermal inertia value. However, including the area fractions of the different surface sections enables us to reconstruct the different thermal inertias to within 10% assuming appropriate measurement noise. The presented approach will increase robustness of the Ryugu thermal inertia determination and results will serve as a ground truth for the global measurements performed by the thermal infrared mapper (TIR) on

  9. Plasma C-type natriuretic peptide as a predictor for therapeutic response to metoprolol in children with postural tachycardia syndrome. (United States)

    Lin, Jing; Han, Zhenhui; Li, Hongxia; Chen, Selena Ying; Li, Xueying; Liu, Ping; Wang, Yuli; Tang, Chaoshu; Du, Junbao; Jin, Hongfang


    POTS is a global public-health disease, but predictor for therapeutic response to metoprolol in children with POTS is lacking. This study was designed to investigate predictive value of plasma C-type natriuretic peptide (CNP) in the therapeutic efficacy of metoprolol on postural tachycardia syndrome (POTS) in children. Totally 34 children with POTS and 27 healthy children were included in the study. The head-up test or head-up tilt test was used to check heart rate and blood pressure from supine to upright in subjects. A double antibody (competitive) sandwich immunoluminometric assay was used to detect plasma CNP. Metoprolol was used to treat children with POTS. The difference in plasma concentrations of CNP between responders and non-responders was compared. An ROC curve was used to analyze plasma CNP to predict efficacy of metoprolol on POTS in children. Plasma CNP in children with POTS was significantly higher than that of healthy children [(51.9 ± 31.4) vs. (25.1 ± 19.1) pg/ml, P metoprolol was significantly higher than non-responders [(59.1 ± 33.5) vs. (34.8 ± 16.7) pg/ml, P = 0.037] before treatment. The ROC curve showed that area under the curve was 0.821 (95% CI 0.642-0.999). The cut-off value of plasma CNP > 32.55 pg/ml yielded a sensitivity of 95.8% and specificity of 70% in predicting therapeutic efficacy of metoprolol on POTS children. Plasma CNP might serve as a useful predictor for the therapeutic efficacy of metoprolol on POTS in children.

  10. Vascular relaxation induced by C-type natriuretic peptide involves the ca2+/NO-synthase/NO pathway.

    Directory of Open Access Journals (Sweden)

    Fernanda A Andrade

    Full Text Available AIMS: C-type natriuretic peptide (CNP and nitric oxide (NO are endothelium-derived factors that play important roles in the regulation of vascular tone and arterial blood pressure. We hypothesized that NO produced by the endothelial NO-synthase (NOS-3 contributes to the relaxation induced by CNP in isolated rat aorta via activation of endothelial NPR-C receptor. Therefore, the aim of this study was to investigate the putative contribution of NO through NPR-C activation in the CNP induced relaxation in isolated conductance artery. MAIN METHODS: Concentration-effect curves for CNP were constructed in aortic rings isolated from rats. Confocal microscopy was used to analyze the cytosolic calcium mobilization induced by CNP. The phosphorylation of the residue Ser1177 of NOS was analyzed by Western blot and the expression and localization of NPR-C receptors was analyzed by immunohistochemistry. KEY FINDINGS: CNP was less potent in inducing relaxation in denuded endothelium aortic rings than in intact ones. L-NAME attenuated the potency of CNP and similar results were obtained in the presence of hydroxocobalamin, an intracellular NO0 scavenger. CNP did not change the phosphorylation of Ser1177, the activation site of NOS-3, when compared with control. The addition of CNP produced an increase in [Ca2+]c in endothelial cells and a decrease in [Ca2+]c in vascular smooth muscle cells. The NPR-C-receptors are expressed in endothelial and adventitial rat aortas. SIGNIFICANCE: These results suggest that CNP-induced relaxation in intact aorta isolated from rats involves NO production due to [Ca2+]c increase in endothelial cells possibly through NPR-C activation expressed in these cells. The present study provides a breakthrough in the understanding of the close relationship between the vascular actions of nitric oxide and CNP.

  11. Macrophage inducible C-type lectin (Mincle) recognizes glycosylated surface (S)-layer of the periodontal pathogen Tannerella forsythia (United States)

    Chinthamani, Sreedevi; Settem, Rajendra P.; Honma, Kiyonobu; Kay, Jason G.


    The oral pathogen Tannerella forsythia is implicated in the development of periodontitis, a common inflammatory disease that leads to the destruction of the gum and tooth supporting tissues, often leading to tooth loss. T. forsythia is a unique Gram-negative organism endowed with an elaborate protein O-glycosylation system that allows the bacterium to express a glycosylated surface (S)-layer comprising two high molecular weight glycoproteins modified with O-linked oligosaccharides. The T. forsythia S-layer has been implicated in the modulation of cytokine responses of antigen presenting cells, such as macrophages, that play a significant role during inflammation associated with periodontitis. The macrophage-inducible C-type lectin receptor (Mincle) is an FcRγ-coupled pathogen recognition receptor that recognizes a wide variety of sugar containing ligands from fungal and bacterial pathogens. In this study, we aimed to determine if Mincle might be involved in the recognition of T. forsythia S-layer and modulation of cytokine response of macrophages against the bacterium. Binding studies using recombinant Mincle-Fc fusion protein indicated a specific Ca2+-dependent binding of Mincle to T. forsythia S-layer. Subsequent experiments with Mincle-expressing and Mincle-knockdown macrophages revealed a role for Mincle/S-layer interaction in the induction of both pro- and anti-inflammatory cytokine secretion in macrophages stimulated with T. forsythia as well as its S-layer. Together, these studies revealed Mincle as an important macrophage receptor involved in the modulation of cytokine responses of macrophages against T. forsythia, and thus may play a critical role in orchestrating the host immune response against the bacterium. PMID:28264048

  12. C-type natriuretic-peptide-potentiated relaxation response of gastric smooth muscle in streptozotocin-induced diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Ying-Lan Cai; Dong-Yuan Xu; Xiang-Lan Li; Zhang-Xun Qiu; Zheng Jin; Wen-Xie Xu


    AIM: To study the sensitivity of gastric smooth muscle to C-type natriuretic peptide (CNP) in streptozotocin (STZ)-induced diabetic rats. METHODS: The spontaneous contraction of a gastric smooth muscle strip was recorded by using physiological methods in rats. The expressions of CNP and natriuretic peptide receptor-B (NPR-B) in gastric tissue were examined by using immunohistochemistry techniques in the diabetic rat. RESULTS: At 4 wk after injection of STZ and vehicle, the frequency of spontaneous contraction of gastric smooth muscle was significantly reduced in diabetic rats, and the frequency was decreased from 3.10 ± 0.14 cycle/min in controls to 2.23 ± 0.13 cycle/min ( n = 8, P < 0.01). However, the ampli tude of spontaneous contraction was not significant different from the normal rat. CNP significantly inhibited spontaneous contraction of gastric smooth muscle in normal and diabetic rats, but the inhibitory effect was significantly potentiated in the diabetic rats. The amplitudes of spontaneous contraction were suppressed by 75.15% ± 0.71% and 58.92% ± 1.32% while the frequencies were decreased by 53.33% ± 2.03% and 26.95% ± 2.82% in diabetic and normal rats, respectively ( n = 8, P < 0.01). The expression of CNP in gastric tissue was not changed in diabetic rats, however the expression of NPR-B was significantly increased in diabetic rats, and the staining indexes of NPR-B were 30.67 ± 1.59 and 17.63 ± 1.49 in diabetic and normal rat, respectively ( n = 8, P < 0.01). CONCLUSION: The results suggest that CNP induced an inhibitory effect on spontaneous contraction of gastric smooth muscle, potentiated in diabetic rat via up-regulation of the natriuretic peptides-NPR-Bparticulate guanylyl cyclase-cyclic GMP signal pathway.

  13. C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression (United States)

    Scotland, Ramona S.; Cohen, Marc; Foster, Paul; Lovell, Matthew; Mathur, Anthony; Ahluwalia, Amrita; Hobbs, Adrian J.


    The multifaceted process of immune cell recruitment to sites of tissue injury is key to the development of an inflammatory response and involved in the pathogenesis of numerous cardiovascular disorders. We recently identified C-type natriuretic peptide (CNP) as an important endothelium-derived mediator that regulates vascular tone and protects against myocardial ischemia/reperfusion injury. Herein, we investigated whether CNP inhibits leukocyte recruitment and platelet aggregation and thereby exerts a potential antiinflammatory influence on the blood vessel wall. We assessed the effects of CNP on leukocyte-endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in animals with high basal leukocyte activation (endothelial nitric oxide synthase knockout mice, eNOS-/-) or under acute inflammatory conditions (induced by interleukin-1 or histamine). CNP suppressed basal leukocyte rolling in eNOS-/- mice in a rapid, reversible, and concentration-dependent manner. These effects of CNP were mimicked by the selective natriuretic peptide receptor-C agonist cANF4-23. CNP also suppressed leukocyte rolling induced by IL-1 or histamine, inhibited platelet-leukocyte interactions, and prevented thrombin-induced platelet aggregation of human blood. Furthermore, analysis of human umbilical vein endothelial cells, leukocytes, and platelets revealed that CNP selectively attenuates expression of P-selectin. Thus, CNP is a modulator of acute inflammation in the blood vessel wall characterized by leukocyte and platelet activation. These antiinflammatory effects appear to be mediated, at least in part, via suppression of P-selectin expression. These observations suggest that endothelial CNP might maintain an anti-atherogenic influence on the blood vessel wall and represent a target for therapeutic intervention in inflammatory cardiovascular disorders. endothelium | natriuretic peptide receptor type C | atherosclerosis | thrombosis

  14. C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and – independent pathways

    Directory of Open Access Journals (Sweden)

    Serra Rosa


    Full Text Available Abstract Background C-type natriuretic peptide (CNP has recently been identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanisms mediating its effects are not completely understood. Results We demonstrate in a tibia organ culture system that pharmacological inhibition of p38 blocks the anabolic effects of CNP. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We also performed Affymetrix microarray analyses on micro-dissected tibiae to identify CNP target genes. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, since many more genes were regulated by CNP in this zone than in the others. While CNP receptors are expressed at similar levels in all three zones, cGMP-dependent kinases I and II, important transducers of CNP signaling, are expressed at much higher levels in hypertrophic cells than in other areas of the tibia, providing a potential explanation for the spatial distribution of CNP effects. In addition, our data show that CNP induces the expression of NPR3, a decoy receptor for natriuretic peptides, suggesting the existence of a feedback loop to limit CNP signaling. Finally, detailed analyses of our microarray data showed that CNP regulates numerous genes involved in BMP signaling and cell adhesion. Conclusion Our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral bone growth, with potential implications for understanding and treatment of numerous skeletal diseases.

  15. Macrophage inducible C-type lectin (Mincle) recognizes glycosylated surface (S)-layer of the periodontal pathogen Tannerella forsythia. (United States)

    Chinthamani, Sreedevi; Settem, Rajendra P; Honma, Kiyonobu; Kay, Jason G; Sharma, Ashu


    The oral pathogen Tannerella forsythia is implicated in the development of periodontitis, a common inflammatory disease that leads to the destruction of the gum and tooth supporting tissues, often leading to tooth loss. T. forsythia is a unique Gram-negative organism endowed with an elaborate protein O-glycosylation system that allows the bacterium to express a glycosylated surface (S)-layer comprising two high molecular weight glycoproteins modified with O-linked oligosaccharides. The T. forsythia S-layer has been implicated in the modulation of cytokine responses of antigen presenting cells, such as macrophages, that play a significant role during inflammation associated with periodontitis. The macrophage-inducible C-type lectin receptor (Mincle) is an FcRγ-coupled pathogen recognition receptor that recognizes a wide variety of sugar containing ligands from fungal and bacterial pathogens. In this study, we aimed to determine if Mincle might be involved in the recognition of T. forsythia S-layer and modulation of cytokine response of macrophages against the bacterium. Binding studies using recombinant Mincle-Fc fusion protein indicated a specific Ca2+-dependent binding of Mincle to T. forsythia S-layer. Subsequent experiments with Mincle-expressing and Mincle-knockdown macrophages revealed a role for Mincle/S-layer interaction in the induction of both pro- and anti-inflammatory cytokine secretion in macrophages stimulated with T. forsythia as well as its S-layer. Together, these studies revealed Mincle as an important macrophage receptor involved in the modulation of cytokine responses of macrophages against T. forsythia, and thus may play a critical role in orchestrating the host immune response against the bacterium.

  16. Distinct structures and environments for the three hemes of the cytochrome bc1 complex from Rhodospirillum rubrum. A resonance Raman study using B-band excitations. (United States)

    Le Moigne, C; Schoepp, B; Othman, S; Verméglio, A; Desbois, A


    The B-band excited resonance Raman (RR) spectra (100-1700 cm-1) of the bacterial cytochrome bc1 complex purified from Rhodospirillum rubrum are reported. Four redox states, i.e., the persulfate-oxidized, "as prepared", and ascorbate- and dithionite-reduced states of the complex, were investigated with the laser excitations at 406.7, 413.1, and 441.6 nm. Following the different absorption properties of the b- and c-type hemes and the different resonance enhancements of the vibrational modes of oxidized and reduced hemes, RR contributions from the b- and c-type hemes were characterized. For the nu2, nu10, and nu8 porphyrin vibrational modes, individual contributions of hemes c1, bH, and bL were determined. The data show that the macrocycle conformation of the three hemes of the cytochrome bc1 complex is different. In particular, the frequencies assigned to ferrous heme bL (1580, 1610, and 352 cm-1, respectively) reveal that its porphyrin is more strongly distorted than that of ferrous heme bH (1584, 1614, and 344 cm-1, respectively). The frequencies of the nu11 modes (1543, 1536, and 1526 cm-1 for ferrous heme c1, heme bH, and heme bL, respectively) confirm that the axial histidylimidazole ligands of heme bL have a marked anionic character. Strong differences in the peripheral interactions of the three hemes with the proteins were also detected through the frequency differences of the nu5, nu13, nu14, and nu42 modes. Considering that hemes bH and bL are inserted into a four-helice bundle, the RR data are interpreted in the frame of a strong protein constraint on heme bL.

  17. Structure-Function of the Cytochrome b6f Complex of Oxygenic Photosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Cramer, W. A.; Yamashita, E.; Baniulis, D.; Whitelegge, J.; Hasan, S. S. [Lithuanian RAF; (UCLA); (Purdue); (Osaka)


    Structure–function of the major integral membrane cytochrome b6f complex that functions in cyanobacteria, algae, and green plants to transfer electrons between the two reaction center complexes in the electron transport chain of oxygenic photosynthesis is discussed in the context of recently obtained crystal structures of the complex and soluble domains of cytochrome f and the Rieske iron–sulfur protein. The energy-transducing function of the complex, generation of the proton trans-membrane electrochemical potential gradient, centers on the oxidation/reduction pathways of the plastoquinol/plastoquinone (QH2/Q), the proton donor/acceptor within the complex. These redox reactions are carried out by five redox prosthetic groups embedded in each monomer, the high potential two iron–two sulfur cluster and the heme of cytochrome f on the electropositive side (p) of the complex, two noncovalently bound b-type hemes that cross the complex and the membrane, and a covalently bound c-type heme (cn) on the electronegative side (n). These five redox-active groups are organized in high- (cyt f/[2Fe–2S] and low-potential (hemes bp, bn, cn) electron transport pathways that oxidize and reduce the quinol and quinone on the p- and n-sides in a Q-cycle-type mechanism, while translocating as many as 2 H+ to the p-side aqueous side for every electron transferred through the high potential chain to the photosystem I reaction center. The presence of heme cn and the connection of the n-side of the membrane and b6f complex to the cyclic electron transport chain indicate that the Q cycle in the oxygenic photosynthetic electron transport chain differs from those connected to the bc1 complex in the mitochondrial respiratory chain and the chain in photosynthetic bacteria. Inferences from the structure and C2 symmetry of the complex for the pathway of QH2/Q transfer

  18. Predicting drug metabolism by cytochrome P450 2C9

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Olsen, Lars


    By the use of knowledge gained through modeling of drug metabolism mediated by the cytochrome P450 2D6 and 3A4 isoforms, we constructed a 2D-based model for site-of-metabolism prediction for the cytochrome P450 2C9 isoform. The similarities and differences between the models for the 2C9 and 2D6...... isoforms are discussed through structural knowledge from the X-ray crystal structures and trends in experimental data. The final model was validated on an independent test set, resulting in an area under the curve value of 0.92, and a site of metabolism was found among the top two ranked atoms for 77...

  19. Structural Diversity of Eukaryotic Membrane Cytochrome P450s*


    Johnson, Eric F.; Stout, C. David


    X-ray crystal structures are available for 29 eukaryotic microsomal, chloroplast, or mitochondrial cytochrome P450s, including two non-monooxygenase P450s. These structures provide a basis for understanding structure-function relations that underlie their distinct catalytic activities. Moreover, structural plasticity has been characterized for individual P450s that aids in understanding substrate binding in P450s that mediate drug clearance.

  20. Special issue: Cytochrome P450 structure and function: introduction. (United States)

    Munro, Andrew W; Leys, David


    The 17th International Conference on Cytochrome P450 Biochemistry, Biophysics and Structure was held in Manchester, UK from 26-30 June 2011. This issue of FEBS J. contains review and primary research articles reflecting the breadth of science covered at this conference, and reflecting the impact of P450-related research in fields as diverse as steroid metabolism, plant biochemistry, structural biology and biotechnology.

  1. Using Cytochrome c{sub 3} to Make Selenium Nanowires

    Energy Technology Data Exchange (ETDEWEB)



    We report on a new method to make nanostructures, in this case selenium nanowires, in aqueous solution at room temperature. We used the protein cytochrome c{sub 3} to reduce selenate (SeO{sub 4}{sup 2{minus}}) to selenium (Se{sup 0}). Cytochrome c{sub 3} is known for its ability to catalyze reduction of metals including U{sup VI} {yields} U{sup IV}, Cr{sup VI} {yields} Cr{sup III}, Mo{sup VI} {yields} Mo{sup IV}, Cu{sup II} {yields} Cu{sup 0}, Pb{sup II} {yields} Pb{sup 0}, Hg{sup II} {yields} Hg{sup 0}. Nanoparticles of Se{sup 0} precipitated from an aqueous solution at room temperature, followed by spontaneous self-assembling into nanowires. Cytochrome c{sub 3} was extracted from the sulfate-reducing bacteria Desulfovibrio vulgaris (strain Holdenborough) and isolated by the procedure of DerVartanian and Legall.

  2. Studies of multi-heme cytochromes from Geobacter sulfurreducens

    Energy Technology Data Exchange (ETDEWEB)

    Pokkuluri, P. Raj; Londer, Yuri, Y.; Orshonsky, Valerie; Orshonsky, Lisa; Duke, Norma; Schiffer, Marianne


    The Geobacteraceae family predominates in the reduction of uranium in subsurface environments. We are focusing on the model organism, Geobacter sulfurreducens; its genome contains a large number (>100) of cytochromes c that function in metal reduction pathways. Intensive functional genomics and physiological studies are in progress in Prof. Derek Lovley's laboratory, and the complete genome sequence of this organism has been determined by Methe et al. 2003. We are studying cytochromes from the c{sub 7} family that are required for the reduction of Fe(III). Previously, we expressed in E. coli (Londer et al., 2002) and determined the three-dimensional structure at 1.45 {angstrom} resolution (Pokkuluri et al., 2004a) of the three-heme cytochrome c{sub 7} (PpcA, coded by ORF01023) characterized by Lloyd et al., 2003. Further we identified in the G. sulfurreducens genome ORFs for several of its homologs (Pokkuluri et al., 2004a). Four of the ORFs are the same size as PpcA; three other ORFs are polymers of c7-type domains, two of which consist of four domains and one of nine domains, that contain 12 and 27 hemes respectively.

  3. Hydration Dependence of Energy Relaxation Time for Cytochrome C (United States)

    Ye, Shuji; Chen, Jing-Yin; Knab, Joseph R.; Markelz, Andrea


    Hydration plays a critical role in protein dynamics. Here we consider the effects of hydration on energy relaxation for an electronically excited heme protein cytochrome c. We measure the hydration dependence of energy relaxation time of cytochrome C films after photoexcitation in the Soret regionusing two-color pump/probe time resolved transmission measurements. Thin films were prepared from cytochrome C/ Trizma buffer solutions and mounted in a hydration controlled cell. We used 400nm (˜3 mW) to pump the B band and 800 nm (˜1 mW) to probe the III band. The III band corresponds to the charge-transfer transition between heme π and iron d orbital, and is assigned to the ground electronic state of the heme. Therefore this band can be used to probe the ground state population. Three separate dynamic components were observed: a very fast transient τ1 ˜ 200 fs; a several hundred femtosecond component (τ2); and a recovery of the ground state absorption(τ3). We find τ3 apparently decreases with decreasing hydration while τ1 and τ2 are independent of hydration.

  4. Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450

    DEFF Research Database (Denmark)

    Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg


    The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR...... to serve as an effective electron transferring "nano-machine"....

  5. Disruption of protein-protein interactions: design of a synthetic receptor that blocks the binding of cytochrome c to cytochrome c peroxidase. (United States)

    Wei, Y; McLendon, G L; Hamilton, A D; Case, M A; Purring, C B; Lin, Q; Park, H S; Lee, C S; Yu, T


    Synthetic receptor 1 has been found via fluorescence titration to compete effectively with cytochrome c peroxidase for binding cytochrome c (Cc), forming 1:1 Cc:1 complex with a binding constant of (3 +/- 1) x 10(8) M-1, and to disrupt Cc: Apaf-1 complex, a key adduct in apoptosis.

  6. Immune response of four dual-CRD C-type lectins to microbial challenges in giant freshwater prawn Macrobrachium rosenbergii. (United States)

    Ren, Qian; Li, Meng; Du, Jie; Zhang, Chi-Yu; Wang, Wen


    C-type lectins (CTLs) are believed to play important roles in the innate immunity of invertebrates and serve as pattern recognition receptors, opsonins, or effector molecules. In this study, the full-lengths cDNA of 4 CTL genes from giant freshwater prawn Macrobrachium rosenbergii were cloned and designated as MrLec1, MrLec2, MrLec3, and MrLec4. All of these 4 lectin cDNAs encode proteins with 2 carbohydrate recognition domains (CRDs). While MrLec1, MrLec3, and MrLec4 had signal peptides, no signal peptide was detected in MrLec2. Two carbohydrate recognition motifs within two CRDs of each lectin were predicted (QPE, EPG in MrLec1; EPT, EPA in MrLec2; QPT, NPR in MrLec3; KPN, EPD in MrLec4). Phylogenetic analysis showed that MrLec4 belongs to group A whereas MrLec1, MrLec2, and MrLec3 belong to group B. Positive selection in dual-CRD lectins suggested their probable roles in innate immunity, and positively selected induced amino acid diversity of lectins may confer their ability to recognize a broad range of microbes. The qRT-PCR analysis in adult prawns showed that MrLec1 is mainly expressed in the hepatopancreas, gills, and stomach, MrLec2 and MrLec4 are mainly distributed in the hepatopancreas, and MrLec3 is mainly expressed in the hepatopancreas and stomach. Time-course analysis using qRT-PCR showed that MrLec1 to MrLec4 are all upregulated by the Vibrio anguillarum challenge. MrLec1 is upregulated after 2, 12, and 24 h of white spot syndrome virus (WSSV) challenge. The expression of MrLec2 increases after 12 and 24 h of WSSV challenge, and the transcript of MrLec3 and MrLec4 are downregulated after 2 h of WSSV challenge. The results suggest the potential roles of dual-CRD lectins in the innate immunity of M. rosenbergii.

  7. Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide

    Directory of Open Access Journals (Sweden)

    Chrisman Ted D


    Full Text Available Abstract Background Guanylyl cyclase-B (GC-B; NPR-B, the receptor for C-type natriuretic peptide (CNP is rapidly and effectively desensitized by a factor(s in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s is of significant medical importance. Results Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2, a known component of high density lipoprotein (HDL. Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P. We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5–10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK or Akt/protein kinase B (Akt/PKB. Conclusion Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this

  8. Subunit analysis of mitochondrial cytochrome c oxidase and cytochrome bc1 by reversed-phase high-performance liquid chromatography. (United States)

    Kesa, Peter; Bhide, Mangesh; Lysakova, Veronika; Musatov, Andrey


    A rapid separation of the ten nuclearly-encoded subunits of mitochondrial cytochrome c oxidase, and ten out of the eleven subunits of cytochrome bc1, was achieved using a short, 50 mm C18-reversed-phase column. The short column decreased the elution time 4-7 fold while maintaining the same resolution quality. Elution was similar to a previously published protocol, i.e., a water/acetonitrile elution gradient containing trifluoroacetic acid. Isolated subunits were identified by MALDI-TOF. The rapidity of the described method makes it extremely useful for determining the subunit composition of isolated mitochondrial complexes. The method can be used for both analytical and micro-preparative purposes.

  9. Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa 3 *


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


    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 bc 1 complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, maki...

  10. New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants. (United States)

    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


    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.

  11. cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

    NARCIS (Netherlands)

    H.R. de Jonge (Hugo); B.C. Tilly (Bernard); B.M. Hogema (Boris); D.J. Pfau (Daniel); C.A. Kelley (Catherine); M.H. Kelley (Megan); A.M. Melita (August); M.T. Morris (Montana); M.S. Viola (Maria); J.N. Forrest Jr. (John)


    textabstractThe in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secr

  12. The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x.

    NARCIS (Netherlands)

    Die, van I.M.; Vliet, van SJ; Nyame, AK; Cummings, RD; Bank, CM; Appelmelk, B.J.; Geijtenbeek, T.B.H.; Kooijk, van Y.


    Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies agai

  13. Prognostic value of N-terminal pro C-type natriuretic peptide in heart failure patients with preserved and reduced ejection fraction

    NARCIS (Netherlands)

    Lok, Dirk J.; Klip, IJsbrand T.; Voors, Adriaan A.; Lok, Sjoukje I.; de la Porte, Pieta W. Bruggink-Andre; Hillege, Hans L.; Jaarsma, Tiny; van Veldhuisen, Dirk J.; van der Meer, Peter


    AimsA-type and B-type natriuretic peptides are established markers in chronic heart failure (HF). C-type natriuretic peptide (CNP) belongs to the same peptide family, but is predominantly localized in the endothelium. The prognostic role of CNP in heart failure has not been established. The aim of t

  14. Molecular cloning and characterization of a C-type lectin from Ancylostoma ceylanicum: evidence for a role in hookworm reproductive physiology. (United States)

    Brown, Allison C.; Harrison, Lisa M.; Kapulkin, Wadim; Jones, Brian F.; Sinha, Anindita; Savage, Amy; Villalon, Nicholas; Cappello, Michael


    Lectins comprise a family of related proteins that mediate essential cell functions through binding to carbohydrates. Within this protein family, C-type lectins are defined by the requirement of calcium for optimal biologic activity. Using reverse transcription PCR, a cDNA corresponding to a putative C-type lectin has been amplified from the hookworm parasite Ancylostoma ceylanicum. The 550 nucleotide open reading frame of the Ancylostoma ceylanicum C-type Lectin-1 (AceCTL-1) cDNA corresponds to a 167 amino acid mature protein (18706 Da) preceded by a 17 amino acid secretory signal sequence. The recombinant protein (rAceCTL-1) was expressed in Drosophila S2 cells and purified using a combination of affinity chromatography and reverse phase HPLC. Using in vitro carbohydrate binding studies, it was determined that rAceCTL-1 binds N-acetyl-D-glucosamine, a common component of eukaryotic egg cell membranes. Using a polyclonal IgG raised against the recombinant protein, the native AceCTL-1 was identified in sperm and soluble protein extracts of adult male A. ceylanicum by immunoblot. Probing of adult hookworm sections with the polyclonal IgG demonstrated localization to the testes in males, as well as the spermatheca and developing embryos in females, consistent with its role as a sperm protein. Together, these data strongly suggest that AceCTL-1 is a male gender-specific C-type lectin with a function in hookworm reproductive physiology. PMID:17129620

  15. cDNA cloning,sequence analysis,and recombinant expression of akitonin beta,a C-type lectin-like protein from Agkistrodon acutus

    Institute of Scientific and Technical Information of China (English)

    Xiang-dong ZHA; Jing LIU; Kang-sen XU


    AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structurefunction relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template.The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBADTOPO as vector and transformed into E. coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin β was found and accepted by GenBank (accession number AF387100). Akitonin β consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin β expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was established for its production.

  16. CLEC4F Is an Inducible C-Type Lectin in F4/80-Positive Cells and Is Involved in Alpha-Galactosylceramide Presentation in Liver

    NARCIS (Netherlands)

    Yang, C.Y.; Chen, J.B.; Tsai, T.F.; Tsai, Y.C.; Tsai, C.Y.; Liang, P.H.; Hsu, T.L.; Wu, C.Y.; Netea, M.G.; Wong, C.H.; Hsieh, S.L.


    CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated.

  17. Understanding the role of multiheme cytochromes in iron(III) reduction and arsenic mobilization by Shewanella sp. ANA-3 (United States)

    Reyes, C.; Duenas, R.; Saltikov, C.


    The reduction of Fe (III) to Fe (II) and of arsenate (As (V)) to arsenite (As (III)) by Fe (III) reducing and As (V) respiring prokaryotes such as the bacterium Shewanella sp. ANA-3 may contribute to arsenic mobilization in aquifers contaminated with arsenic, specifically in places such as Bangladesh. Under oxic conditions As (V) predominates and is often adsorbed onto mineral surfaces such as amorphous ferrihydrite. However, under anoxic conditions As (III) predominates, sorbs to fewer minerals, and has a greater hydrologic mobility compared to As (V). The genetic mechanism underlying arsenic release from subsurface material most likely involves a combination of respiratory gene clusters (e.g. mtr/omc and arr). In this study, we are investigating the genetic pathways underlying arsenic mobilization. We have generated various mutations in the mtr/omc gene cluster, which encodes several outermembrane decaheme c-type cytochromes. Deletions in one mtr/omc gene did not eliminate iron reduction. However, strains carrying multiple gene deletions were greatly impaired in iron reduction abilities. Work is currently underway to generate combinations of iron reduction and arsenate reduction single and double mutants that will be used to investigate microbial mobilization of arsenic in flow-through columns containing As (V)-HFO coated sand. This work will address the importance of arsenate reduction and iron reduction in the mobilization of arsenic.

  18. Cox26 is a novel stoichiometric subunit of the yeast cytochrome c oxidase. (United States)

    Levchenko, Maria; Wuttke, Jan-Moritz; Römpler, Katharina; Schmidt, Bernhard; Neifer, Klaus; Juris, Lisa; Wissel, Mirjam; Rehling, Peter; Deckers, Markus


    The cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain. The complex accepts electrons from cytochrome c and passes them onto molecular oxygen. This process contributes to energy capture in the form of a membrane potential across the inner membrane. The enzyme complex assembles in a stepwise process from the three mitochondria-encoded core subunits Cox1, Cox2 and Cox3, which associate with nuclear-encoded subunits and cofactors. In the yeast Saccharomyces cerevisiae, the cytochrome c oxidase associates with the bc1-complex into supercomplexes, allowing efficient energy transduction. Here we report on Cox26 as a protein found in respiratory chain supercomplexes containing cytochrome c oxidase. Our analyses reveal Cox26 as a novel stoichiometric structural subunit of the cytochrome c oxidase. A loss of Cox26 affects cytochrome c oxidase activity and respirasome organization.

  19. Cytochrome c binding to Apaf-1: The effects of dATP and ionic strength (United States)

    Purring-Koch, Cherie; McLendon, George


    In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. Here, we show through fluorescence polarization experiments that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c·Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along with the discovery that the exposed heme edge of cytochrome c is involved in the cytochrome c·Apaf-1 interaction, are confirmed through enhanced chemiluminescence visualization of native PAGE gels and through acrylamide fluorescence quenching experiments. We also report here that the cytochrome c·Apaf-1 interaction depends highly on ionic strength, indicating that there is a strong electrostatic interaction between the two proteins. PMID:11035811

  20. Comparative study in the effect of C-type natriuretic peptide on gastric motility in various animals

    Institute of Scientific and Technical Information of China (English)

    Hui-Shu Guo; Zheng Jin; Zheng-Yuan Jin; Zhe-Hao Li; Yi-Feng Cui; Zuo-Yu Wang; Wen-Xie Xu


    AIM: To investigate the effect of natriuretic peptides on gastric motility in various animals, and the effect of C-type natriuretic peptide (CNP) on spontaneous contraction of gastric smooth muscle in rat, guinea-pig and human in vitro was compared.METHODS: Spontaneous contraction of gastric smooth muscle was recorded by four channel physiograph.RESULTS: In the guinea-pig and rat gastric antral circular smooth muscle, CNP markedly decreased the amplitude of spontaneous contraction but it didn't affect the frequency,however, the contractile activity was completely inhibited by CNP in gastric antral longitudinal smooth muscle. In the human gastric antral circular and longitudinal smooth musie, CNP completely inhibited spontaneous contraction. In the circular smooth muscle of guinea-pig and rat gastric fundus,CNP obviously decreased the amplitude of spontaneous contraction but it didn't affect the frequency, however, the contractile activity was completely inhibited by CNP in smooth muscle of fundus longitudinal. In the circular and longitudinal smooth muscle of guinea-pig gastric body, CNP at first induced a relaxation and then an increase in amplitude of spontaneous contraction (rebound contraction), but the frequency was not changed. After the circular smooth muscle of gastric body was pretreated with atropine, an M receptor blocker, the rebound contraction was abolished; In circular and longitudinal smooth muscle of rat gastric body, CNP induced a transient and slight relaxation and successively followed by the recovery in amplitude of spontaneous contraction but it also didn't affect the frequency. After the smooth muscle was pretreated with atropine, the transient and slight relaxation was replaced by long term and complete inhibition; The percentage of CNP-induced inhibition was 76.77±6.21% (fundus), 67.21±5.32 % (body) and 58.23±6.21% (antral) in the gastric circular muscle, however, the inhibitory percentage was 100±0.00 % (fundus), 68.66±3.55 % (body

  1. Molecular Underpinnings of Fe(III Oxide Reduction by Shewanella oneidensis MR-1

    Directory of Open Access Journals (Sweden)

    Liang eShi


    Full Text Available In the absence of O2 and other electron acceptors, the Gram-negative bacterium Shewanella oneidensis MR-1 can use ferric [Fe(III] (oxy(hydroxide minerals as the terminal electron acceptors for anaerobic respiration. At circumneutral pH and in the absence of strong complexing ligands, Fe(III oxides are relatively insoluble and thus are external to the bacterial cells. S. oneidensis MR-1 has evolved the machinery (i.e., metal-reducing or Mtr pathway for transferring electrons across the entire cell envelope to the surface of extracellular Fe(III oxides. The protein components identified to date for the Mtr pathway include CymA, MtrA, MtrB, MtrC and OmcA. CymA is an inner-membrane tetraheme c-type cytochrome (c-Cyt that is proposed to oxidize the quinol in the inner-membrane and transfers the released electrons to redox proteins in the periplasm. Although the periplasmic proteins receiving electrons from CymA during Fe(III oxidation have not been identified, they are believed to relay the electrons to MtrA. A decaheme c-Cyt, MtrA is thought to be embedded in the trans outer-membrane and porin-like protein MtrB. Together, MtrAB deliver the electrons across the outer-membrane to the MtrC and OmcA on the outmost bacterial surface. Functioning as terminal reductases, the outer membrane and decaheme c-Cyts MtrC and OmcA can bind the surface of Fe(III oxides and transfer electrons directly to these minerals. To increase their reaction rates, MtrC and OmcA can use the flavins secreted by S. oneidensis MR-1 cells as diffusible co-factors for reduction of Fe(III oxides. MtrC and OmcA can also serve as the terminal reductases for soluble forms of Fe(III. Although our understanding of the Mtr pathway is still far from complete, it is the best characterized microbial pathway used for extracellular electron exchange. Characterizations of the Mtr pathway have made significant contributions to the molecular understanding of microbial reduction of Fe(III oxides.

  2. Virtual Screening and Prediction of Site of Metabolism for Cytochrome P450 1A2 Ligands

    DEFF Research Database (Denmark)

    Vasanthanathan, P.; Hritz, Jozef; Taboureau, Olivier


    With the availability of an increasing number of high resolution 3D structures of human cytochrome P450 enzymes, structure-based modeling tools are more readily used. In this study we explore the possibilities of using docking and scoring experiments on cytochrome P450 1A2. Three different...... and earlier classification data using machine learning methods. The possibilities and limitations of using structure-based drug design tools for cytochrome P450 1A2 come to light and are discussed....

  3. Structural insights into electron transfer in caa 3-type cytochrome oxidase


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


    Summary Paragraph Cytochrome c oxidase is a member of the heme copper oxidase superfamily (HCO) 1 . HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme’s function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome ...

  4. Reconstruction of Extracellular Respiratory Pathways for Iron(III Reduction in Shewanella oneidensis strain MR-1

    Directory of Open Access Journals (Sweden)

    Dan eCoursolle


    Full Text Available Shewanella oneidensis strain MR-1 is a facultative anaerobic bacterium capable of respiring a multitude of electron acceptors, many of which require the Mtr respiratory pathway. The core Mtr respiratory pathway includes a periplasmic c-type cytochrome (MtrA, an integral outer membrane β-barrel protein (MtrB and an outer membrane-anchored c-type cytochrome (MtrC. Together, these components facilitate transfer of electrons from the c-type cytochrome CymA in the cytoplasmic membrane to electron acceptors at and beyond the outer membrane. The genes encoding these core proteins have paralogs in the S. oneidensis genome (mtrB and mtrA each have four while mtrC has three and some of the paralogs of mtrC and mtrA are able to form functional Mtr complexes. We demonstrate that of the additional three mtrB paralogs found in the S. oneidensis genome, only MtrE can replace MtrB to form a functional respiratory pathway to soluble iron(III citrate. We also evaluate which mtrC / mtrA paralog pairs (a total of 12 combinations are able to form functional complexes with endogenous levels of mtrB paralog expression. Finally, we reconstruct all possible functional Mtr complexes and test them in a S. oneidensis mutant strain where all paralogs have been eliminated from the genome. We find that each combination tested with the exception of MtrA / MtrE / OmcA is able to reduce iron(III citrate at a level significantly above background. The results presented here have implications towards the evolution of anaerobic extracellular respiration in Shewanella and for future studies looking to increase the rates of substrate reduction for water treatment, bioremediation, or electricity production.

  5. Kinetic modelling of cytochrome c adsorption on SBA-15. (United States)

    Yokogawa, Yoshiyuki; Yamauchi, Rie; Saito, Akira; Yamato, Yuta; Toma, Takeshi


    The adsorption capacity of mesoporous silicate (MPS) materials as an adsorbent for protein adsorption from the aqueous phase and the mechanism of the adsorption processes by comparative analyses of the applicability of five kinetic transfer models, pseudo-first-order model, pseudo-second-order model, Elovich kinetic model, Bangham's equation model, and intraparticle diffusion model, were investigated. A mixture of tetraethyl orthosilicate (TEOS) and triblock copolymer as a template was stirred, hydrothermally treated to form the mesoporous SBA-15 structure, and heat-treated at 550°C to form the MPS material, SBA-15. The synthesized SBA-15 was immersed in a phosphate buffered saline (PBS) solution containing cytochrome c for 2, 48, and 120 hours at 4°C. The TEM observations of proteins on/in mesoporous SBA-15 revealed the protein behaviors. The holes of the MPS materials were observed to overlap those of the stained proteins for the first 2 hours of immersion. The stained proteins were observed between primary particles and partly inside the mesoporous channels in the MPS material when it had been immersed for 48 hours. For MPS when it had been immersed for 120 hours, stained proteins were observed in almost all meso-scale channels of MPS. The time profiles for adsorption of proteins can be described well by Bangham's equation model and the intraparticle diffusion model. The Bangham's equation model is based on the assumption that pore diffusion was the only rate controlling step during adsorption, whose contribution to the overall mechanism of cytochrome c adsorption on SBA-15 should not be neglected. The kinetic curves obtained from the experiment for cytochrome c adsorption on SBA-15 could show the three steps: the initial rapid increase of the adsorbed amount of cytochrome c, the second gradual increase, and the final equilibrium stage. These three adsorption steps can be interpreted well by the multi-linearity of the intraparticle diffusion model

  6. Cumene hydroperoxide effected hydroperoxidation by cytochrome P-450. (United States)

    Chen, C; Gurka, D P


    9-Methylfluorene was found to be oxygenated to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene by cytochrome P-450 in the presence of cumene hydroperoxide. Molecular oxygen is required and carbon monoxide is inhibitory. The reaction is inhibited by SKF-525A and metyrapone. Metyrapone and cumene hydroperoxide also retard the conversion of 9-hydroperoxy-9-methylfluorene to 9-hydroxy-9-methylfluorene. The reaction is different from hydroperoxide-supported oxygenation, since the cumene hydroperoxide appears to act as an effector of the enzyme rather than oxygen donor. It is suggested that substrates with stable radicals can be dioxygenated in this manner.

  7. Fast prediction of cytochrome P450 mediated drug metabolism

    DEFF Research Database (Denmark)

    Rydberg, Patrik Åke Anders; Poongavanam, Vasanthanathan; Oostenbrink, Chris


    Cytochrome P450 mediated metabolism of drugs is one of the major determinants of their kinetic profile, and prediction of this metabolism is therefore highly relevant during the drug discovery and development process. A new rule-based method, based on results from density functional theory...... % of substrates for a set of 20 substrates. In combination with docking, it can predict isoform-specific metabolism, and we apply this on CYP1A2 with very good results on 81 substrates, for which we find a major metabolite ranked in the top three for 90 % of the substrates (100 % in the training set and 87...

  8. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Ling; Mills, Denise A.; Buhrow, Leann; Hiser, Carrie; Ferguson-Miller, Shelagh (Michigan)


    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

  9. Effects of methotrexate on rat P-450 cytochrome mono-oxygenases; Action du methotrexate sur les monooxygenases a cytochromes P-450 chez le rat

    Energy Technology Data Exchange (ETDEWEB)

    Guitton, J.; Guilluy, R.; Brazier, J.L. [Faculte de Pharmacie, 69 - Lyon (France); Souillet, G. [Hopital Debrousse, 69 - Lyon (France); Riviere, J.L. [INRA, 69 - Marcy l`Etoile (France); Gerard, F. [Institut Pasteur, 69 - Lyon (France)


    Methotrexate, an anti-cancerous agent, acts as an anti-metabolite of the nucleic acids which synthesis is then inhibited. Using aminopyrine breath test after methotrexate processing, the effects of the molecule on activities of the hepatocyte P-450 cytochrome mono-oxygenases, are studied. Breath micro-tests with carbon 13-labelled aminopyrine have been carried out to observe the metabolism evolution. Micro-test results have been compared to microsomal enzymatic activities for various substrates, and also to P-450 cytochrome ratio. Results show that methotrexate induces a reduction in the P-450 cytochrome ratio, and thus reduce the hepatic biotransformation process. 1 fig., 30 refs.

  10. Resolution of cor pulmonale after medical management in a patient with cblC-type methylmalonic aciduria and homocystinuria: a case report. (United States)

    Profitlich, Laurie; Kirmse, Brian; Wasserstein, Melissa P; Diaz, George; Srivastava, Shubhika


    We describe a 3-year-old Hispanic male with cblC-type methylmalonic aciduria and homocystinuria who presented to the emergency department with progressive tachypnea, vomiting, and edema secondary to pulmonary embolism and cor pulmonale. With aggressive medical management, there was complete resolution of right heart failure and pulmonary hypertension after 3 months. Pulmonary embolism is rare in the pediatric population. Children with cblC-type methylmalonic aciduria and homocystinuria may be at increased risk for thrombus formation and pulmonary embolism due to chronic hyperhomocystinemia, a risk factor for thrombus formation in the adult population. Aspirin therapy may be indicated in children with inborn errors of metabolism that predispose to hyperhomocystinemia.

  11. A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination. (United States)

    Mu, Changkao; Song, Xiaoyan; Zhao, Jianmin; Wang, Lingling; Qiu, Limei; Zhang, Huan; Zhou, Zhi; Wang, Mengqiang; Song, Linsheng; Wang, Chunlin


    C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.

  12. [Cytochrome P450 enzymes and their role in drug interactions]. (United States)

    Papp-Jámbor, C; Jaschinski, U; Forst, H


    One of the factors that can alter the response to drugs is the concurrent administration of other drugs. There are several mechanisms by which drugs may interact, but most can be categorised as pharmacokinetic (absorption, distribution, metabolism, excretion), pharmacodynamic, or combined toxicity. Knowledge of the mechanism by which a given drug interaction occurs is often clinically useful and may help to avoid serious adverse events and perioperative morbidity. Although every tissue has some ability to metabolise drugs, the liver is the principal organ of drug metabolism and at the subcellular level the cytochrome P450 enzyme system is the main source of drug interaction. This article reviews the basic principles of drug metabolism and the role of cytochrome P450 in this scenario. Drugs frequently used in anaesthesia and critical care medicine such as benzodiazepines, opioid analgesics, antihypertensive and antiarrhythmic agents, antibiotics and antifungal drugs, antiemetics, histamine-receptor-antagonists, theopylline and paracetamol will be considered. The development of methods and tools which are practical and also economic, are of utmost importance since drug interaction is predictable if the metabolic pathway and the activity (genetic polymorphism) of the enzyme is known.

  13. Cytochrome P450 structure, function and clinical significance: A review. (United States)

    Palrasu, Manikandan; Nagini, Siddavaram


    The cytochrome P450 (CYP) enzymes are membrane-bound hemoproteins that play a pivotal role in the detoxification of xenobiotics, cellular metabolism and homeostasis. Induction or inhibition of CYP enzymes is a major mechanism that underlies drug-drug interactions. CYP enzymes can be transcriptionally activated by various xenobiotics and endogenous substrates through receptor-dependent mechanisms. CYP enzyme inhibition is a principal mechanism for metabolism-based drug-drug interactions. Many chemotherapeutic drugs can cause drug interactions due to their ability to either inhibit or induce the CYP enzyme system. Predictions based on in silico analyses followed by validation have identified several microRNAs that regulate CYPs. Genetic polymorphisms and epigenetic changes in CYP genes may be responsible for inter-individual and inter-ethnic variations in disease susceptibility and the therapeutic efficacy of drugs. Knowledge about the substrates, inducers, inhibitors of CYP isoforms, and the polymorphisms of CYP enzymes may be used as an aid by clinicians to determine therapeutic strategy, and treatment doses for drugs that are metabolized by CYP gene products. The present review is a comprehensive compilation of cytochrome P450 structure, function, pharmacogenetics, and pharmacoepigenetics and clinical significance.

  14. Cytochromes P450 of insects: the tip of the iceberg. (United States)

    Scott, J G; Wen, Z


    The cytochrome P450-dependent monooxygenases are an extremely important metabolic system involved in the metabolism of endogenous compounds and xenobiotics. Collectively, P450 monooxygenases can metabolize numerous substrates and carry out multiple oxidative reactions. The large number of substrates metabolized is due to the plethora of P450 isoforms and to the broad substrate specificity of some isoforms. Monooxygenases of insects have several functional roles, including growth, development, feeding and protection against xenobiotics, including resistance to pesticides and tolerance to plant toxins. This review begins with background information about P450s and their evolution, followed by a discussion of the extraordinary diversity of insect P450s. Given the enormous interest in studying individual P450s, we then provide a synopsis of the different methods that have been used in their isolation and the substrates that are known to be metabolized. We conclude by summarizing the lessons we have learned from the study of individual insect P450s, including their roles in insecticide resistance, plant-insect interactions and insect physiology. However, these studies are just the 'tip of the iceberg'. Our knowledge continues to expand at a rapid pace, suggesting that the next decade will outpace the last in terms of improving our understanding of the cytochromes P450 of insects.

  15. Mutations induced by dacarbazine activated with cytochrome P-450. (United States)

    Mudipalli, A; Nadadur, S S; Maccubbin, A E; Gurtoo, H L


    The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to alkylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system. In the present study, we used an in vitro shuttle vector assay to study the base and sequence specificity of mutagenesis by DTIC. The shuttle vector plasmid pSP189 was treated with DTIC (1-2.5 mM) in vitro in a reconstituted cytochrome P-450 system at 37 degrees C for either 30 or 60 min. SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplification in indicator bacteria. Mutagenesis of DTIC in this system was dependent upon the presence of the cytochrome P-450 reconstituted system and NADPH. Mutations induced by DTIC included single base substitutions (35%), single base deletions (30.5%), single base insertions (19.4%) and large deletions (13.8%). Among the substitutions, transversions and transitions were in the ratio of 1:0.7. Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots.

  16. Theoretical study of the cytochrome P450 mediated metabolism of phosphorodithioate pesticides

    DEFF Research Database (Denmark)

    Rydberg, Patrik


    The toxicity of phosphorodithioate pesticides is due to the formation of the active oxane product through desulfurization by cytochrome P450 enzymes, both in humans and insects. During this desulfurization, inhibition of cytochrome P450 and a loss of heme has been observed. Here, we study...

  17. Acute hypoxia and cytochrome P450-mediated hepatic drug metabolism in humans

    DEFF Research Database (Denmark)

    Jürgens, Gesche; Christensen, Hanne Rolighed; Brøsen, Kim;


    Our objective was to investigate the effect of acute hypoxia on the activity of hepatic cytochrome P450 (CYP) enzymes.......Our objective was to investigate the effect of acute hypoxia on the activity of hepatic cytochrome P450 (CYP) enzymes....

  18. On the role of phospholipids in the cytochrome P450 enzyme system.

    NARCIS (Netherlands)

    Balvers, W.G.


    The cytochrome P450 enzyme system is involved in the metabolism and elimination of an almost unlimited number of endogenous and exogenous substrates. Biotransformation by cytochromes P450 plays a role in the conversion xenobiotics into more hydrophilic products. Generally, this process of biotransfo

  19. Electrochemical determination of hydrogen peroxide using Rhodobacter capsulatus cytochrome c peroxidase at a gold electrode

    NARCIS (Netherlands)

    De Wael, K.; Buschop, H.; Heering, H.A.; De Smet, L.; Van Beeumen, J.; Devreese, B.; Adriaens, A.


    We describe the redox behaviour of horse heart cytochrome c (HHC) and Rhodobacter capsulatus cytochrome c peroxidase (RcCCP) at a gold electrode modified with 4,4′-bipyridyl. RcCCP shows no additional oxidation or reduction peaks compared to the electrochemistry of only HHC, which indicates that it

  20. Regulation of expression of the Aspergillus niger benzoate para-hydroxylase cytochrome P450 system

    NARCIS (Netherlands)

    Brink, J.M. van den; Punt, P.J.; Gorcom, R.F.M. van; Hondel, C.A.M.J.J. van den


    Cytochrome P450 enzyme systems are found throughout nature and are involved in many different, often complex, bioconversions. In the endoplasmic reticulum of the filamentous fungus Aspergillus niger a cytochrome P450 enzyme system is present that is capable of the para-hydroxylation of benzoate. The

  1. Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein

    NARCIS (Netherlands)

    Bengtsson, J; Tjalsma, H; Rivolta, C; Hederstedt, L


    The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochrome caa(3), which is a cytochrome c oxidase. Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a

  2. Characterization and biosynthesis of cytochrome b5 in rat liver microsomes (United States)

    Sargent, J. R.; Vadlamudi, B. P.


    1. Cytochrome b5 is released from rat liver microsomes by both proteolytic enzymes and by treatments that disrupt phospholipids. Cytochrome P-420 is only released to a marked extent by treatments that disrupt phospholipids. 2. Cytochrome b5 was isolated in a pure state from both the rough and smooth fractions of rat liver microsomes after treatment with trypsin, and was shown to contain two cytochrome components with identical spectral properties. 3. Amino acid analyses of the two components are presented, together with peptide `fingerprint' patterns of tryptic digests of the two components. 4. Studies based on the direct isolation of cytochrome b5 after administration of a single dose of radioactive amino acid to rats demonstrate that the cytochrome is synthesized initially in the rough fraction of microsomes and only subsequently appears in the smooth fraction. 5. Isolated rat liver microsomes are capable of incorporating radioactive amino acids into cytochrome b5 under standard conditions. 6. Under these conditions the amino acid is incorporated into peptide linkage in the cytochrome. PMID:16742610

  3. Bioconversion of Mono- and Sesquiterpenoids by Recombinant Human Cytochrome P450 Monooxygenases

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Fichera, Mario A.; Malz, Frank; Ebbelaar, Monique; Bos, Rein; Woerdenbag, Herman J.; Quax, Wim J.; Kayser, Oliver


    Cytochrome P450 monooxygenases play an important role in the biosynthesis and metabolism of terpenoids. We explored the potential of recombinant human liver cytochrome P450 monooxygenases CYP1A2, CYP2C9, and CYP3A4, heterologously expressed in Escherichia coli, to convert mono- and sesquiterpenoids

  4. Construction of a 3D model of cytochrome P450 2B4. (United States)

    Chang, Y T; Stiffelman, O B; Vakser, I A; Loew, G H; Bridges, A; Waskell, L


    A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.

  5. In situ Raman study of redox state changes of mitochondrial cytochromes in a perfused rat heart

    DEFF Research Database (Denmark)

    Brazhe, Nadezda; Treiman, Marek; Faricelli, Barbara;


    surface of the isolated heart at normal and hypoxic conditions. Raman spectra of the heart under normal pO2 demonstrate unique peaks attributable to reduced c-and b-type cytochromes and oxymyoglobin (oMb). The cytochrome peaks decreased in intensity upon FCCP treatment, as predicted from uncoupling...

  6. Mitofilin regulates cytochrome c release during apoptosis by controlling mitochondrial cristae remodeling

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Rui-feng; Zhao, Guo-wei; Liang, Shu-ting; Zhang, Yuan; Sun, Li-hong [National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), 5 Dong Dan San Tiao, Beijing 100005 (China); Chen, Hou-zao, E-mail: [National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), 5 Dong Dan San Tiao, Beijing 100005 (China); Liu, De-pei, E-mail: [National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), 5 Dong Dan San Tiao, Beijing 100005 (China)


    Highlights: Black-Right-Pointing-Pointer Mitofilin deficiency caused disruption of the cristae structures in HeLa cells. Black-Right-Pointing-Pointer Mitofilin deficiency reduced cell proliferation and increased cell sensitivity to apoptotic stimuli. Black-Right-Pointing-Pointer Mitofilin deficiency accelerated the release of cytochrome c from mitochondria. Black-Right-Pointing-Pointer Mitofilin deficiency accelerated STS-induced intrinsic apoptotic pathway without interfering with the activation of Bax. -- Abstract: Mitochondria amplify caspase-dependent apoptosis by releasing proapoptotic proteins, especially cytochrome c. This process is accompanied by mitochondrial cristae remodeling. Our studies demonstrated that mitofilin, a mitochondrial inner membrane protein, acted as a cristae controller to regulate cytochrome c release during apoptosis. Knockdown of mitofilin in HeLa cells with RNAi led to fragmentation of the mitochondrial network and disorganization of the cristae. Mitofilin-deficient cells showed cytochrome c redistribution between mitochondrial cristae and the intermembrane space (IMS) upon intrinsic apoptotic stimuli. In vitro cytochrome c release experiments further confirmed that, compared with the control group, tBid treatment led to an increase in cytochrome c release from mitofilin-deficient mitochondria. Furthermore, the cells with mitofilin knockdown were more prone to apoptosis by accelerating cytochrome c release upon the intrinsic apoptotic stimuli than controls. Moreover, mitofilin deficiency did not interfere with the activation of proapoptotic member Bax upon intrinsic apoptotic stimuli. Thus, mitofilin distinctly functions in cristae remodeling and controls cytochrome c release during apoptosis.

  7. Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Marres, C A; Slater, Conor


    1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reo...

  8. Respiratory system of Gluconacetobacter diazotrophicus PAL5. Evidence for a cyanide-sensitive cytochrome bb and cyanide-resistant cytochrome ba quinol oxidases. (United States)

    González, B; Martínez, S; Chávez, J L; Lee, S; Castro, N A; Domínguez, M A; Gómez, S; Contreras, M L; Kennedy, C; Escamilla, J E


    In highly aerobic environments, Gluconacetobacter diazotrophicus uses a respiratory protection mechanism to preserve nitrogenase activity from deleterious oxygen. Here, the respiratory system was examined in order to ascertain the nature of the respiratory components, mainly of the cyanide sensitive and resistant pathways. The membranes of G. diazotrophicus contain Q(10), Q(9) and PQQ in a 13:1:6.6 molar ratios. UV(360 nm) photoinactivation indicated that ubiquinone is the electron acceptor for the dehydrogenases of the outer and inner faces of the membrane. Strong inhibition by rotenone and capsaicin and resistance to flavone indicated that NADH-quinone oxidoreductase is a NDH-1 type enzyme. KCN-titration revealed the presence of at least two terminal oxidases that were highly sensitive and resistant to the inhibitor. Tetrachorohydroquinol was preferentially oxidized by the KCN-sensitive oxidase. Neither the quinoprotein alcohol dehydrogenase nor its associated cytochromes c were instrumental components of the cyanide resistant pathway. CO-difference spectrum and photodissociation of heme-CO compounds suggested the presence of cytochromes b-CO and a(1)-CO adducts. Air-oxidation of cytochrome b (432 nm) was arrested by concentrations of KCN lower than 25 microM while cytochrome a(1) (442 nm) was not affected. A KCN-sensitive (I(50)=5 microM) cytochrome bb and a KCN-resistant (I(50)=450 microM) cytochrome ba quinol oxidases were separated by ion exchange chromatography.

  9. Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode. (United States)

    Shimada, Satoru; Shinzawa-Itoh, Kyoko; Baba, Junpei; Aoe, Shimpei; Shimada, Atsuhiro; Yamashita, Eiki; Kang, Jiyoung; Tateno, Masaru; Yoshikawa, Shinya; Tsukihara, Tomitake


    Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, we determined the structure of the mammalian Cyt.c-CcO complex at 2.0-Å resolution and identified an electron transfer pathway from Cyt.c to CcO. The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between side chains within a small contact surface area. Between the two proteins are three water layers with a long inter-molecular span, one of which lies between the other two layers without significant direct interaction with either protein. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein-protein interaction at the docking interface represent the first known example of a new class of protein-protein interaction, which we term "soft and specific". This interaction is likely to contribute to the rapid association/dissociation of the Cyt.c-CcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

  10. Retinal ganglion cells of high cytochrome oxidase activity in the rat

    Institute of Scientific and Technical Information of China (English)



    Retinal ganglion cells in the rat were studied using the heavy metal intensified cytochrome oxidase and horseradish peroxidase histochemical methods.The results show that a population of large retinal ganglion cells was consistently observed with the cytochrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birth.These cytochrome oxidase rich ganglion cells appeared to have large somata,3-6 primary dendrites and extensive dendritic arbors,and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).However,the morphological details of some of the cells revealed by the cytochrome oxidase staining method are frequently better than those shown by the HRP histochemical method.These results suggest that the mitochondrial enzyme cytochrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal genglion cells with high metabolic rate in the rat.

  11. Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase. (United States)

    Faletto, M B; Koser, P L; Battula, N; Townsend, G K; Maccubbin, A E; Gelboin, H V; Gurtoo, H L


    Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

  12. Cytochrome oxidase as an indicator of ice storage and frozen storage

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming


    The potential of cytochrome oxidase as an indicator of ice storage and frozen storage of fish was investigated. Optimal assay conditions for cytochrome oxidase in a crude homogenate from cod muscle were studied. Maximal cytochrome oxidase activity was found at pH 6.5-7.5 and an assay temperature...... in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used...... as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degreesC....

  13. Crystallization and micro FT-IR spectroscopy investigation of cytochrome bc1 complex

    Institute of Scientific and Technical Information of China (English)

    岳文海; 何季平; 谢荣; 徐建兴; 朱克莉; 翁诗甫


    A simple method to obtain large red crystals of cytochrome bc1 complex from beef heart mitochondria has been developed. These crystals are very stable. Their shapes are retained for a long time in tip-sealed Pasteur pipets placed in a refrigerator. The structure of crystalline cytochrome bc1 complex by micro FT-IR spectroscopy has been investigated. Based on the IR spectra of cytochrome c, the empirical assignments of the major infrared frequencies of cytochrome bc1 complex are given. Infrared frequencies and relative intensities of variable orientation and section of crystal are significantly different. These imply that infrared spectral characterization of the membrane protein crystallization is associated with the variable symmetries and orientations of the structure. Experimental results show that phospholipid exists in the crystal of cytochrome bc1 complex. The membrane protein is probably spanned on the mitochondrial membrane and buried in phospholipid bilayer in an asymmetric manner.

  14. Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Jung Hoon [Cheongju Univ., Cheongju (Korea, Republic of)


    Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD.

  15. Sequence Comparison of Partial Cytochrome b Genes of Two Coilia species

    Institute of Scientific and Technical Information of China (English)

    LIU Jinxian; GAO Tianxiang; WANG Yujiang; ZHANG Yaping


    Sequence variation of partial cytochrome b genes between two Coilia species, C. ectenes and C. mystus, was investigated. Of the 402 nucleotides, twenty-seven (6.72%) are polymorphic and all are synonymous substitutions. At the third positions of genetic condon of cytochrome b gene, the two species show an extreme anti-G bias (< 4 % ) and a pronounced bias towards A and C (>68%). There is no amino acid sequence divergence between the partial cytochrome b genes of the two species, indicating a close genetic relationship between them. The k-2p genetic distance of partial cytochrome b segment of the two species is 0.072, suggesting that the species were separated 3.6 Ma ago, in the middle Pliocene. Our result reveals that the cytochrome b gene is an appropriate marker for studies of population genetic structures and phylogeographic patterns of the two species.

  16. Rcf1 mediates cytochrome oxidase assembly and respirasome formation, revealing heterogeneity of the enzyme complex. (United States)

    Vukotic, Milena; Oeljeklaus, Silke; Wiese, Sebastian; Vögtle, F Nora; Meisinger, Chris; Meyer, Helmut E; Zieseniss, Anke; Katschinski, Doerthe M; Jans, Daniel C; Jakobs, Stefan; Warscheid, Bettina; Rehling, Peter; Deckers, Markus


    The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S. cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.

  17. On the role of cytochrome c8 in photosynthetic electron transfer of the purple non-sulfur bacterium Rhodoferax fermentans

    DEFF Research Database (Denmark)

    Hochkoeppler, Alejandro; Ciurli, Stefano; Kofod, Pauli


    of +285 mV. Partial analysis of the N-terminus amino-acid sequence shows a high similarity with cytochromes of c8 type (formerly called Pseudomonas cytochrome c-551 type). Time-resolved spectrophotometric studies show that this cytochrome c8 reduces the tetraheme subunit of the photosynthetic reaction...

  18. Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy. (United States)

    Horvath, Rita; Kemp, John P; Tuppen, Helen A L; Hudson, Gavin; Oldfors, Anders; Marie, Suely K N; Moslemi, Ali-Reza; Servidei, Serenella; Holme, Elisabeth; Shanske, Sara; Kollberg, Gittan; Jayakar, Parul; Pyle, Angela; Marks, Harold M; Holinski-Feder, Elke; Scavina, Mena; Walter, Maggie C; Coku, Jorida; Günther-Scholz, Andrea; Smith, Paul M; McFarland, Robert; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N; Hirano, Michio; Lochmüller, Hanns; Taylor, Robert W; Chinnery, Patrick F; Tulinius, Mar; DiMauro, Salvatore


    Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as 'benign cytochrome c oxidase deficiency myopathy' is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

  19. Cyanide - Mechanism of Prophylaxis and Effect on Cytochrome Oxidase. (United States)


    152:1074-1077, 1958. Cooperstein, S. J. and Lazaro , A.: A microspectrophotometric method for the determination of cytochrome oxidase. J. biol. Chem...5 mg/kg); A--A CPZ + NaNO (100 mg/kg) + KCN (10 mg/kg); 0 -- 0 CPZ + Na2S203 (1 gm/kg) + KCN (15 mg/kg); A---A CPZ + NaNO2 (100 mg/)Cg) + Na2S203...chlorpromazine (10 mg/kg), NaqO 2 (100 w.g/kg) and A Na S 03 (1gm/kg). G - C Saline control; A - A KCN; S - CPZ + Na 2S 20 3+ KCN; 0 - 0 CPZ + NaNO 2 + KCN

  20. Impacts of diversification of cytochrome P450 on plant metabolism. (United States)

    Mizutani, Masaharu


    Cytochrome P450 monooxygenases (P450s) catalyze a wide variety of monooxygenation reactions in primary and secondary metabolism in plants. The share of P450 genes in each plant genome is estimated to be up to 1%. This implies that the diversification of P450 has made a significant contribution to the ability to acquire the emergence of new metabolic pathways during land plant evolution. The P450 families conserved universally in land plants contribute to their chemical defense mechanisms. Several P450s are involved in the biosynthesis and catabolism of plant hormones. Species-specific P450 families are essential for the biosynthetic pathways of phytochemicals such as terpenoids and alkaloids. Genome wide analysis of the gene clusters including P450 genes will provide a clue to defining the metabolic roles of orphan P450s. Metabolic engineering with plant P450s is an important technology for large-scale production of valuable phytochemicals such as medicines.

  1. Personalized Cancer Therapy Considering Cytochrome P450 Variability. (United States)

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


    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.

  2. [Cytochrome P450 activity and its alteration in different diseases]. (United States)

    Orellana, Myriam; Guajardo, Viviana


    Cytochrome P450 (CYP) enzymes participate in the metabolism of a variety of naturally occurring and foreign compounds by reactions requiring NADPH and O2. The diversity of reactions catalyzed and its extensive substrate specificity render CYP enzymes as one of the most versatile known catalysts. Individual members of the CYP superfamily are expressed in almost every cell type in the body. As compared to hepatic enzymes, the regulation of human extrahepatic CYPs has not been so well studied. In general, the levels of some hepatic CYP enzymes are depressed by diseases, causing potential and documented impairment of drug clearence and clinical drug toxicity. However, modulation of CYPs is enzyme selective and this selectivity differs in different diseases. This article reviews some basic concepts about CYP and its regulation in some disease states such as hypertension, diabetes, obesity and hepatic, infectious and inflammatory diseases.

  3. Highly selective ligand binding by Methylophilus methylotrophus cytochrome c''. (United States)

    Quintas, Pedro O; Catarino, Teresa; Todorovic, Smilja; Turner, David L


    Cytochrome c'' (cyt c'') from Methylophilus methylotrophus is unusual insofar as the heme has two axial histidine ligands in the oxidized form but one is detached when the protein is reduced. Despite cyt c'' having an axial site available for binding small ligands, we show here that only NO binds readily to the ferrous cyt c''. Binding of CO, as well as CN(-), on the other hand requires considerable structural reorganization, or reduction of the disulfide bridge close to the heme. Standard free energies for the binding of NO and CO reveal high selectivity of the ferrous cyt c'' for NO, indicating its putative physiological role. In this work, we characterize in detail the kinetics of NO binding and the structural features of the Fe(2+)-NO adduct by stopped-flow and resonance Raman spectroscopy, respectively.

  4. [Cytochrome P450 enzymes and microbial drug development - A review]. (United States)

    Li, Zhong; Zhang, Wei; Li, Shengying


    Cytochrome P450 enzymes broadly exist in animals, plants and microorganisms. This superfamily of monooxygenases holds the greatest diversity of substrate structures and catalytic reaction types among all enzymes. P450 enzymes play important roles in natural product biosynthesis. In particular, P450 enzymes are capable of catalyzing the regio- and stereospecific oxidation of non-activated C-H bonds in complex organic compounds under mild conditions, which overrides many chemical catalysts. This advantage thus warrants their great potential in microbial drug development. In this review, we introduce a variety of P450 enzymes involved in natural product biosynthesis; provide a brief overview on protein engineering, biotransformation and practical application of P450 enzymes; and discuss the limits, challenges and prospects of industrial application of P450 enzymes.

  5. Cytochrome c peroxidase activity of heme bound amyloid β peptides. (United States)

    Seal, Manas; Ghosh, Chandradeep; Basu, Olivia; Dey, Somdatta Ghosh


    Heme bound amyloid β (Aβ) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aβ, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aβ. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aβ, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aβ complex behaves as CCP.

  6. Structural Models for Cytochrome P450�Mediated Catalysis

    Directory of Open Access Journals (Sweden)

    David F.V. Lewis


    Full Text Available This review focuses on the structural models for cytochrome P450 that are improving our knowledge and understanding of the P450 catalytic cycle, and the way in which substrates bind to the enzyme leading to catalytic conversion and subsequent formation of mono-oxygenated metabolites. Various stages in the P450 reaction cycle have now been investigated using X-ray crystallography and electronic structure calculations, whereas homology modelling of mammalian P450s is currently revealing important aspects of pharmaceutical and other xenobiotic metabolism mediated by P450 involvement. These features are explored in the current review on P450-based catalysis, which emphasises the importance of structural modelling to our understanding of this enzyme's function. In addition, the results of various QSAR analyses on series of chemicals, which are metabolised via P450 enzymes, are presented such that the importance of electronic and other structural factors in explaining variations in rates of metabolism can be appreciated.

  7. Cytochrome P450 as dimerization catalyst in diketopiperazine alkaloid biosynthesis. (United States)

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


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

  8. Cytochrome b nucleotide sequence variation among the Atlantic Alcidae. (United States)

    Friesen, V L; Montevecchi, W A; Davidson, W S


    Analysis of cytochrome b nucleotide sequences of the six extant species of Atlantic alcids and a gull revealed an excess of adenines and cytosines and a deficit of guanines at silent sites on the coding strand. Phylogenetic analyses grouped the sequences of the common (Uria aalge) and Brünnich's (U. lomvia) guillemots, followed by the razorbill (Alca torda) and little auk (Alle alle). The black guillemot (Cepphus grylle) sequence formed a sister taxon, and the puffin (Fratercula arctica) fell outside the other alcids. Phylogenetic comparisons of substitutions indicated that mutabilities of bases did not differ, but that C was much more likely to be incorporated than was G. Imbalances in base composition appear to result from a strand bias in replication errors, which may result from selection on secondary RNA structure and/or the energetics of codon-anticodon interactions.

  9. Active site dynamics of toluene hydroxylation by cytochrome P-450

    Energy Technology Data Exchange (ETDEWEB)

    Hanzlik, R.P.; Kahhiing John Ling (Univ. of Kansas, Lawrence (United States))


    Rat liver cytochrome P-450 hydroxylates toluene to benzyl alcohol plus o-, m-, and p-cresol. Deuterated toluenes were incubated under saturating conditions with liver microsomes from phenobarbital-pretreated rats, and product yields and ratios were measured. Stepwise deuteration of the methyl leads to stepwise decreases in the alcohol/cresol ratio without changing the cresol isomer ratios. Extensive deuterium retention in the benzyl alcohols from PhCH{sub 2}D and PhCHD{sub 2} suggests there is a large intrinsic isotope effect for benzylic hydroxylation. After replacement of the third benzylic H by D, the drop in the alcohol/cresol ratio was particularly acute, suggsting that metabolic switching from D to H within the methyl group was easier than switching from the methyl to the ring. Comparison of the alcohol/cresol ratio for PhCH{sub 3} vs PhCD{sub 3} indicated a net isotope effect of 6.9 for benzylic hydroxylation. From product yield data for PhCH{sub 3} and PhCD{sub 3}, {sup D}V for benzyl alcohol formation is only 1.92, whereas {sup D}V for total product formation is 0.67 (i.e., inverse). From competitive incubations of PhCH{sub 3}/PhCD{sub 3} mixtures {sup D}(V/K) isotope effects on benzyl alcohol formation and total product formation (3.6 and 1.23, respectively) are greatly reduced, implying strong commitment to catalysis. In contrast, {sup D}(V/K) for the alcohol/cresol ratio is 6.3, indicating that the majority of the intrinsic isotope effect is expressed through metabolic switching. Overall, these data are consistent with reversible formation of a complex between toluene and the active oxygen form of cytochrome P-450, which rearranges internally and reacts to form products faster than it dissociates back to release substrate.

  10. Prognostic relevance of cytochrome C oxidase in primary glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Corinne E Griguer

    Full Text Available Patients with primary glioblastoma multiforme (GBM have one of the lowest overall survival rates among cancer patients, and reliable biomarkers are necessary to predict patient outcome. Cytochrome c oxidase (CcO promotes the switch from glycolytic to OXPHOS metabolism, and increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure. Thus, we investigated the relationship between tumor CcO activity and the survival of patients diagnosed with primary GBM. A total of 84 patients with grade IV glioma were evaluated in this retrospective cohort study. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test, and univariate and multivariate analyses were performed with the Cox regression model. Mitochondrial CcO activity was determined by spectrophotometrically measuring the oxidation of cytochrome c. High CcO activity was detected in a subset of glioma tumors (∼30%, and was an independent prognostic factor for shorter progression-free survival and overall survival [P = 0.0087 by the log-rank test, hazard ratio = 3.57 for progression-free survival; P<0.001 by the log-rank test, hazard ratio = 10.75 for overall survival]. The median survival time for patients with low tumor CcO activity was 14.3 months, compared with 6.3 months for patients with high tumor CcO activity. High CcO activity occurs in a significant subset of high-grade glioma patients and is an independent predictor of poor outcome. Thus, CcO activity may serve as a useful molecular marker for the categorization and targeted therapy of GBMs.

  11. Mode of Antifungal Drugs Interaction with Cytochrome P- 450

    Directory of Open Access Journals (Sweden)

    M- Mahmodian


    Full Text Available Computer was used to identify the interactions of substrates and antifungal drugs with the enzyme, Cytochrome P-450; and then Molplot.bas computer program was applied to get three dimensional figures of 5-hydroxy camphor.oxidation products of camphor analogues, and antifungal drugs.Cartesian characteristics of atoms building molecules, are taken from Buildz. for program, which can calculate X,Y,Z coordinates of atoms by Zmatrix data. The other program which can calculate X,Y,Z coordinates, using fractional characteristics, is the Coord, for program that, gives our cartesian characteristics of the atoms of molecule, then by using these data, we obtain three dimensional figures and distance between active atoms in compounds under consideration. Results show that distance between two oxygen atoms in 5-exo-hydroxy- camphor and the other compounds obtained from oxidation of camphor analogues, with the distance of two oxygen atoms in antifungal compounds under discussion are equal. Therefore, we can conclude that, the antifungal molecule also interacts with enzyme's active site, by its own sites, in a similar manner to the 5-hydroxy camphor molecule, which is:"n1. Nitrogen atom (N of Imidazole and Triazole ring in antifungal molecule with Iron atom in heam molecule belonging to Cytochrome P-450 enzyme, are coordinated."n2. The other atoms such as : 0,S or N in structure of the antifungal drug are coordinated with hydrogen atom of hydroxyl group belong ing to Tyr-96 in the structure of enzyme, forming hydrogen bonding.

  12. Protonation State-Dependent Communication in Cytochrome c Oxidase. (United States)

    Helabad, Mahdi Bagherpoor; Ghane, Tahereh; Reidelbach, Marco; Woelke, Anna Lena; Knapp, Ernst Walter; Imhof, Petra


    Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both redox enzyme and proton pump, proton transfer out of either of the channels toward the BNC or into the protein toward a proton loading site, and ultimately through the membrane, must be highly regulated. The O→E intermediate of cytochrome c oxidase is the first redox state in its catalytic cycle, where proton transfer through the K-channel, from K362 to Y288 at the BNC, is important. Molecular dynamics simulations of this intermediate with 16 different combinations of protonation states of key residues in the D- and K-channel show the mutual impact of the two proton-conducting channels to be protonation state-dependent. Strength as well as means of communication, correlations in positions, or connections along the hydrogen-bonded network, change with the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286, which would in principle allow proton transfer to the BNC, but no proton pumping until a proton has reached E286.

  13. The skeletal proteome of the brittle star Ophiothrix spiculata identifies C-type lectins and other proteins conserved in echinoderm skeleton formation

    Directory of Open Access Journals (Sweden)

    Brian T. Livingston


    Full Text Available Determining the identity and functional role of proteins involved in biomineralization and the formation of skeletons is critical to our understanding of the process. Proteomics has allowed rapid characterization of the proteins occluded within mineralized tissue, but the large numbers of proteins detected makes it difficult to assign the relative importance of each protein. We have taken a comparative approach, examining the skeletal proteome of different species of echinoderms in order to identify the proteins that are conserved and likely to be important. Our previous study comparing the skeletal proteome of the brittle star Ophiocoma wendtii to the published proteomes of the sea urchin Strongylocentrotus purpuratus revealed some conservation of proteins, but indicated that the C-type lectin domain-containing spicule matrix proteins abundant in the sea urchin skeletal proteome were not conserved in the brittle star. Here we examine the skeletal proteome of a different species of brittle star, Ophiothrix spiculata. We have isolated the proteins from the skeleton of O. spiculata and performed LC/MS/MS to identify peptides present. Comparison to transcriptome and genome databases revealed the proteins present in the O. spiculata proteome. Despite being diverged for several million years, the two brittle stars have very similar proteins in their skeletons. Included is a fibrinogen C-like lectin and several C-type lectins proteins, which we describe in detail. The unusual number of C-type lectins found in the S. purpuatus skeleton and the repetitive regions seen in those spicule matrix proteins are not present in O. spiculata.

  14. The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Lorentsen, R H; Jacobsen, C


    Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon...... resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site...

  15. DAC is involved in the accumulation of the cytochrome b6/f complex in Arabidopsis. (United States)

    Xiao, Jianwei; Li, Jing; Ouyang, Min; Yun, Tao; He, Baoye; Ji, Daili; Ma, Jinfang; Chi, Wei; Lu, Congming; Zhang, Lixin


    The biogenesis and assembly of photosynthetic multisubunit protein complexes is assisted by a series of nucleus-encoded auxiliary protein factors. In this study, we characterize the dac mutant of Arabidopsis (Arabidopsis thaliana), which shows a severe defect in the accumulation of the cytochrome b(6)/f complex, and provide evidence suggesting that the efficiency of cytochrome b(6)/f complex assembly is affected in the mutant. DAC is a thylakoid membrane protein with two predicted transmembrane domains that is conserved from cyanobacteria to vascular plants. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation analyses revealed a specific interaction between DAC and PetD, a subunit of the cytochrome b(6)/f complex. However, DAC was found not to be an intrinsic component of the cytochrome b(6)/f complex. In vivo chloroplast protein labeling experiments showed that the labeling rates of the PetD and cytochrome f proteins were greatly reduced, whereas that of the cytochrome b(6) protein remained normal in the dac mutant. DAC appears to be a novel factor involved in the assembly/stabilization of the cytochrome b(6)/f complex, possibly through interaction with the PetD protein.

  16. Cytochrome c oxidase-intermediate fibres: importance in understanding the pathogenesis and treatment of mitochondrial myopathy. (United States)

    Murphy, Julie L; Ratnaike, Thiloka E; Shang, Ersong; Falkous, Gavin; Blakely, Emma L; Alston, Charlotte L; Taivassalo, Tanja; Haller, Ronald G; Taylor, Robert W; Turnbull, Doug M


    An important diagnostic muscle biopsy finding in patients with mitochondrial DNA disease is the presence of respiratory-chain deficient fibres. These fibres are detected as cytochrome c oxidase-deficient following a sequential cytochrome c oxidase-succinate dehydrogenase reaction, often in a mosaic pattern within a population of cytochrome c oxidase-normal fibres. Detailed analysis of muscle biopsies from patients with various mitochondrial DNA defects shows that a spectrum of deficiency exists, as there are a large number of fibres which do not correspond to being either completely cytochrome c oxidase-normal (brown staining) or cytochrome c oxidase-deficient (blue staining). We have used a combination of histochemical and immunocytochemical techniques to show that a population of cytochrome c oxidase-intermediate reacting fibres are a gradation between normal and deficient fibres. We show that cytochrome c oxidase-intermediate fibres also have different genetic characteristics in terms of amount of mutated and wild-type mtDNA, and as such, may represent an important transition between respiratory normal and deficient fibres. Assessing changes in intermediate fibres will be crucial to evaluating the responses to treatment and in particular to exercise training regimes in patients with mitochondrial DNA disease.

  17. The interplay between tubulins and P450 cytochromes during Plasmodium berghei invasion of Anopheles gambiae midgut.

    Directory of Open Access Journals (Sweden)

    Rute C Félix

    Full Text Available BACKGROUND: Plasmodium infection increases the oxidative stress inside the mosquito, leading to a significant alteration on transcription of Anopheles gambiae detoxification genes. Among these detoxification genes several P450 cytochromes and tubulins were differently expressed, suggesting their involvement in the mosquito's response to parasite invasion. P450 cytochromes are usually involved in the metabolism and detoxification of several compounds, but are also regulated by several pathogens, including malaria parasite. Tubulins are extremely important as components of the cytoskeleton, which rearrangement functions as a response to malaria parasite invasion. METHODOLOGY/PRINCIPAL FINDINGS: Gene silencing methods were used to uncover the effects of cytochrome P450 reductase, tubulinA and tubulinB silencing on the A. gambiae response to Plasmodium berghei invasion. The role of tubulins in counter infection processes was also investigated by inhibiting their effect. Colchicine, vinblastine and paclitaxel, three different tubulin inhibitors were injected into A. gambiae mosquitoes. Twenty-four hours post injection these mosquitoes were infected with P. berghei through a blood meal from infected CD1 mice. Cytochrome P450 gene expression was measured using RT-qPCR to detect differences in cytochrome expression between silenced, inhibited and control mosquitoes. Results showed that cytochrome P450 reductase silencing, as well as tubulin (A and B silencing and inhibition affected the efficiency of Plasmodium infection. Silencing and inhibition also affected the expression levels of cytochromes P450. CONCLUSIONS: Our results suggest the existence of a relationship between tubulins and P450 cytochromes during A. gambiae immune response to P. berghei invasion. One of the P450 cytochromes in this study, CYP6Z2, stands out as the potential link in this association. Further work is needed to fully understand the role of tubulin genes in the response to

  18. A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect. (United States)

    Abdullaev, Ziedulla Kh; Bodrova, Marina E; Chernyak, Boris V; Dolgikh, Dmitry A; Kluck, Ruth M; Pereverzev, Mikhail O; Arseniev, Alexander S; Efremov, Roman G; Kirpichnikov, Mikhail P; Mokhova, Elena N; Newmeyer, Donald D; Roder, Heinrich; Skulachev, Vladimir P


    A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12. PMID:11879204

  19. Cytochrome P450s and cytochrome P450 reductase in the olfactory organ of the cotton leafworm Spodoptera littoralis. (United States)

    Pottier, M-A; Bozzolan, F; Chertemps, T; Jacquin-Joly, E; Lalouette, L; Siaussat, D; Maïbèche-Coisne, M


    Cytochrome P450 enzymes (P450s) are involved in many physiological functions in insects, such as the metabolism of signal molecules, adaptation to host plants and insecticide resistance. Several P450s have been reported in the olfactory organs of insects, the antennae, and have been proposed to play a role in odorant processing and/or xenobiotic metabolism. Despite recent transcriptomic analyses in several species, the diversity of antennal P450s in insects has not yet been investigated. Here, we report the identification of 37 putative P450s expressed in the antennae of the pest moth Spodoptera littoralis, as well as the characterization of a redox partner, cytochrome P450 reductase (CPR). Phylogenetic analysis revealed that S. littoralis P450s belong to four clades defined by their conservation with vertebrate P450s and their cellular localization. Interestingly, the CYP3 and CYP4 clans, which have been described to be mainly involved in the metabolism of plant compounds and xenobiotics, were largely predominant. More surprisingly, two P450s related to ecdysteroid metabolism were also identified. Expression patterns in adult and larval tissues were studied. Eight P450s appeared to be specific to the chemosensory organs, ie the antennae and proboscis, suggesting a specific role in odorant and tastant processing. Moreover, exposure of males to a plant odorant down-regulated the transcript level of CPR, revealing for the first time the regulation of this gene by odorants within insect antennae. This work suggests that the antennae of insects are a key site for P450-mediated metabolism of a large range of exogenous and endogenous molecules.

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

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


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

  1. Childhood encephalomyopathy with cytochrome c oxidase deficiency, ataxia, muscle wasting, and mental impairment. (United States)

    Angelini, C; Bresolin, N; Pegolo, G; Bet, L; Rinaldo, P; Trevisan, C; Vergani, L


    The son of third cousins was normal until age 2 when he had difficulty walking. At age 8 there was limb weakness, ataxia, loss of tendon reflexes, dislalia, and he was mildly retarded. During fasting, urinary organic acid excretion was abnormally high. Cytochrome c oxidase activity in muscle was 7% of the normal mean. The enzyme in platelets was 16% of controls with a decreased cytochrome aa3 peak. These data suggest an autosomal recessive transmission of this variant of cytochrome c oxidase deficiency.

  2. Purification and characterization of the cytochrome oxidase from alkalophilic Bacillus firmus RAB.


    Kitada, M; Krulwich, T A


    A cytochrome oxidase was purified 52-fold from membranes of alkalophilic Bacillus firmus RAB by extraction with Triton X-100, ion-exchange and hydroxyapatite chromatography, and gel filtration. On denaturing gels, the purified enzyme dissociated into two subunits of 56,000 and 40,000 Mr as well as a cytochrome c with an Mr of approximately 14,000. Heme contents calculated for an enzyme with a molecular weight of 110,000 were found to be 2 mol of heme a and 1 mol of heme c per mol of cytochrom...

  3. The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance.

    Directory of Open Access Journals (Sweden)

    Julia Leclerc

    Full Text Available Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance.

  4. Cytochrome c binding to Apaf-1: The effects of dATP and ionic strength


    Purring-Koch, Cherie; McLendon, George


    In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. Here, we show through fluorescence polarization experiments that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c·Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along w...

  5. Cumene hydroperoxide supported demethylation of N,N-dimethylaniline by cytochrome P-450 from adrenal cortex mitochondria. (United States)

    Akhrem, A A; Khatyleva SYu; Shkumatov, V M; Chashchin, V L; Kiselev, P A


    The interaction of highly purified cytochrome P-450 from bovine adrenal cortex mitochondria (cytochrome P-450scc) with N,N-dimethylaniline (DMA), aniline, N-dimethylcyclohexylamine and cumene hydroperoxide (CHP) has been investigated. The formation of complexes between cytochrome P-450scc and the above listed compounds could be demonstrated. The reaction of oxidative demethylation of DMA by cumene hydroperoxide involving cytochrome P-450scc has been carried out at 37 degrees C; the mechanism of this process is discussed. Incubation of cytochrome P-450scc with negatively charged phospholipids, phosphatidylglycerol (PG), and phosphatidylinosite (PI) exerts an inhibiting effect on the reaction of oxidative demethylation. The interaction of cytochrome P-450scc with CHP is accompanied by hemoprotein destruction in a complex biphasic way. The process of oxidative demethylation of DMA in the system of cytochrome P-450scc-CHP has been concluded to have a predominantly radical character.

  6. Hippocampal mitochondrial cytochrome C oxidase activity and gene expression in a rat model of chronic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Qing Zhao; Yingli Zhang; Mingming Zhao; Yu Wang; Ming Ma; Xinquan Gu; Xia Cao


    The present study established a rat model of chronic cerebral ischemia using bilateral common carotid artery permanent ligation to analyze cytochrome C oxidase activity and mRNA expression in hippocampal mitochondria.Results showed significantly decreased cytochrome C oxidase activity and cytochrome C oxidase II mRNA expression with prolonged ischemia time.Further analysis revealed five mitochondrial cytochrome C oxidase II gene mutations, two newly generated mutations, and four absent mutational sites at 1 month after cerebral ischemia, as well as three mitochondrial cytochrome C oxidase III gene mutations, including two newly generating mutations, and one disappeared mutational site at 1 month after cerebral ischemia.Results demonstrated that decreased cytochrome C oxidase gene expression and mutations, as well as decreased cytochrome C oxidase activity, resulting in energy dysmetabolism, which has been shown to be involved in the pathological process of ischemic brain injury.

  7. E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation. (United States)

    Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin


    C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response.

  8. Specificity analysis of the C-type lectin from rattlesnake venom, and its selectivity towards Gal- or GalNAc-terminated glycoproteins. (United States)

    Young, N Martin; van Faassen, Henk; Watson, David C; Mackenzie, C Roger


    The rattlesnake (Crotalus atrox) venom lectin is a readily-prepared decameric C-type lectin, specific for Gal and GalNAc. Glycan microarray analysis showed it reacted with a wide range of glycans, chiefly recognizing sets of compounds with Galβ1-4GlcNAc (LacNAc), α-Gal or α-GalNAc non-reducing termini. Its array profile was therefore distinctly different from those of four previously studied mammalian C-type lectins with the same Gal/GalNAc monosaccharide specificity, and it was more broadly reactive than several Gal- or GalNAc-specific plant lectins commonly used for glycan blotting. Though a general reactivity towards glycoproteins might be expected from the avidity conferred by its high valence, it showed a marked preference for glycoproteins with multiple glycans, terminated by Gal or GalNAc. Thus its ten closely-spaced sites each with a K(D) for GalNAc of ~2 mM appeared to make RSVL more selective than the four more widely-spaced sites of soybean agglutinin, with a ten-fold better K(D) for GalNAc.

  9. Evolution of the C-Type Lectin-Like Receptor Genes of the DECTIN-1 Cluster in the NK Gene Complex

    Directory of Open Access Journals (Sweden)

    Susanne Sattler


    Full Text Available Pattern recognition receptors are crucial in initiating and shaping innate and adaptive immune responses and often belong to families of structurally and evolutionarily related proteins. The human C-type lectin-like receptors encoded in the DECTIN-1 cluster within the NK gene complex contain prominent receptors with pattern recognition function, such as DECTIN-1 and LOX-1. All members of this cluster share significant homology and are considered to have arisen from subsequent gene duplications. Recent developments in sequencing and the availability of comprehensive sequence data comprising many species showed that the receptors of the DECTIN-1 cluster are not only homologous to each other but also highly conserved between species. Even in Caenorhabditis elegans, genes displaying homology to the mammalian C-type lectin-like receptors have been detected. In this paper, we conduct a comprehensive phylogenetic survey and give an up-to-date overview of the currently available data on the evolutionary emergence of the DECTIN-1 cluster genes.

  10. A C-type lectin isolated from the skin of Japanese bullhead shark (Heterodontus japonicus) binds a remarkably broad range of sugars and induces blood coagulation. (United States)

    Tsutsui, Shigeyuki; Dotsuta, Yuma; Ono, Ayaka; Suzuki, Masanari; Tateno, Hiroaki; Hirabayashi, Jun; Nakamura, Osamu


    The aim of this study was to determine the physiological role of skin lectins of the Japanese bullhead shark (Heterodontus japonicus). A skin extract was subjected to affinity chromatography using seven different sugars as ligands. Molecular mass and N-terminal amino acid sequence analyses indicated elution of the same protein by each of the seven respective cognate ligands from sugar affinity columns. The predicted amino acid sequence encoded by the cDNA of this protein [designated as H. japonicus C-type-lectin (HjCL)] identified it as a novel fish subgroup VII C-type lectin evolutionarily related to snake venom lectins. HjCL was predicted to bind to mannose because of the presence of a Glu-Pro-Asn (EPN) motif; however, haemagglutination inhibition assays and glycoconjugate microarray analysis demonstrated its binding to numerous structurally diverse sugars. Competitive sugar-binding assays using affinity chromatography indicated that HjCL bound multiple sugars via a common carbohydrate-recognition domain. The mRNA encoding HjCL was specifically detected in the skin, and immunohistochemical analysis detected its expression in uncharacterized large cells in the epidermis. HjCL agglutinated the bacterial pathogen Edwardsiella tarda and promoted immediate clotting of shark blood, indicating that HjCL is involved in host defence on the skin surface especially when the shark is injured and bleeds.

  11. Sulfite oxidase activity of cytochrome c: Role of hydrogen peroxide

    Directory of Open Access Journals (Sweden)

    Murugesan Velayutham


    Full Text Available In humans, sulfite is generated endogenously by the metabolism of sulfur containing amino acids such as methionine and cysteine. Sulfite is also formed from exposure to sulfur dioxide, one of the major environmental pollutants. Sulfite is used as an antioxidant and preservative in dried fruits, vegetables, and beverages such as wine. Sulfite is also used as a stabilizer in many drugs. Sulfite toxicity has been associated with allergic reactions characterized by sulfite sensitivity, asthma, and anaphylactic shock. Sulfite is also toxic to neurons and cardiovascular cells. Recent studies suggest that the cytotoxicity of sulfite is mediated by free radicals; however, molecular mechanisms involved in sulfite toxicity are not fully understood. Cytochrome c (cyt c is known to participate in mitochondrial respiration and has antioxidant and peroxidase activities. Studies were performed to understand the related mechanism of oxidation of sulfite and radical generation by ferric cytochrome c (Fe3+cyt c in the absence and presence of H2O2. Electron paramagnetic resonance (EPR spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO were performed with sulfite, Fe3+cyt c, and H2O2. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3- was obtained. The amount of DMPO-SO3- formed from the oxidation of sulfite by the Fe3+cyt c increased with sulfite concentration. In addition, the amount of DMPO-SO3- formed by the peroxidase activity of Fe3+cyt c also increased with sulfite and H2O2 concentration. From these results, we propose a mechanism in which the Fe3+cyt c and its peroxidase activity oxidizes sulfite to sulfite radical. Our results suggest that Fe3+cyt c could have a novel role in the deleterious effects of sulfite in biological systems due to increased production of sulfite radical. It also shows that the increased production of sulfite radical may be responsible for neurotoxicity and some of the injuries which

  12. Conjugation of cytochrome c with hydrogen titanate nanotubes: novel conformational state with implications for apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ray, Moumita; Mazumdar, Shyamalava [Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 (India); Chatterjee, Sriparna; Das, Tanmay; Bhattacharyya, Somnath; Ayyub, Pushan, E-mail:, E-mail:, E-mail: [Department of Condensed Matter Physics and Materials Science, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005 (India)


    We show that hydrogen titanate (H{sub 2}Ti{sub 3}O{sub 7}) nanotubes form strongly associated reversible nano-bio-conjugates with the vital respiratory protein, cytochrome c. Resonance Raman spectroscopy along with direct electrochemical studies indicate that in this nano-bio-conjugate, cytochrome c exists in an equilibrium of two conformational states with distinctly different formal redox potentials and coordination geometries of the heme center. The nanotube-conjugated cytochrome c also showed enhanced peroxidase activity similar to the membrane-bound protein that is believed to be an apoptosis initiator. This suggests that such a nanotube-cytochrome c conjugate may be a good candidate for cancer therapy applications.

  13. Mapping of redox state of mitochondrial cytochromes in live cardiomyocytes using Raman microspectroscopy

    DEFF Research Database (Denmark)

    Brazhe, Nadezda A; Treiman, Marek; Brazhe, Alexey R;


    This paper presents a nonivasive approach to study redox state of reduced cytochromes [Formula: see text], [Formula: see text] and [Formula: see text] of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach we......-shaped cardiomyocytes possess uneven distribution of reduced cytochromes [Formula: see text], [Formula: see text] and [Formula: see text] in cell center and periphery. Moreover, by means of Raman spectroscopy we demonstrated the decrease in the relative amounts of reduced cytochromes [Formula: see text], [Formula: see...... perform studies of rod- and round-shaped cardiomyocytes, representing different morphological and functional states. Raman mapping and cluster analysis reveal that these cardiomyocytes differ in the amounts of reduced cytochromes [Formula: see text], [Formula: see text] and [Formula: see text]. The rod...

  14. Prognostic Value of Cytochrome C and Cytokines in Acute Viral Encephalopathy

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap


    Full Text Available Serum cytochrome c and cytokines were evaluated as prognostic predictors in 29 children (ages 9 mos to 9 yrs 11 mos with viral acute encephalopathies and multiple organ failure at Fukushima Medical University School of Medicine, Japan.

  15. High thermal stability and unique trimer formation of cytochrome c' from thermophilic Hydrogenophilus thermoluteolus. (United States)

    Fujii, Sotaro; Masanari, Misa; Inoue, Hiroki; Yamanaka, Masaru; Wakai, Satoshi; Nishihara, Hirofumi; Sambongi, Yoshihiro


    Sequence analysis indicated that thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) and its mesophilic homolog, Allochromatium vinosum cytochrome c' (AVCP), closely resemble each other in a phylogenetic tree of the cytochrome c' family, with 55% sequence identity. The denaturation temperature of PHCP was 87 °C, 35 °C higher than that of AVCP. Furthermore, PHCP exhibited a larger enthalpy change value during its thermal denaturation than AVCP. While AVCP was dimeric, as observed previously, PHCP was trimeric, and this was the first observation as a cytochrome c'. Dissociation of trimeric PHCP and its protein denaturation reversibly occurred at the same time in a two-state transition manner. Therefore, PHCP is enthalpically more stable than AVCP, perhaps due to its unique trimeric form, in addition to the lower number of Gly residues in its putative α-helical regions.

  16. Nanoscale charge transport in cytochrome c3/DNA network: Comparative studies between redox-active molecules (United States)

    Yamaguchi, Harumasa; Che, Dock-Chil; Hirano, Yoshiaki; Suzuki, Masayuki; Higuchi, Yoshiki; Matsumoto, Takuya


    The redox-active molecule of a cytochrome c3/DNA network exhibits nonlinear current-voltage (I-V) characteristics with a threshold bias voltage at low temperature and zero-bias conductance at room temperature. I-V curves for the cytochrome c3/DNA network are well matched with the Coulomb blockade network model. Comparative studies of the Mn12 cluster, cytochrome c, and cytochrome c3, which have a wide variety of redox potentials, indicate no difference in charge transport, which suggests that the conduction mechanism is not directly related to the redox states. The charge transport mechanism has been discussed in terms of the newly-formed electronic energy states near the Fermi level, induced by the ionic interaction between redox-active molecules with the DNA network.

  17. Kinetics and Mechanistic Studies on the Reaction between Cytochrome c and Tea Catechins


    Lihua Wang; Elizabeth Santos; Desiree Schenk; Montserrat Rabago-Smith


    Green tea is characterized by the presence of an abundance of polyphenolic compounds, also known as catechins, including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (EGC) and epigallocatechin gallate (EGCG). In addition to being a popular beverage, tea consumption has been suggested as a mean of chemoprevention. However, its mode of action is unclear. It was discovered that tea catechins can react with cytochrome c. When oxidized cytochrome c was mixed with catechins commonl...

  18. Secologanin synthase which catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome P450. (United States)

    Yamamoto, H; Katano, N; Ooi, A; Inoue, K


    Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica. The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450. Of the substrates examined, only specificity for loganin was demonstrated. A possible reaction mechanism is described.

  19. Redox reactions of cytochrome c facilitated by silver-imidazole complex

    Institute of Scientific and Technical Information of China (English)

    FAN, Chun-Hai; LI, Gen-Xi; ZHU, De-Xu; ZHU, Jian-Qin


    An imidazole modified silver electrode is prepared by immersing the substrate silver electrode in a 2% imidazole solution of ethanol at 50℃ for 10 min. The modified electrode is then swept in a cytochrome c solution and the modified layer takes off because the modified electrode is very unstable. Although the amount of the silver-imidazole complex is very small compared with the amount of cytochrome c in the protein solution, it greatly facilitates redox reactions involving the biomacromolecules.

  20. Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons. (United States)

    McClelland, Levi J; Steele, Harmen B B; Whitby, Frank G; Mou, Tung-Chung; Holley, David; Ross, J B Alexander; Sprang, Stephen R; Bowler, Bruce E


    Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate. We present three structures at 1.7-2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and ω-undecylenyl-β-d-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6-4.0 Å from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.

  1. Genetic control of a cytochrome P450 metabolism-based herbicide resistance mechanism in Lolium rigidum



    The dynamics of herbicide resistance evolution in plants are influenced by many factors, especially the biochemical and genetic basis of resistance. Herbicide resistance can be endowed by enhanced rates of herbicide metabolism because of the activity of cytochrome P450 enzymes, although in weedy plants the genetic control of cytochrome P450-endowed herbicide resistance is poorly understood. In this study we have examined the genetic control of P450 metabolism-based herbicide resistance in a w...

  2. A predicted structure of the cytochrome c oxidase from Burkholderia pseudomallei


    Mohd. Raih,Mohd. Firdaus; Sailan,Ahmad Tarmidi; Zamrod,Zulkeflie; Embi,Mohd. Noor; Mohamed, Rahmah


    Cytochrome c oxidase, the terminal enzyme of the respiratory chains of mitochondria and aerobic bacteria, catalyzes electron transfer from cytochrome c to molecular oxygen. The enzyme belongs to the haem-copper-containing oxidases superfamily. A recombinant plasmid carrying a 2.0 kb insert from a Burkholderia pseudomallei genomic library was subjected to automated DNA sequencing utilizing a primer walking strategy. Analysis of the 2002 bp insert revealed a 1536 bp open reading frame predicted...

  3. High stability of apo-cytochrome c' from thermophilic Hydrogenophilus thermoluteolus. (United States)

    Fujii, Sotaro; Masanari, Misa; Yamanaka, Masaru; Wakai, Satoshi; Sambongi, Yoshihiro


    Apo-cytochomes c without heme are usually unstructured. Here we showed that apo-form of thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) was a monomeric protein with high helix content. Apo-PHCP was thermally stable, possibly due to the hydrophobic residues and ion pairs. PHCP is the first example of a structured apo-cytochrome c', which will expand our view of hemoprotein structure formation.

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

    Directory of Open Access Journals (Sweden)

    Shilpi Khare


    Full Text Available 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.

  5. Force modulation and electrochemical gating of conductance in a cytochrome (United States)

    Davis, Jason J.; Peters, Ben; Xi, Wang


    Scanning probe methods have been used to measure the effect of electrochemical potential and applied force on the tunnelling conductance of the redox metalloprotein yeast iso-1-cytochrome c (YCC) at a molecular level. The interaction of a proximal probe with any sample under test will, at this scale, be inherently perturbative. This is demonstrated with conductive probe atomic force microscopy (CP-AFM) current-voltage spectroscopy in which YCC, chemically adsorbed onto pristine Au(111) via its surface cysteine residue, is observed to become increasingly compressed as applied load is increased, with concomitant decrease in junction resistance. Electrical contact at minimal perturbation, where probe-molecule coupling is comparable to that in scanning tunnelling microscopy, brings with it the observation of negative differential resistance, assigned to redox-assisted probe-substrate tunnelling. The role of the redox centre in conductance is also resolved in electrochemical scanning tunnelling microscopy assays where molecular conductance is electrochemically gateable through more than an order of magnitude.

  6. Force modulation and electrochemical gating of conductance in a cytochrome

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Jason J; Peters, Ben; Xi Wang [Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA (United Kingdom)], E-mail:


    Scanning probe methods have been used to measure the effect of electrochemical potential and applied force on the tunnelling conductance of the redox metalloprotein yeast iso-1-cytochrome c (YCC) at a molecular level. The interaction of a proximal probe with any sample under test will, at this scale, be inherently perturbative. This is demonstrated with conductive probe atomic force microscopy (CP-AFM) current-voltage spectroscopy in which YCC, chemically adsorbed onto pristine Au(111) via its surface cysteine residue, is observed to become increasingly compressed as applied load is increased, with concomitant decrease in junction resistance. Electrical contact at minimal perturbation, where probe-molecule coupling is comparable to that in scanning tunnelling microscopy, brings with it the observation of negative differential resistance, assigned to redox-assisted probe-substrate tunnelling. The role of the redox centre in conductance is also resolved in electrochemical scanning tunnelling microscopy assays where molecular conductance is electrochemically gateable through more than an order of magnitude.

  7. Upregulation of Mitochondrial Content in Cytochrome c Oxidase Deficient Fibroblasts. (United States)

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


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

  8. Induction of diphenytriazol on cytochrome CYP1A

    Institute of Scientific and Technical Information of China (English)

    Yun-zhen HU; Tong-wei YAO


    AIM: To study the effects of diphenytriazol on cytochrome P-450 (CYP) enzymes. METHODS: SD rats were pretreated with diphenytriazol. The catalytic activities of rat liver microsomes were determined by assaying ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-dealkylase. Phenacetin and aminopyrine were selected as the substrate of CYP1A and CYP2B, respectively. The concentration of remaining substrate in microsomal incubates was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The inhibition of fluvoxamine or α-naphthoflavone on phenacetin metabolism was measured. RESULTS: Phenacetin was significantly metabolized in the diphenytriazol-treated microsomes and the metabolic degree increased according to the diphenytriazol-treatment days. There existed a significant correlation between the metabolic degree of phenacetin and EROD in the microsomes pretreated with diphenytriazol. Both fluvoxamine and α-naphthofiavone inhibited the metabolism of phenacetin significantly, and the inhibition constants (Ki) were (5.4± 1.0) μmol/L and (10.4±0.5)μmol/L, respectively. The activity of microsomes pretreated with diphenytriazol for 4 d was similar to that in β-naphthoflavone group, but was significantly different from those in control group and phenobarbital group.CONCLUSION: These results reveal that diphenytriazol is a novel inducer of CYP1A.

  9. Cytochrome c: A Multifunctional Protein Combining Conformational Rigidity with Flexibility

    Directory of Open Access Journals (Sweden)

    Reinhard Schweitzer-Stenner


    Full Text Available Cytochrome has served as a model system for studying redox reactions, protein folding, and more recently peroxidase activity induced by partial unfolding on membranes. This review illuminates some important aspects of the research on this biomolecule. The first part summarizes the results of structural analyses of its active site. Owing to heme-protein interactions the heme group is subject to both in-plane and out-of-plane deformations. The unfolding of the protein as discussed in detail in the second part of this review can be induced by changes of pH and temperature and most prominently by the addition of denaturing agents. Both the kinetic and thermodynamic folding and unfolding involve intermediate states with regard to all unfolding conditions. If allowed to sit at alkaline pH (11.5 for a week, the protein does not return to its folding state when the solvent is switched back to neutral pH. It rather adopts a misfolded state that is prone to aggregation via domain swapping. On the surface of cardiolipin containing liposomes, the protein can adopt a variety of partially unfolded states. Apparently, ferricytochrome c can perform biological functions even if it is only partially folded.

  10. The Membrane Modulates Internal Proton Transfer in Cytochrome c Oxidase

    DEFF Research Database (Denmark)

    Öjemyr, Linda Nasvik; Ballmoos, Christoph von; Faxén, Kristina;


    The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O-2 reduction....... Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pK(a) in the pH dependence profile of the proton-uptake rate. This pK(a) change resulted in decreased proton-uptake rates in the pH range of similar to 6.5-9.5, which...... is explained in terms of lowering of the pK(a) of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),or the anionic lipid 1,2-dioleoyl-glycero-3-phospho (1-rac...

  11. Cyclooxygenase- and cytochrome P450-derived eicosanoids in stroke. (United States)

    Huang, Hui; Al-Shabrawey, Mohamed; Wang, Mong-Heng


    Arachidonic acid (AA) is metabolized by cyclooxygenase (COX) and cytochrome P450 (CYP) enzymes into eicosanoids, which are involved in cardiovascular diseases and stroke. Evidence has demonstrated the important functions of these eicosanoids in regulating cerebral vascular tone, cerebral blood flow, and autoregulation of cerebral circulation. Although COX-2 inhibitors have been suggested as potential treatments for stroke, adverse events, including an increased risk of stroke, occur following long-term use of coxibs. It is important to note that prolonged treatment with rofecoxib increased circulating levels of 20-hydroxyeicosatetraenoic acid (20-HETE), and 20-HETE blockade is a possible strategy to prevent coxib-induced stroke events. It appears that 20-HETE has detrimental effects in the brain, and that its blockade exerts cerebroprotection against ischemic stroke and subarachnoid hemorrhage (SAH). There is clear evidence that activation of EP2 and EP4 receptors exerts cerebroprotection against ischemic stroke. Several elegant studies have contributed to defining the importance of stabilizing the levels of epoxyeicosatrienoic acids (EETs), by inhibiting or deleting soluble epoxide hydrolase (sEH), in stroke research. These reports support the notion that sEH blockade is cerebroprotective against ischemic stroke and SAH. Here, we summarize recent findings implicating these eicosanoid pathways in cerebral vascular function and stroke. We also discuss the development of animal models with targeted gene deletion and specific enzymatic inhibitors in each pathway to identify potential targets for the treatment of ischemic stroke and SAH.

  12. Midkine Regulates BP through Cytochrome P450-Derived Eicosanoids. (United States)

    Sato, Yuka; Sato, Waichi; Maruyama, Shoichi; Wilcox, Christopher S; Falck, John R; Masuda, Tomohiro; Kosugi, Tomoki; Kojima, Hiroshi; Maeda, Kayaho; Furuhashi, Kazuhiro; Ando, Masahiko; Imai, Enyu; Matsuo, Seiichi; Kadomatsu, Kenji


    The effects of endothelium-derived hyperpolarizing factors have been attributed to cytochrome P450-derived epoxyeicosatrienoic acids (EETs), but the regulation and role of EETs in endothelial dysfunction remain largely unexplored. Hypertension is a primary risk factor for renal dysfunction, which is frequently accompanied by various systemic diseases induced by endothelial dysfunction in the microcirculation. We previously reported that the endothelial growth factor midkine (MK) enhances hypertension in a model of CKD. Here, we investigated the hypothesis that MK regulates EET activity and thereby BP. MK gene-deleted mice were resistant to hypertension and developed less glomerulosclerosis and proteinuria after administration of a nitric oxide synthase (NOS) inhibitor in the setting of uninephrectomy. The hypertension observed in uninephrectomized wild-type mice after NOS inhibition was ameliorated by anti-MK antibody. MK-deficient mice produced higher amounts of EETs, and EETs dominantly regulated BP in these mice. Furthermore, MK administration to MK-deficient mice recapitulated the BP control observed in wild-type mice. EETs also dominantly regulated renal blood flow, which may influence renal function, in MK-deficient mice. Taken together, these results suggest that the MK/EET pathway is physiologically engaged in BP control and could be a target for the treatment of hypertension complicated by endothelial dysfunction.

  13. A spin label study of conformational changes in cytochrome c. (United States)

    Postnikova, G B; Gorbunova, N P; Volkenstein, M V


    Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.

  14. Cytochrome P450 genetic polymorphisms of Mexican indigenous populations. (United States)

    Sosa-Macías, Martha; Llerena, Adrián


    This review focuses on the genetic polymorphisms of the cytochrome P450 (CYP) genes in Mexican indigenous populations, who are a part of the wide ethnic diversity of this country. These native groups have a particular historical trajectory that is different from the Mexican Mestizos. This variability may be reflected in the frequency distribution of polymorphisms in the CYP genes that encode enzymes involved in the metabolism of drugs and other xenobiotics. Therefore, these polymorphisms may affect drug efficacy and safety in indigenous populations in Mexico. The present study aimed to analyze the prevalence of CYP polymorphisms in indigenous Mexicans and to compare the results with studies in Mexican Mestizos. Because the extrapolation of pharmacogenetic data from Mestizos is not applicable to the majority of indigenous groups, pharmacogenetic studies directed at indigenous populations need to be developed. The Amerindians analyzed in this study showed a low phenotypic (CYP2D6) and genotypic (CYP2D6, CYP2C9) diversity, unlike Mexican Mestizos. The frequency of polymorphisms in the CYP1A1, CYP2C19, CYP2E1, and CYP3A4 genes was more similar among the Amerindians and Mexican Mestizos, with the exception of the CYP1A2 gene, whose *1F variant frequency in Mexican Amerindians was the highest described to date.

  15. Alternative Conformations of Cytochrome c: Structure, Function, and Detection. (United States)

    Hannibal, Luciana; Tomasina, Florencia; Capdevila, Daiana A; Demicheli, Verónica; Tórtora, Verónica; Alvarez-Paggi, Damián; Jemmerson, Ronald; Murgida, Daniel H; Radi, Rafael


    Cytochrome c (cyt c) is a cationic hemoprotein of ∼100 amino acid residues that exhibits exceptional functional versatility. While its primary function is electron transfer in the respiratory chain, cyt c is also recognized as a key component of the intrinsic apoptotic pathway, the mitochondrial oxidative protein folding machinery, and presumably as a redox sensor in the cytosol, along with other reported functions. Transition to alternative conformations and gain-of-peroxidase activity are thought to further enable the multiple functions of cyt c and its translocation across cellular compartments. In vitro, direct interactions of cyt c with cardiolipin, post-translational modifications such as tyrosine nitration, phosphorylation, methionine sulfoxidation, mutations, and even fine changes in electrical fields lead to a variety of conformational states that may be of biological relevance. The identification of these alternative conformations and the elucidation of their functions in vivo continue to be a major challenge. Here, we unify the knowledge of the structural flexibility of cyt c that supports functional moonlighting and review biochemical and immunochemical evidence confirming that cyt c undergoes conformational changes during normal and altered cellular homeostasis.

  16. Animal Species Identification by PCR – RFLP of Cytochrome b

    Directory of Open Access Journals (Sweden)

    Tomáš Minarovič


    Full Text Available An alternative DNA detection system is based on the polymerase chain reaction (PCR amplification of a segment of the mitochondrial cytochrome b gene. Subsequent cleavage by a restriction enzymes gives rise to a specie-specific pattern on an agarose gel. We used five animal species (Mustela vison, Mustela putorius furo, Sus scrofa domesticus, Oryctolagus cuninculus, Anser anser. Length of PCR product was 359 bp and we used universal primers. Restriction fragment length polymorphism was analyzed by using the restriction endonuclease AluI. Results of cleavage were visualized by using electrophoresis and UV transiluminator. Every animal specie has a unique combination of restriction fragments i.e. Mustela vison 81 bp, 109 bp and 169 bp, Mustela putorius furo 169 bp and 190 bp, Sus scrofa domesticus 115 bp and 244 bp, Oryctolagus cunninculus is not cleaved by AluI so it has whole 359 bp fragment on agarose gel, Anser anser 130 bp and 229 bp. The results suggest that the method of PCR - RFLP is rapid and simple method for identification of species. PCR – RFLP can reliably identify chosen species. Application of genetic methods is very useful for breeding of livestock and protection of biodiversity.

  17. Glaucoma and Cytochrome P4501B1 Gene Mutations

    Directory of Open Access Journals (Sweden)

    Mukesh Tanwar


    Full Text Available Developmental anomalies of the ocular anterior chamber angle may lead to an incomplete development of the structures that form the conventional aqueous outflow pathway. Thus, disorders that present with such dysfunction tend to be associated with glaucoma. Among them, Axenfeld-Rieger (ARS malformation is a rare clinical entity with an estimated prevalence of one in every 200,000 individuals. The changes in eye morphogenesis in ARS are highly penetrant and are associated with 50% risk of development of glaucoma. Mutations in the cytochrome P4501B1 (CYP1B1 gene have been reported to be associated with primary congenital glaucoma and other forms of glaucoma and mutations in pituitary homeobox 2 (PITX2 gene have been identified in ARS in various studies. This case was negative for PITX2 mutations and compound heterozygote for CYP1B1 mutations. Clinical manifestations of this patient include bilateral elevated intraocular pressure (>40 mmHg with increased corneal diameter (>14 mm and corneal opacity. Patient also had iridocorneal adhesions, anteriorly displaced Schwalbe line, anterior insertion of iris, broad nasal bridge and protruding umbilicus. This is the first study from north India reporting CYP1B1 mutations in Axenfeld-Rieger syndrome with bilateral buphthalmos and early onset glaucoma. Result of this study supports the role of CYP1B1 as a causative gene in ASD disorders and its role in oculogenesis.

  18. High-throughput mass spectrometric cytochrome P450 inhibition screening. (United States)

    Lim, Kheng B; Ozbal, Can C; Kassel, Daniel B


    We describe here a high-throughput assay to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of eight concentrations and against a panel of six cytochrome P450 (CYP) enzymes: 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. The method utilizes automated liquid handling for sample preparation, and online solid-phase extraction/tandem mass spectrometry (SPE/MS/MS) for sample analyses. The system is capable of generating two 96-well assay plates in 30 min, and completes the data acquisition and analysis of both plates in about 30 min. Many laboratories that perform the CYP inhibition screening automate only part of the processes leaving a throughput bottleneck within the workflow. The protocols described in this chapter are aimed to streamline the entire process from assay to data acquisition and processing by incorporating automation and utilizing high-precision instrument to maximize throughput and minimize bottleneck.

  19. Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions. (United States)

    Unitt, David C; Hollis, Veronica S; Palacios-Callender, Miriam; Frakich, Nanci; Moncada, Salvador


    We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O(2)) conditions. The system measures the concentrations of O(2) and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O(2) concentration and electron turnover of the enzyme. At a high O(2) concentration (70 microM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O(2). At low O(2) (15 microM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O(2) consumption. At both high and low O(2) concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.

  20. Ferricytochrome c protects mitochondrial cytochrome c oxidase against hydrogen peroxide-induced oxidative damage. (United States)

    Sedlák, Erik; Fabian, Marian; Robinson, Neal C; Musatov, Andrej


    An excess of ferricytochrome c protects purified mitochondrial cytochrome c oxidase and bound cardiolipin from hydrogen peroxide-induced oxidative modification. All of the peroxide-induced changes within cytochrome c oxidase, such as oxidation of Trp(19,IV) and Trp(48,VIIc), partial dissociation of subunits VIa and VIIa, and generation of cardiolipin hydroperoxide, no longer take place in the presence of ferricytochrome c. Furthermore, ferricytochrome c suppresses the yield of H(2)O(2)-induced free radical detectable by electron paramagnetic resonance spectroscopy within cytochrome c oxidase. These protective effects are based on two mechanisms. The first involves the peroxidase/catalase-like activity of ferricytochrome c, which results in the decomposition of H(2)O(2), with the apparent bimolecular rate constant of 5.1±1.0M(-1)s(-1). Although this value is lower than the rate constant of a specialized peroxidase, the activity is sufficient to eliminate H(2)O(2)-induced damage to cytochrome c oxidase in the presence of an excess of ferricytochrome c. The second mechanism involves ferricytochrome c-induced quenching of free radicals generated within cytochrome c oxidase. These results suggest that ferricytochrome c may have an important role in protection of cytochrome c oxidase and consequently the mitochondrion against oxidative damage.

  1. UV-light effects on cytochrome c modulated by the aggregation state of phenothiazines.

    Directory of Open Access Journals (Sweden)

    Carolina G dos Santos

    Full Text Available The present study shows the factors that modulate the photodamage promoted by phenothiazines. Cytochrome c was irradiated with UV light for 120 min, over a pH range from 4.0 to 8.0, in the absence and in the presence of different concentrations of thioridazine (TR and fluphenazine (FP. In the absence of phenothiazines, the maximal rate of a Soret band blue shift (nm/min from 409 to 406 nm was obtained at pH 4.0 (0.028 nm/min. The presence of phenothiazines at the concentration range 10-25 µmol/L amplified and accelerated a cytochrome c blue shift (409 to 405 nm, at a rate = 0.041 nm/min. Above 25 µmol/L, crescent concentrations of phenothiazines contributed to cytochrome c protection with (maximal at 2500 µmol/L. Scanning electronic microscopy revealed the formation of nanostructures. The pH also influenced the effect of low phenothiazine concentrations on cytochrome c. Thus, the predominance of phenothiazine-promoted cytochrome c damage or protection depends on a balance of the following factors: the yield of photo-generated drug cation radicals, which is favored by acidic pH; the stability of the cation radicals, which is favored by the drug aggregation; and the cytochrome c structure, modulated by the pH.

  2. Structural and biochemical characterization of DHC2, a novel diheme cytochrome c from Geobacter sulfurreducens. (United States)

    Heitmann, Daniel; Einsle, Oliver


    Multiheme cytochromes c constitute a widespread class of proteins with essential functions in electron transfer and enzymatic catalysis. Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs. Understanding the significance of these motifs is crucial for the elucidation of the highly optimized properties of multiheme cytochromes c, but their spectroscopic investigation is often hindered by the large number and efficient coupling of the individual centers and the limited availability of recombinant protein material. We have identified a diheme cytochrome c, DHC2, from the metal-reducing soil bacterium Geobacter sulfurreducens and determined its crystal structure by the method of multiple-wavelength anomalous dispersion (MAD). The two heme groups of DHC2 pack into one of the typical heme interaction motifs observed in larger multiheme cytochromes, but because of the absence of further, interfering cofactors, the properties of this heme packing motif can be conveniently studied in detail. Spectroscopic properties (UV-vis and EPR) of the protein are typical for cytochromes containing low-spin Fe(III) centers with bis-histidinyl coordination. Midpoint potentials for the two heme groups have been determined to be -135 and -289 mV by potentiometric redox titrations. DHC2 has been produced by recombinant expression in Escherichia coli using the accessory plasmid pEC86 and is therefore accessible for systematic mutational studies in further investigating the properties of heme packing interactions in cytochromes c.

  3. Covalent modification of cytochrome c exposed to trans,trans-2,4-decadienal. (United States)

    Sigolo, Carlos A O; Di Mascio, Paolo; Medeiros, Marisa H G


    Modification of biomolecules by reactive aldehydes is believed to play a role in biological processes, including aging, atherosclerosis, and Alzheimer's disease. Here, the modification of cytochrome c promoted by trans, trans-2,4-decadienal (DDE) was investigated. Matrix-assisted laser desorption/ionization time-of-flight experiments indicated increases in the molecular weight of cytochrome c, consistent with the formation of DDE adducts. Our data show that the protein modification was time-, pH-, and DDE concentration-dependent, leading to the formation of at least six adducts after 2 h of incubation at pH 7.4. Electrospray ionization quantitative TOF mass spectrometry analysis of tryptic digests indicated that His-33, Lys-39, Lys-72, and Lys-100 were modified by DDE. These adducts could have significant effects considering that His-33, Lys-72, and Lys-100 are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with cardiolipin in the inner mitochondrial membrane and cytochrome c oxidase. A blue shift in the cytochrome c Soret band from 409 to 406 nm was also observed after DDE reaction, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. The covalent modifications in cytochrome c could play a role in mitochondrial dysfunction associated with oxidative stress.

  4. Multi-heme Cytochromes in Shewanella oneidensis MR-1: Structures, functions and opportunities

    Energy Technology Data Exchange (ETDEWEB)

    Breuer, Marian; Rosso, Kevin M.; Blumberger, Jochen; Butt, Julea N.


    Multi-heme cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometers. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-heme cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-heme cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward there are opportunities to engage multi-heme cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-heme cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-heme cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies.

  5. The novel cytochrome c6 of chloroplasts: a case of evolutionary bricolage? (United States)

    Howe, Christopher J; Schlarb-Ridley, Beatrix G; Wastl, Juergen; Purton, Saul; Bendall, Derek S


    Cytochrome c6 has long been known as a redox carrier of the thylakoid lumen of cyanobacteria and some eukaryotic algae that can substitute for plastocyanin in electron transfer. Until recently, it was widely accepted that land plants lack a cytochrome c6. However, a homologue of the protein has now been identified in several plant species together with an additional isoform in the green alga Chlamydomonas reinhardtii. This form of the protein, designated cytochrome c6A, differs from the 'conventional' cytochrome c6 in possessing a conserved insertion of 12 amino acids that includes two absolutely conserved cysteine residues. There are conflicting reports of whether cytochrome c6A can substitute for plastocyanin in photosynthetic electron transfer. The evidence for and against this is reviewed and the likely evolutionary history of cytochrome c6A is discussed. It is suggested that it has been converted from a primary role in electron transfer to one in regulation within the chloroplast, and is an example of evolutionary 'bricolage'.

  6. Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'. (United States)

    Dar, Tanveer A; Schaeffer, R Dustin; Daggett, Valerie; Bowler, Bruce E


    To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.

  7. Role of the low-affinity binding site in electron transfer from cytochrome C to cytochrome C peroxidase. (United States)

    Mei, Hongkang; Geren, Lois; Miller, Mark A; Durham, Bill; Millett, Francis


    The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1). In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI *yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant. The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other. These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d). Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism. The value of k(d) is 2000 s(-1) for the 2:1 complex at 10 mM ionic strength. The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site. The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCc*CcP crystal structure.

  8. Alternation of plasma c-type natriuretic peptide in cerebral infarction%脑梗死患者血浆c-型利钠肽变化

    Institute of Scientific and Technical Information of China (English)

    赵文凤; 宋利春


    AIM:To investigate the effects of c type natriuretic peptide (CNP) on cerebral infarction.METHODS:Plasma levels of CNP were concomitantly measured by radioimmunoassay in 30 patients with cerebral infarction and in 30 normal controls.RESULTS:Plasma levels of CNP were increased significantly in the acute stage of cerebral infarction than those in the normal controls(P< 0.01) and levels in the moderate and serious cases were lower than those in the slight cases(P< 0.01).CONCLUSION:In cerebral infarction the increase of plasma CNP was in accordance with the severity of the disease .CNP in the pathophysiology of acute cerebral ischemia had a deleterious effect on the evolution of cerebral infarction.

  9. The large C-type asteroids 146 Lucina and 410 Chloris, and the small S-type asteroids 152 Atala and 631 Philippina - Rotation periods and lightcurves (United States)

    Schober, H. J.


    Photoelectric observations are presented for four asteroids. 146 Lucina and 410 Chloris (larger C-type asteroids) were observed in Sep. 1979 at CTIO, the rotation periods are P = 18h54 and P = 32h.50, respectively, both derived by Harris and Young (1982), using my observations prior to publication. 152 Atala was observed in Nov. 1981 at OHP, a rotation period of P - 5h.282 ± 0h.004 was derived with a lightcurve amplitude of 0m.50. 631 Philippina was observed in Feb. 1981 at ESO, and a period of P = 5h.92 ± 0m.01 could be derived with a maximum lightcurve amplitude of 0m.20. 152 Atala and 631 Philippina are both small-sized S-type asteroids and rotate much faster. Colors and magnitudes were measured for the asteroids, excluding 152 Atala for 631 Philippina colors were obtained frequently over all rotational cycles with no variation exceeding the scatter.

  10. C-type lectin-like domain and fibronectin-like type II domain of phospholipase A(2) receptor 1 modulate binding and migratory responses to collagen. (United States)

    Takahashi, Soichiro; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-ei; Kugiyama, Kiyotaka


    Phospholipase A2 receptor 1 (PLA2R) mediates collagen-dependent migration. The mechanisms by which PLA2R interacts with collagen remain unclear. We produced HEK293 cells expressing full-length wild-type PLA2R or a truncated PLA2R that lacks fibronectin-like type II (FNII) domains or several regions of C-type lectin-like domain (CTLD). We show that the CTLD1-2 as well as the FNII domain of PLA2R are responsible for binding to collagen and for collagen-dependent migration. Thus, multiple regions and domains of the extracellular portion of PLA2R participate in the responses to collagen. These data suggest a potentially new mechanism for PLA2R-mediated biological response beyond that of a receptor for secretory PLA2.

  11. Enhancement of 5-iododeoxyuridine-induced endogenous C-type virus activation by polycyclic hydrocarbons: apparent lack of parallelism between enhancement and carcinogenicity. (United States)

    Yoshikura, H; Zajdela, F; Perin, F; Perin-Roussel, O; Jacquignon, P; Latarjet, R


    When mouse MLg cells were treated with 3-methylcholanthrene or 7,12-dimethylbenz[alpha]anthracene in the presence of microsomal enzymes and NADPH after 5-iododeoxyuridine (IUDR) treatment, the induction rate of the endogenous C-type virus was increased fivefold to sixfold in comparison with the culture treated with IUDR only. In this reaction, both the microsomal enzymes and NADPH were indispensable. 7,8-Benzoflavone, an inhibitor of the metabolism of hydrocarbons in hamster embryo cultures, inhibited the reaction. For detecting the enhancing activity, the concentration of IUDR for the pretreatment, the concentration of the test products, and the duration of the treatment with the products were important factors. In screening 30 polycyclic hydrocarbons, we were unable to detect a correlation between the in vivo carcinogenicity in the skin and the enhancing activity in the conditions tested.

  12. Identification of a novel C-type lectin from the shrimp Litopenaeus vannamei and its role in defense against pathogens infection

    Institute of Scientific and Technical Information of China (English)

    LUO Zhan; ZHANG Jiquan; LI Fuhua; ZHANG Xiaojun; LIU Chengzhang; XIANG Jianhai


    Acting as one of the pattern recognition receptors (PRRs),C-type lectin is believed to mediate pathogen recognition and plays an important role in the clearance of pathogens as part of the innate immune system.In this work,a novel C-type lectin gene (named LvLecl) was cloned from the shrimp Litopenaeus vannamei.The ORF of LvLecl is 510 bp,encoding 169 amino acids.The deduced amino acid sequence contains a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain (CRD) at the C-terminal.LvLecl was mainly expressed in the hepatopancreas.Real-time PCR analysis indicated that the level of LvLecl transcripts significantly changed in the hepatopancreas after the shrimp were artificially challenged with LPS,Micrococcus lysodeikticus and white spot syndrome virus (WSSV).RNAi-based silencing of LvLecl resulted in increases in mortality when the shrimp were challenged with WSSV,and the median lethal time was reduced compared with controls.Although there was no characteristic “EPN” (Glu-Pro-Ser) or “QPD” (Gln-Pro-Asp) motif,the recombinant LvLecl,expressed in Escherichia coli BL21 (DE3),could also agglutinate M.lysodeikticus and Vibrio anguillarum.The agglutinating activities were calcium-dependent and could be inhibited by D-mannose,D-glucose,D-galactose and N-Acetyl-D-mannose.These results suggest that LvLecl might be involved in the immune response against WSSV and bacterial infections and contribute to non-self recognition as a pattem recognition receptor in the innate immune system of the shrimp L.vannamei.

  13. CLEC4F is an inducible C-type lectin in F4/80-positive cells and is involved in alpha-galactosylceramide presentation in liver.

    Directory of Open Access Journals (Sweden)

    Chih-Ya Yang

    Full Text Available CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal, N-acetylgalactosamine (GalNAc, and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/- mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5 but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

  14. Structure of a glycomimetic ligand in the carbohydrate recognition domain of C-type lectin DC-SIGN. Structural requirements for selectivity and ligand design. (United States)

    Thépaut, Michel; Guzzi, Cinzia; Sutkeviciute, Ieva; Sattin, Sara; Ribeiro-Viana, Renato; Varga, Norbert; Chabrol, Eric; Rojo, Javier; Bernardi, Anna; Angulo, Jesus; Nieto, Pedro M; Fieschi, Franck


    In genital mucosa, different fates are described for HIV according to the subtype of dendritic cells (DCs) involved in its recognition. This notably depends on the C-type lectin receptor, langerin or DC-SIGN, involved in gp120 interaction. Langerin blocks HIV transmission by its internalization in specific organelles of Langerhans cells. On the contrary, DC-SIGN enhances HIV trans-infection of T lymphocytes. Thus, approaches aiming to inhibit DC-SIGN, without blocking langerin, represent attractive anti-HIV strategies. We previously demonstrated that dendrons bearing multiple copies of glycomimetic compounds were able to block DC-SIGN-dependent HIV infection in cervical explant models. Optimization of such ligand requires detailed characterization of its binding mode. In the present work, we determined the first high-resolution structure of a glycomimetic/DC-SIGN complex by X-ray crystallography. This glycomimetic, pseudo-1,2-mannobioside, shares shape and conformational properties with Manα1-2Man, its natural counterpart. However, it uses the binding epitope previously described for Lewis X, a ligand specific for DC-SIGN among the C-type lectin family. Thus, selectivity gain for DC-SIGN versus langerin is observed with pseudo-1,2-mannobioside as shown by surface plasmon resonance analysis. In parallel, ligand binding was also analyzed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol. These studies demonstrate that the complex, defined by X-ray crystallography, represents the unique binding mode of this ligand as opposed to the several binding orientations described for the natural ligand. This exclusive binding mode and its selective interaction properties position this glycomimetic as a good lead compound for rational improvement based on a structurally driven approach.

  15. Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis. (United States)

    Singtripop, Tippawan; Saeangsakda, Manasawan; Tatun, Nujira; Kaneko, Yu; Sakurai, Sho


    The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.

  16. Disruption of the CYTOCHROME C OXIDASE DEFICIENT1 gene leads to cytochrome c oxidase depletion and reorchestrated respiratory metabolism in Arabidopsis. (United States)

    Dahan, Jennifer; Tcherkez, Guillaume; Macherel, David; Benamar, Abdelilah; Belcram, Katia; Quadrado, Martine; Arnal, Nadège; Mireau, Hakim


    Cytochrome c oxidase is the last respiratory complex of the electron transfer chain in mitochondria and is responsible for transferring electrons to oxygen, the final acceptor, in the classical respiratory pathway. The essentiality of this step makes it that depletion in complex IV leads to lethality, thereby impeding studies on complex IV assembly and respiration plasticity in plants. Here, we characterized Arabidopsis (Arabidopsis thaliana) embryo-lethal mutant lines impaired in the expression of the CYTOCHROME C OXIDASE DEFICIENT1 (COD1) gene, which encodes a mitochondria-localized PentatricoPeptide Repeat protein. Although unable to germinate under usual conditions, cod1 homozygous embryos could be rescued from immature seeds and developed in vitro into slow-growing bush-like plantlets devoid of a root system. cod1 mutants were defective in C-to-U editing events in cytochrome oxidase subunit2 and NADH dehydrogenase subunit4 transcripts, encoding subunits of respiratory complex IV and I, respectively, and consequently lacked cytochrome c oxidase activity. We further show that respiratory oxygen consumption by cod1 plantlets is exclusively associated with alternative oxidase activity and that alternative NADH dehydrogenases are also up-regulated in these plants. The metabolomics pattern of cod1 mutants was also deeply altered, suggesting that alternative metabolic pathways compensated for the probable resulting restriction in NADH oxidation. Being the first complex IV-deficient mutants described in higher plants, cod1 lines should be instrumental to future studies on respiration homeostasis.

  17. Molecular and catalytic properties of the aldehyde dehydrogenase of Gluconacetobacter diazotrophicus, a quinoheme protein containing pyrroloquinoline quinone, cytochrome b, and cytochrome c. (United States)

    Gómez-Manzo, S; Chavez-Pacheco, J L; Contreras-Zentella, M; Sosa-Torres, M E; Arreguín-Espinosa, R; Pérez de la Mora, M; Membrillo-Hernández, J; Escamilla, J E


    Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.

  18. Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus, a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b, and Cytochrome c▿ (United States)

    Gómez-Manzo, S.; Chavez-Pacheco, J. L.; Contreras-Zentella, M.; Sosa-Torres, M. E.; Arreguín-Espinosa, R.; Pérez de la Mora, M.; Membrillo-Hernández, J.; Escamilla, J. E.


    Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone. PMID:20802042

  19. Long-wavelength infrared sensing by cytochrome C protein thin film deposited by the spin coating method. (United States)

    Lai, Bo-Yu; Chu, Chung-Hao; Su, Guo-Dung John


    High infrared absorption, large temperature coefficient of resistance (TCR) and small 1/f noise are preferred characteristics for sensing materials used in bolometers. In this paper, we discuss a cytochrome c protein as a potential sensing material for long-wavelength bolometers. We simulated and experimentally proved high infrared absorption of cytochrome c in the wavelength between 8 μm and 14 μm. Cytochrome c thin films were deposited on a hydrophilic surface using the spin coating method. The resistance variation with temperature is measured and we show that the TCR of cytochrome c thin films is consistently higher than 20%. The measured values of 1/f noise were as low as 2.33 × 10⁻¹³ V²/Hz at 60 Hz. Finally, we test the reliability of cytochrome c by measuring the resistance changes over time under varying conditions. We found that cytochrome c thin films deteriorated significantly without appropriate packaging.

  20. Cytochrome P450-dependent metabolism of caffeine in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Alexandra Coelho

    Full Text Available Caffeine (1, 3, 7-trimethylxanthine, an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents. A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs that were highly overexpressed. Flies treated with metyrapone--an inhibitor of CYP enzymes--showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects.

  1. The planetary biology of cytochrome P450 aromatases

    Directory of Open Access Journals (Sweden)

    Gaucher Eric A


    Full Text Available Abstract Background Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology, and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must any discussion of function within a biomolecular system. Results Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases–enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene. Conclusions This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should

  2. Atypical cytochrome p450 kinetics: implications for drug discovery. (United States)

    Tracy, Timothy S


    The Michaelis-Menten model is commonly used to estimate a drug's potential in vivo hepatic clearance based on in vitro data obtained during drug discovery and development. This paradigm assumes that the drug obeys 'typical' enzyme kinetics and thus can be described by this model. However, it is increasingly being recognised that a number of drugs metabolised not only by the cytochrome P450 enzymes but also by other enzymes and transporters can exhibit atypical kinetic profiles, and thus are not accurately modeled with the Michaelis-Menten model. Application of an incorrect model can then lead to mis-estimation of in vitro intrinsic clearance and thus affect the prediction of in vivo clearance. This review discusses several types of atypical kinetic profiles that may be observed, including examples of homotropic cooperativity (i.e. sigmoidal kinetics, biphasic kinetics and substrate inhibition kinetics) as well as heterotropic cooperativity (i.e. activation). Application of the incorrect kinetic model may profoundly affect estimations of intrinsic clearance. For example, incorrectly applying the Michaelis-Menten model to a kinetic profile exhibiting substrate inhibition kinetics will result in an underestimation of Km (Michaelis-Menten constant) and V(max) (maximal velocity), whereas application of the Michaelis-Menten model to sigmoidal kinetic data typically results in an overestimation of Km and V(max) at the lower substrate concentrations that are typically therapeutically relevant. One must also be careful of potential artefactual causes of atypical kinetic profiles, such as enzyme activation by solvents, buffer dependent kinetic profiles, or altered kinetic parameter estimates due to nonspecific binding of the substrate to proteins. Despite a plethora of data on the effects of atypical kinetic profiles in vitro, only modest effects have been noted in vivo (with the exception of substrate dependent inhibition). Thus, the clinical relevance of these phenomena

  3. The subunit composition and function of mammalian cytochrome c oxidase. (United States)

    Kadenbach, Bernhard; Hüttemann, Maik


    Cytochrome c oxidase (COX) from mammals and birds is composed of 13 subunits. The three catalytic subunits I-III are encoded by mitochondrial DNA, the ten nuclear-coded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIII) by nuclear DNA. The nuclear-coded subunits are essentially involved in the regulation of oxygen consumption and proton translocation by COX, since their removal or modification changes the activity and their mutation causes mitochondrial diseases. Respiration, the basis for ATP synthesis in mitochondria, is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits IV, VIa, VIb, VIIa, VIIb, and VIII, but the holoenzyme in mammals is always composed of 13 subunits. Various proteins and enzymes were shown, e.g., by co-immunoprecipitation, to bind to specific COX subunits and modify its activity, but these interactions are reversible, in contrast to the tightly bound 13 subunits. In addition, the formation of supercomplexes with other oxidative phosphorylation complexes has been shown to be largely variable. The regulatory complexity of COX is increased by protein phosphorylation. Up to now 18 phosphorylation sites have been identified under in vivo conditions in mammals. However, only for a few phosphorylation sites and four nuclear-coded subunits could a specific function be identified. Research on the signaling pathways leading to specific COX phosphorylations remains a great challenge for understanding the regulation of respiration and ATP synthesis in mammalian organisms. This article reviews the function of the individual COX subunits and their isoforms, as well as proteins and small molecules interacting and regulating the enzyme.

  4. Redox-controlled proton gating in bovine cytochrome c oxidase (United States)

    Rousseau, Denis


    Cytochrome c oxidase is the terminal enzyme in the electron transfer chain of essentially all organisms that utilize oxygen to generate energy. It reduces oxygen to water and harnesses the energy to pump protons across the mitochondrial membrane in eukaryotes and the plasma membrane in prokaryotes. The mechanism by which proton pumping is coupled to the oxygen reduction reaction remains unresolved, owing to the difficulty of visualizing proton movement within the massive membrane-associated protein matrix. Here, with a novel hydrogen/deuterium exchange resonance Raman spectroscopy method, we have identified two critical elements of the proton pump: a proton loading site near the propionate groups of heme a, which is capable of transiently storing protons uploaded from the negative-side of the membrane prior to their release into the positive-side of the membrane and a conformational gate that controls proton translocation in response to the change in the redox state of heme a. These findings form the basis for a postulated molecular model describing a detailed mechanism by which unidirectional proton translocation is coupled to electron transfer from heme a to heme a3, associated with oxygen chemistry occurring in the heme a3 site, during enzymatic turnover. Each time heme a undergoes an oxidation-reduction transition a proton is translocated across the membrane accounting for the observation that two protons are translocated during the oxidative phase of the enzymatic cycle and two more are translocated during the reductive phase. This work was done in collaboration with Drs. Tsuyoshi Egawa and Syun-Ru Yeh. This work was supported the National Institutes of Health Grant GM098799 to D.L.R and National Science Foundation Grant NSF 0956358 to S.-R.Y.

  5. Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

    Directory of Open Access Journals (Sweden)

    Thomas H Sanderson

    Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

  6. Giardia intestinalis incorporates heme into cytosolic cytochrome b₅. (United States)

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


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

  7. How hydrogen peroxide is metabolized by oxidized cytochrome c oxidase. (United States)

    Jancura, Daniel; Stanicova, Jana; Palmer, Graham; Fabian, Marian


    In the absence of external electron donors, oxidized bovine cytochrome c oxidase (CcO) exhibits the ability to decompose excess H2O2. Depending on the concentration of peroxide, two mechanisms of degradation were identified. At submillimolar peroxide concentrations, decomposition proceeds with virtually no production of superoxide and oxygen. In contrast, in the millimolar H2O2 concentration range, CcO generates superoxide from peroxide. At submillimolar concentrations, the decomposition of H2O2 occurs at least at two sites. One is the catalytic heme a3-CuB center where H2O2 is reduced to water. During the interaction of the enzyme with H2O2, this center cycles back to oxidized CcO via the intermediate presence of two oxoferryl states. We show that at pH 8.0 two molecules of H2O2 react with the catalytic center accomplishing one cycle. In addition, the reactions at the heme a3-CuB center generate the surface-exposed lipid-based radical(s) that participates in the decomposition of peroxide. It is also found that the irreversible decline of the catalytic activity of the enzyme treated with submillimolar H2O2 concentrations results specifically from the decrease in the rate of electron transfer from heme a to the heme a3-CuB center during the reductive phase of the catalytic cycle. The rates of electron transfer from ferrocytochrome c to heme a and the kinetics of the oxidation of the fully reduced CcO with O2 were not affected in the peroxide-modified CcO.

  8. Cytochrome P450-dependent metabolism of caffeine in Drosophila melanogaster. (United States)

    Coelho, Alexandra; Fraichard, Stephane; Le Goff, Gaëlle; Faure, Philippe; Artur, Yves; Ferveur, Jean-François; Heydel, Jean-Marie


    Caffeine (1, 3, 7-trimethylxanthine), an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents). A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs) that were highly overexpressed. Flies treated with metyrapone--an inhibitor of CYP enzymes--showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects.

  9. Characterizing the proton loading site in cytochrome c oxidase. (United States)

    Lu, Jianxun; Gunner, M R


    Cytochrome c oxidase (CcO) uses the energy released by reduction of O2 to H2O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a3, Cu(B), Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1-4 residues load and release protons. However, the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded.

  10. Controlled adsorption of cytochrome c to nanostructured gold surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Ines [Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, REQUIMTE, Departamento de Quimica (Portugal); Feio, Maria J. [Faculdade de Ciencias da Universidade do Porto, REQUIMTE, Departamento de Quimica e Bioquimica (Portugal); Santos, Nuno C. [Faculdade de Medicina da Universidade de Lisboa, Instituto de Medicina Molecular (Portugal); Eaton, Peter [Faculdade de Ciencias da Universidade do Porto, REQUIMTE, Departamento de Quimica e Bioquimica (Portugal); Serro, Ana Paula; Saramago, Benilde [Centro de Quimica Estrutural, Instituto Superior Tecnico (Portugal); Pereira, Eulalia [Faculdade de Ciencias da Universidade do Porto, REQUIMTE, Departamento de Quimica e Bioquimica (Portugal); Franco, Ricardo, E-mail: [Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, REQUIMTE, Departamento de Quimica (Portugal)


    Controlled electrostatic physisorption of horse heart cytochrome c (Cyt c) onto nanostructured gold surfaces was investigated using Quartz-Crystal Microbalance measurements in planar gold surfaces with or without functionalization using a self-assembled monolayer (SAM) of the alkanethiol mercaptoundecanoic acid (MUA). MUA is a useful functionalization ligand for gold surfaces, shedding adsorbed biomolecules from the excessive electron density of the metal. A parallel analysis was conducted in the corresponding curved surfaces of 15 nm gold nanoparticles (AuNPs), using zeta-potential and UV- visible spectroscopy. Atomic Force Microscopy of both types of functionalized gold surfaces with a MUA SAM, allowed for visualization of Cyt c deposits on the nanostructured gold surface. The amount of Cyt c adsorbed onto the gold surface could be controlled by the solution pH. For the assays conducted at pH 4.5, when MUA SAM- functionalized planar gold surfaces are positive or neutral, and Cyt c has a positive net charge, only 13 % of the planar gold surface area was coated with protein. In contrast, at pH 7.4, when MUA SAM-functionalized planar gold surfaces and Cyt c have opposite charges, a protein coverage of 28 % could be observed implying an adsorption process strongly governed by electrostatic forces. Cyt c adsorption on planar and curved gold surfaces are found to be greatly favored by the presence of a MUA-capping layer. In particular, on the AuNPs, the binding constant is three times larger than the binding constant obtained for the original citrate-capped AuNPs.

  11. Coverage-dependent changes of cytochrome c transverse location in phospholipid membranes revealed by FRET. (United States)

    Domanov, Yegor A; Molotkovsky, Julian G; Gorbenko, Galyna P


    The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.

  12. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. (United States)

    Danielson, P B


    Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s.

  13. Comparative analysis of cytochrome P450-like genes from Locusta migratoria manilensis: expression profiling and response to insecticide exposure

    Institute of Scientific and Technical Information of China (English)

    Yan-Qiong Guo; Jian-Zhen Zhang; Mei-Ling Yang; Liang-Zhen Yan; Kun Yan Zhu; Ya-Ping Guo; En-Bo Ma


    The cytochrome P450 monooxygenase (cytochrome P450) gene superfamily comprises many genes that may be involved in the biotransformations of pesticides and other xenobiotics.To date,very little is known about cytochrome P450 genes in the oriental migratory locust,Locusta migratoria manilensis.In this study,we carried out a genomewide analysis of cytochrome P450 genes of the locust to identify putative cytochrome P450 genes and characterize their expression responses to insecticide exposures.We identified 15 cytochrome P450-1ike genes from a locust expressed sequence tag database (LocustDB).Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that most cytochrome P450-1ike genes displayed different tissue and developmental stage expression patterns.However,most of them were predominantly expressed in the midgut,gastric caeca,fatbodies,and/or hindgut.Biochemical analysis showed that cytochrome P450 was differentially affected by three different insecticides.Deltamethrin caused significant inductions in 12 h at LD30 (dose to kill 30% of the tested individuals) in the nymphs,whereas malathion and carbaryl did not have significant effect on cytochrome P450 enzyme activity.Further RT-PCR analysis showed significant increases of transcriptions of several cytochrome P450 genes in deltamethrin-treated locusts.Thus,the increased cytochrome P450 enzyme activity is likely due to increased transcriptions of multiple cytochrome P450genes in response to deltamethrin exposure.These results are expected to help us better understand the interactions between insecticides and major detoxification enzymes,and possible changes of the susceptibility to other insecticides in deltamethrin-treated insects at various molecular levels.

  14. Disruption of a hydrogen bond network in human versus spider monkey cytochrome c affects heme crevice stability. (United States)

    Goldes, Matthew E; Jeakins-Cooley, Margaret E; McClelland, Levi J; Mou, Tung-Chung; Bowler, Bruce E


    The hypothesis that the recent rapid evolution of primate cytochromes c, which primarily involves residues in the least stable Ω-loop (Ω-loop C, residues 40-57), stabilizes the heme crevice of cytochrome c relative to other mammals, is tested. To accomplish this goal, we have compared the properties of human and spider monkey cytochrome c and a set of four variants produced in the process of converting human cytochrome c into spider monkey cytochrome c. The global stability of all variants has been measured by guanidine hydrochloride denaturation. The stability of the heme crevice has been assessed with the alkaline conformational transition. Structural insight into the effects of the five amino acid substitutions needed to convert human cytochrome c into spider monkey cytochrome c is provided by a 1.15Å resolution structure of spider monkey cytochrome c. The global stability for all variants is near 9.0kcal/mol at 25°C and pH7, which is higher than that observed for other mammalian cytochromes c. The heme crevice stability is more sensitive to the substitutions required to produce spider monkey cytochrome c with decreases of up to 0.5 units in the apparent pKa of the alkaline conformational transition relative to human cytochrome c. The structure of spider monkey cytochrome c indicates that the Y46F substitution destabilizes the heme crevice by disrupting an extensive hydrogen bond network that connects three surface loops including Ω-loop D (residues 70-85), which contains the Met80 heme ligand.

  15. Tile C型骨盆骨折的后路手术治疗%Surgical treatment for Tile C type pelvis fracture through posterior approach

    Institute of Scientific and Technical Information of China (English)

    陈志伟; 杨乐忠; 刘春磊


    目的:探讨经后入路手术内固定治疗Tile C型骨盆骨折的疗效.方法:2005年1月至2009年6月采用单纯后侧入路治疗12例Tile C型骨盆骨折患者,其中男8例,女4例;年龄25-58岁,平均39.5岁.伤后至手术时间7~10d,平均9.5d.入院后均行X线及CT三维立体成像检查,按照Tile分型标准:C1型5例,C2型2例,C1+C2型4例,C3型1例.经抗休克处理,全身情况稳定后,重建钢板固定后环,前环不予内固定.术后常规惠侧下肢行3~4kg皮肤牵引3周.结果:所有患者均获得随访,随访时间6~24个月,平均12.6个月,伤口愈合良好,骨折均愈合,无骨盆畸形愈合、腰骶部疼痛、下肢不等长等并发症.按照Majeed的疗效评定标准:总分(91.50±6.95)分;优10例,良2例.结论:采用单纯后侧入路固定后环治疗TileC型骨盆骨折,可矫正畸形,重建骨盆环的稳定性,效果满意.%Objective: To study the clinical results of surgical treatment for Tile C type pelvis fractures with internal fixation by posterior approach. Methods: From January 2005 to June 2009,12 patients with Tile C type pelvis fracture were treated by open reduction through posterior approach. There were 8 males and 4 females, with an average age of 39.5 years ranging from 25 to 58 years. The time from injury to operation was ranged from 7 to 10 days with an average of 9.5 days. All the patients were given X-ray, 3-D CT examinations before operation. The fracture were classified by Tile classification:Type C 1 in 5 cases,Type C2 in 2 cases,Type C1 and Type C2 in 4 cases,Type C3 in 1 case. All the posterior rings were fixed by re-establishing steel board without anterior ring fixation after stabilization of body condition. All the patients were treated with skin traction for 3 weeks after operation. Results:All 12 patients were followed up for 6 months to 24 months with an average of 12.6 months. All the incisions healed well,and the fractures got union. No pelvic malunion,low back pain or

  16. Isolation of Rhizobium phaseoli Tn5-induced mutants with altered expression of cytochrome terminal oxidases o and aa3. (United States)

    Soberón, M; Membrillo-Hernández, J; Aguilar, G R; Sánchez, F


    Two Rhizobium phaseoli mutants affected in cytochrome expression were obtained by Tn5-mob mutagenesis of the wild-type strain (CE3). Mutant strain CFN031 expressed sevenfold less cytochrome o in culture, expressed cytochrome aa3 under microaerophilic culture conditions, in contrast to strain CE3, and was affected in its vegetative growth properties and proliferation inside plant host cells. Mutant CFN037 expressed cytochrome aa3 under microaerophilic culture conditions, while bacteroid development and nitrogen fixation occurred earlier than in strain CE3. Images FIG. 2 PMID:2155209

  17. Inhalation of butanols: changes in the cytochrome P-450 enzyme system. (United States)

    Aarstad, K; Zahlsen, K; Nilsen, O G


    After inhalation of different butanol isomers for 3 days (2000 ppm) and 5 days (500 ppm), liver and kidney parameters of the microsomal cytochrome P-450 enzyme system were increased. sec-Butanol caused the highest increase in cytochrome P-450 concentration with a 47% rise in the kidneys (500 ppm for 5 days) and 33% in the liver (2000 ppm for 3 days). A concomitant increase of the in vitro n-hexane metabolism in liver microsomes was observed with a 77% increased formation of the preneurotoxic metabolite 2-hexanol compared with control. iso-Butanol did not alter total cytochrome P-450 concentration but caused a significant 30% decrease in the formation of 2-hexanol. Inhalation of all butanols slightly decreased the enzyme levels in the lung. Changes in microsomal enzymes did not correlate with measured serum concentrations of the different butanols showing different inducing capacities among the butanol isomers themselves or the participation of metabolites in the inducing process. As a conclusion sec-butanol, probably through its metabolite methyl-ethyl-ketone, is the most potent inducer of microsomal cytochrome P-450 in liver and kidney while iso-butanol does not alter total cytochrome P-450.

  18. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding. (United States)

    Liko, Idlir; Degiacomi, Matteo T; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V


    Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance.

  19. Amyloid-β peptide binds to cytochrome C oxidase subunit 1. (United States)

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


    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.

  20. Toxic dark effects of protoporphyrin on the cytochrome P-450 system in rat liver microsomes. (United States)

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


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

  1. Infrared sensor by inkjet printing cytochrome c on suspending aluminum electrodes of post CMOS process (United States)

    Liang, Shuo-Feng; Yen, Po-Hsien; Su, Guo-Dung John


    Cytochrome c protein thin film possesses a high temperature coefficient of resistance. In this paper, we systematically investigated the characteristics of cytochrome c, whose absorption coefficient is 65% at wavelengths of 8 12 μm. We found that the changes in resistance resulted from surface roughness. We also discovered that, while cytochrome c improves the temperature coefficient of resistance, a pure protein solution does not conduct well. It needs a buffer solution, acting as an electrolyte, to increase electrical conductance. However, the buffer solution decreases the temperature coefficient. Therefore, optimization of the ratio of cytochrome c protein to buffer solution is required. We determined the best mixing ratio of the protein solution for a sensing material. We then designed a chip for an infrared microbolometer with a MEMS structure of suspended aluminum electrodes. The protein solution was deposited on the sensing pixel using an inkjet printer. The temperature coefficient of resistance, thermal conductance, time constant and responsivity were 25.98%/K, 7.96 × 10-5 W/K, 1.094 ms and 2.57 × 105 V/W at 2 μA bias current, respectively. We experimentally demonstrated integrating cytochrome c protein with a CMOS circuit as a sensing pixel for a longwavelength infrared microbolometer. Based on our experimental results, such a microbolometer array holds promise for the future.

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

    Directory of Open Access Journals (Sweden)

    Luis Fernando Hernandez-Zimbron

    Full Text Available 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.

  3. Control of insulin secretion by cytochrome C and calcium signaling in islets with impaired metabolism. (United States)

    Rountree, Austin M; Neal, Adam S; Lisowski, Mark; Rizzo, Norma; Radtke, Jared; White, Sarah; Luciani, Dan S; Kim, Francis; Hampe, Christiane S; Sweet, Ian R


    The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal.

  4. The C-type lectin-like domain containing proteins Clec-39 and Clec-49 are crucial for Caenorhabditis elegans immunity against Serratia marcescens infection. (United States)

    Miltsch, S M; Seeberger, P H; Lepenies, B


    Caenorhabditis elegans exhibits protective immunity against a variety of fungal and bacterial pathogens. Since C. elegans lacks an adaptive immune system, pathogen recognition is mediated entirely by innate immunity. To date, little is known about the involvement of pattern recognition receptors (PRRs) in pathogen sensing as part of the C. elegans immunity. C-type lectin-like domain (CTLD) containing proteins represent a superfamily of PRRs. A large number of genes encoding for CTLD proteins are present in the C. elegans genome, however the role of CTLD proteins in bacterial recognition and antibacterial immunity has not yet been determined. In this study, we investigated the function of selected C. elegans CTLD proteins during infection with the Gram-negative bacterium Serratia marcescens. Wild-type and CTLD gene-deficient C. elegans strains were compared in their susceptibility to S. marcescens infection. Interestingly, survival and egg laying were significantly reduced in strains deficient for clec-39 and clec-49 indicating a role for both CTLD proteins in C. elegans immune defense against bacteria as evidenced by using S. marcescens infection. Binding studies with recombinantly expressed Clec-39-Fc and Clec-49-Fc fusion proteins revealed that both CTLD proteins recognized live bacteria in a Ca(2+)-independent manner. This study provides insight into the role of CTLD proteins in C. elegans immunity and demonstrates their function during bacterial infection.

  5. C-type lectin receptors Dectin-3 and Dectin-2 form a heterodimeric pattern-recognition receptor for host defense against fungal infection. (United States)

    Zhu, Le-Le; Zhao, Xue-Qiang; Jiang, Changying; You, Yun; Chen, Xiao-Ping; Jiang, Yuan-Ying; Jia, Xin-Ming; Lin, Xin


    C-type lectin receptors (CLRs) play critical roles as pattern-recognition receptors (PRRs) for sensing Candida albicans infection, which can be life-threatening for immunocompromised individuals. Here we have shown that Dectin-3 (also called CLECSF8, MCL, or Clec4d), a previously uncharacterized CLR, recognized α-mannans on the surfaces of C. albicans hyphae and induced NF-κB activation. Mice with either blockade or genetically deleted Dectin-3 were highly susceptible to C. albicans infection. Dectin-3 constantly formed heterodimers with Dectin-2, a well-characterized CLR, for recognizing C. albicans hyphae. Compared to their respective homodimers, Dectin-3 and Dectin-2 heterodimers bound α-mannans more effectively, leading to potent inflammatory responses against fungal infections. Together, our study demonstrates that Dectin-3 forms a heterodimeric PRR with Dectin-2 for sensing fungal infection and suggests that different CLRs may form different hetero- and homodimers, which provide different sensitivity and diversity for host cells to detect various microbial infections.

  6. Sub-millimeter wave spectroscopy of CHD2OH: a-type and asymmetry induced c-type transitions in the lowest three torsional sub-levels (United States)

    Mukhopadhyay, Indra


    The sub-millimeter wave (SMMW) spectral measurements using a fast scan backward wave oscillator based spectrometer have been carried out for asymmetrically deuterated methanol CHD2OH (Methanol-D2). Transition frequencies have an estimated uncertainty of about ±50 kHz. Albeit the complexity in the spectra, assignments were possible for a large number of a-type (ΔK = 0) transitions. In the course of the assignment process a strong c-type (ΔK = 1) Q-branch connecting two states of different symmetry species has been identified. This Q-branch assignment is significant because it is forbidden in the normal parent species CH3OH. It becomes allowed in the current species due to the effects of the asymmetry introduced by the off-axis deuterium in the hindering potential to the internal rotation in the molecule. The assignments are rigorously confirmed using combination relations which required the measurement of some other related lines. To our knowledge this is the first time such symmetry breaking transitions are reported in CHD2OH and in fact this is the first time the SMMW spectrum of CHD2OH is being reported. Detailed spectral study of this molecule in the IR and FIR regions is in progress and will be reported elsewhere. Detailed study of the identification optically pumped FIR laser line is underway.

  7. Structure of the plasminogen kringle 4 binding calcium-free form of the C-type lectin-like domain of tetranectin

    DEFF Research Database (Denmark)

    Nielbo, Steen; Thomsen, Jens K; Graversen, Jonas Heilskov;


    Tetranectin is a homotrimeric protein containing a C-type lectin-like domain. This domain (TN3) can bind calcium, but in the absence of calcium, the domain binds a number of kringle-type protein ligands. Two of the calcium-coordinating residues are also critical for binding plasminogen kringle 4 (K......4). The structure of the calcium free-form of TN3 (apoTN3) has been determined by NMR. Compared to the structure of the calcium-bound form of TN3 (holoTN3), the core region of secondary structural elements is conserved, while large displacements occur in the loops involved in calcium or K4 binding....... A conserved proline, which was found to be in the cis conformation in holoTN3, is in apoTN3 predominantly in the trans conformation. Backbone dynamics indicate that, in apoTN3 especially, two of the three calcium-binding loops and two of the three K4-binding residues exhibit increased flexibility, whereas...

  8. Raman spectra of R{sub 2}O{sub 3} (R—rare earth) sesquioxides with C-type bixbyite crystal structure: A comparative study

    Energy Technology Data Exchange (ETDEWEB)

    Abrashev, M. V., E-mail: [Instituto de Física, UFRGS, Porto Alegre, 91501-970 Rio Grande do Sul (Brazil); Faculty of Physics, University of Sofia, BG-1164 Sofia (Bulgaria); Todorov, N. D. [Faculty of Physics, University of Sofia, BG-1164 Sofia (Bulgaria); Geshev, J. [Instituto de Física, UFRGS, Porto Alegre, 91501-970 Rio Grande do Sul (Brazil)


    Raman spectra of R{sub 2}O{sub 3} (R—Sc, Er, Y, Ho, Gd, Eu, and Sm) powders with C-type bixbyite crystal structure are measured. With the help of these data and ones, previously published for other oxides from the same structural family, general dependencies of the frequencies of the Raman peaks on the cubic crystal unit cell parameter are constructed. Using these dependencies and knowing the symmetry of the peaks for one of the oxides, determined from previous single-crystal measurements, it is possible to find out the symmetry of the peaks from the spectra of all compounds. It was found that the frequency of the six lowest frequency peaks scales with the square root of the mass of the rare earth showing that mainly R ions take part in these vibrations. These results agree with performed here lattice dynamical calculations. The anomalous softening of the frequency of some peaks in the spectra of Eu{sub 2}O{sub 3} is discussed.

  9. Hayabusa2 Sample Catcher and Container: Metal-Seal System for Vacuum Encapsulation of Returned Samples with Volatiles and Organic Compounds Recovered from C-Type Asteroid Ryugu (United States)

    Okazaki, Ryuji; Sawada, Hirotaka; Yamanouchi, Shinji; Tachibana, Shogo; Miura, Yayoi N.; Sakamoto, Kanako; Takano, Yoshinori; Abe, Masanao; Itoh, Shoichi; Yamada, Keita; Yabuta, Hikaru; Okamoto, Chisato; Yano, Hajime; Noguchi, Takaaki; Nakamura, Tomoki; Nagao, Keisuke


    The spacecraft Hayabusa2 was launched on December 3, 2014, to collect and return samples from a C-type asteroid, 162173 Ryugu (provisional designation, 1999 JU3). It is expected that the samples collected contain organic matter and water-bearing minerals and have key information to elucidate the origin and history of the Solar System and the evolution of bio-related organics prior to delivery to the early Earth. In order to obtain samples with volatile species without terrestrial contamination, based on lessons learned from the Hayabusa mission, the sample catcher and container of Hayabusa2 were refined from those used in Hayabusa. The improvements include (1) a mirror finish of the inner wall surface of the sample catcher and the container, (2) adoption of an aluminum metal sealing system, and (3) addition of a gas-sampling interface for gas collection and evacuation. The former two improvements were made to limit contamination of the samples by terrestrial atmosphere below 1 Pa after the container is sealed. The gas-sampling interface will be used to promptly collect volatile species released from the samples in the sample container after sealing of the container. These improvements maintain the value of the returned samples.

  10. The C-type lectin receptor SIGNR3 binds to fungi present in commensal microbiota and influences immune regulation in experimental colitis

    Directory of Open Access Journals (Sweden)

    Magdalena eEriksson


    Full Text Available Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern recognition receptors sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs are pattern recognition receptors recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR SIGNR3 in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homologue of the human DC-SIGN receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze if this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium (DSS-induced colitis model was employed. SIGNR3-/- mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3-/- mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

  11. Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1β in cartilage explants. (United States)

    Peake, Nick J; Pavlov, Anton M; D'Souza, Alveena; Pingguan-Murphy, Belinda; Sukhorukov, Gleb B; Hobbs, Adrian J; Chowdhury, Tina T


    C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1β (IL-1β). SEM showed uniform, 2 to 3 μm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1β and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1β in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

  12. Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

    Energy Technology Data Exchange (ETDEWEB)

    Usami, Katsuaki [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Matsuno, Keita; Igarashi, Manabu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Denda-Nagai, Kaori [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Takada, Ayato [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Irimura, Tatsuro, E-mail: [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan)


    Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.

  13. Using THz Spectroscopy, Evolutionary Network Analysis Methods, and MD Simulation to Map the Evolution of Allosteric Communication Pathways in c-Type Lysozymes. (United States)

    Woods, Kristina N; Pfeffer, Juergen


    It is now widely accepted that protein function is intimately tied with the navigation of energy landscapes. In this framework, a protein sequence is not described by a distinct structure but rather by an ensemble of conformations. And it is through this ensemble that evolution is able to modify a protein's function by altering its landscape. Hence, the evolution of protein functions involves selective pressures that adjust the sampling of the conformational states. In this work, we focus on elucidating the evolutionary pathway that shaped the function of individual proteins that make-up the mammalian c-type lysozyme subfamily. Using both experimental and computational methods, we map out specific intermolecular interactions that direct the sampling of conformational states and accordingly, also underlie shifts in the landscape that are directly connected with the formation of novel protein functions. By contrasting three representative proteins in the family we identify molecular mechanisms that are associated with the selectivity of enhanced antimicrobial properties and consequently, divergent protein function. Namely, we link the extent of localized fluctuations involving the loop separating helices A and B with shifts in the equilibrium of the ensemble of conformational states that mediate interdomain coupling and concurrently moderate substrate binding affinity. This work reveals unique insights into the molecular level mechanisms that promote the progression of interactions that connect the immune response to infection with the nutritional properties of lactation, while also providing a deeper understanding about how evolving energy landscapes may define present-day protein function.

  14. Vixapatin (VP12, a C-Type Lectin-Protein from Vipera xantina palestinae Venom: Characterization as a Novel Anti-angiogenic Compound

    Directory of Open Access Journals (Sweden)

    Philip Lazarovici


    Full Text Available A C-type lectin-like protein (CTL, originally identified as VP12 and lately named Vixapatin, was isolated and characterized from Israeli viper Vipera xantina palestinae snake venom. This CTL was characterized as a selective α2β1 integrin inhibitor with anti-melanoma metastatic activity. The major aim of the present study was to prove the possibility that this protein is also a potent novel anti-angiogenic compound. Using an adhesion assay, we demonstrated that Vixapatin selectively and potently inhibited the α2 mediated adhesion of K562 over-expressing cells, with IC50 of 3 nM. 3 nM Vixapatin blocked proliferation of human dermal microvascular endothelial cells (HDMEC; 25 nM inhibited collagen I induced migration of human fibrosarcoma HT-1080 cells; and 50 nM rat C6 glioma and human breast carcinoma MDA-MB-231 cells. 1 µM Vixapatin reduced HDMEC tube formation by 75% in a Matrigel assay. Furthermore, 1 µM Vixapatin decreased by 70% bFGF-induced physiological angiogenesis, and by 94% C6 glioma-induced pathological angiogenesis, in shell-less embryonic quail chorioallantoic membrane assay. Vixapatin’s ability to inhibit all steps of the angiogenesis process suggest that it is a novel pharmacological tool for studying α2β1 integrin mediated angiogenesis and a lead compound for the development of a novel anti-angiogenic/angiostatic/anti-cancer drug.

  15. Role of protein-protein interactions in cytochrome P450-mediated drug metabolism and toxicity. (United States)

    Kandel, Sylvie E; Lampe, Jed N


    Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein-protein interactions play a critical role in this process. Historically, the study of CYP-protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein-protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein-protein interactions with CYP enzymes.

  16. Role of cytochrome P sub 450 in the control of the production of erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Fandrey, J.; Seydel, F.P.; Siegers, C.P.; Jelkmann, W. (Medical Univ. of Luebeck (West Germany))


    Effects of agents affecting cytochrome P{sub 450} were studied on the production of erythropoietin (Epo) in cultures of the human hepatoma cell line HepG2. Epo was measured by radioimmunoassay of the culture media after 24 h of incubation. The addition of phenobarbital or 3-methylcholanthrene, which induce cytochrome P{sub 450}, significantly enhanced the formation of Epo. Likewise, the thyroid hormones T{sub 3} and T{sub 4} stimulated the rate of the production of Epo. On the other hand, the formation of Epo was lowered following the addition of diethyl-dithiocarbamate or cysteamine chloride, which inhibit cytochrome P{sub 450}. These findings support the idea that O{sub 2} sensitive hemoproteins of the microsomal mixed-functional oxidases play a role in the control of the synthesis of Epo.

  17. Fluorescence line-narrowing spectra of Zn-cytochrome c. Temperature dependence (United States)

    Koloczek, Henryk; Fidy, Judit; Vanderkooi, Jane M.


    Fluorescence site selection spectra of Zn-substituted cytochrome c and Zn-mesoporphyrin were measured as a function of excitation energy and temperature within 5-60 K. The emission energies determined for Zn-cytochrome c were comparable with those of Zn-mesoporphyrin and mesoporphyrin in polar glassy matrices and with resonance Raman spectra of heme proteins. In the site selection spectra of both Zn-cytochrome c and Zn-mesoporphyrin in polar organic glass an increase in the intensity of zero phonon emission lines was observed with an increase of temperature between 5 and 30 K. The irregular spectral changes were interpreted on the basis of the temperature dependence of the linewidth as well as possible hole-burning phenomena.

  18. A Novel Redox State Heme a Marker in Cytochrome c Oxidase Revealed by Raman Spectroscopy (United States)

    Piccoli, C.; Perna, G.; Scrima, R.; Cela, O.; Rinaldi, R.; Boffoli, D.; Capozzi, V.; Capitanio, N.


    This study was aimed to characterize by Raman spectroscopy (excitation line 633 nm) different redox states of the mitochondrial cytochrome c oxidase. The results obtained from a systematic analysis carried out on the mitochondrial enzyme prepared under redox conditions, differently affecting the valence state of the metal prosthetic groups, and a comparison with homologous bacterial heme-copper oxidases, cytochrome c and pyridine hemo-chrome extract revealed a novel redox state marker specifically linked to the redox transition of heme a, peaking at 1645 cm-1, and tentatively assigned to the C=C and/or C=N streching mode of the imidazole ring of a proxymal histidine ligand. The possible involvment of this redox-linked conformational change in the catalytic activity of cytochrome oxidase is discussed.

  19. SMARTCyp: A 2D Method for Prediction of Cytochrome P450-Mediated Drug Metabolism. (United States)

    Rydberg, Patrik; Gloriam, David E; Zaretzki, Jed; Breneman, Curt; Olsen, Lars


    SMARTCyp is an in silico method that predicts the sites of cytochrome P450-mediated metabolism of druglike molecules. The method is foremost a reactivity model, and as such, it shows a preference for predicting sites that are metabolized by the cytochrome P450 3A4 isoform. SMARTCyp predicts the site of metabolism directly from the 2D structure of a molecule, without requiring calculation of electronic properties or generation of 3D structures. This is a major advantage, because it makes SMARTCyp very fast. Other advantages are that experimental data are not a prerequisite to create the model, and it can easily be integrated with other methods to create models for other cytochrome P450 isoforms. Benchmarking tests on a database of 394 3A4 substrates show that SMARTCyp successfully identifies at least one metabolic site in the top two ranked positions 76% of the time. SMARTCyp is available for download at

  20. Electrochemical and Spectroscopic Study on the Interaction of Cytochrome c with Anionic Lipid Vesicles

    Institute of Scientific and Technical Information of China (English)

    JING,Wei-Guo; LIU,Chang-Wei; TANG,Ji-Lin; WU,Zheng-Yan; DONG,Shao-Jun; WANG,Er-kang


    The structure and the electron-transfer of cytochrome c binding on the anionic lipid vesicles wrer analyzed by electrochemical and various spectroscopic methods.It was found that upon binding to anionic lipid membrane,the formal potential of cytochrome c shifted 30 mV negtively indicating an easier redox interaction than that in its native state.This is due to the local alteration of the coordination and the heme crevice.The structural perturbation in which a molten globule-like state is formed during binding to anionic lipid vesicles is more important.This study may help to understand the mechanism of the electron-transfer reactions of cytochrome c at the mitochondrial membrane.

  1. Reduction of U(VI) and Toxic Metals by Desulfovibrio Cytochrome C3

    Energy Technology Data Exchange (ETDEWEB)

    Wall, Judy D


    The central objective of our proposed research was twofold: 1) to investigate the structure-function relationship of Desulfovibrio desulfuricans (now Desulfovibrio alaskensis G20) cytochrome c3 with uranium and 2) to elucidate the mechanism for uranium reduction in vitro and in vivo. Physiological analysis of a mutant of D. desulfuricans with a mutation of the gene encoding the type 1 tetraheme cytochrome c3 had demonstrated that uranium reduction was negatively impacted while sulfate reduction was not if lactate were the electron donor. This was thought to be due to the presence of a branched pathway of electron flow from lactate leading to sulfate reduction. Our experimental plan was to elucidate the structural and mechanistic details of uranium reduction involving cytochrome c3.

  2. Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis. (United States)

    Domanski, T L; Halpert, J R


    Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

  3. Kinetics and Mechanistic Studies on the Reaction between Cytochrome c and Tea Catechins

    Directory of Open Access Journals (Sweden)

    Lihua Wang


    Full Text Available Green tea is characterized by the presence of an abundance of polyphenolic compounds, also known as catechins, including epicatechin (EC, epigallocatechin (EGC, epicatechin gallate (EGC and epigallocatechin gallate (EGCG. In addition to being a popular beverage, tea consumption has been suggested as a mean of chemoprevention. However, its mode of action is unclear. It was discovered that tea catechins can react with cytochrome c. When oxidized cytochrome c was mixed with catechins commonly found in green tea under non-steady-state conditions, a reduction of cytochrome c was observed. The reaction rate of the catechins was dependent on the pH and the nature of the catechin. The pseudo-first order rate constant obtained increased in the order of EC < ECG < EGC < EGCG, which is consistent with previously reported superoxide reduction activities and Cu2+ reduction activities of tea catechins.

  4. RNA fragments mimicking tRNA analogs interact with cytochrome c. (United States)

    Pawlowska, Roza; Janicka, Magdalena; Jedrzejczyk, Dominika; Chworos, Arkadiusz


    In times, when drug seeking assays focus on the natural molecular triggers and their analogs, a deeper insight into molecular mechanisms governing the initial step of intrinsic apoptosis (cytochrome c release) is essential to suppress the immortality of pathologically changed cells. In this study, we examined RNA molecules mimicking mitochondrial tRNAs interacting with cytochrome c and possibly affecting its cellular function. tRNA analogs were designed and synthesized prior to the conformational analysis and gel assays clearly stating the nucleic acid-protein complex formation. The circular dichroism spectroscopic (CD) and microscale thermophoresis examination revealed the structural and conformational differences between four tRNA analogs in their interactions with cytochrome c. Obtained CD spectra and gel studies resulted in the complex ratio estimation and conclusion that not only the complex formation may be preferential towards specific tRNAs present in the cell, but nucleobase modifications are not essential for such interaction.

  5. Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles. (United States)

    Musatov, Andrey; Siposova, Katarina; Kubovcikova, Martina; Lysakova, Veronika; Varhac, Rastislav


    Bilayered long- and short-chain phospholipid assemblies, known as bicelles, have been widely used as model membranes in biological studies. However, to date, there has been no demonstration of structural or functional viability for the fundamental mitochondrial electron transport complexes reconstituted into or interacting with bicelles. In the present work, bicelles were formed from the mixture of long- and short-chain phospholipids, specifically 14:0 and 6:0 phosphatidylcholines (1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine, (DHPC)). Isolated from bovine heart, cytochrome c oxidase was successfully incorporated into bicelles. Bicelles and cytochrome c oxidase incorporated into bicelles ("proteobicelles") were characterized by absorption spectroscopy, dynamic light scattering, atomic force microscopy, sedimentation velocity and differential scanning calorimetry. It was demonstrated that at total concentration of phospholipids CL = 24 mM and the molar ratio (q) of long-chain DMPC over short-chain DHPC equal to 0.4, the diameter of bicelles formed at neutral pH is in the range of 30-60 nm with the thickness of bicelles of about 4 nm. Adding cytochrome c oxidase to bicelles unified the size of the resulting proteobicelles to about 160 nm. Cytochrome c oxidase in bicelles was fully reducible by artificial donors of electrons, exhibited "normal" reaction with external ligands, and was fully active. Both, sedimentation velocity analysis and temperature-induced denaturation indicated that enzyme in bicelles is monomeric. We concluded that cytochrome c oxidase in bicelles maintains its structural and functional integrity, and that bicelles can be used for more comprehensive investigation of cytochrome c oxidase and most likely other mitochondrial electron transfer complexes.

  6. Isolation and analysis of the genes for cytochrome c oxidase in Paracoccus denitrificans


    Raitio, Mirja; Jalli, Tuulikki; Saraste, Matti


    Synthetic oligonucleotide probes were used to clone two loci from the chromosomal DNA of Paracoccus denitrificans that contain the genes for cytochrome c oxidase (cytochrome aa3). One locus seems to contain four or five genes probably forming an operon. Two of these code for the oxidase subunits II and III. Three open reading frames are found between the COII and COIII genes. The other locus codes for the subunit I. A short open reading frame is found upstream of this gene. All three subunits...



    Taanman, J.W.; Capaldi, R A


    Yeast cytochrome c oxidase has been isolated by ion exchange chromatography using lauryl maltoside (n-dodecyl beta-D-maltoside) as the solubilizing detergent. The enzyme prepared in this way has a heme aa3 concentration of 8-9 nmol/mg of protein and a turnover number in the range of 180-210 s-1 at pH 6.2 in 0.01% lauryl maltoside at 20-degrees-C. Yeast cytochrome c oxidase prepared by any of several previously published methods which use Triton X-100 contains nine subunits. The enzyme isolate...

  8. Hepatocarcinogenic heterocyclic aromatic amines that induce cytochrome P-448 isozymes, mainly cytochrome P-448H (P-450IA2), responsible for mutagenic activation of the carcinogens in rat liver. (United States)

    Degawa, M; Tanimura, S; Agatsuma, T; Hashimoto, Y


    Male F344 rats were treated with hepatocarcinogenic heterocyclic aromatic amines such as amino acid- and protein-pyrolysate components (Trp P-1, Trp P-2, Glu P-1, Glu P-2, A alpha C, MeA alpha C, IQ and MeIQx) and changes in microsomal cytochrome P-450 isozymes in the livers were examined by means of immuno-Western blotting using anti-rat cytochrome P-450 monoclonal antibodies. The results suggested that all chemicals tested induce cytochrome P-448 isozymes, particularly cytochrome P-448H (P-450IA2), which efficiently mediate mutagenic activation of the carcinogens. This was substantiated by the enzymatic analyses with the substrates showing different characters to rat cytochrome P-450 isozyme-mediated mutagenesis.

  9. Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance. (United States)

    Hou, Yuting; An, Jianhong; Deng, Chunyan; Chen, Shu; Xiang, Juan


    The interactions between the redox couple of cytochrome c (Cyt c) and cytochrome c oxidase (COX) were investigated at a mimic redox-modulated interface by using an electrochemical surface plasmon resonance (EC-SPR) system. Although early studies of the binding between COX and Cyt c have been conducted using several techniques in homogeneous solutions, a problem still inherent is that ferro-cytochrome c (Cyt c red), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt c ox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt c red is quantitatively electro-generated from Cyt c ox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt c red/Cyt c ox can be evaluated accurately. In this study, the values of association/dissociation rate constants (k a, k d) for both COX/Cyt c red and COX/Cyt c ox were obtained. The dissociation constants, K D, were finally calculated as 3.33 × 10(-8) mol · L(-1) for COX/Cyt c red and 4.25 × 10(-5) mol · L(-1) for COX/Cyt c ox, respectively. In-situ EC-SPR is promising for better mimicking the in vivo condition that COX is embedded in the inner mitochondrial membrane and Cyt c acts as an electron shuttle in the mobile phase. It is an effective method for the investigation of redox-dependent biomolecular interactions. Graphical Abstract Schematic representation of the experimental designs using EC-SPR system. (a) the Au-Cys-COX SPR chip with SAM layers. (b) redox-modulated Cyt c and its binding onto pre-immobilized COX.

  10. HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity (United States)

    Furge, Laura Lowe; Fletke, Kyle J.


    Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

  11. Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

    NARCIS (Netherlands)

    Schallmey, Anett; den Besten, Gijs; Teune, Ite G. P.; Kembaren, Roga F.; Janssen, Dick B.


    Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzym


    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  13. Characterization of a western North American carnivore community using PCR-RFLP of cytochrome b obtained from fecal samples

    NARCIS (Netherlands)

    Bidlack, Allison L.; Reed, Sarah E.; Palsboll, Per J.; Getz, Wayne M.


    We developed a simple and reliable method to identify carnivore scats to species using PCR and RFLP of a portion of the mtDNA cytochrome b gene, which works for seven of the most common carnivores in western North America. We identified a short (196 bp) polymorphic region of cytochrome b which would

  14. Macrophage-inducible C-type lectin is associated with anti-cyclic citrullinated peptide antibodies-positive rheumatoid arthritis in men

    Institute of Scientific and Technical Information of China (English)

    WU Xin-yu; GUO Jian-ping; YIN Fang-rui; LU Xiao-lan; LI Ru; HE Jing; LIU Xu; LI Zhan-guo


    Background Macrophage-inducible C-type lectin (MINCLE) is an important member of C-type lectin superfamily,which has been shown evidence for susceptibility to arthritis in animal models.We aimed to investigate the possible association of MINCLE with rheumatoid arthritis (RA) susceptibility in Chinese Hart population.Methods Haplotypes from HapMap database (Chinese Hart Beijing,CHB) were used to select tag-single nucleotide polymorphism (SNP) (r2=0.8) residing in MINCLE gene.A total of 563 patients with RA and 404 healthy controls were TagMan genotyped for SNP rs10841845.Association analyses were performed on the whole data set and on RA subsets based on gender difference and the status of anti-cyclic citrullinated peptide (anti-CCP) antibody in RA patients.Association statistics were calculated by age and sex adjusted logistic regression.Results Overall,MINCLE SNP rs10841845 was not associated with susceptibility to RA.However,following anti-CCP stratification,rs10841845 GG genotypes conferred a significantly protective effects against anti-CCP-positive RA (OR 0.65,95% CI 0.430-0.995,P=0.048).Following gender stratification,SNP rs10841845 G allele appeared to insert its RA protective effect only in male patients,both at allele level (G vs.A OR 0.66,95% CI 0.46-0.93,P=0.018) and at genotype level (GG vs.AA+AG,OR 0.429,95% CI 0.20-0.95,P=0.036).Notably,the male RA protective effect of rs10841845 G allele was only seen in anti-CCP-positive RA (G vs.A:OR 0.64,95% CI 0.43-0.96,P=0.029; GG vs.AA+AG:OR 0.375,95% Cl 0.14-0.94,P=0.038).Furthermore,we observed a significant reduction of Disease Activity Score (DAS) 28 score (3.91±0.70 vs.5.66±0.31,P=0.022) and serum C-reactive protein levels (31.64±24.13 vs.91.80±12.02,P=0.012)in male anti-CCP-positive RA patients carrying rs10841845 GG genotype,compared with patients carrying AA+AG genotypes.Conclusions Our study provides the evidence for a gender specific association between MINCLE rs10841845 and RA

  15. A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation. (United States)

    Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd


    Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.

  16. Ammonia inhibits the C-type natriuretic peptide-dependent cyclic GMP synthesis and calcium accumulation in a rat brain endothelial cell line. (United States)

    Konopacka, Agnieszka; Zielińska, Magdalena; Albrecht, Jan


    Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.

  17. A single-CRD C-type lectin from oyster Crassostrea gigas mediates immune recognition and pathogen elimination with a potential role in the activation of complement system. (United States)

    Li, Hui; Zhang, Huan; Jiang, Shuai; Wang, Weilin; Xin, Lusheng; Wang, Hao; Wang, Lingling; Song, Linsheng


    C-type lectins (CTLs), serving as pattern recognition receptors (PRRs), are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins that participate in nonself-recognition and pathogen elimination. In the present study, a single carbohydrate-recognition domain (CRD) CTL was identified from oyster Crassostrea gigas (designated as CgCLec-2). There was only one CRD within the deduced amino acid sequence of CgCLec-2 consisting of 129 amino acid residues. A conserved EPN (Glu246-Pro247-Asn248) motif was found in Ca(2+)-binding site 2 of CgCLec-2. The CgCLec-2 mRNA could be detected in all the examined tissues at different expression levels in oysters. The mRNA expression of CgCLec-2 in hemocytes was up-regulated significantly at 6 h post Vibrio splendidus challenge. The recombinant CgCLec-2 (rCgCLec-2) could bind various Pathogen-Associated Molecular Patterns (PAMPs), including lipopolysaccharide, mannan and peptidoglycan, and displayed strong binding abilities to Vibrio anguillarum, V. splendidus and Yarrowiali polytica and week binding ability to Staphylococcus aureus. It could also enhance the phagocytic activity of oyster hemocytes to V. splendidus and exhibited growth suppression activity against gram-positive bacteria S. aureus but no effect on gram-negative bacteria V. splendidus. Furthermore, the interaction between rCgCLec-2 and rCgMASPL-1 was confirmed by GST Pull down. The results suggested that CgCLec-2 served as not only a PRR in immune recognition but also a regulatory factor in pathogen elimination, and played a potential role in the activation of complement system.

  18. Super-resolution imaging of C-type lectin spatial rearrangement within the dendritic cell plasma membrane at fungal microbe contact sites

    Directory of Open Access Journals (Sweden)

    Michelle S Itano


    Full Text Available Dendritic cells express DC-SIGN and CD206, C-type lectins (CTLs that bind a variety of pathogens and may facilitate pathogen uptake for subsequent antigen presentation. Both proteins form punctate membrane nanodomains (~80 nm on naïve cells. We analyzed the spatiotemporal distribution of CTLs following host-fungal particle contact using confocal microscopy and three distinct methods of cluster identification and measurement of receptor clusters in super-resolution datasets: DBSCAN, Pair Correlation and a custom implementation of the Getis spatial statistic. Quantitative analysis of confocal and super-resolution images demonstrated that CTL nanodomains become concentrated in the contact site relative to non-contact membrane after the first hour of exposure and established that this recruitment is sustained out to four hours. DC-SIGN nanodomains in fungal contact sites exhibit a 70% area increase and a 38% decrease in interdomain separation. Contact site CD206 nanodomains possess 90% greater area and 42% lower interdomain separation relative to non-contact regions. Contact site CTL clusters appear as disk-shaped domains of approximately 150-175 nm in diameter. The increase in length scale of CTL nanostructure in contact sites suggests that the smaller nanodomains on resting membranes may merge during fungal nanodomain structure, or that they become packed closely enough to achieve sub-resolution inter-domain edge separations of < 30 nm. This study provides evidence of local receptor spatial rearrangements on the nanoscale that occur in the plasma membrane upon pathogen binding and may direct important signaling interactions required to recognize and respond to the presence of a relatively large pathogen.

  19. Vascular Tone Regulation Induced by C-Type Natriuretic Peptide: Differences in Endothelium-Dependent and -Independent Mechanisms Involved in Normotensive and Spontaneously Hypertensive Rats (United States)

    Caniffi, Carolina; Cerniello, Flavia M.; Gobetto, María N.; Sueiro, María L.; Arranz, Cristina


    Given that the role of C-type natriuretic peptide (CNP) in the regulation of vascular tone in hypertensive states is unclear, we hypothesized that impaired response of the nitric oxide system to CNP in spontaneously hypertensive rats (SHR) could affect vascular relaxation induced by the peptide in this model of hypertension, and that other endothelial systems or potassium channels opening could also be involved. We examined the effect of CNP on isolated SHR aortas, and the hindlimb vascular resistance (HVR) in response to CNP administration compared to normotensive rats. Aortas were mounted in an isometric organ bath and contracted with phenylephrine. CNP relaxed arteries in a concentration-dependent manner but was less potent in inducing relaxation in SHR. The action of CNP was diminished by removal of the endothelium, inhibition of nitric oxide synthase by Nω-nitro-L-arginine methyl ester, and inhibition of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one in both groups. In contrast, blockade of cyclooxygenase or subtype 2 bradykinin receptor increased CNP potency only in SHR. In both Wistar and SHR, CNP relaxation was blunted by tetraethylammonium and partially inhibited by BaCl2 and iberiotoxin, indicating that it was due to opening of the Kir and BKCa channels. However, SHR seem to be more sensitive to Kir channel blockade and less sensitive to BKCa channel blockade than normotensive rats. In addition, CNP decreases HVR in Wistar and SHR, but the effect of CNP increasing blood flow was more marked in SHR. We conclude that CNP induces aorta relaxation by activation of the nitric oxide system and opening of potassium channels, but the response to the peptide is impaired in conductance vessel of hypertensive rats. PMID:27936197

  20. The C-type natriuretic peptide induces thermal hyperalgesia through a noncanonical Gβγ-dependent modulation of TRPV1 channel. (United States)

    Loo, Lipin; Shepherd, Andrew J; Mickle, Aaron D; Lorca, Ramón A; Shutov, Leonid P; Usachev, Yuriy M; Mohapatra, Durga P


    Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain essentially unknown. Many biological effects of NPs are mediated by guanylate cyclase (GC)-coupled NP receptors, NPR-A and NPR-B, whereas the third NP receptor, NPR-C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR-C can couple to specific Gα(i)-Gβγ-mediated intracellular signaling cascades in numerous cell types. We found that NPR-C is coexpressed in transient receptor potential vanilloid-1 (TRPV1)-expressing mouse dorsal root ganglia (DRG) neurons. NPR-C can be coimmunoprecipitated with Gα(i), and C-type natriuretic peptide (CNP) treatment induced translocation of protein kinase Cε (PKCε) to the plasma membrane of these neurons, which was inhibited by pertussis toxin pretreatment. Application of CNP potentiated capsaicin- and proton-activated TRPV1 currents in cultured mouse DRG neurons and increased their firing frequency, an effect that was absent in DRG neurons from TRPV1(-/-) mice. CNP-induced sensitization of TRPV1 activity was attenuated by pretreatment of DRG neurons with the specific inhibitors of Gβγ, phospholipase C-β (PLCβ), or PKC, but not of protein kinase A, and was abolished by mutations at two PKC phosphorylation sites in TRPV1. Furthermore, CNP injection into mouse hindpaw led to the development of thermal hyperalgesia that was attenuated by administration of specific inhibitors of Gβγ or TRPV1 and was also absent in TRPV1(-/-) mice. Thus, our work identifies the Gβγ-PLCβ-PKC-dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity.

  1. Simulating the slow to fast switch in cytochrome c oxidase catalysis by introducing a loop flip near to the enzyme's cytochrome c (substrate) binding site. (United States)

    Alleyne, Trevor; Ignacio, Diane N; Sampson, Valerie B; Ashe, Damian; Wilson, Michael


    The mitochondrial enzyme cytochrome c oxidase catalyses the reduction of molecular oxygen in the critical step of oxidative phosphorylation that links the oxidation of food consumed to ATP production in cells. The enzyme catalyses the reduction of oxygen at two vastly different rates that are thought to be linked to two different conformations but the conformation of the 'fast enzyme' remains obscure. In this study we demonstrated how oxygen binding at haem a3 could trigger long distance conformational changes and then simulated a conformational change in an eight residue loop near to the enzyme's substrate (cytochrome c) binding site. We then used this modified COX to simulate a stable COX-cytochrome c ES-complex. Compared to ES-complexes formed in the absence of the conformation change, the distance between the redox centres of the two proteins was reduced by half and instead of nine, only four COX amino acid residues were found along the axis linking the electron entry point and the CuA redox centre of COX: We proposed that intramolecular electron transfer in COX occurs via a charge/hydrogen relay system involving these four residues. We suggest that the conformational change and resulting shortened electron pathway are features of fast-acting COX. This article is protected by copyright. All rights reserved.

  2. [Multiphasic character of the kinetics of cytochrome P-450 destruction in microsomal LM2- and LM4-forms in the reaction with cumene hydroperoxide]. (United States)

    Akhrem, A A; Eremin, A N; Usanov, S A; Metelitsa, D I


    Cytochrome P-450 destruction kinetics by cumene hydroperoxide has been studied in LM2 and LM4 microsomal and purified forms. Three destruction phases of cytochrome P-450 were shown to be observed irrespective of the source and integration degree, cytochrome P-450 pseudomonomolecular consumption rate constants being dependent in a complex way upon the cumene hydroperoxide initial concentration. The radical character of cytochrome P-450 destruction was proved by experiments with 1-naphtol. The mechanism of radicals formation is discussed.

  3. The role of cytochrome c on apoptosis induced by Anagrapha falcifera multiple nuclear polyhedrosis virus in insect Spodoptera litura cells.

    Directory of Open Access Journals (Sweden)

    Kaiyu Liu

    Full Text Available There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line. In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell-free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.

  4. Single-molecule Mapping of Long-range Electron Transfer for a Cytochrome b562 Variant

    DEFF Research Database (Denmark)

    Della Pia, Eduardo Antonio; Chi, Qijin; Jones, D. Dafydd;


    Cytochrome b562 was engineered to introduce a cysteine residue at a surface-exposed position to facilitate direct self-assembly on a Au(111) surface. The confined protein exhibited reversible and fast electron exchange with a gold substrate over a distance of 20 Å between the heme redox center an...

  5. Redox tuning of cytochrome b562 through facile metal porphyrin substitution

    DEFF Research Database (Denmark)

    Della Pia, Eduardo Antonio; Chi, Qijin; Elliott, Martin;


    The biologically and nanotechnologically important heme protein cytochrome b562 was reconstructed with zinc and copper porphyrins, leading to significant changes in the spectral, redox and electron transfer properties. The Cu form shifts the redox potential by +300 mV and exhibits high electron t...

  6. Cytochrome P450-mediated hepatic metabolism of new fluorescent substrates in cats and dogs.

    NARCIS (Netherlands)

    van Beusekom, C.D.; Schipper, L.; Fink-Gremmels, J.


    This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities

  7. Effects of nuclear translocation of tissue transglutaminase and the release of cytochrome C on hepatocyte apoptosis

    Institute of Scientific and Technical Information of China (English)

    宋良文; 马宪梅; 李扬; 崔雪梅; 王晓民


    Objective To assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes. Methods Hepatocytes isolated from tissue transglutaminase gene knock-out rats and mice were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by 14 C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.Results TTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.Conclusions Increased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

  8. Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego. (United States)

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


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

  9. Mitochondrial cytochrome b sequence variations and phylogeny of the East Asian bagrid catfishes

    Institute of Scientific and Technical Information of China (English)


    The mitochondrial DNA cytochrome b gene was sequenced from 8 bagrid catfishes in China. Aligned with cytochrome b sequences from 9 bagrid catfishes in Japan, Korea and Russia retrieved from GenBank, and selected Silurus meridionalis, Liobagrus anguillicauda, Liobagrus reini and Phenacogrammus interruptus as outgroups, we constructed a matrix of 21 DNA sequences. The Kimura's two-parameter distances were calculated and molecular phylogenetic trees were constructed by using the maximum parsimony (MP) and neighbor-joining (NJ) methods. The results show that (i) there exist 3-bp deletions of mitochondrial cytochrome b gene compared with cypriniforms and characiforms; (ii) the molecular phylogenetic tree suggests that bagrid catfishes form a monophyletic group, and the genus Mystus is the earliest divergent in the East Asian bagrid catfishes, as well as the genus Pseudobagrus is a monophyletic group but the genus Pelteobagrus and Leiocassis are complicated; and (iii) the evolution rate of the East Asian bagrids mitochondrial cytochrome b gene is about 0.18%~0.30% sequence divergence per million years.

  10. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jian-Ching; Rebrin, Igor [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States); Klichko, Vladimir; Orr, William C. [Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275 (United States); Sohal, Rajindar S., E-mail: [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States)


    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  11. The reaction of Pseudomonas aeurginosa cytochrome c oxidase with sodium metabisulphite. (United States)

    Parr, S R; Wilson, M T; Greenwood, C


    Spectrophotometric evidence is presented for the formation of a complex between metabisulphite and reduced Pseudomonas aeruginosa cytochrome c oxidase. The effects of metabisulphite on the recombination of CO with the reduced enzyme are discussed in terms of alternate binding sites for S(2)O(5) (2-) and CO.

  12. Permethrin Induces Overexpression of Cytochrome c Oxidase Subunit 3 in Aedes aegypti (United States)

    Using quantitative PCR (QPCR), the relative transcriptional levels of cytochrome c oxidase subunit 3 (CO3) were studied in Aedes aegypti (L.) in response to treatments with acetone, permethrin, or fipronil. The transcriptional levels of CO3 were significantly (p <0.05) higher in acetone-treated Ae. ...

  13. The binding of cytochrome c to neuroglobin: A docking and surface plasmon resonance study

    DEFF Research Database (Denmark)

    Bønding, Signe Helbo; Henty, K.; Dingley, A.J.;


    It has recently been proposed that the role of neuroglobin in the protection of neurons from ischaemia induced cell death requires the formation of a transient complex with cytochrome c. No such complex has yet been isolated. Here, we present the results of soft docking calculations, which indica...

  14. Nitrogen inversion barriers affect the N-oxidation of tertiary alkylamines by cytochromes P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Jørgensen, Martin S.; Jacobsen, T.A.;


    Calculations: Cytochrome P450 enzymes facilitate a number of chemically different reactions. For example, amines can be either N-dealkylated or N-oxidized, but it is complex to rationalize which of these competing reactions occurs. It is shown that the barrier for inversion of the alkylamine nitr...

  15. Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c(4)

    DEFF Research Database (Denmark)

    Andersen, Niels Højmark; Jensen, Thomas Jon; Nørgaard, Allan;


    F stutzeri cytochrome c. is a di-haem protein, composed of two globular domains each with His-Met coordinated haem. and a hydrogen bond network between the domains. The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination, and ...

  16. Covalently Immobilised Cytochrome C Imaged by In Situ Scanning Tunnelling Microscopy

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov; Olesen, Klaus G.; Danilov, Alexey I.;


    In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms...


    Metabolism of two triazole-containing antifungal azoles was studied using expressed human and rat cytochrome P450s (CYP) and liver microsomes. Substrate depletion methods were used due to the complex array of metabolites produced from myclobutanil and triadimefon. Myclobutanil wa...

  18. Correlates of Cytochrome P450 1A1 Expression in Bottlenose Dolphin (Tursiops truncatus) Integument Biopsies

    NARCIS (Netherlands)

    Wilson, J.Y.; Wells, R.; Anguilar, A.; Borrell, A.; Tornero, V.; Reijnders, P.J.H.; Moore, M.


    Integument biopsy is a nondestructive method for sampling free-ranging cetaceans, which allows for the determination of both contaminant concentrations and biomarker responses. Cytochrome P450 1A1 (CYP1A1) expression is induced by polycyclic aromatic hydrocarbons and planar halogenated aromatic hydr


    We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

  20. Electron transfer patterns of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri

    DEFF Research Database (Denmark)

    Raffalt, Anders Christer; Schmidt, L.; Christensen, Hans Erik Mølager;


    We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c(4) by [Co(bipy)(3)](2- 3-) (bipy = 2,2'-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential ...

  1. Screening and identification of novel cytochrome P450s in ticks (United States)

    Cytochrome P450s are the major phase I drug metabolizing enzymes found in most species, including those belonging to the phylum Arthropoda. Much of the work within the area of xenobiotic metabolism in this phylum has centered on mosquito species such as Anopheles gambiae due to their role as vectors...

  2. In vitro metabolism of genistein and tangeretin by human and murine cytochrome p450s

    DEFF Research Database (Denmark)

    Breinholt, Vibeke; Rasmussen, Salka; Brøsen, Kim;


    Recombinant cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6 enzymes obtained from Escherichia coli and human liver microsomes samples were used to investigate the ability of human CYP enzymes to metabolize the two dietary flavonoids, genistein and tangeretin. Analysis of the metabolic profile from inc...

  3. Prediction of activation energies for aromatic oxidation by cytochrome P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Ryde, Ulf; Olsen, Lars


    We have estimated the activation energy for aromatic oxidation by compound I in cytochrome P450 for a diverse set of 17 substrates using state-of-the-art density functional theory (B3LYP) with large basis sets. The activation energies vary from 60 to 87 kJ/mol. We then test if these results can...

  4. Consequences of Vitamin D and xenobiotic metabolism by cytochrome P450 in HIV infection

    NARCIS (Netherlands)

    Bout-van den Beukel, C. van den


    Antiretroviral drugs (ARVs) and medicinal herbs as well as vitamin D are metabolized by the cytochrome P450 enzyme system in the liver. This thesis focuses on the interaction between these compounds. We also explored the hypothesis that HIV-patients might develop insufficiënt vitamin D levels as a r

  5. Functional implications of pigments bound to a cyanobacterial cytochrome b6f complex. (United States)

    Wenk, Stephan-Olav; Schneider, Dirk; Boronowsky, Ute; Jäger, Cornelia; Klughammer, Christof; de Weerd, Frank L; van Roon, Henny; Vermaas, Wim F J; Dekker, Jan P; Rögner, Matthias


    A highly purified cytochrome b(6)f complex from the cyanobacterium Synechocystis sp. PCC 6803 selectively binds one chlorophyll a and one carotenoid in analogy to the recent published structure from two other b(6)f complexes. The unknown function of these pigments was elucidated by spectroscopy and site-directed mutagenesis. Low-temperature redox difference spectroscopy showed red shifts in the chlorophyll and carotenoid spectra upon reduction of cytochrome b(6), which indicates coupling of these pigments with the heme groups and thereby with the electron transport. This is supported by the correlated kinetics of these redox reactions and also by the distinct orientation of the chlorophyll molecule with respect to the heme cofactors as shown by linear dichroism spectroscopy. The specific role of the carotenoid echinenone for the cytochrome b(6)f complex of Synechocystis 6803 was elucidated by a mutant lacking the last step of echinenone biosynthesis. The isolated mutant complex preferentially contained a carotenoid with 0, 1 or 2 hydroxyl groups (most likely 9-cis isomers of beta-carotene, a monohydroxy carotenoid and zeaxanthin, respectively) instead. This indicates a substantial role of the carotenoid - possibly for strucure and assembly - and a specificity of its binding site which is different from those in most other oxygenic photosynthetic organisms. In summary, both pigments are probably involved in the structure, but may also contribute to the dynamics of the cytochrome b(6)f complex.

  6. Human cytochrome p450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Punt, A.; Boersma, M.G.; Bogaards, J.J.P.; Fiamegos, Y.C.; Schilter, B.; Bladeren, van P.J.; Cnubben, N.H.P.; Rietjens, I.M.C.M.


    Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1 '-hydroxyestragole were identified and compared to the enzymes of importance for 1'-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that

  7. Human cytochrome P450 enzyme specificity for bioactivation of safrole to the proximate carcinogen 1'-hydroxysafrole

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Bogaards, J.J.P.; Awad, H.M.; Boersma, M.G.; Brand, W.; Fiamegos, Y.C.; Beek, van T.A.; Alink, G.M.; Sudhölter, E.J.R.; Cnubben, N.H.P.; Rietjens, I.M.C.M.


    In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1'-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1'-hydroxylation of safrole was characterized in a variety of in vitro te

  8. Human cytochrome P450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Punt, A.; Boersma, M.G.; Bogaards, J.J.P.; Fiamegos, Y.C.; Schilter, B.; Bladeren, P.J. van; Cnubben, N.H.P.; Rietjens, I.M.C.M.


    Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1′-hydroxyestragole were identified and compared to the enzymes of importance for 1′-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that


    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  10. Comparative kinetics of Qi site inhibitors of cytochrome bc1 complex: picomolar antimycin and micromolar cyazofamid. (United States)

    Li, Hui; Zhu, Xiao-Lei; Yang, Wen-Chao; Yang, Guang-Fu


    Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate-cytochrome c reductase (SCR) with Ki  = 0.033 ± 0.00027 nm and non-competitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of SCR with Ki  = 12.90 ± 0.91 μm and a non-competitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2 . Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63 kcal/mol. Antimycin formed an H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow tight-binding inhibitor, whereas cyazofamid is a classic inhibitor.

  11. Electrochemistry in the Mimicry of Oxidative Drug Metabolism by Cytochrome P450s

    NARCIS (Netherlands)

    Nouri-Nigjeh, Eslam; Bischoff, Rainer; Bruins, Andries P.; Permentier, Hjalmar P.


    Prediction of oxidative drug metabolism at the early stages of drug discovery and development requires fast and accurate analytical techniques to mimic the in vivo oxidation reactions by cytochrome P450s (CYP). Direct electrochemical oxidation combined with mass spectrometry, although limited to the

  12. Vectorial nature of redox Bohr effects in bovine heart cytochrome c oxidase. (United States)

    Capitanio, N; Capitanio, G; De Nitto, E; Papa, S


    The vectorial nature of redox Bohr effects (redox-linked pK shifts) in cytochrome c oxidase from bovine heart incorporated in liposomes has been analyzed. The Bohr effects linked to oxido-reduction of heme a and CuB display membrane vectorial asymmetry. This provides evidence for involvement of redox Bohr effects in the proton pump of the oxidase.

  13. The role of cytochrome P450s in polycyclic aromatic hydrocarbon carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Polzer, R.J.


    Metabolic activation of polycyclic aromatic hydrocarbons (PAH) to carcinogenic diol epoxides has been determined to be a critical step in tumor initiation by PAH. The key enzyme(s) involved in the metabolic activation are members of the cytochrome P450 superfamily. Two distinct isoforms of cytochrome P450 have been determined to be induced upon treatment of cells in culture with benzo(a)pyrene (B(a)P) by use of Immobilized Artificial Membrane Column High Performance Liquid Chromatography, Western blotting, Northern blotting, and in vitro metabolism studies. Cytochrome P4501A is involved in the metabolism of PAH in the human hepatoma cell line, HepG2; the human mammary carcinoma cell line, MCF-7; and the mouse hepatoma cell line; Hepa-1; whereas cytochrome P450EF is involved in this metabolism in both secondary hamster and mouse embryo cell cultures. Induction of cytochrome P450s by B(a)P generally leads to an increased metabolism of tritiated B(a)P, DMBA, and DB(a,1)P to water-soluble metabolities and to the formation of PAH-DNA adducts, suggesting that induction by B(a)P alters the metabolism of PAH to metabolic activation. DMBA induction of cytochrome P450s leads to various changes in metabolism and PAH-DNA binding and these changes were both cell and PAH specific. These results suggest that DMBA can shift metabolism of certain PAH towards metabolic activation in some cells, while in other cells DMBA or one of its metabolities can compete with other PAH for metabolic activation. UDP-glucuronosyl-transferase and epoxide hydrase do not have significant roles in detoxifying proximate or ultimate carcinogenic PAH metabolites, however, sulfotransferase and glutathione-S-transferase do detoxify proximate and ultimate carcinogenic metabolities in the HepG2 cell line. Finally, attempts to inhibit B(a)P metabolism and DNA-binding in intact cells in culture through conjugation of inhibitory cytochrome P4501A1 antibodies to insulin or folic acid were examined.

  14. Photoaffinity labeling of cytochrome P4501A1 with azidocumene: identification of cumene hydroperoxide binding region. (United States)

    Cvrk, T; Strobel, H W


    Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which cumene hydroperoxide binds, we prepared an analog of cumene hydroperoxide for use as a photoaffinity label. p-Azido-isopro-pylbenzene (azidocumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [3H]-azidocumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492-503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494-512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501-504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501-L502-K503, is the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A

  15. 老年DM2患者认知功能障碍与C型利钠肽的关系%The Relationship Between C-type Natriuretic Peptide and Cognitive Impairment in Older Patients with Type 2 Diabetes

    Institute of Scientific and Technical Information of China (English)

    李新玲; 朱向阳; 黄怀宇; 金艳


    目的:研究老年2型糖尿病(DM2)患者认知障碍与C型利钠肽(C-type natriuretic peptide,CNP)的关系,进一步探讨糖尿病认知障碍的发病机制.方法 80例60岁以上的DM2患者,根据蒙特利尔认知评估量表(Montreal Cognitive Assessment,MoCA)分为认知障碍组(31例)和非认知障碍组(49例).另取80例正常对照组.采用放射免疫分析血浆CNP水平,分析各组间血浆CNP水平的变化及相关因素.结果 与正常对照组比较,DM2非认知障碍组血浆CNP水平明显增高,差异存在统计学意义(P<0.01).而DM2认知障碍组与正常对照组及非认知障碍组比较,血浆CNP水平明显降低,差异均存在统计学意义(P<0.01).老年DM2认知障碍患者的MoCA评分与血浆CNP水平呈正相关(r=0.513,P<0.01),具有统计学意义.结论 CNP在DM2早期可代偿性分泌增多,在糖尿病合并认知障碍时则失代偿性分泌减少.CNP与糖尿病认知障碍密切相关,糖尿病血管病变是糖尿病认知障碍的重要发病机制.%Objective To investigate the relationship between C-type natriuretic peptide and cognitive impairment in older patients with type 2 diabetes, and to explore the pathogenesis of diabetic cognitive impairment. Methods According to the Montreal Cognitive Assessment (MoCA) scores,80 type 2 diabetic patients over the age of 60 years were divided into two groups,one group including 31 cases with cognitive impairment,the other 49 patients with non-cognitive impairment. And 80 normal participants were selected as the control group. Plasma level of C-type natriuretic peptide was measured by radio-immunity assay in all subjects. The changes and associations of the plasma C-type natriuretie peptide level among three groups was analyzed. R6SUlt In the non-cognitive impairment group, plasma level of C-type natriuretic peptide was higher than that in the control group(P <0. 01) . But the plasma level of C-type natriuretic peptide in the cognitive impairment group was

  16. A novel C-type lectin, Nattectin-like protein, with a wide range of bacterial agglutination activity in large yellow croaker Larimichthys crocea. (United States)

    Lv, Changhuan; Zhang, Dongling; Wang, Zhiyong


    C-type lectins (CTLs) are generally recognized as a superfamily of Ca(2+)-dependent carbohydrate-binding proteins, which serve as pattern recognition receptors (PRRs) in innate immunity of vertebrates. In this study, the molecular characterization and immune roles of a novel CTL from Larimichthys crocea (designated as LcNTC) were investigated. LcNTC is a novel protein that shared 33%-49% homology with other teleosts CTLs. The full-length cDNA of LcNTC was composed of 859 bp with a 465 bp open reading frame encoding a putative protein of 154 residues. LcNTC contained a single CRD with four conserved disulfide-bonded cysteine residues (Cys(57)-Cys(148), Cys(126)-Cys(140)) and EPN/AND motifs instead of invariant EPN/WND motifs required for carbohydrate-binding specificity and constructing Ca(2+)-binding sites. LcNTC mRNA was detected in all examined tissues with the most abundant in the gill. After challenged with poly I:C and Vibrio parahaemolyticus, the temporal expression of LcNTC was significantly up-regulated in the liver, spleen and head-kidney. LcNTC transcripts were also induced in the gill, skin, spleen and head-kidney post-infection with Cryptocaryon irritans. The recombinant LcNTC (rLcNTC) purified from Escherichia coli BL21 (DE3) exhibited strong agglutination activity against erythrocytes from human, rabbit and large yellow croaker in a Ca(2+)-dependent manner, and the agglutination could be inhibited by D-Mannose, D-Glucose, D-Fructose, α-Lactose, D-Maltose and LPS. Positive microbial agglutination activities of rLcNTC were observed against all tested bacteria in the presence of Ca(2+), including Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus and Micrococcus lysoleikticus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila). These findings collectively indicated that LcNTC might be involved in the innate immunity of L. crocea as a PRR.

  17. Molecular cloning of a new secreted sulfated mucin-like protein with a C-type lectin domain that is expressed in lymphoblastic cells. (United States)

    Bannwarth, S; Giordanengo, V; Lesimple, J; Lefebvre, J C


    We have previously demonstrated hyposialylation of the two major CD45 and leukosialin (CD43) molecules at the surface of latently human immunodeficiency virus type 1-infected CEM T cells (CEMLAI/NP), (Lefebvre, J. C., Giordanengo, V., Doglio, A., Cagnon, L., Breittmayer, J. P., Peyron, J. F., and Lesimple, J. (1994) Virology 199, 265-274; Lefebvre, J. C., Giordanengo, V., Limouse, M., Doglio, A., Cucchiarini, M., Monpoux, F., Mariani, R., and Peyron, J. F. (1994) J. Exp. Med. 180, 1609-1617). Searching to clarify mechanism(s) of hyposialylation, we observed two sulfated secreted glycoproteins (molecular mass approximately 47 and approximately 40 kDa) (P47 and P40), which were differentially sulfated and/or differentially secreted in the culture supernatants of CEMLAI/NP cells when compared with parental CEM cells. A hybridoma clone (7H1) resulting from the fusion between CEMLAI/NP and human embryonic fibroblasts MRC5 cells produced very large amounts of P47 that was purified using Jacalin lectin (specific for O-glycans) and microsequenced. Cloning of P47 was achieved using a CEMLAI/NP cDNA library screened with a degenerate oligonucleotide probe based on its NH2-terminal amino acid sequence. A single open reading frame encoding a protein of 323 amino acids was deduced from the longest isolated recombinant (1.4 kilobase). P47 is a secreted sulfated protein. It carries an NH2-terminal RGD (Arg-Gly-Asp) triplet, a striking alpha-helical leucine zipper composed of six heptads, and a C-terminal C-type lectin domain. The NH2-terminal portion is rich in glutamic acids with a predicted pI of 3.9. In addition, a hinge region with numerous condensed potential sites for O-glycan side chains, which are also the most likely sulfation sites, is located between the RGD and leucine zipper domains. Transcripts were detected in lymphoid tissues (notably bone marrow) and abundantly in T and B lymphoblastoid but very faintly in monocytoid cell lines.

  18. Proton-pumping mechanism of cytochrome c oxidase: a kinetic master-equation approach. (United States)

    Kim, Young C; Hummer, Gerhard


    Cytochrome c oxidase is an efficient energy transducer that reduces oxygen to water and converts the released chemical energy into an electrochemical membrane potential. As a true proton pump, cytochrome c oxidase translocates protons across the membrane against this potential. Based on a wealth of experiments and calculations, an increasingly detailed picture of the reaction intermediates in the redox cycle has emerged. However, the fundamental mechanism of proton pumping coupled to redox chemistry remains largely unresolved. Here we examine and extend a kinetic master-equation approach to gain insight into redox-coupled proton pumping in cytochrome c oxidase. Basic principles of the cytochrome c oxidase proton pump emerge from an analysis of the simplest kinetic models that retain essential elements of the experimentally determined structure, energetics, and kinetics, and that satisfy fundamental physical principles. The master-equation models allow us to address the question of how pumping can be achieved in a system in which all reaction steps are reversible. Whereas proton pumping does not require the direct modulation of microscopic reaction barriers, such kinetic gating greatly increases the pumping efficiency. Further efficiency gains can be achieved by partially decoupling the proton uptake pathway from the active-site region. Such a mechanism is consistent with the proposed Glu valve, in which the side chain of a key glutamic acid shuttles between the D channel and the active-site region. We also show that the models predict only small proton leaks even in the absence of turnover. The design principles identified here for cytochrome c oxidase provide a blueprint for novel biology-inspired fuel cells, and the master-equation formulation should prove useful also for other molecular machines. .

  19. Mitochondrial cytochrome c oxidase: mechanism of action and role in regulating oxidative phosphorylation. (United States)

    Wilson, David F; Vinogradov, Sergei A


    Mitochondrial oxidative phosphorylation has a central role in eukaryotic metabolism, providing the energy (ATP) required for survival. Regulation of this important pathway is, however, still not understood, largely due to limitations in the ability to measure the essential metabolites, including oxygen (pO2, oxygen pressure), ADP, and AMP. In addition, neither the mechanism of oxygen reduction by mitochondrial cytochrome c oxidase nor how its rate is controlled is understood, although this enzyme determines the rate of oxygen consumption and thereby the rate of ATP synthesis. Cytochrome c oxidase is responsible for reduction of molecular oxygen to water using reducing equivalents donated by cytochrome c and for site 3 energy coupling in oxidative phosphorylation. A mechanism-based model of the cytochrome c oxidase reaction is presented in which transfer of reducing equivalents from the lower- to the higher-potential region of the coupling site occurs against an opposing energy barrier, Q. The steady-state rate equation is fitted to data for the dependence of mitochondrial respiratory rate on cytochrome c reduction, oxygen pressure (pO2), and [ATP]/[ADP][Pi] at pH 6.5 to 8.35 (where Pi is inorganic phosphate). The fit of the rate expression to the experimental data is very good for all experimental conditions. Levels of the intermediates in oxygen reduction in the oxidase reaction site have been calculated. An intermediate in the reaction, tentatively identified as peroxide, bridged between the iron and copper atoms of the reaction site has a central role in coupling mitochondrial respiration to the [ATP]/[ADP][Pi].

  20. Modification of the spectral properties of cytochrome b in mutants of Saccharomyces cerevisiae resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Mapping at two distinct genetic loci of the split mitochondrial gene of cytochrome b. (United States)

    Briquet, M; Goffeau, A


    The effects of five inhibitors of the cytochrome bc1 complex: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), 2-n-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO), antimycin A, funiculosin and mucidin were measured in submitochondrial particles of strains of the yeast Saccharomyces cerevisiae belonging to two classes of diuron-resistant mutants Diu 1 and Diu 2 which are modified in different exons of the split mitochondrial gene of cytochrome b. 1. The oxidation of NADH and of cytochrome b-561 exhibits a similar resistance to diuron and HpHOQnO in Diu 1 and Diu 2 mutants. 2. No extra reduction of cytochrome b-561 and cytochrome b-565 is observed in the presence of diuron and HpHOQnO. 3. Both Diu 1 and Diu 2 mutants exhibit the red shift of cytochrome b-561 induced by concentrations of HpHOQno 2 -- 3-times higher than those required in the parental strains. 4. The spectral and respiratory effects of antimycin A, funiculosin and mucidin and generally similar in the diuron-resistant mutants and in their parental strains. However a cross-resistance between diuron and antimycin A is indicated in one Diu 2 mutant. 5. From the combined genetic and biochemical data it is concluded that the interaction of diuron and HpHOQnO with cytochrome b is mediated by at least two specific amino acids located apart in the central region of the apocytochrome b peptide coded by mitochondrial DNA. These two amino acids control tightly the extra reduction of cytochromes b-565 and b-561 as well as the flow of electrons through the bc1 complex. However the binding of HpHOQnO required for the expression of the red shift of cytochrome b-561 is only slightly affected by the diu-1 and diu-2 mutations.

  1. Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

    Directory of Open Access Journals (Sweden)

    Manik C Ghosh

    Full Text Available Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

  2. The role of highly purified forms of rat liver cytochrome P-450 in the dimethylation of dimethylnitrosamine and its activation to mutagens. (United States)

    Masson, H A; Ioannides, C; Gibson, G G


    Highly purified NADPH-cytochrome P-450 reductase and the major phenobarbital (PB) and beta-naphthoflavone (beta NF) forms of cytochrome P-450 were used in reconstituted systems to study the demethylation and subsequent activation of dimethylnitrosamine (DMN) to mutagenic intermediates. Both forms of cytochrome P-450 were active in the demethylation of DMN, cytochrome P-450 from PB-treated animals being more efficient, generating nearly twice as much formaldehyde per nmol of haemoprotein. Neither form of the cytochrome could activate DMN to mutagens in the Ames test. These findings indicate that DMN demethylation does not lead to its activation to mutagenic products.

  3. A temperature-jump study of the reaction between azurin and cytochrome c oxidase from Pseudomonas aeruginosa. (United States)

    Brunori, M; Parr, S R; Greenwood, C; Wilson, M T


    The electron-transfer reaction between azurin and the cytochrome oxidase from Pseudomonas aeruginosa was investigated by temperature-jump relaxation in the absence of O2 and in the presence of CO. The results show that: (i) reduced azurin exists in two forms in equilibrium, only one of which is capable of exchanging electrons with the Pseudomonas cytochrome oxidase, in agreement with M. T. Wilson, C. Greenwood, M. Brunori & E. Antonini (1975) (Biochem. J. 145, 449-457); (ii) the electron transfer between azurin and Pseudomonas cytochrome oxidase occurs within a molecular complex of the two proteins; this internal transfer becomes rate-limiting at high reagent concentrations.

  4. 树突状细胞相关凝集素受体1和2在真菌免疫领域的研究进展%DC-associated C-type lecxin-1 and DC-associated C-type lecxin-2 in antifungal dfences

    Institute of Scientific and Technical Information of China (English)

    黄晓强; 陈剑


    树突状细胞相关凝集素受体1和2(Dectin1和2)是2C型凝集素受体家族(CLR)的重要成员,作为模式识别受体( PRRs),其有效地识别病原体相关分子模式(PAMPs);Dectin-1识别β-葡聚糖,通过自身免疫受体络氨酸激活基序( ITAM)向胞内转导信号;Dectin-2识别α-甘露聚糖,通过耦联FcRγ链上的ITAM结构转导信号.ITAM招募并激活非受体络氨酸蛋白激酶(Syk),后者激活MAPKs或介导CARD9-Malt1-Bcl10复合体组装,激活核因子-κB(NF-κB),诱导合成炎症因子等一系列细胞活动.其配体β-葡聚糖和甘露聚糖都是真菌细胞壁的主要成分.近年来研究表明,Dectin-1和Dectin-2受体在真菌免疫防御中具有重要作用.%DC-associated C-type lectin-1 and 2 (Dectin-1 and Dectin-2) are pivotal C-type lectin family members,both of them function as pattern recognition receptors (PRRs) to initiate innate and adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs).Dectin-1 recognizes β-glucans and signals with its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-like motif,whereas Dectin-2 recognizes α-mannans and signals through association with the ITAM-containing Fc receptor γ chain.Upon ligand ligation,spleen tyrosine kinase (Syk) is recruited to the ITAM.Syk mediates the activation of MAPKs;or induces the formation of the CARD9-Malt1-Bcl10 complex,resulting in the activation of nuclear factor-κB (NF-κB).Recert studies indicate that Dectin-1 and Dectin-2 play important roles in immune defense against fungi.

  5. Neoplastic lesions of the human liver in relation to the activity of the cytochrome P-450 dependent monooxygenase system. (United States)

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


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

  6. Mitochondrial cytochrome b sequence data are not an improvement for species identification in scleractinian corals

    Directory of Open Access Journals (Sweden)

    John P. Wares


    Full Text Available There are well-known difficulties in using the cytochrome oxidase I (COI mitochondrial gene region for population genetics and DNA barcoding in corals. A recent study of species divergence in the endemic Caribbean genus Agaricia reinforced such knowledge. However, the growing availability of whole mitochondrial genomes may help indicate more promising gene regions for species delineation. I assembled the whole mitochondrial genome for Agaricia fragilis from Illumina single-end 250 bp reads and compared this sequence to that of the congener A. humilis. Although these data suggest that the cytochrome b (CYB gene region is more promising, comparison of available CYB sequence data from scleractinian and other reef-building corals indicates that multilocus approaches are still probably necessary for phylogenetic and population genetic analysis of recently-diverged coral taxa.

  7. Characterization of triptolide hydroxylation by cytochrome P450 in human and rat liver microsomes. (United States)

    Li, W; Liu, Y; He, Y-Q; Zhang, J-W; Gao, Y; Ge, G-B; Liu, H-X; Huo, H; Liu, H-T; Wang, L-M; Sun, J; Wang, Q; Yang, L


    Triptolide, the primary active component of a traditional Chinese medicine Tripterygium wilfordii Hook F, has a wide range of pharmacological activities. In the present study, the metabolism of triptolide by cytochrome P450s was investigated in human and rat liver microsomes. Triptolide was converted to four metabolites (M-1, M-2, M-3, and M-4) in rat liver microsomes and three (M-2, M-3, and M-4) in human liver microsomes. All the products were identified as mono-hydroxylated triptolides by liquid chromatography-mass spectrometry (LC-MS). The studies with chemical selective inhibitors, complementary DNA-expressed human cytochrome P450s, correlation analysis, and enzyme kinetics were also conducted. The results demonstrate that CYP3A4 and CYP2C19 could be involved in the metabolism of triptolide in human liver, and that CYP3A4 was the primary isoform responsible for its hydroxylation.

  8. Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase. (United States)

    Lehninger, A L; Reynafarje, B; Costa, L


    The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.

  9. New cytochrome P450 isoforms as cancer biomarkers and targets for chemopreventive and chemotherapeutic agents

    Directory of Open Access Journals (Sweden)

    Hanna Szaefer


    Full Text Available Cytochromes P450 (P450 are a multigene family of enzymes possessing a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, and endogenous compounds. The activity of different P450 isoforms varies within specific tissues and cell types and is selectively regulated together with their gene expression. Moreover, differential expression of certain P450 isoforms’ genes in tumor cells compared to normal tissues can be observed. This creates the potential for the use of these isozymes as tumor markers or selective prodrug activators. This article discusses the characteristics and function of five isoforms of cytochrome P450 (P450 1B1, P450 2W1, P450 2S1, P450 2R1, P450 2U1 that could be potential targets for tumor therapeutic and preventive strategies. These isoforms have been chosen because their level of expression in tumor tissues is definitely higher than in normal tissues.

  10. Effect of Intestinal Cytochrome P450 3A on Phytochemical Presystemic Metabolism

    Institute of Scientific and Technical Information of China (English)


    Phytochemicals, orally administered substances, are found to undergo presystemic metabolism mainly in the intestine. Although early researches confirmed the role of intestinal bacteria in phytochemical presystemic metabolism, along with the development of molecular biology in investigating intestinal metabolism, a breakthrough has been won in research into metabolizing enzymes and transporters in intestine,which demands more attention and further studies. Recently, Cytochrome P450 3A has been found to be the most effective enzyme in mediating both oxidative (Phase Ⅰ) and conjugative (Phase Ⅱ ) metabolism in the intestine. The present review summarizes the current findings correlated with the effect of intestinal cytochrome P450 3A on phytochemical presystemic metabolism, which provides a good basis for further research on phytochemical pharmacokinetics.

  11. Discoid lupus erythematosus in an X-linked cytochrome-positive carrier of chronic granulomatous disease. (United States)

    Yeaman, G R; Froebel, K; Galea, G; Ormerod, A; Urbaniak, S J


    A 13-year-old female presented with photosensitivity, recurrent aphthous ulcers and discoid lupus erythematosus (DLE)-like skin lesions. These symptoms have been linked to the carrier status of chronic granulomatous disease (CGD). Neutrophil (PMN) function was investigated by nitroblue tetrazolium reduction test and chemiluminescence. A severe impairment of PMN oxidative burst activity was revealed in spite of supranormal levels of cytochrome b245. Glucose-6-phosphate dehydrogenase activity was deficient. Her mother and two sisters also showed reduced PMN function. These findings are consistent with a cytochrome positive X-linked form of CGD with variable lyonization. DLE in association with the carrier status of this CGD variant has not been reported previously.

  12. Ex vivo inhibition of rat brain cytochrome P-450 activity by stiripentol. (United States)

    Mesnil, M; Testa, B; Jenner, P


    Stiripentol is an anti-epileptic drug of novel structure with previously demonstrated strong in vitro inhibitory activity on rat cerebral cytochrome P-450 mediated naphthalene hydroxylation [6]. When administered to rats as a single i.p. dose, the drug is presently shown to have the same in vitro effect. Maximal inhibition is seen 2 hr after administration, but at this time the brain concentrations of intact drug, although peaking, appear too low (ca. 11 micrograms/g tissue) to account for the intensity of the effect seen in vitro. This suggests in vivo activation to a metabolic intermediate forming a complex with cerebral cytochrome P-450, which 2 hr after dosing is fully insensitive to stiripentol added to incubates. Restoration of enzymic activity and of sensitivity to added stiripentol occurs progressively and is practically complete 24 hr after dosing.

  13. Global (Q)SAR models on substrates for human Cytochrome P450 3A4

    DEFF Research Database (Denmark)

    Ringsted, Tine; Nikolov, Nikolai Georgiev; Wedebye, Eva Bay;

    The Cytochrome P450 (CYP) is a superfamily of enzymes which catalyze the metabolism of a wide range of endobiotics and xenobiotics. The latter category comprises drugs and about 75% of marketed drugs are metabolised by CYP enzymes. Besides drugs, CYP enzymes detoxify environmental compounds...... domain. Domain coverage of EINECS chemicals and number of predicted substrates are discussed. Reference: C.W. Yap and Y.Z. Chen, Prediction of cytochrome p450 3A4, 2D6, and 2C9 inhibitors and substrates by using support vector machines, J. Chem. Inf. Model. 45 (2005), pp. 982–992....... but paradoxically they also have the ability to form reactive intermediates which can damage DNA, lipids and proteins. It is therefore important to gain knowledge on which substrates that can potentially be metabolised by CYP. The CYP 3A4 isoenzyme plays a dominant role by the metabolic elimination of up to 35...

  14. SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes.

    Directory of Open Access Journals (Sweden)

    Célia M Silveira

    Full Text Available Cytochrome cd1 nitrite reductases (cd1NiRs catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1 depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552, we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1.

  15. Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons (United States)

    Chalmers, G. R.; Edgerton, V. R.


    The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

  16. Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study

    Energy Technology Data Exchange (ETDEWEB)

    Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia [Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena (Italy); Tavagnacco, Claudio [Department of Chemistry, University of Trieste, via Giorgieri 1, 34127 Trieste (Italy); Borsari, Marco, E-mail: [Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena (Italy)


    Highlights: > Denaturation involves intermediate and partially unfolded forms. > An unfolded species displaying the haem with Fe coordinated by two His is observed. > Under unfolding conditions the nature of the SAM influences conformation of protein. > Concentration of the unfolding agent affects redox properties of immobilized protein. - Abstract: The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E{sup o}' of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E{sup o}' value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.

  17. Therapeutic photobiomodulation: nitric oxide and a novel function of mitochondrial cytochrome c oxidase. (United States)

    Poyton, Robert O; Ball, Kerri A


    Currently, light therapies are widely used in both human and veterinarian medicine. The application of light to clinical therapeutics includes: photodynamic therapy, used to kill cancer cells; UVA therapies, used to treat a variety of skin diseases; and photobiomodulation, used to promote cell growth and recovery from injury. Photobiomodu-lation uses light emitting diodes (LEDs) or low energy lasers, which emit light in the visible red to near infrared range. Light in this range penetrates tissue reasonably well, lacks the carcinogenic/mutagenic properties of UV light, and acts on an endogenous photoreceptor which likely acts to initiate light-altered signaling pathways. Although early studies identified mitochondrial cytochrome c oxidase as an endogenous photoreceptor for photobiomodulation, the cellular and molecular mechanisms underlying photobiomodulation have not been clear. Three recent findings provide important new insight. First, nitric oxide has been implicated. Second, cytochrome c oxidase, an enzyme known to reduce oxygen to water at the end of the mitochondrial respiratory chain, has been shown to have a new enzymatic activity--the reduction of nitrite to nitric oxide. This nitrite reductase activity is elevated under hypoxic conditions but also occurs under normoxia. And third, low intensity light enhances nitric oxide synthesis by cytochrome c oxidase without altering its ability to reduce oxygen. From these findings, we propose that cytochrome c oxidase functions in photobiomodulation by producing nitric oxide, a signaling molecule which can then function in both intra- and extracellular signaling pathways. We also propose that the effectiveness of photobiomodulation is under the control of tissue oxygen and nitrite levels.

  18. Identification of ubiquinol cytochrome c reductase hinge (UQCRH) as a potential diagnostic biomarker for lung adenocarcinoma


    Gao, Feng; Liu, Qicai; Li, Guoping; Dong, Feng; Qiu, Minglian; Lv, Xiaoting; Zhang, Sheng; Guo, Zheng


    Ubiquinol cytochrome c reductase hinge (UQCRH) is a novel protein that localizes in the mitochondrial membrane and induces mitochondrial reactive oxygen species (ROS) generation. It had a high expression rate of 87.10% (108/124) in lung adenocarcinoma. Moreover, serum UQCRH level in patients with lung adenocarcinoma was significantly increased compared with that of pneumonia patients (p < 0.0001) and normal control subjects (p < 0.0001). Receiver operating characteristic curve analysis using ...

  19. Potential inhibition of cytochrome P450 3A4 by propofol in human primary hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Li-Qun Yang; Wei-Feng Yu; Yun-Fei Cao; Bin Gong; Qing Chang; Guang-Shun Yang


    AIM: Hepatic cytochrome P450 isoenzymes constitute a superfamily of hemoproteins that play a major role in the metabolism of endogenous compounds and in the detoxification of xenobiotic molecules. P450 3A4 is one of the most important forms in human being, and mediates the metabolism of around 70% of therapeutic drugs and endogenous compounds. Propofol, a widely used intravenous anesthetic drug, is known to inhibit cytochrome P450activities in isolated rat hepatocytes. The goal of this study was to evaluate the potential efficacy of propofol on P4503A4 in a dose-dependent manner to understand its drugdrug interaction.METHODS: Hepatocytes were isolated from liver specimens from hepatic angioma patients undergone hepatic surgery.Primary incubated hepatocytes were treated with 0, 0.01,0.05, 0.1, 0.5, and 1.0 mM propofol for 24 hours. P450 3A4activity was measured with Nash′s colorimetry. The protein expression was assessed by Western blot analysis.RESULTS: A dose-dependent inhibitory effect of propofol was observed in cytochrome P450 3A4 activity. A minimal dosage of propofol (0.01 mM) induced a significant inhibition of P450 3A4 activity, although its regular dosages (0.01-0.1mM) showed no inhibitory effect on the cellular protein expression of P450 3A4.CONCLUSION: Propofol may be a potential CYP3A4 inhibitor as this anesthetic can inhibit isoenzyme activity significantly and reduce the metabolic rate of CYP3A4 substrates. This inhibition occurs at post-expression level, and concentration of propofol used clinically does not affect CYP3A4 protein expression. propofol may thus induce drug interaction of cytochrome P450 3A4 activity at the dosage used clinically.

  20. Intestinal cytochromes P450 regulating the intestinal microbiota and its probiotic profile

    Directory of Open Access Journals (Sweden)

    Eugenia Elefterios Venizelos Bezirtzoglou


    Full Text Available Cytochromes P450 (CYPs enzymes metabolize a large variety of xenobiotic substances. In this vein, a plethora of studies were conducted to investigate their role, as cytochromes are located in both liver and intestinal tissues. The P450 profile of the human intestine has not been fully characterized. Human intestine serves primarily as an absorptive organ for nutrients, although it has also the ability to metabolize drugs. CYPs are responsible for the majority of phase I drug metabolism reactions. CYP3A represents the major intestinal CYP (80% followed by CYP2C9. CYP1A is expressed at high level in the duodenum, together with less abundant levels of CYP2C8-10 and CYP2D6. Cytochromes present a genetic polymorphism intra- or interindividual and intra- or interethnic. Changes in the pharmacokinetic profile of the drug are associated with increased toxicity due to reduced metabolism, altered efficacy of the drug, increased production of toxic metabolites, and adverse drug interaction. The high metabolic capacity of the intestinal flora is due to its enormous pool of enzymes, which catalyzes reactions in phase I and phase II drug metabolism. Compromised intestinal barrier conditions, when rupture of the intestinal integrity occurs, could increase passive paracellular absorption. It is clear that high microbial intestinal charge following intestinal disturbances, ageing, environment, or food-associated ailments leads to the microbial metabolism of a drug before absorption. The effect of certain bacteria having a benefic action on the intestinal ecosystem has been largely discussed during the past few years by many authors. The aim of the probiotic approach is to repair the deficiencies in the gut flora and establish a protective effect. There is a tentative multifactorial association of the CYP (P450 cytochrome role in the different diseases states, environmental toxic effects or chemical exposures and nutritional status.

  1. Dynamic Water Networks in Cytochrome c Oxidase from Paracoccus denitrificans Investigated by Molecular Dynamics Simulations


    Olkhova, Elena; Hutter, Michael C; Lill, Markus A.; Helms, Volkhard; Michel, Hartmut


    We present a molecular dynamics study of cytochrome c oxidase from Paracoccus denitrificans in the fully oxidized state, embedded in a fully hydrated dimyristoylphosphatidylcholine lipid bilayer membrane. Parallel simulations with different levels of protein hydration, 1.125 ns each in length, were carried out under conditions of constant temperature and pressure using three-dimensional periodic boundary conditions and full electrostatics to investigate the distribution and dynamics of water ...

  2. Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase.


    Hay, M; Richards, J. H.; Lu, Y.


    A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA...

  3. Dimethylnitrosamine genotoxicity in rat liver primary cell cultures with low cytochrome P-450 levels. (United States)

    Mendoza-Figueroa, T


    Liver primary cell cultures (LPCC) with decreasing concentrations of cytochrome P-450 were used to investigate the genotoxicity of the hepatic carcinogen dimethylnitrosamine (DMN) and the correlation between DMN genotoxicity and cytochrome P-450 levels. Hepatocytes were isolated from partially hepatectomized rats and incubated with [3H]thymidine; single-strand DNA molecular weight was determined by alkaline sucrose sedimentation. The molecular weight of DNA decreased 50% in LPCC plated either 2 or 24 h before being treated for 24 h with 70 micron DMN. Cytochrome P-450 content was 188 pmol per mg protein in freshly isolated hepatocytes, whereas it was 70 and 32 pmol per mg protein in hepatocytes that had been cultured 24 and 48 h, respectively. Incorporation of 14C into acid-insoluble material was the same in LPCC exposed 24 h to [14C]DMN starting either 2 or 24 h after cell plating. At non-toxic concentrations (0.01-1 microM), SKF 525-A, an inhibitor of mixed-function oxidase enzymes, inhibited approximately 20% of the binding of 14C from [14C]DMN to acid-insoluble material in LPCC plated either 2 or 24 h before they were exposed to DMN for 24 h. Hepatocyte cultures exposed to the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (at concentrations ranging between 6.8 X 10(-8) and 6.8 X 10(-5) M) starting 2 and 24 h after plating, exhibited significant unscheduled DNA synthesis. These results indicate that DMN genotoxicity was similar in LPCC differing considerably in cytochrome P-450 levels, and they suggest that DMN genotoxicity in these cultures is due mainly to similar DMN activation than to decreased DNA repair.

  4. The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide. (United States)

    Parr, S R; Wilson, M T; Greenwood, C


    The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.

  5. Aminoglycoside-Resistant Mutation of Pseudomonas aeruginosa Defective in Cytochrome c552 and Nitrate Reductase


    Bryan, L E; Nicas, Thalia; Holloway, B W; Crowther, Carol


    A gentamicin-resistant mutant of Pseudomonas aeruginosa PAO503 was selected after ethyl methane sulfonate mutagenesis. The strain, P. aeruginosa PAO2401 had increased resistance to all aminoglycosides tested but exhibited no change for other antibiotics. The mutation designated aglA (aminoglycoside resistance) was 50% cotransducible with the 8-min ilvB,C marker on the P. aeruginosa chromosome. It showed a marked reduction in cytochrome c552 and nitrate reductase (Nar) and a change in terminal...

  6. Proton transport through D- and H-channels in the bovine heart cytochrome c oxidase (United States)

    Ishmail, S.; Ivanushkin, P.; Mourokh, L.


    We study proton transport in the cytochrome c oxidase complex of bovine heart. A simple model of proton motion based on Langevin equation is combined with the determination of the electrostatic potential using molecular dynamics software packages. We compare the proton transfer rates in the fully oxidized and fully reduced forms of the enzyme and conclude that the H-channel becomes more open in the reduced form, but the D-channel does not, in agreement with experimental results.

  7. Rat liver microsomal cytochrome P450-dependent oxidation of 3,5-disubstituted analogues of paracetamol

    NARCIS (Netherlands)

    Bessems, J.G.M.; Koppele, J.M. te; Dijk, P.A. van; Stee, L.L.P. van; Commandeur, J.N.M.; Vermeulen, N.P.E.


    1. The cytochrome P450-dependent binding of paracetamol and a series of 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -C(H)3, -C2H5, -iC3H7) have been determined with β-naphthoflavone (βNF)-induced rat liver microsomes and produced reverse type I spectral changes. K(s,app) varied fr

  8. Structural and spectroscopic studies on the porin-cytochrome complex from Shewanella oneidensis MR-1


    Lawes, Matthew


    The"outer"membrane,"hetero!trimeric"multi!heme"cytochrome"complex"MtrCAB,"enables"the" process"of"dissimilatory"metal"reduction"(DMR)"in"Shewanella(oneidensis." "" The"properties"of" the"decaheme"protein"MtrA"as"well"as"a" truncated"version"of" this"protein" were" investigated" using" analytical" ultracentrifugation" (AUC)," small" angle" X!ray" scattering" (SAXS)" and" spectropotentiometric" techniques." MtrA" and" a" truncated"N!terminal" MtrA" construct"containing" the" f...

  9. Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.


    Reynafarje, B; Alexandre, A.; Davies, P.; Lehninger, A L


    The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 i...

  10. The effects of selected flavonoids on cytochromes P450 in rat liver and small intestine


    Křížková, Jitka; Burdová, Kamila; Stiborová, Marie; Křen, Vladimír; Hodek, Petr


    In recent years, the consumption and use of dietary supplements containing concentrated phytochemicals (e.g. flavonoids) increased dramatically. Flavonoids, as foreign compounds (xenobiotics), have great potential to modulate the activity of cytochrome P450s (CYPs), xenobiotic-metabolizing enzymes involved in the activation and detoxification of food and environmental carcinogens. Thus, the aim of this study was to investigate the effects of model glycosylated and deglycosylated flavonoids on...

  11. FASTKD2 Nonsense Mutation in an Infantile Mitochondrial Encephalomyopathy Associated with Cytochrome C Oxidase Deficiency



    In two siblings we found a mitochondrial encephalomyopathy, characterized by developmental delay, hemiplegia, convulsions, asymmetrical brain atrophy, and low cytochrome c oxidase (COX) activity in skeletal muscle. The disease locus was identified on chromosome 2 by homozygosity mapping; candidate genes were prioritized for their known or predicted mitochondrial localization and then sequenced in probands and controls. A homozygous nonsense mutation in the KIAA0971 gene segregated with the di...

  12. Cytochrome P-450 dependent monooxygenase activity in rat nasal epithelial membranes

    Energy Technology Data Exchange (ETDEWEB)

    Hadley, W.M.; Dahl, A.R.


    Cytochrome P-450 was found in nasal epithelial membranes (NEM) of the rat. The quantity was 12% that of liver on a per mg of microsomal protein basis and 1.6 times that of the lung on the same basis. Metabolism of p-nitroanisole was faster by microsomes from NEM than by microsomes from liver or lungs while the metabolism rate of aniline by microsomes from NEM was between that of microsomes from liver and lung.

  13. Protein engineering of the cytochrome P450 monooxygenase from bacillus megaterium


    Urlacher, Vlada B.; Schmid, Rolf D


    The role and importance of cytochrome P450 enzymes (CYP) in drug development, biodegradation processes and biocatalysis has been widely acknowledged. P450 monooxygenases exhibit an extremely wide substrate spectrum which is the basis of their ability to activate or detoxify a large variety of target molecules. P450 monooxygenases have been isolated from bacteria, yeasts, insects, as well as mammalian and plant tissues. Currently, the enzyme family is one of the best known gene subfamilies wit...

  14. Cytochrome P450 CYP1B1 activity in renal cell carcinoma. (United States)

    McFadyen, M C E; Melvin, W T; Murray, G I


    Renal cell carcinoma (RCC) is the most common malignancy of the kidney and has a poor prognosis due to its late presentation and resistance to current anticancer drugs. One mechanism of drug resistance, which is potentially amenable to therapeutic intervention, is based on studies in our laboratory. CYP1B1 is a cytochrome P450 enzyme overexpressed in a variety of malignant tumours. Our studies are now elucidating a functional role for CYP1B1 in drug resistance. Cytochrome P450 reductase (P450R) is required for optimal metabolic activity of CYP1B1. Both CYP1B1 and P450R can catalyse the biotransformation of anticancer drugs at the site of the tumour. In this investigation, we determined the expression of CYP1B1 and P450R in samples of normal kidney and RCC (11 paired normal and tumour and a further 15 tumour samples). The O-deethylation of ethoxyresorufin to resorufin was used to measure CYP1B1 activity in RCC. Cytochrome P450 reductase activity was determined by following the reduction of cytochrome c at 550 nm. The key finding of this study was the presence of active CYP1B1 in 70% of RCC. Coincubation with the CYP1B1 inhibitor alpha-naphthoflavone (10 nM) inhibited this activity. No corresponding CYP1B1 activity was detected in any of the normal tissue examined (n=11). Measurable levels of active P450R were determined in all normal (n=11) and tumour samples (n=26). The presence of detectable CYP1B1, which is capable of metabolising anticancer drugs in tumour cells, highlights a novel target for therapeutic intervention.

  15. Resonance Raman Optical Activity and Surface Enhanced Resonance Raman Optical Activity analysis of Cytochrome C

    DEFF Research Database (Denmark)

    Johannessen, Christian; Abdali, Salim; White, Peter C.


    to resonance enhanced skeletal porphyrin vibrations, more pronounced than any contribution from the protein back-bone. Combining the intrinsic resonance enhancement of cytochrome c with surface plasmon enhancement by colloidal silver particles, the Surface Enhanced Resonance Raman Scattering (SERRS) and Chiral......, while some disparities were observed between the resonance ROA and ChERS spectra. The difference can be ascribed perturbations of the physical properties of the protein upon adhesion to the surface of the silver colloids....

  16. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. (United States)

    Folmer, O; Black, M; Hoeh, W; Lutz, R; Vrijenhoek, R


    We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

  17. Cytochrome P450 Drug Metabolizing Enzymes in Roma Population Samples: Systematic Review of the Literature. (United States)

    Szalai, Renata; Hadzsiev, Kinga; Melegh, Bela


    The cytochrome P450 drug metabolizing enzymes are highly polymorphic and show inter-individual differences in variability in drug response, which varies widely also with ethnicity. This study aims to summarize the available data on genetic polymorphisms associated with cytochrome enzymes conducted on Roma populations. Our goal was to compare the frequency of the variant alleles, genotypes and predicted phenotypes with corresponding rates from other populations. We carried out a systematic review including the papers published on the pharmacogenetically relevant variants of cytochrome P450 genes related to Roma population. The study was performed using several articles, websites and databases, including PubMed, Ensembl, dbSNP, HapMap and 1000 Genomes Project. This review attempts to summarize and discuss our current knowledge about the frequency distribution of the ever investigated 20 allelic variants of 9 cytochrome genes (CYP1A2, CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP2C8, CYP2D6, CYP3A5, CYP4F2) in Roma DNA samples and compare them with other populations. Differences between Roma and Hungarian samples are reported for 7 variant genotypes. CYP2C9 *2/*3 and CYP2C19 *2/*2 genotypes showed more than 3-fold differences. Additional differences are displayed for allele frequency of 7 variants (rs762551, rs3745274, rs1058930, rs1065852, rs3892097, rs1057910 and rs4244285) in Roma population samples. The interethnic variability in clinically relevant genetic polymorphisms of drug metabolizing enzymes, which may explain distinct drug response, highlights the need to allow for the ancestry of participants in pharmacogenetic studies.

  18. Inhibition of rat brain microsomal cytochrome P450-dependent dealkylation activities by an oxidative stress. (United States)

    Lagrange, P; El-Bachá, R D; Netter, P; Minn, A


    There is increasing evidence that an oxidative stress not only alters cellular lipids and nucleic acids, but also numerous proteins. This oxidation results in alterations of some cellular functions, either by reversible modifications allowing a post-transcriptional regulation of enzyme activities or receptor affinities, or by irreversible modifications of the protein, triggering its inactivation and destruction. In the present work, we examined the effects of an experimental oxidative stress on rat brain microsomal cytochrome P450-dependent dealkylation activities. For that purpose, superoxide anions were produced either by the NADPH-dependent redox cycling of a quinine, menadione, or by the addition of apomorphine, which produces by autoxidation both superoxide anions and apomorphine-derived quinones. The inhibition of brain cytochrome P450-dependent alkoxyresorufin O-dealkylase activities was dependent on both menadione or apomorphine concentrations. Simultaneously, an increase of microsomal carbonyl groups was recorded. Immunoblotting characterization of brain microsomal oxidized protein was carried out, using antibodies raised against 2,4-dinitrophenylhydrazine as a reagent of protein carbonyl groups, and a revelation by a chemiluminescence method. We observed an increase in cerebral CYP1A protein oxidation, related to menadione concentration, suggesting that oxidation of cytochrome P450 protein may result in its catalytic inactivation.

  19. Electorchemical Studies of Cytochrome c on Electodes Modified by Single—Wall Carbon Naotubes

    Institute of Scientific and Technical Information of China (English)

    程发良; 杜珊; 金葆康


    Single-wall carbon nanotubes(SWNTs) modified gold electrodes were prepared by using two different methods.The electrochemical behavior of cytochrome c on the modified gold electrodes was investigated.The first kind of SWNT-modified electrode (noted as SWNT/Au electrode)was prepared by the adsorption of carboxylterminated SWNTs from DMF dispersion on the gold electrode.The oxidatively processed SWNT tips were covalently modified by coupling with amines (AET) to form amide linkage.Via Au-S chemical bonding,the self-assembled monolayer of thiol-unctionalized nanotubes on gold surface was fabricated so as to prepare the others SWNT-modified electrode (noted as SWNT/AET/Au electrode).It was shown from cyclic voltammetry cxperiments that cytochrome c exhibited direct electrochemical responses on the both electrodes, but only the current of controlled diffusion existed on the SWNT/Au electrode while both the currents of controlled diffusion and adsorption of cytochrome c occurred on the SWNT/AET/Au electrode.Photoelastic Modulation Infared Reflection Absorpthion Spectroscopy (PEM-IRRAS) and Quartz Crystal Microbalance (QCM) were employed to verify the adsorption of SWNTs on the gold electrodes.The results proved that SWNTs could enhance the direct electron transfer proecss between the electrodes and redox proteins.

  20. Tumor-specific expression of cytochrome P450 CYP1B1. (United States)

    Murray, G I; Taylor, M C; McFadyen, M C; McKay, J A; Greenlee, W F; Burke, M D; Melvin, W T


    Cytochrome P450 CYP1B1 is a recently cloned dioxin-inducible form of the cytochrome P450 family of xenobiotic metabolizing enzymes. An antibody raised against a peptide specific for CYP1B1 was found to recognize CYP1B1 expressed in human lymphoblastoid cells but not to recognize other forms of cytochrome P450, particularly CYP1A1 and CYP1A2. Using this antibody, the cellular distribution and localization of CYP1B1 were investigated by immunohistochemistry in a range of malignant tumors and corresponding normal tissues. CYP1B1 was found to be expressed at a high frequency in a wide range of human cancers of different histogenetic types, including cancers of the breast, colon, lung, esophagus, skin, lymph node, brain, and testis. There was no detectable immunostaining for CYP1B1 in normal tissues. These results provide the basis for the development of novel methods of cancer diagnosis based on the identification of CYP1B1 in tumor cells and the development of anticancer drugs that are selectively activated in tumors by CYP1B1.

  1. Circular-dichroic properties and secondary structure of Pseudomonas aeruginosa soluble cytochrome c oxidase. (United States)

    Tordi, M G; Silvestrini, M C; Colosimo, A; Provencher, S; Brunori, M


    The c.d. spectra of Pseudomonas aeruginosa cytochrome c oxidase in the oxidized state and the reduced state are reported in the visible- and u.v. absorption regions. In the visible region the comparison between the spectra of reduced cytochrome c oxidase and ferrocytochrome c-551 allows the identification of the c.d. bands mainly due to the d1 haem chromophore in cytochrome c oxidase. In the near-u.v. region the assignment of some of the observed peaks to the haem groups and to the aromatic amino acid residues is proposed. A careful analysis of the data in the far-u.v. region leads to the determination of the relative amounts of alpha-helix and beta-sheet in the enzyme, giving for the first time a picture of its secondary structure. A significant difference in this respect between the reduced and the oxidized species is observed and discussed in the light of similar conclusions reported by other workers.

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

    Börsch, Michael


    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.

  3. Redox-Dependent Conformational Changes in Cytochrome c Oxidase Suggest a Gating Mechanism for Proton Uptake

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Ling; Liu, Jian; Mills, Denise A.; Proshlyakov, Denis A.; Hiser, Carrie; Ferguson-Miller, Shelagh; (MSU)


    A role for conformational change in the coupling mechanism of cytochrome c oxidase is the subject of controversy. Relatively small conformational changes have been reported in comparisons of reduced and oxidized crystal structures of bovine oxidase but none in bacterial oxidases. Comparing the X-ray crystal structures of the reduced (at 2.15 {angstrom} resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, we observe a displacement of heme a3 involving both the porphyrin ring and the hydroxyl farnesyl tail, accompanied by protein movements in nearby regions, including the mid part of helix VIII of subunit I which harbors key residues of the K proton uptake path, K362 and T359. The conformational changes in the reduced form are reversible upon reoxidation. They result in an opening of the top of the K pathway and more ordered waters being resolved in that region, suggesting an access path for protons into the active site. In all high-resolution structures of oxidized R. sphaeroides cytochrome c oxidase, a water molecule is observed in the hydrophobic region above the top of the D path, strategically positioned to facilitate the connection of residue E286 of subunit I to the active site or to the proton pumping exit path. In the reduced and reduced plus cyanide structures, this water molecule disappears, implying disruption of proton conduction from the D path under conditions when the K path is open, thus providing a mechanism for alternating access to the active site.

  4. Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1. (United States)

    Bareth, Bettina; Nikolov, Miroslav; Lorenzi, Isotta; Hildenbeutel, Markus; Mick, David U; Helbig, Christin; Urlaub, Henning; Ott, Martin; Rehling, Peter; Dennerlein, Sven


    The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.

  5. Novel cytochrome P450, cyp6a17, is required for temperature preference behavior in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jongkyun Kang

    Full Text Available Perception of temperature is an important brain function for organisms to survive. Evidence suggests that temperature preference behavior (TPB in Drosophila melanogaster, one of poikilothermal animals, is regulated by cAMP-dependent protein kinase (PKA signaling in mushroom bodies of the brain. However, downstream targets for the PKA signaling in this behavior have not been identified. From a genome-wide search for the genes regulated by PKA activity in the mushroom bodies, we identified the cyp6a17 Cytochrome P450 gene as a new target for PKA. Our detailed analysis of mutants by genetic, molecular and behavioral assays shows that cyp6a17 is essential for temperature preference behavior. cyp6a17 expression is enriched in the mushroom bodies of the adult brain. Tissue-specific knockdown and rescue experiments demonstrate that cyp6a17 is required in the mushroom bodies for normal temperature preference behavior. This is the first study, to our knowledge, to show PKA-dependent expression of a cytochrome P450 gene in the mushroom bodies and its role as a key factor for temperature preference behavior. Taken together, this study reveals a new PKA-Cytochrome P450 pathway that regulates the temperature preference behavior.

  6. Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells. (United States)

    Contran, N; Cerana, R; Crosti, P; Malerba, M


    Programmed cell death plays a vital role in normal plant development, response to environmental stresses, and defense against pathogen attack. Different types of programmed cell death occur in plants and the involvement of mitochondria is still under investigation. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin induces cell death that shows apoptotic features, including chromatin condensation, DNA fragmentation, and release of cytochrome c from mitochondria. In this work, we show that cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria, inhibits the cell death, DNA fragmentation, and cytochrome c release induced by fusicoccin. In addition, we show that fusicoccin induces a change in the shape of mitochondria which is not prevented by cyclosporin A. These results suggest that the release of cytochrome c induced by fusicoccin occurs through a cyclosporin A-sensitive system that is similar to the permeability transition pore of animal mitochondria and they make it tempting to speculate that this release may be involved in the phytotoxin-induced programmed cell death of sycamore cells.

  7. Dual Targeting of a Mitochondrial Protein: The Case Study of Cytochrome C1

    Institute of Scientific and Technical Information of China (English)

    Anja R(o)diger; Bianca Baudisch; Uwe Langner; Ralf Bernd Kl(o)sgen


    As a result of the endosymbiotic gene transfer, the majority of proteins of mitochondria and chloroplasts is encoded in the nucleus and synthesized in the cytosol as precursor molecules carrying N-terminal transit peptides for the transport into the respective target organelle. In most instances, transport takes place into either mitochondria or chlor-oplasts, although a few examples of dual targeting into both organelles have been described. Here, we show by a com-bination of three different experimental strategies that also cytochrome c of potato, a component of the respiratory electron transport chain, is imported not only into mitochondria, but also into plastids. In organello import experiments with isolated mitochondria and chloroplasts, which were analyzed in both single and mixed organelle assays, demonstrate that the processing products accumulating after import within the two endosymbiotic organelles are different in size. Dual targeting of cytochrome c is observed also in vivo, after biolistic transformation of leaf epidermal cells with suitable reporter constructions. Finally, Western analyses employing cytochrome c-specific antiserum provide evidence that the protein accumulates in significant amounts in mitochondria and chloroplasts of both pea and spinach. The possible consequences of our findings on the relevance of the dual targeting phenomenon are discussed.

  8. Water-soluble phosphate-functionalized polyfluorene as fluorescence biosensors toward cytochrome c

    Institute of Scientific and Technical Information of China (English)


    An anionic water-soluble polyfluorene derivative, poly(9,9-bis(6′-phosphatehexyl)fluorene-alt-1,4-pheny lene) sodium salt (PFHPNa), was synthesized by Suzuki coupling reaction in DMF/water. Polymer PFHPNa was well soluble in water with a strong blue fluorescence emission. Effect of the side chain length on fluorescence sensory properties was studied by comparing quenching efficiencies toward different quenchers of PFHPNa with a reported polymer poly(9,9-bis(3′-phosphatepropyl)fluorene-alt-1,4-phenylene) sodium salt (PFPPNa), which have different side chains in length. For small molecular quenchers (methylviologen, MV2+) and meso-5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4), polymer PFHPNa had lower sensitivity due to the much longer side chain length. The positively charged metalloprotein cytochrome c could quench fluorescence of conjugated polymers via energy transfer and electron transfer. Moreover, polymer PFHPNa showed higher fluorescence quenching toward large biomolecules than PFPPNa. The corresponding Stern-Volmer (Ksv) value of polymer PFHPNa was determined to be 2.1×108 M-1 for cytochrome c. It could be used as a sensitive and selective fluorescence sensor for protein cytochrome c.

  9. Kinetic Monte Carlo Simulations and Molecular Conductance Measurements of the Bacterial Decaheme Cytochrome MtrF

    Energy Technology Data Exchange (ETDEWEB)

    Byun, H. S.; Pirbadian, S.; Nakano, Aiichiro; Shi, Liang; El-Naggar, Mohamed Y.


    Microorganisms overcome the considerable hurdle of respiring extracellular solid substrates by deploying large multiheme cytochrome complexes that form 20 nanometer conduits to traffic electrons through the periplasm and across the cellular outer membrane. Here we report the first kinetic Monte Carlo simulations and single-molecule scanning tunneling microscopy (STM) measurements of the Shewanella oneidensis MR-1 outer membrane decaheme cytochrome MtrF, which can perform the final electron transfer step from cells to minerals and microbial fuel cell anodes. We find that the calculated electron transport rate through MtrF is consistent with previously reported in vitro measurements of the Shewanella Mtr complex, as well as in vivo respiration rates on electrode surfaces assuming a reasonable (experimentally verified) coverage of cytochromes on the cell surface. The simulations also reveal a rich phase diagram in the overall electron occupation density of the hemes as a function of electron injection and ejection rates. Single molecule tunneling spectroscopy confirms MtrF's ability to mediate electron transport between an STM tip and an underlying Au(111) surface, but at rates higher than expected from previously calculated heme-heme electron transfer rates for solvated molecules.

  10. Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.). (United States)

    Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng


    Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification.

  11. Cytochrome P450 CYP1B1 over-expression in primary and metastatic ovarian cancer. (United States)

    McFadyen, M C; Cruickshank, M E; Miller, I D; McLeod, H L; Melvin, W T; Haites, N E; Parkin, D; Murray, G I


    Ovarian cancer is the most frequent cause of death from gynaecological malignancies world wide. Little improvement has been made in the long-term outcome of this disease, with the 5-year survival of patients only 30%. This poor prognosis is due to the late presentation of the disease and to the unpredictable response of ovarian cancer to chemotherapy. The cytochrome P450 enzymes are a superfamily of haemoproteins, known to be involved in the metabolic activation and/or detoxification of a number of anti-cancer drugs. CYP1B1 is a tumour-related form of cytochrome P450 which is over expressed in a wide variety of primary tumours of different histological type. The presence of CYP1B1 may be of importance in the modulation of these tumours to anti-cancer drugs. We have conducted a comprehensive immunohistochemical investigation, into the presence of cytochrome P450 CYP1B1 in primary and metastatic ovarian cancer. The key findings of this study are the increased expression of CYP1B1 in the majority of ovarian cancers investigated (92%), with a strong correlation demonstrated between CYP1B1 expression in both primary and metastatic ovarian cancer (P = 0.005 Spearman's rank correlation test). In contrast no detectable CYP1B1 was found in normal ovary.

  12. An ultrasensitive performance enhanced novel cytochrome c biosensor for the detection of rebaudioside A. (United States)

    Bathinapatla, Ayyappa; Kanchi, Suvardhan; Singh, Parvesh; Sabela, Myalowenkosi I; Bisetty, Krishna


    In this study a novel cyctochrome c modified nanocomposite electrochemical biosensor was developed for the electrochemical determination of rebaudioside A in different food samples. The electrode surface was fabricated with graphene oxide assimilated with gold nanoparticles decorated on multiwalled carbon nanotubes/cytochrome c. The developed biosensor exhibited a 10-fold enhancement in the differential pulse voltammetry signal carried out at pH 11.0 in a 0.1M borate buffer. Under the optimized conditions, Ip (µA) was proportional to the rebaudioside A concentration in the range of 0.001-0.05 mM (R(2)=0.8308) and 0.075-1.25 mM (R(2)=0.9920) with a detection limit (S/N=3) of 0.264 µM. Results of this study revealed that cyctochrome c was adsorbed tightly onto the surface of the modified electrode and showed an enzymatic catalytic activity towards the quasi-reversible reduction of rebaudioside A at -0.1 V (vs Ag/AgCl). The direct electron transfer by cytochrome c was further supported by HOMO-LUMO calculations performed at the density functional theory level. Additionally, the molecular docking simulations predicted a stronger binding affinity of rebaudioside A towards cytochrome c, thus supporting their host-guest relationship. The use of novel electrode materials in this study demonstrates the application of the electrochemical biosensor in the food industry.

  13. Osteomalacia in an HIV-infected man receiving rifabutin, a cytochrome P450 enzyme inducer: a case report

    Directory of Open Access Journals (Sweden)

    Horne Anne M


    Full Text Available Abstract Introduction People infected with human immunodeficiency virus are frequently treated with medications that can induce or inhibit cytochrome P450 enzymes. Case presentation A 59 year old man treated with zidovudine, lamivudine, indinavir, and ritonavir for infection with human immunodeficiency virus volunteered to take part in a study of bone loss. He was found to have vitamin D insufficiency with secondary hyperparathyroidism and received vitamin D and calcium supplementation. He suffered a recurrence of infection with Mycobacterium avium intracellulare for which he received treatment with ciprofloxacin, rifabutin, and ethambutol. Subsequently, he developed worsening vitamin D deficiency with hypocalcaemia, secondary hyperparathyroidism and elevated markers of bone turnover culminating in an osteomalacic vertebral fracture. Correction of the vitamin D deficiency required 100,000 IU of cholecalciferol monthly. Rifabutin is a cytochrome P450 inducer, and vitamin D and its metabolites are catabolised by cytochrome P450 enzymes. We therefore propose that treatment with rifabutin led to the induction of cytochrome P450 enzymes catabolising vitamin D, thereby causing vitamin D deficiency and osteomalacia. This process might be mediated through the steroid and xenobiotic receptor (SXR. Conclusion Treatment with rifabutin induces the cytochrome P450 enzymes that metabolise vitamin D and patients treated with rifabutin might be at increased risk of vitamin D deficiency. In complex medication regimens involving agents that induce or inhibit cytochrome P450 enzmyes, consultation with a clinical pharmacist or pharmacologist may be helpful in predicting and/or preventing potentially harmful interactions.

  14. Molecular Characterization and Functional Analysis of Three Pathogenesis-Related Cytochrome P450 Genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea

    Directory of Open Access Journals (Sweden)

    Xiao-Lu Xu


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

  15. Green algal cytochrome b6-f complexes: isolation and characterization from Dunaliella saline, Chlamydomonas reinhardtii and Scenedesmus obliquus. (United States)

    Wynn, R M; Bertsch, J; Bruce, B D; Malkin, R


    Cytochrome b6-f complexes have been isolated from Chlamydomonas reinhardtii, Dunaliella saline and Scenedesmus obliquus. Each complex is essentially free of chlorophyll and carotenoids and contains cytochrome b6 and cytochrome f hemes in a 2:1 molar ratio. C. reinhardtii and S. obliquus complexes contain the Rieske iron-sulfur protein (present in approx 1:1 molar ratio to cytochrome f) and each catalyzes a DBMIB- and DNP-INT-sensitive electron transfer from duroquinol to spinach plastocyanin. Immunological assays using antibodies to the peptides from the spinach cytochrome complex show varying cross-reactivity patterns except for the complete absence of binding to the Rieske proteins in any of the three complexes, suggesting little structural similarity between the Rieske proteins of algae with those from higher plants. One complex (D. salina) has been uniformly labeled by growth in NaH14CO3 to determine stoichiometries of constituent polypeptide subunits. Results from these studies indicate that all functionally active cytochrome b6-f complexes contain four subunits which occur in equimolar amounts.

  16. The redox state of cytochrome c modulates resistance to methotrexate in human MCF7 breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Susana Barros

    Full Text Available BACKGROUND: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. RESULTS: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexate-induced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. CONCLUSIONS: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

  17. Development and validation of an analytical method for the quantification of cytochrome c in skin transport studies. (United States)

    Bachhav, Yogeshwar G; Kalia, Yogeshvar N


    A simple isocratic HPLC method for the quantification of Cytochrome c in skin permeation samples was developed and validated. The mobile phase comprised a 41 : 59 mixture of an organic phase A (0.1% trifluoroacetic acid in a 90 : 10 mixture of MeCN-H(2)O) and an aqueous phase B (0.1% trifluoroacetic acid in H(2)O). The Cytochrome c retention and run times were 2.62 and 8.0 min, respectively--much shorter than those for existing gradient methods. The response was accurate, precise and linear from 2.5 to 25 microg/mL. The mean recoveries for intra-day and inter-day analysis ranged from 88.5 to 103.8% and the RSD varied from 0.05 to 1.55%. The assay was used to quantify transport of Cytochrome c across intact and laser-microporated porcine skin in vitro. Cytochrome c permeation and the amount of protein retained within the membrane over 24 h were quantified as a function of the number of micropores. Although no Cytochrome c permeation was observed across intact skin, laser microporation enabled delivery of 22.9 +/- 3.3 and 56.0 +/- 15.9 microg/cm(2) of the protein across skin samples with 300 and 1800 micropores, respectively. In conclusion, the HPLC method provided a fast, efficient means to quantify Cytochrome c in samples from skin transport studies.

  18. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans. (United States)

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


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

  19. A novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U: expression, molecular features and properties of redox potential. (United States)

    Ogami, Shinichi; Hijikata, Shoichi; Tsukahara, Tamotsu; Mie, Yasuhiro; Matsuno, Toshihide; Morita, Naoki; Hara, Isao; Yamazaki, Koji; Inoue, Norio; Yokota, Atsushi; Hoshino, Tamotsu; Yoshimune, Kazuaki; Yumoto, Isao


    A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly(22)-Asn(34), in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C(15), C(16) and C(17) chain lengths and C(15) chain length, respectively, and it was considered that these fatty acids are bound to glycerol-Cys(18). Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys(18) --> Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.

  20. A critical role for the cccA gene product, cytochrome c2, in diverting electrons from aerobic respiration to denitrification in Neisseria gonorrhoeae. (United States)

    Hopper, Amanda C; Li, Ying; Cole, Jeffrey A


    Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c2, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c4 and c5 transfer electrons to cytochrome oxidase cbb3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c4 or c5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc2), cycA (Δc4), cycB (Δc5), and ccoP (ccoP-C368A), we demonstrate that cytochrome c2 is required for electron transfer from cytochrome c4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae.