Regulatory interactions in the dimeric cytochrome bc(1) complex: the advantages of being a twin.

PubMed

Covian, Raul; Trumpower, Bernard L

2008-09-01

The dimeric cytochrome bc(1) complex catalyzes the oxidation-reduction of quinol and quinone at sites located in opposite sides of the membrane in which it resides. We review the kinetics of electron transfer and inhibitor binding that reveal functional interactions between the quinol oxidation site at center P and quinone reduction site at center N in opposite monomers in conjunction with electron equilibration between the cytochrome b subunits of the dimer. A model for the mechanism of the bc(1) complex has emerged from these studies in which binding of ligands that mimic semiquinone at center N regulates half-of-the-sites reactivity at center P and binding of ligands that mimic catalytically competent binding of ubiquinol at center P regulates half-of-the-sites reactivity at center N. An additional feature of this model is that inhibition of quinol oxidation at the quinone reduction site is avoided by allowing catalysis in only one monomer at a time, which maximizes the number of redox acceptor centers available in cytochrome b for electrons coming from quinol oxidation reactions at center P and minimizes the leakage of electrons that would result in the generation of damaging oxygen radicals.

Cytochrome bc1-cy Fusion Complexes Reveal the Distance Constraints for Functional Electron Transfer Between Photosynthesis Components*

PubMed Central

Lee, Dong-Woo; Öztürk, Yavuz; Osyczka, Artur; Cooley, Jason W.; Daldal, Fevzi

2008-01-01

Photosynthetic (Ps) growth of purple non-sulfur bacteria such as Rhodobacter capsulatus depends on the cyclic electron transfer (ET) between the ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductases (cyt bc1 complex), and the photochemical reaction centers (RC), mediated by either a membrane-bound (cyt cy) or a freely diffusible (cyt c2) electron carrier. Previously, we constructed a functional cyt bc1-cy fusion complex that supported Ps growth solely relying on membrane-confined ET (Lee, D.-W., Ozturk, Y., Mamedova, A., Osyczka, A., Cooley, J. W., and Daldal, F. (2006) Biochim. Biophys. Acta1757 ,346 -35216781662). In this work, we further characterized this cyt bc1-cy fusion complex, and used its derivatives with shorter cyt cy linkers as “molecular rulers” to probe the distances separating the Ps components. Comparison of the physicochemical properties of both membrane-embedded and purified cyt bc1-cy fusion complexes established that these enzymes were matured and assembled properly. Light-activated, time-resolved kinetic spectroscopy analyses revealed that their variants with shorter cyt cy linkers exhibited fast, native-like ET rates to the RC via the cyt bc1. However, shortening the length of the cyt cy linker decreased drastically this electronic coupling between the cyt bc1-cy fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt cy linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc1-cy fusion complexes and the RC and their surrounding light harvesting proteins was very short. These findings support the notion that membrane-bound Ps components form large, active structural complexes that are “hardwired” for cyclic ET. PMID:18343816

Electronic connection between the quinone and cytochrome C redox pools and its role in regulation of mitochondrial electron transport and redox signaling.

PubMed

Sarewicz, Marcin; Osyczka, Artur

2015-01-01

Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria. Copyright © 2015 the American Physiological Society.

Electronic Connection Between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

PubMed Central

Sarewicz, Marcin; Osyczka, Artur

2015-01-01

Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria. PMID:25540143

Modeling the molecular basis of atovaquone resistance in parasites and pathogenic fungi.

PubMed

Kessl, Jacques J; Meshnick, Steven R; Trumpower, Bernard L

2007-10-01

Atovaquone is a substituted hydroxynaphthoquinone that is used therapeutically for treating Plasmodium falciparum malaria, Pneumocystis jirovecii pneumonia and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting parasite and fungal respiration by binding to the cytochrome bc1 complex. The recent, growing failure of atovaquone treatment and increased mortality of patients with malaria or Pneumocystis pneumonia has been linked to the appearance of mutations in the cytochrome b gene. To better understand the molecular basis of drug resistance, we have developed the yeast and bovine bc1 complexes as surrogates to model the molecular interaction of atovaquone with human and resistant pathogen enzymes.

Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes.

PubMed

Blackwell, M F; Whitmarsh, J

1990-11-01

PYRENE FLUORESCENCE QUENCHING BY PLASTOQUINONE WAS USED TO ESTIMATE THE RATE OF PLASTOQUINONE LATERAL DIFFUSION IN SOYBEAN PHOSPHATIDYLCHOLINE PROTEOLIPOSOMES CONTAINING THE FOLLOWING INTEGRAL MEMBRANE PROTEINS: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc(1), and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 . 10(-7) cm(2) s(-1) in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc(1), and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration.

Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes

PubMed Central

Blackwell, Mary F.; Whitmarsh, John

1990-01-01

Pyrene fluorescence quenching by plastoquinone was used to estimate the rate of plastoquinone lateral diffusion in soybean phosphatidylcholine proteoliposomes containing the following integral membrane proteins: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc1, and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 · 10-7 cm2 s-1 in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc1, and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration. PMID:19431774

Computational discovery of picomolar Q(o) site inhibitors of cytochrome bc1 complex.

PubMed

Hao, Ge-Fei; Wang, Fu; Li, Hui; Zhu, Xiao-Lei; Yang, Wen-Chao; Huang, Li-Shar; Wu, Jia-Wei; Berry, Edward A; Yang, Guang-Fu

2012-07-11

A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Q(o) site inhibitors of the cytochrome bc(1) complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (K(i) = 881.80 nM, porcine bc(1)), the most potent compound 4f displayed 20 507-fold improved binding affinity (K(i) = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (K(i) = 83.00 pM) bound to the chicken bc(1) at 2.70 Å resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques.

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

PubMed

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

2016-08-01

Endochin-like quinolones (ELQs) are potent and specific inhibitors of cytochrome bc1 from Plasmodium falciparum and Toxoplasma gondii and show promise for novel antiparasitic drug development. To determine whether the mitochondrial electron transport chain of Leishmania parasites could be targeted similarly for drug development, we investigated the activity of 134 structurally diverse ELQs. A cohort of ELQs was selectively toxic to amastigotes of Leishmania mexicana and L. donovani, with 50% inhibitory concentrations (IC50s) in the low micromolar range, but the structurally similar hydroxynaphthoquinone buparvaquone was by far the most potent inhibitor of electron transport, ATP production, and intracellular amastigote growth. Cytochrome bc1 is thus a promising target for novel antileishmanial drugs, and further improvements on the buparvaquone scaffold are warranted for development of enhanced therapeutics. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Quantum Computational Studies of Electron Transfer in Respiratory Complex III and its Application for Designing New Mitocan Drugs

NASA Astrophysics Data System (ADS)

Hagras, Muhammad Ahmed

Electron transfer occurs in many biological systems which are imperative to sustain life; oxidative phosphorylation in prokaryotes and eukaryotes, and photophosphorylation in photosynthetic and plant cells are well-balanced and complementary processes. Investigating electron transfer in those natural systems provides detailed knowledge of the atomistic events that lead eventually to production of ATP, or harvesting light energy. Ubiquinol:cytochrome c oxidoreductase complex (also known as bc 1 complex, or respiratory complex III) is a middle player in the electron transport proton pumping orchestra, located in the inner-mitochondrial membrane in eukaryotes or plasma membrane in prokaryotes, which converts the free energy of redox reactions to electrochemical proton gradient across the membrane, following the fundamental chemiosmotic principle discovered by Peter Mitchell 1. In humans, the malfunctioned bc1 complex plays a major role in many neurodegenerative diseases, stress-induced aging, and cancer development, because it produces most of the reactive oxygen species, which are also involved in cellular signaling 2. The mitochondrial bc1 complex has an intertwined dimeric structure comprised of 11 subunits in each monomer, but only three of them have catalytic function, and those are the only domains found in bacterial bc1 complex. The core subunits include: Rieske domain, which incorporates iron-sulfur cluster [2Fe-2S]; trans-membrane cytochrome b domain, incorporating low-potential heme group (heme b L) and high-potential heme group (heme b H); and cytochrome c1 domain, containing heme c1 group and two separate binding sites, Qo (or QP) site where the hydrophobic electron carrier ubihydroquinol QH2 is oxidized, and Qi (or QN) site where ubiquinone molecule Q is reduced 3. Electrons and protons in the bc1 complex flow according to the proton-motive Q-cycle proposed by Mitchell, which includes a unique electron flow bifurcation at the Qo site. At this site, one electron of a bound QH2 molecule transfers to [2Fe-2S] cluster of the Rieske domain, docked at the proximal docking site, and another electron transfers to heme b L , which subsequently passes it to heme bH , and finally to Q or SQ molecule bound at the Qi-site 4. Rieske domain undergoes a domain movement 22 A to bind at the distal docking site, where [2Fe-2S] cluster passes its electron to heme c1, which in turn passes it to heme c of the water-soluble cytochrome c carrier 3c, 5 (which shuttles it to cytochrome c oxidase, complex IV). In the current compiled work presented in the subsequent chapters, we deployed a stacking tiers hierarchy where each chapter's work presents a foundation for the next one. In chapter 1, we first present different methods to calculate tunneling currents in proteins including a new derivation method for the inter-atomic tunneling current method. In addition, we show the results of the inter-atomic tunneling current theory on models based on heme bL-heme bH redox pair system in bc1 complex. Afterwards, in chapter 2, we examine the electron tunneling pathways 6 between different intra-monomeric and inter-monomeric redox centers of bc1 complex, including its electron carriers - ubiquinol, ubiquinone, and cytochrome c molecules, using the well-studied coarse-grained interatomic method of the tunneling current theory 7. Going through the different tunneling pathways in bc1 complex, we discovered a pair of internal switches that modulate the electron transfer rate which we discuss in full details in chapter 3. Motivated by the discovery of those internal switches, we discuss in chapter 4 the discovery of a new binding pocket (designated as NonQ-site or NQ-site for short) in bc 1 complex which is located at the opposite side of the enzyme with respect to Qo site. In contrast to Qo site, however, the NQ-site penetrates deeply in the cytochrome b domain and reaches very closely the LH region. Hence the NQ-site provides a suitable binding pocket for ligands that can influence the orientation of Phe90 residue, and hence modulate the corresponding ET rate between heme b L and heme bH. Finally we present in chapter 5 our unique integrated software package (called Electron Tunneling in Proteins Program or ETP) which provides an environment with different capabilities such as tunneling current calculation, semi-empirical quantum mechanical calculation and molecular modeling simulation for calculation and analysis of electron transfer reactions in proteins.

Reaction Intermediates of Quinol Oxidation in a Photoactivatable System that Mimics Electron Transfer in the Cytochrome bc1 Complex

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

Cape, Jonathan L.; Bowman, Michael K.; Kramer, David M.

2005-03-30

Current competing models for the two-electron oxidation of quinol (QH{sub 2}) at the cytochrome bc{sub 1} complex and related complexes have different requirements for the reaction intermediate. At present, the intermediate species of the enzymatic oxidation process have not been observed or characterized, probably due to their transient nature. Here, we use a biomimetic oxidant, Ru(bpy){sub 2}(pbim)(PF6)2 (bpy = 2,2'-dipyridyl, pbim = 2-(2-benzimidazolate)pyridine) in an aprotic medium to probe the oxidation of the ubiquinol analogue, 2,3-dimethoxy-5-methyl-1,4-benzoquinol (UQH{sub 2}-0), an the plastoquinol analogue, trimethyl-1,4-benzoquinol (TMQH{sub 2}-0), using time-resolved and steady state spectroscopic techniques. This system qualitatively reproduces key features observed duringmore » ubiquinol oxidation by the mitochondrial cytochrome bc1 complex. Comparison of isotope dependent activation properties in the native and synthetic systems, as well as, analysis of the time-resolved direct-detection electron para magnetic resonance signals in the synthetic system allows us to conclude that: (1) the initial and rate-limiting step in quinol oxidation, both in the biological and biomimetic systems, involves electron and proton transfer, probably via a proton coupled electron transfer mechanism; (2) a neutral semiquinone intermediate is formed in the biomimetic system; and (3) oxidation of the QH*/QH{sub 2} couple for UQH{sub 2}-0, but not TMQH{sub 2}-0, exhibits a non-classical primary deuterium kinetic isotope effect on its Arrhenius activation energy ({Delta}G{sup TS}), where {Delta}G{sup TS} for the protiated form is larger than for the deuterated form. The same behavior is observed during steady state turnover of the cyt bc{sub 1} complex using ubiquinol, but not plastoquinol, as a substrate, leading to the conclusion that similar chemical pathways are involved in both systems. The synthetic system is an unambiguous n=1 electron acceptor and it is thus inferred that sequential oxidation of ubiquinol (by two sequential n=1 processes) is more rapid than a truly concerted (n=2) oxidation in the cyt bc{sub 1} complex.« less

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase.

PubMed

Woronowicz, Kamil; Sha, Daniel; Frese, Raoul N; Sturgis, James N; Nanda, Vikas; Niederman, Robert A

2011-08-01

Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.

Ab initio single and multideterminant methods used in the determination of reduction potentials and magnetic properties of Rieske ferredoxins

NASA Astrophysics Data System (ADS)

Powers, Nathan Lee

2008-10-01

The [Fe2S2]2+/[Fe2S 2]+ electronic structure of seven Rieske protein active sites (bovine mitochondrial cytochrome bc1 complex, spinach chloroplast cytochrome b6f complex, Rieske-type ferredoxin associated with biphenyl dioxygenase from Burkholderia cepacia, yeast cytochrome bcl complex from Saccharomyces cerevisiae, Rieske subunit of arsenite oxidase from Alcaligenes faecalis, respiratory-type Rieske protein from Thermus thermophilus, and Rieske protein II (soxF) from Sulfolobus acidocaldarius), which lie in a reduction potential range from -150 mV to 375 mV, have been studied by both single and multi-determinant quantum mechanical methods. Calculated reduction potentials and magnetic properties are found comparable to experimental values.

Lipid functions in cytochrome bc complexes: an odd evolutionary transition in a membrane protein structure

PubMed Central

Hasan, S. Saif; Cramer, William A.

2012-01-01

Lipid-binding sites and properties were compared in the hetero-oligomeric cytochrome (cyt) b6f and the yeast bc1 complexes that function, respectively, in photosynthetic and respiratory electron transport. Seven lipid-binding sites in the monomeric unit of the dimeric cyanobacterial b6f complex overlap four sites in the Chlamydomonas reinhardtii algal b6f complex and four in the yeast bc1 complex. The proposed lipid functions include: (i) interfacial–interhelix mediation between (a) the two 8-subunit monomers of the dimeric complex, (b) between the core domain (cyt b, subunit IV) and the six trans membrane helices of the peripheral domain (cyt f, iron–sulphur protein (ISP), and four small subunits in the boundary ‘picket fence’); (ii) stabilization of the ISP domain-swapped trans-membrane helix; (iii) neutralization of basic residues in the single helix of cyt f and of the ISP; (iv) a ‘latch’ to photosystem I provided by the β-carotene chain protruding through the ‘picket fence’; (v) presence of a lipid and chlorophyll a chlorin ring in b6f in place of the eighth helix in the bc1 cyt b polypeptide. The question is posed of the function of the lipid substitution in relation to the evolutionary change between the eight and seven helix structures of the cyt b polypeptide. On the basis of the known n-side activation of light harvesting complex II (LHCII) kinase by the p-side level of plastoquinol, one possibility is that the change was directed by the selective advantage of p- to n-side trans membrane signalling functions in b6f, with the lipid either mediating this function or substituting for the trans membrane helix of a signalling protein lost in crystallization. PMID:23148267

Mechanism of ubiquinol oxidation by the bc(1) complex: role of the iron sulfur protein and its mobility.

PubMed

Crofts, A R; Guergova-Kuras, M; Huang, L; Kuras, R; Zhang, Z; Berry, E A

1999-11-30

Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and structures with inhibitors in place, show a 16-22 A displacement of the [2Fe-2S] cluster and the position of the C-terminal extrinsic domain of the iron sulfur protein. None of the structures shows a static configuration that would allow catalysis of all partial reactions of quinol oxidation. We have suggested that the different conformations reflect a movement of the subunit necessary for catalysis. The displacement from an interface with cytochrome c(1) in native crystals to an interface with cytochrome b is induced by stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and involves ligand formation between His-161 of the [2Fe-2S] binding cluster and the inhibitor. The movement is a rotational displacement, so that the same conserved docking surface on the iron sulfur protein interacts with cytochrome c(1) and with cytochrome b. The mobile extrinsic domain retains essentially the same tertiary structure, and the anchoring N-terminal tail remains in the same position. The movement occurs through an extension of a helical segment in the short linking span. We report details of the protein structure for the two main configurations in the chicken heart mitochondrial complex and discuss insights into mechanism provided by the structures and by mutant strains in which the docking at the cytochrome b interface is impaired. The movement of the iron sulfur protein represents a novel mechanism of electron transfer, in which a tethered mobile head allows electron transfer through a distance without the entropic loss from free diffusion.

Rational Design of Highly Potent and Slow-Binding Cytochrome bc1 Inhibitor as Fungicide by Computational Substitution Optimization

PubMed Central

Hao, Ge-Fei; Yang, Sheng-Gang; Huang, Wei; Wang, Le; Shen, Yan-Qing; Tu, Wen-Long; Li, Hui; Huang, Li-Shar; Wu, Jia-Wei; Berry, Edward A.; Yang, Guang-Fu

2015-01-01

Hit to lead (H2L) optimization is a key step for drug and agrochemical discovery. A critical challenge for H2L optimization is the low efficiency due to the lack of predictive method with high accuracy. We described a new computational method called Computational Substitution Optimization (CSO) that has allowed us to rapidly identify compounds with cytochrome bc1 complex inhibitory activity in the nanomolar and subnanomolar range. The comprehensively optimized candidate has proved to be a slow binding inhibitor of bc1 complex, ~73-fold more potent (Ki = 4.1 nM) than the best commercial fungicide azoxystrobin (AZ; Ki = 297.6 nM) and shows excellent in vivo fungicidal activity against downy mildew and powdery mildew disease. The excellent correlation between experimental and calculated binding free-energy shifts together with further crystallographic analysis confirmed the prediction accuracy of CSO method. To the best of our knowledge, CSO is a new computational approach to substitution-scanning mutagenesis of ligand and could be used as a general strategy of H2L optimisation in drug and agrochemical design.

From chemolithoautotrophs to electrolithoautotrophs: CO2 fixation by Fe(II)-oxidizing bacteria coupled with direct uptake of electrons from solid electron sources.

PubMed

Ishii, Takumi; Kawaichi, Satoshi; Nakagawa, Hirotaka; Hashimoto, Kazuhito; Nakamura, Ryuhei

2015-01-01

At deep-sea vent systems, hydrothermal emissions rich in reductive chemicals replace solar energy as fuels to support microbial carbon assimilation. Until recently, all the microbial components at vent systems have been assumed to be fostered by the primary production of chemolithoautotrophs; however, both the laboratory and on-site studies demonstrated electrical current generation at vent systems and have suggested that a portion of microbial carbon assimilation is stimulated by the direct uptake of electrons from electrically conductive minerals. Here we show that chemolithoautotrophic Fe(II)-oxidizing bacterium, Acidithiobacillus ferrooxidans, switches the electron source for carbon assimilation from diffusible Fe(2+) ions to an electrode under the condition that electrical current is the only source of energy and electrons. Site-specific marking of a cytochrome aa3 complex (aa3 complex) and a cytochrome bc1 complex (bc1 complex) in viable cells demonstrated that the electrons taken directly from an electrode are used for O2 reduction via a down-hill pathway, which generates proton motive force that is used for pushing the electrons to NAD(+) through a bc1 complex. Activation of carbon dioxide fixation by a direct electron uptake was also confirmed by the clear potential dependency of cell growth. These results reveal a previously unknown bioenergetic versatility of Fe(II)-oxidizing bacteria to use solid electron sources and will help with understanding carbon assimilation of microbial components living in electronically conductive chimney habitats.

From chemolithoautotrophs to electrolithoautotrophs: CO2 fixation by Fe(II)-oxidizing bacteria coupled with direct uptake of electrons from solid electron sources

PubMed Central

Ishii, Takumi; Kawaichi, Satoshi; Nakagawa, Hirotaka; Hashimoto, Kazuhito; Nakamura, Ryuhei

2015-01-01

At deep-sea vent systems, hydrothermal emissions rich in reductive chemicals replace solar energy as fuels to support microbial carbon assimilation. Until recently, all the microbial components at vent systems have been assumed to be fostered by the primary production of chemolithoautotrophs; however, both the laboratory and on-site studies demonstrated electrical current generation at vent systems and have suggested that a portion of microbial carbon assimilation is stimulated by the direct uptake of electrons from electrically conductive minerals. Here we show that chemolithoautotrophic Fe(II)-oxidizing bacterium, Acidithiobacillus ferrooxidans, switches the electron source for carbon assimilation from diffusible Fe2+ ions to an electrode under the condition that electrical current is the only source of energy and electrons. Site-specific marking of a cytochrome aa3 complex (aa3 complex) and a cytochrome bc1 complex (bc1 complex) in viable cells demonstrated that the electrons taken directly from an electrode are used for O2 reduction via a down-hill pathway, which generates proton motive force that is used for pushing the electrons to NAD+ through a bc1 complex. Activation of carbon dioxide fixation by a direct electron uptake was also confirmed by the clear potential dependency of cell growth. These results reveal a previously unknown bioenergetic versatility of Fe(II)-oxidizing bacteria to use solid electron sources and will help with understanding carbon assimilation of microbial components living in electronically conductive chimney habitats. PMID:26500609

Photo-Initiated Electron Transfer Within the P. denitrificans Cytochrome bc1 Complex: The mobility of the Iron Sulfur Protein is modulated by the occupant of the Qo site†

PubMed Central

Havens, Jeffrey; Castellani, Michela; Kleinschroth, Thomas; Ludwig, Bernd; Durham, Bill; Millett, Francis

2011-01-01

Domain rotation of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c1 redox centers plays a key role in the mechanism of the cyt bc1 complex. Electron transfer within the cyt bc1 complex of P. denitrificans was studied using a ruthenium dimer to rapidly photo-oxidize cyt c1 within 1 μs and initiate the reaction. In the absence of any added quinol or inhibitor of the bc1 complex at pH 8.0, electron transfer from reduced ISP to cyt c1 was biphasic with rate constants of k1f = 6300 ± 3000 s−1 and k1s = 640 ± 300 s−1 and amplitudes of 10 ± 3% and 16 ± 4 % of the total amount of cyt c1 photooxidized. Upon addition of any of the Pm type inhibitors MOA-stilbene, myxothiazol, or azoxystrobin to cyt bc1 in the absence of quinol, the total amplitude increased 2-fold, consistent with a decrease in redox potential of the ISP. In addition, the relative amplitude of the fast phase increased significantly, consistent with a change in the dynamics of the ISP domain rotation. In contrast, addition of the Pf type inhibitors JG-144 and famoxadone decreased the rate constant k1f by 5 to 10-fold, and increased the amplitude over 2-fold. Addition of quinol substrate in the absence of inhibitors led to a two-fold increase in the amplitude of the k1f phase. The effect of QH2 on the kinetics of electron transfer from reduced ISP to cyt c1 was thus similar to that of the Pm inhibitors and very different from that of the Pf inhibitors. The current results indicate that the species occupying the Qo site has a significant conformational influence on the dynamics of the ISP domain rotation. PMID:22026826

Atomistic determinants of co-enzyme Q reduction at the Qi-site of the cytochrome bc1 complex

NASA Astrophysics Data System (ADS)

Postila, Pekka A.; Kaszuba, Karol; Kuleta, Patryk; Vattulainen, Ilpo; Sarewicz, Marcin; Osyczka, Artur; Róg, Tomasz

2016-09-01

The cytochrome (cyt) bc1 complex is an integral component of the respiratory electron transfer chain sustaining the energy needs of organisms ranging from humans to bacteria. Due to its ubiquitous role in the energy metabolism, both the oxidation and reduction of the enzyme’s substrate co-enzyme Q has been studied vigorously. Here, this vast amount of data is reassessed after probing the substrate reduction steps at the Qi-site of the cyt bc1 complex of Rhodobacter capsulatus using atomistic molecular dynamics simulations. The simulations suggest that the Lys251 side chain could rotate into the Qi-site to facilitate binding of half-protonated semiquinone - a reaction intermediate that is potentially formed during substrate reduction. At this bent pose, the Lys251 forms a salt bridge with the Asp252, thus making direct proton transfer possible. In the neutral state, the lysine side chain stays close to the conserved binding location of cardiolipin (CL). This back-and-forth motion between the CL and Asp252 indicates that Lys251 functions as a proton shuttle controlled by pH-dependent negative feedback. The CL/K/D switching, which represents a refinement to the previously described CL/K pathway, fine-tunes the proton transfer process. Lastly, the simulation data was used to formulate a mechanism for reducing the substrate at the Qi-site.

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

PubMed

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

2005-03-18

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

Role of the PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 2. PufX is required for efficient ubiquinone/ubiquinol exchange between the reaction center QB site and the cytochrome bc1 complex.

PubMed

Barz, W P; Verméglio, A; Francia, F; Venturoli, G; Melandri, B A; Oesterhelt, D

1995-11-21

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides because it is required for multiple-turnover electron transfer under anaerobic conditions [see accompanying article; Barz, W. P., Francia, F., Venturoli, G., Melandri, B. A., Verméglio, A., & Oesterhelt, D. (1995) Biochemistry 34, 15235-15247]. In order to understand the molecular role of PufX, light-induced absorption spectroscopy was performed using a pufX- mutant, a pufX+ strain, and two suppressor mutants. We show that the reaction center (RC) requires PufX for its functionality under different redox conditions than the cytochrome bc1 complex: When the kinetics of flash-induced reduction of cytochrome b561 were monitored in chromatophores, we observed a requirement of PufX for turnover of the cytochrome bc1 complex only at high redox potential (Eh > 140 mV), suggesting a function of PufX in lateral ubiquinol transfer from the RC. In contrast, PufX is required for multiple turnover of the RC only under reducing conditions: When the Q pool was partially oxidized in vivo using oxygen or electron acceptors like dimethyl sulfoxide or trimethylamine N-oxide, the deletion of PufX had no effect on light-driven electron flow through the RC. Flash train experiments under anaerobic in vivo conditions revealed that RC photochemistry does not depend on PufX for the first two flash excitations. Following the third and subsequent flashes, however, efficient charge separation requires PufX, indicating an important role of PufX for fast Q/QH2 exchange at the QB site of the RC. We show that the Q/QH2 exchange rate is reduced approximately 500-fold by the deletion of PufX when the Q pool is nearly completely reduced, demonstrating an essential role of PufX for the access of ubiquinone to the QB site. The fast ubiquinone/ubiquinol exchange is partially restored by suppressor mutations altering the macromolecular antenna structure. These results suggest an indirect role of PufX in structurally organizing a functional photosynthetic apparatus.

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

PubMed

Yoshimatsu, Katsuhiko; Araya, Osamu; Fujiwara, Taketomo

2007-01-01

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

Atovaquone and ELQ-300 Combination Therapy as a Novel Dual-Site Cytochrome bc1 Inhibition Strategy for Malaria.

PubMed

Stickles, Allison M; Smilkstein, Martin J; Morrisey, Joanne M; Li, Yuexin; Forquer, Isaac P; Kelly, Jane X; Pou, Sovitj; Winter, Rolf W; Nilsen, Aaron; Vaidya, Akhil B; Riscoe, Michael K

2016-08-01

Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone (ATV) blocks the quinol oxidase (Qo) site of cyt bc1, while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Qi) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Qo site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc1 is a valuable strategy for antimalarial combination therapy and that Qi site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor ATV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Atovaquone and ELQ-300 Combination Therapy as a Novel Dual-Site Cytochrome bc1 Inhibition Strategy for Malaria

PubMed Central

Smilkstein, Martin J.; Morrisey, Joanne M.; Li, Yuexin; Forquer, Isaac P.; Kelly, Jane X.; Pou, Sovitj; Winter, Rolf W.; Nilsen, Aaron; Vaidya, Akhil B.

2016-01-01

Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone (ATV) blocks the quinol oxidase (Qo) site of cyt bc1, while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Qi) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Qo site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc1 is a valuable strategy for antimalarial combination therapy and that Qi site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor ATV. PMID:27270285

Genome-Enabled Studies of Anaerobic, Nitrate-Dependent Iron Oxidation in the Chemolithoautotrophic Bacterium Thiobacillus denitrificans

NASA Astrophysics Data System (ADS)

Beller, H. R.; Zhou, P.; Legler, T. C.; Chakicherla, A.; O'Day, P. A.

2013-12-01

Thiobacillus denitrificans is a chemolithoautotrophic bacterium capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation, both of which can strongly influence the long-term efficacy of in situ reductive immobilization of uranium in contaminated aquifers. We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II)-oxidizing bacteria. Here we report on efforts to identify genes associated with nitrate-dependent Fe(II) oxidation, namely (a) whole-genome transcriptional studies [using FeCO3, Fe2+, and U(IV) oxides as electron donors under denitrifying conditions], (b) Fe(II) oxidation assays performed with knockout mutants targeting primarily highly expressed or upregulated c-type cytochromes, and (c) random transposon-mutagenesis studies with screening for Fe(II) oxidation. Assays of mutants for 26 target genes, most of which were c-type cytochromes, indicated that none of the mutants tested were significantly defective in nitrate-dependent Fe(II) oxidation. The non-defective mutants included the c1-cytochrome subunit of the cytochrome bc1 complex (complex III), which has relevance to a previously proposed role for this complex in nitrate-dependent Fe(II) oxidation and to current concepts of reverse electron transfer. Of the transposon mutants defective in Fe(II) oxidation, one mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I) was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor. Overall, our results indicate that nitrate-dependent Fe(II) oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV) oxidation, nor have other c-type cytochromes yet been implicated in the process.

Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans

PubMed Central

Beller, Harry R.; Zhou, Peng; Legler, Tina C.; Chakicherla, Anu; Kane, Staci; Letain, Tracy E.; A. O’Day, Peggy

2013-01-01

Thiobacillus denitrificans is a chemolithoautotrophic bacterium capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation, both of which can strongly influence the long-term efficacy of in situ reductive immobilization of uranium in contaminated aquifers. We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II)-oxidizing bacteria. Here we report on efforts to identify genes associated with nitrate-dependent Fe(II) oxidation, namely (a) whole-genome transcriptional studies [using FeCO3, Fe2+, and U(IV) oxides as electron donors under denitrifying conditions], (b) Fe(II) oxidation assays performed with knockout mutants targeting primarily highly expressed or upregulated c-type cytochromes, and (c) random transposon-mutagenesis studies with screening for Fe(II) oxidation. Assays of mutants for 26 target genes, most of which were c-type cytochromes, indicated that none of the mutants tested were significantly defective in nitrate-dependent Fe(II) oxidation. The non-defective mutants included the c1-cytochrome subunit of the cytochrome bc1 complex (complex III), which has relevance to a previously proposed role for this complex in nitrate-dependent Fe(II) oxidation and to current concepts of reverse electron transfer. A transposon mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I) was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor. Overall, our results indicate that nitrate-dependent Fe(II) oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV) oxidation, nor have other c-type cytochromes yet been implicated in the process. PMID:24065960

Atomic force microscopy reveals multiple patterns of antenna organization in purple bacteria: implications for energy transduction mechanisms and membrane modeling.

PubMed

Sturgis, James N; Niederman, Robert A

2008-01-01

Recent topographs of the intracytoplasmic membrane (ICM) of purple bacteria obtained by atomic force microscopy (AFM) have provided the first surface views of the native architecture of a multicomponent biological membrane at submolecular resolution, representing an important landmark in structural biology. A variety of species-dependent, closely packed arrangements of light-harvesting (LH) complexes was revealed: the most highly organized was found in Rhodobacter sphaeroides in which the peripheral LH2 antenna was seen either in large clusters or in fixed rows interspersed among ordered arrays of dimeric LH1-reaction center (RC) core complexes. A more random organization was observed in other species containing both the LH1 and LH2 complexes, as typified by Rhododspirillum photometricum with randomly packed monomeric LH1-RC core complexes intermingled with large, paracrystalline domains of LH2 antenna. Surprisingly, no structures that could be identified as the ATP synthase or cytochrome bc (1) complexes were observed, which may reflect their localization at ICM vesicle poles or in curved membrane areas, out of view from the flat regions imaged by AFM. This possible arrangement of energy transducing complexes has required a reassessment of energy tranduction mechanisms which place the cytochrome bc (1) complex in close association with the RC. Instead, more plausible proposals must account for the movement of quinone redox species over considerable membrane distances on appropriate time scales. AFM, together with atomic resolution structures are also providing the basis for molecular modeling of the ICM that is leading to an improved picture of the supramolecular organization of photosynthetic complexes, as well as the forces that drive their segregation into distinct domains.

Partial kinetoplast-mitochondrial gene organization and expression in the respiratory deficient plant trypanosomatid Phytomonas serpens.

PubMed

Maslov, D A; Nawathean, P; Scheel, J

1999-04-30

In plant-dwelling trypanosomatids from the genus Phytomonas, mitochondrial functions, such as cytochrome mediated respiration, ATP production and Krebs cycle, are missing, and cell energetics is based on the glycolysis. Using Blue Native/Tricine-SDS two-dimensional gel electrophoretic analysis, we observed that mitochondrial respiratory Complexes III (cytochrome bc1) and IV (cytochrome c oxidase) were absent in Phytomonas serpens; however, Complex V (ATPase) was present. A deletion of the genes for cytochrome c oxidase subunit III (COIII) and apocytochrome b (Cyb) was identified within the 6234 bp sequenced region of the 31 kb maxicircle kinetoplast DNA. Genes, found in this region, include 12S and 9S ribosomal RNAs, subunits 7, 8 and 9 of NADH dehydrogenase (ND7, ND8 and ND9) and subunit 6 of ATPase (A6 or MURF4), as well as the genes (MURF1, MURF5 and G3) with unknown function. Most genes are actively transcribed and some mRNAs are edited. Fully edited mRNAs for A6 and G3 were abundant, while edited ND7 transcripts were rare, and only partially edited and pre-edited transcripts for ND8 were detected. The data show that the mitochondrial genome of P. serpens is functional, although its functions may be limited to expressing the ATPase and, possibly, NADH dehydrogenase complexes.

Synthetic chromanol derivatives and their interaction with complex III in mitochondria from bovine, yeast, and Leishmania.

PubMed

Monzote, L; Stamberg, W; Patel, A; Rosenau, T; Maes, L; Cos, P; Gille, L

2011-10-17

Synthetic chromanol derivatives (TMC4O, 6-hydroxy-2,2,7,8-tetramethyl-chroman-4-one; TMC2O, 6-hydroxy-4,4,7,8-tetramethyl-chroman-2-one; and Twin, 1,3,4,8,9,11-hexamethyl-6,12-methano-12H-dibenzo[d,g][1,3]dioxocin-2,10-diol) share structural elements with the potent inhibitor of the mitochondrial cytochrome (cyt) bc(1) complex stigmatellin. Studies with isolated bovine cyt bc(1) complex demonstrated that these compounds partially inhibit the mammalian enzyme. The aim of this work was to comparatively investigate these toxicological aspects of synthetic vitamin E derivatives in mitochondria of different species. The chromanols and atovaquone as reference compound were evaluated for their inhibition of the cyt bc(1) activity in mitochondrial fractions from bovine hearts, yeast, and Leishmania. In addition, compounds were evaluated in vitro for their inhibitory activity against whole-cell Leishmania and mouse peritoneal macrophages. In these organisms, the chromanols showed a species-selective inhibition of the cyt bc(1) activity different from that of atovaquone. While in atovaquone the side chain mediates species-selectivity, the marked differences for TMC2O and TMC4O in cyt bc(1) inhibition suggests that direct substitution of the chromanol headgroup will control selectivity in these compounds. Low micromolar concentrations of TMC2O (IC(50) = 9.5 ± 0.5 μM) inhibited the growth of Leishmania, and an esterified TMC2CO derivative inhibited the cyt bc(1) activity with an IC(50) of 4.9 ± 0.9 μM. These findings suggest that certain chromanols also exhibit beyond their antioxidative properties antileishmanial activities and that TMC2O derivatives could be useful toward the development of highly active antiprotozoal compounds.

Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c.

PubMed

Antalík, Marián; Bágel'ová, Jaroslava; Gazová, Zuzana; Musatov, Andrej; Fedunová, Diana

2003-03-21

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent pK(a) for the alkaline transition of cytochrome c. The change in pK(a) is comparable to the value when cytochrome c is complexed with cytochrome bc(1). Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met(80)-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3+/-0.2 to 66.9+/-0.2 degrees C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2+/-0.2 to 68.8+/-0.2 degrees C in the presence of polyglutamates with number of glutamate monomers n >or approximately equal 8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by approximately 42.5% with n>or approximately equal 8. The binding constant for the binding of octaglutamate (m.w. approximately 1000) to cyt c was found to be 1.15 x 10(5) M(-1) at pH 8.0 and low ionic strength. The results indicate that the polyglutamate (n>or approximately equal 8) is able to increase the stability of the methionine sulfur-heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.

Evidence from the structure and function of cytochromes c(2) that nonsulfur purple bacterial photosynthesis followed the evolution of oxygen respiration.

PubMed

Meyer, Terry; Van Driessche, Gonzalez; Ambler, Richard; Kyndt, John; Devreese, Bart; Van Beeumen, Jozef; Cusanovich, Michael

2010-10-01

Cytochromes c(2) are the nearest bacterial homologs of mitochondrial cytochrome c. The sequences of the known cytochromes c(2) can be placed in two subfamilies based upon insertions and deletions, one subfamily is most like mitochondrial cytochrome c (the small C2s, without significant insertions and deletions), and the other, designated large C2, shares 3- and 8-residue insertions as well as a single-residue deletion. C2s generally function between cytochrome bc(1) and cytochrome oxidase in respiration (ca 80 examples known to date) and between cytochrome bc(1) and the reaction center in nonsulfur purple bacterial photosynthesis (ca 21 examples). However, members of the large C2 subfamily are almost always involved in photosynthesis (12 of 14 examples). In addition, the gene for the large C2 (cycA) is associated with those for the photosynthetic reaction center (pufBALM). We hypothesize that the insertions in the large C2s, which were already functioning in photosynthesis, allowed them to replace the membrane-bound tetraheme cytochrome, PufC, that otherwise mediates between the small C2 or other redox proteins and photosynthetic reaction centers. Based upon our analysis, we propose that the involvement of C2 in nonsulfur purple bacterial photosynthesis was a metabolic feature subsequent to the evolution of oxygen respiration.

Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants.

PubMed

Santos-Ocaña, Carlos; Do, Thai Q; Padilla, Sergio; Navas, Placido; Clarke, Catherine F

2002-03-29

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q(6) by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains. Q(6) supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains. Following culture in medium containing dextrose, the added Q(6) was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q(6) in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q(6). These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q(6) into the plasma membrane. The two coq7 mutant strains rescued by Q(6) supplementation for growth on ethanol contained mitochondrial Q(6) levels similar to wild type. However, the content of Q(6) in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q(6), succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q(6) content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c(1) polypeptide. These studies show that transport of extracellular Q(6) to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc(1) complex is unstable. These results indicate that delivery of exogenous Q(6) to mitochondria is required fore activity and stability of the bc(1) complex in yeast coq mutants.

Visualizing changes in electron distribution in coupled chains of cytochrome bc(1) by modifying barrier for electron transfer between the FeS cluster and heme c(1).

PubMed

Cieluch, Ewelina; Pietryga, Krzysztof; Sarewicz, Marcin; Osyczka, Artur

2010-02-01

Cytochrome c(1) of Rhodobacter (Rba.) species provides a series of mutants which change barriers for electron transfer through the cofactor chains of cytochrome bc(1) by modifying heme c(1) redox midpoint potential. Analysis of post-flash electron distribution in such systems can provide useful information about the contribution of individual reactions to the overall electron flow. In Rba. capsulatus, the non-functional low-potential forms of cytochrome c(1) which are devoid of the disulfide bond naturally present in this protein revert spontaneously by introducing a second-site suppression (mutation A181T) that brings the potential of heme c(1) back to the functionally high levels, yet maintains it some 100 mV lower from the native value. Here we report that the disulfide and the mutation A181T can coexist in one protein but the mutation exerts a dominant effect on the redox properties of heme c(1) and the potential remains at the same lower value as in the disulfide-free form. This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme c(1) without breaking disulfide. A comparison of the flash-induced electron transfers in native and mutated cytochrome bc(1) revealed significant differences in the post-flash equilibrium distribution of electrons only when the connection of the chains with the quinone pool was interrupted at the level of either of the catalytic sites by the use of specific inhibitors, antimycin or myxothiazol. In the non-inhibited system no such differences were observed. We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains. It follows a rather simple description in which the direction of electron flow through the coupled chains of cytochrome bc(1) exclusively depends on the rates of all reversible partial reactions, including the Q/QH2 exchange rate to/from the catalytic sites. 2009 Elsevier B.V. All rights reserved.

Possible roles of two quinone molecules in direct and indirect proton pumps of bovine heart NADH-quinone oxidoreductase (complex I).

PubMed

Ohnishi, S Tsuyoshi; Salerno, John C; Ohnishi, Tomoko

2010-12-01

In many energy transducing systems which couple electron and proton transport, for example, bacterial photosynthetic reaction center, cytochrome bc(1)-complex (complex III) and E. coli quinol oxidase (cytochrome bo(3) complex), two protein-associated quinone molecules are known to work together. T. Ohnishi and her collaborators reported that two distinct semiquinone species also play important roles in NADH-ubiquinone oxidoreductase (complex I). They were called SQ(Nf) (fast relaxing semiquinone) and SQ(Ns) (slow relaxing semiquinone). It was proposed that Q(Nf) serves as a "direct" proton carrier in the semiquinone-gated proton pump (Ohnishi and Salerno, FEBS Letters 579 (2005) 4555), while Q(Ns) works as a converter between one-electron and two-electron transport processes. This communication presents a revised hypothesis in which Q(Nf) plays a role in a "direct" redox-driven proton pump, while Q(Ns) triggers an "indirect" conformation-driven proton pump. Q(Nf) and Q(Ns) together serve as (1e(-)/2e(-)) converter, for the transfer of reducing equivalent to the Q-pool. Copyright © 2010 Elsevier B.V. All rights reserved.

Genome-wide identification of 31 cytochrome P450 (CYP) genes in the freshwater rotifer Brachionus calyciflorus and analysis of their benzo[α]pyrene-induced expression patterns.

PubMed

Han, Jeonghoon; Kim, Duck-Hyun; Kim, Hui-Su; Kim, Hee-Jin; Declerck, Steven A J; Hagiwara, Atsushi; Lee, Jae-Seong

2018-03-01

While marine invertebrate cytochrome P450 (CYP) genes and their roles in detoxification mechanisms have been studied, little information is available regarding freshwater rotifer CYPs and their functions. Here, we used genomic sequences and RNA-seq databases to identify 31 CYP genes in the freshwater rotifer Brachionus calyciflorus. The 31 Bc-CYP genes with a few tandem duplications were clustered into CYP 2, 3, 4, mitochondrial, and 46 clans with two marine rotifers Brachionus plicatilis and Brachionus koreanus. To understand the molecular responses of these 31 Bc-CYP genes, we also examined their expression patterns in response to benzo[α]pyrene (B[α]P). Three Bc-CYP genes (Bc-CYP3044B3, Bc-CYP3049B4, Bc-CYP3049B6) were significantly upregulated (P<0.05) in response to B[α]P, suggesting that these CYP genes can be involved in detoxification in response to B[α]P exposure. These genes might be useful as biomarkers of B[α]P exposure in B. calyciflorus. Overall, our findings expand the repertoire of known CYPs and shed light on their potential roles in xenobiotic detoxification in rotifers. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutational Analysis of the QRRQ Motif in the Yeast Hig1 Type 2 Protein Rcf1 Reveals a Regulatory Role for the Cytochrome c Oxidase Complex*

PubMed Central

Garlich, Joshua; Strecker, Valentina; Wittig, Ilka; Stuart, Rosemary A.

2017-01-01

The yeast Rcf1 protein is a member of the conserved family of proteins termed the hypoxia-induced gene (domain) 1 (Hig1 or HIGD1) family. Rcf1 interacts with components of the mitochondrial oxidative phosphorylation system, in particular the cytochrome bc1 (complex III)-cytochrome c oxidase (complex IV) supercomplex (termed III-IV) and the ADP/ATP carrier proteins. Rcf1 plays a role in the assembly and modulation of the activity of complex IV; however, the molecular basis for how Rcf1 influences the activity of complex IV is currently unknown. Hig1 type 2 isoforms, which include the Rcf1 protein, are characterized in part by the presence of a conserved motif, (Q/I)X3(R/H)XRX3Q, termed here the QRRQ motif. We show that mutation of conserved residues within the Rcf1 QRRQ motif alters the interactions between Rcf1 and partner proteins and results in the destabilization of complex IV and alteration of its enzymatic properties. Our findings indicate that Rcf1 does not serve as a stoichiometric component, i.e. as a subunit of complex IV, to support its activity. Rather, we propose that Rcf1 serves to dynamically interact with complex IV during its assembly process and, in doing so, regulates a late maturation step of complex IV. We speculate that the Rcf1/Hig1 proteins play a role in the incorporation and/or remodeling of lipids, in particular cardiolipin, into complex IV and. possibly, other mitochondrial proteins such as ADP/ATP carrier proteins. PMID:28167530

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

PubMed Central

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

2009-01-01

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

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

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

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

2009-06-08

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

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

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

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

2014-03-20

Structure–function of the major integral membrane cytochrome b 6f complex that functions in cyanobacteria, algae, and green plants to transfer electrons between the two reaction center complexes in the electron transport chain of oxygenic photosynthesis is discussed in the context of recently obtained crystal structures of the complex and soluble domains of cytochrome f and the Rieske iron–sulfur protein. The energy-transducing function of the complex, generation of the proton trans-membrane electrochemical potential gradient, centers on the oxidation/reduction pathways of the plastoquinol/plastoquinone (QH 2/Q), the proton donor/acceptor within the complex. These redox reactions are carried out by five redox prosthetic groupsmore » embedded in each monomer, the high potential two iron–two sulfur cluster and the heme of cytochrome f on the electropositive side (p) of the complex, two noncovalently bound b-type hemes that cross the complex and the membrane, and a covalently bound c-type heme (c n) on the electronegative side (n). These five redox-active groups are organized in high- (cyt f/[2Fe–2S] and low-potential (hemes b p, b n, c n) electron transport pathways that oxidize and reduce the quinol and quinone on the p- and n-sides in a Q-cycle-type mechanism, while translocating as many as 2 H + to the p-side aqueous side for every electron transferred through the high potential chain to the photosystem I reaction center. The presence of heme c n and the connection of the n-side of the membrane and b 6f complex to the cyclic electron transport chain indicate that the Q cycle in the oxygenic photosynthetic electron transport chain differs from those connected to the bc 1 complex in the mitochondrial respiratory chain and the chain in photosynthetic bacteria. Inferences from the structure and C2 symmetry of the complex for the pathway of QH 2/Q transfer within the complex, problems posed by the presence of lipid in the inter-monomer cavity, and the narrow portal for QH2 passage through the p-side oxidation site proximal to the [2Fe–2S] cluster are discussed.« less

Structure of the alternative complex III in a supercomplex with cytochrome oxidase.

PubMed

Sun, Chang; Benlekbir, Samir; Venkatakrishnan, Padmaja; Wang, Yuhang; Hong, Sangjin; Hosler, Jonathan; Tajkhorshid, Emad; Rubinstein, John L; Gennis, Robert B

2018-05-01

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria 1-3 . Like complex III (also known as the bc 1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity 4-7 . Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily 8 , including the proton pump polysulfide reductase 9,10 . We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer 11-14 , both as an independent enzyme and as a functional 1:1 supercomplex with an aa 3 -type cytochrome c oxidase (cyt aa 3 ). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa 3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.

Effect of H bond removal and changes in the position of the iron–sulphur head domain on the spin–lattice relaxation properties of the [2Fe–2S]2+ Rieske cluster in cytochrome bc1† †Electronic supplementary information (ESI) available: Fig. S1: results of equilibrium redox titration of the Fe–S cluster for different forms of cytochrome bc1; Fig. S2: selected results of simulations of X-band CW EPR spectra of the Rieske cluster recorded at 25 K using a single-component model; Fig. S3: temperature dependence of the spin–lattice relaxation rate (1/T1) of the Rieske cluster for the tds-inhibited WT in linearized form for Raman and Orbach processes; Table S1: values of g and g-strain components determined from fitting the two-component model to X-band CW EPR spectra (25 K) for different forms of cytochrome bc1. See DOI: 10.1039/c5cp02815a

PubMed Central

Sarewicz, Marcin; Dutka, Małgorzata; Pietras, Rafał; Borek, Arkadiusz

2015-01-01

Here, comparative electron spin–lattice relaxation studies of the 2Fe–2S iron–sulphur (Fe–S) cluster embedded in a large membrane protein complex – cytochrome bc1 – are reported. Structural modifications of the local environment alone (mutations S158A and Y160W removing specific H bonds between Fe–S and amino acid side chains) or in combination with changes in global protein conformation (mutations/inhibitors changing the position of the Fe–S binding domain within the protein complex) resulted in different redox potentials as well as g-, g-strain and the relaxation rates (T1–1) for the Fe–S cluster. The relaxation rates for T < 25 K were measured directly by inversion recovery, while for T > 60 K they were deduced from simulation of continuous wave EPR spectra of the cluster using a model that included anisotropy of Lorentzian broadening. In all cases, the relaxation rate involved contributions from direct, second-order Raman and Orbach processes, each dominating over different temperature ranges. The analysis of T1–1 (T) over the range 5–120 K yielded the values of the Orbach energy (EOrb), Debye temperature θD and Raman process efficiency CRam for each variant of the protein. As the Orbach energy was generally higher for mutants S158A and Y160W, compared to wild-type protein (WT), it is suggested that H bond removal influences the geometry leading to increased strength of antiferromagnetic coupling between two Fe ions of the cluster. While θD was similar for all variants (∼107 K), the efficiency of the Raman process generally depends on the spin–orbit coupling that is lower for S158A and Y160W mutants, when compared to the WT. However, in several cases CRam did not only correlate with spin–orbit coupling but was also influenced by other factors – possibly the modification of protein rigidity and therefore the vibrational modes around the Fe–S cluster that change upon the movement of the iron–sulphur head domain. PMID:26355649

Inhalation of Roman chamomile essential oil attenuates depressive-like behaviors in Wistar Kyoto rats.

PubMed

Kong, Yingying; Wang, Ting; Wang, Rong; Ma, Yichuan; Song, Shanshan; Liu, Juan; Hu, Weiwei; Li, Shengtian

2017-06-01

The idea of aromatherapy, using essential oils, has been considered as an alternative antidepressant treatment. In the present study, we investigated the effect of Roman chamomile essential oil inhalation for two weeks on depressive-like behaviors in Wistar-Kyoto (WKY) rats. We found that inhalation of either Roman chamomile or one of its main components α-pinene, attenuated depressive-like behavior in WKY rats in the forced swim test. Using isobaric tags for relative and absolute quantitation analysis (iTRAQ), we found that inhalation of α-pinene increased expression of proteins that are involved in oxidative phosphorylation, such as cytochrome c oxidase subunit 6C-2, cytochrome c oxidase subunit 7A2, ATPase inhibitor in the hippocampus, and cytochrome c oxidase subunit 6C-2, ATP synthase subunit e, Acyl carrier protein, and Cytochrome b-c1 complex subunit 6 in the PFC (prefrontal cortex). In addition, using the quantitative real-time polymerase chain reaction technique, we confirmed an increase of parvalbumin mRNA expression in the hippocampus, which was shown to be upregulated by 2.8-fold in iTRAQ analysis, in α-pinene treated WKY rats. These findings collectively suggest the involvement of mitochondrial functions and parvalbumin-related signaling in the antidepressant effect of α-pinene inhalation.

Structure and function of the tetraheme cytochrome associated to the reaction center of Roseobacter denitrificans.

PubMed

Garcia, D; Richaud, P; Breton, J; Verméglio, A

1994-01-01

We have characterized the tetrahemic RC bound cytochrome isolated from the quasi-photosynthetic bacterium Roseobacter denitrificans in terms of absorption spectrum, redox property and orientation with respect to the membrane plane. The heme, designated H1, which possesses the highest redox midpoint potential (+290 mV), absorbs at 555 nm. Its plane makes an angle of 40 degrees with the membrane plane. The second high potential heme, H2 (+240 mV), peaks at 554 nm and makes a tilt of 55 degrees with the membrane. The two low potential hemes, L1 and L2, present a similar and rather high redox midpoint potential (+90 mV). They absorb at 553 nm and 550 nm. One of these hemes is oriented at 40 degrees while the other makes an angle of 90 degrees with the membrane plane. The soluble cytochrome c551 completes the cyclic electron transfer between the RC and the bc1 complex. Both the oxidation and the re-reduction of cytochrome c551 are diffusible processes. Under semi-aerobic conditions, one of the low potential hemes is photo-oxidized under illumination but only extremely slowly re-reduced. This explains the requirement of high aerobic conditions for growth of Roseobacter denitrificans cells in the light.

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

PubMed

Rottenberg, Hagai

2014-10-01

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

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

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

Kolodkin-Gal, I; Elsholz, AKW; Muth, C

2013-04-29

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa(3) and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showedmore » that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.« less

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

PubMed Central

Kolodkin-Gal, Ilana; Elsholz, Alexander K.W.; Muth, Christine; Girguis, Peter R.; Kolter, Roberto; Losick, Richard

2013-01-01

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration. PMID:23599347

The diheme cytochrome c4 from Vibrio cholerae is a natural electron donor to the respiratory cbb3 oxygen reductase

PubMed Central

Chang, Hsin-Yang; Ahn, Young; Pace, Laura A.; Lin, Myat T.; Lin, Yun-Hui; Gennis, Robert B.

2010-01-01

The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb3 oxygen reductase. The cbb3 oxygen reductase has been previously isolated and characterized, however the natural mobile electron donor(s) which shuttles electrons between the bc1 complex and the cbb3 oxygen reductase is not known. The most likely candidates are the diheme cytochrome c4 and mono-heme cytochrome c5, which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c4 and c5 from V. cholerae have been cloned and expressed heterologously in E. coli. It is shown that reduced cytochrome c4 is a substrate for the purified cbb3 oxygen reductase and can support steady state oxygen reductase activity of at least 300 e−1/s. In contrast, reduced cytochrome c5 is not a good substrate for the cbb3 oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c4 does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c4 indicates that the apparent lack of saturation is due to the strong dependence of KM and Vmax on the concentration of oxidized cytochrome c4. Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c4 are 240 mV and 340 mV (vs SHE), similar to the electrochemical properties of other c4-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c4-type cytochrome and a cbb3 oxygen reductase within the β- and γ- proteobacterial clades, suggesting that cytochrome c4 is the likely natural electron donor to the cbb3 oxygen reductases within these organisms. These would include the β-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb3 oxygen reductases are the only terminal oxidases in their respiratory chains, and the γ- proteobacterium Pseudomonas stutzeri. PMID:20715760

Generator-specific targets of mitochondrial reactive oxygen species.

PubMed

Bleier, Lea; Wittig, Ilka; Heide, Heinrich; Steger, Mirco; Brandt, Ulrich; Dröse, Stefan

2015-01-01

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for "signaling" and "damaging" ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc1 complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H2O2) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Elevated expression of esterase and cytochrome P450 are related with lambda-cyhalothrin resistance and lead to cross resistance in Aphis glycines Matsumura.

PubMed

Xi, Jinghui; Pan, Yiou; Bi, Rui; Gao, Xiwu; Chen, Xuewei; Peng, Tianfei; Zhang, Min; Zhang, Hua; Hu, Xiaoyue; Shang, Qingli

2015-02-01

A resistant strain of the Aphis glycines Matsumura (CRR) has developed 76.67-fold resistance to lambda-cyhalothrin compared with the susceptible (CSS) strain. Synergists piperonyl butoxide (PBO), S,S,S-Tributyltrithiophosphate (DEF) and triphenyl phosphate (TPP) dramatically increased the toxicity of lambda-cyhalothrin to the resistant strain. Bioassay results indicated that the CRR strain had developed high levels of cross-resistance to chlorpyrifos (11.66-fold), acephate (8.20-fold), cypermethrin (53.24-fold), esfenvalerate (13.83-fold), cyfluthrin (9.64-fold), carbofuran (14.60-fold), methomyl (9.32-fold) and bifenthrin (4.81-fold), but did not have cross-resistance to chlorfenapyr, imidacloprid, diafenthiuron, abamectin. The transcriptional levels of CYP6A2-like, CYP6A14-like and cytochrome b-c1 complex subunit 9-like increased significantly in the resistant strain than that in the susceptible. Similar trend were observed in the transcripts and DNA copy number of CarE and E4 esterase. Overall, these results demonstrate that increased esterase hydrolysis activity, combined with elevated cytochrome P450 monooxygenase detoxicatication, plays an important role in the high levels of lambda-cyhalothrin resistance and can cause cross-resistance to other insecticides in the CRR strain. Copyright © 2014 Elsevier Inc. All rights reserved.

Phylogenomic reconstruction of archaeal fatty acid metabolism

PubMed Central

Dibrova, Daria V.; Galperin, Michael Y.; Mulkidjanian, Armen Y.

2014-01-01

While certain archaea appear to synthesize and/or metabolize fatty acids, the respective pathways still remain obscure. By analyzing the genomic distribution of the key lipid-related enzymes, we were able to identify the likely components of the archaeal pathway of fatty acid metabolism, namely, a combination of the enzymes of bacterial-type β-oxidation of fatty acids (acyl-CoA-dehydrogenase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase) with paralogs of the archaeal acetyl-CoA C-acetyltransferase, an enzyme of the mevalonate biosynthesis pathway. These three β-oxidation enzymes working in the reverse direction could potentially catalyze biosynthesis of fatty acids, with paralogs of acetyl-CoA C-acetyltransferase performing addition of C2 fragments. The presence in archaea of the genes for energy-transducing membrane enzyme complexes, such as cytochrome bc complex, cytochrome c oxidase, and diverse rhodopsins, was found to correlate with the presence of the proposed system of fatty acid biosynthesis. We speculate that because these membrane complexes functionally depend on fatty acid chains, their genes could have been acquired via lateral gene transfer from bacteria only by those archaea that already possessed a system of fatty acid biosynthesis. The proposed pathway of archaeal fatty acid metabolism operates in extreme conditions and therefore might be of interest in the context of biofuel production and other industrial applications. PMID:24818264

The Alternative complex III: properties and possible mechanisms for electron transfer and energy conservation.

PubMed

Refojo, Patrícia N; Teixeira, Miguel; Pereira, Manuela M

2012-10-01

Alternative complexes III (ACIII) are recently identified membrane-bound enzymes that replace functionally the cytochrome bc(1/)b(6)f complexes. In general, ACIII are composed of four transmembrane proteins and three peripheral subunits that contain iron-sulfur centers and C-type hemes. ACIII are built by a combination of modules present in different enzyme families, namely the complex iron-sulfur molybdenum containing enzymes. In this article a historical perspective on the investigation of ACIII is presented, followed by an overview of the present knowledge on these enzymes. Electron transfer pathways within the protein are discussed taking into account possible different locations (cytoplasmatic or periplasmatic) of the iron-sulfur containing protein and their contribution to energy conservation. In this way several hypotheses for energy conservation modes are raised including linear and bifurcating electron transfer pathways. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

A complex of cardiac cytochrome c1 and cytochrome c.

PubMed

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

1976-01-10

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of cytochrome c on the methionine-80 side of the heme crevice.

Proton pumping in the bc1 complex: a new gating mechanism that prevents short circuits.

PubMed

Crofts, Antony R; Lhee, Sangmoon; Crofts, Stephanie B; Cheng, Jerry; Rose, Stuart

2006-08-01

The Q-cycle mechanism of the bc1 complex explains how the electron transfer from ubihydroquinone (quinol, QH2) to cytochrome (cyt) c (or c2 in bacteria) is coupled to the pumping of protons across the membrane. The efficiency of proton pumping depends on the effectiveness of the bifurcated reaction at the Q(o)-site of the complex. This directs the two electrons from QH2 down two different pathways, one to the high potential chain for delivery to an electron acceptor, and the other across the membrane through a chain containing heme bL and bH to the Qi-site, to provide the vectorial charge transfer contributing to the proton gradient. In this review, we discuss problems associated with the turnover of the bc1 complex that center around rates calculated for the normal forward and reverse reactions, and for bypass (or short-circuit) reactions. Based on rate constants given by distances between redox centers in known structures, these appeared to preclude conventional electron transfer mechanisms involving an intermediate semiquinone (SQ) in the Q(o)-site reaction. However, previous research has strongly suggested that SQ is the reductant for O2 in generation of superoxide at the Q(o)-site, introducing an apparent paradox. A simple gating mechanism, in which an intermediate SQ mobile in the volume of the Q(o)-site is a necessary component, can readily account for the observed data through a coulombic interaction that prevents SQ anion from close approach to heme bL when the latter is reduced. This allows rapid and reversible QH2 oxidation, but prevents rapid bypass reactions. The mechanism is quite natural, and is well supported by experiments in which the role of a key residue, Glu-295, which facilitates proton transfer from the site through a rotational displacement, has been tested by mutation.

Purification and properties of two terminal oxidase complexes of Escherichia coli aerobic respiratory chain.

PubMed

Kita, K; Konishi, K; Anraku, Y

1986-01-01

Two terminal oxidase complexes, cytochrome b-562-o complex and cytochrome b-558-d complex, are isolated in highly purified forms which show ubiquinol oxidase activities. From the result of steady-state kinetics of cytochromes in the membrane and E'm values of purified cytochromes, we propose a branched arrangement of the late exponential phase of aerobic growth, as shown in Fig. 10. Cytochrome b-556 is reduced by several dehydrogenases and the gene for this cytochrome (cybA) is located in the sdh gene cluster. Recently, we found another low-potential b-type cytochrome, cytochrome b-561 (Em' = 20 mV), which is also reduced by dehydrogenases. The position of this new cytochrome in the aerobic respiratory chain is under investigation. Two terminal oxidase complexes branch at the site of ubiquinone-8, and the Km value for oxygen of the purified cytochrome b-558-d complex is about 8-fold lower than that of the purified cytochrome b-562-o complex when ubiquinol-1 is used as substrate. This result is consistent with the idea that the cytochrome b-558-d complex is synthesized as an alternative oxidase for more efficient utilization of oxygen at low oxygen concentration. Thus, E. coli cells can maintain efficient oxidative energy conservation over a wide range of oxygen pressures by simply changing the contents of the two terminal oxidases, each of which functions as a coupling site.

Electronic and magnetic properties of Fe-, Co-, and Ni-decorated BC3: A first-principles study

NASA Astrophysics Data System (ADS)

Zhu, Jingzhong; Zhao, Yinchang; Zulfiqar, Muhammad; Zeng, Shuming; Ni, Jun

2018-05-01

The electronic and magnetic properties of Fe-, Co-, and Ni-decorated two dimensional (2D) BC3 are systematically investigated by first-principles calculations. We find that the Fe, Co, and Ni atoms can be strongly adsorbed on the hollow sites of 2D BC3. Fe and Co adatoms are more stable when adsorbed on the hollow sites of the carbon rings in the 2D BC3, while the hollow sites of boron-carbon rings in the 2D BC3 are the most stable sites for the adsorption of Ni adatoms. These proposed metal-BC3 complexes exhibit interesting electronic and magnetic behaviors. In particular, the Fe-BC3 and Co-BC3 complexes are metals with magnetic ground states , while the Ni-BC3 complex behaves as a nonmagnetic semiconductor with a direct bandgap. Furthermore, our magnetic analysis reveals that induced magnetism in the Fe-BC3 and Co-BC3 complexes arises from their local magnetic moments. Functionalization of 2D BC3 through these metal-adatom adsorption appears to be a promising way to extend its applications.

Comparison of environmental and isolate Sulfobacillus genomes reveals diverse carbon, sulfur, nitrogen, and hydrogen metabolisms

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

Justice, Nicholas B.; Norman, Anders; Brown, Christopher T.

Bacteria of the genus Sulfobacillus are found worldwide as members of microbial communities that accelerate sulfide mineral dissolution in acid mine drainage environments (AMD), acid-rock drainage environments (ARD), as well as in industrial bioleaching operations. Despite their frequent identification in these environments, their role in biogeochemical cycling is poorly understood. Here we report draft genomes of five species of the Sulfobacillus genus (AMDSBA1-5) reconstructed by cultivation-independent sequencing of biofilms sampled from the Richmond Mine (Iron Mountain, CA). Three of these species (AMDSBA2, AMDSBA3, and AMDSBA4) have no cultured representatives while AMDSBA1 is a strain of S. benefaciens, and AMDSBA5 amore » strain of S. thermosulfidooxidans. We analyzed the diversity of energy conservation and central carbon metabolisms for these genomes and previously published Sulfobacillus genomes. Pathways of sulfur oxidation vary considerably across the genus, including the number and type of subunits of putative heterodisulfide reductase complexes likely involved in sulfur oxidation. The number and type of nickel-iron hydrogenase proteins varied across the genus, as does the presence of different central carbon pathways. Only the AMDSBA3 genome encodes a dissimilatory nitrate reducatase and only the AMDSBA5 and S. thermosulfidooxidans genomes encode assimilatory nitrate reductases. Lastly, within the genus, AMDSBA4 is unusual in that its electron transport chain includes a cytochrome bc type complex, a unique cytochrome c oxidase, and two distinct succinate dehydrogenase complexes. Overall, the results significantly expand our understanding of carbon, sulfur, nitrogen, and hydrogen metabolism within the Sulfobacillus genus.« less

Comparison of environmental and isolate Sulfobacillus genomes reveals diverse carbon, sulfur, nitrogen, and hydrogen metabolisms

DOE PAGES

Justice, Nicholas B.; Norman, Anders; Brown, Christopher T.; ...

2014-12-15

Bacteria of the genus Sulfobacillus are found worldwide as members of microbial communities that accelerate sulfide mineral dissolution in acid mine drainage environments (AMD), acid-rock drainage environments (ARD), as well as in industrial bioleaching operations. Despite their frequent identification in these environments, their role in biogeochemical cycling is poorly understood. Here we report draft genomes of five species of the Sulfobacillus genus (AMDSBA1-5) reconstructed by cultivation-independent sequencing of biofilms sampled from the Richmond Mine (Iron Mountain, CA). Three of these species (AMDSBA2, AMDSBA3, and AMDSBA4) have no cultured representatives while AMDSBA1 is a strain of S. benefaciens, and AMDSBA5 amore » strain of S. thermosulfidooxidans. We analyzed the diversity of energy conservation and central carbon metabolisms for these genomes and previously published Sulfobacillus genomes. Pathways of sulfur oxidation vary considerably across the genus, including the number and type of subunits of putative heterodisulfide reductase complexes likely involved in sulfur oxidation. The number and type of nickel-iron hydrogenase proteins varied across the genus, as does the presence of different central carbon pathways. Only the AMDSBA3 genome encodes a dissimilatory nitrate reducatase and only the AMDSBA5 and S. thermosulfidooxidans genomes encode assimilatory nitrate reductases. Lastly, within the genus, AMDSBA4 is unusual in that its electron transport chain includes a cytochrome bc type complex, a unique cytochrome c oxidase, and two distinct succinate dehydrogenase complexes. Overall, the results significantly expand our understanding of carbon, sulfur, nitrogen, and hydrogen metabolism within the Sulfobacillus genus.« less

Effect of H bond removal and changes in the position of the iron-sulphur head domain on the spin-lattice relaxation properties of the [2Fe-2S](2+) Rieske cluster in cytochrome bc(1).

PubMed

Sarewicz, Marcin; Dutka, Małgorzata; Pietras, Rafał; Borek, Arkadiusz; Osyczka, Artur

2015-10-14

Here, comparative electron spin-lattice relaxation studies of the 2Fe-2S iron-sulphur (Fe-S) cluster embedded in a large membrane protein complex - cytochrome bc1 - are reported. Structural modifications of the local environment alone (mutations S158A and Y160W removing specific H bonds between Fe-S and amino acid side chains) or in combination with changes in global protein conformation (mutations/inhibitors changing the position of the Fe-S binding domain within the protein complex) resulted in different redox potentials as well as g-, g-strain and the relaxation rates (T1(-1)) for the Fe-S cluster. The relaxation rates for T < 25 K were measured directly by inversion recovery, while for T > 60 K they were deduced from simulation of continuous wave EPR spectra of the cluster using a model that included anisotropy of Lorentzian broadening. In all cases, the relaxation rate involved contributions from direct, second-order Raman and Orbach processes, each dominating over different temperature ranges. The analysis of T1(-1) (T) over the range 5-120 K yielded the values of the Orbach energy (EOrb), Debye temperature θD and Raman process efficiency CRam for each variant of the protein. As the Orbach energy was generally higher for mutants S158A and Y160W, compared to wild-type protein (WT), it is suggested that H bond removal influences the geometry leading to increased strength of antiferromagnetic coupling between two Fe ions of the cluster. While θD was similar for all variants (∼107 K), the efficiency of the Raman process generally depends on the spin-orbit coupling that is lower for S158A and Y160W mutants, when compared to the WT. However, in several cases CRam did not only correlate with spin-orbit coupling but was also influenced by other factors - possibly the modification of protein rigidity and therefore the vibrational modes around the Fe-S cluster that change upon the movement of the iron-sulphur head domain.

[Impact of Pax-8 gene interference on mitochondrial function and cardiomyocyte apoptosis].

PubMed

Dai, Xiao-chun; Zhou, Xi; Huang, Xiao-yan; Wang, Liang-guo; Lin, Su; Yang, De-ye

2013-01-01

To observe the effects of paired box gene 8 (Pax-8) silencing by RNA interference on mitochondrial function and cardiomyocytes apoptosis. The cultured H9C2 (2-1) myocytes were divided into 3 groups: short interference RNA targeting Pax-8 (Pax-8 siRNA) group, non-specific siRNA group as the negative control (NC siRNA), and blank control group (BC siRNA). Fluorescence spectrophotometry was used to detect the activity of caspase-3. RT-PCR was performed to detect mRNA expression of Bcl2 and Bax. The protein expression of Bcl2, Bax and cytoplasm of Cytochrome was examined by Western blot. Changes of ΔΨm were detected by flow cytometry.ΔΨm with JC-1 monomer/polymer ratio was calculated for measuring mitochondrial depolarization proportion. Compared to NC siRNA and BC siRNA group (0.075 ± 0.021, 0.072 ± 0.019), the activity of caspase-3 in Pax-8 siRNA group (0.167 ± 0.012) was significantly increased (P < 0.05); Bcl2 mRNA and protein expression in Pax-8 siRNA group (0.61 ± 0.06, 0.94 ± 0.11) were significantly downregulated compared with NC siRNA group (0.90 ± 0.070, 1.39 ± 0.15) and BC siRNA group (0.94 ± 0.087, 1.49 ± 0.20) (P < 0.05); Bax mRNA and protein expression in Pax-8 siRNA group (1.05 ± 0.10, 1.25 ± 0.12) were markedly upregulated compared with NC siRNA group (0.72 ± 0.03, 0.99 ± 0.12) and BC siRNA group (0.64 ± 0.03, 0.92 ± 0.06), P < 0.05; cytosolic cytochrome expression in Pax-8 siRNA group (0.75 ± 0.14) was significantly upregulated compared with NC siRNA group (0.51 ± 0.06) and BC siRNA group (0.48 ± 0.07) (P < 0.05); JC-1 monomer/polymer ratio in Pax-8 siRNA group (0.163 ± 0.011) was significantly increased compared with NC siRNA group (0.092 ± 0.015) and BC siRNA group (0.072 ± 0.025) (P < 0.05) indicating mitochondrial membrane potential was significantly reduced in Pax-8 siRNA group. Above parameters were similar between NC siRNA group and BC siRNA group (P > 0.05). Inhibiting Pax-8 results in enhanced cardiomyocytes apoptosis through the mitochondrial pathway.

A Novel Uncultured Bacterium of the Family Gallionellaceae: Description and Genome Reconstruction Based on the Metagenomic Analysis of Microbial Community in Acid Mine Drainage.

PubMed

Kadnikov, V V; Ivasenko, D A; Beletsky, A V; Mardanov, A V; Danilova, E V; Pimenov, N V; Karnachuk, O V; Ravin, N V

2016-07-01

Drainage waters at the metal mining areas often have low pH and high content of dissolved metals due to oxidation of sulfide minerals. Extreme conditions limit microbial diversity in- such ecosystems. A drainage water microbial community (6.5'C, pH 2.65) in an open pit at the Sherlovaya Gora polymetallic open-cast mine (Transbaikal region, Eastern Siberia, Russia) was studied using metagenomic techniques. Metagenome sequencing provided information for taxonomic and functional characterization of the micro- bial community. The majority of microorganisms belonged to a single uncultured lineage representing a new Betaproteobacteria species of the genus Gallionella. While no.acidophiles are known among the cultured members of the family Gallionellaceae, similar 16S rRNA gene sequences were detected in acid mine drain- ages. Bacteria ofthe genera Thiobacillus, Acidobacterium, Acidisphaera, and Acidithiobacillus,-which are com- mon in acid mine drainage environments, were the minor components of the community. Metagenomic data were -used to determine the almost complete (-3.4 Mb) composite genome of the new bacterial. lineage desig- nated Candidatus Gallionella acididurans ShG14-8. Genome analysis revealed that Fe(II) oxidation probably involved the cytochromes localized on the outer membrane of the cell. The electron transport chain included NADH dehydrogenase, a cytochrome bc1 complex, an alternative complex III, and cytochrome oxidases of the bd, cbb3, and bo3 types. Oxidation of reduced sulfur compounds probably involved the Sox system, sul- fide-quinone oxidoreductase, adenyl sulfate reductase, and sulfate adenyltransferase. The genes required for autotrophic carbon assimilation via the Calvin cycle were present, while no pathway for nitrogen fixation was revealed. High numbers of RND metal transporters and P type ATPases were probably responsible for resis- tance to heavy metals. The new microorganism was an aerobic chemolithoautotroph of the group of psychrotolerant iron- and sulfur-oxidizing acidophiles of the family Gallionellaceae, which are common in acid mine drainages.

Redox pathways of the mitochondrion.

PubMed

Koehler, Carla M; Beverly, Kristen N; Leverich, Edward P

2006-01-01

The mitochondrion houses a variety of redox pathways, utilized for protection from oxidative damage and assembly of the organelle. The glutathione/glutaredoxin and thioredoxin systems function in the mitochondrial matrix. The intermembrane space is protected from oxidative damage via superoxide dismutase and glutathione. Subunits in the cytochrome bc (1) complex utilize disulfide bonds for enzymatic activity, whereas cytochrome oxidase relies on disulfide linkages for copper acquisition. A redox pathway (Mia40p and Erv1p) mediates the import of intermembrane space proteins such as the small Tim proteins, Cox17p, and Cox19p, which have disulfide bonds. Many of the candidate proteins with disulfide bridges possess a twin CX3C motif or CX9C motif and utilize both metal binding and disulfide linkages for function. It may seem surprising that the intermembrane space has developed redox pathways, considering that the buffered environment should be reducing like the cytosol. However, the prokaryotic origin of the mitochondrion suggests that the intermembrane space may be akin to the oxidative environment of the bacterial periplasm. Although the players forming disulfide bonds are not conserved between mitochondria and prokaryotes, the mitochondrion may have maintained redox chemistry as an assembly mechanism in the intermembrane space for the import of proteins and metals and enzymatic activity.

Theoretical Study of Free Energy in Docking Stability of Azurin(II)-Cytochrome c551(II) Complex System

NASA Astrophysics Data System (ADS)

Yamamoto, Tetsunori; Nishikawa, Keigo; Sugiyama, Ayumu; Purqon, Acep; Mizukami, Taku; Shimahara, Hideto; Nagao, Hidemi; Nishikawa, Kiyoshi

2008-02-01

The docking structure of the Azurin-Cytochrome C551 is presented. We investigate a complex system of Azurin(II)-Cytochrome C551(II) by using molecular dynamics simulation. We estimate some physical properties, such as root-mean-square deviation (RMSD), binding energy between Azurin and Cytochrome C551, distance between Azurin(II) and Cytochrome C551(II) through center of mass and each active site. We also discuss docking stability in relation to the configuration by free energy between Azurin(II)-Cytochrome C551(II) and Azurin(I)-Cytochrome C551(III).

Determining and understanding the control of flux. An illustration in submitochondrial particles of how to validate schemes of metabolic control.

PubMed

Moreno-Sánchez, R; Bravo, C; Westerhoff, H V

1999-09-01

Two complementary methods were used to determine how the rate of respiration and that of ATP hydrolysis were controlled in rat liver submitochondrial particles. In the first, 'direct control analysis' method, respiration was titrated with malonate, antimycin or cyanide at 20, 30 and 37 degrees C, to determine the flux control exerted by succinate dehydrogenase, cytochrome bc1 complex and cytochrome c oxidase, respectively. Together, the three respiratory complexes only controlled the flux by about 50%, leaving the other 50% of flux control to the H+ leak. In the second, 'elasticity based' method, the elasticity coefficients of the respiratory chain or the H+-ATPase and the H+ leak towards the H+ gradient were determined. Then, the flux control coefficients were calculated using the connectivity and summation laws of metabolic control theory. The correspondence between the flux control coefficients determined in the two ways validated the two methods. This allowed us to use the second method to analyse what was the kinetic origin of the observed distribution of control. Control of ATP hydrolysis by the ATPase decreased with increasing ATPase activity; hence, the control exerted by the H+ leak increased with increasing ATPase activity, due to a diminishing elasticity towards the H+ gradient. Reverse electron transport was mainly controlled by the ATPase; the sum of flux control coefficients of succinate dehydrogenase, NADH-CoQ oxidoreductase, and H+-ATPase yielded a value greater than one, indicating that the H+ leak exerted a significant negative control on this pathway.

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

PubMed

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

1981-02-15

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

Therapeutic efficacy of AS2077715 against experimental tinea pedis in guinea pigs in comparison with terbinafine.

PubMed

Ohsumi, Keisuke; Murai, Hidetsugu; Nakamura, Ikko; Watanabe, Masato; Fujie, Akihiko

2014-10-01

AS2077715 is a novel antifungal metabolite produced by the newly isolated fungal strain Capnodium sp. 339855. This compound has potent inhibitory activity against Trichophyton mentagrophytes mitochondrial cytochrome bc1 complex (complex III) and potent fungicidal activity against T. mentagrophytes, as measured in vitro. Here, we compared the effects of AS2077715 and terbinafine in a guinea pig model of tinea pedis. In a treatment regimen started from the day 7 after infection, 10 daily oral doses of 10 and 20 mg kg(-1) AS2077715 and 20 mg kg(-1) of terbinafine significantly decreased fungal colony-forming units (CFUs) in foot pad skin. In a treatment regimen started from the day 11 after infection, 20 mg kg(-1) AS2077715 significantly reduced fungal CFUs in foot pad skin after 7 daily doses in comparison with 20 mg kg(-1) terbinafine-treated guinea pigs. Our findings suggest that in vivo potency and efficacy of AS2077715 are equal to or greater than that of terbinafine, positioning AS2077715 as a good candidate for use in treating trichophytosis.

Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

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

Chen, Qun; Yin, Guotian; Stewart, Sarah

2010-07-09

Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 {sup o}C. Amobarbital (2.5 mM) or azide (5 mM) was used tomore » block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.« less

Anaerobic humus and Fe(III) reduction and electron transport pathway by a novel humus-reducing bacterium, Thauera humireducens SgZ-1.

PubMed

Ma, Chen; Yu, Zhen; Lu, Qin; Zhuang, Li; Zhou, Shun-Gui

2015-04-01

In this study, an anaerobic batch experiment was conducted to investigate the humus- and Fe(III)-reducing ability of a novel humus-reducing bacterium, Thauera humireducens SgZ-1. Inhibition tests were also performed to explore the electron transport pathways with various electron acceptors. The results indicate that in anaerobic conditions, strain SgZ-1 possesses the ability to reduce a humus analog, humic acids, soluble Fe(III), and Fe(III) oxides. Acetate, propionate, lactate, and pyruvate were suitable electron donors for humus and Fe(III) reduction by strain SgZ-1, while fermentable sugars (glucose and sucrose) were not. UV-visible spectra obtained from intact cells of strain SgZ-1 showed absorption peaks at 420, 522, and 553 nm, characteristic of c-type cytochromes (cyt c). Dithionite-reduced cyt c was reoxidized by Fe-EDTA and HFO (hydrous ferric oxide), which suggests that cyt c within intact cells of strain SgZ-1 has the ability to donate electrons to extracellular Fe(III) species. Inhibition tests revealed that dehydrogenases, quinones, and cytochromes b/c (cyt b/c) were involved in reduction of AQS (9, 10-anthraquinone-2-sulfonic acid, humus analog) and oxygen. In contrast, only NADH dehydrogenase was linked to electron transport to HFO, while dehydrogenases and cyt b/c were found to participate in the reduction of Fe-EDTA. Thus, various different electron transport pathways are employed by strain SgZ-1 for different electron acceptors. The results from this study help in understanding the electron transport processes and environmental responses of the genus Thauera.

Role of CYP1A2 polymorphisms in breast cancer risk in women.

PubMed

Ayari, Imene; Fedeli, Ugo; Saguem, Saad; Hidar, Samir; Khlifi, Saida; Pavanello, Sofia

2013-01-01

Cytochrome P4501A2 (CYP1A2) is a key enzyme in the etiology of breast cancer (BC). It is involved in breast carcinogen activation [aromatic (AAs) and heterocyclic amines (HAs), polycyclic aromatic hydrocarbons (PAHs)], in the production of beneficial oestrogen [2-hydroxyestrone (2-OHE1)] and in converting arachidonic acid (AAc) to epoxyeicosatrienoic acids (EETs), which have anti-inflammatory properties. Within a hospital-based case-control study, the effect of functional CYP1A2 variants [-3860G/A (rs2069514), -2467T/delT (rs3569413), -163C/A (rs762551)] and their interactions with environmental factors in BC risk was investigated. The study population included 125 BC cases and 43 non-cancer controls. Genotyping was performed in RT-PCR using Taqman assays. The gene-environment interaction was appraised using a case-only study design. We found that the -3860A variant, independently from environmental factors, as well as by interacting with fried foods (p=0.025) and indoor exposure to pollutants (p=0.050), reduced the risk of BC (p=0.025), whereas its interaction with coffee (p=0.045) increased the BC risk. This is the first study indicating that the -3860A variant, by decreasing CYP1A2 activity, modifies BC risk by interacting with environmental factors, thereby supporting the hypothesis that reduced CYP1A2 activity contributes to BC risk in different ways, for example, it may be protective by reducing the activation of pro-carcinogens such as AAs, HAs and PAHs, but would increase risk by reducing the beneficial formation of 2-OHE1 and EETs.

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

PubMed

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

2018-05-01

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

Relation Between the Molopo Farms and Bushveld Complexes: An Analysis of Pyroxene Exsolution Lamellae

NASA Astrophysics Data System (ADS)

Moore, I.; Feineman, M. D.; Nyblade, A.

2017-12-01

The Molopo Farms Complex (MFC) is a layered igneous intrusion in Botswana, considered to be related to the nearby South African Bushveld Complex (BC) due to their similarities. The BC has been researched in depth for its economic deposits of platinum group elements (PGEs), while the under-researched MFC has no PGEs and is under 200 m of sediment. This study aims to increase knowledge about the MFC regarding the theory that the BC and MFC come from the same parental magma body by showing similar cooling history in the exsolution of pyroxenes. Using optical microscopy and scanning electron microscopy (SEM) paired with an energy-dispersive detector (EDS), thin sections of pyroxenes with exsolution lamellae from both complexes were observed in terms of chemical composition and microtextures. MFC pyroxenes were then compared to literature data of BC pyroxenes. The pyroxenes are closely related, indicating that the MFC and the BC cooled at a similar rate and come from the same parental magma body. Further research can expand on these findings to prove that the MFC and BC are from the same magma.

The cytochrome complex SoxXA of Paracoccus pantotrophus is produced in Escherichia coli and functional in the reconstituted sulfur-oxidizing enzyme system.

PubMed

Rother, Dagmar; Friedrich, Cornelius G

2002-07-29

The heterodimeric c-type cytochrome complex SoxXA of Paracoccus pantotrophus was produced in Escherichia coli. The soxX and soxA genes, separated by two genes in the sox gene cluster of P. pantotrophus, were fused with ribosome binding sites optimal for E. coli and combined to give soxXA in pRD133.27. The cytochrome complex SoxXA was produced in E. coli M15 containing pRD133.27, pREP4 encoding the Lac repressor and plasmid pEC86, carrying essential cytochrome c maturation genes. SoxX and SoxA were formed in a ratio of about 2.5:1. SoxA appeared to be unstable when not complexed with SoxX. The cytochrome complex SoxXA, purified to homogeneity from periplasmic extracts of E. coli M15 (pRD133.27, pREP4, pEC86), exhibited identical biochemical and biophysical properties as compared to SoxXA of P. pantotrophus. Moreover, this cytochrome complex was shown to be equally catalytically active with respect to rates and reactivity with different sulfur substrates in the reconstituted sulfur-oxidizing enzyme system using homogeneous Sox-proteins of P. pantotrophus. Homogeneous SoxX was catalytically inactive.

Co-expression, purification and characterization of the lipase and foldase of Burkholderia contaminans LTEB11.

PubMed

Alnoch, Robson Carlos; Stefanello, Adriano Alves; Paula Martini, Viviane; Richter, Jeferson Luiz; Mateo, Cesar; Souza, Emanuel Maltempi de; Mitchell, David Alexander; Muller-Santos, Marcelo; Krieger, Nadia

2018-05-30

Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase subfamily I.2. The genes lipA and lipB were amplified and cloned into expression vectors pET28a(+) and pT7-7, respectively. His-tagged LipBC and native LifBC were co-expressed in Escherichia coli and purified. LipBC and LifBC have molecular weights of 35.9 kDa and 37 kDa, respectively, and remain complexed after purification. The Lip-LifBC complex was active and stable over a wide range of pH values (6.5-10) and temperatures (25-45 °C), with the highest specific activity (1426 U mg -1 ) being against tributyrin. The Lip-LifBC complex immobilized on Sepabeads was able to catalyze the synthesis of ethyl-oleate in n‑hexane with an activity of 4 U g -1 , maintaining high conversion (>80%) over 5 reaction cycles of 6 h at 45 °C. The results obtained in this work provide a basis for the development of applications of recombinant LipBC in biocatalysis. Copyright © 2018 Elsevier B.V. All rights reserved.

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

PubMed Central

Braun, H P; Emmermann, M; Kruft, V; Schmitz, U K

1992-01-01

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase. Images PMID:1324169

Overall energy conversion efficiency of a photosynthetic vesicle

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

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in amore » quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.« less

Overall energy conversion efficiency of a photosynthetic vesicle

PubMed Central

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek; Hunter, C Neil; Schulten, Klaus

2016-01-01

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytb⁢c1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12–0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination. DOI: http://dx.doi.org/10.7554/eLife.09541.001 PMID:27564854

Overall energy conversion efficiency of a photosynthetic vesicle

DOE PAGES

Sener, Melih; Strumpfer, Johan; Singharoy, Abhishek; ...

2016-08-26

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in amore » quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.« less

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

PubMed

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

2002-04-01

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

Evidence for a ternary complex formed between flavodoxin and cytochrome c3: 1H-NMR and molecular modeling studies.

PubMed

Palma, P N; Moura, I; LeGall, J; Van Beeumen, J; Wampler, J E; Moura, J J

1994-05-31

Small electron-transfer proteins such as flavodoxin (16 kDa) and the tetraheme cytochrome c3 (13 kDa) have been used to mimic, in vitro, part of the complex electron-transfer chain operating between substrate electron donors and respiratory electron acceptors, in sulfate-reducing bacteria (Desulfovibrio species). The nature and properties of the complex formed between these proteins are revealed by 1H-NMR and molecular modeling approaches. Our previous study with the Desulfovibrio vulgaris proteins [Moura, I., Moura, J.J. G., Santos, M.H., & Xavier, A. V. (1980) Cienc. Biol. (Portugal) 5, 195-197; Stewart, D.E. LeGall, J., Moura, I., Moura, J. J. G., Peck, H.D. Jr., Xavier, A. V., Weiner, P. K., & Wampler, J.E. (1988) Biochemistry 27, 2444-2450] indicated that the complex between cytochrome c3 and flavodoxin could be monitored by changes in the NMR signals of the heme methyl groups of the cytochrome and that the electrostatic surface charge (Coulomb's law) on the two proteins favored interaction between one unique heme of the cytochrome with flavodoxin. If the interaction is indeed driven by the electrostatic complementarity between the acidic flavodoxin and a unique positive region of the cytochrome c3, other homologous proteins from these two families of proteins might be expected to interact similarly. In this study, three homologous Desulfovibrio cytochromes c3 were used, which show a remarkable variation in their individual isoelectric points (ranging from 5.5 to 9.5). On the basis of data obtained from protein-protein titrations followed at specific proton NMR signals (i.e., heme methyl resonances), a binding model for this complex has been developed with evaluation of stoichiometry and binding constants. This binding model involves one site on the cytochromes c3 and two sites on the flavodoxin, with formation of a ternary complex at saturation. In order to understand the potential chemical form of the binding model, a structural model for the hypothetical ternary complex, formed between one molecule of Desulfovibrio salexigens flavodoxin and two molecules of cytochrome c3, is proposed. These molecular models of the complexes were constructed on the basis of complementarity of Coulombic electrostatic surface potentials, using the available X-ray structures of the isolated proteins and, when required, model structures (D. salexigens flavodoxin and Desulfovibrio desulfuricans ATCC 27774 cytochrome c3) predicted by homology modeling.

Binding of the respiratory chain inhibitor ametoctradin to the mitochondrial bc1 complex.

PubMed

Fehr, Marcus; Wolf, Antje; Stammler, Gerd

2016-03-01

Ametoctradin is an agricultural fungicide that inhibits the mitochondrial bc1 complex of oomycetes. The bc1 complex has two quinone binding sites that can be addressed by inhibitors. Depending on their binding sites and binding modes, the inhibitors show different degrees of cross-resistance that need to be considered when designing spray programmes for agricultural fungicides. The binding site of ametoctradin was unknown. Cross-resistance analyses, the reduction of isolated Pythium sp. bc1 complex in the presence of different inhibitors and molecular modelling studies were used to analyse the binding site and binding mode of ametoctradin. All three approaches provide data supporting the argument that ametoctradin binds to the Pythium bc1 complex similarly to stigmatellin. The binding mode of ametoctradin differs from other agricultural fungicides such as cyazofamid and the strobilurins. This explains the lack of cross-resistance with strobilurins and related inhibitors, where resistance is mainly caused by G143A amino acid exchange. Accordingly, mixtures or alternating applications of these fungicides and ametoctradin can help to minimise the risk of the emergence of new resistant isolates. © 2015 Society of Chemical Industry.

H2BC: a new technique for NMR analysis of complex carbohydrates.

PubMed

Petersen, Bent O; Vinogradov, Evguenii; Kay, William; Würtz, Peter; Nyberg, Nils T; Duus, Jens Ø; Sørensen, Ole W

2006-03-20

It is demonstrated that the H2BC NMR pulse sequence (J. Am. Chem. Soc.2005, 127, 6154, Magn. Reson. Chem.2005, 43, 971-974) offers unambiguous assignments and significant simplification of NMR spectra of large and complex carbohydrates compared to other techniques for the establishment of correlations over more than one bond. H2BC almost exclusively correlates protons and proton-bearing carbon spins separated by two covalent bonds and is independent of occasionally vanishing (2)J(CH) coupling constants, which alleviates the problem of missing two-bond correlations in HMBC spectra. H2BC also solves the problem of distinguishing two- and three-bond correlations in HSQC-TOCSY or HMBC. It is a further asset of H2BC that the experiment is significantly shorter than HMBC and HSQC-TOCSY, and hence less sensitive to transverse relaxation. The H2BC experiment is demonstrated on an approximately 30-residue oligosaccharide from Francisella victoria.

Molecular Dynamics Analysis Reveals Structural Insights into Mechanism of Nicotine N-Demethylation Catalyzed by Tobacco Cytochrome P450 Mono-Oxygenase

PubMed Central

Wang, Shan; Yang, Shuo; An, Baiyi; Wang, Shichen; Yin, Yuejia; Lu, Yang; Xu, Ying; Hao, Dongyun

2011-01-01

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys–trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase. PMID:21858078

A Novel Role for Cytochrome c: Efficient Catalysis of S-Nitrosothiol Formation

PubMed Central

Basu, Swati; Keszler, Agnes; Azarova, Natalia A.; Nwanze, Nneka; Perlegas, Andreas; Shiva, Sruti; Broniowska, Katarzyna A.; Hogg, Neil; Kim-Shapiro, Daniel B.

2009-01-01

While S-nitrosothiols are regarded as important elements of many NO-dependent signal transduction pathways, the physiological mechanism of their formation remains elusive. Here, we demonstrate a novel mechanism by which cytochrome c may represent an efficient catalyst of S-nitrosation in vivo. In this mechanism, initial binding of GSH to ferric cytochrome c is followed by reaction of NO with this complex, yielding ferrous cytochrome c and GSNO. We show that when submitochondrial particles or cell lysates are exposed to NO in the presence of cytochrome c, there is a robust formation of protein S-nitrosothiols. In the case of submitochondrial particles protein S-nitrosation is paralleled with an inhibition of mitochondrial complex I. These observations raise the possibility that cytochrome c is a mediator of S-nitrosation in biological systems, particularly during hypoxia, and that release of cytochrome c in to the cytosol during apoptosis potentially releases a GSNO synthase activity which could modulate apoptotic signaling. PMID:19879353

Mapping of Redox State of Mitochondrial Cytochromes in Live Cardiomyocytes Using Raman Microspectroscopy

PubMed Central

Brazhe, Nadezda A.; Treiman, Marek; Brazhe, Alexey R.; Find, Ninett L.; Maksimov, Georgy V.; Sosnovtseva, Olga V.

2012-01-01

This paper presents a nonivasive approach to study redox state of reduced cytochromes , and of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach we 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 , and . The rod-shaped cardiomyocytes possess uneven distribution of reduced cytochromes , and in cell center and periphery. Moreover, by means of Raman spectroscopy we demonstrated the decrease in the relative amounts of reduced cytochromes , and in the rod-shaped cardiomyocytes caused by H2O2-induced oxidative stress before any visible changes. Results of Raman mapping and time-dependent study of reduced cytochromes of complexes II and III and cytochrome in cardiomyocytes are in a good agreement with our fluorescence indicator studies and other published data. PMID:22957018

The photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum.

PubMed

Bernal-Bayard, Pilar; Puerto-Galán, Leonor; Yruela, Inmaculada; García-Rubio, Inés; Castell, Carmen; Ortega, José M; Alonso, Pablo J; Roncel, Mercedes; Martínez, Jesús I; Hervás, Manuel; Navarro, José A

2017-09-01

The photosynthetic cytochrome c 550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c 550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c 550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c 550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c 550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c 550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c 550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c 550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre.

Soluble Variants of Rhodobacter capsulatus Membrane-anchored Cytochrome cy Are Efficient Photosynthetic Electron Carriers*

PubMed Central

Öztürk, Yavuz; Lee, Dong-Woo; Mandaci, Sevnur; Osyczka, Artur; Prince, Roger C.; Daldal, Fevzi

2008-01-01

Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt cy. In species like R. capsulatus, cyt cy functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt cy (S-cy), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt cy. This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-cy regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-cy variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-cy was about 340 mV, similar to that of cyts c2 or cy. Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-cy was distinct from those seen with the cyt c2 or cyt cy. The kinetics exhibited a pronounced slow phase, suggesting that cyt S-cy interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-cy revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt cy, whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers. PMID:18343817

Identification of protein W, the elusive sixth subunit of the Rhodopseudomonas palustris reaction center-light harvesting 1 core complex.

PubMed

Jackson, Philip J; Hitchcock, Andrew; Swainsbury, David J K; Qian, Pu; Martin, Elizabeth C; Farmer, David A; Dickman, Mark J; Canniffe, Daniel P; Hunter, C Neil

2018-02-01

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc 1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Characterisation of Black Carbon (BC) mixing state and flux in Beijing using single particle measurements.

NASA Astrophysics Data System (ADS)

Joshi, Rutambhara; Liu, Dantong; Allan, James; Coe, Hugh; Flynn, Michael; Broda, Kurtis; Olfert, Jason; Irwin, Martin; Sun, Yele; Fu, Pingqing; Wang, Junfeng; Ge, Xinlei; Langford, Ben; Nemitz, Eiko; Mullinger, Neil

2017-04-01

BC is generated by the incomplete combustion of carbonaceous fuels and it is an important component of fine PM2.5. In the atmosphere BC particles have a complex structure and its mixing state has crucial impact on optical properties. Quantifying the sources and emissions of black carbon in urban environments is important and presently uncertain, particularly in megacities undergoing rapid growth and change in emissions. During the winter of 2016 (10th Nov-10th Dec) the BC was characterised as part of a large joint UK-China field experiment in Beijing. This paper focuses on understanding the mixing state of BC as well as identification and quantification of BC sources. We used a combination of a Centrifugal Particle Mass Analyser (CPMA) and a Single Particle Soot Photometer (SP2) to uniquely quantify the morphology independent mass of single refractory BC particles and their coating content. The CPMA allows us to select pre-charged aerosol particles according to their mass to charge ratio and the SP2 provides information on the mass of refractory BC through a laser-induced incandescence method. Furthermore, another SP2 was used to measure the BC flux at 100m height using the Eddy Covariance method. We have successfully gathered 4 weeks of continuous measurements which include several severe pollution events in Beijing. Here we present preliminary results, characterising the distribution of coating mass on BC particles in Beijing and linking this to the main sources of BC in the city. We will provide initial estimates of the BC flux over a several kilometre footprint. Such analysis will provide important information for the further investigation of source distribution, emission, lifetime and optical properties of BC under complex environments in Beijing.

T-matrix modeling of linear depolarization by morphologically complex soot and soot-containing aerosols

NASA Astrophysics Data System (ADS)

Mishchenko, Michael I.; Liu, Li; Mackowski, Daniel W.

2013-07-01

We use state-of-the-art public-domain Fortran codes based on the T-matrix method to calculate orientation and ensemble averaged scattering matrix elements for a variety of morphologically complex black carbon (BC) and BC-containing aerosol particles, with a special emphasis on the linear depolarization ratio (LDR). We explain theoretically the quasi-Rayleigh LDR peak at side-scattering angles typical of low-density soot fractals and conclude that the measurement of this feature enables one to evaluate the compactness state of BC clusters and trace the evolution of low-density fluffy fractals into densely packed aggregates. We show that small backscattering LDRs measured with ground-based, airborne, and spaceborne lidars for fresh smoke generally agree with the values predicted theoretically for fluffy BC fractals and densely packed near-spheroidal BC aggregates. To reproduce higher lidar LDRs observed for aged smoke, one needs alternative particle models such as shape mixtures of BC spheroids or cylinders.

T-Matrix Modeling of Linear Depolarization by Morphologically Complex Soot and Soot-Containing Aerosols

NASA Technical Reports Server (NTRS)

Mishchenko, Michael I.; Liu, Li; Mackowski, Daniel W.

2013-01-01

We use state-of-the-art public-domain Fortran codes based on the T-matrix method to calculate orientation and ensemble averaged scattering matrix elements for a variety of morphologically complex black carbon (BC) and BC-containing aerosol particles, with a special emphasis on the linear depolarization ratio (LDR). We explain theoretically the quasi-Rayleigh LDR peak at side-scattering angles typical of low-density soot fractals and conclude that the measurement of this feature enables one to evaluate the compactness state of BC clusters and trace the evolution of low-density fluffy fractals into densely packed aggregates. We show that small backscattering LDRs measured with groundbased, airborne, and spaceborne lidars for fresh smoke generally agree with the values predicted theoretically for fluffy BC fractals and densely packed near-spheroidal BC aggregates. To reproduce higher lidar LDRs observed for aged smoke, one needs alternative particle models such as shape mixtures of BC spheroids or cylinders.

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

PubMed

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

2003-05-01

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

A Critical Role for the cccA Gene Product, Cytochrome c2, in Diverting Electrons from Aerobic Respiration to Denitrification in Neisseria gonorrhoeae

PubMed Central

Hopper, Amanda C.; Li, Ying

2013-01-01

Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c2, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c4 and c5 transfer electrons to cytochrome oxidase cbb3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c4 or c5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc2), cycA (Δc4), cycB (Δc5), and ccoP (ccoP-C368A), we demonstrate that cytochrome c2 is required for electron transfer from cytochrome c4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae. PMID:23543713

Structural insights into electron transfer in caa3-type cytochrome oxidase

PubMed Central

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

2012-01-01

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

Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

PubMed Central

Glerum, M; Robinson, B H; Spratt, C; Wilson, J; Patrick, D

1987-01-01

Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient. PMID:2821802

The Stromal Contribution to the Development of Resistance to New-Generation Drugs by Castration-Resistant Prostate Cancer

DTIC Science & Technology

2015-10-01

against PCa, such as Enzalutamide or Abiraterone (abi), which target the AR or cytochrome p450 (CYP17)A1, respectively (4). By the way of the “seed and...Castration-Resistant Prostate Cancer PRINCIPAL INVESTIGATOR: Amy Lubik RECIPIENT: University of British Columbia Vancouver, BC Canada V6H 3Z6 REPORT...Generation Drugs by Castration-Resistant Prostate Cancer 5b. GRANT NUMBER W81XWH-13-1-0379 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Lubik, Amy Anne, PhD 5d

Arachidonic Acid Metabolite as a Novel Therapeutic Target in Breast Cancer Metastasis

PubMed Central

Borin, Thaiz F.; Angara, Kartik; Rashid, Mohammad H.; Achyut, Bhagelu R.; Arbab, Ali S.

2017-01-01

Metastatic breast cancer (BC) (also referred to as stage IV) spreads beyond the breast to the bones, lungs, liver, or brain and is a major contributor to the deaths of cancer patients. Interestingly, metastasis is a result of stroma-coordinated hallmarks such as invasion and migration of the tumor cells from the primary niche, regrowth of the invading tumor cells in the distant organs, proliferation, vascularization, and immune suppression. Targeted therapies, when used as monotherapies or combination therapies, have shown limited success in decreasing the established metastatic growth and improving survival. Thus, novel therapeutic targets are warranted to improve the metastasis outcomes. We have been actively investigating the cytochrome P450 4 (CYP4) family of enzymes that can biosynthesize 20-hydroxyeicosatetraenoic acid (20-HETE), an important signaling eicosanoid involved in the regulation of vascular tone and angiogenesis. We have shown that 20-HETE can activate several intracellular protein kinases, pro-inflammatory mediators, and chemokines in cancer. This review article is focused on understanding the role of the arachidonic acid metabolic pathway in BC metastasis with an emphasis on 20-HETE as a novel therapeutic target to decrease BC metastasis. We have discussed all the significant investigational mechanisms and put forward studies showing how 20-HETE can promote angiogenesis and metastasis, and how its inhibition could affect the metastatic niches. Potential adjuvant therapies targeting the tumor microenvironment showing anti-tumor properties against BC and its lung metastasis are discussed at the end. This review will highlight the importance of exploring tumor-inherent and stromal-inherent metabolic pathways in the development of novel therapeutics for treating BC metastasis. PMID:29292756

Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

PubMed Central

Liko, Idlir; Degiacomi, Matteo T.; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V.

2016-01-01

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. PMID:27364008

Dimerization of the Bacterial Biotin Carboxylase Subunit Is Required for Acetyl Coenzyme A Carboxylase Activity In Vivo

PubMed Central

Smith, Alexander C.

2012-01-01

Acetyl coenzyme A (acteyl-CoA) carboxylase (ACC) is the first committed enzyme of the fatty acid synthesis pathway. Escherichia coli ACC is composed of four different proteins. The first enzymatic activity of the ACC complex, biotin carboxylase (BC), catalyzes the carboxylation of the protein-bound biotin moiety of another subunit with bicarbonate in an ATP-dependent reaction. Although BC is found as a dimer in cell extracts and the carboxylase activities of the two subunits of the dimer are interdependent, mutant BC proteins deficient in dimerization are reported to retain appreciable activity in vitro (Y. Shen, C. Y. Chou, G. G. Chang, and L. Tong, Mol. Cell 22:807–818, 2006). However, in vivo BC must interact with the other proteins of the complex, and thus studies of the isolated BC may not reflect the intracellular function of the enzyme. We have tested the abilities of three BC mutant proteins deficient in dimerization to support growth and report that the two BC proteins most deficient in dimerization fail to support growth unless expressed at high levels. In contrast, the wild-type protein supports growth at low expression levels. We conclude that BC must be dimeric to fulfill its physiological function. PMID:22037404

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

Treesearch

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

1999-01-01

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

Modeling of interaction between cytochrome c and the WD domains of Apaf-1: bifurcated salt bridges underlying apoptosome assembly.

PubMed

Shalaeva, Daria N; Dibrova, Daria V; Galperin, Michael Y; Mulkidjanian, Armen Y

2015-05-27

Binding of cytochrome c, released from the damaged mitochondria, to the apoptotic protease activating factor 1 (Apaf-1) is a key event in the apoptotic signaling cascade. The binding triggers a major domain rearrangement in Apaf-1, which leads to oligomerization of Apaf-1/cytochrome c complexes into an apoptosome. Despite the availability of crystal structures of cytochrome c and Apaf-1 and cryo-electron microscopy models of the entire apoptosome, the binding mode of cytochrome c to Apaf-1, as well as the nature of the amino acid residues of Apaf-1 involved remain obscure. We investigated the interaction between cytochrome c and Apaf-1 by combining several modeling approaches. We have applied protein-protein docking and energy minimization, evaluated the resulting models of the Apaf-1/cytochrome c complex, and carried out a further analysis by means of molecular dynamics simulations. We ended up with a single model structure where all the lysine residues of cytochrome c that are known as functionally-relevant were involved in forming salt bridges with acidic residues of Apaf-1. This model has revealed three distinctive bifurcated salt bridges, each involving a single lysine residue of cytochrome c and two neighboring acidic resides of Apaf-1. Salt bridge-forming amino acids of Apaf-1 showed a clear evolutionary pattern within Metazoa, with pairs of acidic residues of Apaf-1, involved in bifurcated salt bridges, reaching their highest numbers in the sequences of vertebrates, in which the cytochrome c-mediated mechanism of apoptosome formation seems to be typical. The reported model of an Apaf-1/cytochrome c complex provides insights in the nature of protein-protein interactions which are hard to observe in crystallographic or electron microscopy studies. Bifurcated salt bridges can be expected to be stronger than simple salt bridges, and their formation might promote the conformational change of Apaf-1, leading to the formation of an apoptosome. Combination of structural and sequence analyses provides hints on the evolution of the cytochrome c-mediated apoptosis.

Studies of molecular species of the human androgen receptor (AR): comparison of the physicochemical properties of the (/sup 3/H)methyltrienolone-AR complex formed in cytosol to the complex produced in intact genital skin fibroblasts

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

Keenan, B.S.; Greger, N.C.; Hedge, A.M.

1986-07-01

Two forms of the human genital skin fibroblast (GSF) androgen receptor (AR) complexed with (/sup 3/H)17 alpha-methyltrienolone were compared: 1) the intact complex formed in cytosol at 4 C (broken cell or B/C complex); and 2) the complex formed in the whole cell at 37 C (W/C complex). The intact form of the B/C complex was distinguished from partly degraded forms by the gel filtration profile in 0.5 M KCl. The W/C complex was considered to represent the transformed state of the receptor. The W/C complex had a smaller molecular radius than the B/C complex by gel filtration (Kav =more » 0.26-0.28 vs. 0.11-0.18). By low salt density gradient centrifugation, the B/C complex sedimented at 8.8S and the W/C complex at 6.6S. However, in 0.5 M KCl, each sedimented at 5.1S, and they were homogeneous, indicating that the monomeric forms differed markedly in molecular radius, but by only about 20,000 daltons in calculated mol wt (134,500 vs. 114,300 daltons). The complexes were separated from DNA, desalted, and compared by chromatography on DEAE-Sephacel and hydroxylapatite (HAP). The B/C complex bound readily to both column matrices and eluted from each as a sharp homogeneous peak: from DEAE at 172-190 mM KCl and from HAP at 123 mM phosphate. The W/C complex, however, was heterogeneous. One component did not bind to DEAE, and one eluted at 22-40 mM KCl. The W/C complex eluted from HAP as a peak at 42 mM, with a shoulder at 102 mM phosphate. Thus, transformation of the human genital skin fibroblast androgen receptor involves a major decrease in molecular radius and loss of negative charge with a possible loss of a 20,000-dalton macromolecular component.« less

Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure–Activity Relationship and Target Identification Studies

PubMed Central

2017-01-01

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell–wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure–activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties. PMID:29148755

Mode of action of the qcr9 and cat3 mutations in restoring the ability of Saccharomyces cerevisiae tps1 mutants to grow on glucose.

PubMed

Blázquez, M A; Gancedo, C

1995-12-20

Mutations in the TPS1 gene, which encodes trehalose-6-P synthase, cause a glucose-negative phenotype in Saccharomyces cerevisiae. Antimycin A or disruption of the QCR9 gene, which encodes one subunit of the cytochrome bc1 complex, restore the ability to grow in glucose-containing media. Under these conditions the cell excreted a large amount of glycerol, corresponding to about 20% of the glucose taken up. Suppression appears to be achieved by diversion of accumulated glycolytic intermediates to the production of glycerol, thereby providing NAD+ and phosphate for the glyceraldehyde-3-P dehydrogenase reaction. Analysis of the mutation sci1-1, which also suppresses the glucose-negative phenotype of tps1 mutants, showed that glucose transport was decreased in sci1-1 mutants. The gene SCI1 was cloned and its nucleotide sequence revealed it to be identical to CAT3/SNF4. The suppression mediated by sci1-1 is attributable to a decrease in glycolytic flux.

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

PubMed Central

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

2014-01-01

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

Identifying involvement of Lys251/Asp252 pair in electron transfer and associated proton transfer at the quinone reduction site of Rhodobacter capsulatus cytochrome bc1.

PubMed

Kuleta, Patryk; Sarewicz, Marcin; Postila, Pekka; Róg, Tomasz; Osyczka, Artur

2016-10-01

Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc1, one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH2) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Qi site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Qi-site mediated oxidation of heme bH, reverse reduction of heme bH by quinol and heme bH/Qi semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH2 triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Two hydrophobic subunits are essential for the heme b ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli.

PubMed

Nakamura, K; Yamaki, M; Sarada, M; Nakayama, S; Vibat, C R; Gennis, R B; Nakayashiki, T; Inokuchi, H; Kojima, S; Kita, K

1996-01-05

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b556. An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b556 in its CO reactivity and red shift of the alpha absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinate-ubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquitnone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.

Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4BC74A in live cells using Förster resonance energy transfer and fluorescence recovery after photobleaching

NASA Astrophysics Data System (ADS)

Kraft, Lewis J.; Kenworthy, Anne K.

2012-01-01

The protein microtubule-associated protein 1, light chain 3 (LC3) functions in autophagosome formation and plays a central role in the autophagy pathway. Previously, we found LC3 diffuses more slowly in cells than is expected for a freely diffusing monomer, suggesting it may constitutively associate with a macromolecular complex containing other protein components of the pathway. In the current study, we used Förster resonance energy transfer (FRET) microscopy and fluorescence recovery after photobleaching (FRAP) to investigate the interactions of LC3 with Atg4BC74A, a catalytically inactive mutant of the cysteine protease involved in lipidation and de-lipidation of LC3, as a model system to probe protein complex formation in the autophagy pathway. We show Atg4BC74A is in FRET proximity with LC3 in both the cytoplasm and nucleus of living cells, consistent with previous biochemical evidence that suggests these proteins directly interact. In addition, overexpressed Atg4BC74A diffuses significantly more slowly than predicted based on its molecular weight, and its translational diffusion coefficient is significantly slowed upon coexpression with LC3 to match that of LC3 itself. Taken together, these results suggest Atg4BC74A and LC3 are contained within the same multiprotein complex and that this complex exists in both the cytoplasm and nucleoplasm of living cells.

Interaction of photosystem I from Phaeodactylum tricornutum with plastocyanins as compared with its native cytochrome c6: Reunion with a lost donor.

PubMed

Bernal-Bayard, Pilar; Pallara, Chiara; Carmen Castell, M; Molina-Heredia, Fernando P; Fernández-Recio, Juan; Hervás, Manuel; Navarro, José A

2015-12-01

In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself. Copyright © 2015 Elsevier B.V. All rights reserved.

Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

PubMed

Thomas, Jemima C; Matak-Vinkovic, Dijana; Van Molle, Inge; Ciulli, Alessio

2013-08-06

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Membrane cytochromes of Escherichia coli chl mutants.

PubMed Central

Hackett, N R; Bragg, P D

1983-01-01

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

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the Cu(A) center of cytochrome c oxidase.

PubMed

Hill, Bruce C; Andrews, Diann

2012-06-01

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the Cu(A) center contained within subunit II of the oxidase complex. The Cu(A) center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a(3) and Cu(B). Cu(A) consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of Cu(A) present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in Cu(A) assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-Cu(A) site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes. Copyright © 2011 Elsevier B.V. All rights reserved.

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

DOE PAGES

Cartron, Michaël L.; Olsen, John D.; Sener, Melih; ...

2014-02-13

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH 2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc 1 (cytbc 1) complexes that oxidise QH 2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc 1 complexes with gold nanobeads, each attached by a Histidine 10 (His 10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesiclesmore » showed that the majority of the cytbc 1 complexes occur as dimers in the membrane. The cytbc 1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc 1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc 1-RC-LH1- PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc 1, ATP synthase, cytaa 3 and cytcbb 3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc 1 dimers and 2 ATP synthases. In conclusion, simulation of the interconnected energy, electron and proton transfer processes showed a halfmaximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.« less

Activation of EVI1 transcription by the LEF1/β-catenin complex with p53-alteration in myeloid blast crisis of chronic myeloid leukemia

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

Manachai, Nawin; Saito, Yusuke; Nakahata, Shingo

The presence of a BCR-ABL1 fusion gene is necessary for the pathogenesis of chronic myeloid leukemia (CML) through t(9;22)(q34;q11) translocation. Imatinib, an ABL tyrosine kinase inhibitor, is dramatically effective in CML patients; however, 30% of CML patients will need further treatment due to progression of CML to blast crisis (BC). Aberrant high expression of ecotropic viral integration site 1 (EVI1) is frequently observed in CML during myeloid-BC as a potent driver with a CML stem cell signature; however, the precise molecular mechanism of EVI1 transcriptional regulation during CML progression is poorly defined. Here, we demonstrate the transcriptional activity of EVI1more » is dependent on activation of lymphoid enhancer-binding factor 1 (LEF1)/β-catenin complex by BCR-ABL with loss of p53 function during CML-BC. The activation of β-catenin is partly dependent on BCR-ABL expression through enhanced GSK3β phosphorylation, and EVI1 expression is directly enhanced by the LEF1/β-catenin complex bound to the EVI1 promoter region. Moreover, the loss of p53 expression is inversely correlated with high expression of EVI1 in CML leukemia cells with an aggressive phase of CML, and a portion of the activation mechanism of EVI1 expression is dependent on β-catenin activation through GSK3β phosphorylation by loss of p53. Therefore, we found that the EVI1 activation in CML-BC is dependent on LEF1/β-catenin activation by BCR-ABL expression with loss of p53 function, representing a novel selective therapeutic approach targeting myeloid blast crisis progression. - Highlights: • Transcriptional regulation of EVI1 in CML-BC is proposed. • EVI1 transcription is directly regulated by LEF1/β-catenin complex in CML-BC. • Loss of p53 function as a key regulator for β-catenin-EVI1 in CML myeloid-BC. • The LEF1/β-catenin binding site on the EVI1 promoter is a new target for CML-BC.« less

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

PubMed

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

2016-04-01

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

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

PubMed Central

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

1990-01-01

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

Chemiosmotic Energy Conservation in Dinoroseobacter shibae: Proton Translocation Driven by Aerobic Respiration, Denitrification, and Photosynthetic Light Reaction.

PubMed

Kirchhoff, Christian; Ebert, Matthias; Jahn, Dieter; Cypionka, Heribert

2018-01-01

Dinoroseobacter shibae is an aerobic anoxygenic phototroph and able to utilize light energy to support its aerobic energy metabolism. Since the cells can also grow anaerobically with nitrate and nitrite as terminal electron acceptor, we were interested in how the cells profit from photosynthesis during denitrification and what the steps of chemiosmotic energy conservation are. Therefore, we conducted proton translocation experiments and compared O 2 - , NO 3 - , and NO 2 - respiration during different light regimes and in the dark. We used wild type cells and transposon mutants with knocked-out nitrate- and nitrite- reductase genes ( napA and nirS ), as well as a mutant ( ppsR ) impaired in bacteriochlorophyll a synthesis. Light had a positive impact on proton translocation, independent of the type of terminal electron acceptor present. In the absence of an electron acceptor, however, light did not stimulate proton translocation. The light-driven add-on to proton translocation was about 1.4 H + /e - for O 2 respiration and about 1.1 H + /e - for NO 3 - and NO 2 - . We could see that the chemiosmotic energy conservation during aerobic respiration involved proton translocation, mediated by the NADH dehydrogenase, the cytochrome bc 1 complex, and the cytochrome c oxidase. During denitrification the last proton translocation step of the electron transport was missing, resulting in a lower H + /e - ratio during anoxia. Furthermore, we studied the type of light-harvesting and found that the cells were able to channel light from the green-blue spectrum most efficiently, while red light has only minor impact. This fits well with the depth profiles for D. shibae abundance in the ocean and the penetration depth of light with different wavelengths into the water column.

Recent progress in heteronuclear long-range NMR of complex carbohydrates: 3D H2BC and clean HMBC.

PubMed

Meier, Sebastian; Petersen, Bent O; Duus, Jens Ø; Sørensen, Ole W

2009-11-02

The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of (13)C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the (1)H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution.

Ecosystem features determine seagrass community response to sea otter foraging

USGS Publications Warehouse

Hessing-Lewis, Margot; Rechsteiner, Erin U.; Hughes, Brent B.; Tinker, M. Tim; Monteith, Zachary L.; Olson, Angeleen M.; Henderson, Matthew Morgan; Watson, Jane C.

2017-01-01

Comparing sea otter recovery in California (CA) and British Columbia (BC) reveals key ecosystem properties that shape top-down effects in seagrass communities. We review potential ecosystem drivers of sea otter foraging in CA and BC seagrass beds, including the role of coastline complexity and environmental stress on sea otter effects. In BC, we find greater species richness across seagrass trophic assemblages. Furthermore, Cancer spp. crabs, an important link in the seagrass trophic cascade observed in CA, are less common. Additionally, the more recent reintroduction of sea otters, more complex coastline, and reduced environmental stress in BC seagrass habitats supports the hypotheses that sea otter foraging pressure is currently reduced there. In order to manage the ecosystem features that lead to regional differences in top predator effects in seagrass communities, we review our findings, their spatial and temporal constraints, and present a social-ecological framework for future research.

Superhard BC(3) in cubic diamond structure.

PubMed

Zhang, Miao; Liu, Hanyu; Li, Quan; Gao, Bo; Wang, Yanchao; Li, Hongdong; Chen, Changfeng; Ma, Yanming

2015-01-09

We solve the crystal structure of recently synthesized cubic BC(3) using an unbiased swarm structure search, which identifies a highly symmetric BC(3) phase in the cubic diamond structure (d-BC(3)) that contains a distinct B-B bonding network along the body diagonals of a large 64-atom unit cell. Simulated x-ray diffraction and Raman peaks of d-BC(3) are in excellent agreement with experimental data. Calculated stress-strain relations of d-BC(3) demonstrate its intrinsic superhard nature and reveal intriguing sequential bond-breaking modes that produce superior ductility and extended elasticity, which are unique among superhard solids. The present results establish the first boron carbide in the cubic diamond structure with remarkable properties, and these new findings also provide insights for exploring other covalent solids with complex bonding configurations.

CYP1A2 polymorphisms, occupational and environmental exposures and risk of bladder cancer.

PubMed

Pavanello, Sofia; Mastrangelo, Giuseppe; Placidi, Donatella; Campagna, Marcello; Pulliero, Alessandra; Carta, Angela; Arici, Cecilia; Porru, Stefano

2010-07-01

Cytochrome P4501A2 (CYP1A2) is a key enzyme for activation of bladder carcinogens. Polymorphisms in the 5'-noncoding promoter region of CYP1A2 gene [mainly -2467T/delT(rs35694136) and -163C/A(rs762551)], are crucial in modifying CYP1A2 activity in smokers. Within the framework of a hospital-based case/control study, we investigated the relationship between CYP1A2 polymorphisms, occupational/environmental exposures and bladder cancer (BC) risk. The study population included 185 BC cases and 180 non-cancer controls, all Caucasian males. Data were collected on lifetime smoking, coffee drinking, dietary habits and lifetime occupation, with particular reference to exposure to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs). A case-only design was applied to study the interaction between CYP1A2 -2467T/delT (or -163C/A) and occupational and environmental factors. Multiple logistic regression showed a significantly increased risk among heavy smokers (> or =50 packyears; OR 5.6, 95% CI: 2.5-12.5) and heavy coffee drinkers (>5 cups/day; OR 3.1, 95% CI: 1.2-7.9). Exposure to AAs showed a significant trend of BC risk with increasing cumulative exposure (CE) (P = 0.04), with heavy smoking as possible confounder. A decreased risk was noted for large leaf vegetable consumption, with significant trend from <1/month to >3 times/week (P = 0.008). The case-only analysis showed an interaction between -2467T/delT and tobacco smoking >25 packyears (P = 0.04); no interaction was detected between such polymorphisms and coffee consumption, dietary habits and occupational exposure to AAs. No effects were shown with -163C/A genotype as well as no overall effect of CYP1A2 by itself on BC risk. This is the first study suggesting that CYP1A2 -2467T/delT modifies the effect of cigarette smoking on BC risk.

Reconstructing the Qo Site of Plasmodium falciparum bc 1 Complex in the Yeast Enzyme

PubMed Central

Vallières, Cindy; Fisher, Nicholas; Meunier, Brigitte

2013-01-01

The bc 1 complex of the mitochondrial respiratory chain is essential for Plasmodium falciparum proliferation, the causative agent of human malaria. Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Qo pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Qo sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Qo mutants. We measured the bc 1 complex sensitivity to atovaquone, azoxystrobin, a Qo site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc 1 complex.The data obtained highlighted variations in the Qo site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Qo mutants could be useful and easy-to-use tools for the study of that class of antimalarials. PMID:23951230

Arsenic removal by perilla leaf biochar in aqueous solutions and groundwater: An integrated spectroscopic and microscopic examination.

PubMed

Niazi, Nabeel Khan; Bibi, Irshad; Shahid, Muhammad; Ok, Yong Sik; Burton, Edward D; Wang, Hailong; Shaheen, Sabry M; Rinklebe, Jörg; Lüttge, Andreas

2018-01-01

In this study, we examined the removal of arsenite (As(III)) and arsenate (As(V)) by perilla leaf-derived biochars produced at 300 and 700 °C (referred as BC300 and BC700) in aqueous environments. Results revealed that the Langmuir isotherm model provided the best fit for As(III) and As(V) sorption, with the sorption affinity following the order: BC700-As(III) > BC700-As(V) > BC300-As(III) > BC300-As(V) (Q L  = 3.85-11.01 mg g -1 ). In general, As removal decreased (76-60%) with increasing pH from 7 to 10 except for the BC700-As(III) system, where notably higher As removal (88-90%) occurred at pH from 7 to 9. Surface functional moieties contributed to As sequestration by the biochars examined here. However, significantly higher surface area and aromaticity of BC700 favored a greater As removal compared to BC300, suggesting that surface complexation/precipitation dominated As removal by BC700. Arsenic K-edge X-ray absorption near edge structure (XANES) spectroscopy demonstrated that up to 64% of the added As(V) was reduced to As(III) in BC700- and BC300-As(V) sorption experiments, and in As(III) sorption experiments, partial oxidation of As(III) to As(V) occurred (37-39%). However, XANES spectroscopy was limited to precisely quantify As binding with sulfur species as As 2 S 3 -like phase. Both biochars efficiently removed As from natural As-contaminated groundwater (As: 23-190 μg L -1 ; n = 12) despite in the presence of co-occurring anions (e.g., CO 3 2- , PO 4 3- , SO 4 2- ) with the highest levels of As removal observed for BC700 (97-100%). Overall, this study highlights that perilla leaf biochars, notably BC700, possessed the greatest ability to remove As from solution and groundwater (drinking water). Significantly, the integrated spectroscopic techniques advanced our understanding to examine complex redox transformation of As(III)/As(V) with biochar, which are crucial to determine fate of As on biochar in aquatic environments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats.

PubMed

Murray, M

1988-05-01

Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear to be complexed to any extent in microsomes from SKF 525-A-induced rats.

A study of H-alpha velocities in NGC 1499, NGC 7000, and IC 1318B/C

NASA Technical Reports Server (NTRS)

Fountain, W. F.; Gary, G. A.; Odell, C. R.

1983-01-01

Multiple slit echelle spectrograph observations of the H-alpha emission line are used to map the radial velocities of the California Nebula (NGC 1499), the North American Nebula complex (NGC 7000 and IC 5070), and IC 1318B/C. The California Nebula is singularly constant in velocity, considering its geometry. The North American Nebula complex reflects a very simple, classical dynamical picture. The expansion discovered earlier in IC 1318B/C is confirmed, detailed, and the model refined. The new data, along with that in earlier papers of this series, show that stellar wind acceleration and champagne flow mechanisms both play important roles in determining the evolution of H II regions.

Mechanism of action of coumarin and silver(I)-coumarin complexes against the pathogenic yeast Candida albicans.

PubMed

Thati, Bhumika; Noble, Andy; Rowan, Raymond; Creaven, Bernadette S; Walsh, Maureen; McCann, Malachy; Egan, Denise; Kavanagh, Kevin

2007-08-01

The anti-fungal activity and mode of action of a range of silver(I)-coumarin complexes was examined. The most potent silver(I)-coumarin complexes, namely 7-hydroxycoumarin-3-carboxylatosilver(I), 6-hydroxycoumarin-3-carboxylatosilver(I) and 4-oxy-3-nitrocoumarinbis(1,10-phenanthroline)silver(I), had MIC80 values of between 69.1 and 4.6 microM against the pathogenic yeast Candida albicans. These compounds also reduced respiration, lowered the ergosterol content of cells and increased the trans-membrane leakage of amino acids. A number of the complexes disrupted cytochrome synthesis in the cell and induced the appearance of morphological features consistent with cell death by apoptosis. These compounds appear to act by disrupting the synthesis of cytochromes which directly affects the cell's ability to respire. A reduction in respiration leads to a depletion in ergosterol biosynthesis and a consequent disruption of the integrity of the cell membrane. Disruption of cytochrome biosynthesis may induce the onset of apoptosis which has been shown previously to be triggered by alteration in the location of cytochrome c. Silver(I)-coumarin complexes demonstrate good anti-fungal activity and manifest a mode of action distinct to that of the conventional azole and polyene drugs thus raising the possibility of their use when resistance to conventional drug has emerged or in combination with such drugs.

Superhard BC 3 in cubic diamond structure

DOE PAGES

Zhang, Miao; Liu, Hanyu; Li, Quan; ...

2015-01-06

We solve the crystal structure of recently synthesized cubic BC 3 using an unbiased swarm structure search, which identifies a highly symmetric BC 3 phase in the cubic diamond structure (d–BC3) that contains a distinct B-B bonding network along the body diagonals of a large 64-atom unit cell. Simulated x-ray diffraction and Raman peaks of d–BC 3 are in excellent agreement with experimental data. Calculated stress-strain relations of d–BC 3 demonstrate its intrinsic superhard nature and reveal intriguing sequential bond-breaking modes that produce superior ductility and extended elasticity, which are unique among superhard solids. Here, the present results establish themore » first boron carbide in the cubic diamond structure with remarkable properties, and these new findings also provide insights for exploring other covalent solids with complex bonding configurations.« less

The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

PubMed

Castelle, Cindy J; Roger, Magali; Bauzan, Marielle; Brugna, Myriam; Lignon, Sabrina; Nimtz, Manfred; Golyshina, Olga V; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

2015-08-01

The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma. Copyright © 2015 Elsevier B.V. All rights reserved.

Haloacyl complexes of boron, [(CF3)3BC(O)Hal]- (Hal=F, Cl, Br, I).

PubMed

Finze, Maik; Bernhardt, Eduard; Willner, Helge; Lehmann, Christian W

2005-11-04

The haloacyltris(trifluoromethyl)borate anions [(CF3)3BC(O)Hal]- (Hal=F, Cl, Br, I) have been synthesized by reacting (CF3)3BCO with either MHal (M=K, Cs; Hal=F) in SO2 or MHal (M=[nBu4N]+, [Et4N]+, [Ph4P]+; Hal=Cl, Br, I) in dichloromethane. Metathesis reactions of the fluoroacyl complex with Me3SiHal (Hal=Cl, Br, I) led to the formation of its higher homologues. The thermal stabilities of the haloacyltris(trifluoromethyl)borates decrease from the fluorine to the iodine derivative. The chemical reactivities decrease in the same order as demonstrated by a series of selected reactions. The new [(CF3)3BC(O)Hal]- (Hal=F, Cl, Br) salts are used as starting materials in the syntheses of novel compounds that contain the (CF3)3B-C fragment. All borate anions [(CF3)3BC(O)Hal]- (Hal=F, Cl, Br, I) have been characterized by multinuclear NMR spectroscopy (11B, 13C, 17O, 19F) and vibrational spectroscopy. [PPh4][(CF3)3BC(O)Br] crystallizes in the monoclinic space group P2/c (no. 13) and the bond parameters are compared with those of (CF3)3BCO and K[(CF3)3BC(O)F]. The interpretation of the spectroscopic and structural data are supported by DFT calculations [B3LYP/6-311+G(d)].

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

PubMed

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

2016-08-25

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

[Efficacy and safety of heptral, vitamin B6 and folic acid during toxic hepatitis induced by CCL4].

PubMed

Antelava, N A; Gogoluari, M I; Gogoluari, L I; Pirtskhalaĭshvili, N N; Okudzhava, M V

2007-09-01

The aim of this work was to evaluate of efficacy and safety of complex Heptral, Vitamin B6 and Folic Acid in experimental hepatitis therapy compared with monotherapy. Experiments were carried out on pubertal rats. Eperimental hepatitis models were induced by Tetrachlormethane. The tetrachlormethane intoxication was reproduced by subcutaneous injection of CCL(4) 1ml/kg dissolved in 1ml of olive oil. Cytochrome P450, cytochrome b5, reduced glutation,activity of glutationetranspherase and content of ATP in hepatocytes were measured by the spectrophotometric techniques,but content of homocysteine by chromophtography techniques. Under CCL(4) intoxication disturbance of liver detoxication function, energy deficit and surplus of homocysteine were observed. Treatment of the toxic hepatitis with heptral increased the level of cytochrome P450, cytochrome b5, glutation activity of glutationetranspherase glutathione and reduced content of homocysteine. Complex therapy with Heptral and B6 and folic acid reveal more expressive hepatoprotective effect and safety than monotherapy with Heptral. Complex therapy improves not only the parameters of biotransformation (metabolic and conjugation phase), but also normalizes the level of ATP and homocystein. Vitamins B6 and folic acid increases the efficacy and safety of Heptral. This complex was recomended for treatment of hepatitis.

Rapid kill of malaria parasites by artemisinin and semi-synthetic endoperoxides involves ROS-dependent depolarization of the membrane potential

PubMed Central

Antoine, Thomas; Fisher, Nicholas; Amewu, Richard; O'Neill, Paul M.; Ward, Stephen A.; Biagini, Giancarlo A.

2014-01-01

Objectives Artemisinin and artemisinin semi-synthetic derivatives (collectively known as endoperoxides) are first-line antimalarials for the treatment of uncomplicated and severe malaria. Endoperoxides display very fast killing rates and are generally recalcitrant to parasite resistance development. These key pharmacodynamic features are a result of a complex mechanism of action, the details of which lack consensus. Here, we report on the primary physiological events leading to parasite death. Methods Parasite mitochondrial (ΔΨm) and plasma membrane (ΔΨp) electrochemical potentials were measured using real-time single-cell imaging following exposure to pharmacologically relevant concentrations of endoperoxides (artemisinin, dihydroartemisinin, artesunate and the synthetic tetraoxane RKA182). In addition, mitochondrial electron transport chain components NADH:quinone oxidoreductase (alternative complex I), bc1 (complex III) and cytochrome oxidase (complex IV) were investigated to determine their functional sensitivity to the various endoperoxides. Results Parasite exposure to endoperoxides resulted in rapid depolarization of parasite ΔΨm and ΔΨp. The rate of depolarization was decreased in the presence of a reactive oxygen species (ROS) scavenger and Fe3+ chelators. Depolarization of ΔΨm by endoperoxides is not believed to be through the inhibition of mitochondrial electron transport chain components, owing to the lack of significant inhibition when assayed directly. Conclusions The depolarization of ΔΨm and ΔΨp is shown to be mediated via the generation of ROS that are initiated by iron bioactivation of endoperoxides and/or catalysed by iron-dependent oxidative stress. These data are discussed in the context of current hypotheses concerning the mode of action of endoperoxides. PMID:24335485

Limitation of using Angstrom exponent for source apportionment of black carbon in complex environments - A case study from the North West Indo- Gangetic plain

NASA Astrophysics Data System (ADS)

Garg, S.; Sinha, B.; Sinha, V.; Chandra, P.; Sarda Esteve, R.; Gros, V.

2015-12-01

Determining the contribution of different sources to the total BC is necessary for targeted mitigation. Absorption Angstrom exponent (αabs) measurements of black carbon (BC) have recently been introduced as a novel tool to apportion the contribution of biomass burning sources to BC. Two-component Aethalometer model for apportioning BC to biomass burning sources and fossil fuel combustion sources, which uses αabs as a generic indicator of the source type, is widely used for determining the contribution of the two types of sources to the total BC. Our work studies BC emissions in the highly-populated, anthropogenic emissions-dominated Indo-Gangetic Plain and demonstrates that the αabs cannot be used as a generic tracer for biomass burning emissions in a complex environment. Simultaneously collected high time resolution data from a 7-wavelength Aethalometer (AE 42, Magee Scientific, USA) and a high sensitivity Proton Transfer Reaction- Quadrupole Mass Spectrometer (PTR-MS) installed at a sub-urban site in Mohali (Punjab), India, were used to identify a number of biomass combustion plumes during which BC enhancements correlated strongly with an increase in acetonitrile (a well-established biomass burning tracer) mixing ratio. Each type of biomass combustion is classified and characterized by distinct emission ratios of aromatic compounds and oxygenated VOCs to acetonitrile. The identified types of biomass combustion include two different types of crop residue burning (paddy and wheat), burning of leaf-litter, and garbage burning. Traffic (fossil-fuel burning) plumes were also selected for comparison. We find that the two-component Aethalometer source-apportionment method cannot be extrapolated to all types of biomass combustion and αabs of traffic plumes can be >1 in developing countries like India, where use of adulterated fuel in vehicles is common. Thus in a complex environment, where multiple anthropogenic BC sources and air masses of variable photochemical age impact a receptor site, the angstrom exponent is not representative of the combustion type and therefore, cannot be used as a generic tracer to constrain source contributions.

Regulation of NADH/CoQ oxidoreductase: do phosphorylation events affect activity?

PubMed

Maj, Mary C; Raha, Sandeep; Myint, Tomoko; Robinson, Brian H

2004-01-01

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.

Binuclear transition-metal complexes as new reagents for selective cross-linking of proteins. Coordination of cytochrome c to dirhodium(II). mu. -tetraacetate

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

Chen, J.; Kostic, N.M.

1988-07-27

This study introduces binuclear transition-metal complexes as reagents for selective covalent cross-linking of proteins. Incubation of horse cytochrome c (designated cyt) with Rh{sub 2}(OAc){sub 4} under mild conditions yields the diprotein complex, Rh{sub 2}(OAc){sub 4}(cyt){sub 2}, whose composition is established by size-exclusion chromatography, uv-vis spectroscopy, and {sup 1}H NMR spectroscopy. The protein molecules are coordinated to the Rh atoms via the imidazole (Im) rings of their His 33 residues, as shown by uv difference and {sup 1}H NMR spectroscopy, by the pH effect on the complex formation, and by the control experiments with tuna cytochrome c. The diprotein complex ismore » stable under ordinary conditions, and yet it can be cleaved, and the native protein recovered, by treatment with a suitable strong nucleophile. Spectroscopic and electrochemical measurements show that the structural and redox properties of cytochrome c are not perturbed significantly by cross-linking. Comparison between Rh{sub 2}(OAc){sub 4}(Im){sub 2} and Rh{sub 2}(OAc){sub 4}(cyt){sub 2} shows that the complex containing small ligands is not an entirely realistic model of the complex containing proteins. In particular, the enhanced stability of the latter toward hydrolysis may be due to steric bulk of the protein ligands and to hydrogen bonds that amino acid side chains may form with the inorganic link. Some of the findings of this study may pertain to the mechanism of antitumor action of the Rh{sub 2}(RCOO){sub 4} complexes. 86 refs., 2 tabs.« less

The Respiratory Arsenite Oxidase: Structure and the Role of Residues Surrounding the Rieske Cluster

PubMed Central

Warelow, Thomas P.; Oke, Muse; Schoepp-Cothenet, Barbara; Dahl, Jan U.; Bruselat, Nicole; Sivalingam, Ganesh N.; Leimkühler, Silke; Thalassinos, Konstantinos; Kappler, Ulrike; Naismith, James H.; Santini, Joanne M.

2013-01-01

The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a −20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc 1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc 1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter. PMID:24023621

Rheological performance of bacterial cellulose based nonmineralized and mineralized hydrogel scaffolds

NASA Astrophysics Data System (ADS)

Basu, Probal; Saha, Nabanita; Bandyopadhyay, Smarak; Saha, Petr

2017-05-01

Bacterial cellulose (BC) based hydrogels (BC-PVP and BC-CMC) are modified with β-tri-calcium phosphate (β-TCP) and hydroxyapatite (HA) to improve the structural and functional properties of the existing hydrogel scaffolds. The modified hydrogels are then biomineralized with CaCO3 following liquid diffusion technique, where salt solutions of Na2CO3 (5.25 g/100 mL) and CaCl2 (7.35 g/100 mL) were involved. The BC-PVP and BC-CMC are being compared with the non-mineralized (BC-PVP-β-TCP/HA and BC-CMC-β-TCP/HA) and biomineralized (BC-PVP-β-TCP/HA-CaCO3 and BC-CMC-β-TCP/HA-CaCO3) hydrogels on the basis of their structural and rheological properties. The Fourier Transform Infrared (FTIR) spectral analysis demonstrated the presence of BC, CMC, PVP, β-TCP, HA in the non-mineralized and BC, CMC, PVP, β-TCP, HA and CaCO3 in the biomineralized samples. Interestingly, the morphological property of non-mineralized and biomineralized, hydrogels are different than that of BC-PVP and BC-CMC based novel biomaterials. The Scanning Electron Microscopic (SEM) images of the before mentioned samples reveal the denser structures than BC-PVP and BC-CMC, which exhibits the changes in their pore sizes. Concerning rheological analysis point of view, all the non-mineralized and biomineralized hydrogel scaffolds have shown significant elastic property. Additionally, the complex viscosity (η*) values have also found in decreasing order with the increase of angular frequency (ω) 0.1 rad.sec-1 to 100 rad.sec-1. All these BC based hydrogel scaffolds are elastic in nature, can be recommended for their application as an implant for bone tissue engineering.

Facile synthesis of Cu(II) impregnated biochar with enhanced adsorption activity for the removal of doxycycline hydrochloride from water.

PubMed

Liu, Su; Xu, Wei-Hua; Liu, Yun-Guo; Tan, Xiao-Fei; Zeng, Guang-Ming; Li, Xin; Liang, Jie; Zhou, Zan; Yan, Zhi-Li; Cai, Xiao-Xi

2017-08-15

In this study, the effect factors and mechanisms of doxycycline hydrochloride (DOX) adsorption on copper nitrate modified biochar (Cu-BC) was investigated. Cu-BC absorbent was synthesized through calcination of peanut shells biomass at 450°C and then impregnation with copper nitrate. The Cu-BC has exhibited excellent sorption efficiency about 93.22% of doxycycline hydrochloride from aqueous solution, which was double higher than that of the unmodified biochar. The experimental results suggest that the adsorption efficiency of DOX on the Cu-BC is dominated by the strong complexation, electrostatic interactions between DOX molecules and the Cu-BC samples. Comprehensively considering the cost, efficiency and the application to realistic water, the Cu-BC hold the significant potential for enhancing the effectiveness to remove DOX from water. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter.

PubMed

Bao, X; Shorrosh, B S; Ohlrogge, J B

1997-11-01

In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits. To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter. Fifteen introns were identified. The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity). BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis. GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf. Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression. A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron. In addition, several conserved regulatory elements were identified in the BC promoter. Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

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

C Chou; L Tong

2011-12-31

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADPmore » molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.« less

Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

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

Chou, Chi-Yuan; Tong, Liang

2012-06-19

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADPmore » molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.« less

Molecular dynamics simulations give insight into the conformational change, complex formation, and electron transfer pathway for cytochrome P450 reductase

PubMed Central

Sündermann, Axel; Oostenbrink, Chris

2013-01-01

Cytochrome P450 reductase (CYPOR) undergoes a large conformational change to allow for an electron transfer to a redox partner to take place. After an internal electron transfer over its cofactors, it opens up to facilitate the interaction and electron transfer with a cytochrome P450. The open conformation appears difficult to crystallize. Therefore, a model of a human CYPOR in the open conformation was constructed to be able to investigate the stability and conformational change of this protein by means of molecular dynamics simulations. Since the role of the protein is to provide electrons to a redox partner, the interactions with cytochrome P450 2D6 (2D6) were investigated and a possible complex structure is suggested. Additionally, electron pathway calculations with a newly written program were performed to investigate which amino acids relay the electrons from the FMN cofactor of CYPOR to the HEME of 2D6. Several possible interacting amino acids in the complex, as well as a possible electron transfer pathway were identified and open the way for further investigation by site directed mutagenesis studies. PMID:23832577

Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies.

PubMed

Dell'acqua, Simone; Pauleta, Sofia R; Monzani, Enrico; Pereira, Alice S; Casella, Luigi; Moura, José J G; Moura, Isabel

2008-10-14

The multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center.

Functional Analysis of BcBem1 and Its Interaction Partners in Botrytis cinerea: Impact on Differentiation and Virulence

PubMed Central

Schumacher, Julia; Kokkelink, Leonie; Tudzynski, Paul

2014-01-01

In phytopathogenic fungi the establishment and maintenance of polarity is not only essential for vegetative growth and differentiation, but also for penetration and colonization of host tissues. We investigated orthologs of members of the yeast polarity complex in the grey mould fungus Botrytis cinerea: the scaffold proteins Bem1 and Far1, the GEF (guanine nucleotide exchange factor) Cdc24, and the formin Bni1 (named Sep1 in B. cinerea). BcBem1 does not play an important role in regular hyphal growth, but has significant impact on spore formation and germination, on the establishment of conidial anastomosis tubes (CATs) and on virulence. As in other fungi, BcBem1 interacts with the GEF BcCdc24 and the formin BcSep1, indicating that in B. cinerea the apical complex has a similar structure as in yeast. A functional analysis of BcCdc24 suggests that it is essential for growth, since it was not possible to obtain homokaryotic deletion mutants. Heterokaryons of Δcdc24 (supposed to exhibit reduced bccdc24 transcript levels) already show a strong phenotype: an inability to penetrate the host tissue, a significantly reduced growth rate and malformation of conidia, which tend to burst as observed for Δbcbem1. Also the formin BcSep1 has significant impact on hyphal growth and development, whereas the role of the putative ortholog of the yeast scaffold protein Far1 remains open: Δbcfar1 mutants have no obvious phenotypes. PMID:24797931

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

PubMed Central

Leong, Ta-Yan; Briggs, Winslow R.

1982-01-01

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

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

PubMed

Noor, Mohamed Radzi; Soulimane, Tewfik

2013-05-01

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

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

PubMed

Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

2008-05-02

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

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

PubMed

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

2011-06-01

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

Diagnostic value of succinate ubiquinone reductase activity in the identification of patients with mitochondrial DNA depletion.

PubMed

Hargreaves, P; Rahman, S; Guthrie, P; Taanman, J W; Leonard, J V; Land, J M; Heales, S J R

2002-02-01

Mitochondrial DNA (mtDNA) depletion syndrome (McKusick 251880) is characterized by a progressive quantitative loss of mtDNA resulting in severe mitochondrial dysfunction. A diagnosis of mtDNA depletion can only be confirmed after Southern blot analysis of affected tissue. Only a limited number of centres have the facilities to offer this service, and this is frequently on an irregular basis. There is therefore a need for a test that can refine sample selection as well as complementing the molecular analysis. In this study we compared the activities of the nuclear-encoded succinate ubiquinone reductase (complex II) to the activities of the combined mitochondrial and nuclear-encoded mitochondrial electron transport chain (ETC) complexes; NADH:ubiquinone reductase (complex I), ubiquinol-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV), in skeletal muscle biopsies from 7 patients with confirmed mtDNA depletion. In one patient there was no evidence of an ETC defect. However, the remaining 6 patients exhibited reduced complex I and IV activities. Five of these patients also displayed reduced complex II-III (succinate:cytochrome-c reductase) activity. Individual measurement of complex II and complex III activities demonstrated normal levels of complex II activity compared to complex III, which was reduced in the 5 biopsies assayed. These findings suggest a possible diagnostic value for the detection of normal levels of complex II activity in conjunction with reduced complex I, III and IV activity in the identification of likely candidates for mtDNA depletion syndrome

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

NASA Astrophysics Data System (ADS)

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

2014-02-01

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

The absorption Ångström exponent of black carbon: from numerical aspects

NASA Astrophysics Data System (ADS)

Liu, Chao; Eddy Chung, Chul; Yin, Yan; Schnaiter, Martin

2018-05-01

The absorption Ångström exponent (AAE) is an important aerosol optical parameter used for aerosol characterization and apportionment studies. The AAE of black carbon (BC) particles is widely accepted to be 1.0, although observational estimates give quite a wide range of 0.6-1.3. With considerable uncertainties related to observations, a numerical study is a powerful method, if not the only one, to provide a better and more accurate understanding on BC AAE. This study calculates BC AAE using realistic particle geometries based on fractal aggregate and an accurate numerical optical model (namely the multiple-sphere T-matrix method), and considers bulk properties of an ensemble of BC particles following lognormal size distributions. At odds with the expectations, BC AAE is not 1.0, even when BC is assumed to have small sizes and a wavelength-independent refractive index. With a wavelength-independent refractive index, the AAE of fresh BC is approximately 1.05 and relatively insensitive to particle size. For BC with geometric mean diameters larger than 0.12 µm, BC AAE becomes smaller when BC particles are aged (compact structures or coated by other non-absorptive materials). For coated BC, we prescribe the coating fraction variation based on a laboratory study, where smaller BC cores are shown to develop larger coating fractions than those of bigger BC cores. For both compact and coated BC, the AAE is highly sensitive to particle size distribution, ranging from approximately 0.8 to even over 1.4 with wavelength-independent refractive index. When the refractive index is allowed to vary with wavelength, a feature with observational backing, the BC AAE may show an even wider range. For different BC morphologies, we derive simple empirical equations on BC AAE based on our numerical results, which can serve as a guide for the response of BC AAE to BC size and refractive index. Due to its complex influences, the effects of BC geometry is better to be discussed at certain BC properties, i.e., known size and refractive index.

Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c

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

Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it; Ciaccio, Chiara; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, I-70126 Bari

2011-01-07

Research highlights: {yields} Cardiolipin binding to cytochrome c. {yields} Cardiolipin-dependent peroxynitrite isomerization by cytochrome c. {yields} Cardiolipin-cytochrome c complex plays pro-apoptotic effects. {yields} Cardiolipin-cytochrome c complex plays anti-apoptotic effects. -- Abstract: Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does notmore » catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k{sub on}) is (3.2 {+-} 0.4) x 10{sup 5} M{sup -1} s{sup -1}. The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1 {+-} 0.8) x 10{sup -5} M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.« less

Crystal structure of the Leishmania major peroxidase–cytochrome c complex

PubMed Central

Jasion, Victoria S.; Doukov, Tzanko; Pineda, Stephanie H.; Li, Huiying; Poulos, Thomas L.

2012-01-01

The causative agent of leishmaniasis is the protozoan parasite Leishmania major. Part of the host protective mechanism is the production of reactive oxygen species including hydrogen peroxide. In response, L. major produces a peroxidase, L. major peroxidase (LmP), that helps to protect the parasite from oxidative stress. LmP is a heme peroxidase that catalyzes the peroxidation of mitochondrial cytochrome c. We have determined the crystal structure of LmP in a complex with its substrate, L. major cytochrome c (LmCytc) to 1.84 Å, and compared the structure to its close homolog, the yeast cytochrome c peroxidase–cytochrome c complex. The binding interface between LmP and LmCytc has one strong and one weak ionic interaction that the yeast system lacks. The differences between the steady-state kinetics correlate well with the Lm redox pair being more dependent on ionic interactions, whereas the yeast redox pair depends more on nonpolar interactions. Mutagenesis studies confirm that the ion pairs at the intermolecular interface are important to both kcat and KM. Despite these differences, the electron transfer path, with respect to the distance between hemes, along the polypeptide chain is exactly the same in both redox systems. A potentially important difference, however, is the side chains involved. LmP has more polar groups (Asp and His) along the pathway compared with the nonpolar groups (Leu and Ala) in the yeast system, and as a result, the electrostatic environment along the presumed electron transfer path is substantially different. PMID:23100535

Theoretical study on interaction of cytochrome f and plastocyanin complex by a simple coarse-grained model with molecular crowding effect

NASA Astrophysics Data System (ADS)

Nakagawa, Satoshi; Kurniawan, Isman; Kodama, Koichi; Arwansyah, Muhammad Saleh; Kawaguchi, Kazutomo; Nagao, Hidemi

2018-03-01

We present a simple coarse-grained model with the molecular crowding effect in solvent to investigate the structure and dynamics of protein complexes including association and/or dissociation processes and investigate some physical properties such as the structure and the reaction rate from the viewpoint of the hydrophobic intermolecular interactions of protein complex. In the present coarse-grained model, a function depending upon the density of hydrophobic amino acid residues in a binding area of the complex is introduced, and the function involves the molecular crowding effect for the intermolecular interactions of hydrophobic amino acid residues between proteins. We propose a hydrophobic intermolecular potential energy between proteins by using the density-dependent function. The present coarse-grained model is applied to the complex of cytochrome f and plastocyanin by using the Langevin dynamics simulation to investigate some physical properties such as the complex structure, the electron transfer reaction rate constant from plastocyanin to cytochrome f and so on. We find that for proceeding the electron transfer reaction, the distance between metals in their active sites is necessary within about 18 Å. We discuss some typical complex structures formed in the present simulation in relation to the molecular crowding effect on hydrophobic interactions.

Dual Nature of Translational Control by Regulatory BC RNAs ▿

PubMed Central

Eom, Taesun; Berardi, Valerio; Zhong, Jun; Risuleo, Gianfranco; Tiedge, Henri

2011-01-01

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3′ stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems. PMID:21930783

The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells.

PubMed

Orecchioni, Stefania; Reggiani, Francesca; Talarico, Giovanna; Mancuso, Patrizia; Calleri, Angelica; Gregato, Giuliana; Labanca, Valentina; Noonan, Douglas M; Dallaglio, Katiuscia; Albini, Adriana; Bertolini, Francesco

2015-03-15

The human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression. The biguanide Metformin (Met), commonly used for Type 2 diabetes, might have activity against BC and was found to inhibit angiogenesis in vivo. We studied Met and another biguanide, phenformin (Phe), in vitro and in vivo in BC models. In vitro, biguanides activated AMPK, inhibited Complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. In coculture, biguanides inhibited the production of several angiogenic proteins. In vivo, biguanides inhibited local and metastatic growth of triple negative and HER2+ BC in immune-competent and immune-deficient mice orthotopically injected with BC. Biguanides inhibited local and metastatic BC growth in a genetically engineered murine model model of HER2+ BC. In vivo, biguanides increased pimonidazole binding (but not HIF-1 expression) of WAT progenitors, reduced tumor microvessel density and altered the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. Phe was significantly more active than Met both in vitro and in vivo. Considering their safety profile, biguanides deserve to be further investigated for BC prevention in high-risk subjects, in combination with chemo and/or targeted therapy and/or as post-therapy consolidation or maintenance therapy for the prevention of BC recurrence. © 2014 UICC.

Preface.

PubMed

Villarreal-Garza, Cynthia; Castro-Sánchez, Andrea

2017-01-01

Young women with breast cancer (BC) are a special population with distinctive genetic, clinical, and psychosocial features and requirements based on at least three considerations: (1) Cancer presentation at young age is often diagnosed at advanced stages, hence its prognosis is worse compared to their older counterparts1,2; (2) young BC patients often receive aggressive and prolonged systemic treatments that can be associated with significant long-term morbidity3; and (3) young women with BC experience substantial psychosocial vulnerability resulting from high levels of distress and depression associated with oncological interventions4. These factors pose complex issues for young patients with BC, particularly in regard to their family dynamics, social and professional lives, and self-development, thereby substantially undermining their quality of life.

Immobilization of heavy metals in electroplating sludge by biochar and iron sulfide.

PubMed

Lyu, Honghong; Gong, Yanyan; Tang, Jingcshun; Huang, Yao; Wang, Qilin

2016-07-01

Electroplating sludge (ES) containing large quantities of heavy metals is regarded as a hazardous waste in China. This paper introduced a simple method of treating ES using environmentally friendly fixatives biochar (BC) and iron sulfide (FeS), respectively. After 3 days of treatment with FeS at a FeS-to-ES mass ratio of 1:5, the toxicity characteristic leaching procedure (TCLP)-based leachability of total Cr (TCr), Cu(II), Ni(II), Pb(II), and Zn(II) was decreased by 59.6, 100, 63.8, 73.5, and 90.5 %, respectively. After 5 days of treatment with BC at a BC-to-ES mass ratio of 1:2, the TCLP-based leachability was declined by 35.1, 30.6, 22.3, 23.1, and 22.4 %, respectively. Pseudo first-order kinetic model adequately simulated the sorption kinetic data. Structure and morphology analysis showed that adsorption, electrostatic attraction, surface complexation, and chemical precipitation were dominant mechanisms for heavy metals immobilization by BC, and that chemical precipitation (formation of metal sulfide and hydroxide precipitates), iron exchange (formation of CuFeS2), and surface complexation were mainly responsible for heavy metals removal by FeS. Economic costs of BC and FeS were 500 and 768 CNY/t, lower than that of Na2S (940 CNY/t). The results suggest that BC and FeS are effective, economic, and environmentally friendly fixatives for immobilization of heavy metals in ES before landfill disposal.

Crystal Structure of Biotin Carboxylase in Complex with Substrates and Implications for Its Catalytic Mechanism

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

Chou, C.; Yu, L; Tong, L

2009-01-01

Biotin-dependent carboxylases are widely distributed in nature and have important functions in many cellular processes. These enzymes share a conserved biotin carboxylase (BC) component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the donor. Despite the availability of a large amount of biochemical and structural information on BC, the molecular basis for its catalysis is currently still poorly understood. We report here the crystal structure at 2.0 {angstrom} resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu{sup 296} is the general base that extracts the proton frommore » bicarbonate, and Arg{sup 338} is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of BC is positioned closer to the active site, leading to a 2-{angstrom} shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygen atoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This oxygen is also located close to the N1' atom of biotin, providing strong evidence that the phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1' atom. The structural observations are supported by mutagenesis and kinetic studies. Overall, this first structure of BC in complex with substrates offers unprecedented insights into the molecular mechanism for the catalysis by this family of enzymes.« less

Light Absorption Enhancement of Black Carbon Aerosol Constrained by Particle Morphology.

PubMed

Wu, Yu; Cheng, Tianhai; Liu, Dantong; Allan, James D; Zheng, Lijuan; Chen, Hao

2018-06-19

The radiative forcing of black carbon aerosol (BC) is one of the largest sources of uncertainty in climate change assessments. Contrasting results of BC absorption enhancement ( E abs ) after aging are estimated by field measurements and modeling studies, causing ambiguous parametrizations of BC solar absorption in climate models. Here we quantify E abs using a theoretical model parametrized by the complex particle morphology of BC in different aging scales. We show that E abs continuously increases with aging and stabilizes with a maximum of ∼3.5, suggesting that previous seemingly contrast results of E abs can be explicitly described by BC aging with corresponding particle morphology. We also report that current climate models using Mie Core-Shell model may overestimate E abs at a certain aging stage with a rapid rise of E abs , which is commonly observed in the ambient. A correction coefficient for this overestimation is suggested to improve model predictions of BC climate impact.

METABOLISM OF MYCLOBUTANIL AND TRIADIMEFON BY HUMAN AND RAT CYTOCHROME P450 ENZYMES AND LIVER MICROSOMES.

EPA Science Inventory

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...

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients.

PubMed

Pequignot, M O; Desguerre, I; Dey, R; Tartari, M; Zeviani, M; Agostino, A; Benelli, C; Fouque, F; Prip-Buus, C; Marchant, D; Abitbol, M; Marsac, C

2001-05-04

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.

Complex Relationships between Occupation, Environment, DNA Adducts, Genetic Polymorphisms and Bladder Cancer in a Case-Control Study Using a Structural Equation Modeling

PubMed Central

Porru, Stefano; Pavanello, Sofia; Carta, Angela; Arici, Cecilia; Simeone, Claudio; Izzotti, Alberto; Mastrangelo, Giuseppe

2014-01-01

DNA adducts are considered an integrate measure of carcinogen exposure and the initial step of carcinogenesis. Their levels in more accessible peripheral blood lymphocytes (PBLs) mirror that in the bladder tissue. In this study we explore whether the formation of PBL DNA adducts may be associated with bladder cancer (BC) risk, and how this relationship is modulated by genetic polymorphisms, environmental and occupational risk factors for BC. These complex interrelationships, including direct and indirect effects of each variable, were appraised using the structural equation modeling (SEM) analysis. Within the framework of a hospital-based case/control study, study population included 199 BC cases and 213 non-cancer controls, all Caucasian males. Data were collected on lifetime smoking, coffee drinking, dietary habits and lifetime occupation, with particular reference to exposure to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs). No indirect paths were found, disproving hypothesis on association between PBL DNA adducts and BC risk. DNA adducts were instead positively associated with occupational cumulative exposure to AAs (p = 0.028), whereas XRCC1 Arg 399 (p<0.006) was related with a decreased adduct levels, but with no impact on BC risk. Previous findings on increased BC risk by packyears (p<0.001), coffee (p<0.001), cumulative AAs exposure (p = 0.041) and MnSOD (p = 0.009) and a decreased risk by MPO (p<0.008) were also confirmed by SEM analysis. Our results for the first time make evident an association between occupational cumulative exposure to AAs with DNA adducts and BC risk, strengthening the central role of AAs in bladder carcinogenesis. However the lack of an association between PBL DNA adducts and BC risk advises that these snapshot measurements are not representative of relevant exposures. This would envisage new scenarios for biomarker discovery and new challenges such as repeated measurements at different critical life stages. PMID:24722645

Black carbon, a 'hidden' player in the global C cycle

NASA Astrophysics Data System (ADS)

Santín, C.; Doerr, S. H.

2012-04-01

During the 2011 alone more than 600 scientific papers about black carbon (BC) were published, half of them dealing with soils (ISI Web of Knowledge, accessed 15/01/2012). If the search is extended to the other terms by which BC is commonly named (i.e. biochar, charcoal, pyrogenic C or soot), the number of 2011 publications increases to >2400, 20% of them also related to soils. These figures confirm BC as a well-known feature in the scientific literature and, thus, in our research community. In fact, there is a wide variety of research topics where BC is currently studied: from its potential as long-term C reservoir in soils (man-made biochar), to its effects on the Earth's radiation balance (soot-BC), including its value as indicator in paleoenvironmental studies (charcoal) or, even surprisingly, its use in suicide attempts. BC is thus relevant to many aspects of our environment, making it a very far-reaching, but also very complex topic. When focusing 'only' on the role of BC in the global C cycle, numerous questions arise. For example: (i) how much BC is produced by different sources (i.e. vegetation fires, fossil fuel and biofuel combustion); (ii) what are the main BC forms and their respective proportions generated (i.e. proportion of atmospheric BC [BC-soot] and the solid residues [char-BC]); (iii) where does this BC go (i.e. main mobilization pathways and sinks); (iv) how long does BC stay in the different systems (i.e. residence times in soils, sediments, water and atmosphere); (v) which are the BC stocks and its main transformations within and between the different systems (i.e. BC preservation, alteration and mineralization); (vi) what is the interaction of BC with other elements and how does this influence BC half-life (i.e. physical protection, interaction with pollutants, priming effects in other organic materials)? These questions, and some suggestions about how to tackle these, will be discussed in this contribution. It will focus in particular on the role of black carbon within soil system sciences, but will also consider it from an integrated atmosphere-marine-terrestrial perspective.

Flux, Budget and Sources of Black Carbon (BC) in the Continental Shelf of the Bohai and Yellow Seas, China

NASA Astrophysics Data System (ADS)

Fang, Y.; Chen, Y.; Tian, C.

2015-12-01

Black carbon (BC) derived from incomplete combustion of fossil fuels and biomass has received increasing attention due to their potential importance in a wide range of biogeochemical processes. China has been generally considered as the world's largest BC emitter. Due to a combination of the prevailing East Asia monsoon and large amounts of riverine outflow, BC released from China can be transported to the adjacent continental shelf seas, the Bohai Sea (BS) and Yellow Sea (YS). Based on measurements of BC in 191 surface sediments, 36 riverine water, and 2 seawater samples, as well as the reported BC data set of the aerosol samples in the Bohai Rim, the concentration, flux, and budget of BC in the BS and YS were investigated. The spatial distribution of the BC concentration in surface sediments was largely influenced by the regional hydrodynamic conditions, with high values mainly occurring in the central mud areas. The BC burial flux in the BS and YS ranged from 4 to 1100 μg/cm2/yr, and averaged 166 ± 200 μg/cm2/yr. The area-integrated sedimentary BC sink flux in the entire BS and YS was ~325 Gg/yr. The BC budget calculated in the BS showed that atmospheric deposition and riverine discharge played comparable importance in delivering BC to the BS, and sequestration to bottom sediments was the major BC output pattern, accounting for ~88% of the total input BC. Besides, we attempted to apportion the BC sources in the BS and YS surface sediments using PAHs (organic molecular proxies cogenerated with BC) and BC as an input data to the Positive Matrix Factorization (PMF) receptor model. Results showed that ~83% of the sediment BC was attributed to the combustion of fossil fuels, and the remaining ~17% was from biomass burning. Due to the differences in their production mechanisms and therefore physicochemical properties, the above distinction and quantification would help us better understand their different environmental behaviors in the complex continental shelf regimes.

Formal modeling and analysis of ER-α associated Biological Regulatory Network in breast cancer.

PubMed

Khalid, Samra; Hanif, Rumeza; Tareen, Samar H K; Siddiqa, Amnah; Bibi, Zurah; Ahmad, Jamil

2016-01-01

Breast cancer (BC) is one of the leading cause of death among females worldwide. The increasing incidence of BC is due to various genetic and environmental changes which lead to the disruption of cellular signaling network(s). It is a complex disease in which several interlinking signaling cascades play a crucial role in establishing a complex regulatory network. The logical modeling approach of René Thomas has been applied to analyze the behavior of estrogen receptor-alpha (ER- α ) associated Biological Regulatory Network (BRN) for a small part of complex events that leads to BC metastasis. A discrete model was constructed using the kinetic logic formalism and its set of logical parameters were obtained using the model checking technique implemented in the SMBioNet software which is consistent with biological observations. The discrete model was further enriched with continuous dynamics by converting it into an equivalent Petri Net (PN) to analyze the logical parameters of the involved entities. In-silico based discrete and continuous modeling of ER- α associated signaling network involved in BC provides information about behaviors and gene-gene interaction in detail. The dynamics of discrete model revealed, imperative behaviors represented as cyclic paths and trajectories leading to pathogenic states such as metastasis. Results suggest that the increased expressions of receptors ER- α , IGF-1R and EGFR slow down the activity of tumor suppressor genes (TSGs) such as BRCA1, p53 and Mdm2 which can lead to metastasis. Therefore, IGF-1R and EGFR are considered as important inhibitory targets to control the metastasis in BC. The in-silico approaches allow us to increase our understanding of the functional properties of living organisms. It opens new avenues of investigations of multiple inhibitory targets (ER- α , IGF-1R and EGFR) for wet lab experiments as well as provided valuable insights in the treatment of cancers such as BC.

Fractal scaling laws of black carbon aerosol and their influence on spectral radiative properties

NASA Astrophysics Data System (ADS)

Tiwari, S.; Chakrabarty, R. K.; Heinson, W.

2016-12-01

Current estimates of the direct radiative forcing for Black Carbon (BC) aerosol span over a poorly constrained range between 0.2 and 1 W.m-2. To improve this large uncertainty, tighter constraints need to be placed on BC's key wavelength-dependent optical properties, namely, the absorption (MAC) and scattering (MSC) cross sections per unit mass and hemispherical upscatter fraction (β; a dimensionless scattering directionality parameter). These parameters are very sensitive to changes in particle morphology and complex refractive index nindex. Their interplay determines the magnitude of net positive or negative radiative forcing efficiencies. The current approach among climate modelers for estimating MAC and MSC values of BC is from their optical cross-sections calculated assuming spherical particle morphology with homogeneous, constant-valued refractive index in the visible solar spectrum. The β values are typically assumed to be a constant across this spectrum. This approach, while being computationally inexpensive and convenient, ignores the inherent fractal morphology of BC and its scaling behaviors, and resulting optical properties. In this talk, I will present recent results from my laboratory on determination of the fractal scaling laws of BC aggregate packing density and its complex refractive index for size spanning across three orders of magnitude, and their effects on spectral (Visible-infrared wavelength) scaling of MAC, MSC, and β values. Our experiments synergistically combined novel BC generation techniques, aggregation models, contact-free multi-wavelength optical measurements, and electron microscopy analysis. The scale dependence of nindex on aggregate size followed power-law exponents of -1.4 and -0.5 for sub- and super-micron size aggregates, respectively. The spherical Rayleigh-optics approximation limits, used by climate models for spectral extrapolation of BC optical cross-sections and deconvolution of multi-species mixing ratios, are redefined using the concept of phase shift parameter. I will highlight the importance of size-dependent β values and its role in offsetting the strong light absorbing nature of BC. Finally, the errors introduced in forcing efficiency calculations of BC by assuming spherical homogeneous morphology will be evaluated.

Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone

PubMed Central

Lawres, Lauren A.; Garg, Aprajita; Kumar, Vidya; Bruzual, Igor; Forquer, Isaac P.; Renard, Isaline; Virji, Azan Z.; Boulard, Pierre; Rodriguez, Eduardo X.; Allen, Alexander J.; Pou, Sovitj; Wegmann, Keith W.; Winter, Rolf W.; Nilsen, Aaron; Mao, Jialing; Preston, Douglas A.; Belperron, Alexia A.; Bockenstedt, Linda K.; Hinrichs, David J.; Riscoe, Michael K.; Doggett, J. Stone

2016-01-01

Human babesiosis is a tick-borne multisystem disease caused by Babesia species of the apicomplexan phylum. Most clinical cases and fatalities of babesiosis are caused by Babesia microti. Current treatment for human babesiosis consists of two drug combinations, atovaquone + azithromycin or quinine + clindamycin. These treatments are associated with adverse side effects and a significant rate of drug failure. Here, we provide evidence for radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone (ELQ) prodrug and atovaquone. In vivo efficacy studies in mice using ELQ-271, ELQ-316, and the ELQ-316 prodrug, ELQ-334, demonstrated excellent growth inhibitory activity against the parasite, with potency equal to that of orally administered atovaquone at 10 mg/kg. Analysis of recrudescent parasites after ELQ or atovaquone monotherapy identified genetic substitutions in the Qi or Qo sites, respectively, of the cytochrome bc1 complex. Impressively, a combination of ELQ-334 and atovaquone, at doses as low as 5.0 mg/kg each, resulted in complete clearance of the parasite with no recrudescence up to 122 d after discontinuation of therapy. These results will set the stage for future clinical evaluation of ELQ and atovaquone combination therapy for treatment of human babesiosis. PMID:27270894

CMS-G from Beta vulgaris ssp. maritima is maintained in natural populations despite containing an atypical cytochrome c oxidase.

PubMed

Meyer, Etienne H; Lehmann, Caroline; Boivin, Stéphane; Brings, Lea; De Cauwer, Isabelle; Bock, Ralph; Kühn, Kristina; Touzet, Pascal

2018-02-23

While mitochondrial mutants of the respiratory machinery are rare and often lethal, cytoplasmic male sterility (CMS), a mitochondrially inherited trait that results in pollen abortion, is frequently encountered in wild populations. It generates a breeding system called gynodioecy. In Beta vulgaris ssp. maritima , a gynodioecious species, we found CMS-G to be widespread across the distribution range of the species. Despite the sequencing of the mitochondrial genome of CMS-G, the mitochondrial sterilizing factor causing CMS-G is still unknown. By characterizing biochemically CMS-G, we found that the expression of several mitochondrial proteins is altered in CMS-G plants. In particular, Cox1, a core subunit of the cytochrome c oxidase (complex IV), is larger but can still assemble into complex IV. However, the CMS-G-specific complex IV was only detected as a stabilized dimer. We did not observe any alteration of the affinity of complex IV for cytochrome c ; however, in CMS-G, complex IV capacity is reduced. Our results show that CMS-G is maintained in many natural populations despite being associated with an atypical complex IV. We suggest that the modified complex IV could incur the associated cost predicted by theoretical models to maintain gynodioecy in wild populations. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Functionalized poly(3-hydroxybutyric acid) bodies as new in vitro biocatalysts.

PubMed

Stenger, Benjamin; Gerber, Adrian; Bernhardt, Rita; Hannemann, Frank

2018-01-01

Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

SEASONAL HEPATIC CYTOCHROME P-450 INDUCTION IN COTTON RATS (SIGMODON HISPIDUS) INHABITING PETROCHEMICAL WASTE SITES. (R826242)

EPA Science Inventory

Abstract

Wildlife species inhabiting contaminated sites are often exposed to complex mixtures of chemicals that have known effects on physiological and biochemical function. We evaluated the induction of major hepatic cytochrome P-450 isoenzymes through O-dealky...

CROSS-SPECIES COMPARISON OF CONAZOLE FUNGICIDE METABOLITES USING RAT AND RAINBOW TROUT (ONCHORHYNCHUS MYKISS) HEPATIC MICROSOMES AND PURIFIED HUMAN CYTOCHROME P450 3A4

EPA Science Inventory

Conazoles represent a unique class of azole-containing fungicides that are widely used in both pharmaceutical and agriculture applications. The antifungal property of conazoles occurs via complexation with cytochrome P450 monooxygenases (CYP) responsible for mediating fungal cell...

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye‐Sensitized TiO2

PubMed Central

Ainsworth, Emma V.; Lockwood, Colin W. J.; White, Gaye F.; Hwang, Ee Taek; Sakai, Tsubasa; Gross, Manuela A.; Richardson, David J.; Clarke, Thomas A.

2016-01-01

Abstract The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar‐assisted chemical synthesis. In Shewanella oneidensis MR‐1 (MR‐1), trans‐outer‐membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer‐membrane‐spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR‐1. We show photoreduction of MtrC and OmcA adsorbed on RuII‐dye‐sensitized TiO2 nanoparticles and that these protein‐coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer‐membrane‐associated cytochromes of MR‐1 for solar‐driven microbial synthesis in natural and engineered bacteria. PMID:27685371

A hypothetical complex between crystalline flavocytochrome b2 and cytochrome c.

PubMed

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

1993-08-01

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

Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase)

PubMed Central

Miyadera, Hiroko; Shiomi, Kazuro; Ui, Hideaki; Yamaguchi, Yuichi; Masuma, Rokuro; Tomoda, Hiroshi; Miyoshi, Hideto; Osanai, Arihiro; Kita, Kiyoshi; Ōmura, Satoshi

2003-01-01

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues. PMID:12515859

Detection of intra-brain cytoplasmic 1 (BC1) long noncoding RNA using graphene oxide-fluorescence beacon detector.

PubMed

Kim, Mee Young; Hwang, Do Won; Li, Fangyuan; Choi, Yoori; Byun, Jung Woo; Kim, Dongho; Kim, Jee-Eun; Char, Kookheon; Lee, Dong Soo

2016-03-21

Detection of cellular expression of long noncoding RNAs (lncRNAs) was elusive due to the ambiguity of exposure of their reactive sequences associated with their secondary/tertiary structures and dynamic binding of proteins around lncRNAs. Herein, we developed graphene-based detection techniques exploiting the quenching capability of graphene oxide (GO) flakes for fluorescent dye (FAM)-labeled single-stranded siRNAs and consequent un-quenching by their detachment from GO by matching lncRNAs. A brain cytoplasmic 1 (BC1) lncRNA expression was significantly decreased by a siRNA, siBC1-1. GO quenched the FAM-labeled siBC1-1 peptide nucleic acid (PNA) probe, and this quenching was recovered by BC1. While FAM-siBC1-1-PNA-GO complex transfected spontaneously mouse or human neural stem cells, fluorescence was recovered only in mouse cells having high BC1 expression. Fluorescent dye-labeled single-stranded RNA-GO probe could detect the reactive exposed nucleic acid sequence of a cytoplasmic lncRNA expressing in the cytoplasm, which strategy can be used as a detection method of lncRNA expression.

Complex Landscape of Germline Variants in Brazilian Patients With Hereditary and Early Onset Breast Cancer.

PubMed

Torrezan, Giovana T; de Almeida, Fernanda G Dos Santos R; Figueiredo, Márcia C P; Barros, Bruna D de Figueiredo; de Paula, Cláudia A A; Valieris, Renan; de Souza, Jorge E S; Ramalho, Rodrigo F; da Silva, Felipe C C; Ferreira, Elisa N; de Nóbrega, Amanda F; Felicio, Paula S; Achatz, Maria I; de Souza, Sandro J; Palmero, Edenir I; Carraro, Dirce M

2018-01-01

Pathogenic variants in known breast cancer (BC) predisposing genes explain only about 30% of Hereditary Breast Cancer (HBC) cases, whereas the underlying genetic factors for most families remain unknown. Here, we used whole-exome sequencing (WES) to identify genetic variants associated to HBC in 17 patients of Brazil with familial BC and negative for causal variants in major BC risk genes ( BRCA1/2, TP53 , and CHEK2 c.1100delC). First, we searched for rare variants in 27 known HBC genes and identified two patients harboring truncating pathogenic variants in ATM and BARD1 . For the remaining 15 negative patients, we found a substantial vast number of rare genetic variants. Thus, for selecting the most promising variants we used functional-based variant prioritization, followed by NGS validation, analysis in a control group, cosegregation analysis in one family and comparison with previous WES studies, shrinking our list to 23 novel BC candidate genes, which were evaluated in an independent cohort of 42 high-risk BC patients. Rare and possibly damaging variants were identified in 12 candidate genes in this cohort, including variants in DNA repair genes ( ERCC1 and SXL4 ) and other cancer-related genes ( NOTCH2, ERBB2, MST1R , and RAF1 ). Overall, this is the first WES study applied for identifying novel genes associated to HBC in Brazilian patients, in which we provide a set of putative BC predisposing genes. We also underpin the value of using WES for assessing the complex landscape of HBC susceptibility, especially in less characterized populations.

Unique Flexibility in Energy Metabolism Allows Mycobacteria to Combat Starvation and Hypoxia

PubMed Central

Berney, Michael; Cook, Gregory M.

2010-01-01

Mycobacteria are a group of obligate aerobes that require oxygen for growth, but paradoxically have the ability to survive and metabolize under hypoxia. The mechanisms responsible for this metabolic plasticity are unknown. Here, we report on the adaptation of Mycobacterium smegmatis to slow growth rate and hypoxia using carbon-limited continuous culture. When M. smegmatis is switched from a 4.6 h to a 69 h doubling time at a constant oxygen saturation of 50%, the cells respond through the down regulation of respiratory chain components and the F1Fo-ATP synthase, consistent with the cells lower demand for energy at a reduced growth rate. This was paralleled by an up regulation of molecular machinery that allowed more efficient energy generation (i.e. Complex I) and the use of alternative electron donors (e.g. hydrogenases and primary dehydrogenases) to maintain the flow of reducing equivalents to the electron transport chain during conditions of severe energy limitation. A hydrogenase mutant showed a 40% reduction in growth yield highlighting the importance of this enzyme in adaptation to low energy supply. Slow growing cells at 50% oxygen saturation subjected to hypoxia (0.6% oxygen saturation) responded by switching on oxygen scavenging cytochrome bd, proton-translocating cytochrome bc1-aa3 supercomplex, another putative hydrogenase, and by substituting NAD+-dependent enzymes with ferredoxin-dependent enzymes thus highlighting a new pattern of mycobacterial adaptation to hypoxia. The expression of ferredoxins and a hydrogenase provides a potential conduit for disposing of and transferring electrons in the absence of exogenous electron acceptors. The use of ferredoxin-dependent enzymes would allow the cell to maintain a high carbon flux through its central carbon metabolism independent of the NAD+/NADH ratio. These data demonstrate the remarkable metabolic plasticity of the mycobacterial cell and provide a new framework for understanding their ability to survive under low energy conditions and hypoxia. PMID:20062806

Complement Activation in Relation to Capillary Leakage in Children with Septic Shock and Purpura

PubMed Central

Hazelzet, Jan A.; de Groot, Ronald; van Mierlo, Gerard; Joosten, Koen F. M.; van der Voort, Edwin; Eerenberg, Anke; Suur, Marja H.; Hop, Wim C. J.; Hack, C. Erik

1998-01-01

To assess the relationship between capillary leakage and inflammatory mediators during sepsis, blood samples were taken on hospital admission, as well as 24 and 72 h later, from 52 children (median age, 3.3 years) with severe meningococcal sepsis, of whom 38 survived and 14 died. Parameters related to cytokines (interleukin 6 [IL-6] IL-8, plasma phospholipase A2, and C-reactive protein [CRP]), to neutrophil degranulation (elastase and lactoferrin), to complement activation (C3a, C3b/c, C4b/c, and C3- and C4-CRP complexes), and to complement regulation (functional and inactivated C1 inhibitor and C4BP) were determined. The degree of capillary leakage was derived from the amount of plasma infused and the severity of disease by assessing the pediatric risk of mortality (PRISM) score. Levels of IL-6, IL-8, C3b/c, C3-CRP complexes, and C4BP on admission, adjusted for the duration of skin lesions, were significantly different in survivors and nonsurvivors (C3b/c levels were on average 2.2 times higher in nonsurvivors, and C3-CRP levels were 1.9 times higher in survivors). Mortality was independently related to the levels of C3b/c and C3-CRP complexes. In agreement with this, levels of complement activation products correlated well with the PRISM score or capillary leakage. Thus, these data show that complement activation in patients with severe meningococcal sepsis is associated with a poor outcome and a more severe disease course. Further studies should reveal whether complement activation may be a target for therapeutical intervention in this disease. PMID:9784543

Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm.

PubMed

Cervelli, Manuela; Bellavia, Gabriella; Fratini, Emiliano; Amendola, Roberto; Polticelli, Fabio; Barba, Marco; Federico, Rodolfo; Signore, Fabrizio; Gucciardo, Giacomo; Grillo, Rosalba; Woster, Patrick M; Casero, Robert A; Mariottini, Paolo

2010-10-14

Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

PubMed Central

2010-01-01

Background Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. Methods BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. Results Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. Conclusions This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials. PMID:20946629

Apolipoprotein E4 (1-272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells.

PubMed

Nakamura, Toshiyuki; Watanabe, Atsushi; Fujino, Takahiro; Hosono, Takashi; Michikawa, Makoto

2009-08-20

Apolipoprotein E allele epsilon4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1-272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1-272) more strongly than intact apoE4(1-299). Further analysis showed that in Neuro-2a cells expressing apoE4(1-272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1-299). ApoE4(1-272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1-272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

PubMed Central

Moreno-Beltrán, Blas; Guerra-Castellano, Alejandra; Del Conte, Rebecca; García-Mauriño, Sofía M.; Díaz-Moreno, Sofía; González-Arzola, Katiuska; Santos-Ocaña, Carlos; Velázquez-Campoy, Adrián; De la Rosa, Miguel A.; Turano, Paola; Díaz-Moreno, Irene

2017-01-01

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects. PMID:28348229

Black Carbon and Precipitation: An Energetics Perspective

NASA Astrophysics Data System (ADS)

Sand, M.; Samset, B. H.; Stjern, C.; Tsigaridis, K.; Myhre, G.

2017-12-01

Airborne Black Carbon (BC) can affect precipitation rates, both globally and regionally, through a number of mechanisms. Many studies have investigated the impact of the direct radiative effect, indirect modification of cloud properties and rapid adjustments (the semidirect effect), individually or in combination, but the net climate impacts of anthropogenic and natural BC are still highly uncertain. A particular problem is the complex behavior of BC-climate interactions with altitude. Since the atmospheric residence time, ageing and removal processes for BC are also poorly known, differences in vertical BC concentration profiles between models and intercomparison experiments greatly complicate the picture. Recently, precipitation changes predicted by climate models have been studied in the framework of changes to the global and regional energy balance. Here, we employ such an energetics perspective to simulations of BC inserted at isolated altitudes, in two major climate models (NCAR CESM1, NASA GISS). We show the resulting regional and global changes to precipitation, and analyze it in both in terms of individual components of radiative forcing, and the atmospheric energy balance. The results are presented in the context of recent literature.

Electron transfer between cytochrome. alpha. and copper A in cytochrome c oxidase: A perturbed equilibrium study

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

Morgan, J.E.; Li, P.M.; Jang, D.J.

1989-08-22

Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by the perturbed equilibrium method. The authors have prepared a three-electron-reduced, CO-inhibited form of the enzyme in which cytochrome a and copper A are partially reduced and in an intramolecular redox equilibrium. When these samples were irradiated with a nitrogen laser to photodissociate the bound CO, changes in absorbance at 598 and 830 nm were observed which were consistent with a fast electron transfer from cytochrome a to copper A. The absorbance changes at 598 nm gave an apparent rate of 17,000 {plus minus} 2,000 s{sup {minus}1} (1more » {sigma}), at pH 7.0 and 25.5{degree}C. These changes were not observed in either the CO mixed-valence or the CO-inhibited fully reduced forms of the enzyme. The rate was fastest at about pH 8.0, falling off toward both lower and higher pHs. There was a small but clear temperature dependence. The process was also observed in the cytochrome c-cytochrome c oxidase high-affinity complex. The electron equilibration measured between cytochrome {alpha} and copper A is far faster than any rate measured or inferred previously for this process.« less

Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

PubMed Central

Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

1999-01-01

Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

Long-Range Superexchange in Electron Transport Proteins

NASA Astrophysics Data System (ADS)

Gruschus, James Michael

A new Hamiltonian model for the calculation of long-range electronic couplings in complex molecular systems is presented. These couplings make possible the electron transfers occurring at several critical steps in photosynthesis and respiration. The couplings studied are demonstrated to arise from a mechanism known as superexchange, where the electrons of the insulating medium are intimately involved in the delocalization of the donor wavefunction tail, allowing significant interaction with the acceptor at much greater separations than could be achieved were the medium absent. Superexchange phenomena in molecules of moderate complexity are first compared to couplings calculated with the model Hamiltonian, with very encouraging results. The method is then applied to several cytochrome c proteins where electron transfer has been measured between a zinc-substituted porphyrin and a ruthenium complex ligated to several sites at the protein surface. The calculated couplings are in unprecedented agreement with experiment. Novel, analytical derivatives of the superexchange coupling with respect to the orbital energies and interactions are then carried out on these proteins yielding the general, chemically relevant result that the entire three-dimensional zone between redox sites is important in mediating the superexchange coupling, in contrast to the prevailing assumption that the coupling can be characterized by a one-dimensional pathway consisting primarily of chains of bonded atoms. In addition, the derivatives provide the most comprehensive ever, atom-by -atom visualization of the superexchange process. Using AMBER molecular dynamics trajectories of the cytochrome c proteins, the effect of structural fluctuations on superexchange is examined. The calculated couplings show a substantial variability, a result contrary to the constant coupling implicit in most present-day transfer rate theory. Couplings are also calculated on surfaces enveloping several variants of cytochrome c, as well as plastocyanin, cytochrome b _5, and cytochrome c peroxidase. The surfaces reveal important clues as to which conformations of the electron transport protein complexes actually give rise to electron transfer, a subject of broad biological interest.

Isolation and Purification of Complex II from Proteus Mirabilis Strain ATCC 29245

PubMed Central

Shabbiri, Khadija; Ahmad, Waqar; Syed, Quratulain; Adnan, Ahmad

2010-01-01

A respiratory complex was isolated from plasma membrane of pathogenic Proteus mirabilis strain ATCC 29245. It was identified as complex II consisting of succinate:quinone oxidoreductase (EC 1.3.5.1) containing single heme b. The complex II was purified by ion-exchange chromatography and gel filtration. The molecular weight of purified complex was 116.5 kDa and it was composed of three subunits with molecular weights of 19 kDa, 29 kDa and 68.5 kDa. The complex II contained 9.5 nmoles of cytochrome b per mg protein. Heme staining indicated that the 19 kDa subunit was cytochrome b. Its reduced form showed absorptions peaks at 557.0, 524.8 and 424.4 nm. The α-band was shifted from 557.0 nm to 556.8 nm in pyridine ferrohemochrome spectrum. The succinate: quinone oxidoreductase activity was found to be high in this microorganism. PMID:24031557

Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Kojima, S; Kita, K

1997-01-01

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction. The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit. From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex. Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit. The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).

An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.

PubMed

Brixius-Anderko, Simone; Hannemann, Frank; Ringle, Michael; Khatri, Yogan; Bernhardt, Rita

2017-05-01

Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 μM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Catalysis by Methylamine Dehydrogenase and Electron Transfer to Amicyanin and Cytochrome C(551I) from Paracoccus Denitrificans.

NASA Astrophysics Data System (ADS)

Brooks, Harold Burns

1995-01-01

The quinoprotein methylamine dehydrogenase (MADH), a type I copper protein, amicyanin, and cytochrome c _{55li} form a physiologic ternary complex (Chen et al. (1994) Science 264, 86-90) in which electrons are transferred from tryptophan tryptophylquinone to copper to heme. The reduction of MADH by rm H_3- and rm D_3 -methylamine, the reoxidation of MADH by amicyanin, and the reduction of cytochrome c_{55li } by reduced amicyanin in the presence of MADH have been studied by stopped-flow spectroscopy. When rm CD_3NH_2 was used as a substrate for MADH a deuterium kinetic isotope effect of 17.2 was measured for the hydrogen abstraction step. The maximum deuterium kinetic isotope effect that was measured in steady-state kinetic experiments was 3.0. The temperature dependencies of the rate constants for the reaction of methylamine with MADH were also determined. An iminosemiquinone intermediate for the oxidation of substrate-reduced MADH by amicyanin was detected using stopped-flow spectroscopy, and the presence of the substrate derived nitrogen was confirmed by electron spin echo envelope modulation (ESEEM) spectroscopy. Marcus theory, which was used to analyze the electron transfer reaction between the dithionite-generated redox forms of MADH and amicyanin, gave values of 218 kJ rm mol^{ -1} (2.3 eV) for the reorganizational energy (lambda ) and 11.6 rm cm^{-1} for the coupling rm (H_{AB}). In contrast, the oxidation of substrate-reduced MADH by amicyanin was a gated electron transfer reaction with values for DeltaH* of 76 kJ rm mol^ {-1} and DeltaS* of -41 J rm mol^{ -1} ^circ K^ {-1}. These studies are consistent with the formation of transient unstable intermediates preceeding electron transfer between MADH and amicyanin. Preliminary investigations of the ternary complex of MADH, amicyanin, and cytochrome c_{55li } suggest two distinct cytochrome c _{55li} binding sites on amicyanin. This conclusion is supported by the biphasic nature of the stopped -flow trace, the inhibition of the rm k^ {fast}_{obs} by MADH, and the ionic strength dependence of the two phases. The slow phase had a rate of 3.1 rm s^ {-1} which is consistent with electron transfer between amicyanin and cytochrome c_ {55li} within the ternary complex. The fast phase does not exhibit saturation behavior, must have an electron transfer rate greater than 1000 rm s^{-1}, and likely involves a complex of amicyanin and cytochrome c_{55li } near the hydrophobic patch of amicyanin.

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

DOE PAGES

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

2014-09-24

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

IMAC fractionation in combination with LC-MS reveals H2B and NIF-1 peptides as potential bladder cancer biomarkers.

PubMed

Frantzi, Maria; Zoidakis, Jerome; Papadopoulos, Theofilos; Zürbig, Petra; Katafigiotis, Ioannis; Stravodimos, Konstantinos; Lazaris, Andreas; Giannopoulou, Ioanna; Ploumidis, Achilles; Mischak, Harald; Mullen, William; Vlahou, Antonia

2013-09-06

Improvement in bladder cancer (BC) management requires more effective diagnosis and prognosis of disease recurrence and progression. Urinary biomarkers attract special interest because of the noninvasive means of urine collection. Proteomic analysis of urine entails the adoption of a fractionation methodology to reduce sample complexity. In this study, we applied immobilized metal affinity chromatography in combination with high-resolution LC-MS/MS for the discovery of native urinary peptides potentially associated with BC aggressiveness. This approach was employed toward urine samples from patients with invasive BC, noninvasive BC, and benign urogenital diseases. A total of 1845 peptides were identified, corresponding to a total of 638 precursor proteins. Specific enrichment for proteins involved in nucleosome assembly and for zinc-finger transcription factors was observed. The differential expression of two candidate biomarkers, histone H2B and NIF-1 (zinc finger 335) in BC, was verified in independent sets of urine samples by ELISA and by immunohistochemical analysis of BC tissue. The results collectively support changes in the expression of both of these proteins with tumor progression, suggesting their potential role as markers for discriminating BC stages. In addition, the data indicate a possible involvement of NIF-1 in BC progression, likely as a suppressor and through interactions with Sox9 and HoxA1.

N-doping effectively enhances the adsorption capacity of biochar for heavy metal ions from aqueous solution.

PubMed

Yu, Wenchao; Lian, Fei; Cui, Guannan; Liu, Zhongqi

2018-02-01

N-doping was successfully employed to improve the adsorption capacity of biochar (BC) for Cu 2+ and Cd 2+ by direct annealing of crop straws in NH 3 . The surface N content of BC increased more than 20 times by N-doping; meanwhile the content of oxidized-N was gradually diminished but graphitic-N was formed and increased with increasing annealing temperature and duration time. After N-doping, a high graphitic-N percentage (46.4%) and S BET (418.7 m 2 /g) can be achieved for BC. As a result, the N-doped BC exhibited an excellent adsorption capacity for Cu 2+ (1.63 mmol g -1 ) and Cd 2+ (1.76 mmol g -1 ), which was up to 4.0 times higher than that of the original BC. Furthermore, the adsorption performance of the N-doped BC remained stable even at acidic conditions. A positive correlation can be found between adsorption capacity with the graphitic N content on BC surface. The surface chemistry of N-doped BC before and after the heavy metal ions adsorption was carefully examined by XPS and FTIR techniques, which indicated that the adsorption mechanisms mainly included cation-π bonding and complexation with graphitic-N and hydroxyl groups of carbon surfaces. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lactate oxidation coupled to energy production in mitochondria like particles from Setaria digitata, a filarial parasite.

PubMed

Sivan, V M; Raj, R K

1994-10-14

In the filarial parasite, Setaria digitata, the mitochondria like particles (MLP) show NAD reduction with sodium lactate. The MLP also reduces dye and ferricyanide with lactate. The ferricyanide reduction by lactate is found to be sensitive to the cytochrome o inhibitor orthohydroxy diphenyl (OHD) and complex I inhibitor rotenone, modulated by ADP (+) and ATP (-) and inhibited by pyruvate and oxaloacetate. MLP shows lactate oxidation sensitive to OHD, rotenone and sodium malonate. Thus, the lactate utilizing complex system, consisting of an NADH generating MLP bound lactate dehydrogenase and a lactate flavocytochrome reductase tightly linked to complex I and cytochrome o, produces ATP in functional association with fumarate reductase complex and other enzyme systems. Hence, this study provides new dimensions to the study of metabolism in filarial parasites.

Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy.

PubMed

Hallmann, Kerstin; Kudin, Alexei P; Zsurka, Gábor; Kornblum, Cornelia; Reimann, Jens; Stüve, Burkhard; Waltz, Stephan; Hattingen, Elke; Thiele, Holger; Nürnberg, Peter; Rüb, Cornelia; Voos, Wolfgang; Kopatz, Jens; Neumann, Harald; Kunz, Wolfram S

2016-02-01

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient's fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Approval summary for imatinib mesylate capsules in the treatment of chronic myelogenous leukemia.

PubMed

Cohen, Martin H; Williams, Grant; Johnson, John R; Duan, John; Gobburu, Jogarao; Rahman, Atiqur; Benson, Kimberly; Leighton, John; Kim, Sung K; Wood, Rebecca; Rothmann, Mark; Chen, Gang; U, Khin Maung; Staten, Ann M; Pazdur, Richard

2002-05-01

Chronic myelogenous leukemia (CML) results from the breakpoint cluster region-Abl fusion gene product, a tyrosine kinase involved in cell division and apoptosis. Imatinib, an orally administered inhibitor of the breakpoint cluster region-Abl tyrosine kinase, is capable of blocking proliferation and inducing apoptosis in CML cell lines. In this report, we describe the preclinical profile of imatinib and the data submitted in the New Drug Application that led to its marketing approval. Chemistry manufacturing and controls, animal toxicology, and biopharmaceutical data are described. Results of Phase I and Phase II clinical studies in patients with CML in blast crisis (CML-BC), in accelerated phase (CML-AP), and in chronic phase disease-resistant or intolerant to IFN-alpha (CML-CP) are summarized. The basis for marketing approval and postmarketing commitments by the pharmaceutical company are discussed. Toxicology studies in the rat, dog, and monkey show the hematological, renal, and hepatobiliary toxicity of imatinib. Pharmacokinetic studies in patients with CML demonstrate 98% imatinib bioavailability. The elimination half-lives of the parent drug and the major active metabolite, CGP74588, from plasma are approximately 18 and 40 h, respectively. Approximately 81% of the drug is eliminated in 7 days, 68% in the feces and 13% in the urine. Cytochrome P-450 3A4 is the main enzyme responsible for imatinib metabolism. Phase I and II clinical studies were conducted. The Phase I study, in 83 CML patients, evaluated oral imatinib doses from 25 to 1000 mg/day. Dose-limiting toxicity was not observed. The three Phase II studies, in CML-CP, CML-AP, and CML-BC, enrolled 1027 patients. CML-CP patients received 400 mg/day imatinib, whereas CML-AP and CML-BC patients generally received 600 mg/day imatinib. Primary study endpoints were cytogenetic response rate (CML-CP) and hematological response rate (CML-AP and CML-BC). The cytogenetic response rate for CML-CP patients was 49%. The hematological response rate of CML-AP and CML-BC patients was 63 and 26%, respectively. The most common imatinib adverse events were nausea, vomiting, myalgia, edema, and diarrhea. Elevated liver enzymes and/or bilirubin were reported in 27 patients (2.6%). On May 10, 2001, imatinib mesylate (Gleevec, formerly known as STI-571 and Glivec), manufactured and distributed by Novartis Pharmaceuticals, East Hanover, NJ, was approved by the United States Food and Drug Administration for the treatment of CML in three clinical settings: CML-BC, CML-AP, and CML-CP. This report summarizes the Food and Drug Administration's review of the New Drug Application.

Arsenic removal by Japanese oak wood biochar in aqueous solutions and well water: Investigating arsenic fate using integrated spectroscopic and microscopic techniques.

PubMed

Niazi, Nabeel Khan; Bibi, Irshad; Shahid, Muhammad; Ok, Yong Sik; Shaheen, Sabry M; Rinklebe, Jörg; Wang, Hailong; Murtaza, Behzad; Islam, Ejazul; Farrakh Nawaz, M; Lüttge, Andreas

2018-04-15

In this study, we examined the sorption of arsenite (As(III)) and arsenate (As(V)) to Japanese oak wood-derived biochar (OW-BC) in aqueous solutions, and determined its efficiency to remove As from As-contaminated well water. Results revealed that, among the four sorption isotherm models, Langmuir model showed the best fit to describe As(III) and As(V) sorption on OW-BC, with slightly greater sorption affinity for As(V) compared to As(III) (Q L =3.89 and 3.16mgg -1 ; R 2 =0.91 and 0.85, respectively). Sorption edge experiments indicated that the maximum As removal was 81% and 84% for As(III)- and As(V)-OW-BC systems at pH7 and 6, respectively, which decreased above these pH values (76-69% and 80-58%). Surface functional groups, notably OH, COOH, CO, CH 3 , were involved in As sequestration by OW-BC, suggesting the surface complexation/precipitation and/or electrostatic interaction of As on OW-BC surface. Arsenic K-edge X-ray absorption near edge structure (XANES) spectroscopy indicated that 36% of the added As(III) was partially oxidized to As(V) in the As(III) sorption experiment, and in As(V) sorption experiment, 48% of As(V) was, albeit incompletely, reduced to As(III) on OW-BC surface. Application of OW-BC to As-contaminated well water (As: 27-144μgL -1 ; n=10) displayed that 92 to 100% of As was depleted despite in the presence of co-occurring competing anions (e.g., SO 4 2- , CO 3 2- , PO 4 3- ). This study shows that OW-BC has a great potential to remove As from solution and drinking (well) water. Overall, the combination of macroscopic sorption data and integrated spectroscopic and microscopic techniques highlight that the fate of As on biochar involves complex redox transformation and association with surface functional moieties in aquatic systems, thereby providing crucial information required for implication of biochar in environmental remediation programs. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of sedentary communities in the Maya lowlands: coexisting mobile groups and public ceremonies at Ceibal, Guatemala.

PubMed

Inomata, Takeshi; MacLellan, Jessica; Triadan, Daniela; Munson, Jessica; Burham, Melissa; Aoyama, Kazuo; Nasu, Hiroo; Pinzón, Flory; Yonenobu, Hitoshi

2015-04-07

Our archaeological investigations at Ceibal, a lowland Maya site located in the Pasión region, documented that a formal ceremonial complex was built around 950 B.C. at the onset of the Middle Preclassic period, when ceramics began to be used in the Maya lowlands. Our refined chronology allowed us to trace the subsequent social changes in a resolution that had not been possible before. Many residents of Ceibal appear to have remained relatively mobile during the following centuries, living in ephemeral post-in-ground structures and frequently changing their residential localities. In other parts of the Pasión region, there may have existed more mobile populations who maintained the traditional lifestyle of the preceramic period. Although the emerging elite of Ceibal began to live in a substantial residential complex by 700 B.C., advanced sedentism with durable residences rebuilt in the same locations and burials placed under house floors was not adopted in most residential areas until 500 B.C., and did not become common until 300 B.C. or the Late Preclassic period. During the Middle Preclassic period, substantial formal ceremonial complexes appear to have been built only at a small number of important communities in the Maya lowlands, and groups with different levels of sedentism probably gathered for their constructions and for public rituals held in them. These collaborative activities likely played a central role in socially integrating diverse groups with different lifestyles and, eventually, in developing fully established sedentary communities.

Kinetic Monte Carlo Simulations and Molecular Conductance Measurements of the Bacterial Decaheme Cytochrome MtrF

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

Byun, H. S.; Pirbadian, S.; Nakano, Aiichiro

2014-09-05

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, asmore » 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.« less

Catalytic reduction of O2 by cytochrome C using a synthetic model of cytochrome C oxidase.

PubMed

Collman, James P; Ghosh, Somdatta; Dey, Abhishek; Decréau, Richard A; Yang, Ying

2009-04-15

Cytochrome c oxidase (CcO) catalyzes the four-electron reduction of oxygen to water, the one-electron reductant Cytochrome c (Cytc) being the source of electrons. Recently we reported a functional model of CcO that electrochemically catalyzes the four-electron reduction of O(2) to H(2)O (Collman et al. Science 2007, 315, 1565). The current paper shows that the same functional CcO model catalyzes the four-electron reduction of O(2) using the actual biological reductant Cytc in a homogeneous solution. Both single and steady-state turnover kinetics studies indicate that O(2) binding is rate-determining and that O-O bond cleavage and electron transfer from reduced Cytc to the oxidized model complex are relatively fast.

The Expression Alteration of BC1 RNA and its Interaction with Eukaryotic Translation Initiation Factor eIF4A Post-Status Epilepticus.

PubMed

Zeng, Xiangchang; Zong, Wenjing; Gao, Qing; Chen, Siyu; Chen, Lulu; Zeng, Guirong; Huang, Weihua; Li, Zhenyu; Zeng, Chang; Xie, Yuanyuan; Li, Xiaohui; Xiao, Bo; Dongsheng-Ouyang; Hu, Kai

2018-05-17

Abnormal dendritic sprouting and synaptic remodelling are important pathological features of temporal lobe epilepsy. BC1 RNA is a translation repressor involved in the regulation of the dendritic protein synthesis and mRNA transport, which is essential for dendritic development and plasticity. The expression alteration of BC1 RNA in the pilocarpine induced epilepsy model remains unknown. It is unclear if the interactions between BC1 RNA and eukaryotic initiation factor 4A (eIF4A) exists in this model. The purpose of this study was to investigate the expression changes of BC1 RNA and its interactions with eIF4A post-status epilepticus (SE). Chloride lithium and pilocarpine were used to induce the SE rat model. Either a whole brain or hippocampus tissues were collected at different time points after SE. The expression patterns of BC1 was detected by qPCR and in situ hybridization. The levels of eIF4AI/II protein expression were analyzed via western blotting and immunohistochemistry. The BC1 RNA-eIF4AI/II interaction was determined by electrophoretic mobility shift assay (EMSA). We found that the BC1 RNA levels decreased in hippocampus 3d, 1w and 2w post-SE before the levels recovered. The eIF4AI/II began to rise 3d post-SE and reached the maximum level 1w post-SE. After 1w post-SE the levels decreased in the hippocampal CA1, CA3 and DG subregions. EMSA analysis showed that BC1 RNA specifically interacted with the eIF4AI/II. The BC1 RNA-eIF4AI/II complex reduced to the lowest level 1w post-SE. Our results suggested that BC1 has a negative regulatory correlation with eIF4AI/II, where BC1 RNA could be involved in epileptogenesis by regulating dendritic protein synthesis.

Joint measurements of black carbon and particle mass for heavy-duty diesel vehicles using a portable emission measurement system

NASA Astrophysics Data System (ADS)

Zheng, Xuan; Wu, Ye; Zhang, Shaojun; Baldauf, Richard W.; Zhang, K. Max; Hu, Jingnan; Li, Zhenhua; Fu, Lixin; Hao, Jiming

2016-09-01

The black carbon (BC) emitted from heavy-duty diesel vehicles (HDDVs) is an important source of urban atmospheric pollution and creates strong climate-forcing impacts. The emission ratio of BC to total particle mass (PM) (i.e., BC/PM ratio) is an essential variable used to estimate total BC emissions from historical PM data; however, these ratios have not been measured using portable emission measurement systems (PEMS) in order to obtain real-world measurements over a wide range of driving conditions. In this study, we developed a PEMS platform by integrating two Aethalometers and an electric low pressure impactor to realize the joint measurement of real-world BC and PM emissions for ten HDDVs in China. Test results showed that the average BC/PM ratio for five HDDVs equipped with mechanical fuel injection (MI) engines was 0.43 ± 0.06, significantly lower (P < 0.05) than another five HDDVs equipped with electronically-controlled fuel injection (EI) engines (0.56 ± 0.12). Traffic conditions also affected the BC/PM ratios with higher ratios on freeway routes than on local roads. Furthermore, higher ratios were observed for HDDVs equipped with EI engines than for the MI engines for the highway and local road routes. With an operating mode binning approach, we observed that the instantaneous BC/PM ratios of EI engine vehicles were above those of the MI engine vehicles in all operating modes except for the braking mode (i.e., Bin 0). Therefore, the complex impacts from engine technology and traffic conditions on BC/PM ratios should be carefully considered when estimating real-world BC emissions from HDDVs based on overall PM emissions data.

"Best Case/Worst Case": Training Surgeons to Use a Novel Communication Tool for High-Risk Acute Surgical Problems.

PubMed

Kruser, Jacqueline M; Taylor, Lauren J; Campbell, Toby C; Zelenski, Amy; Johnson, Sara K; Nabozny, Michael J; Steffens, Nicole M; Tucholka, Jennifer L; Kwekkeboom, Kris L; Schwarze, Margaret L

2017-04-01

Older adults often have surgery in the months preceding death, which can initiate postoperative treatments inconsistent with end-of-life values. "Best Case/Worst Case" (BC/WC) is a communication tool designed to promote goal-concordant care during discussions about high-risk surgery. The objective of this study was to evaluate a structured training program designed to teach surgeons how to use BC/WC. Twenty-five surgeons from one tertiary care hospital completed a two-hour training session followed by individual coaching. We audio-recorded surgeons using BC/WC with standardized patients and 20 hospitalized patients. Hospitalized patients and their families participated in an open-ended interview 30 to 120 days after enrollment. We used a checklist of 11 BC/WC elements to measure tool fidelity and surgeons completed the Practitioner Opinion Survey to measure acceptability of the tool. We used qualitative analysis to evaluate variability in tool content and to characterize patient and family perceptions of the tool. Surgeons completed a median of 10 of 11 BC/WC elements with both standardized and hospitalized patients (range 5-11). We found moderate variability in presentation of treatment options and description of outcomes. Three months after training, 79% of surgeons reported BC/WC is better than their usual approach and 71% endorsed active use of BC/WC in clinical practice. Patients and families found that BC/WC established expectations, provided clarity, and facilitated deliberation. Surgeons can learn to use BC/WC with older patients considering acute high-risk surgical interventions. Surgeons, patients, and family members endorse BC/WC as a strategy to support complex decision making. Copyright © 2017 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.

RNA recognition by a human antibody against brain cytoplasmic 200 RNA

PubMed Central

Jung, Euihan; Lee, Jungmin; Hong, Hyo Jeong; Park, Insoo; Lee, Younghoon

2014-01-01

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation. PMID:24759090

Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System.

PubMed

Chojnacka, Magdalena; Gornicka, Agnieszka; Oeljeklaus, Silke; Warscheid, Bettina; Chacinska, Agnieszka

2015-06-12

The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Subcellular fractionation by zonal centrifugation of glucose-repressed anaerobically grown Saccharomyces carlsbergensis

PubMed Central

Cartledge, T. G.; Lloyd, D.

1972-01-01

1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl2. 2. These homogenates contained no detectable cytochrome c oxidase, succinate–cytochrome c oxidoreductase, succinate–ferricyanide oxidoreductase, l(+)-lactate–cytochrome c oxidoreductase or catalase. Cytochromes a+a3 and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH– and NADPH–cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 105g-min left 80–90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S1) together with 65 and 56% of the NADH– and NADPH–cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S1 revealed the presence of a class of small particles containing NADPH–cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to `promitochondria' as described by other workers. ImagesPLATE 1PLATE 2PLATE 3 PMID:4405573

A New Method to Obtain the Black Carbon Mixing State of Biomass and Combustion Aerosols

NASA Astrophysics Data System (ADS)

Irwin, M.; Liu, D.; Joshi, R.; Allan, J. D.; Coe, H.; Flynn, M.; Olfert, J. S.; Broda, K.; Fu, P.; Sun, Y.; Ge, X.; Wang, J.

2017-12-01

Black carbon particles (BC) significantly contribute to warming effects in the atmosphere, altering weather systems, and pose significant health risks. These impacts are especially efficient at regional hotspots with high emissions of pollutants, such as in fast-developing megacities. These urban environments have the most population exposure, and improving the understanding of the sources and the processing of pollutants in these environments is critical in guiding policy making. Here we present the results of BC characterization in Beijing during the winter of 2016 (10th Nov-10th Dec), as part of a large joint UK-China field experiment. During this experiment, we successfully gathered 4 weeks of continuous measurements, including several severe pollution events in Beijing. MethodologyThe mixing state of BC, which is how BC is associated with non-BC material (its coating) within a particle, is crucial to determine its lifetime in the atmosphere and also its optical properties. However precisely quantifying the BC mixing state has posed a challenge, in part due to complex particle morphology. We have applied morphology-independent measurements of BC mixing state on a single-particle basis throughout this experiment: mono-dispersed particle mass (MP) is selected using a Centrifugal Particle Mass Analyser (CPMA, Cambustion Ltd) and a single particle soot photometer (SP2, DMT inc.) was used downstream of the CPMA to measure the refractory BC mass (MrBC). The full scan of CPMA masses (21 mass bins covering most of MP) are performed every half hour, following polydispersed particles measured without running CPMA.

Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1

PubMed Central

Alves, Mónica N.; Neto, Sónia E.; Alves, Alexandra S.; Fonseca, Bruno M.; Carrêlo, Afonso; Pacheco, Isabel; Paquete, Catarina M.; Soares, Cláudio M.; Louro, Ricardo O.

2015-01-01

The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB–OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC. PMID:26175726

Identification of Novel Mitochondrial Protein Components of Chlamydomonas reinhardtii. A Proteomic Approach1

PubMed Central

van Lis, Robert; Atteia, Ariane; Mendoza-Hernández, Guillermo; González-Halphen, Diego

2003-01-01

Pure mitochondria of the photosynthetic alga Chlamydomonas reinhardtii were analyzed using blue native-polyacrylamide gel electrophoresis (BN-PAGE). The major oxidative phosphorylation complexes were resolved: F1F0-ATP synthase, NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase. The oligomeric states of these complexes were determined. The F1F0-ATP synthase runs exclusively as a dimer, in contrast to the C. reinhardtii chloroplast enzyme, which is present as a monomer and subcomplexes. The sequence of a 60-kD protein, associated with the mitochondrial ATP synthase and with no known counterpart in any other organism, is reported. This protein may be related to the strong dimeric character of the algal F1F0-ATP synthase. The oxidative phosphorylation complexes resolved by BN-PAGE were separated into their subunits by second dimension sodium dodecyl sulfate-PAGE. A number of polypeptides were identified mainly on the basis of their N-terminal sequence. Core I and II subunits of complex III were characterized, and their proteolytic activities were predicted. Also, the heterodimeric nature of COXIIA and COXIIB subunits in cytochrome c oxidase was demonstrated. Other mitochondrial proteins like the chaperone HSP60, the alternative oxidase, the aconitase, and the ADP/ATP carrier were identified. BN-PAGE was also used to approach the analysis of the major chloroplast protein complexes of C. reinhardtii. PMID:12746537

Electrostatic orientation of the electron-transfer complex between plastocyanin and cytochrome c.

PubMed

Roberts, V A; Freeman, H C; Olson, A J; Tainer, J A; Getzoff, E D

1991-07-15

To understand the specificity and efficiency of protein-protein interactions promoting electron transfer, we evaluated the role of electrostatic forces in precollision orientation by the development of two new methods, computer graphics alignment of protein electrostatic fields and a systematic orientational search of intermolecular electrostatic energies for two proteins at present separation distances. We applied these methods to the plastocyanin/cytochrome c interaction, which is faster than random collision, but too slow for study by molecular dynamics techniques. Significant electrostatic potentials were concentrated on one-fourth (969 A2) of the plastocyanin surface, with the greatest negative potential centered on the Tyr-83 hydroxyl within the acidic patch, and on one-eighth (632 A2) of the cytochrome c surface, with the greatest positive potential centered near the exposed heme edge. Coherent electrostatic fields occurred only over these regions, suggesting that local, rather than global, charge complementarity controls productive recognition. The three energetically favored families of pre-collision orientations all directed the positive region surrounding the heme edge of cytochrome c toward the acidic patch of plastocyanin but differed in heme plane orientation. Analysis of electrostatic fields, electrostatic energies of precollision orientations with 12 and 6 A separation distances, and surface topographies suggested that the favored orientations should converge to productive complexes promoting a single electron-transfer pathway from the cytochrome c heme edge to Tyr-83 of plastocyanin. Direct interactions of the exposed Cu ligand in plastocyanin with the cytochrome c heme edge are not unfavorable sterically or electrostatically but should occur no faster than randomly, indicating that this is not the primary pathway for electron transfer.

Transient binding of CO to Cu(B) in cytochrome c oxidase is dynamically linked to structural changes around a carboxyl group: a time-resolved step-scan Fourier transform infrared investigation.

PubMed Central

Heitbrink, Dirk; Sigurdson, Håkan; Bolwien, Carsten; Brzezinski, Peter; Heberle, Joachim

2002-01-01

The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the mid-infrared range (2300-950 cm(-1)). Difference spectra of the bovine complex were obtained at -20 degrees C with 5 micros time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu(B) and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK(a) of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme. PMID:11751290

Multi-Mode Binding of Cellobiohydrolase Cel7A from Trichoderma reesei to Cellulose

PubMed Central

Jalak, Jürgen; Väljamäe, Priit

2014-01-01

Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-β-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was exclusively active site mediated and was consistent with Langmuir's one binding site model with K d and A max values of 2.9 nM and 126 nmol/g BC, respectively. This is the strongest binding observed with non-complexed cellulases and apparently represents the productive binding of TrCel7A to cellulose chain ends on the hydrophobic face of BC microfibril. With increasing free TrCel7A concentrations the isotherm gradually deviated from the Langmuir's one binding site model. This was caused by the increasing contribution of lower affinity binding modes that included both active site mediated binding and non-productive binding with active site free from cellulose chain. The binding of TrCel7A to BC was found to be only partially reversible. Furthermore, the isotherm was dependent on the concentration of BC with more efficient binding observed at lower BC concentrations. The phenomenon can be ascribed to the BC concentration dependent aggregation of BC microfibrils with concomitant reduction of specific surface area. PMID:25265511

Exploring the potential of needle trap microextraction combined with chromatographic and statistical data to discriminate different types of cancer based on urinary volatomic biosignature.

PubMed

Porto-Figueira, Priscilla; Pereira, Jorge A M; Câmara, José S

2018-09-06

The worldwide high cancer incidence and mortality demands for more effective and specific diagnostic strategies. In this study, we evaluated the efficiency of an innovative methodology, Needle Trap Microextraction (NTME), combined with gas chromatography-mass spectrometry (GC-MS), for the establishment of the urinary volatomic biosignature from breast (BC), and colon (CC) cancer patients as well as healthy individuals (CTL). To achieve this, 40 mL of the headspace of acidified urine (4 mL, 20% NaCl, pH = 2), equilibrated at 50 °C during 40 min, were loaded through the DVB/Car1000/CarX sorbent inside the NTD, and subjected to a GC-MS analysis. This allowed the identification of 130 VOMs from different chemical families that were further processed using discriminant analysis through the partial least squares method (PLS-DA). Several pathways are over activated in cancer patients, being phenylalanine pathway in BC and limonene and pinene degradation pathway in CC the most relevant. Butanoate metabolism is also highly activated in both cancers, as well as tyrosine metabolism in a lesser extension. In BC the xenobiotics metabolism by cytochrome P450 and fatty acid biosynthesis are also differentially activated. Different clusters corresponding to the groups recruited allowed to define sets of volatile organic metabolites (VOMs fingerprints) that exhibit high classification rates, sensitivity and specificity in the discrimination of the selected cancers. As far as we are aware, this is the first time that NTME is used for isolation urinary volatile metabolites, being the obtained results very promising. Copyright © 2018 Elsevier B.V. All rights reserved.

Structural changes of cytochrome c(552) from Thermus thermophilus adsorbed on anionic and hydrophobic surfaces probed by FTIR and 2D-FTIR spectroscopy.

PubMed

Lecomte, S; Hilleriteau, C; Forgerit, J P; Revault, M; Baron, M H; Hildebrandt, P; Soulimane, T

2001-03-02

The structural changes of cytochrome c(552) bound to anionic and hydrophobic clay surfaces have been investigated by Fourier transform infrared spectroscopy. Binding to the anionic surface of montmorillonite is controlled by electrostatic interactions since addition of electrolyte (0.5 mol L(-1) KCl) causes desorption of more than 2/3 of the protein molecules. Electrostatic binding occurs through the back side of the protein (i.e., remote from the heme site) and is associated only with subtle changes of the secondary structure. In contrast, adsorption to the hydrophobic surface of talc leads to a decrease in alpha-helical structure by ca. 5% and an increase in beta-sheet structure by ca. 6%. These structural changes are attributed to a hydrophobic region on the front surface of cytochrome c(552) close to the partially exposed heme edge. This part on the protein surface is identified as the interaction domain for talc and most likely also serves for binding to the natural reaction partner, a ba(3)-oxidase. Fourier transform infrared spectra of cytochrome c(552) and the clay-cytochrome c(552) complexes have been measured as a function of time following dissolution and suspension in deuterated buffer, respectively. A two-dimensional correlation analysis was applied to these spectra to investigate the dynamics of the structural changes in the protein. For both complexes, adsorption and subsequent unfolding processes in the binding domains are faster than the time resolution of the spectroscopic experiments. Thus, the processes that could be monitored are refolding of peptide segments and side chain rearrangements following the adsorption-induced perturbation of the protein structure and the solvation of the adsorbed protein. In each case, side chain alterations of solvent-exposed tyrosine, aspartate, and glutamate residues were observed. For the cytochrome c(552)-talc complex, these changes are followed by a slow refolding of the peptide chain in the binding domain and, subsequently, a further H/D exchange of amide group protons.

Biochemical and structural characterization of CYP109A2, a vitamin D3 25-hydroxylase from Bacillus megaterium.

PubMed

Abdulmughni, Ammar; Jóźwik, Ilona K; Brill, Elisa; Hannemann, Frank; Thunnissen, Andy-Mark W H; Bernhardt, Rita

2017-11-01

Cytochrome P450 enzymes are increasingly investigated due to their potential application as biocatalysts with high regio- and/or stereo-selectivity and under mild conditions. Vitamin D 3 (VD 3 ) metabolites are of pharmaceutical importance and are applied for the treatment of VD 3 deficiency and other disorders. However, the chemical synthesis of VD 3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD 3 with high regio-selectivity. The in vitro conversion, using cytochrome P450 reductase (BmCPR) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis-Menten reaction kinetics. A whole-cell system in B. megaterium overexpressing CYP109A2 reached 76 ± 5% conversion after 24 h and allowed to identify the main product by NMR analysis as 25-hydroxylated VD 3 . Product yield amounted to 54.9 mg·L -1 ·day -1 , rendering the established whole-cell system as a highly promising biocatalytic route for the production of this valuable metabolite. The crystal structure of substrate-free CYP109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP109A2 uses a highly similar set of residues for VD 3 binding as the related VD 3 hydroxylases CYP109E1 from B. megaterium and CYP107BR1 (Vdh) from Pseudonocardia autotrophica. However, the folds and sequences of the BC loops in these three P450s are highly divergent, leading to differences in the shape and apolar/polar surface distribution of their active site pockets, which may account for the observed differences in substrate specificity and the regio-selectivity of VD 3 hydroxylation. The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 5OFQ (substrate-free CYP109A2). Cytochrome P450 monooxygenase CYP109A2, EC 1.14.14.1, UniProt ID: D5DF88, Ferredoxin, UniProt ID: D5DFQ0, cytochrome P450 reductase, EC 1.8.1.2, UniProt ID: D5DGX1. © 2017 Federation of European Biochemical Societies.

Apolipoprotein E4 (1–272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells

PubMed Central

Nakamura, Toshiyuki; Watanabe, Atsushi; Fujino, Takahiro; Hosono, Takashi; Michikawa, Makoto

2009-01-01

Background Apolipoprotein E allele ε4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1–272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. Results To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1–272) more strongly than intact apoE4(1–299). Further analysis showed that in Neuro-2a cells expressing apoE4(1–272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1–299). Conclusion ApoE4(1–272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1–272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration. PMID:19695092

CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

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

Villarreal, Seth A.; Pattanayek, Rekha; Williams, Dewight R.

The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. Themore » KaiB–KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein–protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.« less

Climatic Effects of Black Carbon Aerosols Over the Tibetan Plateau

NASA Astrophysics Data System (ADS)

He, Cenlin

Black carbon (BC), also known as soot, has been identified as the second most important anthropogenic emissions in terms of global climate forcing in the current atmosphere. Ample evidence has shown that BC deposition is an important driver of rapid snow melting and glacier retreat over the Tibetan Plateau, which holds the largest snow/ice mass outside polar regions. However, the climatic effects of BC over the Tibetan Plateau have not been thoroughly investigated in such a manner as to understand, quantify, and reduce large uncertainties in the estimate of radiative and hydrological effects. Thus, this Ph.D. study seeks to understand and improve key processes controlling BC life cycle in global and regional models and to quantify BC radiative effects over the Tibetan Plateau. First, the capability of a state-of-the-art global chemical transport model (CTM), GEOS-Chem, and the associated model uncertainties are systematically evaluated in simulating BC over the Tibetan Plateau, using in situ measurements of BC in surface air, BC in snow, and BC absorption optical depth. The effects of three key factors on the simulation are also delineated, including Asian anthropogenic emissions, BC aging process, and model resolution. Subsequently, a microphysics-based BC aging scheme that accounts for condensation, coagulation, and heterogeneous chemical oxidation processes is developed and examined in GEOS-Chem by comparing with aircraft measurements. Compared to the default aging scheme, the microphysical scheme reduces model-observation discrepancies by a factor of 3, particularly in the middle and upper troposphere. In addition, a theoretical BC aging-optics model is developed to account for three typical evolution stages, namely, freshly emitted aggregates, coated BC by soluble material, and BC particles undergoing further hygroscopic growth. The geometric-optics surface-wave (GOS) approach is employed to compute the BC single-scattering properties at each aging stage, which are subsequently compared with laboratory measurements. Results show large variations in BC optical properties caused by coating morphology and aging stages. Furthermore, a comprehensive intercomparison of the GOS approach, the superposition T-matrix method, and laboratory measurements is performed for optical properties of BC with complex structures during aging. Moreover, a new snow albedo model is developed for widely-observed close-packed snow grains internally mixed with BC. Results indicate that albedo simulations that account for snow close packing match closer to observations. Close packing enhances BC-induced snow albedo reduction and associated surface radiative forcing by up to 15% (20%) for fresh (old) snow, which suggests that BC-snow albedo forcing is underestimated in previous modeling studies without accounting for close packing. Finally, the snow albedo forcing and direct radiative forcing (DRF) of BC in the Tibetan Plateau are estimated using GEOS-Chem in conjunction with a stochastic snow model and a radiative transfer model. This, for the first time, accounts for realistic non-spherical snow grain shape and stochastic multiple inclusions of BC within snow in assessing BC-snow interactions. The annual mean BC snow albedo forcing is 2.9 W m-2 over snow-covered Plateau regions. BC-snow internal mixing increases the albedo forcing by 40-60% compared with external mixing, whereas Koch snowflakes reduce the forcing by 20-40% relative to spherical snow grains. BC DRF at the top of the atmosphere is 2.3 W m-2 with uncertainties of -70% - +85% in the Plateau. The BC forcings are further attributed to emissions from different regions.

Arsenic sorption by red mud-modified biochar produced from rice straw.

PubMed

Wu, Chuan; Huang, Liu; Xue, Sheng-Guo; Huang, Yu-Ying; Hartley, William; Cui, Meng-Qian; Wong, Ming-Hung

2017-08-01

Red mud-modified biochar (RM-BC) has been produced to be utilized as a novel adsorbent to remove As because it can effectively combine the beneficial features of red mud (rich metal oxide composition and porous structure) and biochar (large surface area and porous structure properties). SEM-EDS and XRD analyses demonstrated that red mud had loaded successfully on the surface of biochar. With the increasing of pH in solution, arsenate (As(V)) adsorption on RM-BC decreased while arsenite (As(III)) increased. Arsenate adsorption kinetics process on RM-BC fitted the pseudo-second-order model, while that of As(III) favored the Elovich model. All sorption isotherms produced superior fits with the Langmuir model. RM-BC exhibited improved As removal capabilities, with a maximum adsorption capacity (Q max ) for As(V) of 5923 μg g -1 , approximately ten times greater than that of the untreated BC (552.0 μg g -1 ). Furthermore, it has been indicated that the adsorption of As(V) on RM-BC may be strongly associated with iron oxides (hematite and magnetite) and aluminum oxides (gibbsite) by X-ray absorption near-edge spectroscopy (XANES), which was possibly because of surface complexation and electrostatic interactions. RM-BC may be used as a valuable adsorbent for removing As in the environment due to the waste materials being relatively abundant.

Membrane development in purple photosynthetic bacteria in response to alterations in light intensity and oxygen tension.

PubMed

Niederman, Robert A

2013-10-01

Studies on membrane development in purple bacteria during adaptation to alterations in light intensity and oxygen tension are reviewed. Anoxygenic phototrophic such as the purple α-proteobacterium Rhodobacter sphaeroides have served as simple, dynamic, and experimentally accessible model organisms for studies of the photosynthetic apparatus. A major landmark in photosynthesis research, which dramatically illustrates this point, was provided by the determination of the X-ray structure of the reaction center (RC) in Blastochloris viridis (Deisenhofer and Michel, EMBO J 8:2149-2170, 1989), once it was realized that this represented the general structure for the photosystem II RC present in all oxygenic phototrophs. This seminal advance, together with a considerable body of subsequent research on the light-harvesting (LH) and electron transfer components of the photosynthetic apparatus has provided a firm basis for the current understanding of how phototrophs acclimate to alterations in light intensity and quality. Oxygenic phototrophs adapt to these changes by extensive thylakoid membrane remodeling, which results in a dramatic supramolecular reordering to assure that an appropriate flow of quinone redox species occurs within the membrane bilayer for efficient and rapid electron transfer. Despite the high level of photosynthetic unit organization in Rba. sphaeroides as observed by atomic force microscopy (AFM), fluorescence induction/relaxation measurements have demonstrated that the addition of the peripheral LH2 antenna complex in cells adapting to low-intensity illumination results in a slowing of the rate of electron transfer turnover by the RC of up to an order of magnitude. This is ascribed to constraints in quinone redox species diffusion between the RC and cytochrome bc1 complexes arising from the increased packing density as the intracytoplasmic membrane (ICM) bilayer becomes crowded with LH2 rings. In addition to downshifts in light intensity as a paradigm for membrane development studies in Rba. sphaeroides, the lowering of oxygen tension in chemoheterotropically growing cells results in a gratuitous formation of the ICM by an extensive membrane biogenesis process. These membrane alterations in response to lowered illumination and oxygen levels in purple bacteria are under the control of a number of interrelated two-component regulatory circuits reviewed here, which act at the transcriptional level to regulate the formation of both the pigment and apoprotein components of the LH, RC, and respiratory complexes. We have performed a proteomic examination of the ICM development process in which membrane proteins have been identified that are temporally expressed both during adaptation to low light intensity and ICM formation at low aeration and are spatially localized in both growing and mature ICM regions. For these proteomic analyses, membrane growth initiation sites and mature ICM vesicles were isolated as respective upper-pigmented band (UPB) and chromatophore fractions and subjected to clear native electrophoresis for isolation of bands containing the LH2 and RC-LH1 core complexes. In chromatophores, increasing levels of LH2 polypeptides relative to those of the RC-LH1 complex were observed as ICM membrane development proceeded during light-intensity downshifts, along with a large array of other associated proteins including high spectral counts for the F1FO-ATP synthase subunits and the cytochrome bc1 complex, as well as RSP6124, a protein of unknown function, that was correlated with increasing LH2 spectral counts. In contrast, the UPB was enriched in cytoplasmic membrane (CM) markers, including electron transfer and transport proteins, as well as general membrane protein assembly factors confirming the origin of the UPB from both peripheral respiratory membrane and sites of active CM invagination that give rise to the ICM. The changes in ICM vesicles were correlated to AFM mapping results (Adams and Hunter, Biochim Biophys Acta 1817:1616-1627, 2012), in which the increasing LH2 levels were shown to form densely packed LH2-only domains, representing the light-responsive antenna complement formed under low illumination. The advances described here could never have been envisioned when the author was first introduced in the mid-1960s to the intricacies of the photosynthetic apparatus during a lecture delivered in a graduate Biochemistry course at the University of Illinois by Govindjee, to whom this volume is dedicated on the occasion of his 80th birthday.

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

PubMed

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

2015-08-11

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

Developing leadership in nurse managers: the British Columbia Nursing Leadership Institute.

PubMed

MacPhee, Maura; Bouthillette, France

2008-01-01

The British Columbia Nursing Administrative Leadership Institute for First Line Nurse Leaders (BC NLI) is a collaborative partnership among British Columbia's Chief Nursing Officers, the Ministry of Health Nursing Directorate and the University of British Columbia School of Nursing. This initiative consists of a four-day residential program and a year-long leadership project between BC NLI participants and their organizational mentors. The evidence-based curriculum covers universal leadership and management concepts, but it also addresses leadership issues of relevance to nurse leaders in today's complex healthcare environments. The BC NLI is part of a provincial health human resources endeavour to ensure sufficient nursing leaders - for now and in the future. This paper will discuss the development, implementation and evaluation of the BC NLI. Unique aspects of the program, such as its online networking component, will be described, and its role in nursing leadership research will be briefly examined.

EZH2 in Bladder Cancer, a Promising Therapeutic Target

PubMed Central

Martínez-Fernández, Mónica; Rubio, Carolina; Segovia, Cristina; López-Calderón, Fernando F.; Dueñas, Marta; Paramio, Jesús M.

2015-01-01

Bladder Cancer (BC) represents a current clinical and social challenge. The recent studies aimed to describe the genomic landscape of BC have underscored the relevance of epigenetic alterations in the pathogenesis of these tumors. Among the epigenetic alterations, histone modifications occupied a central role not only in cancer, but also in normal organism homeostasis and development. EZH2 (Enhancer of Zeste Homolog 2) belongs to the Polycomb repressive complex 2 as its catalytic subunit, which through the trimethylation of H3 (Histone 3) on K27 (Lysine 27), produces gene silencing. EZH2 is frequently overexpressed in multiple tumor types, including BC, and plays multiple roles besides the well-recognized histone mark generation. In this review, we summarize the present knowledge on the oncogenic roles of EZH2 and its potential use as a therapeutic target, with special emphasis on BC pathogenesis and management. PMID:26580594

Orientations of Iron-Sulfur Clusters FA and FB in the Homodimeric Type-I Photosynthetic Reaction Center of Heliobacterium modesticaldum.

PubMed

Kondo, Toru; Matsuoka, Masahiro; Azai, Chihiro; Itoh, Shigeru; Oh-Oka, Hirozo

2016-05-12

Orientations of the FA and FB iron-sulfur (FeS) clusters in a structure-unknown type-I homodimeric heriobacterial reaction center (hRC) were studied in oriented membranes of the thermophilic anaerobic photosynthetic bacterium Heliobacterium modesticaldum by electron paramagnetic resonance (EPR), and compared with those in heterodimeric photosystem I (PS I). The Rieske-type FeS center in the cytochrome b/c complex showed a well-oriented EPR signal. Illumination at 14 K induced an FB(-) signal with g-axes of gz = 2.066, gy = 1.937, and gx = 1.890, tilted at angles of 60°, 60°, and 45°, respectively, with respect to the membrane normal. Chemical reduction with dithionite produced an additional signal of FA(-), which magnetically interacted with FB(-), with gz = 2.046, gy = 1.942, and gx = 1.911 at 30°, 60°, and 90°, respectively. The angles and redox properties of FA(-) and FB(-) in hRC resemble those of FB(-) and FA(-), respectively, in PS I. Therefore, FA and FB in hRC, named after their g-value similarities, seem to be located like FB and FA, not like FA and FB, respectively, in PS I. The reducing side of hRC could resemble those in PS I, if the names of FA and FB are interchanged with each other.

Determination of Highly Sensitive Biological Cell Model Systems to Screen BPA-Related Health Hazards Using Pathway Studio.

PubMed

Ryu, Do-Yeal; Rahman, Md Saidur; Pang, Myung-Geol

2017-09-06

Bisphenol-A (BPA) is a ubiquitous endocrine-disrupting chemical. Recently, many issues have arisen surrounding the disease pathogenesis of BPA. Therefore, several studies have been conducted to investigate the proteomic biomarkers of BPA that are associated with disease processes. However, studies on identifying highly sensitive biological cell model systems in determining BPA health risk are lacking. Here, we determined suitable cell model systems and potential biomarkers for predicting BPA-mediated disease using the bioinformatics tool Pathway Studio. We compiled known BPA-mediated diseases in humans, which were categorized into five major types. Subsequently, we investigated the differentially expressed proteins following BPA exposure in several cell types, and analyzed the efficacy of altered proteins to investigate their associations with BPA-mediated diseases. Our results demonstrated that colon cancer cells (SW480), mammary gland, and Sertoli cells were highly sensitive biological model systems, because of the efficacy of predicting the majority of BPA-mediated diseases. We selected glucose-6-phosphate dehydrogenase (G6PD), cytochrome b-c1 complex subunit 1 (UQCRC1), and voltage-dependent anion-selective channel protein 2 (VDAC2) as highly sensitive biomarkers to predict BPA-mediated diseases. Furthermore, we summarized proteomic studies in spermatozoa following BPA exposure, which have recently been considered as another suitable cell type for predicting BPA-mediated diseases.

Determination of Highly Sensitive Biological Cell Model Systems to Screen BPA-Related Health Hazards Using Pathway Studio

PubMed Central

Ryu, Do-Yeal

2017-01-01

Bisphenol-A (BPA) is a ubiquitous endocrine-disrupting chemical. Recently, many issues have arisen surrounding the disease pathogenesis of BPA. Therefore, several studies have been conducted to investigate the proteomic biomarkers of BPA that are associated with disease processes. However, studies on identifying highly sensitive biological cell model systems in determining BPA health risk are lacking. Here, we determined suitable cell model systems and potential biomarkers for predicting BPA-mediated disease using the bioinformatics tool Pathway Studio. We compiled known BPA-mediated diseases in humans, which were categorized into five major types. Subsequently, we investigated the differentially expressed proteins following BPA exposure in several cell types, and analyzed the efficacy of altered proteins to investigate their associations with BPA-mediated diseases. Our results demonstrated that colon cancer cells (SW480), mammary gland, and Sertoli cells were highly sensitive biological model systems, because of the efficacy of predicting the majority of BPA-mediated diseases. We selected glucose-6-phosphate dehydrogenase (G6PD), cytochrome b-c1 complex subunit 1 (UQCRC1), and voltage-dependent anion-selective channel protein 2 (VDAC2) as highly sensitive biomarkers to predict BPA-mediated diseases. Furthermore, we summarized proteomic studies in spermatozoa following BPA exposure, which have recently been considered as another suitable cell type for predicting BPA-mediated diseases. PMID:28878155

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

PubMed

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

2014-10-28

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

Development of sedentary communities in the Maya lowlands: Coexisting mobile groups and public ceremonies at Ceibal, Guatemala

PubMed Central

Inomata, Takeshi; MacLellan, Jessica; Triadan, Daniela; Munson, Jessica; Burham, Melissa; Aoyama, Kazuo; Nasu, Hiroo; Pinzón, Flory; Yonenobu, Hitoshi

2015-01-01

Our archaeological investigations at Ceibal, a lowland Maya site located in the Pasión region, documented that a formal ceremonial complex was built around 950 B.C. at the onset of the Middle Preclassic period, when ceramics began to be used in the Maya lowlands. Our refined chronology allowed us to trace the subsequent social changes in a resolution that had not been possible before. Many residents of Ceibal appear to have remained relatively mobile during the following centuries, living in ephemeral post-in-ground structures and frequently changing their residential localities. In other parts of the Pasión region, there may have existed more mobile populations who maintained the traditional lifestyle of the preceramic period. Although the emerging elite of Ceibal began to live in a substantial residential complex by 700 B.C., advanced sedentism with durable residences rebuilt in the same locations and burials placed under house floors was not adopted in most residential areas until 500 B.C., and did not become common until 300 B.C. or the Late Preclassic period. During the Middle Preclassic period, substantial formal ceremonial complexes appear to have been built only at a small number of important communities in the Maya lowlands, and groups with different levels of sedentism probably gathered for their constructions and for public rituals held in them. These collaborative activities likely played a central role in socially integrating diverse groups with different lifestyles and, eventually, in developing fully established sedentary communities. PMID:25831523

Patterns and processes in the genetic differentiation of the Brachionus calyciflorus complex, a passively dispersing freshwater zooplankton.

PubMed

Xiang, Xian-ling; Xi, Yi-long; Wen, Xin-li; Zhang, Gen; Wang, Jin-xia; Hu, Ke

2011-05-01

Elucidating the evolutionary patterns and processes of extant species is an important objective of any research program that seeks to understand population divergence and, ultimately, speciation. The island-like nature and temporal fluctuation of limnetic habitats create opportunities for genetic differentiation in rotifers through space and time. To gain further understanding of spatio-temporal patterns of genetic differentiation in rotifers other than the well-studied Brachionus plicatilis complex in brackish water, a total of 318 nrDNA ITS sequences from the B. calyciflorus complex in freshwater were analysed using phylogenetic and phylogeographic methods. DNA taxonomy conducted by both the sequence divergence and the GMYC model suggested the occurrence of six potential cryptic species, supported also by reproductive isolation among the tested lineages. The significant genetic differentiation and non-significant correlation between geographic and genetic distances existed in the most abundant cryptic species, BcI-W and Bc-SW. The large proportion of genetic variability for cryptic species Bc-SW was due to differences between sampling localities within seasons, rather than between different seasons. Nested Clade Analysis suggested allopatric or past fragmentation, contiguous range expansion and long-distance colonization possibly coupled with subsequent fragmentation as the probable main forces shaping the present-day phylogeographic structure of the B. calyciflorus species complex. Copyright © 2011 Elsevier Inc. All rights reserved.

Ancient palace complex (300–100 BC) discovered in the Valley of Oaxaca, Mexico

PubMed Central

Redmond, Elsa M.; Spencer, Charles S.

2017-01-01

Recently completed excavations at the site of El Palenque in Mexico’s Valley of Oaxaca have recovered the well-preserved remains of a palace complex dated by associated radiocarbon samples and ceramics to the Late Formative period or Late Monte Albán I phase (300–100 BC), the period of archaic state emergence in the region. The El Palenque palace exhibits certain architectural and organizational features similar to the royal palaces of much later Mesoamerican states described by Colonial-period sources. The excavation data document a multifunctional palace complex covering a maximum estimated area of 2,790 m2 on the north side of the site’s plaza and consisting of both governmental and residential components. The data indicate that the palace complex was designed and built as a single construction. The palace complex at El Palenque is the oldest multifunctional palace excavated thus far in the Valley of Oaxaca. PMID:28348218

Direct effect of Taxol on free radical formation and mitochondrial permeability transition.

PubMed

Varbiro, G; Veres, B; Gallyas, F; Sumegi, B

2001-08-15

To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the microM range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (DeltaPsi) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 microM cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN(-), azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of DeltaPsi and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity.

Modeling of the Nitric Oxide Transport in the Human Lungs.

PubMed

Karamaoun, Cyril; Van Muylem, Alain; Haut, Benoît

2016-01-01

In the human lungs, nitric oxide (NO) acts as a bronchodilatator, by relaxing the bronchial smooth muscles and is closely linked to the inflammatory status of the lungs, owing to its antimicrobial activity. Furthermore, the molar fraction of NO in the exhaled air has been shown to be higher for asthmatic patients than for healthy patients. Multiple models have been developed in order to characterize the NO dynamics in the lungs, owing to their complex structure. Indeed, direct measurements in the lungs are difficult and, therefore, these models are valuable tools to interpret experimental data. In this work, a new model of the NO transport in the human lungs is proposed. It belongs to the family of the morphological models and is based on the morphometric model of Weibel (1963). When compared to models published previously, its main new features are the layered representation of the wall of the airways and the possibility to simulate the influence of bronchoconstriction (BC) and of the presence of mucus on the NO transport in lungs. The model is based on a geometrical description of the lungs, at rest and during a respiratory cycle, coupled with transport equations, written in the layers composing an airway wall and in the lumen of the airways. First, it is checked that the model is able to reproduce experimental information available in the literature. Second, the model is used to discuss some features of the NO transport in healthy and unhealthy lungs. The simulation results are analyzed, especially when BC has occurred in the lungs. For instance, it is shown that BC can have a significant influence on the NO transport in the tissues composing an airway wall. It is also shown that the relation between BC and the molar fraction of NO in the exhaled air is complex. Indeed, BC might lead to an increase or to a decrease of this molar fraction, depending on the extent of the BC and on the possible presence of mucus. This should be confirmed experimentally and might provide an interesting way to characterize the extent of BC in unhealthy patients.

L-pyroglutamic acid inhibits energy production and lipid synthesis in cerebral cortex of young rats in vitro.

PubMed

Silva, A R; Silva, C G; Ruschel, C; Helegda, C; Wyse, A T; Wannmacher, C M; Wajner, M; Dutra-Filho, C S

2001-12-01

In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and gamma-glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO2 production and lipid synthesis from [U-14C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO2 production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5-3.0 mM and cytochrome c oxidase activity by 22-30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases.

Photoprotection Conferred by Changes in Photosynthetic Protein Levels and Organization during Dehydration of a Homoiochlorophyllous Resurrection Plant1

PubMed Central

Charuvi, Dana; Nevo, Reinat; Shimoni, Eyal; Naveh, Leah; Zia, Ahmad; Adam, Zach; Farrant, Jill M.; Kirchhoff, Helmut; Reich, Ziv

2015-01-01

During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b6f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state. PMID:25713340

The folding energy landscape and free energy excitations of cytochrome c.

PubMed

Weinkam, Patrick; Zimmermann, Jörg; Romesberg, Floyd E; Wolynes, Peter G

2010-05-18

The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Because of the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case, following principles different from those used to describe the folding of proteins without cofactors. Recent investigations, however, demonstrate that common models describing folding for many proteins work well for cytochrome c when heme is explicitly introduced, generally providing results that agree with experimental observations. In this Account, we first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Because attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as "perfectly funneled". In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are denoted sources of "frustration", which cause specific transient intermediates to be populated. Native structure-based models do, however, include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold so that folding can proceed. The ability of native structure-based models to capture this kind of motion is partly responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe the sequence of folding events for cytochrome c well (as inferred from hydrogen-exchange experiments), thereby justifying their use as a starting point. At low pH, the experimentally observed folding sequence of cytochrome c deviates from that at pH 7 and from models with perfectly funneled energy landscapes. Here, alternate folding pathways are a result of "chemical frustration". This frustration arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH. Beginning with native structure-based terms, we construct more complex models by adding chemical frustration. These more complex models only modestly perturb the energy landscape, which remains, overall, well funneled. These perturbed models can accurately describe how alternative folding pathways are used at low pH. At alkaline pH, cytochrome c populates distinctly different structural ensembles. For instance, lysine residues are deprotonated and compete for the heme ligation site. The same models that can describe folding at low pH also predict well the structures and relative stabilities of intermediates populated at alkaline pH. The success of models based on funneled energy landscapes suggest that cytochrome c folding is driven primarily by native contacts. The presence of heme appears to add chemical complexity to the folding process, but it does not require fundamental modification of the general principles used to describe folding. Moreover, its added complexity provides a valuable means of probing the folding energy landscape in greater detail than is possible with simpler systems.

Tomato Fruit Chromoplasts Behave as Respiratory Bioenergetic Organelles during Ripening1[W][OPEN

PubMed Central

Renato, Marta; Pateraki, Irini; Boronat, Albert; Azcón-Bieto, Joaquín

2014-01-01

During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts. It was recently reported that tomato chromoplasts can synthesize ATP through a respiratory process called chromorespiration. Here we show that chromoplast oxygen consumption is stimulated by the electron donors NADH and NADPH and is sensitive to octyl gallate (Ogal), a plastidial terminal oxidase inhibitor. The ATP synthesis rate of isolated chromoplasts was dependent on the supply of NAD(P)H and was fully inhibited by Ogal. It was also inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting the involvement of a chemiosmotic gradient. In addition, ATP synthesis was sensitive to 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a cytochrome b6f complex inhibitor. The possible participation of this complex in chromorespiration was supported by the detection of one of its components (cytochrome f) in chromoplasts using immunoblot and immunocytochemical techniques. The observed increased expression of cytochrome c6 during ripening suggests that it could act as electron acceptor of the cytochrome b6f complex in chromorespiration. The effects of Ogal on respiration and ATP levels were also studied in tissue samples. Oxygen uptake of mature green fruit and leaf tissues was not affected by Ogal, but was inhibited increasingly in fruit pericarp throughout ripening (up to 26% in red fruit). Similarly, Ogal caused a significant decrease in ATP content of red fruit pericarp. The number of energized mitochondria, as determined by confocal microscopy, strongly decreased in fruit tissue during ripening. Therefore, the contribution of chromoplasts to total fruit respiration appears to increase in late ripening stages. PMID:25125503

Effects of humic acid and heavy metals on the sorption of polar and apolar organic pollutants onto biochars.

PubMed

Wang, Fei; Sun, Hongwen; Ren, Xinhao; Liu, Yarui; Zhu, Hongkai; Zhang, Peng; Ren, Chao

2017-12-01

The effects of humic acid (HA) and heavy metals (Cu 2+ and Ag + ) on the sorption of polar and apolar organic pollutants onto biochars that were produced at temperatures of 200 °C (BC200) and 700 °C (BC700) were studied. Due to the plentiful polar functional groups on BC200, cationic propranolol exhibited higher levels of sorption than naphthalene on BC200 while naphthalene and propranolol showed similar sorption capacities on BC700. HA changed the characteristics of biochars and generally inhibited the sorption of target organic pollutants on biochars; however, enhancement occurred in some cases depending on the pollutants involved and their concentrations, biochars used and the addition sequences and concentrations of HA. On BC200, HA modifications mainly influenced sorption by decreasing its polarity and increasing its aromaticity, while on BC700, the surface area and pore volume greatly decreased due to the pore-blocking effects of HA. Residue dissolved HA in solution may also contribute to sorption inhibition. Complexation between polar functional groups on BC200 and heavy metals slightly enhanced the sorption of neutral naphthalene and significantly enhanced that of anionic 4-nitro-1-naphtol, while limited the sorption of cationic propranolol. Heavy metals together with their associated water molecules decreased the sorption of target chemicals on BC700 via pore-filling or pore-mouth-covering. Inhibition of heavy metals for 4-nitro-1-naphthol was found to be the weakest due to the bridge effects of heavy metals between 4-nitro-1-naphtol and BC700. The higher polarizability of Ag + led to the increase of its sorption on biochars in the presence of organic aromatic pollutants. The results of the present study shed light on the sorption mechanisms of bi-solute systems and enable us to select suitable biochar sorbents when chemicals co-exist. Copyright © 2017. Published by Elsevier Ltd.

A new chemotherapy agent-free theranostic system composed of graphene oxide nano-complex and aptamers for treatment of cancer cells.

PubMed

Bahreyni, Amirhossein; Yazdian-Robati, Rezvan; Hashemitabar, Shirin; Ramezani, Mohammad; Ramezani, Pouria; Abnous, Khalil; Taghdisi, Seyed Mohammad

2017-06-30

The common cancer treatment strategies like chemotherapy and radiotherapy are nonspecific and can trigger severe side effects by damaging normal cells. So, targeted cancer therapies, such as apoptosis induction, have attracted great attention in recent years. In this project, two nano-complexes, MUC1 aptamer-NAS-24 aptamer-Graphene oxide (GO) and MUC1 aptamer-Cytochrome C aptamer-GO, were designed to induce cell programmed death in MDA-MB-231 and MCF-7 cells (breast cancer cell lines) and to verify the level of apoptosis in both cell lines. MUC1 aptamer was a molecular recognition probe that led the internalization of two nano-complexes into MDA-MB-231 and MCF-7 cells (MUC1 positive cells) but not into HepG2 cell (liver cancer cell line, MUC1 negative cells). The apoptosis induction relied on binding of NAS-24 aptamer to its target, vimentin, in MDA-MB-231 and MCF-7 (target cells) with different levels of vimentin content. The function of first nano-complex was confirmed by binding of FAM-labeled cytochrome C aptamer to its target (cytochrome C) which was released from mitochondria, based on the function of the first nano-complex. Fluorometric analysis and gel retardation assay proved the formation of nano-complexes. The results of flow cytometry and fluorescence microscopy indicated efficient apoptosis induction just in target cells (MDA-MB-231 and MCF-7 cells) but not in non-target cells (HepG2 cell). The results of MTT assay also confirmed cell death process. Overall, our results proved excellent targeted apoptosis in breast cancer cells by designed nano-complexes which can be applied as an efficient cancer therapy method. Copyright © 2017 Elsevier B.V. All rights reserved.

Applicability of bacterial cellulose as an alternative to paper points in endodontic treatment.

PubMed

Yoshino, Aya; Tabuchi, Mari; Uo, Motohiro; Tatsumi, Hiroto; Hideshima, Katsumi; Kondo, Seiji; Sekine, Joji

2013-04-01

Dental root canal treatment is required when dental caries progress to infection of the dental pulp. A major goal of this treatment is to provide complete decontamination of the dental root canal system. However, the morphology of dental root canal systems is complex, and many human dental roots have inaccessible areas. In addition, dental reinfection is fairly common. In conventional treatment, a cotton pellet and paper point made from plant cellulose is used to dry and sterilize the dental root canal. Such sterilization requires a treatment material with high absorbency to remove any residue, the ability to improve the efficacy of intracanal medication and high biocompatibility. Bacterial cellulose (BC) is produced by certain strains of bacteria. In this study, we developed BC in a pointed form and evaluated its applicability as a novel material for dental canal treatment with regard to solution absorption, expansion, tensile strength, drug release and biocompatibility. We found that BC has excellent material and biological characteristics compared with conventional materials, such as paper points (plant cellulose). BC showed noticeably higher absorption and expansion than paper points, and maintained a high tensile strength even when wet. The cumulative release of a model drug was significantly greater from BC than from paper points, and BC showed greater compatibility than paper points. Taken together, BC has great potential for use in dental root canal treatment. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Decoding the usefulness of non-coding RNAs as breast cancer markers.

PubMed

Amorim, Maria; Salta, Sofia; Henrique, Rui; Jerónimo, Carmen

2016-09-15

Although important advances in the management of breast cancer (BC) have been recently accomplished, it still constitutes the leading cause of cancer death in women worldwide. BC is a heterogeneous and complex disease, making clinical prediction of outcome a very challenging task. In recent years, gene expression profiling emerged as a tool to assist in clinical decision, enabling the identification of genetic signatures that better predict prognosis and response to therapy. Nevertheless, translation to routine practice has been limited by economical and technical reasons and, thus, novel biomarkers, especially those requiring non-invasive or minimally invasive collection procedures, while retaining high sensitivity and specificity might represent a significant development in this field. An increasing amount of evidence demonstrates that non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), are aberrantly expressed in several cancers, including BC. miRNAs are of particular interest as new, easily accessible, cost-effective and non-invasive tools for precise management of BC patients because they circulate in bodily fluids (e.g., serum and plasma) in a very stable manner, enabling BC assessment and monitoring through liquid biopsies. This review focus on how ncRNAs have the potential to answer present clinical needs in the personalized management of patients with BC and comprehensively describes the state of the art on the role of ncRNAs in the diagnosis, prognosis and prediction of response to therapy in BC.

Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation

PubMed Central

Meeks, Shannon L.; Healey, John F.; Parker, Ernest T.; Barrow, Rachel T.

2007-01-01

The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/Phe2200 phospholipid binding β-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 β-sandwich. MAbs from groups A, AB, and B inhibit the binding of fVIIIa to phospholipid membranes. Group BC was the most common group and displayed the highest specific fVIII inhibitor activities. MAbs in this group are type II inhibitors that inhibit the activation of fVIII by either thrombin or factor Xa and poorly inhibit the binding of fVIII to phospholipid membranes or von Willebrand factor (VWF). Group BC MAbs are epitopically and mechanistically distinct from the extensively studied group C MAb, ESH8. These results reveal the structural and functional complexity of the anti-C2 domain antibody response and indicate that interference with fVIII activation is a major attribute of the inhibitor landscape. PMID:17848617

Long and spatially variable Neolithic Demographic Transition in the North American Southwest

PubMed Central

Kohler, Timothy A.; Reese, Kelsey M.

2014-01-01

In many places of the world, a Neolithic Demographic Transition (NDT) is visible as a several-hundred-year period of increased birth rates coupled with stable mortality rates, resulting in dramatic population growth that is eventually curtailed by increased mortality. Similar processes can be reconstructed in particular detail for the North American Southwest, revealing an anomalously long and spatially variable NDT. Irrigation-dependent societies experienced relatively low birth rates but were quick to achieve a high degree of sociopolitical complexity, whereas societies dependent on dry or rainfed farming experienced higher birth rates but less initial sociopolitical complexity. Low birth rates after A.D. 1200 mark the beginning of the decline of the Hohokam. Overall in the Southwest, birth rates increased slowly from 1100 B.C. to A.D. 500, and remained at high levels with some fluctuation until decreasing rapidly beginning A.D. 1300. Life expectancy at 15 increased slowly from 900 B.C. to A.D. 700, and then increased rapidly for 200 y before fluctuating and then declining after A.D. 1400. Life expectancy at birth, on the other hand, generally declined from 1100 B.C. to A.D. 1100/1200, before rebounding. Farmers took two millennia (∼1100 B.C. to ∼A.D. 1000) to reach the carrying capacity of the agricultural niche in the Southwest. PMID:24982134

Atomic force microscopy studies of native photosynthetic membranes.

PubMed

Sturgis, James N; Tucker, Jaimey D; Olsen, John D; Hunter, C Neil; Niederman, Robert A

2009-05-05

In addition to providing the earliest surface images of a native photosynthetic membrane at submolecular resolution, examination of the intracytoplasmic membrane (ICM) of purple bacteria by atomic force microscopy (AFM) has revealed a wide diversity of species-dependent arrangements of closely packed light-harvesting (LH) antennae, capable of fulfilling the basic requirements for efficient collection, transmission, and trapping of radiant energy. A highly organized architecture was observed with fused preparations of the pseudocrystalline ICM of Blastochloris viridis, consiting of hexagonally packed monomeric reaction center light-harvesting 1 (RC-LH1) core complexes. Among strains which also form a peripheral LH2 antenna, images of ICM patches from Rhodobacter sphaeroides exhibited well-ordered, interconnected networks of dimeric RC-LH1 core complexes intercalated by rows of LH2, coexisting with LH2-only domains. Other peripheral antenna-containing species, notably Rhodospirillum photometricum and Rhodopseudomonas palustris, showed a less regular organization, with mixed regions of LH2 and RC-LH1 cores, intermingled with large, paracrystalline domains. The ATP synthase and cytochrome bc(1) complex were not observed in any of these topographs and are thought to be localized in the adjacent cytoplasmic membrane or in inaccessible ICM regions separated from the flat regions imaged by AFM. The AFM images have served as a basis for atomic-resolution modeling of the ICM vesicle surface, as well as forces driving segregation of photosynthetic complexes into distinct domains. Docking of atomic-resolution molecular structures into AFM topographs of Rsp. photometricum membranes generated precise in situ structural models of the core complex surrounded by LH2 rings and a region of tightly packed LH2 complexes. A similar approach has generated a model of the highly curved LH2-only membranes of Rba. sphaeroides which predicts that sufficient space exists between LH2 complexes for quinones to diffuse freely. Measurement of the intercomplex distances between adjacent LH2 rings of Phaeospirillum molischianum has permitted the first calculation of the separation of bacteriochlorophyll a molecules in the native ICM. A recent AFM analysis of the organization of green plant photosystem II (PSII) in grana thylakoids revealed the protruding oxygen-evolving complex, crowded together in parallel alignment at three distinct levels of stacked membranes over the lumenal surface. The results also confirmed that PSII-LHCII supercomplexes are displaced relative to one another in opposing grana membranes.

Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

PubMed

Szczesna-Skorupa, E; Chen, C D; Rogers, S; Kemper, B

1998-12-08

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

Multimeric Complexes among Ankyrin-Repeat and SOCS-box Protein 9 (ASB9), ElonginBC, and Cullin 5: Insights into the Structure and Assembly of ECS-type Cullin-RING E3 Ubiquitin Ligases

PubMed Central

2013-01-01

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC–Cullin–SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM–MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9–EloBC–Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM–MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems. PMID:23837592

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

PubMed

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

1994-06-01

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

Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c

PubMed Central

González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A.

2015-01-01

Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ’s histone chaperone activity. PMID:26216969

Different structure of the complexes of two cytochrome P-450 isozymes with acetanilide by 1H-NMR relaxation and spectrophotometry.

PubMed

Woldman YaYu; Weiner, L M; Lyakhovich, V V

1993-05-28

The functional and spectral characteristics of the interaction of acetanilide with phenobarbital- and methylcholanthrene- induced rat liver microsomes, as well as with corresponding major isozymes (cytochromes P-450b and P-450c) have been compared. The magnitude of the reverse 1st type binding spectra proved to be negatively correlated with the acetanilide oxidation on isozymes under study. The data on paramagnetic relaxation of acetanilide protons in the presence of P-450 have shown the structure of the enzyme-substrate complex to be different for two isozymes, acetanilide molecule being closer to Fe ion in the active site in the case of P-450c, which is active towards acetanilide oxidation. For the P-450c-acetanilide complex the group oxidized (phenyl) is the closest to Fe ion.

Biochar composites with nano zerovalent iron and eggshell powder for nitrate removal from aqueous solution with coexisting chloride ions.

PubMed

Ahmad, Munir; Ahmad, Mahtab; Usman, Adel R A; Al-Faraj, Abdullah S; Abduljabbar, Adel S; Al-Wabel, Mohammad I

2017-09-18

Biochar (BC) was produced from date palm tree leaves and its composites were prepared with nano zerovalent iron (nZVI-BC) and hen eggshell powder (EP-BC). The produced BC and its composites were characterized by SEM, XRD, BET, and FTIR for surface structural, mineralogical, and chemical groups and tested for their efficiency for nitrate removal from aqueous solutions in the presence and absence of chloride ions. The incidence of graphene and nano zerovalent iron (Fe 0 ) in the nZVI-BC composite was confirmed by XRD. The nZVI-BC composite possessed highest surface area (220.92 m 2  g -1 ), carbon (80.55%), nitrogen (3.78%), and hydrogen (11.09%) contents compared to other materials. Nitrate sorption data was fitted well to the Langmuir (R 2  = 0.93-0.98) and Freundlich (R 2  = 0.90-0.99) isotherms. The sorption kinetics was adequately explained by the pseudo-second-order, power function, and Elovich models. The nZVI-BC composite showed highest Langmuir predicted sorption capacity (148.10 mg g -1 ) followed by EP-BC composite (72.77 mg g -1 ). In addition to the high surface area, the higher nitrate removal capacity of nZVI-BC composite could be attributed to the combination of two processes, i.e., chemisorption (outer-sphere complexation) and reduction of nitrate to ammonia or nitrogen by Fe 0 . The appearance of Fe-O stretching and N-H bonds in post-sorption FTIR spectra of nZVI-BC composite suggested the occurrence of redox reaction and formation of Fe compound with N, such as ferric nitrate (Fe(NO 3 ) 3 ·9H 2 O). Coexistence of chloride ions negatively influenced the nitrate sorption. The decrease in nitrate sorption with increasing chloride ion concentration was observed, which could be due to the competition of free active sites on the sorbents between nitrate and chloride ions. The nZVI-BC composite exhibited higher nitrate removal efficiency compared to other materials even in the presence of highest concentration (100 mg L -1 ) of coexisting chloride ion.

Activation of EVI1 transcription by the LEF1/β-catenin complex with p53-alteration in myeloid blast crisis of chronic myeloid leukemia.

PubMed

Manachai, Nawin; Saito, Yusuke; Nakahata, Shingo; Bahirvani, Avinash Govind; Osato, Motomi; Morishita, Kazuhiro

2017-01-22

The presence of a BCR-ABL1 fusion gene is necessary for the pathogenesis of chronic myeloid leukemia (CML) through t(9;22)(q34;q11) translocation. Imatinib, an ABL tyrosine kinase inhibitor, is dramatically effective in CML patients; however, 30% of CML patients will need further treatment due to progression of CML to blast crisis (BC). Aberrant high expression of ecotropic viral integration site 1 (EVI1) is frequently observed in CML during myeloid-BC as a potent driver with a CML stem cell signature; however, the precise molecular mechanism of EVI1 transcriptional regulation during CML progression is poorly defined. Here, we demonstrate the transcriptional activity of EVI1 is dependent on activation of lymphoid enhancer-binding factor 1 (LEF1)/β-catenin complex by BCR-ABL with loss of p53 function during CML-BC. The activation of β-catenin is partly dependent on BCR-ABL expression through enhanced GSK3β phosphorylation, and EVI1 expression is directly enhanced by the LEF1/β-catenin complex bound to the EVI1 promoter region. Moreover, the loss of p53 expression is inversely correlated with high expression of EVI1 in CML leukemia cells with an aggressive phase of CML, and a portion of the activation mechanism of EVI1 expression is dependent on β-catenin activation through GSK3β phosphorylation by loss of p53. Therefore, we found that the EVI1 activation in CML-BC is dependent on LEF1/β-catenin activation by BCR-ABL expression with loss of p53 function, representing a novel selective therapeutic approach targeting myeloid blast crisis progression. Copyright © 2016 Elsevier Inc. All rights reserved.

A comparative DFT study on CO oxidation reaction over Si-doped BC2N nanosheet and nanotube

NASA Astrophysics Data System (ADS)

Nematollahi, Parisa; Neyts, Erik C.

2018-05-01

In this study, we performed density functional theory (DFT) calculations to investigate different reaction mechanisms of CO oxidation catalyzed by the Si atom embedded defective BC2N nanostructures as well as the analysis of the structural and electronic properties. The structures of all the complexes are optimized and characterized by frequency calculations at the M062X/6-31G∗ computational level. Also, The electronic structures and thermodynamic parameters of adsorbed CO and O2 molecules over Si-doped BC2N nanostructures are examined in detail. Moreover, to investigate the curvature effect on the CO oxidation reaction, all the adsorption and CO oxidation reactions on a finite-sized armchair (6,6) Si-BC2NNT are also studied. Our results indicate that there can be two possible pathways for the CO oxidation with O2 molecule: O2(g) + CO(g) → O2(ads) + CO(ads) → CO2(g) + O(ads) and O(ads) + CO(g) → CO2(g). The first reaction proceeds via the Langmuir-Hinshelwood (LH) mechanism while the second goes through the Eley-Rideal (ER) mechanism. On the other hand, by increasing the tube diameter, the energy barrier increases due to the strong adsorption energy of the O2 molecule which is related to its dissociation over the tube surface. Our calculations indicate that the two step energy barrier of the oxidation reaction over Si-BC2NNS is less than that over the Si-BC2NNT. Hence, Si-BC2NNS may serve as an efficient and highly activated substrate to CO oxidation rather than (4,4) Si-BC2NNT.

Effect of vitamin supplements on HIV shedding in breast milk.

PubMed

Villamor, Eduardo; Koulinska, Irene N; Aboud, Said; Murrin, Clare; Bosch, Ronald J; Manji, Karim P; Fawzi, Wafaie W

2010-10-01

Supplementation in lactating HIV-1-infected women with preformed vitamin A and β-carotene (VA/BC) increases the risk of mother-to-child transmission of HIV through breastfeeding. Identifying a biological mechanism to explain this unexpected finding would lend support to a causal effect. The aim of the study was to evaluate the effect of VA/BC or multivitamin (B complex, vitamin C, and vitamin E) supplementation of HIV-infected women on HIV shedding in breast milk during the first 2 y postpartum. We quantified viral (cell-free) and proviral (cell-associated) HIV loads in breast-milk samples collected ≤15 d after delivery and every 3 mo thereafter from 594 Tanzanian HIV-1-infected women who participated in a randomized trial. Women received 1 of the following 4 daily oral regimens in a 2 × 2 factorial fashion during pregnancy and throughout the first 2 y postpartum: multivitamin, VA/BC, multivitamin including VA/BC, or placebo. The proportion of breast-milk samples with detectable viral load was significantly higher in women who received VA/BC (51.3%) than in women who were not assigned to VA/BC (44.8%; P = 0.02). The effect was apparent ≥6 mo postpartum (relative risk: 1.34; 95% CI: 1.04, 1.73). No associations with proviral load were observed. The multivitamin had no effects. In observational analyses, β-carotene but not retinol breast-milk concentrations were significantly associated with an increased viral load in milk. VA/BC supplementation in lactating women increases the HIV load in breast milk. This finding contributes to explaining the adverse effect of VA/BC on mother-to-child transmission. β-Carotene appears to have an effect on breast-milk viral load, independent of preformed vitamin A. This trial was registered at clinicaltrials.gov as NCT00197756.

Structure and variation of the mitochondrial genome of fishes.

PubMed

Satoh, Takashi P; Miya, Masaki; Mabuchi, Kohji; Nishida, Mutsumi

2016-09-07

The mitochondrial (mt) genome has been used as an effective tool for phylogenetic and population genetic analyses in vertebrates. However, the structure and variability of the vertebrate mt genome are not well understood. A potential strategy for improving our understanding is to conduct a comprehensive comparative study of large mt genome data. The aim of this study was to characterize the structure and variability of the fish mt genome through comparative analysis of large datasets. An analysis of the secondary structure of proteins for 250 fish species (248 ray-finned and 2 cartilaginous fishes) illustrated that cytochrome c oxidase subunits (COI, COII, and COIII) and a cytochrome bc1 complex subunit (Cyt b) had substantial amino acid conservation. Among the four proteins, COI was the most conserved, as more than half of all amino acid sites were invariable among the 250 species. Our models identified 43 and 58 stems within 12S rRNA and 16S rRNA, respectively, with larger numbers than proposed previously for vertebrates. The models also identified 149 and 319 invariable sites in 12S rRNA and 16S rRNA, respectively, in all fishes. In particular, the present result verified that a region corresponding to the peptidyl transferase center in prokaryotic 23S rRNA, which is homologous to mt 16S rRNA, is also conserved in fish mt 16S rRNA. Concerning the gene order, we found 35 variations (in 32 families) that deviated from the common gene order in vertebrates. These gene rearrangements were mostly observed in the area spanning the ND5 gene to the control region as well as two tRNA gene cluster regions (IQM and WANCY regions). Although many of such gene rearrangements were unique to a specific taxon, some were shared polyphyletically between distantly related species. Through a large-scale comparative analysis of 250 fish species mt genomes, we elucidated various structural aspects of the fish mt genome and the encoded genes. The present results will be important for understanding functions of the mt genome and developing programs for nucleotide sequence analysis. This study demonstrated the significance of extensive comparisons for understanding the structure of the mt genome.

Bioceramic/poly (glycolic)-poly (lactic acid) composite induces mineralized barrier after direct capping of rat tooth pulp tissue.

PubMed

Gala-Garcia, Alfonso; Teixeira, Karina Imaculada Rosa; Wykrota, Francisco Henrique Lana; Sinisterra, Rubén Dario; Cortés, Maria Esperanza

2010-01-01

The aim of this study was to observe the histopathological pulp response following direct pulp capping of mechanically exposed teeth in rats with a composite of beta-tricalcium phosphate-hydroxyapatite bioceramic (BC) and poly (glycolic)-poly (lactic acid) (PLGA) material or a calcium hydroxide [Ca(OH)2] material, compared to BC alone and a negative control of water. Pulp of the maxillary molars was exposed, followed by capping with the experimental material. The pulpal tissue response was assessed post-operatively at 1, 7, 14 and 30 d, followed by histological analysis. The Ca(OH)2 group exhibited severe acute inflammatory cell infiltration at day 14. However after 30 d, a new hard tissue with macro porous obliteration of the pulp chamber and a characteristic necrotic area had appeared. BC and Ca(OH)2 capping were associated with moderate inflammation and dentinal bridge similar. Meanwhile, in the BC/PLGA composite group, there was moderate inflammatory infiltrate and formation of a dense and complete dentinal bridge. In conclusion, the BC/PLGA composite material showed a large zone of tertiary dentin, and effectively reorganized the dentin-pulp complex.

Supramolecular organization of cytochrome c oxidase- and alternative oxidase-dependent respiratory chains in the filamentous fungus Podospora anserina.

PubMed

Krause, Frank; Scheckhuber, Christian Q; Werner, Alexandra; Rexroth, Sascha; Reifschneider, Nicole H; Dencher, Norbert A; Osiewacz, Heinz D

2004-06-18

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Schägger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I(2) and I(2)III(2)), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

A novel, kinetically stable, catalytically active, all-ferric, nitrite-bound complex of Paracoccus pantotrophus cytochrome cd1.

PubMed Central

Allen, James W A; Higham, Christopher W; Zajicek, Richard S; Watmough, Nicholas J; Ferguson, Stuart J

2002-01-01

The oxidized form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase, as isolated, has bis-histidinyl co-ordination of the c haem and His/Tyr co-ordination of the d(1) haem. On reduction, the haem co-ordinations change to His/Met and His/vacant respectively. If the latter form of the enzyme is reoxidized, a conformer is generated in which the ferric c haem is His/Met co-ordinated; this can revert to the 'as isolated' state of the enzyme over approx. 20 min at room temperature. However, addition of nitrite to the enzyme after a cycle of reduction and reoxidation produces a kinetically stable, all-ferric complex with nitrite bound to the d(1) haem and His/Met co-ordination of the c haem. This complex is catalytically active with the physiological electron donor protein pseudoazurin. The effective dissociation constant for nitrite is 2 mM. Evidence is presented that d(1) haem is optimized to bind nitrite, as opposed to other anions that are commonly good ligands to ferric haem. The all-ferric nitrite bound state of the enzyme could not be generated stoichiometrically by mixing nitrite with the 'as isolated' conformer of cytochrome cd(1) without redox cycling. PMID:12086580

Preparation and characterization of a novel graphene/biochar composite for aqueous phenanthrene and mercury removal.

PubMed

Tang, Jingchun; Lv, Honghong; Gong, Yanyan; Huang, Yao

2015-11-01

A graphene/biochar composite (G/BC) was synthesized via slow pyrolysis of graphene (G) pretreated wheat straw, and tested for the sorption characteristics and mechanisms of representative aqueous contaminants (phenanthrene and mercury). Structure and morphology analysis showed that G was coated on the surface of biochar (BC) mainly through π-π interactions, resulting in a larger surface area, more functional groups, greater thermal stability, and higher removal efficiency of phenanthrene and mercury compared to BC. Pseudo second-order model adequately simulated sorption kinetics, and sorption isotherms of phenanthrene and mercury were simulated well by dual-mode and BET models, respectively. FTIR and SEM analysis suggested that partitioning and surface sorption were dominant mechanisms for phenanthrene sorption, and that surface complexation between mercury and C-O, CC, -OH, and OC-O functional groups was responsible for mercury removal. The results suggested that the G/BC composite is an efficient, economic, and environmentally friendly multifunctional adsorbent for environmental remediation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Probabilistic Geoacoustic Inversion in Complex Environments

DTIC Science & Technology

2015-09-30

Probabilistic Geoacoustic Inversion in Complex Environments Jan Dettmer School of Earth and Ocean Sciences, University of Victoria, Victoria BC...long-range inversion methods can fail to provide sufficient resolution. For proper quantitative examination of variability, parameter uncertainty must...project aims to advance probabilistic geoacoustic inversion methods for complex ocean environments for a range of geoacoustic data types. The work is

Relationship between Telomere Length, Genetic Traits and Environmental/Occupational Exposures in Bladder Cancer Risk by Structural Equation Modelling.

PubMed

Pavanello, Sofia; Carta, Angela; Mastrangelo, Giuseppe; Campisi, Manuela; Arici, Cecilia; Porru, Stefano

2017-12-21

Background : Telomere length (TL) maintenance plays an important role in bladder cancer (BC) and prognosis. However the manifold influence of everyday life exposures and genetic traits on leucocyte TL (LTL), is not fully elucidated. Methods : Within the framework of a hospital-based case ( n = 96)/control ( n = 94) study (all Caucasian males), we investigated the extent to which LTL and BC risk were modulated by genetic polymorphisms and environmental and occupational exposures. Data on lifetime smoking, alcohol and coffee drinking, dietary habits and occupational exposures, pointing to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs) were collected. Structural equation modelling (SEM) analysis appraised this complex relationships. Results : The SEM analysis indicates negative direct links ( p < 0.05) between LTL with age, DNA adducts, alcohol and NAT2, and positive ones with coffee, MPO and XRCC3; and between BC risk ( p < 0.01) with cigarettes, cumulative exposure to AAs and coffee, while are negative with LTL and age. There was evidence of indirect effects ( p < 0.05) on BC risk, probably via LTL reduction, by age and NAT2 (positive link), MPO and XRCC3 (negative link). Our study supports evidence that LTL attrition is a critical event in BC. The new finding that LTL erosion depends on some preventable everyday life exposures genetically modulated, opens new perspectives in BC prevention.

Relationship between Telomere Length, Genetic Traits and Environmental/Occupational Exposures in Bladder Cancer Risk by Structural Equation Modelling

PubMed Central

Pavanello, Sofia; Carta, Angela; Mastrangelo, Giuseppe; Campisi, Manuela; Porru, Stefano

2017-01-01

Background: Telomere length (TL) maintenance plays an important role in bladder cancer (BC) and prognosis. However the manifold influence of everyday life exposures and genetic traits on leucocyte TL (LTL), is not fully elucidated. Methods: Within the framework of a hospital-based case (n = 96)/control (n = 94) study (all Caucasian males), we investigated the extent to which LTL and BC risk were modulated by genetic polymorphisms and environmental and occupational exposures. Data on lifetime smoking, alcohol and coffee drinking, dietary habits and occupational exposures, pointing to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs) were collected. Structural equation modelling (SEM) analysis appraised this complex relationships. Results: The SEM analysis indicates negative direct links (p < 0.05) between LTL with age, DNA adducts, alcohol and NAT2, and positive ones with coffee, MPO and XRCC3; and between BC risk (p < 0.01) with cigarettes, cumulative exposure to AAs and coffee, while are negative with LTL and age. There was evidence of indirect effects (p < 0.05) on BC risk, probably via LTL reduction, by age and NAT2 (positive link), MPO and XRCC3 (negative link). Conclusions: Our study supports evidence that LTL attrition is a critical event in BC. The new finding that LTL erosion depends on some preventable everyday life exposures genetically modulated, opens new perspectives in BC prevention. PMID:29267235

Patient-Centered Care in Breast Cancer Genetic Clinics

PubMed Central

Brédart, Anne; Anota, Amélie; Kuboth, Violetta; Lareyre, Olivier; Cano, Alejandra; Stoppa-Lyonnet, Dominique; Schmutzler, Rita; Dolbeault, Sylvie

2018-01-01

With advances in breast cancer (BC) gene panel testing, risk counseling has become increasingly complex, potentially leading to unmet psychosocial needs. We assessed psychosocial needs and correlates in women initiating testing for high genetic BC risk in clinics in France and Germany, and compared these results with data from a literature review. Among the 442 counselees consecutively approached, 212 (83%) in France and 180 (97%) in Germany, mostly BC patients (81% and 92%, respectively), returned the ‘Psychosocial Assessment in Hereditary Cancer’ questionnaire. Based on the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) BC risk estimation model, the mean BC lifetime risk estimates were 19% and 18% in France and Germany, respectively. In both countries, the most prevalent needs clustered around the “living with cancer” and “children-related issues” domains. In multivariate analyses, a higher number of psychosocial needs were significantly associated with younger age (b = −0.05), higher anxiety (b = 0.78), and having children (b = 1.51), but not with country, educational level, marital status, depression, or loss of a family member due to hereditary cancer. These results are in line with the literature review data. However, this review identified only seven studies that quantitatively addressed psychosocial needs in the BC genetic counseling setting. Current data lack understandings of how cancer risk counseling affects psychosocial needs, and improves patient-centered care in that setting. PMID:29439543

Patient-Centered Care in Breast Cancer Genetic Clinics.

PubMed

Brédart, Anne; Anota, Amélie; Dick, Julia; Kuboth, Violetta; Lareyre, Olivier; De Pauw, Antoine; Cano, Alejandra; Stoppa-Lyonnet, Dominique; Schmutzler, Rita; Dolbeault, Sylvie; Kop, Jean-Luc

2018-02-12

With advances in breast cancer (BC) gene panel testing, risk counseling has become increasingly complex, potentially leading to unmet psychosocial needs. We assessed psychosocial needs and correlates in women initiating testing for high genetic BC risk in clinics in France and Germany, and compared these results with data from a literature review. Among the 442 counselees consecutively approached, 212 (83%) in France and 180 (97%) in Germany, mostly BC patients (81% and 92%, respectively), returned the 'Psychosocial Assessment in Hereditary Cancer' questionnaire. Based on the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) BC risk estimation model, the mean BC lifetime risk estimates were 19% and 18% in France and Germany, respectively. In both countries, the most prevalent needs clustered around the "living with cancer" and "children-related issues" domains. In multivariate analyses, a higher number of psychosocial needs were significantly associated with younger age (b = -0.05), higher anxiety (b = 0.78), and having children (b = 1.51), but not with country, educational level, marital status, depression, or loss of a family member due to hereditary cancer. These results are in line with the literature review data. However, this review identified only seven studies that quantitatively addressed psychosocial needs in the BC genetic counseling setting. Current data lack understandings of how cancer risk counseling affects psychosocial needs, and improves patient-centered care in that setting.

Cooperative Lewis pairs based on late transition metals: activation of small molecules by platinum(0) and B(C6 F5 )3.

PubMed

Forrest, Sebastian J K; Clifton, Jamie; Fey, Natalie; Pringle, Paul G; Sparkes, Hazel A; Wass, Duncan F

2015-02-09

A Lewis basic platinum(0)-CO complex supported by a diphosphine ligand and B(C6 F5 )3 act cooperatively, in a manner reminiscent of a frustrated Lewis pair, to activate small molecules such as hydrogen, CO2 , and ethene. This cooperative Lewis pair facilitates the coupling of CO and ethene in a new way. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermodynamics of interactions between mammalian cytochromes P450 and b5.

PubMed

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

2017-04-01

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

An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

PubMed

Shiota, Masaki; Yamazaki, Tomohiko; Yoshimatsu, Keiichi; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2016-12-01

Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor. Copyright © 2016 Elsevier B.V. All rights reserved.

Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site, Cys-XX-Cys-His*

PubMed Central

Babbitt, Shalon E.; San Francisco, Brian; Mendez, Deanna L.; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; Bretsnyder, Eric C.; Kranz, Robert G.

2014-01-01

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19. PMID:25170082

Tomato fruit chromoplasts behave as respiratory bioenergetic organelles during ripening.

PubMed

Renato, Marta; Pateraki, Irini; Boronat, Albert; Azcón-Bieto, Joaquín

2014-10-01

During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts. It was recently reported that tomato chromoplasts can synthesize ATP through a respiratory process called chromorespiration. Here we show that chromoplast oxygen consumption is stimulated by the electron donors NADH and NADPH and is sensitive to octyl gallate (Ogal), a plastidial terminal oxidase inhibitor. The ATP synthesis rate of isolated chromoplasts was dependent on the supply of NAD(P)H and was fully inhibited by Ogal. It was also inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting the involvement of a chemiosmotic gradient. In addition, ATP synthesis was sensitive to 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a cytochrome b6f complex inhibitor. The possible participation of this complex in chromorespiration was supported by the detection of one of its components (cytochrome f) in chromoplasts using immunoblot and immunocytochemical techniques. The observed increased expression of cytochrome c6 during ripening suggests that it could act as electron acceptor of the cytochrome b6f complex in chromorespiration. The effects of Ogal on respiration and ATP levels were also studied in tissue samples. Oxygen uptake of mature green fruit and leaf tissues was not affected by Ogal, but was inhibited increasingly in fruit pericarp throughout ripening (up to 26% in red fruit). Similarly, Ogal caused a significant decrease in ATP content of red fruit pericarp. The number of energized mitochondria, as determined by confocal microscopy, strongly decreased in fruit tissue during ripening. Therefore, the contribution of chromoplasts to total fruit respiration appears to increase in late ripening stages. © 2014 American Society of Plant Biologists. All Rights Reserved.

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

PubMed

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

2014-01-01

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

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

PubMed

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

1994-03-15

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

Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes

NASA Astrophysics Data System (ADS)

Kobayashi, Yasuhito; Motohashi, Yutaka; Miyazaki, Yoshifumi; Yatagai, Mitsuyoshi; Takano, Takehito

1991-12-01

Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive Leydig cells was found to undergo significant time-of-day variation with a peak time of 0000 hours (light phase from 0800 to 2000 hours). Injection of cadinenes (300 mg/kg per day intraperitoneally at 48 and 96 h before sacrifice) induced PB-P-450 isozyme but did not induce MC-P-448 isozyme. The induction of PB-P-450 isozyme by cadinenes was time dependent, and the early dark phase (2000 and 0000 hours) was most sensitive. These results suggest that temporal variation of cytochrome P-450 isozymes is one of the important physiological variations in detoxification and activation of various xenobiotics and chemicals in the testis.

Effects of Limited Aeration and of the ArcAB System on Intermediary Pyruvate Catabolism in Escherichia coli

PubMed Central

Alexeeva, Svetlana; de Kort, Bart; Sawers, Gary; Hellingwerf, Klaas J.; de Mattos, M. Joost Teixeira

2000-01-01

The capacity of Escherichia coli to adapt its catabolism to prevailing redox conditions resides mainly in three catabolic branch points involving (i) pyruvate formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHc), (ii) the exclusively fermentative enzymes and those of the Krebs cycle, and (iii) the alternative terminal cytochrome bd and cytochrome bo oxidases. A quantitative analysis of the relative catabolic fluxes through these pathways is presented for steady-state glucose-limited chemostat cultures with controlled oxygen availability ranging from full aerobiosis to complete anaerobiosis. Remarkably, PFL contributed significantly to the catabolic flux under microaerobic conditions and was found to be active simultaneously with PDHc and cytochrome bd oxidase-dependent respiration. The synthesis of PFL and cytochrome bd oxidase was found to be maximal in the lower microaerobic range but not in a ΔArcA mutant, and we conclude that the Arc system is more active with respect to regulation of these two positively regulated operons during microaerobiosis than during anaerobiosis. PMID:10940038

Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs.

PubMed

Novak, David; Mojovic, Milos; Pavicevic, Aleksandra; Zatloukalova, Martina; Hernychova, Lenka; Bartosik, Martin; Vacek, Jan

2018-02-01

Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electron-transfer properties and ability to regulate the processes involved in homeostasis or apoptosis. Here we report an electrochemical strategy for investigating the electroactivity of cyt c and its analogs with a disrupted heme moiety, i.e. apocytochrome c (acyt c) and porphyrin cytochrome c (pcyt c). The electrochemical data are supplemented with low-temperature and spin-probe electron paramagnetic resonance (EPR) spectroscopy. The main contribution of this report is a complex evaluation of cyt c reduction and oxidation at the level of surface-localized amino acid residues and the heme moiety in a single electrochemical scan. The electrochemical pattern of cyt c is substantially different to both analogs acyt c and pcyt c, which could be applicable in further studies on the redox properties and structural stability of cytochromes and other hemeproteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytochrome P450 drug interactions with statin therapy.

PubMed

Goh, Ivanna Xin Wei; How, Choon How; Tavintharan, Subramaniam

2013-03-01

Statins are commonly used in the treatment of hyperlipidaemia. Although the benefits of statins are well-documented, they have the potential to cause myopathy and rhabdomyolysis due to the complex interactions of drugs, comorbidities and genetics. The cytochrome P450 family consists of major enzymes involved in drug metabolism and bioactivation. This article aims to highlight drug interactions involving statins, as well as provide updated recommendations and approaches regarding the safe and appropriate use of statins in the primary care setting.

The effects of detergents DDM and beta-OG on the singlet excited state lifetime of the chlorophyll a in cytochrome b6f complex from spinach chloroplasts.

PubMed

Chen, XiaoBo; Zhao, XiaoHui; Zhang, JianPing; Li, LiangBi; Kuang, TingYun

2007-08-01

The singlet excited state lifetime of the chlorophyll a (Chl a) in cytochrome b(6)f (Cyt b(6)f) complex was reported to be shorter than that of free Chl a in methanol, but the value was different for Cyt b(6)f complexes from different sources ( approximately 200 and approximately 600 ps are the two measured results). The present study demonstrated that the singlet excited state lifetime is associated with the detergents n-dodecyl-beta-D-maltoside (DDM) and n-octyl-beta-D-glucopyranoside (beta-OG), but has nothing to do with the different sources of Cyt b(6)f complexes. Compared with the Cyt b(6)f dissolved in beta-OG, the Cyt b(6)f in DDM had a lower fluorescence yield, a lower photodegradation rate of Chl a, and a shorter lifetime of Chl a excited state. In short, the singlet excited state lifetime, approximately 200 ps, of the Chl a in Cyt b(6)f complex in DDM is closer to the true in vivo.

Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB

PubMed Central

2017-01-01

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure–activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis. PMID:29035551

Characterization of Mycobacterium tuberculosis Complex DNAs from Egyptian Mummies by Spoligotyping

PubMed Central

Zink, Albert R.; Sola, Christophe; Reischl, Udo; Grabner, Waltraud; Rastogi, Nalin; Wolf, Hans; Nerlich, Andreas G.

2003-01-01

Bone and soft tissue samples from 85 ancient Egyptian mummies were analyzed for the presence of ancient Mycobacterium tuberculosis complex DNA (aDNA) and further characterized by spoligotyping. The specimens were obtained from individuals from different tomb complexes in Thebes West, Upper Egypt, which were used for upper social class burials between the Middle Kingdom (since ca. 2050 BC) and the Late Period (until ca. 500 BC). A total of 25 samples provided a specific positive signal for the amplification of a 123-bp fragment of the repetitive element IS6110, indicating the presence of M. tuberculosis DNA. Further PCR-based tests for the identification of subspecies failed due to lack of specific amplification products in the historic tissue samples. Of these 25 positive specimens, 12 could be successfully characterized by spoligotyping. The spoligotyping signatures were compared to those in an international database. They all show either an M. tuberculosis or an M. africanum pattern, but none revealed an M. bovis-specific pattern. The results from a Middle Kingdom tomb (used exclusively between ca. 2050 and 1650 BC) suggest that these samples bear an M. africanum-type specific spoligotyping signature. The samples from later periods provided patterns typical for M. tuberculosis. This study clearly demonstrates that spoligotyping can be applied to historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type but, rather, suggest that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum. PMID:12517873

DNA synthesis involving a complexes form of DNA polymerase I in extracts of Escherichia coli.

PubMed Central

Hendler, R W; Pereira, M; Scharff, R

1975-01-01

DNA polymerase I (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) has been recovered as a complex of about 390,000 molecular weight. The complex displays an ATP-stimulated DNA-synthesizing activity that prefers native to heat-denatured DNA. Genetic evidence indicates that the recBC enzyme is associated with the polymerase in the complex. Preliminary evidence for complexes involving DNA polymerases II and III is also presented. PMID:1094453

Efficiency and mechanisms of Cd removal from aqueous solution by biochar derived from water hyacinth (Eichornia crassipes).

PubMed

Zhang, Feng; Wang, Xin; Yin, Daixia; Peng, Bo; Tan, Changyin; Liu, Yunguo; Tan, Xiaofei; Wu, Shixue

2015-04-15

This study investigated the efficiency and mechanisms of Cd removal by biochar pyrolyzed from water hyacinth (BC) at 250-550 °C. BC450 out-performed the other BCs at varying Cd concentrations and can remove nearly 100% Cd from aqueous solution within 1 h at initial Cd ≤ 50 mg l(-1). The process of Cd sorption by BC450 followed the pseudo-second order kinetics with the equilibrium being achieved after 24 h with initial Cd ranging from 100 to 500 mg l(-1). The maximum Cd sorption capacity of BC450 was estimated to be 70.3 mg g(-1) based on Langmuir model, which is prominent among a range of low-cost sorbents. Based on the balance analysis between cations released and Cd sorbed onto BC450 in combination with SEM-EDX and XPS data, ion-exchange followed by surface complexation is proposed as the dominant mechanism responsible for Cd immobilization by BC450. In parallel, XRD analysis also suggested the formation of insoluble Cd minerals (CdCO3, Cd3P2, Cd3(PO4)2 and K4CdCl6) from either (co)-precipitation or ion exchange. Results from this study highlighted that the conversion of water hyacinth into biochar is a promising method to achieve effective Cd immobilization and improved management of this highly problematic invasive species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genome-wide association study to identify common variants associated with brachial circumference: a meta-analysis of 14 cohorts.

PubMed

Boraska, Vesna; Day-Williams, Aaron; Franklin, Christopher S; Elliott, Katherine S; Panoutsopoulou, Kalliope; Tachmazidou, Ioanna; Albrecht, Eva; Bandinelli, Stefania; Beilin, Lawrence J; Bochud, Murielle; Cadby, Gemma; Ernst, Florian; Evans, David M; Hayward, Caroline; Hicks, Andrew A; Huffman, Jennifer; Huth, Cornelia; James, Alan L; Klopp, Norman; Kolcic, Ivana; Kutalik, Zoltán; Lawlor, Debbie A; Musk, Arthur W; Pehlic, Marina; Pennell, Craig E; Perry, John R B; Peters, Annette; Polasek, Ozren; St Pourcain, Beate; Ring, Susan M; Salvi, Erika; Schipf, Sabine; Staessen, Jan A; Teumer, Alexander; Timpson, Nicholas; Vitart, Veronique; Warrington, Nicole M; Yaghootkar, Hanieh; Zemunik, Tatijana; Zgaga, Lina; An, Ping; Anttila, Verneri; Borecki, Ingrid B; Holmen, Jostein; Ntalla, Ioanna; Palotie, Aarno; Pietiläinen, Kirsi H; Wedenoja, Juho; Winsvold, Bendik S; Dedoussis, George V; Kaprio, Jaakko; Province, Michael A; Zwart, John-Anker; Burnier, Michel; Campbell, Harry; Cusi, Daniele; Smith, George Davey; Frayling, Timothy M; Gieger, Christian; Palmer, Lyle J; Pramstaller, Peter P; Rudan, Igor; Völzke, Henry; Wichmann, H-Erich; Wright, Alan F; Zeggini, Eleftheria

2012-01-01

Brachial circumference (BC), also known as upper arm or mid arm circumference, can be used as an indicator of muscle mass and fat tissue, which are distributed differently in men and women. Analysis of anthropometric measures of peripheral fat distribution such as BC could help in understanding the complex pathophysiology behind overweight and obesity. The purpose of this study is to identify genetic variants associated with BC through a large-scale genome-wide association scan (GWAS) meta-analysis. We used fixed-effects meta-analysis to synthesise summary results across 14 GWAS discovery and 4 replication cohorts comprising overall 22,376 individuals (12,031 women and 10,345 men) of European ancestry. Individual analyses were carried out for men, women, and combined across sexes using linear regression and an additive genetic model: adjusted for age and adjusted for age and BMI. We prioritised signals for follow-up in two-stages. We did not detect any signals reaching genome-wide significance. The FTO rs9939609 SNP showed nominal evidence for association (p<0.05) in the age-adjusted strata for men and across both sexes. In this first GWAS meta-analysis for BC to date, we have not identified any genome-wide significant signals and do not observe robust association of previously established obesity loci with BC. Large-scale collaborations will be necessary to achieve higher power to detect loci underlying BC.

Genome-Wide Association Study to Identify Common Variants Associated with Brachial Circumference: A Meta-Analysis of 14 Cohorts

PubMed Central

Boraska, Vesna; Day-Williams, Aaron; Franklin, Christopher S.; Elliott, Katherine S.; Panoutsopoulou, Kalliope; Tachmazidou, Ioanna; Albrecht, Eva; Bandinelli, Stefania; Beilin, Lawrence J.; Bochud, Murielle; Cadby, Gemma; Ernst, Florian; Evans, David M.; Hayward, Caroline; Hicks, Andrew A.; Huffman, Jennifer; Huth, Cornelia; James, Alan L.; Klopp, Norman; Kolcic, Ivana; Kutalik, Zoltán; Lawlor, Debbie A.; Musk, Arthur W.; Pehlic, Marina; Pennell, Craig E.; Perry, John R. B.; Peters, Annette; Polasek, Ozren; Pourcain, Beate St; Ring, Susan M.; Salvi, Erika; Schipf, Sabine; Staessen, Jan A.; Teumer, Alexander; Timpson, Nicholas; Vitart, Veronique; Warrington, Nicole M.; Yaghootkar, Hanieh; Zemunik, Tatijana; Zgaga, Lina; An, Ping; Anttila, Verneri; Borecki, Ingrid B.; Holmen, Jostein; Ntalla, Ioanna; Palotie, Aarno; Pietiläinen, Kirsi H.; Wedenoja, Juho; Winsvold, Bendik S.; Dedoussis, George V.; Kaprio, Jaakko; Province, Michael A.; Zwart, John-Anker; Burnier, Michel; Campbell, Harry; Cusi, Daniele; Davey Smith, George; Frayling, Timothy M.; Gieger, Christian; Palmer, Lyle J.; Pramstaller, Peter P.; Rudan, Igor; Völzke, Henry; Wichmann, H. -Erich; Wright, Alan F.; Zeggini, Eleftheria

2012-01-01

Brachial circumference (BC), also known as upper arm or mid arm circumference, can be used as an indicator of muscle mass and fat tissue, which are distributed differently in men and women. Analysis of anthropometric measures of peripheral fat distribution such as BC could help in understanding the complex pathophysiology behind overweight and obesity. The purpose of this study is to identify genetic variants associated with BC through a large-scale genome-wide association scan (GWAS) meta-analysis. We used fixed-effects meta-analysis to synthesise summary results across 14 GWAS discovery and 4 replication cohorts comprising overall 22,376 individuals (12,031 women and 10,345 men) of European ancestry. Individual analyses were carried out for men, women, and combined across sexes using linear regression and an additive genetic model: adjusted for age and adjusted for age and BMI. We prioritised signals for follow-up in two-stages. We did not detect any signals reaching genome-wide significance. The FTO rs9939609 SNP showed nominal evidence for association (p<0.05) in the age-adjusted strata for men and across both sexes. In this first GWAS meta-analysis for BC to date, we have not identified any genome-wide significant signals and do not observe robust association of previously established obesity loci with BC. Large-scale collaborations will be necessary to achieve higher power to detect loci underlying BC. PMID:22479309

Drug-resistant pulmonary tuberculosis in the Baja California-San Diego County border population.

PubMed Central

Peter, C R; Schultz, E; Moser, K; Cox, M; Freeman, R; Ramirez-Zetina, M; Lomeli, M R

1998-01-01

A study was conducted to determine the frequency of, and risk factors for, drug-resistant pulmonary tuberculosis (TB) among Baja California (BC) and San Diego County (SDC) residents. Another purpose was to document the amount of contact between pulmonary TB patients and residents of the opposite side of the the border. During the period from February 1995 to May 1996, pulmonary TB patients from BC (n = 427) and SDC (n = 331) were evaluated with cultures, drug susceptibility tests, and questionnaires. Drug resistance was found in 41% of the BC Mycobacterium tuberculosis complex (MTB) isolates and 20% of the SDC isolates. Resistance to both isoniazid (INH) and rifampin (RIF) varied from 1% of isolates from SDC patients to 17% of isolates from BC patients. Patients with a history of previous treatment had increased odds of drug-resistant disease. Older BC patients were more likely to have INH- or RIF-resistant TB. Although 42% of Tijuana TB patients reported recent contact with residents from SDC, travel to Mexico and contact with residents from Mexico were not significant risk factors for drug-resistant TB among SDC residents. However, the demonstrated contact between TB patients and residents on opposite sides of the border indicates the importance of coordinating efforts internationally to control TB. PMID:9795580

Molecular Dynamics Simulations of Insulin: Elucidating the Conformational Changes that Enable Its Binding.

PubMed

Papaioannou, Anastasios; Kuyucak, Serdar; Kuncic, Zdenka

2015-01-01

A sequence of complex conformational changes is required for insulin to bind to the insulin receptor. Recent experimental evidence points to the B chain C-terminal (BC-CT) as the location of these changes in insulin. Here, we present molecular dynamics simulations of insulin that reveal new insights into the structural changes occurring in the BC-CT. We find three key results: 1) The opening of the BC-CT is inherently stochastic and progresses through an open and then a "wide-open" conformation--the wide-open conformation is essential for receptor binding, but occurs only rarely. 2) The BC-CT opens with a zipper-like mechanism, with a hinge at the Phe24 residue, and is maintained in the dominant closed/inactive state by hydrophobic interactions of the neighboring Tyr26, the critical residue where opening of the BC-CT (activation of insulin) is initiated. 3) The mutation Y26N is a potential candidate as a therapeutic insulin analogue. Overall, our results suggest that the binding of insulin to its receptor is a highly dynamic and stochastic process, where initial docking occurs in an open conformation and full binding is facilitated through interactions of insulin receptor residues with insulin in its wide-open conformation.

A revision of the Schinia Volupia (Fitch) species complex Lepidoptera: Noctuidae: Heliothinae)

USDA-ARS?s Scientific Manuscript database

DNA barcode analysis of cytochrome oxidase I (COI) could not differentiate between the species of the Schinia volupia (Fitch) complex including, S. volupia, S. masoni Smith, S. fulleri (McElvare), S. sanrafaeli (Opler), S. miniana (Grote), and S. biforma Smith. Genitalic characters could only differ...

ATP Synthase Repression in Tobacco Restricts Photosynthetic Electron Transport, CO2 Assimilation, and Plant Growth by Overacidification of the Thylakoid Lumen[OA

PubMed Central

Rott, Markus; Martins, Nádia F.; Thiele, Wolfram; Lein, Wolfgang; Bock, Ralph; Kramer, David M.; Schöttler, Mark A.

2011-01-01

Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b6f complex to the metabolic demand for ATP and NADPH. While the cytochrome b6f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclear-encoded γ-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastid-encoded atpB gene (encoding the essential β-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b6f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO2 fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed. PMID:21278125

The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.

PubMed

Ulfig, Agnes; Freudl, Roland

2018-05-11

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N -oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC. © 2018 Ulfig and Freudl.

TP53 mutation-correlated genes predict the risk of tumor relapse and identify MPS1 as a potential therapeutic kinase in TP53-mutated breast cancers.

PubMed

Győrffy, Balázs; Bottai, Giulia; Lehmann-Che, Jacqueline; Kéri, György; Orfi, László; Iwamoto, Takayuki; Desmedt, Christine; Bianchini, Giampaolo; Turner, Nicholas C; de Thè, Hugues; André, Fabrice; Sotiriou, Christos; Hortobagyi, Gabriel N; Di Leo, Angelo; Pusztai, Lajos; Santarpia, Libero

2014-05-01

Breast cancers (BC) carry a complex set of gene mutations that can influence their gene expression and clinical behavior. We aimed to identify genes driven by the TP53 mutation status and assess their clinical relevance in estrogen receptor (ER)-positive and ER-negative BC, and their potential as targets for patients with TP53 mutated tumors. Separate ROC analyses of each gene expression according to TP53 mutation status were performed. The prognostic value of genes with the highest AUC were assessed in a large dataset of untreated, and neoadjuvant chemotherapy treated patients. The mitotic checkpoint gene MPS1 was the most significant gene correlated with TP53 status, and the most significant prognostic marker in all ER-positive BC datasets. MPS1 retained its prognostic value independently from the type of treatment administered. The biological functions of MPS1 were investigated in different BC cell lines. We also assessed the effects of a potent small molecule inhibitor of MPS1, SP600125, alone and in combination with chemotherapy. Consistent with the gene expression profiling and siRNA assays, the inhibition of MPS1 by SP600125 led to a reduction in cell viability and a significant increase in cell death, selectively in TP53-mutated BC cells. Furthermore, the chemical inhibition of MPS1 sensitized BC cells to conventional chemotherapy, particularly taxanes. Our results collectively demonstrate that TP53-correlated kinase MPS1, is a potential therapeutic target in BC patients with TP53 mutated tumors, and that SP600125 warrant further development in future clinical trials. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Spatiotemporal Association of Real-Time Concentrations of Black Carbon (BC) with Fine Particulate Matters (PM2.5) in Urban Hotspots of South Korea.

PubMed

Kim, Sungroul; Yu, Sol; Yun, Dongmin

2017-11-06

We evaluated the spatiotemporal distributions of black carbon (BC) and particulate matters with aerodynamic diameters of less than 2.5 m (PM 2.5 ) concentrations at urban diesel engine emission (DEE) hotspots of South Korea. Concentrations of BC and PM 2.5 were measured at the entrance gate of two diesel bus terminals and a train station, in 2014. Measurements were conducted simultaneously at the hotspot (Site 1) and at its adjacent, randomly selected, residential areas, apartment complex near major roadways, located with the same direction of 300 m (Site 2) and 500 m (Site 3) away from Site 1 on 4 different days over the season, thrice per day; morning ( n = 120 measurements for each day and site), evening ( n = 120), and noon ( n = 120). The median (interquartile range) PM 2.5 ranged from 12.6 (11.3-14.3) to 60.1 (47.0-76.0) μg/m³ while those of BC concentrations ranged from 2.6 (1.9-3.7) to 6.3 (4.2-10.3) μg/m³. We observed a strong relationship of PM 2.5 concentrations between sites (slopes 0.89-0.9, the coefficient of determination 0.89-0.96) while the relationship for BC concentrations between sites was relatively weak (slopes 0.76-0.85, the coefficient of determination 0.54-0.72). PM 2.5 concentrations were changed from 4% to 140% by unit increase of BC concentration, depending on site and time while likely supporting the necessity of monitoring of BC as well as PM 2.5 , especially at urban DEE related hotspot areas.

Artificial intelligence for breast cancer screening: Opportunity or hype?

PubMed

Houssami, Nehmat; Lee, Christoph I; Buist, Diana S M; Tao, Dacheng

2017-12-01

Interpretation of mammography for breast cancer (BC) screening can confer a mortality benefit through early BC detection, can miss a cancer that is present or fast growing, or can result in false-positives. Efforts to improve screening outcomes have mostly focused on intensifying imaging practices (double instead of single-reading, more frequent screens, or supplemental imaging) that may add substantial resource expenditures and harms associated with population screening. Less attention has been given to making mammography screening practice 'smarter' or more efficient. Artificial intelligence (AI) is capable of advanced learning using large complex datasets and has the potential to perform tasks such as image interpretation. With both highly-specific capabilities, and also possible un-intended (and poorly understood) consequences, this viewpoint considers the promise and current reality of AI in BC detection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interaction between obesity-related genes, FTO and MC4R, associated to an increase of breast cancer risk.

PubMed

da Cunha, Patrícia Amorim; de Carlos Back, Lia Kubelka; Sereia, Aline Fernanda Rodrigues; Kubelka, Clara; Ribeiro, Maria Cecíia Menks; Fernandes, Bráulio Leal; de Souza, Ilíada Rainha

2013-12-01

Breast cancer (BC) is a complex disease and obesity is a well-known risk factor for its development, especially after menopause. Several studies have shown Single Nucleotide Polymorphisms (SNPs) linked to overweight and obesity, such as: rs1121980 (T/C) and rs9939609 (A/T) in Fat Mass and Obesity Associated gene (FTO) and rs17782313 (T/C) in Melanocortin 4 Receptor gene (MC4R). Thus, we aimed to investigate the association between these obesity-related SNPs and BC risk. One hundred BC patients and 148 healthy women from Santa Catarina, Brazil entered the study. SNPs were genotyped using Taqman assays. For statistical analyses SNPStats and SPSS softwares were used. Association analyses were performed by logistic regression and were adjusted for age and Body mass index (BMI). Multiple SNPs inheritance models (log-additive, dominant, recessive, codominant) were performed to determine odds ratios (ORs), assuming 95 % confidence interval (CI) and P value = 0.05 as the significance limit. When analyzed alone, FTO rs1121980 and rs9939609 did not show significant associations with BC development, however MC4R rs17782313 showed increased risk for BC even after adjustments (P-value = 0.032). Interestingly, the interaction of FTO and MC4R polymorphisms showed a powerful association with BC. We observed a 4.59-fold increased risk for woman who have the allele combination C/T/C (FTO rs1121980/FTO rs9939609/MC4R rs17782313) (P-value = 0.0011, adjusted for age and BMI). We found important and unpublished associations between these obesity-related genes and BC risk. These associations seem to be independent of their effect on BMI, indicating a direct role of the interaction between FTO and MC4R polymorphisms in BC development.

Effect of vitamin supplements on HIV shedding in breast milk123

PubMed Central

Koulinska, Irene N; Aboud, Said; Murrin, Clare; Bosch, Ronald J; Manji, Karim P; Fawzi, Wafaie W

2010-01-01

Background: Supplementation in lactating HIV-1–infected women with preformed vitamin A and β-carotene (VA/BC) increases the risk of mother-to-child transmission of HIV through breastfeeding. Identifying a biological mechanism to explain this unexpected finding would lend support to a causal effect. Objective: The aim of the study was to evaluate the effect of VA/BC or multivitamin (B complex, vitamin C, and vitamin E) supplementation of HIV-infected women on HIV shedding in breast milk during the first 2 y postpartum. Design: We quantified viral (cell-free) and proviral (cell-associated) HIV loads in breast-milk samples collected ≤15 d after delivery and every 3 mo thereafter from 594 Tanzanian HIV-1–infected women who participated in a randomized trial. Women received 1 of the following 4 daily oral regimens in a 2 × 2 factorial fashion during pregnancy and throughout the first 2 y postpartum: multivitamin, VA/BC, multivitamin including VA/BC, or placebo. Results: The proportion of breast-milk samples with detectable viral load was significantly higher in women who received VA/BC (51.3%) than in women who were not assigned to VA/BC (44.8%; P = 0.02). The effect was apparent ≥6 mo postpartum (relative risk: 1.34; 95% CI: 1.04, 1.73). No associations with proviral load were observed. The multivitamin had no effects. In observational analyses, β-carotene but not retinol breast-milk concentrations were significantly associated with an increased viral load in milk. Conclusions: VA/BC supplementation in lactating women increases the HIV load in breast milk. This finding contributes to explaining the adverse effect of VA/BC on mother-to-child transmission. β-Carotene appears to have an effect on breast-milk viral load, independent of preformed vitamin A. This trial was registered at clinicaltrials.gov as NCT00197756. PMID:20739426

Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex.

PubMed

Cardote, Teresa A F; Gadd, Morgan S; Ciulli, Alessio

2017-06-06

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2 VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

The Beaker phenomenon and the genomic transformation of northwest Europe.

PubMed

Olalde, Iñigo; Brace, Selina; Allentoft, Morten E; Armit, Ian; Kristiansen, Kristian; Booth, Thomas; Rohland, Nadin; Mallick, Swapan; Szécsényi-Nagy, Anna; Mittnik, Alissa; Altena, Eveline; Lipson, Mark; Lazaridis, Iosif; Harper, Thomas K; Patterson, Nick; Broomandkhoshbacht, Nasreen; Diekmann, Yoan; Faltyskova, Zuzana; Fernandes, Daniel; Ferry, Matthew; Harney, Eadaoin; de Knijff, Peter; Michel, Megan; Oppenheimer, Jonas; Stewardson, Kristin; Barclay, Alistair; Alt, Kurt Werner; Liesau, Corina; Ríos, Patricia; Blasco, Concepción; Miguel, Jorge Vega; García, Roberto Menduiña; Fernández, Azucena Avilés; Bánffy, Eszter; Bernabò-Brea, Maria; Billoin, David; Bonsall, Clive; Bonsall, Laura; Allen, Tim; Büster, Lindsey; Carver, Sophie; Navarro, Laura Castells; Craig, Oliver E; Cook, Gordon T; Cunliffe, Barry; Denaire, Anthony; Dinwiddy, Kirsten Egging; Dodwell, Natasha; Ernée, Michal; Evans, Christopher; Kuchařík, Milan; Farré, Joan Francès; Fowler, Chris; Gazenbeek, Michiel; Pena, Rafael Garrido; Haber-Uriarte, María; Haduch, Elżbieta; Hey, Gill; Jowett, Nick; Knowles, Timothy; Massy, Ken; Pfrengle, Saskia; Lefranc, Philippe; Lemercier, Olivier; Lefebvre, Arnaud; Martínez, César Heras; Olmo, Virginia Galera; Ramírez, Ana Bastida; Maurandi, Joaquín Lomba; Majó, Tona; McKinley, Jacqueline I; McSweeney, Kathleen; Mende, Balázs Gusztáv; Modi, Alessandra; Kulcsár, Gabriella; Kiss, Viktória; Czene, András; Patay, Róbert; Endrődi, Anna; Köhler, Kitti; Hajdu, Tamás; Szeniczey, Tamás; Dani, János; Bernert, Zsolt; Hoole, Maya; Cheronet, Olivia; Keating, Denise; Velemínský, Petr; Dobeš, Miroslav; Candilio, Francesca; Brown, Fraser; Fernández, Raúl Flores; Herrero-Corral, Ana-Mercedes; Tusa, Sebastiano; Carnieri, Emiliano; Lentini, Luigi; Valenti, Antonella; Zanini, Alessandro; Waddington, Clive; Delibes, Germán; Guerra-Doce, Elisa; Neil, Benjamin; Brittain, Marcus; Luke, Mike; Mortimer, Richard; Desideri, Jocelyne; Besse, Marie; Brücken, Günter; Furmanek, Mirosław; Hałuszko, Agata; Mackiewicz, Maksym; Rapiński, Artur; Leach, Stephany; Soriano, Ignacio; Lillios, Katina T; Cardoso, João Luís; Pearson, Michael Parker; Włodarczak, Piotr; Price, T Douglas; Prieto, Pilar; Rey, Pierre-Jérôme; Risch, Roberto; Rojo Guerra, Manuel A; Schmitt, Aurore; Serralongue, Joël; Silva, Ana Maria; Smrčka, Václav; Vergnaud, Luc; Zilhão, João; Caramelli, David; Higham, Thomas; Thomas, Mark G; Kennett, Douglas J; Fokkens, Harry; Heyd, Volker; Sheridan, Alison; Sjögren, Karl-Göran; Stockhammer, Philipp W; Krause, Johannes; Pinhasi, Ron; Haak, Wolfgang; Barnes, Ian; Lalueza-Fox, Carles; Reich, David

2018-03-08

From around 2750 to 2500 bc, Bell Beaker pottery became widespread across western and central Europe, before it disappeared between 2200 and 1800 bc. The forces that propelled its expansion are a matter of long-standing debate, and there is support for both cultural diffusion and migration having a role in this process. Here we present genome-wide data from 400 Neolithic, Copper Age and Bronze Age Europeans, including 226 individuals associated with Beaker-complex artefacts. We detected limited genetic affinity between Beaker-complex-associated individuals from Iberia and central Europe, and thus exclude migration as an important mechanism of spread between these two regions. However, migration had a key role in the further dissemination of the Beaker complex. We document this phenomenon most clearly in Britain, where the spread of the Beaker complex introduced high levels of steppe-related ancestry and was associated with the replacement of approximately 90% of Britain's gene pool within a few hundred years, continuing the east-to-west expansion that had brought steppe-related ancestry into central and northern Europe over the previous centuries.

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

PubMed

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

2001-11-01

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

Photo-excitation of electrons in cytochrome c oxidase as a theory of the mechanism of the increase of ATP production in mitochondria by laser therapy

NASA Astrophysics Data System (ADS)

Zielke, Andrzej

2014-02-01

The hypothesis explains the molecular basis for restoring mitochondrial function by laser therapy. It also explains how laser therapy reverses both excessive oxidation (lack of NADH/FADH2) and excessive reduction (lack of O2) states of cytochrome c oxidase complex. It is proposed that photons interact with heme molecules of cytochrome c oxidase. A molecule of heme contains a porphyrin ring and an atom of iron in the center. The iron atom (Fe) can switch oxidation states back and forth between ferrous (Fe2+) and ferric (Fe3+) by accepting or releasing an electron. The porphyrin ring is a complex aromatic molecule that has 26 pi electrons which are "delocalized", spinning in the carbon rings creating a resonating electromagnetic cloud. Photons with similar wavelengths are absorbed by the cloud increasing its energy. The energy is then passed on to the centrally located atom of iron existing in a reduced state (Fe2+). The electrons on the orbits of the iron atom accept this electromagnetic energy, and change orbitals to a higher energetic level. If the energy is sufficient, electrons leave the atom entirely. If this occurs, Fe2+ become oxidized to Fe3+ releasing electrons, thus restoring electron flow and the production of ATP. At the same time, electrons freed from complex IV may have sufficient energy to be picked by NAD+/FADH and re-enter the chain at the complex I or II amplifying the flow of electrons.

Mitochondrial Dysfunction and Oxidative Stress Promote Apoptotic Cell Death in the Striatum via Cytochrome c/Caspase-3 Signaling Cascade Following Chronic Rotenone Intoxication in Rats

PubMed Central

Lin, Tsu-Kung; Cheng, Ching-Hsiao; Chen, Shang-Der; Liou, Chia-Wei; Huang, Chi-Ren; Chuang, Yao-Chung

2012-01-01

Parkinson’s disease (PD) is a progressive neurological disorder marked by nigrostriatal dopaminergic degeneration. Evidence suggests that mitochondrial dysfunction may be linked to PD through a variety of different pathways, including free-radical generation and dysfunction of the mitochondrial Complex I activity. In Lewis rats, chronic systemic administration of a specific mitochondrial Complex I inhibitor, rotenone (3 mg/kg/day) produced parkinsonism-like symptoms. Increased oxidized proteins and peroxynitrite, and mitochondrial or cytosol translocation of Bim, Bax or cytochrome c in the striatum was observed after 2–4 weeks of rotenone infusion. After 28 days of systemic rotenone exposure, imunohistochemical staining for tyrosine hydroxylase indicated nigrostriatal dopaminergic neuronal cell degeneration. Characteristic histochemical (TUNEL or activated caspase-3 staining) or ultrastructural (electron microscopy) features of apoptotic cell death were present in the striatal neuronal cell after chronic rotenone intoxication. We conclude that chronic rotenone intoxication may enhance oxidative and nitrosative stress that induces mitochondrial dysfunction and ultrastructural damage, resulting in translocation of Bim and Bax from cytosol to mitochondria that contributes to apoptotic cell death in the striatum via cytochrome c/caspase-3 signaling cascade. PMID:22942730

Separate Fe-S Scaffold And Carrier Functions For SufB2C2 And SufA During In Vitro Maturation Of [2Fe-2S] Fdx

PubMed Central

Chahal, Harsimranjit K.; Outten, F. Wayne

2012-01-01

Iron-sulfur (Fe-S) clusters are inorganic cofactors required for a variety of biological processes. In vivo biogenesis of Fe-S clusters proceeds via complex pathways involving multiple protein complexes. In the Suf Fe-S cluster biogenesis system, SufB may be a scaffold for nascent Fe-S cluster assembly whereas SufA is proposed to act as either a scaffold or an Fe-S cluster carrier from the scaffold to target apo-proteins. However, SufB can form multiple stable complexes with other Suf proteins, such as SufB2C2 and SufBC2D and the specific functions of these complexes in Fe-S cluster assembly are not clear. Here we compare the ability of the SufB2C2 and SufBC2D complexes as well as SufA to promote in vitro maturation of the [2Fe-2S] ferredoxin (Fdx). We found that SufB2C2 was most proficient as a scaffold for de novo assembly of holo-Fdx using sulfide and iron as freely available building blocks while SufA was best at direct transfer of a pre-formed Fe-S cluster to Fdx. Furthermore, cluster transfer from [4Fe-4S] SufB2C2 or SufBC2D to Fdx will proceed through a SufA intermediate to Fdx is SufA is present. Finally, addition of ATP repressed cluster transfer from [4Fe-4S] SufB2C2 to Fdx and from SufBC2D to [2Fe-2S] SufA or Fdx. These studies indicate that SufB2C2 can serve as a terminal scaffold to load the SufA Fe-S cluster carrier for in vitro maturation of [2Fe-2S] enzymes like Fdx. This work is the first to systematically compare the cluster transfer rates of a scaffold (SufB) to the transfer rates of a carrier (SufA) under the same conditions to the same target enzyme and is also the first to reconstitute the full transfer pathway (from scaffold to carrier to target enzyme) in a single reaction. PMID:23018275

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection.

PubMed

Shepherd, Mark; Achard, Maud E S; Idris, Adi; Totsika, Makrina; Phan, Minh-Duy; Peters, Kate M; Sarkar, Sohinee; Ribeiro, Cláudia A; Holyoake, Louise V; Ladakis, Dimitrios; Ulett, Glen C; Sweet, Matthew J; Poole, Robert K; McEwan, Alastair G; Schembri, Mark A

2016-10-21

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.

Screening for bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, complex vertebral malformation, bovine citrullinaemia, and factor XI deficiency in Holstein cows reared in Turkey.

PubMed

Meydan, Hasan; Yildiz, Mehmet A; Agerholm, Jørgen S

2010-10-07

Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins. Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not. Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID). This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.

The h-region of twin-arginine signal peptides supports productive binding of bacterial Tat precursor proteins to the TatBC receptor complex.

PubMed

Ulfig, Agnes; Fröbel, Julia; Lausberg, Frank; Blümmel, Anne-Sophie; Heide, Anna Katharina; Müller, Matthias; Freudl, Roland

2017-06-30

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in their binding to the Tat translocase, but some facets of this interaction remain unclear. Here, we investigated the role of the hydrophobic (h-) region of the Escherichia coli trimethylamine N -oxide reductase (TorA) signal peptide in TatBC receptor binding in vivo and in vitro We show that besides the RR motif, a minimal, functional h-region in the signal peptide is required for Tat-dependent export in Escherichia coli Furthermore, we identified mutations in the h-region that synergistically suppressed the export defect of a TorA[KQ]-30aa-MalE Tat reporter protein in which the RR motif was replaced with a lysine-glutamine pair. Strikingly, all suppressor mutations increased the hydrophobicity of the h-region. By systematically replacing a neutral residue in the h-region with various amino acids, we detected a positive correlation between the hydrophobicity of the h-region and the translocation efficiency of the resulting reporter variants. In vitro cross-linking of residues located in the periplasmically-oriented part of the TatBC receptor to TorA[KQ]-30aa-MalE reporter variants harboring a more hydrophobic h-region in their signal peptides confirmed that unlike in TorA[KQ]-30aa-MalE with an unaltered h-region, the mutated reporters moved deep into the TatBC-binding cavity. Our results clearly indicate that, besides the Tat motif, the h-region of the Tat signal peptides is another important binding determinant that significantly contributes to the productive interaction of Tat precursor proteins with the TatBC receptor complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Coarse graining flow of spin foam intertwiners

NASA Astrophysics Data System (ADS)

Dittrich, Bianca; Schnetter, Erik; Seth, Cameron J.; Steinhaus, Sebastian

2016-12-01

Simplicity constraints play a crucial role in the construction of spin foam models, yet their effective behavior on larger scales is scarcely explored. In this article we introduce intertwiner and spin net models for the quantum group SU (2 )k×SU (2 )k, which implement the simplicity constraints analogous to four-dimensional Euclidean spin foam models, namely the Barrett-Crane (BC) and the Engle-Pereira-Rovelli-Livine/Freidel-Krasnov (EPRL/FK) model. These models are numerically coarse grained via tensor network renormalization, allowing us to trace the flow of simplicity constraints to larger scales. In order to perform these simulations we have substantially adapted tensor network algorithms, which we discuss in detail as they can be of use in other contexts. The BC and the EPRL/FK model behave very differently under coarse graining: While the unique BC intertwiner model is a fixed point and therefore constitutes a two-dimensional topological phase, BC spin net models flow away from the initial simplicity constraints and converge to several different topological phases. Most of these phases correspond to decoupling spin foam vertices; however we find also a new phase in which this is not the case, and in which a nontrivial version of the simplicity constraints holds. The coarse graining flow of the BC spin net models indicates furthermore that the transitions between these phases are not of second order. The EPRL/FK model by contrast reveals a far more intricate and complex dynamics. We observe an immediate flow away from the original simplicity constraints; however, with the truncation employed here, the models generically do not converge to a fixed point. The results show that the imposition of simplicity constraints can indeed lead to interesting and also very complex dynamics. Thus we need to further develop coarse graining tools to efficiently study the large scale behavior of spin foam models, in particular for the EPRL/FK model.

Screening for bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, complex vertebral malformation, bovine citrullinaemia, and factor XI deficiency in Holstein cows reared in Turkey

PubMed Central

2010-01-01

Background Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins. Methods Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not. Results Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID). Conclusion This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population. PMID:20929557

Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

DOEpatents

Craft, David L.; Madduri, Krishna M.; Loper, John C.

2003-01-01

A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

Complexation of iron hexacyanides by cytochrome c. Evidence for electron exchange at the exposed heme edge.

PubMed

Stellwagen, E; Cass, R D

1975-03-25

Electrostatic binding of at least two anionic iron hexacyanides to cationic horse heart cytochrome c was demonstrated by equilibrium dialysis measurements. No binding was detected following trifluoroacetylation of all of the 19 lysine residues. Replacement of the natural heme iron ligand methionine 80 by the alternative intrinsic ligand lysine 79 but not the extrinsic ligand imidazole resulted in the loss of one hexacyanide binding site. It is proposed that this site is located at the exposed heme edge and is functional in electron exchange.

Nasal Bone Shape Is under Complex Epistatic Genetic Control in Mouse Interspecific Recombinant Congenic Strains

PubMed Central

Burgio, Gaétan; Baylac, Michel; Heyer, Evelyne; Montagutelli, Xavier

2012-01-01

Background Genetic determinism of cranial morphology in the mouse is still largely unknown, despite the localization of putative QTLs and the identification of genes associated with Mendelian skull malformations. To approach the dissection of this multigenic control, we have used a set of interspecific recombinant congenic strains (IRCS) produced between C57BL/6 and mice of the distant species Mus spretus (SEG/Pas). Each strain has inherited 1.3% of its genome from SEG/Pas under the form of few, small-sized, chromosomal segments. Results The shape of the nasal bone was studied using outline analysis combined with Fourier descriptors, and differential features were identified between IRCS BcG-66H and C57BL/6. An F2 cross between BcG-66H and C57BL/6 revealed that, out of the three SEG/Pas-derived chromosomal regions present in BcG-66H, two were involved. Segments on chromosomes 1 (∼32 Mb) and 18 (∼13 Mb) showed additive effect on nasal bone shape. The three chromosomal regions present in BcG-66H were isolated in congenic strains to study their individual effect. Epistatic interactions were assessed in bicongenic strains. Conclusions Our results show that, besides a strong individual effect, the QTL on chromosome 1 interacts with genes on chromosomes 13 and 18. This study demonstrates that nasal bone shape is under complex genetic control but can be efficiently dissected in the mouse using appropriate genetic tools and shape descriptors. PMID:22662199

Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors.

PubMed

Giastas, Petros; Andreou, Athena; Papakyriakou, Athanasios; Koutsioulis, Dimitris; Balomenou, Stavroula; Tzartos, Socrates J; Bouriotis, Vassilis; Eliopoulos, Elias E

2018-02-06

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.

Pseudoazurin dramatically enhances the reaction profile of nitrite reduction by Paracoccus pantotrophus cytochrome cd1 and facilitates release of product nitric oxide.

PubMed

Sam, Katharine A; Fairhurst, Shirley A; Thorneley, Roger N F; Allen, James W A; Ferguson, Stuart J

2008-05-02

Cytochrome cd(1) is a respiratory nitrite reductase found in the periplasm of denitrifying bacteria. When fully reduced Paracoccus pantotrophus cytochrome cd(1) is mixed with nitrite in a stopped-flow apparatus in the absence of excess reductant, a kinetically stable complex of enzyme and product forms, assigned as a mixture of cFe(II) d(1)Fe(II)-NO(+) and cFe(III) d(1)Fe(II)-NO (cd(1)-X). However, in order for the enzyme to achieve steady-state turnover, product (NO) release must occur. In this work, we have investigated the effect of a physiological electron donor to cytochrome cd(1), the copper protein pseudoazurin, on the mechanism of nitrite reduction by the enzyme. Our data clearly show that initially oxidized pseudoazurin causes rapid further turnover by the enzyme to give a final product that we assign as all-ferric cytochrome cd(1) with nitrite bound to the d(1) heme (i.e. from which NO had dissociated). Pseudoazurin catalyzed this effect even when present at only one-tenth the stoichiometry of cytochrome cd(1). In contrast, redox-inert zinc pseudoazurin did not affect cd(1)-X, indicating a crucial role for electron movement between monomers or individual enzyme dimers rather than simply a protein-protein interaction. Furthermore, formation of cd(1)-X was, remarkably, accelerated by the presence of pseudoazurin, such that it occurred at a rate consistent with cd(1)-X being an intermediate in the catalytic cycle. It is clear that cytochrome cd(1) functions significantly differently in the presence of its two substrates, nitrite and electron donor protein, than in the presence of nitrite alone.

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

PubMed Central

Rott, M A; Donohue, T J

1990-01-01

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

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

PubMed

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

2015-03-01

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

Complex formation between chlorophyll a and cytochrome c: surface properties at the air-water interface. Absorbance, fluorescence and fluorescence-lifetime in Langmuir-Blodgett films.

PubMed

Lamarche, F; Picard, G; Téchy, F; Aghion, J; Leblanc, R M

1991-04-23

The binding of cytochrome c to an insoluble monolayer of chlorophyll a was studied. Surface pressure (II), surface potential (delta V) and [14C]cytochrome c surface-concentration (gamma) isotherms were measured versus molecular area (sigma) in mixed films. Compared to the successive-addition method, this procedure allows the formation of homogeneous mixed films. The cytochrome c is incorporated into a chlorophyll a monolayer, compressed at a surface pressure of 20 mN.m-1. On expansion, the quantity of protein incorporated into the monolayer gradually increases. Subsequent compression-expansion cycles result in similar isotherms, distinct from that measured during the first expansion. All surface properties measured, but more specifically the surface radioactivity of [14C]cytochrome c, indicate the irreversibility of protein incorporation into the chlorophyll a monolayer. In fact, surface properties of the binary film are completely different from the properties of either of the pure components. As a result, calculated values of surface potentials for mixed films using the additivity law deviate from experimentally measured potentials. The absorption and fluorescence spectra of mixed films transferred onto a solid substrate by the Langmuir-Blodgett technique, indicate a dilution effect of chlorophyll a by cytochrome c. However, the dilution effect cannot be detected by the fluorescence lifetimes of pure chlorophyll a and mixed chlorophyll a-cytochrome c films, both shorter than 0.2 ns. This provides support for the existence of an energy-transfer mechanism between chlorophyll a monomer and chlorophyll a aggregates which could serve as an energy trap. The role of the protein could be related to that of the matrix.

A framework for stochastic simulations and visualization of biological electron-transfer dynamics

NASA Astrophysics Data System (ADS)

Nakano, C. Masato; Byun, Hye Suk; Ma, Heng; Wei, Tao; El-Naggar, Mohamed Y.

2015-08-01

Electron transfer (ET) dictates a wide variety of energy-conversion processes in biological systems. Visualizing ET dynamics could provide key insight into understanding and possibly controlling these processes. We present a computational framework named VizBET to visualize biological ET dynamics, using an outer-membrane Mtr-Omc cytochrome complex in Shewanella oneidensis MR-1 as an example. Starting from X-ray crystal structures of the constituent cytochromes, molecular dynamics simulations are combined with homology modeling, protein docking, and binding free energy computations to sample the configuration of the complex as well as the change of the free energy associated with ET. This information, along with quantum-mechanical calculations of the electronic coupling, provides inputs to kinetic Monte Carlo (KMC) simulations of ET dynamics in a network of heme groups within the complex. Visualization of the KMC simulation results has been implemented as a plugin to the Visual Molecular Dynamics (VMD) software. VizBET has been used to reveal the nature of ET dynamics associated with novel nonequilibrium phase transitions in a candidate configuration of the Mtr-Omc complex due to electron-electron interactions.

Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc1 complex

NASA Astrophysics Data System (ADS)

Martin, Daniel R.; Matyushov, Dmitry V.

2015-04-01

Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc1 bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ˜0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

Emergence of fractal scaling in complex networks

NASA Astrophysics Data System (ADS)

Wei, Zong-Wen; Wang, Bing-Hong

2016-09-01

Some real-world networks are shown to be fractal or self-similar. It is widespread that such a phenomenon originates from the repulsion between hubs or disassortativity. Here we show that this common belief fails to capture the causality. Our key insight to address it is to pinpoint links critical to fractality. Those links with small edge betweenness centrality (BC) constitute a special architecture called fractal reference system, which gives birth to the fractal structure of those reported networks. In contrast, a small amount of links with high BC enable small-world effects, hiding the intrinsic fractality. With enough of such links removed, fractal scaling spontaneously arises from nonfractal networks. Our results provide a multiple-scale view on the structure and dynamics and place fractality as a generic organizing principle of complex networks on a firmer ground.

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

PubMed Central

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

1992-01-01

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

Gene expression patterns combined with network analysis identify hub genes associated with bladder cancer.

PubMed

Bi, Dongbin; Ning, Hao; Liu, Shuai; Que, Xinxiang; Ding, Kejia

2015-06-01

To explore molecular mechanisms of bladder cancer (BC), network strategy was used to find biomarkers for early detection and diagnosis. The differentially expressed genes (DEGs) between bladder carcinoma patients and normal subjects were screened using empirical Bayes method of the linear models for microarray data package. Co-expression networks were constructed by differentially co-expressed genes and links. Regulatory impact factors (RIF) metric was used to identify critical transcription factors (TFs). The protein-protein interaction (PPI) networks were constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and clusters were obtained through molecular complex detection (MCODE) algorithm. Centralities analyses for complex networks were performed based on degree, stress and betweenness. Enrichment analyses were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Co-expression networks and TFs (based on expression data of global DEGs and DEGs in different stages and grades) were identified. Hub genes of complex networks, such as UBE2C, ACTA2, FABP4, CKS2, FN1 and TOP2A, were also obtained according to analysis of degree. In gene enrichment analyses of global DEGs, cell adhesion, proteinaceous extracellular matrix and extracellular matrix structural constituent were top three GO terms. ECM-receptor interaction, focal adhesion, and cell cycle were significant pathways. Our results provide some potential underlying biomarkers of BC. However, further validation is required and deep studies are needed to elucidate the pathogenesis of BC. Copyright © 2015 Elsevier Ltd. All rights reserved.

Simulation of the water balance of boreal watersheds of northeastern British Columbia, Canada using MIKE SHE, an integrated hydrological model

NASA Astrophysics Data System (ADS)

Abadzadesahraei, S.; Déry, S.; Rex, J. F.

2016-12-01

Northeastern British Columbia (BC) is undergoing rapid development for oil and gas extraction, largely depending on subsurface hydraulic fracturing (fracking), which relies on available freshwater. Even though this industrial activity has made substantial contributions to regional and provincial economies, it is important to ensure that sufficient and sustainable water supplies are available for all those dependent on the resource, including ecological systems. Further, BC statistics predict that the northeastern region's population will increase by 30% over the next 25 years, thereby amplifying the demands of domestic and industrial water usage. Hence, given the increasing demands for surface water in the complex wetlands of northeastern BC, obtaining accurate long-term water balance information is of vital importance. Thus, this study aims to simulate the 1979-2014 water balance at two boreal watersheds using the MIKE SHE model. More specifically, this research intends to quantify the historical, and regional, water budgets and their associated hydrological processes at two boreal watersheds—the Coles Lake and Tsea Lake watersheds—in northeastern BC. The development of coupled groundwater and surface water model of these watersheds are discussed. The model setup, calibration process, and results are presented, focusing on the water balance of boreal watersheds. Hydrological components within these watersheds are quantified through a combination of intensive fieldwork, observational data, analysis and numerical modeling. The output from the model provides important information for decision makers to manage water resources in northeastern BC. Keywords: Northeastern BC; boreal watershed; water balance; MIKE SHE hydrological model.

The crystal structure of the Rv0301-Rv0300 VapBC-3 toxin-antitoxin complex from M. tuberculosis reveals a Mg 2+ ion in the active site and a putative RNA-binding site

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

Min, Andrew B; Miallau, Linda; Sawaya, Michael R

VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA.more » The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg2+ ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg2+ ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.« less

Four Closely Related HIV-1 CRF01_AE/CRF07_BC Recombinant Forms Identified in East China.

PubMed

Li, Fan; Li, Yuxueyun; Feng, Yi; Hu, Jing; Ruan, Yuhua; Xing, Hui; Shao, Yiming

2017-07-01

Five near full-length genomes of novel second-generation HIV-1 recombinant virus (JS150021, JS150029, JS150129, JS150132, and AH150183) were identified from five HIV-positive people in Jiangsu and Anhui province, east China. Phylogenic analyses showed that these five sequences are all composed of two well-established circulating recombinant forms (CRFs) CRF07_BC and CRF01_AE, grouped into four new discovered recombinant forms, which show several very similar but not identical recombinant breakpoints. The four recombinant forms are also identified to be a sort of family or related viruses, seems to be the results of different recombination events. The emergence of a serious new closely related CRF07_BC/CRF01_AE recombinant strain indicates the increasing complexity of sexual transmission of the HIV-1 epidemic in China.

Adsorption of Cd(II) from aqueous solutions by rape straw biochar derived from different modification processes.

PubMed

Li, Bing; Yang, Lan; Wang, Chang-Quan; Zhang, Qing-Pei; Liu, Qing-Cheng; Li, Yi-Ding; Xiao, Rui

2017-05-01

In order to deal with cadmium (Cd(II)) pollution, three modified biochar materials: alkaline treatment of biochar (BC-NaOH), KMnO 4 impregnation of biochar (BC-MnO x ) and FeCl 3 magnetic treatment of biochar (BC-FeO x ), were investigated. Nitrogen adsorption-desorption isotherms, Fourier transform infrared spectroscopy (FTIR), Boehm titration, and scanning electron microscopy (SEM) were used to determine the characteristics of adsorbents and explore the main adsorption mechanism. The results show that manganese oxide particles are carried successfully within the biochar, contributing to micropore creation, boosting specific surface area and forming innersphere complexes with oxygen-containing groups, while also increasing the number of oxygen-containing groups. The adsorption sites created by the loaded manganese oxide, rather than specific surface areas, play the most important roles in cadmium adsorption. Batch adsorption experiments demonstrate a Langmuir model fit for Cd(II), and BC-MnO x provided the highest sorption capacity (81.10 mg g -1 ). The sorption kinetics of Cd(II) on adsorbents follows pseudo-second-order kinetics and the adsorption rate of the BC-MnO x material was the highest (14.46 g (mg·h) -1 ). Therefore, biochar modification methods involving KMnO 4 impregnation may provide effective ways of enhancing Cd(II) removal from aqueous solutions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cohort Profile: HAART Observational Medical Evaluation and Research (HOMER) Cohort

PubMed Central

Patterson, Sophie; Cescon, Angela; Samji, Hasina; Cui, Zishan; Yip, Benita; Lepik, Katherine J; Moore, David; Lima, Viviane D; Nosyk, Bohdan; Harrigan, P Richard; Montaner, Julio SG; Shannon, Kate; Wood, Evan; Hogg, Robert S

2015-01-01

Since 1986, antiretroviral therapy (ART) has been available free of charge to individuals living with HIV in British Columbia (BC), Canada, through the BC Centre of Excellence in HIV/AIDS (BC-CfE) Drug Treatment Program (DTP). The Highly Active Antiretroviral Therapy (HAART) Observational Medical Evaluation and Research (HOMER) cohort was established in 1996 to maintain a prospective record of clinical measurements and medication profiles of a subset of DTP participants initiating HAART in BC. This unique cohort provides a comprehensive data source to investigate mortality, prognostic factors and treatment response among people living with HIV in BC from the inception of HAART. Currently over 5000 individuals are enrolled in the HOMER cohort. Data captured include socio-demographic characteristics (e.g. sex, age, ethnicity, health authority), clinical variables (e.g. CD4 cell count, plasma HIV viral load, AIDS-defining illness, hepatitis C co-infection, mortality) and treatment variables (e.g. HAART regimens, date of treatment initiation, treatment interruptions, adherence data, resistance testing). Research findings from the HOMER cohort have featured in numerous high-impact peer-reviewed journals. The HOMER cohort collaborates with other HIV cohorts on both national and international scales to answer complex HIV-specific research questions, and welcomes input from external investigators regarding potential research proposals or future collaborations. For further information please contact the principal investigator, Dr Robert Hogg (robert_hogg@sfu.ca). PMID:24639444

The Chemical Interplay between Nitric Oxide and Mitochondrial Cytochrome c Oxidase: Reactions, Effectors and Pathophysiology

PubMed Central

Sarti, Paolo; Forte, Elena; Giuffrè, Alessandro; Mastronicola, Daniela; Magnifico, Maria Chiara; Arese, Marzia

2012-01-01

Nitric oxide (NO) reacts with Complex I and cytochrome c oxidase (CcOX, Complex IV), inducing detrimental or cytoprotective effects. Two alternative reaction pathways (PWs) have been described whereby NO reacts with CcOX, producing either a relatively labile nitrite-bound derivative (CcOX-NO2  −, PW1) or a more stable nitrosyl-derivative (CcOX-NO, PW2). The two derivatives are both inhibited, displaying different persistency and O2 competitiveness. In the mitochondrion, during turnover with O2, one pathway prevails over the other one depending on NO, cytochrome c 2+ and O2 concentration. High cytochrome c 2+, and low O2 proved to be crucial in favoring CcOX nitrosylation, whereas under-standard cell-culture conditions formation of the nitrite derivative prevails. All together, these findings suggest that NO can modulate physiologically the mitochondrial respiratory/OXPHOS efficiency, eventually being converted to nitrite by CcOX, without cell detrimental effects. It is worthy to point out that nitrite, far from being a simple oxidation byproduct, represents a source of NO particularly important in view of the NO cell homeostasis, the NO production depends on the NO synthases whose activity is controlled by different stimuli/effectors; relevant to its bioavailability, NO is also produced by recycling cell/body nitrite. Bioenergetic parameters, such as mitochondrial ΔΨ, lactate, and ATP production, have been assayed in several cell lines, in the presence of endogenous or exogenous NO and the evidence collected suggests a crucial interplay between CcOX and NO with important energetic implications. PMID:22811713

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

PubMed

Tikhonov, Alexander N

2018-01-01

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

Changes in mitochondrial electron transport chain activity during insect metamorphosis.

PubMed

Chamberlin, M E

2007-02-01

The midgut of the tobacco hornworm (Manduca sexta) is a highly aerobic tissue that is destroyed by programmed cell death during larval-pupal metamorphosis. The death of the epithelium begins after commitment to pupation, and the oxygen consumption of isolated midgut mitochondria decreases soon after commitment. To assess the role of the electron transport chain in this decline in mitochondrial function, the maximal activities of complexes I-IV of the respiratory chain were measured in isolated midgut mitochondria. Whereas there were no developmental changes in the activity of complex I or III, activities of complexes II and IV [cytochrome c oxidase (COX)] were higher in mitochondria from precommitment than postcommitment larvae. This finding is consistent with a higher rate of succinate oxidation in mitochondria isolated from precommitment larvae and reveals that the metamorphic decline in mitochondrial respiration is due to the targeted destruction or inactivation of specific sites within the mitochondria, rather than the indiscriminate destruction of the organelles. The COX turnover number (e- x s(-1) x cytochrome aa3(-1)) was greater for the enzyme from precommitment than postcommitment larvae, indicating a change in the enzyme structure and/or its lipid environment during the early stages of metamorphosis. The turnover number of COX in the intact mitochondria (in organello COX) was also lower in postcommitment larvae. In addition to changes in the protein or membrane phospholipids, the metamorphic decline in this rate constant may be a result of the observed loss of endogenous cytochrome c.

Phylogeny of Rieske/cytb Complexes with a Special Focus on the Haloarchaeal Enzymes

PubMed Central

Baymann, Frauke; Schoepp-Cothenet, Barbara; Lebrun, Evelyne; van Lis, Robert; Nitschke, Wolfgang

2012-01-01

Rieske/cytochrome b (Rieske/cytb) complexes are proton pumping quinol oxidases that are present in most bacteria and Archaea. The phylogeny of their subunits follows closely the 16S-rRNA phylogeny, indicating that chemiosmotic coupling was already present in the last universal common ancestor of Archaea and bacteria. Haloarchaea are the only organisms found so far that acquired Rieske/cytb complexes via interdomain lateral gene transfer. They encode two Rieske/cytb complexes in their genomes; one of them is found in genetic context with nitrate reductase genes and has its closest relatives among Actinobacteria and the Thermus/Deinococcus group. It is likely to function in nitrate respiration. The second Rieske/cytb complex of Haloarchaea features a split cytochrome b sequence as do Cyanobacteria, chloroplasts, Heliobacteria, and Bacilli. It seems that Haloarchaea acquired this complex from an ancestor of the above-mentioned phyla. Its involvement in the bioenergetic reaction chains of Haloarchaea is unknown. We present arguments in favor of the hypothesis that the ancestor of Haloarchaea, which relied on a highly specialized bioenergetic metabolism, that is, methanogenesis, and was devoid of quinones and most enzymes of anaerobic or aerobic bioenergetic reaction chains, integrated laterally transferred genes into its genome to respond to a change in environmental conditions that made methanogenesis unfavorable. PMID:22798450

Properties of refractory BC containing particles during the ACRIDICON-CHUVA aircraft campaign in the Amazon basin

NASA Astrophysics Data System (ADS)

Holanda, Bruna; Pöhlker, Mira; Klimach, Thomas; Saturno, Jorge; Ditas, Florian; Ditas, Jeannine; Ma, Nan; Zhang, Yuxuan; Cheng, Yafang; Wendisch, Manfred; Machado, Luiz; Barbosa, Henrique; Pöhlker, Christopher; Artaxo, Paulo; Pöschl, Ulrich; Andreae, Meinrat

2017-04-01

Black carbon (BC) particles are emitted directly into the atmosphere by processes of incomplete combustion and therefore can be used as a tracer of atmospheric pollution. BC is considered one of the drivers of global warming due to its efficient absorption of solar and infra-red radiation (Bond et al., 2013). Depending on abundance and size, aerosols can also modify the characteristics of clouds and enhance or suppress precipitation (Pöschl et al., 2010). The BC particles can gain surface coatings by condensation of low and semi-volatile compounds, coagulation, and cloud processing. The inclusion of a non-absorbing coating influences the way that BC particles act as cloud nuclei and may increase their absorption through the lensing effect (Fuller et al., 1999). These aging processes change significantly the optical, chemical and physical properties of the particles, as well as their atmospheric lifetime, making BC a source of large uncertainties in current atmospheric models. Taking into account the complex dynamics of BC particles in the atmosphere, we are analyzing data from the ACRIDICON-CHUVA aircraft campaign, which took place in the Amazon basin, Brazil, during the dry season of 2014 (Wendisch et al., 2016). A detailed characterization of BC particles was done using the Single Particle Soot Photometer (SP2) instrument, which directly measures the mass of individual refractory BC particles (rBC). Additionally, the SP2 provides information about the size distribution of rBC cores and their associated coatings. These properties were measured covering a wide geographic area with different pollution conditions and at several levels of the atmosphere at high time resolution. The rBC concentrations change significantly with altitude and with the source of pollution, being a few nanograms per cubic meter for altitudes higher that 5 km. In the surroundings of Manaus city, the mean BC concentration was 0.7 μg/m3, with core sizes peaking at 180 nm. The highest BC mass values were observed over fresh biomass burning plumes (6 μg/m3) with smaller core sizes ( 150 nm). Moreover, in a specific flight (AC19) we identified an extended layer of pollution at 4 km altitude. Backward trajectories calculated using FLEXPART suggest that this pollution layer originated in Africa and has aged few days during its travel over the Atlantic. Similarities in the properties of rBC particles within the pollution and boundary layers suggest that the long range transport of pollution from Africa can be an important source of BC to the Amazonian atmosphere. Here we present first results from our analyses that characterize the various pollution aerosols and their properties in the Amazon basin. References Bond, T.C. et al., 2013. Bounding the role of black carbon in the climate system: A scientific assessment. Journal of Geophysical Research: Atmospheres, 118(11), pp.5380-5552. Fuller, K. A. et al., 1999. Effects of mixing on extinction by carbonaceous particles. Journal of Geophysical Research: Atmospheres, 104(D13), 15941-15954. Pöschl, U. et al., 2010. Rainforest aerosols as biogenic nuclei of clouds and precipitation in the Amazon. Science 329, 1513. Wendisch, M. et al., 2016. The ACRIDICON-CHUVA campaign: Studying tropical deep convective clouds and precipitation over Amazonia using the new German research aircraft HALO. Bull. Amer. Meteor. Soc.

Formation of a cytochrome c-nitrous oxide reductase complex is obligatory for N2O reduction by Paracoccus pantotrophus.

PubMed

Rasmussen, Tim; Brittain, Thomas; Berks, Ben C; Watmough, Nicholas J; Thomson, Andrew J

2005-11-07

Nitrous oxide reductase (N2OR) catalyses the final step of bacterial denitrification, the two-electron reduction of nitrous oxide (N2O) to dinitrogen (N2). N2OR contains two metal centers; a binuclear copper center, CuA, that serves to receive electrons from soluble donors, and a tetranuclear copper-sulfide center, CuZ, at the active site. Stopped flow experiments at low ionic strengths reveal rapid electron transfer (kobs=150 s-1) between reduced horse heart (HH) cytochrome c and the CuA center in fully oxidized N2OR. When fully reduced N2OR was mixed with oxidized cytochrome c, a similar rate of electron transfer was recorded for the reverse reaction, followed by a much slower internal electron transfer from CuZ to CuA(kobs=0.1-0.4 s-1). The internal electron transfer process is likely to represent the rate-determining step in the catalytic cycle. Remarkably, in the absence of cytochrome c, fully reduced N2OR is inert towards its substrate, even though sufficient electrons are stored to initiate a single turnover. However, in the presence of reduced cytochrome c and N2O, a single turnover occurs after a lag-phase. We propose that a conformational change in N2OR is induced by its specific interaction with cytochrome c that in turn either permits electron transfer between CuA and CuZ or controls the rate of N2O decomposition at the active site.

Competitive seeds-selection in complex networks

NASA Astrophysics Data System (ADS)

Zhao, Jiuhua; Liu, Qipeng; Wang, Lin; Wang, Xiaofan

2017-02-01

This paper investigates a competitive diffusion model where two competitors simultaneously select a set of nodes (seeds) in the network to influence. We focus on the problem of how to select these seeds such that, when the diffusion process terminates, a competitor can obtain more supports than its opponent. Instead of studying this problem in the game-theoretic framework as in the existing work, in this paper we design several heuristic seed-selection strategies inspired by commonly used centrality measures-Betweenness Centrality (BC), Closeness Centrality (CC), Degree Centrality (DC), Eigenvector Centrality (EC), and K-shell Centrality (KS). We mainly compare three centrality-based strategies, which have better performances in competing with the random selection strategy, through simulations on both real and artificial networks. Even though network structure varies across different networks, we find certain common trend appearing in all of these networks. Roughly speaking, BC-based strategy and DC-based strategy are better than CC-based strategy. Moreover, if a competitor adopts CC-based strategy, then BC-based strategy is a better strategy than DC-based strategy for his opponent, and the superiority of BC-based strategy decreases as the heterogeneity of the network decreases.

In vivo photosystem I reduction in thermophilic and mesophilic cyanobacteria: the thermal resistance of the process is limited by factors other than the unfolding of the partners.

PubMed

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

2005-08-19

Photosystem I reduction by plastocyanin and cytochrome c(6) in cyanobacteria has been extensively studied in vitro, but much less information is provided on this process inside the cell. Here, we report an analysis of the electron transfer from both plastocyanin and cytochrome c(6) to photosystem I in intact cells of several cyanobacterial species, including a comparative study of the temperature effect in mesophilic and thermophilic organisms. Our data show that cytochrome c(6) reduces photosystem I by following a reaction mechanism involving complex formation, whereas the copper-protein follows a simpler collisional mechanism. These results contrast with previous kinetic studies in vitro. The effect of temperature on photosystem I reduction leads us to conclude that the thermal resistance of this process is determined by factors other than the proper stability of the protein partners.

Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): characterization of a 'missing link' in the multihaem cytochrome c family.

PubMed

Haase, Doreen; Hermann, Bianca; Einsle, Oliver; Simon, Jörg

2017-07-01

Members of the multihaem cytochrome c family such as pentahaem cytochrome c nitrite reductase (NrfA) or octahaem hydroxylamine oxidoreductase (Hao) are involved in various microbial respiratory electron transport chains. Some members of the Hao subfamily, here called εHao proteins, have been predicted from the genomes of nitrate/nitrite-ammonifying bacteria that usually lack NrfA. Here, εHao proteins from the host-associated Epsilonproteobacteria Campylobacter fetus and Campylobacter curvus and the deep-sea hydrothermal vent bacteria Caminibacter mediatlanticus and Nautilia profundicola were purified as εHao-maltose binding protein fusions produced in Wolinella succinogenes. All four proteins were able to catalyze reduction of nitrite (yielding ammonium) and hydroxylamine whereas hydroxylamine oxidation was negligible. The introduction of a tyrosine residue at a position known to cause covalent trimerization of Hao proteins did neither stimulate hydroxylamine oxidation nor generate the Hao-typical absorbance maximum at 460 nm. In most cases, the εHao-encoding gene haoA was situated downstream of haoC, which predicts a tetrahaem cytochrome c of the NapC/NrfH family. This suggested the formation of a membrane-bound HaoCA assembly reminiscent of the menaquinol-oxidizing NrfHA complex. The results indicate that εHao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a 'missing link' in the evolution of NrfA and Hao enzymes. © 2017 John Wiley & Sons Ltd.

Subcellular fractionation by differential and zonal centrifugation of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis

PubMed Central

Cartledge, T. G.; Lloyd, D.

1972-01-01

1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 105g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH– and NADPH–cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH–cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions. ImagesPLATE 2PLATE 1 (cont.)PLATE 1PLATE 2 (cont.) PMID:4400904

Properties of calcium silicate-monobasic calcium phosphate materials for endodontics containing tantalum pentoxide and zirconium oxide.

PubMed

Zamparini, Fausto; Siboni, Francesco; Prati, Carlo; Taddei, Paola; Gandolfi, Maria Giovanna

2018-05-08

The aim of the study was to evaluate chemical-physical properties and apatite-forming ability of three premixed calcium silicate materials containing monobasic calcium phosphate (CaH 4 P 2 O 8 ) bioceramic, tantalum pentoxide and zirconium oxide, recently marketed for endodontics (TotalFill BC-Sealer, BC-RRM-Paste, BC-RRM-Putty). Microchemical and micromorphological analyses, radiopacity, initial and final setting times, calcium release and alkalising activity were tested. The nucleation of calcium phosphates (CaPs) and/or apatite after 28 days ageing was evaluated by ESEM-EDX and micro-Raman spectroscopy. BC-Sealer and BC-RRM-Paste showed similar initial (23 h), prolonged final (52 h) setting times and good radiopacity (> 7 mm Al); BC-RRM-Putty showed fast initial (2 h) and final setting times (27 h) and excellent radiopacity (> 9 mm Al). All materials induced a marked alkalisation (pH 11-12) up to 28 days and showed the release of calcium ions throughout the entire test period (cumulative calcium release 641-806 ppm). After 28 days ageing, a well-distributed mineral layer was present on all samples surface; EDX demonstrated relevant calcium and phosphorous peaks. B-type carbonated apatite and calcite deposits were identified by micro-Raman spectroscopy on all the 28-day-aged samples; the deposit thickness was higher on BC-RRM-Paste and BC-RRM-Putty, in agreement with calcium release data. These materials met the required chemical and physical standards and released biologically relevant ions. The CaSi-CaH 4 P 2 O 8 system present in the materials provided Ca and OH ions release with marked abilities to nucleate a layer of B-type carbonated apatite favoured/accelerated by the bioceramic presence. The ability to nucleate apatite may lead many clinical advantages: In orthograde endodontics, it may improve the sealing ability by the deposition of CaPs at the material-root dentine interface, and in endodontic surgery, it could promote bone and periodontal tissue regeneration. As premixed materials, their application in endodontics may result easier in several complex endodontic situations (apicoectomy, root perforation, presence of wide/wet apices).

Role of PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 1. PufX is required for efficient light-driven electron transfer and photophosphorylation under anaerobic conditions.

PubMed

Barz, W P; Francia, F; Venturoli, G; Melandri, B A; Verméglio, A; Oesterhelt, D

1995-11-21

The pufX gene is essential for photoheterotrophic growth of the purple bacterium Rhodobacter sphaeroides. In order to analyze the molecular function of the PufX membrane protein, we constructed a chromosomal pufX deletion mutant and phenotypically compared it to a pufX+ control strain and to two suppressor mutants which are able to grow photosynthetically in the absence of pufX. Using this genetic background, we confirmed that PufX is required for photoheterotrophic growth under anaerobic conditions, although all components of the photosynthetic apparatus were present in similar amounts in all strains investigated. We show that the deletion of PufX is not lethal for illuminated pufX- cells, suggesting that PufX is required for photosynthetic cell division. Since chromatophores isolated from the pufX- mutant were found to be unsealed vesicles, the role of PufX in photosynthetic energy transduction was studied in vivo. We show that PufX is essential for light-induced ATP synthesis (photophosphorylation) in anaerobically incubated cells. Measurements of absorption changes induced by a single turnover flash demonstrated that PufX is not required for electron flow through the reaction center and the cytochrome bc1 complex under anaerobic conditions. During prolonged illumination, however, PufX is essential for the generation of a sufficiently large membrane potential to allow photosynthetic growth. These in vivo results demonstrate that under anaerobic conditions PufX plays an essential role in facilitating effective interaction of the components of the photosynthetic apparatus.

Anaerobic Sulfur Metabolism Coupled to Dissimilatory Iron Reduction in the Extremophile Acidithiobacillus ferrooxidans

PubMed Central

Osorio, Héctor; Mangold, Stefanie; Denis, Yann; Ñancucheo, Ivan; Esparza, Mario; Johnson, D. Barrie; Bonnefoy, Violaine; Dopson, Mark

2013-01-01

Gene transcription (microarrays) and protein levels (proteomics) were compared in cultures of the acidophilic chemolithotroph Acidithiobacillus ferrooxidans grown on elemental sulfur as the electron donor under aerobic and anaerobic conditions, using either molecular oxygen or ferric iron as the electron acceptor, respectively. No evidence supporting the role of either tetrathionate hydrolase or arsenic reductase in mediating the transfer of electrons to ferric iron (as suggested by previous studies) was obtained. In addition, no novel ferric iron reductase was identified. However, data suggested that sulfur was disproportionated under anaerobic conditions, forming hydrogen sulfide via sulfur reductase and sulfate via heterodisulfide reductase and ATP sulfurylase. Supporting physiological evidence for H2S production came from the observation that soluble Cu2+ included in anaerobically incubated cultures was precipitated (seemingly as CuS). Since H2S reduces ferric iron to ferrous in acidic medium, its production under anaerobic conditions indicates that anaerobic iron reduction is mediated, at least in part, by an indirect mechanism. Evidence was obtained for an alternative model implicating the transfer of electrons from S0 to Fe3+ via a respiratory chain that includes a bc1 complex and a cytochrome c. Central carbon pathways were upregulated under aerobic conditions, correlating with higher growth rates, while many Calvin-Benson-Bassham cycle components were upregulated during anaerobic growth, probably as a result of more limited access to carbon dioxide. These results are important for understanding the role of A. ferrooxidans in environmental biogeochemical metal cycling and in industrial bioleaching operations. PMID:23354702

A spectroscopic method for observing the domain movement of the Rieske iron–sulfur protein

PubMed Central

Brugna, Myriam; Rodgers, Simon; Schricker, Anna; Montoya, Guillermo; Kazmeier, Michael; Nitschke, Wolfgang; Sinning, Irmgard

2000-01-01

The g-tensor orientation of the chemically reduced Rieske cluster in cytochrome bc1 complex from Rhodovulum sulfidophilum with respect to the membrane was determined in the presence and absence of inhibitors and in the presence of oxidized and reduced quinone in the quinol-oxidizing-site (Qo-site) by EPR on two-dimensionally ordered samples. Almost identical orientations were observed when oxidized or reduced quinone, stigmatellin, or 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole was present. Occupancy of the Qo-site by myxothiazole induced appearance of a minority population with a substantially differing conformation and presence of E-β-methoxyacrylate-stilbene significantly reduced the contribution of the major conformation observed in the other cases. Furthermore, when the oxidized iron–sulfur cluster was reduced at cryogenic temperatures by the products of radiolysis, the orientation of its magnetic axes was found to differ significantly from that of the chemically reduced center. The “irradiation-induced” conformation converts to that of the chemically reduced center after thawing of the sample. These results confirm the effects of Qo-site inhibitors on the equilibrium conformation of the Rieske iron–sulfur protein and provide evidence for a reversible redox-influenced interconversion between conformational states. Moreover, the data obtained with the iron—sulfur protein demonstrate that the conformation of “EPR-inaccessible” reduction states of redox centers can be studied by inducing changes of redox state at cryogenic temperatures. This technique appears applicable to a wide range of comparable electron transfer systems performing redox-induced conformational changes. PMID:10681446

An alternative ionospheric correction model for global navigation satellite systems

NASA Astrophysics Data System (ADS)

Hoque, M. M.; Jakowski, N.

2015-04-01

The ionosphere is recognized as a major error source for single-frequency operations of global navigation satellite systems (GNSS). To enhance single-frequency operations the global positioning system (GPS) uses an ionospheric correction algorithm (ICA) driven by 8 coefficients broadcasted in the navigation message every 24 h. Similarly, the global navigation satellite system Galileo uses the electron density NeQuick model for ionospheric correction. The Galileo satellite vehicles (SVs) transmit 3 ionospheric correction coefficients as driver parameters of the NeQuick model. In the present work, we propose an alternative ionospheric correction algorithm called Neustrelitz TEC broadcast model NTCM-BC that is also applicable for global satellite navigation systems. Like the GPS ICA or Galileo NeQuick, the NTCM-BC can be optimized on a daily basis by utilizing GNSS data obtained at the previous day at monitor stations. To drive the NTCM-BC, 9 ionospheric correction coefficients need to be uploaded to the SVs for broadcasting in the navigation message. Our investigation using GPS data of about 200 worldwide ground stations shows that the 24-h-ahead prediction performance of the NTCM-BC is better than the GPS ICA and comparable to the Galileo NeQuick model. We have found that the 95 percentiles of the prediction error are about 16.1, 16.1 and 13.4 TECU for the GPS ICA, Galileo NeQuick and NTCM-BC, respectively, during a selected quiet ionospheric period, whereas the corresponding numbers are found about 40.5, 28.2 and 26.5 TECU during a selected geomagnetic perturbed period. However, in terms of complexity the NTCM-BC is easier to handle than the Galileo NeQuick and in this respect comparable to the GPS ICA.

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

PubMed Central

Zayas, Margarita; Long, Gang; Madan, Vanesa; Bartenschlager, Ralf

2016-01-01

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles. PMID:26727512

Imaging and the completion of the omics paradigm in breast cancer.

PubMed

Leithner, D; Horvat, J V; Ochoa-Albiztegui, R E; Thakur, S; Wengert, G; Morris, E A; Helbich, T H; Pinker, K

2018-06-08

Within the field of oncology, "omics" strategies-genomics, transcriptomics, proteomics, metabolomics-have many potential applications and may significantly improve our understanding of the underlying processes of cancer development and progression. Omics strategies aim to develop meaningful imaging biomarkers for breast cancer (BC) by rapid assessment of large datasets with different biological information. In BC the paradigm of omics technologies has always favored the integration of multiple layers of omics data to achieve a complete portrait of BC. Advances in medical imaging technologies, image analysis, and the development of high-throughput methods that can extract and correlate multiple imaging parameters with "omics" data have ushered in a new direction in medical research. Radiogenomics is a novel omics strategy that aims to correlate imaging characteristics (i. e., the imaging phenotype) with underlying gene expression patterns, gene mutations, and other genome-related characteristics. Radiogenomics not only represents the evolution in the radiology-pathology correlation from the anatomical-histological level to the molecular level, but it is also a pivotal step in the omics paradigm in BC in order to fully characterize BC. Armed with modern analytical software tools, radiogenomics leads to new discoveries of quantitative and qualitative imaging biomarkers that offer hitherto unprecedented insights into the complex tumor biology and facilitate a deeper understanding of cancer development and progression. The field of radiogenomics in breast cancer is rapidly evolving, and results from previous studies are encouraging. It can be expected that radiogenomics will play an important role in the future and has the potential to revolutionize the diagnosis, treatment, and prognosis of BC patients. This article aims to give an overview of breast radiogenomics, its current role, future applications, and challenges.

TaABC1, a member of the activity of bc1 complex protein kinase family from common wheat, confers enhanced tolerance to abiotic stresses in Arabidopsis

PubMed Central

Wang, Caixiang; Jing, Ruilian; Mao, Xinguo; Chang, Xiaoping; Li, Ang

2011-01-01

Abiotic stresses such as drought, salinity, and low temperature have drastic effects on plant growth and development. However, the molecular mechanisms regulating biochemical and physiological changes in response to stresses are not well understood. Protein kinases are major signal transduction factors among the reported molecular mechanisms mediating acclimation to environmental changes. Protein kinase ABC1 (activity of bc1 complex) is involved in regulating coenzyme Q biosynthesis in mitochondria in yeast (Saccharomyces cersvisiae), and in balancing oxidative stress in chloroplasts in Arabidopsis thaliana. In the current study, TaABC1 (Triticum aestivum L. activity of bc1 complex) protein kinase was localized to the cell membrane, cytoplasm, and nucleus. The effects of overexpressing TaABC1 in transgenic Arabidopsis plants on responses to drought, salt, and cold stress were further investigated. Transgenic Arabidopsis overexpressing the TaABC1 protein showed lower water loss and higher osmotic potential, photochemistry efficiency, and chlorophyll content, while cell membrane stability and controlled reactive oxygen species homeostasis were maintained. In addition, overexpression of TaABC1 increased the expression of stress-responsive genes, such as DREB1A, DREB2A, RD29A, ABF3, KIN1, CBF1, LEA, and P5CS, detected by real-time PCR analysis. The results suggest that TaABC1 overexpression enhances drought, salt, and cold stress tolerance in Arabidopsis, and imply that TaABC1 may act as a regulatory factor involved in a multiple stress response pathways. PMID:21115661

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

PubMed

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

2016-08-01

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

High-valent manganese–oxo valence tautomers and the influence of Lewis/Brönsted acids on C–H bond cleavage

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

Baglia, Regina A.; Krest, Courtney M.; Yang, Tzuhsiung

The addition of Lewis or Brönsted acids (LA = Zn(OTf) 2, B(C 6F 5) 3, HBAr F, TFA) to the high-valent manganese–oxo complex Mn V(O)(TBP 8Cz) results in the stabilization of a valence tautomer Mn IV(O-LA)(TBP 8Cz •+). The Zn II and B(C 6F 5) 3 complexes were characterized by manganese K-edge X-ray absorption spectroscopy (XAS). The position of the edge energies and the intensities of the pre-edge (1s to 3d) peaks confirm that the Mn ion is in the +4 oxidation state. Fitting of the extended X-ray absorption fine structure (EXAFS) region reveals 4 N/O ligands at Mn–N avemore » = 1.89 Å and a fifth N/O ligand at 1.61 Å, corresponding to the terminal oxo ligand. This Mn–O bond length is elongated compared to the Mn V(O) starting material (Mn–O = 1.55 Å). The reactivity of Mn IV(O-LA)(TBP 8Cz •+) toward C–H substrates was examined, and it was found that H • abstraction from C–H bonds occurs in a 1:1 stoichiometry, giving a Mn IV complex and the dehydrogenated organic product. The rates of C–H cleavage are accelerated for the Mn IV(O-LA)(TBP 8Cz •+) valence tautomer as compared to the MnV(O) valence tautomer when LA = Zn II, B(C 6F 5) 3, and HBArF, whereas for LA = TFA, the C–H cleavage rate is slightly slower than when compared to MnV(O). A large, nonclassical kinetic isotope effect of k H/ k D = 25–27 was observed for LA = B(C 6F 5) 3 and HBAr F, indicating that H-atom transfer (HAT) is the rate-limiting step in the C–H cleavage reaction and implicating a potential tunneling mechanism for HAT. Furthermore, the reactivity of Mn IV(O-LA)(TBP 8Cz •+) toward C–H bonds depends on the strength of the Lewis acid. The HAT reactivity is compared with the analogous corrole complex Mn IV(O–H)(tpfc •+) recently reported.« less

Silylene extrusion from organosilanes via double geminal Si-H bond activation by a Cp*Ru(kappa2-P,N)+ complex: observation of a key stoichiometric step in the glaser-tilley alkene hydrosilylation mechanism.

PubMed

Rankin, Matthew A; MacLean, Darren F; Schatte, Gabriele; McDonald, Robert; Stradiotto, Mark

2007-12-26

Treatment of Cp*RuCl(kappa2-P,N-2b) (2b = 2-NMe2-3-PiPr2-indene) with TlSO3CF3 produced the cyclometalated complex [4]+SO3CF3- in 94% isolated yield. Exposure of [4]+X- (X = B(C6F5)4 or SO3CF3) to Ph2SiH2 (10 equiv) or PhSiH3 afforded the corresponding [Cp*(mu-P,N-2b)(H)2Ru=SiRPh]+X- complexes, [5]+X- (R = Ph; X = B(C6F5)4, 82%; X = SO3CF3, 39%) and [6]+X- (R = H; X = B(C6F5)4, 94%; X = SO3CF3, 95%). Notably, these transformations represent the first documented examples of Ru-mediated silylene extrusion via double geminal Si-H bond activation of an organosilane-a key step in the recently proposed Glaser-Tilley (G-T) alkene hydrosilylation mechanism. Treatment of [5]+B(C6F5)4- with KN(SiMe3)2 or [6]+SO3CF3- with NaN(SiMe3)2 afforded the corresponding zwitterionic Cp*(mu-2-NMe2-3-PiPr2-indenide)(H)2Ru=SiRPh complex in 69% (R = Ph, 7) or 86% (R = H, 8) isolated yield. Both [6]+X- and 8 proved unreactive toward 1-hexene and styrene and provided negligible catalytic turnover in the attempted metal-mediated hydrosilylation of these substrates with PhSiH3, thereby providing further empirical evidence for the required intermediacy of base-free Ru=Si species in the G-T mechanism. Isomerization of the P,N-indene ligand backbone in [6]+X-, giving rise to [Cp*(mu-1-PiPr2-2-NMe2-indene)(H)2Ru=SiHPh]+X- ([9]+X-), was observed. In the case of [9]+SO3CF3-, net intramolecular addition of the Ru=Si-H group across the styrene-like C=C unit within the ligand backbone to give 10 (96% isolated yield) was observed. Crystallographic characterization data are provided for [4]+X-, [5]+X-, [6]+X-, 8, and 10.

High-valent manganese–oxo valence tautomers and the influence of Lewis/Brönsted acids on C–H bond cleavage

DOE PAGES

Baglia, Regina A.; Krest, Courtney M.; Yang, Tzuhsiung; ...

2016-09-30

The addition of Lewis or Brönsted acids (LA = Zn(OTf) 2, B(C 6F 5) 3, HBAr F, TFA) to the high-valent manganese–oxo complex Mn V(O)(TBP 8Cz) results in the stabilization of a valence tautomer Mn IV(O-LA)(TBP 8Cz •+). The Zn II and B(C 6F 5) 3 complexes were characterized by manganese K-edge X-ray absorption spectroscopy (XAS). The position of the edge energies and the intensities of the pre-edge (1s to 3d) peaks confirm that the Mn ion is in the +4 oxidation state. Fitting of the extended X-ray absorption fine structure (EXAFS) region reveals 4 N/O ligands at Mn–N avemore » = 1.89 Å and a fifth N/O ligand at 1.61 Å, corresponding to the terminal oxo ligand. This Mn–O bond length is elongated compared to the Mn V(O) starting material (Mn–O = 1.55 Å). The reactivity of Mn IV(O-LA)(TBP 8Cz •+) toward C–H substrates was examined, and it was found that H • abstraction from C–H bonds occurs in a 1:1 stoichiometry, giving a Mn IV complex and the dehydrogenated organic product. The rates of C–H cleavage are accelerated for the Mn IV(O-LA)(TBP 8Cz •+) valence tautomer as compared to the MnV(O) valence tautomer when LA = Zn II, B(C 6F 5) 3, and HBArF, whereas for LA = TFA, the C–H cleavage rate is slightly slower than when compared to MnV(O). A large, nonclassical kinetic isotope effect of k H/ k D = 25–27 was observed for LA = B(C 6F 5) 3 and HBAr F, indicating that H-atom transfer (HAT) is the rate-limiting step in the C–H cleavage reaction and implicating a potential tunneling mechanism for HAT. Furthermore, the reactivity of Mn IV(O-LA)(TBP 8Cz •+) toward C–H bonds depends on the strength of the Lewis acid. The HAT reactivity is compared with the analogous corrole complex Mn IV(O–H)(tpfc •+) recently reported.« less

Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism.

PubMed

Liu, Zhenhua; Tavares, Raquel; Forsythe, Evan S; André, François; Lugan, Raphaël; Jonasson, Gabriella; Boutet-Mercey, Stéphanie; Tohge, Takayuki; Beilstein, Mark A; Werck-Reichhart, Danièle; Renault, Hugues

2016-10-07

Expansion of the cytochrome P450 gene family is often proposed to have a critical role in the evolution of metabolic complexity, in particular in microorganisms, insects and plants. However, the molecular mechanisms underlying the evolution of this complexity are poorly understood. Here we describe the evolutionary history of a plant P450 retrogene, which emerged and underwent fixation in the common ancestor of Brassicales, before undergoing tandem duplication in the ancestor of Brassicaceae. Duplication leads first to gain of dual functions in one of the copies. Both sister genes are retained through subsequent speciation but eventually return to a single copy in two of three diverging lineages. In the lineage in which both copies are maintained, the ancestral functions are split between paralogs and a novel function arises in the copy under relaxed selection. Our work illustrates how retrotransposition and gene duplication can favour the emergence of novel metabolic functions.

Cohort Profile: HAART Observational Medical Evaluation and Research (HOMER) cohort.

PubMed

Patterson, Sophie; Cescon, Angela; Samji, Hasina; Cui, Zishan; Yip, Benita; Lepik, Katherine J; Moore, David; Lima, Viviane D; Nosyk, Bohdan; Harrigan, P Richard; Montaner, Julio S G; Shannon, Kate; Wood, Evan; Hogg, Robert S

2015-02-01

Since 1986, antiretroviral therapy (ART) has been available free of charge to individuals living with HIV in British Columbia (BC), Canada, through the BC Centre of Excellence in HIV/AIDS (BC-CfE) Drug Treatment Program (DTP). The Highly Active Antiretroviral Therapy (HAART) Observational Medical Evaluation and Research (HOMER) cohort was established in 1996 to maintain a prospective record of clinical measurements and medication profiles of a subset of DTP participants initiating HAART in BC. This unique cohort provides a comprehensive data source to investigate mortality, prognostic factors and treatment response among people living with HIV in BC from the inception of HAART. Currently over 5000 individuals are enrolled in the HOMER cohort. Data captured include socio-demographic characteristics (e.g. sex, age, ethnicity, health authority), clinical variables (e.g. CD4 cell count, plasma HIV viral load, AIDS-defining illness, hepatitis C co-infection, mortality) and treatment variables (e.g. HAART regimens, date of treatment initiation, treatment interruptions, adherence data, resistance testing). Research findings from the HOMER cohort have featured in numerous high-impact peer-reviewed journals. The HOMER cohort collaborates with other HIV cohorts on both national and international scales to answer complex HIV-specific research questions, and welcomes input from external investigators regarding potential research proposals or future collaborations. For further information please contact the principal investigator, Dr Robert Hogg (robert_hogg@sfu.ca). © The Author 2014; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.

Movement variability in the golf swing of male and female skilled golfers.

PubMed

Horan, Sean A; Evans, Kerrie; Kavanagh, Justin J

2011-08-01

Despite the complexity of movement, the swings of skilled golfers are considered to be highly consistent. Interestingly, no direct investigation of movement variability or coupling variability during the swings of skilled golfers has occurred. To determine whether differences in movement variability exist between male and female skilled golfers during the downswing of the full golf swing. Three-dimensional thorax, pelvis, hand, and clubhead data were collected from 19 male (mean ± SD: age = 26 ± 7 yr) and 19 female (age = 25 ± 7 yr) skilled golfers. Variability of segmental movement and clubhead trajectory were examined at three phases of the downswing using discrete (SD) and continuous analyses (spanning set), whereas variability of intersegment coupling was examined using average coefficient of correspondence. Compared with males, females exhibited higher thorax and pelvis variability for axial rotation at the midpoint of the downswing and ball contact (BC). Similarly, thorax-pelvis coupling variability was higher for females than males at both the midpoint of the downswing and BC. Regardless of thorax and pelvis motion, the variability of hand and clubhead trajectory sequentially decreased from the top of the backswing to BC for both males and females. Male and female skilled golfers use different upper body movement strategies during the downswing while achieving similarly low levels of clubhead trajectory variability at BC. It is apparent that the priority of skilled golfers is to progressively minimize hand and clubhead trajectory variability toward BC, despite the individual motion or coupling of the thorax and pelvis.

Visceral Crisis Means Short Survival Among Patients With Luminal A Metastatic Breast Cancer: A Retrospective Cohort Study.

PubMed

Sbitti, Yassir; Slimani, Khaoula; Debbagh, Adil; Mokhlis, Anouar; Kadiri, Habiba; Laraqui, Abdelilah; Errihani, Hassan; Ichou, Mohamed

2017-08-01

Patients with visceral crisis from luminal metastatic breast cancer (mBC) are often treated with palliative chemotherapy. No studies have analyzed the aggressiveness of the care in visceral crisis from luminal mBC patients. The objective of this study was to assess practices in this setting in a university medical oncology department. This retrospective study included all patients who were managed for luminal mBC between January 2013 and April 2016. The analysis focused on the characteristics of the patients, the modalities of cancer treatment and delays between visceral crisis and death. Thirty-five patients pre-treated with two hormonal therapy lines were enrolled retrospectively. Worse performance status and a higher proportion of severe organ dysfunction for luminal mBC were observed among patients with visceral crisis. Sixty-five percent of patients received cytotoxic treatment. One cycle of chemotherapy was administrated in the majority of patients. Palliative care was performed in 35% of patients. Chemotherapy did not have any significant effect on patient outcome in the present study. The mean time between visceral crisis and death was 4.7 weeks (standard deviation = 1.9). Our study showed that visceral crisis in patients with luminal mBC is a complex problem. We need more comprehension of molecular pathogenesis to visceral crisis disease to propose efficacious treatments for these patients and to identify subgroup of patients who need chemotherapy followed by maintenance endocrine therapy.

Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae)

PubMed Central

2014-01-01

Background The Triatomini and Rhodniini (Hemiptera: Reduviidae) tribes include the most diverse Chagas disease vectors; however, the phylogenetic relationships within the tribes remain obscure. This study provides the most comprehensive phylogeny of Triatomini reported to date. Methods The relationships between all of the Triatomini genera and representatives of the three Rhodniini species groups were examined in a novel molecular phylogenetic analysis based on the following six molecular markers: the mitochondrial 16S; Cytochrome Oxidase I and II (COI and COII) and Cytochrome B (Cyt B); and the nuclear 18S and 28S. Results Our results show that the Rhodnius prolixus and R. pictipes groups are more closely related to each other than to the R. pallescens group. For Triatomini, we demonstrate that the large complexes within the paraphyletic Triatoma genus are closely associated with their geographical distribution. Additionally, we observe that the divergence within the spinolai and flavida complex clades are higher than in the other Triatoma complexes. Conclusions We propose that the spinolai and flavida complexes should be ranked under the genera Mepraia and Nesotriatoma. Finally, we conclude that a thorough morphological investigation of the paraphyletic genera Triatoma and Panstrongylus is required to accurately assign queries to natural genera. PMID:24685273

Effects of ancillary ligands on selectivity of protein labeling with platinum(II) chloro complexes

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

Zhou, Xia-Ying.

1990-02-01

Potassium (2,6-pyridinedicarboxylato)chloroplatinate(II) was synthesized. The molecular structure of the complex in (n-Bu){sub 4}N(Pt(dipic)Cl){center dot}0.5H{sub 2}O was determined by x-ray crystallography. The (Pt(dipic)Cl){sup {minus}} is essentially planar and contains a Pt(II) atom, a tridentate dipicolinate dianion ligand, and a unidentate Cl{sup {minus}} ligand. The bis(bidentate) complex trans-(Pt(dipic){sub 2}){sup 2{minus}} was also observed by {sup 1}H NMR. A red gel-like substance was observed when the yellow aqueous solution of K(Pt(dipic)Cl) was cooled or concentrated. The K(Pt(dipic)Cl) molecules form stacks in the solid state and gel-like substance but remain monomeric over a wide range of concentrations and temperatures. The reactivity and selectivity of(Pt(dipic)Cl){supmore » {minus}} toward cytochromes c from horse and tuna were studied. The new transition-metal reagent is specific for methionine residues. Di(2-pyridyl-{beta}-ethyl)sulfidochloroplatinum(II) chloride dihydrate was also synthesized. This complex labels histidine and methionine residues in cytochrome c. The ancillary ligands in these platinum(II) complexes clearly determine the selectivity of protein labeling. 106 refs., 10 figs., 11 tabs.« less

Transition-metal chromophore as a new, sensitive spectroscopic tag for proteins. Selective covalent labeling of histidine residues in cytochromes c with chloro(2,2':6',2''-terpyridine)platinum(II) chloride

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

Ratilla, E.M.A.; Brothers, H.M. II; Kostic, N.M.

1987-07-22

Reactivity and selectivity of Pt(trpy)Cl/sup +/ toward proteins are studied with cytochromes c from horse and tuna as examples. The new transition-metal reagent is specific for histidine residues at pH 5. The reaction, facile one-step displacement of the Cl/sup -/ ligand by imidazole, produces good yield. The binding sites, His 26 and His 33 in the horse protein and His 26 in the tuna protein, are identified by UV-vis spectrophotometry and by peptide-mapping experiments. Model complexes with imidazole, histidine, histidine derivatives, and histidine-containing peptides are prepared and characterized. The covalently attached Pt(trpy)/sup 2 +/ labels allow easy separation of themore » protein derivatives by cation-exchange chromatography. The labels do not perturb the conformation and reduction potential of cytochrome c, as shown by UV-vis spectrophotometry, cyclic voltammetry, differential-pulse voltammetry, EPR spectroscopy, and /sup 1/H NMR spectroscopy. The selectivity of Pt(trpy)Cl/sup +/ is entirely opposite from that of PtCl/sub 4//sup 2 -/ although both of them are platinum(II)-chloro complexes. Owing to an interplay between the steric and electronic effects of the terpyridyl ligand, the new reagent is unreactive toward methionine (a thio ether) and cystine (a disulfide), which are otherwise highly nucleophilic ligands, but very reactive toward imidazole, which is otherwise a relatively weak ligand. Unusual and useful selectivity of preformed transition-metal complexes toward proteins evidently can be achieved by a judicious choice of ancillary ligands.« less

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

PubMed Central

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

1991-01-01

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

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances.

PubMed

Dantas, Joana M; Ferreira, Marisa R; Catarino, Teresa; Kokhan, Oleksandr; Raj Pokkuluri, P; Salgueiro, Carlos A

2018-05-16

The bacterium Geobacter sulfurreducens can transfer electrons to the quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH 2 QDS) can also be used as energy source G. sulfurreducens. Such bi-directional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bi-functional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH 2 QDS. Using stopped-flow measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same bi-functional behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for 13 C, 15 N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and 1 H heme methyl group signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex with AQDS through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens. Copyright © 2018. Published by Elsevier B.V.

Mitochondrial mechanisms of neural cell death and neuroprotective interventions in Parkinson's disease.

PubMed

Fiskum, Gary; Starkov, Anatoly; Polster, Brian M; Chinopoulos, Christos

2003-06-01

Mitochondrial dysfunction, due to either environmental or genetic factors, can result in excessive production of reactive oxygen species, triggering the apoptotic death of dopaminergic cells in Parkinson's disease. Mitochondrial free radical production is promoted by the inhibition of electron transport at any point distal to the sites of superoxide production. Neurotoxins that induce parkinsonian neuropathology, such as MPP(+) and rotenone, stimulate superoxide production at complex I of the electron transport chain and also stimulate free radical production at proximal redox sites including mitochondrial matrix dehydrogenases. The oxidative stress caused by elevated mitochondrial production of reactive oxygen species promotes the expression and (or) intracellular distribution of the proapoptotic protein Bax to the mitochondrial outer membrane. Interactions between Bax and BH3 death domain proteins such as tBid result in Bax membrane integration, oligomerization, and permeabilization of the outer membrane to intermembrane proteins such as cytochrome c. Once released into the cytosol, cytochrome c together with other proteins activates the caspase cascade of protease activities that mediate the biochemical and morphological alterations characteristic of apoptosis. In addition, loss of mitochondrial cytochrome c stimulates mitochondrial free radical production, further promoting cell death pathways. Excessive mitochondrial Ca(2+) accumulation can also release cytochrome c and promote superoxide production through a mechanism distinctly different from that of Bax. Ca(2+) activates a mitochondrial inner membrane permeability transition causing osmotic swelling, rupture of the outer membrane, and complete loss of mitochondrial structural and functional integrity. While amphiphilic cations, such as dibucaine and propranolol, inhibit Bax-mediated cytochrome c release, transient receptor potential channel inhibitors inhibit mitochondrial swelling and cytochrome c release induced by the inner membrane permeability transition. These advances in the knowledge of mitochondrial cell death mechanisms and their inhibitors may lead to neuroprotective interventions applicable to Parkinsons's disease.

The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea

PubMed Central

Siewers, Verena; Smedsgaard, Jørn; Tudzynski, Paul

2004-01-01

The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this “direct” pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: ΔBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene. PMID:15240257

Nutrient Needs of Young Athletes.

ERIC Educational Resources Information Center

Willenberg, Barbara; Hemmelgarn, Melinda

1991-01-01

Explains the nutritional requirements of children and adolescents, and the physiological roles of the major nutrients. Details the nutrient needs of young athletes, including pre- and postgame meals and fluid replacement. Discusses eating disorders and obesity. Advocates a diet rich in complex carbohydrates. (BC)

Dependence of columnar aerosol size distribution, optical properties, and chemical components on regional transport in Beijing

NASA Astrophysics Data System (ADS)

Wang, Shuo; Zhao, Weixiong; Xu, Xuezhe; Fang, Bo; Zhang, Qilei; Qian, Xiaodong; Zhang, Weijun; Chen, Weidong; Pu, Wei; Wang, Xin

2017-11-01

Seasonal dependence of the columnar aerosol optical and chemical properties on regional transport in Beijing over 10 years (from January 2005 to December 2014) were analyzed by using the ground-based remote sensing combined with backward trajectory analysis. Daily air mass backward trajectories terminated in Beijing were computed with HYSPLIT-4 model and were categorized into five clusters. The columnar mass concentrations of black carbon (BC), brown carbon (BrC), dust (DU), aerosol water content (AW), and ammonium sulfate like aerosol (AS) of each cluster were retrieved from the optical data obtained from the Aerosol Robotic NETwork (AERONET) with five-component model. It was found that the columnar aerosol properties in different seasons were changed, and they were related to the air mass origins. In spring, aerosol was dominated by coarse particles. Summer was characterized by higher single scattering albedo (SSA), lower real part of complex refractive index (n), and obvious hygroscopic growth due to humid air from the south. During autumn and winter, there was an observable increase in absorption aerosol optical thickness (AAOT) and the imaginary part of complex refraction (k), with high levels of retrieved BC and BrC. However, concentrations of BC showed less dependence on the clusters during the two seasons owing to the widely spread coal heating in north China.

Sunflower (Helianthus annuus L.) as a pre-Columbian domesticate in Mexico

PubMed Central

Lentz, David L.; Pohl, Mary DeLand; Alvarado, José Luis; Tarighat, Somayeh; Bye, Robert

2008-01-01

Mexico has long been recognized as one of the world's cradles of domestication with evidence for squash (Cucurbita pepo) cultivation appearing as early as 8,000 cal B.C. followed by many other plants, such as maize (Zea mays), peppers (Capsicum annuum), common beans (Phaseolus vulgaris), and cotton (Gossypium hirsutum). We present archaeological, linguistic, ethnographic, and ethnohistoric data demonstrating that sunflower (Helianthus annuus) had entered the repertoire of Mexican domesticates by ca. 2600 cal B.C., that its cultivation was widespread in Mexico and extended as far south as El Salvador by the first millennium B.C., that it was well known to the Aztecs, and that it is still in use by traditional Mesoamerican cultures today. The sunflower's association with indigenous solar religion and warfare in Mexico may have led to its suppression after the Spanish Conquest. The discovery of ancient sunflower in Mexico refines our knowledge of domesticated Mesoamerican plants and adds complexity to our understanding of cultural evolution. PMID:18443289

Characterization of a New HIV-1 CRF01_AE/ CRF07_BC recombinant virus in Tianjin, China.

PubMed

Zhou, Zhehua; Ma, Ping; Feng, Yi; Ou, Weidong; Qian, Jing; Gao, Liying; Zhang, Defa; Shao, Yiming; Wei, Min

2018-05-04

Human immunodeficiency virus (HIV) is notorious for its rapid evolving since its transmissions from money to human. Currently, HIV contains multiple subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Here, from an HIV-positive mother and her child in Tianjin, China, we identified a novel HIV-1 second-generation recombinant virus (TJ20170316 and TJ20170317) between CRF01_AE and CRF07_BC. Near full-length genomes were obtained from both samples, and they shared very close sequences, except some point mutations. Phylogenetic analyses of the near full-length genomes showed that they consist of CRF01_AE backbone and part CRF07_BC sequences. Recombinant Identification Program (RIP) and Simplot software identified four breakpoints in gag, pol, vif, tat genes in TJ20170316, totally different from other reported CRFs and URFs. The emergence of such URF in Tianjin, China, highlights the complexity of HIV-1 epidemic and more measures should be taken to prevent HIV transmissions.

Combustion-derived substances in deep basins of Puget Sound: historical inputs from fossil fuel and biomass combustion.

PubMed

Kuo, Li-Jung; Louchouarn, Patrick; Herbert, Bruce E; Brandenberger, Jill M; Wade, Terry L; Crecelius, Eric

2011-04-01

Reconstructions of 250 years historical inputs of two distinct types of black carbon (soot/graphitic black carbon (GBC) and char-BC) were conducted on sediment cores from two basins of the Puget Sound, WA. Signatures of polycyclic aromatic hydrocarbons (PAHs) were also used to support the historical reconstructions of BC to this system. Down-core maxima in GBC and combustion-derived PAHs occurred in the 1940s in the cores from the Puget Sound Main Basin, whereas in Hood Canal such peak was observed in the 1970s, showing basin-specific differences in inputs of combustion byproducts. This system showed relatively higher inputs from softwood combustion than the northeastern U.S. The historical variations in char-BC concentrations were consistent with shifts in climate indices, suggesting an influence of climate oscillations on wildfire events. Environmental loading of combustion byproducts thus appears as a complex function of urbanization, fuel usage, combustion technology, environmental policies, and climate conditions. Copyright © 2010 Elsevier Ltd. All rights reserved.

[Brownian dynamics simulations of protein-protein interactions in photosynthetic electron transport chain].

PubMed

Khruschev, S S; Abaturova, A M; Diakonova, A N; Fedorov, V A; Ustinin, D M; Kovalenko, I B; Riznichenko, G Yu; Rubin, A B

2015-01-01

The application of Brownian dynamics for simulation of transient protein-protein interactions is reviewed. The review focuses on theoretical basics of Brownian dynamics method, its particular implementations, advantages and drawbacks of the method. The outlook for future development of Brownian dynamics-based simulation techniques is discussed. Special attention is given to analysis of Brownian dynamics trajectories. The second part of the review is dedicated to the role of Brownian dynamics simulations in studying photosynthetic electron transport. Interactions of mobile electron carriers (plastocyanin, cytochrome c6, and ferredoxin) with their reaction partners (cytochrome b6f complex, photosystem I, ferredoxin:NADP-reductase, and hydrogenase) are considered.

Recessive resistance to Bean common mosaic virus conferred by the bc-1 and bc-2 genes in common bean (Phaseolus vulgaris L.) affects long distance movement of the virus.

PubMed

Feng, Xue; Orellana, Gardenia; Myers, James; Karasev, Alexander V

2018-04-12

Recessive resistance to Bean common mosaic virus (BCMV) in common bean (Phaseolus vulgaris L.) is governed by four genes that include one strain-nonspecific helper gene bc-u, and three strain-specific genes bc-1, bc-2, and bc-3. The bc-3 gene was identified as an eIF4E translation initiation factor gene mediating resistance through disruption of the interaction between this protein and the VPg protein of the virus. The mode of action of bc-1 and bc-2 in expression of BCMV resistance is unknown, although bc-1 gene was found to affect systemic spread of a related potyvirus, Bean common mosaic necrosis virus. To investigate the possible role of both bc-1 and bc-2 genes in replication, cell-to-cell, and long distance movement of BCMV in P. vulgaris, we tested virus spread of eight BCMV isolates representing pathogroups I, IV, VI, VII, and VIII, in a set of bean differentials expressing different combinations of six resistance alleles including bc-u, bc-1, bc-1 2 , bc-2, bc-2 2 , and bc-3. All studied BCMV isolates were able to replicate and spread in inoculated leaves of bean cultivars harboring bc-u, bc-1, bc-1 2 , bc-2, and bc-2 2 alleles and their combinations, while no BCMV replication was found in inoculated leaves of 'IVT7214' carrying the bc-u, bc-2 and bc-3 genes, except for isolate 1755a capable of overcoming the resistance conferred by bc-2 and bc-3. In contrast, the systemic spread of all BCMV isolates from pathogroups I, IV,VI, VII, and VIII was impaired in common bean cultivars carrying bc-1, bc-1 2 , bc-2, and bc-2 2 alleles. The data suggest that bc-1 and bc-2 recessive resistance genes have no effect on the replication and cell-to-cell movement of BCMV, but affect systemic spread of BCMV in common bean. The BCMV resistance conferred by bc-1 and bc-2 and affecting systemic spread was found only partially effective when these two genes were expressed singly. The efficiency of the restriction of the systemic spread of the virus was greatly enhanced when the alleles of bc-1 and bc-2 genes were combined together.

Numerical calculation of protein-ligand binding rates through solution of the Smoluchowski equation using smoothed particle hydrodynamics

DOE PAGES

Pan, Wenxiao; Daily, Michael; Baker, Nathan A.

2015-05-07

Background: The calculation of diffusion-controlled ligand binding rates is important for understanding enzyme mechanisms as well as designing enzyme inhibitors. Methods: We demonstrate the accuracy and effectiveness of a Lagrangian particle-based method, smoothed particle hydrodynamics (SPH), to study diffusion in biomolecular systems by numerically solving the time-dependent Smoluchowski equation for continuum diffusion. Unlike previous studies, a reactive Robin boundary condition (BC), rather than the absolute absorbing (Dirichlet) BC, is considered on the reactive boundaries. This new BC treatment allows for the analysis of enzymes with “imperfect” reaction rates. Results: The numerical method is first verified in simple systems and thenmore » applied to the calculation of ligand binding to a mouse acetylcholinesterase (mAChE) monomer. Rates for inhibitor binding to mAChE are calculated at various ionic strengths and compared with experiment and other numerical methods. We find that imposition of the Robin BC improves agreement between calculated and experimental reaction rates. Conclusions: Although this initial application focuses on a single monomer system, our new method provides a framework to explore broader applications of SPH in larger-scale biomolecular complexes by taking advantage of its Lagrangian particle-based nature.« less

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

Liu, Xiu-Li, E-mail: usually.158@163.com; Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, No 169 Donghu Road, Wuchang District, Wuhan 430071; Peng, Chun-Wei, E-mail: pqc278@163.com

Highlights: {yields} HER2 level is closely related to the biologic behaviors of breast cancer cells. {yields} A new method to simultaneously image HER2 and type IV collagen was established. {yields} HER2 status and type IV collagen degradation predict breast cancer invasion. {yields} The complex interactions between tumor and its environment were revealed. -- Abstract: It has been well recognized that human epidermal growth factor receptor 2 (HER2) level in breast cancer (BC) is closely related to the malignant biologic behaviors of the tumor, including invasion and metastasis. Yet, there has been a lack of directly observable evidence to support suchmore » notion. Here we report a quantum dots (QDs)-based double-color imaging technique to simultaneously show the HER2 level on BC cells and the type IV collagen in the tumor matrix. In benign breast tumor, the type IV collagen was intact. With the increasing of HER2 expression level, there has been a progressive decrease in type IV collagen around the cancer nest. At HER2 (3+) expression level, there has virtually been a total destruction of type IV collagen. Moreover, HER2 (3+) BC cells also show direct invasion into the blood vessels. This novel imaging method provides direct observable evidence to support the theory that the HER2 expression level is directly related to BC invasion.« less

The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis.

PubMed

Cugno, Graziano; Parreira, José R; Ferlizza, Enea; Hernández-Castellano, Lorenzo E; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan; Campos, Alexandre M O; Almeida, André M

2016-01-01

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer's incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15-20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.

The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis

PubMed Central

Cugno, Graziano; Parreira, José R.; Ferlizza, Enea; Hernández-Castellano, Lorenzo E.; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan

2016-01-01

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer’s incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15–20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss. PMID:27031334

The Folding Energy Landscape and Free Energy Excitations of Cytochrome c

PubMed Central

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

2014-01-01

The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Due to the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case that follows different principles than those used to describe folding of proteins without cofactors. Recent investigations, however, demonstrate that models which are commonly used to describe folding for many proteins work well for cytochrome c when heme is explicitly introduced and generally provide results that agree with experimental observations. We will first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Since attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as “perfectly funneled.” In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are sources of frustration which cause specific transient intermediates to be populated. Native structure-based models do include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold in order for folding to proceed. The ability of native structure-based models to capture these type of motions is in part responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe well the sequence of folding events for cytochrome c inferred from hydrogen exchange experiments thereby justifying their use as a starting point. At low pH, the folding sequence of cytochrome c deviates from that at pH 7 and from those predicted from models with perfectly funneled energy landscapes. Alternate folding pathways are a result of “chemical frustration.” This frustration arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH. We construct more complex models that include chemical frustration, in addition to the native structure-based terms. These more complex models only modestly perturb the energy landscape which remains overall well funneled. These perturbed models can accurately describe how alternative folding pathways are used at low pH. At alkaline pH, cytochrome c populates distinctly different structural ensembles. For instance, lysine residues are deprotonated and compete for the heme ligation site. The same models that can describe folding at low pH also predict well the structures and relative stabilities of intermediates populated at alkaline pH. PMID:20143816

Peroxisome proliferator-activated receptors γ/mitochondrial uncoupling protein 2 signaling protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus

PubMed Central

2012-01-01

Background Status epilepticus induces subcellular changes that may lead to neuronal cell death in the hippocampus. However, the mechanism of seizure-induced neuronal cell death remains unclear. The mitochondrial uncoupling protein 2 (UCP2) is expressed in selected regions of the brain and is emerged as an endogenous neuroprotective molecule in many neurological disorders. We evaluated the neuroprotective role of UCP2 against seizure-induced hippocampal neuronal cell death under experimental status epilepticus. Methods In Sprague–Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Oxidized protein level, translocation of Bcl-2, Bax and cytochrome c between cytosol and mitochondria, and expression of peroxisome proliferator-activated receptors γ (PPARγ) and UCP2 were examined in the hippocampal CA3 subfield following KA-induced status epilepticus. The effects of microinjection bilaterally into CA3 area of a PPARγ agonist, rosiglitazone or a PPARγ antagonist, GW9662 on UCP2 expression, induced superoxide anion (O2· -) production, oxidized protein level, mitochondrial respiratory chain enzyme activities, translocation of Bcl-2, Bax and cytochrome c, and DNA fragmentation in bilateral CA3 subfields were examined. Results Increased oxidized proteins and mitochondrial or cytosol translocation of Bax or cytochrome c in the hippocampal CA3 subfield was observed 3–48 h after experimental status epilepticus. Expression of PPARγ and UCP2 increased 12–48 h after KA-induced status epilepticus. Pretreatment with rosiglitazone increased UCP2 expression, reduced protein oxidation, O2· - overproduction and dysfunction of mitochondrial Complex I, hindered the translocation of Bax and cytochrome c, and reduced DNA fragmentation in the CA3 subfield. Pretreatment with GW9662 produced opposite effects. Conclusions Activation of PPARγ upregulated mitochondrial UCP2 expression, which decreased overproduction of reactive oxygen species, improved mitochondrial Complex I dysfunction, inhibited mitochondrial translocation of Bax and prevented cytosolic release of cytochrome c by stabilizing the mitochondrial transmembrane potential, leading to amelioration of apoptotic neuronal cell death in the hippocampus following status epilepticus. PMID:22849356

Magnetic dipole transitions of Bc and Bc* mesons in the relativistic independent quark model

NASA Astrophysics Data System (ADS)

Patnaik, Sonali; Dash, P. C.; Kar, Susmita; Patra, Sweta P.; Barik, N.

2017-12-01

We study M1-transitions involving mesons: Bc(1 s ), Bc*(1 s ), Bc(2 s ), Bc*(2 s ), Bc(3 s ), and Bc*(3 s ) in the relativistic independent quark (RIQ) model based on a flavor independent average potential in the scalar-vector harmonic form. The transition form factor for Bc*→Bcγ is found to have analytical continuation from spacelike to physical timelike region. Our predicted coupling constant gBc*Bc=0.34 GeV-1 and decay width Γ (Bc*→Bcγ )=23 eV agree with other model predictions. In view of possible observation of Bc and Bc* s-wave states at LHC and Z-factory and potential use of theoretical estimate on M1-transitions, we investigate the allowed as well as hindered transitions of orbitally excited Bc-meson states and predict their decay widths in overall agreement with other model predictions. We consider the typical case of Bc*(1 s )→Bc(1 s )γ , where our predicted decay width which is found quite sensitive to the mass difference between Bc* and Bc mesons may help in determining the mass of Bc* experimentally.

CW EPR parameters reveal cytochrome P450 ligand binding modes.

PubMed

Lockart, Molly M; Rodriguez, Carlo A; Atkins, William M; Bowman, Michael K

2018-06-01

Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-H 2 O-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-bridged complexes are remarkably common among a range of nitrogenous drugs or drug fragments that bind to CYP3A4 or CYP2C9. Principal component analysis reveals a distinct clustering of CW EPR spectral parameters for water-bridged complexes. CW EPR reveals heterogeneous mixtures of ligated states, including multiple directly-coordinated complexes and water-bridged complexes. These results suggest that water-bridged complexes are under-represented in CYP structural databases and can have energies similar to other ligation modes. The data indicates that water-bridged binding modes can be identified and distinguished from directly-coordinated binding by CW EPR. Copyright © 2018 Elsevier Inc. All rights reserved.

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

PubMed Central

Presley, Tennille; Vedam, Kaushik; Liu, Xiaoping; Zweier, Jay L.

2009-01-01

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO2. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO2, but it was unaffected at low pO2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42°C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O2 binding at CcO at complex IV. PMID:19412660

Atrazine and Diuron partitioning within a soil-water-surfactant system

NASA Astrophysics Data System (ADS)

Wang, P.; Keller, A.

2006-12-01

The interaction between pesticide and soil and water is even more complex in the presence of surfactants. In this study, batch equilibrium was employed to study the sorption of surfactants and the partitioning behaviors of Atrazine and Diuron within a soil-water-surfactant system. Five soils and four surfactants (nonionic Triton- 100, cationic Benzalkonium Chloride (BC), anionic Linear Alkylbenzenesulfonate (LAS), and anionic Sodium Dodecyl Sulfate (SDS)) were used. All surfactant sorption isotherms exhibited an initial linear increase at low surfactant concentrations but reached an asymptotic value as the surfactant concentrations increased. Among the surfactants, BC had the highest sorption onto all soils, followed by Triton-100 and then by LAS and SDS, implying that the nature of the charge significantly influences surfactant sorption. Sorption of either Triton-100 or BC was highly correlated with soil Cation Exchange Capacity (CEC) while that of LAS and SDS was complicated by the presence of Ca2+ and Mg2+ in the aqueous phase and the CEC sites. Both LAS and SDS formed complexes with Ca2+ and Mg2+, resulting in a significant decrease in the detergency of the surfactants. At high surfactant concentrations and with micelles present in the aqueous phase, the micelles formed a more competitive partitioning site for the pesticides, resulting in less pesticide sorbed to the soil. At low Triton-100 and BC concentration, the sorption of the surfactants first resulted in less Atrazine sorption but more Diuron sorption, implying competition between the surfactants and Atrazine, which serves as an indirect evidence that there is a different sorption mechanism for Atrazine. Atrazine is a weak base and it protonates and becomes positively charged near particle surfaces where the pH is much lower than in the bulk solution. The protonated Atrazine may then be held on the CEC sites via electrostatic attraction. Triton-100, LAS and SDS sorbed on the soil showed similar sequestration efficiency for Atrazine and Diuron while BC sorbed on the soils with lower CECs showed much higher Atrazine and Diuron sequestration efficiency than the other surfactants, suggesting that the sorbed BC on these soils with less CEC forms bulk-like partitioning media more easily than the soils with higher CECs. These results significantly improve our understanding of partitioning of pesticides within soil-water-surfactant systems. These findings can serve to improve the pesticide removal efficiency of soil washing systems. Also, our results show that by studying the effect of surfactants on pesticide sorption can serve as a new method to study pesticide sorption mechanisms.

Synthesis and structure of the first discrete dinuclear cationic aluminum complexes.

PubMed

Wang, Xingbao; Dorcet, Vincent; Luo, Yi; Carpentier, Jean-Francois; Kirillov, Evgueni

2016-08-02

The reactions of the charge neutral dinuclear aluminum tetraalkyl complexes of di-Schiff base ligands, i.e. [AlMe2{ON}-R-{ON}AlMe2] (1a, R = 1,3-propylene; 1b, R = 1,3-cyclohexylene) with B(C6F5)3 and [H(Et2O)2](+)[H2N{B(C6F5)3}2](-) were investigated. When B(C6F5)3 was used as the cationizing agent (1 or 2 equiv. vs. Al), only monocationic dinuclear complexes [2a,b]+[MeB(C6F5)3]- were obtained. In contrast, with [H(Et2O)2](+)[H2N{B(C6F5)3}2](-), both mixed-dicationic [3a,b·(OEt2)2]2+[MeB(C6F5)3]-[H2N{B(C6F5)3}2]- and homo-dicationic [3a,b·(OEt2)2]2+[H2N{B(C6F5)3}2]-2 ion-pairs were prepared. All cationic complexes were characterized by (1)H, (13)C, (19)F and (11)B NMR spectroscopy, and an X-ray diffraction study was performed for [3b·(OEt2)2]2+[H2N{B(C6F5)3}2]-2.

The Herbaspirillum seropedicae SmR1 Fnr orthologs controls the cytochrome composition of the electron transport chain.

PubMed

Batista, Marcelo B; Sfeir, Michelle Z T; Faoro, Helisson; Wassem, Roseli; Steffens, Maria B R; Pedrosa, Fábio O; Souza, Emanuel M; Dixon, Ray; Monteiro, Rose A

2013-01-01

The transcriptional regulatory protein Fnr, acts as an intracellular redox sensor regulating a wide range of genes in response to changes in oxygen levels. Genome sequencing of Herbaspirillum seropedicae SmR1 revealed the presence of three fnr-like genes. In this study we have constructed single, double and triple fnr deletion mutant strains of H. seropedicae. Transcriptional profiling in combination with expression data from reporter fusions, together with spectroscopic analysis, demonstrates that the Fnr1 and Fnr3 proteins not only regulate expression of the cbb3-type respiratory oxidase, but also control the cytochrome content and other component complexes required for the cytochrome c-based electron transport pathway. Accordingly, in the absence of the three Fnr paralogs, growth is restricted at low oxygen tensions and nitrogenase activity is impaired. Our results suggest that the H. seropedicae Fnr proteins are major players in regulating the composition of the electron transport chain in response to prevailing oxygen concentrations.

The Herbaspirillum seropedicae SmR1 Fnr orthologs controls the cytochrome composition of the electron transport chain

PubMed Central

Batista, Marcelo B.; Sfeir, Michelle Z. T.; Faoro, Helisson; Wassem, Roseli; Steffens, Maria B. R.; Pedrosa, Fábio O.; Souza, Emanuel M.; Dixon, Ray; Monteiro, Rose A.

2013-01-01

The transcriptional regulatory protein Fnr, acts as an intracellular redox sensor regulating a wide range of genes in response to changes in oxygen levels. Genome sequencing of Herbaspirillum seropedicae SmR1 revealed the presence of three fnr-like genes. In this study we have constructed single, double and triple fnr deletion mutant strains of H. seropedicae. Transcriptional profiling in combination with expression data from reporter fusions, together with spectroscopic analysis, demonstrates that the Fnr1 and Fnr3 proteins not only regulate expression of the cbb3-type respiratory oxidase, but also control the cytochrome content and other component complexes required for the cytochrome c-based electron transport pathway. Accordingly, in the absence of the three Fnr paralogs, growth is restricted at low oxygen tensions and nitrogenase activity is impaired. Our results suggest that the H. seropedicae Fnr proteins are major players in regulating the composition of the electron transport chain in response to prevailing oxygen concentrations. PMID:23996052

Evaluation of thermal optical analysis method of elemental carbon for marine fuel exhaust.

PubMed

Lappi, Maija K; Ristimäki, Jyrki M

2017-12-01

The awareness of black carbon (BC) as the second largest anthropogenic contributor in global warming and an ice melting enhancer has increased. Due to prospected increase in shipping especially in the Arctic reliability of BC emissions and their invented amounts from ships is gaining more attention. The International Maritime Organization (IMO) is actively working toward estimation of quantities and effects of BC especially in the Arctic. IMO has launched work toward constituting a definition for BC and agreeing appropriate methods for its determination from shipping emission sources. In our study we evaluated the suitability of elemental carbon (EC) analysis by a thermal-optical transmittance (TOT) method to marine exhausts and possible measures to overcome the analysis interferences related to the chemically complex emissions. The measures included drying with CaSO 4, evaporation at 40-180ºC, H 2 O treatment, and variation of the sampling method (in-stack and diluted) and its parameters (e.g., dilution ratio, Dr). A reevaluation of the nominal organic carbon (OC)/EC split point was made. Measurement of residual carbon after solvent extraction (TC-C SOF ) was used as a reference, and later also filter smoke number (FSN) measurement, which is dealt with in a forthcoming paper by the authors. Exhaust sources used for collecting the particle sample were mainly four-stroke marine engines operated with variable loads and marine fuels ranging from light to heavy fuel oils (LFO and HFO) with a sulfur content range of <0.1-2.4% S. The results were found to be dependent on many factors, namely, sampling, preparation and analysis method, and fuel quality. It was found that the condensed H 2 SO 4 + H 2 O on the particulate matter (PM) filter had an effect on the measured EC content, and also promoted the formation of pyrolytic carbon (PyC) from OC, affecting the accuracy of EC determination. Thus, uncertainty remained regarding the EC results from HFO fuels. The work supports one part of the decision making in black carbon (BC) determination methodology. If regulations regarding BC emissions from marine engines will be implemented in the future, a well-defined and at best unequivocal method of BC determination is required for coherent and comparable emission inventories and estimating BC effects. As the aerosol from marine emission sources may be very heterogeneous and low in BC, special attention to the effects of sampling conditions and sample pretreatments on the validity of the results was paid in developing the thermal-optical analysis methodology (TOT).

Historical and Future Black Carbon Deposition on the Three Ice Caps: Ice Core Measurements and Model Simulations from 1850 to 2100

NASA Technical Reports Server (NTRS)

Bauer, Susanne E.; Bausch, Alexandra; Nazarenko, Larissa; Tsigaridis, Kostas; Xu, Baiqing; Edwards. Ross; Bisiaux, Marion; McConnell, Joe

2013-01-01

Ice core measurements in conjunction with climate model simulations are of tremendous value when examining anthropogenic and natural aerosol loads and their role in past and future climates. Refractory black carbon (BC) records from the Arctic, the Antarctic, and the Himalayas are analyzed using three transient climate simulations performed with the Goddard Institute for Space Studies ModelE. Simulations differ in aerosol schemes (bulk aerosols vs. aerosol microphysics) and ocean couplings (fully coupled vs. prescribed ocean). Regional analyses for past (1850-2005) and future (2005-2100) carbonaceous aerosol simulations focus on the Antarctic, Greenland, and the Himalayas. Measurements from locations in the Antarctic show clean conditions with no detectable trend over the past 150 years. Historical atmospheric deposition of BC and sulfur in Greenland shows strong trends and is primarily influenced by emissions from early twentieth century agricultural and domestic practices. Models fail to reproduce observations of a sharp eightfold BC increase in Greenland at the beginning of the twentieth century that could be due to the only threefold increase in the North American emission inventory. BC deposition in Greenland is about 10 times greater than in Antarctica and 10 times less than in Tibet. The Himalayas show the most complicated transport patterns, due to the complex terrain and dynamical regimes of this region. Projections of future climate based on the four CMIP5 Representative Concentration Pathways indicate further dramatic advances of pollution to the Tibetan Plateau along with decreasing BC deposition fluxes in Greenland and the Antarctic.

A Novel Approach for Determining Source-Receptor Relationships of Aerosols in Model Simulations

NASA Astrophysics Data System (ADS)

Ma, P.; Gattiker, J.; Liu, X.; Rasch, P. J.

2013-12-01

The climate modeling community usually performs sensitivity studies in the 'one-factor-at-a-time' fashion. However, owing to the a-priori unknown complexity and nonlinearity of the climate system and simulation response, it is computationally expensive to systematically identify the cause-and-effect of multiple factors in climate models. In this study, we use a Gaussian Process emulator, based on a small number of Community Atmosphere Model Version 5.1 (CAM5) simulations (constrained by meteorological reanalyses) using a Latin Hypercube experimental design, to demonstrate that it is possible to characterize model behavior accurately and very efficiently without any modifications to the model itself. We use the emulator to characterize the source-receptor relationships of black carbon (BC), focusing specifically on describing the constituent burden and surface deposition rates from emissions in various regions. Our results show that the emulator is capable of quantifying the contribution of aerosol burden and surface deposition from different source regions, finding that most of current Arctic BC comes from remote sources. We also demonstrate that the sensitivity of the BC burdens to emission perturbations differs for various source regions. For example, the emission growth in Africa where dry convections are strong results in a moderate increase of BC burden over the globe while the same emission growth in the Arctic leads to a significant increase of local BC burdens and surface deposition rates. These results provide insights into the dynamical, physical, and chemical processes of the climate model, and the conclusions may have policy implications for making cost-effective global and regional pollution management strategies.

Preventing a data breach from becoming a disaster.

PubMed

Goldberg, Ed

2013-01-01

Organisations have traditionally dealt with data breaches by investing in protective measures without a great deal of attention to mitigation of breach consequences and response. Conversely, business continuity (BC) planning has traditionally focused on mitigating disasters, not on preventing them. From a BC planning perspective, organisations need to assume that a data breach is inevitable and plan accordingly. The spate of data breaches in these past few years hit many organisations that were well protected. Those that suffered disastrous consequences as a result of a data breach lacked effective mitigation and response, not protection. The complexity and speed of an effective data breach response require that detailed planning takes place in advance of a breach.

Nitric Oxide–Triggered Remodeling of Chloroplast Bioenergetics and Thylakoid Proteins upon Nitrogen Starvation in Chlamydomonas reinhardtii[W

PubMed Central

Wei, Lili; Derrien, Benoit; Gautier, Arnaud; Houille-Vernes, Laura; Boulouis, Alix; Saint-Marcoux, Denis; Malnoë, Alizée; Rappaport, Fabrice; de Vitry, Catherine; Vallon, Olivier; Choquet, Yves; Wollman, Francis-André

2014-01-01

Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b6f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b6f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Clp chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b6f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b6f degradation pathway is activated. PMID:24474630

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

PubMed Central

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

2015-01-01

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

Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

NASA Astrophysics Data System (ADS)

Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

2001-06-01

An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (<20 μM). In this study, we present evidence for a consumption of NO in mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

Acute toxicity and associated mechanisms of four strobilurins in algae.

PubMed

Liu, Xiaoxu; Wang, Yu; Chen, Hao; Zhang, Junli; Wang, Chengju; Li, Xuefeng; Pang, Sen

2018-06-01

Strobilurins have been reported highly toxic to non-target aquatic organisms but few illustrated how they cause toxic effects on algae. This study investigated the acute toxicity of Kresoxim-methy (KRE), Pyraclostrobin (PYR), Trifloxystrobin (TRI) and Picoxystrobin (PIC) on two algae and their toxicity mechanisms. Four strobilurins showed lower toxic effects on Chlorella pyrenoidsa but higher on Chlorella vulgaris. bc1 complex activities in C. vulgaris were significantly inhibited by all strobilurins, suggesting bc 1 complex might be the target of strobilurin toxicity in algae. Moreover, SOD, CAT and POD activities were significantly up-regulated by all doses of KRE, PYR and PIC. In contrast, low concentrations of TRI stimulated SOD and POD activities but highest concentration significantly inhibited those activities. Comet assays showed damaged DNA in C. vulgaris by four strobulirins, suggesting their potential genotoxic threats to algae. The results illustrated acute toxicity by strobulirins on algae and their possible toxicity mechanisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Distinct oxidative stabilities of char versus soot black carbon: Implications for quantification and environmental recalcitrance

NASA Astrophysics Data System (ADS)

Elmquist, Marie; Cornelissen, Gerard; Kukulska, Zofia; Gustafsson, Örjan

2006-06-01

Sequestration in sediments of black carbon (BC) from vegetation fires and fuel combustion may constitute a significant sink of otherwise rapidly cycling carbon from the atmosphere-biosphere cycle. It also has the potential to provide a historical record of atmospheric BC loadings. Previous treatments of BC as one homogeneous entity are being replaced with the growing awareness of a BC combustion continuum, a range spanning from slightly charred biomass to soot and graphite. Here the relative recalcitrance of different BC forms is evaluated, and implications for both BC quantification and environmental stability are considered. The stabilities of four BC reference materials against thermal oxidation in air were quite distinct with T50%BC values (i.e., the temperature where 50% BC remained in the residue) of 444°C (diesel soot-BC), 388°C (n-hexane soot-BC), 338°C (wood char-BC), and 266°C (grass char-BC). The implications for BC quantification have been illustrated for a thermal oxidation (the CTO-375) method commonly applied to study BC in sediments. This technique measured BC:TOC ratios of 78.3 ± 1.3% for the diesel soot-BC and 45.3 ± 6.1% for n-hexane soot-BC, whereas no CTO375-BC was detected for the two analyzed char-BC materials. The greater lability of char-BC compared to soot-BC likely reflects higher accessibility to internal microporosity in char-BC, facilitating internal O2 transfer. Decreasing the temperature cutoff below 375°C to also include char-BC is not possible as thermograms of nonpyrogenic reference materials indicated that such material would then be artifactually quantified as BC. The presence of mineral oxides in the sediment matrix may lead to a catalytically mediated lowering of the activation energy for soot-BC oxidation but not for char-BC or nonpyrogenic organic material. Several recent studies combine to challenge the proposition of complete recalcitrance of BC. Particularly, the thermal lability of char-BC from grassland fires deserves further attention in order to improve the understanding of BC in the global carbon cycle.

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

PubMed

Tikhonov, Alexander N

2014-08-01

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

Cytochrome c Oxidase Biogenesis and Metallochaperone Interactions: Steps in the Assembly Pathway of a Bacterial Complex

PubMed Central

Ludwig, Bernd

2017-01-01

Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes. PMID:28107462

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

PubMed

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

2010-04-01

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

Molecular interaction studies revealed the bifunctional behavior of triheme cytochrome PpcA from Geobacter sulfurreducens toward the redox active analog of humic substances

DOE PAGES

Dantas, Joana M.; Kokhan, Oleksandr; Pokkuluri, P. Raj; ...

2015-06-09

Humic substances (HS) constitute a significant fraction of natural organic matter in terrestrial and aquatic environments and can act as terminal electron acceptors in anaerobic microbial respiration. Geobacter sulfurreducens has a remarkable respiratory versatility and can utilize the HS analog anthraquinone-2,6-disulfonate (AQDS) as a terminal electron acceptor or its reduced form (AH 2QDS) as an electron donor. Previous studies set the triheme cytochrome PpcA as a key component for HS respiration in G. sulfurreducens, but the process is far from fully understood. In this work, NMR chemical shift perturbation measurements were used to map the interaction region between PpcA andmore » AH 2QDS, and to measure their binding affinity. The results showed that the AH 2QDS binds reversibly to the more solvent exposed edge of PpcA heme IV. The NMR and visible spectroscopies coupled to redox measurements were used to determine the thermodynamic parameters of the PpcA:quinol complex. The higher reduction potential of heme IV (- 127 mV) compared to that of AH 2QDS (- 184 mV) explains why the electron transfer is more favorable in the case of reduction of the cytochrome by the quinol. The clear evidence obtained for the formation of an electron transfer complex between AH 2QDS and PpcA, combined with the fact that the protein also formed a redox complex with AQDS, revealed for the first time the bifunctional behavior of PpcA toward an analog of the HS. In conclusion, such behavior might confer selective advantage to G. sulfurreducens, which can utilize the HS in any redox state available in the environment for its metabolic needs.« less

Effect of secondary organic aerosol coating thickness on the real-time detection and characterization of biomass-burning soot by two particle mass spectrometers

DOE PAGES

Ahern, Adam T.; Subramanian, Ramachandran; Saliba, Georges; ...

2016-12-22

Biomass burning is a large source of light-absorbing refractory black carbon (rBC) particles with a wide range of morphologies and sizes. The net radiative forcing from these particles is strongly dependent on the amount and composition of non-light-absorbing material internally mixed with the rBC and on the morphology of the mixed particles. Understanding how the mixing state and morphology of biomass-burning aerosol evolves in the atmosphere is critical for constraining the influence of these particles on radiative forcing and climate. We investigated the response of two commercial laser-based particle mass spectrometers, the vacuum ultraviolet (VUV) ablation LAAPTOF and the IRmore » vaporization SP-AMS, to monodisperse biomass-burning particles as we sequentially coated the particles with secondary organic aerosol (SOA) from α-pinene ozonolysis. We studied three mobility-selected soot core sizes, each with a number of successively thicker coatings of SOA applied. Using IR laser vaporization, the SP-AMS had different changes in sensitivity to rBC compared to potassium as a function of applied SOA coatings. We show that this is due to different effective beam widths for the IR laser vaporization region of potassium versus black carbon. The SP-AMS's sensitivity to black carbon (BC) mass was not observed to plateau following successive SOA coatings, despite achieving high OA : BC mass ratios greater than 9. We also measured the ion fragmentation pattern of biomass-burning rBC and found it changed only slightly with increasing SOA mass. The average organic matter ion signal measured by the LAAPTOF demonstrated a positive correlation with the condensed SOA mass on individual particles, despite the inhomogeneity of the particle core compositions. This demonstrates that the LAAPTOF can obtain quantitative mass measurements of aged soot-particle composition from realistic biomass-burning particles with complex morphologies and composition.« less

Natural oxidation of a temperature series of biochars: opposite effect on the sorption of aromatic cationic herbicides.

PubMed

Shi, Kaishun; Xie, Ya; Qiu, Yuping

2015-04-01

The natural oxidation of biochar in the environment has been widely observed. However, its influence on the sorption of organic contaminants remains poorly understood. In the present study, a series of wood-based biochars prepared between 300 and 600°C (referred to as BC300-BC600) was abiotically incubated for one year to examine the aging effect of the temperature series of biochars on their sorption of aromatic cationic herbicides (ACHs, paraquat and diquat) as well as a nonpolar reference adsorbate (naphthalene). One year of oxidation showed no obvious effect on the surface area, but distinct increases in the O/C elemental ratio, density of the surface groups and cation exchange capacity (CEC) were observed. Therefore, these properties were significantly affected by the charring temperature. After incubation, high-temperature biochars (BC500 and BC600) displayed a 14.1-36.3% decrease in the sorption (qm) of ACHs. The alteration of their sorption tendency was similar to the reduced sorption of naphthalene on oxidized biochars, in which the increased surface groups lowered the surface area accessible to adsorbates because of blockage by adsorbed water molecule clusters. Conversely, a pronounced increase of ACHs sorption by 121.7-201.1% on the low-temperature biochar (BC300) was observed, presumably due to the increase of CEC values after oxidation. This result was further demonstrated by a significant linear relationship between the paraquat sorption (qm) and CEC values (R(2)=0.9895) of oxidized biochars. Interestingly, one year of oxidation simultaneously resulted in an enhanced sorption of paraquat and a reduced sorption of diquat on BC400, which indicated that the oxidation-induced sorption change of ACHs is a complex function of changes in the surface properties of the biochars as well as the molecular structure of the solute. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of secondary organic aerosol coating thickness on the real-time detection and characterization of biomass-burning soot by two particle mass spectrometers

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

Ahern, Adam T.; Subramanian, Ramachandran; Saliba, Georges

Biomass burning is a large source of light-absorbing refractory black carbon (rBC) particles with a wide range of morphologies and sizes. The net radiative forcing from these particles is strongly dependent on the amount and composition of non-light-absorbing material internally mixed with the rBC and on the morphology of the mixed particles. Understanding how the mixing state and morphology of biomass-burning aerosol evolves in the atmosphere is critical for constraining the influence of these particles on radiative forcing and climate. We investigated the response of two commercial laser-based particle mass spectrometers, the vacuum ultraviolet (VUV) ablation LAAPTOF and the IRmore » vaporization SP-AMS, to monodisperse biomass-burning particles as we sequentially coated the particles with secondary organic aerosol (SOA) from α-pinene ozonolysis. We studied three mobility-selected soot core sizes, each with a number of successively thicker coatings of SOA applied. Using IR laser vaporization, the SP-AMS had different changes in sensitivity to rBC compared to potassium as a function of applied SOA coatings. We show that this is due to different effective beam widths for the IR laser vaporization region of potassium versus black carbon. The SP-AMS's sensitivity to black carbon (BC) mass was not observed to plateau following successive SOA coatings, despite achieving high OA : BC mass ratios greater than 9. We also measured the ion fragmentation pattern of biomass-burning rBC and found it changed only slightly with increasing SOA mass. The average organic matter ion signal measured by the LAAPTOF demonstrated a positive correlation with the condensed SOA mass on individual particles, despite the inhomogeneity of the particle core compositions. This demonstrates that the LAAPTOF can obtain quantitative mass measurements of aged soot-particle composition from realistic biomass-burning particles with complex morphologies and composition.« less

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

PubMed

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

2015-11-01

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

Investigating protein-protein interaction surfaces using a reduced stereochemical and electrostatic model.

PubMed

Warwicker, J

1989-03-20

A method of calculating the electrostatic potential energy between two molecules, using finite difference potential, is presented. A reduced charge set is used so that the interaction energy can be calculated as the two static molecules explore their full six-dimensional configurational space. The energies are contoured over surfaces fixed to each molecule with an interactive computer graphics program. For two crystal structures (trypsin-trypsin inhibitor and anti-lysozyme Fab-lysozyme), it is found that the complex corresponds to highly favourable interacting regions in the contour plots. These matches arise from a small number of protruding basic residues interacting with enhanced negative potential in each case. The redox pair cytochrome c peroxidase-cytochrome c exhibits an extensive favourably interacting surface within which a possible electron transfer complex may be defined by an increased electrostatic complementarity, but a decreased electrostatic energy. A possible substrate transfer configuration for the glycolytic enzyme pair glyceraldehyde phosphate dehydrogenase-phosphoglycerate kinase is presented.

Positive and purifying selection in mitochondrial genomes of a bird with mitonuclear discordance.

PubMed

Morales, Hernán E; Pavlova, Alexandra; Joseph, Leo; Sunnucks, Paul

2015-06-01

Diversifying selection on metabolic pathways can reduce intraspecific gene flow and promote population divergence. An opportunity to explore this arises from mitonuclear discordance observed in an Australian bird Eopsaltria australis. Across >1500 km, nuclear differentiation is low and latitudinally structured by isolation by distance, whereas two highly divergent, parapatric mitochondrial lineages (>6.6% in ND2) show a discordant longitudinal geographic pattern and experience different climates. Vicariance, incomplete lineage sorting and sex-biased dispersal were shown earlier to be unlikely drivers of the mitonuclear discordance; instead, natural selection on a female-linked trait was the preferred hypothesis. Accordingly, here we tested for signals of positive, divergent selection on mitochondrial genes in E. australis. We used codon models and physicochemical profiles of amino acid replacements to analyse complete mitochondrial genomes of the two mitochondrial lineages in E. australis, its sister species Eopsaltria griseogularis, and outgroups. We found evidence of positive selection on at least five amino acids, encoded by genes of two oxidative phosphorylation pathway complexes NADH dehydrogenase (ND4 and ND4L) and cytochrome bc1 (cyt-b) against a background of widespread purifying selection on all mitochondrial genes. Three of these amino acid replacements were fixed in ND4 of the geographically most widespread E. australis lineage. The other two replacements were fixed in ND4L and cyt-b of the geographically more restricted E. australis lineage. We discuss whether this selection may reflect local environmental adaptation, a by-product of other selective processes, or genetic incompatibilities, and propose how these hypotheses can be tested in future. © 2015 John Wiley & Sons Ltd.

Defining protein electrostatic recognition processes

NASA Astrophysics Data System (ADS)

Getzoff, Elizabeth D.; Roberts, Victoria A.

The objective is to elucidate the nature of electrostatic forces controlling protein recognition processes by using a tightly coupled computational and interactive computer graphics approach. The TURNIP program was developed to determine the most favorable precollision orientations for two molecules by systematic search of all orientations and evaluation of the resulting electrostatic interactions. TURNIP was applied to the transient interaction between two electron transfer metalloproteins, plastocyanin and cytochrome c. The results suggest that the productive electron-transfer complex involves interaction of the positive region of cytochrome c with the negative patch of plastocyanin, consistent with experimental data. Application of TURNIP to the formation of the stable complex between the HyHEL-5 antibody and its protein antigen lysozyme showed that long-distance electrostatic forces guide lysozyme toward the HyHEL-5 binding site, but do not fine tune its orientation. Determination of docked antigen/antibody complexes requires including steric as well as electrostatic interactions, as was done for the U10 mutant of the anti-phosphorylcholine antibody S107. The graphics program Flex, a convenient desktop workstation program for visualizing molecular dynamics and normal mode motions, was enhanced. Flex now has a user interface and was rewritten to use standard graphics libraries, so as to run on most desktop workstations.

Mitochondrial function in diaphragm of emphysematous hamsters after treatment with nandrolone.

PubMed

Wijnhoven, Hanneke J H; Ennen, Leo; Rodenburg, Richard J T; Dekhuijzen, P N Richard

2006-01-01

Respiratory failure in patients with COPD may be caused by insufficient force production or insufficient endurance capacity of the respiratory muscles. Anabolic steroids may improve respiratory muscle function in COPD. The effect of anabolic steroids on mitochondrial function in the diaphragm in emphysema is unknown. In an emphysematous male hamster model, we investigated whether administration of the anabolic steroid nandrolone decanoate (ND) altered the activity of mitochondrial respiratory chain complexes in the diaphragm. The bodyweight of hamsters treated with ND was decreased after treatment compared with initial values, and serum testosterone levels were significantly lower in hamsters treated with ND than in control hamsters. No difference in the activity of mitochondrial respiratory chain complexes in the diaphragm between normal and emphysematous hamsters was observed. Treatment with ND did not change the activity of mitochondrial respiratory chain complexes in the diaphragm of both normal and emphysematous hamsters. In emphysematous hamsters, administration of ND decreased the activity of succinate:cytochrome c oxidoreductase compared with ND treatment in normal hamsters. We conclude that anabolic steroids have negative effects on the activity of succinate:cytochrome c oxidoreductase and anabolic status in this emphysematous hamster model.

Mitochondrial function in diaphragm of emphysematous hamsters after treatment with nandrolone

PubMed Central

Wijnhoven, Hanneke JH; Ennen, Leo; Rodenburg, Richard JT; Dekhuijzen, PN Richard

2006-01-01

Respiratory failure in patients with COPD may be caused by insufficient force production or insufficient endurance capacity of the respiratory muscles. Anabolic steroids may improve respiratory muscle function in COPD. The effect of anabolic steroids on mitochondrial function in the diaphragm in emphysema is unknown. In an emphysematous male hamster model, we investigated whether administration of the anabolic steroid nandrolone decanoate (ND) altered the activity of mitochondrial respiratory chain complexes in the diaphragm. The bodyweight of hamsters treated with ND was decreased after treatment compared with initial values, and serum testosterone levels were significantly lower in hamsters treated with ND than in control hamsters. No difference in the activity of mitochondrial respiratory chain complexes in the diaphragm between normal and emphysematous hamsters was observed. Treatment with ND did not change the activity of mitochondrial respiratory chain complexes in the diaphragm of both normal and emphysematous hamsters. In emphysematous hamsters, administration of ND decreased the activity of succinate:cytochrome c oxidoreductase compared with ND treatment in normal hamsters. We conclude that anabolic steroids have negative effects on the activity of succinate:cytochrome c oxidoreductase and anabolic status in this emphysematous hamster model. PMID:18046906

Cell-free NADPH oxidase activation assays: "in vitro veritas".

PubMed

Pick, Edgar

2014-01-01

The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD).

Degradation of dimethyl disulphide in soil with or without biochar amendment.

PubMed

Han, Dawei; Yan, Dongdong; Cao, Aocheng; Fang, Wensheng; Liu, Pengfei; Li, Yuan; Ouyang, Canbin; Wang, Qiuxia

2017-09-01

Dimethyl disulphide (DMDS) is a new and effective alternative to methyl bromide for soil fumigation. The effect of biochar on the fate of DMDS in soil is not fully understood. The objective of this study was to determine the degradation kinetics of DMDS in different soils and evaluate the effect of biochar amendment on DMDS degradation using incubation experiments. The degradation half-life of DMDS was between 1.05 and 6.66 days under non-sterile conditions, and 12.63 to 22.67 days under sterile conditions in five types of soil. Seven out of the eight tested biochar amendments (BC-2 to BC-8) delayed the degradation of DMDS in soil, increasing the half-life of DMDS in Fangshan soil from 1.05 to 1.16-5.87 days following amendment with 1% (w/w) biochar. The degradation rate of DMDS in Fangshan soil accelerated as the amendment rate of BC-1 increased, and decreased as the amendment rate of BC-7 increased. Biodegradation is an important degradation route for DMDS in soil, and DMDS degraded faster in alkaline soil. The effects of biochar amendments on DMDS degradation in soil are determined by complex multiple factors (such as surface area, pH and physicochemical composition), rather than by any single property of biochar. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Paleopathology of Human Tuberculosis and the Potential Role of Climate

PubMed Central

Nerlich, Andreas G.; Lösch, Sandra

2009-01-01

Both origin and evolution of tuberculosis and its pathogens (Mycobacterium tuberculosis complex) are not fully understood. The paleopathological investigation of human remains offers a unique insight into the molecular evolution and spread including correlative data of the environment. The molecular analysis of material from Egypt (3000–500 BC), Sudan (200–600 AD), Hungary (600–1700 AD), Latvia (1200–1600 AD), and South Germany (1400–1800 AD) urprisingly revealed constantly high frequencies of tuberculosis in all different time periods excluding significant environmental influence on tuberculosis spread. The typing of various mycobacteria strains provides evidence for ancestral M. tuberculosis strains in Pre- to early Egyptian dynastic material (3500–2650 BC), while typical M. africanum signatures were detected in a Middle Kingdom tomb (2050–1650 BC). Samples from the New Kingdom to Late Period (1500–500 BC) indicated modern M. tuberculosis strains. No evidence was seen for M. bovis in Egyptian material while M. bovis signatures were first identified in Siberian biomaterial dating 2000 years before present. These results contraindicates the theory that M. tuberculosis evolved from M. bovis during early domestication in the region of the “Fertile Crescent,” but supports the scenario that M. tuberculosis probably derived from an ancestral progenitor strain. The environmental influence of this evolutionary scenario deserves continuing intense evaluation. PMID:19360109

Characteristics and source apportionment of black carbon aerosols over an urban site.

PubMed

Rajesh, T A; Ramachandran, S

2017-03-01

Aethalometer based source apportionment model using the measured aerosol absorption coefficients at different wavelengths is used to apportion the contribution of fossil fuel and wood burning sources to the total black carbon (BC) mass concentration. Temporal and seasonal variabilities in BC mass concentrations, equivalent BC from fossil fuel (BC f f ), and wood burning (BC w b ) are investigated over an urban location in western India during January 2014 to December 2015. BC, BC f f , and BC w b mass concentrations exhibit strong diurnal variation and are mainly influenced by atmospheric dynamics. BC f f was higher by a factor of 2-4 than BC w b and contributes maximum to BC mass throughout the day, confirming consistent anthropogenic activities. Diurnal contribution of BC f f and BC w b exhibits opposite variation due to differences in emission sources over Ahmedabad. Night time BC values are about a factor of 1.4 higher than day time BC values. The annual mean percentage contributions of day time and night time are 42 and 58 %, respectively. BC, BC f f , and BC w b mass concentrations exhibit large and significant variations during morning, afternoon, evening, and night time. During afternoon, mass concentration values are minimum throughout the year because of the fully evolved boundary layer and reduced anthropogenic activities. BC exhibits a strong seasonal variability with postmonsoon high (8.3 μg m -3 ) and monsoon low (1.9 μg m -3 ). Annual mean BC f f and BC w b contributions are 80 and 20 %, respectively, to total BC, which suggests that major contribution of BC in Ahmedabad comes from fossil fuel emissions. The results show that the study location is dominated by fossil fuel combustion as compared to the emissions from wood burning. The results obtained represent a regional value over an urban regime which can be used as inputs on source apportionment to model BC emissions in regional and global climate models.

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature.

PubMed

Ishigami, Izumi; Zatsepin, Nadia A; Hikita, Masahide; Conrad, Chelsie E; Nelson, Garrett; Coe, Jesse D; Basu, Shibom; Grant, Thomas D; Seaberg, Matthew H; Sierra, Raymond G; Hunter, Mark S; Fromme, Petra; Fromme, Raimund; Yeh, Syun-Ru; Rousseau, Denis L

2017-07-25

Cytochrome c oxidase (C c O), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C c O (bC c O) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a 3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near Cu B , which, in turn, moves closer to the heme a 3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3 , thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

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

Ishigami, Izumi; Zatsepin, Nadia A.; Hikita, Masahide

Here, cytochrome c oxidase (C cO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C cO (bC cO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-raymore » crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ~142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near Cu B, which, in turn, moves closer to the heme a 3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.« less

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

DOE PAGES

Ishigami, Izumi; Zatsepin, Nadia A.; Hikita, Masahide; ...

2017-07-11

Here, cytochrome c oxidase (C cO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C cO (bC cO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-raymore » crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ~142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near Cu B, which, in turn, moves closer to the heme a 3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.« less

Fumonisins, Tortillas and Neural Tube Defects: Untangling a Complex Issue

USDA-ARS?s Scientific Manuscript database

Fumonisin mycotoxins are found in corn and corn-based foods. Fumonisin B1 (FB1), the most common, disrupts sphingolipid metabolism thereby causing species-specific diseases in animals that include cancer in rodents and (birth) neural tube defects (NTD) in LM/Bc mice. Fumonisins’ affect on human heal...

Integrative Models in Environmental Planning and Policy Making.

ERIC Educational Resources Information Center

Kyler, David Clinton

1984-01-01

Discusses conceptual models of thought that have recently emerged to confront the conventional approaches to analysis and solution to complex environmental problems. In addition to a critical attack on the tradition of specialization and reductionism, several models are summarized that originated from ecology, cybernetics, and system theory. (BC)

A History of Instructional Technology.

ERIC Educational Resources Information Center

Saettler, Paul

Theoretical and methodological foundations of the modern audiovisual/radio/television/programed instruction complex have been provided by educational theorists from the Elder Sophists of the fifth century B.C., to the medieval scholars who taught in the monastic or cathedral schools, to the reformers of 1700-1900, to the psychologists of the 20th…

Direct targeting of SUZ12/ROCK2 by miR-200b/c inhibits cholangiocarcinoma tumourigenesis and metastasis

PubMed Central

Peng, F; Jiang, J; Yu, Y; Tian, R; Guo, X; Li, X; Shen, M; Xu, M; Zhu, F; Shi, C; Hu, J; Wang, M; Qin, R

2013-01-01

Background: The multidrug resistance and distant metastasis of cholangiocarcinoma result in high postoperative recurrence and low long-term survival rates. It has been demonstrated that the ectopic expression of miR-200 suppresses the multidrug resistance and metastasis of cancer. However, the expression and function of miR-200 in cholangiocarcinoma has not yet been described. Methods: In this study, we identified dysregulated microRNAs (miRNAs, miR) in cholangiocarcinoma tissue by microarray analysis, and subsequent real-time PCR and northern blot analyses validated the expression of candidate miR. We performed functional analyses and investigated the relationship between miR-200b/c expression and the properties of cholangiocarcinoma cells. A dual luciferase assay was applied to examine the effect of miRNAs on the 3′-UTR of target genes, and we demonstrated the function of the target gene by siRNA transfection identifying the downstream pathway via western blotting. Results: We found significantly downregulated expression of four miR-200 family members (miR-200a/b/c/429) and then confirmed that ectopic miR-200b/200c inhibits the migration and invasion of cholangiocarcinoma cells both in vitro and in vivo. We found that miR-200b/c influenced the tumourigenesis of cholangiocarcinoma cells including their tumour-initiating capacity, sphere formation, and drug resistance. We further found that miR-200b/c regulated migration and invasion capacities by directly targeting rho-kinase 2 and regulated tumorigenic properties by directly targeting SUZ12 (a subunit of a polycomb repressor complex). Conclusion: Our study shows that miR-200b/c has a critical role in the regulation of the tumorigenic and metastatic capacity of cholangiocarcinoma and reveals the probable underlying mechanisms. PMID:24169343

Advanced Backcross QTL Analysis of Fiber Strength and Fineness in a Cross between Gossypium hirsutum and G. mustelinum.

PubMed

Wang, Baohua; Zhuang, Zhimin; Zhang, Zhengsheng; Draye, Xavier; Shuang, Lan-Shuan; Shehzad, Tariq; Lubbers, Edward L; Jones, Don; May, O Lloyd; Paterson, Andrew H; Chee, Peng W

2017-01-01

The molecular genetic basis of cotton fiber strength and fineness in crosses between Gossypium mustelinum and Gossypium hirsutum (Upland cotton) was dissected using 21 BC 3 F 2 and 12 corresponding BC 3 F 2:3 and BC 3 F 2:4 families. The BC 3 F 2 families were genotyped with simple sequence repeat markers from a G. hirsutum by G. mustelinum linkage map, and the three generations of BC 3 -derived families were phenotyped for fiber strength (STR) and fineness (Micronaire, MIC). A total of 42 quantitative trait loci (QTLs) were identified through one-way analysis of variance, including 15 QTLs for STR and 27 for MIC, with the percentage of variance explained by individual loci averaging 13.86 and 14.06%, respectively. Eighteen of the 42 QTLs were detected at least twice near the same markers in different generations/families or near linked markers in the same family, and 28 of the 42 QTLs were identified in both mixed model-based composite interval mapping and one-way variance analyses. Alleles from G. mustelinum increased STR for eight of 15 and reduced MIC for 15 of 27 QTLs. Significant among-family genotypic effects ( P < 0.001) were detected in 13 and 10 loci for STR and MIC respectively, and five loci showed significant ( P < 0.001) genotype × family interaction for MIC. These results support the hypothesis that fiber quality improvement for Upland cotton could be realized by introgressing G. mustelinum alleles although complexities due to the different effects of genetic background on introgressed chromatin might be faced. Building on prior work with G. barbadense, G. tomentosum , and G. darwinii , QTL mapping involving introgression of G. mustelinum alleles offers new allelic variation to Upland cotton germplasm.

Measuring persistence to hormonal therapy in patients with breast cancer: accounting for temporary treatment discontinuation.

PubMed

Huiart, Laetitia; Ferdynus, Cyril; Dell'Aniello, Sophie; Bakiri, Naciba; Giorgi, Roch; Suissa, Samy

2014-08-01

Several studies have been conducted to estimate persistence to hormonal therapy among women with breast cancer (BC). Most studies focus on first treatment discontinuation. Patients, however, can have numerous periods of treatment discontinuation or treatment exposure. Our objective is to estimate persistence to tamoxifen in patients with BC while accounting for temporary treatment discontinuations and this by using multi-state (MS) models. A cohort of 10,806 women with BC having received at least one prescription of tamoxifen between 1998 and 2008 was constituted from the UK General Practice Research Database. We fitted a semi-Markov model with three states to estimate the probability of being off treatment over a 5-year period while accounting for temporary treatment discontinuations (transition between on treatment and off treatment) and competing risks (recurrence of BC or death). Non-persistence, as estimated from the MS model, ranged from 12.1% (95% confidence interval [95%CI]: 9.2-15.1) at 1 year to 14.9% (95%CI: 11.7-18.1) at 5 years. Estimations of non-persistence based on the Kaplan-Meier model were higher, i.e., 29.3% (95%CI: 28.1-30.6) at 5 years, as well as those obtained from a competing risk model, i.e., 24.0% (95%CI: 22.9-25.1). Most temporary discontinuations (94.7%) lasted less than 6 months. Temporary treatment discontinuations are frequent and should be accounted for when measuring adherence to treatment. MS models can provide a useful framework for this sort of analysis insofar as they help describe patients' complex behavior. This may help tailor interventions that improve persistence to hormonal therapy among women with BC. Copyright © 2014 John Wiley & Sons, Ltd.

Modeling spatial variations of black carbon particles in an urban highway-building environment.

PubMed

Tong, Zheming; Wang, Yan Jason; Patel, Molini; Kinney, Patrick; Chrillrud, Steven; Zhang, K Max

2012-01-03

Highway-building environments are prevalent in metropolitan areas. This paper presents our findings in investigating pollutant transport in a highway-building environment by combing field measurement and numerical simulations. We employ and improve the Comprehensive Turbulent Aerosol Dynamics and Gas Chemistry (CTAG) model to simulate the spatial variations of black carbon (BC) concentrations near highway I-87 and an urban school in the South Bronx, New York. The results of CTAG simulations are evaluated against and agree adequately with the measurements of wind speed, wind directions, and BC concentrations. Our analysis suggests that the BC concentration at the measurement point of the urban school could decrease by 43-54% if roadside buildings were absent. Furthermore, we characterize two generalized conditions in a highway-building environment, i.e., highway-building canyon and highway viaduct-building. The former refers to the canyon between solid highway embankment and roadside buildings, where the spatial profiles of BC depend on the equivalent canyon aspect ratio and flow recirculation. The latter refers to the area between a highway viaduct (i.e., elevated highway with open space underneath) and roadside buildings, where strong flow recirculation is absent and the spatial profiles of BC are determined by the relative heights of the highway and buildings. The two configurations may occur at different locations or in the same location with different wind directions when highway geometry is complex. Our study demonstrates the importance of incorporating highway-building interaction into the assessment of human exposure to near-road air pollution. It also calls for active roles of building and highway designs in mitigating near-road exposure of urban population.

Modeling spatial variations of black carbon particles in an urban highway-buildings environment

PubMed Central

Tong, Zheming; Wang, Yan; Patel, Molini; Kinney, Patrick; Chrillrud, Steven; Zhang, K. Max

2011-01-01

Highway-building environments are prevalent in metropolitan areas. This paper presents our findings in investigating pollutant transport in a highway-building environment by combing field measurement and numerical simulations. We employ and improve the Comprehensive Turbulent Aerosol Dynamics and Gas Chemistry (CTAG) model to simulate the spatial variations of black carbon (BC) concentrations near highway I-87 and an urban school in the South Bronx, New York. The results of CTAG simulations are evaluated against and agree adequately with the measurements of wind speed, wind directions and BC concentrations. Our analysis suggests that the BC concentration at the measurement point of the urban school could decrease by 43–54% if roadside buildings were absent. Furthermore, we characterize two generalized conditions in a highway-building environment, i.e., highway-building canyon and highway viaduct-building. The former refers to the canyon between solid highway embankment and roadside buildings, where the spatial profiles of BC depend on the equivalent canyon aspect ratio and flow recirculation. The latter refers to the area between a highway viaduct (i.e., elevated highway with open space underneath) and roadside buildings, where strong flow recirculation is absent and the spatial profiles of BC are determined by the relative heights of the highway and buildings. The two configurations may occur at different locations or in the same location with different wind directions when highway geometry is complex. Our study demonstrates the importance of incorporating highway-building interaction into the assessment of human exposure to near-road air pollution. It also calls for active roles of building and highway designs in mitigating near-road exposure of urban population. PMID:22084971

Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components.

PubMed

Sato, Kanna; Suzuki, Ryu; Nishikubo, Nobuyuki; Takenouchi, Sachi; Ito, Sachiko; Nakano, Yoshimi; Nakaba, Satoshi; Sano, Yuzou; Funada, Ryo; Kajita, Shinya; Kitano, Hidemi; Katayama, Yoshihiro

2010-06-01

The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.

Enhanced light absorptivity of black carbon with air pollution development in urban Beijing, China

NASA Astrophysics Data System (ADS)

Zhang, Y.; Zhang, Q.; Cheng, Y.; Su, H.; He, K.

2017-12-01

The impacts of black carbon (BC) aerosols on air quality and climate are dependent on BC light absorptivity. However, the light absorptivity of ambient BC-containing particles remains conflicting. In this work, we investigated the evolution of BC light absorptivity with pollution development in urban Beijing, China. We found that the mass absorption cross-section (MAC) of ambient BC-containing particles measured during the campaign increased with BC mass concentration, which can be attributed to more coating materials on BC surface with pollution development. A single-particle soot photometer (SP2) measurement showed that the coating thickness (CT) of BC-containing particles increased by 48% with PM1 and BC mass concentration increasing from 10 μg m-3 and 0.3 μg m-3 to 230 μg m-3 and 12 μg m-3. Based on Mie calculation, the CT increase could led to light absorption enhancement (Eab) of BC-containing particles increasing by 22%, consistent with the increase of measured MAC. The relationship between growth rate of BC light absorptivity (kEab) and that of PM1 or rBC concentration (kPM1 or krBC) showed that kEab ≈ 4.8% kPM1 or kEab ≈ 2.5% krBC. The analysis of effective emission intensity (EEI) for BC revealed that the enhancement of BC light absorptivity with increasing pollution levels was dominated by regional transport. During the pollution period, 63% of BC over Beijing originated from regional sources. The aging of these regional BC during atmospheric transport controlled the increase of coating materials for BC-containing particles observed in Beijing. As a result of enhanced light absorptivity with pollution development, BC forcing efficiency could increase by 20% during polluted period. Our work identified the importance of BC on radiative forcing under polluted environment, which is determined by not only the increase of BC mass concentration, but also the enhancement of BC forcing efficiency due to more coating materials.

Isolation and Functional Characterization of a Floral Repressor, BcMAF1, From Pak-choi (Brassica rapa ssp. Chinensis).

PubMed

Huang, Feiyi; Liu, Tongkun; Hou, Xilin

2018-01-01

MADS-box genes form a large gene family in plants and are involved in multiple biological processes, such as flowering. However, the regulation mechanism of MADS-box genes in flowering remains unresolved, especially under short-term cold conditions. In the present study, we isolated BcMAF1 , a Pak-choi ( Brassica rapa ssp. Chinensis ) MADS AFFECTING FLOWERING ( MAF ), as a floral repressor and functionally characterized BcMAF1 in Arabidopsis and Pak-choi. Subcellular localization and sequence analysis indicated that BcMAF1 was a nuclear protein and contained a conserved MADS-box domain. Expression analysis revealed that BcMAF1 had higher expression levels in leaves, stems, and petals, and could be induced by short-term cold conditions in Pak-choi. Overexpressing BcMAF1 in Arabidopsis showed that BcMAF1 had a negative function in regulating flowering, which was further confirmed by silencing endogenous BcMAF1 in Pak-choi. In addition, qPCR results showed that AtAP3 expression was reduced and AtMAF2 expression was induced in BcMAF1 -overexpressing Arabidopsis . Meanwhile, BcAP3 transcript was up-regulated and BcMAF2 transcript was down-regulated in BcMAF1 -silencing Pak-choi. Yeast one-hybrid and dual luciferase transient assays showed that BcMAF1 could bind to the promoters of BcAP3 and BcMAF2 . These results indicated that BcAP3 and BcMAF2 might be the targets of BcMAF1. Taken together, our results suggested that BcMAF1 could negatively regulate flowering by directly activating BcMAF2 and repressing BcAP3 .

Radiation Nanomedicine for EGFR-Positive Breast Cancer: Panitumumab-Modified Gold Nanoparticles Complexed to the β-Particle-Emitter, (177)Lu.

PubMed

Yook, Simmyung; Cai, Zhongli; Lu, Yijie; Winnik, Mitchell A; Pignol, Jean-Philippe; Reilly, Raymond M

2015-11-02

Our objective was to construct a novel radiation nanomedicine for treatment of breast cancer (BC) expressing epidermal growth factor receptors (EGFR), particularly triple-negative tumors (TNBC). Gold nanoparticles (AuNP; 30 nm) were modified with polyethylene glycol (PEG) chains (4 kDa) derivatized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators for complexing the β-emitter, (177)Lu and with PEG chains (5 kDa) linked to panitumumab for targeting BC cells expressing EGFR. The AuNP were further coated with PEG chains (2 kDa) to stabilize the particles to aggregation. The binding and internalization of EGFR-targeted AuNP ((177)Lu-T-AuNP) into BC cells was studied and compared to nontargeted (177)Lu-NT-AuNP. The cytotoxicity of (177)Lu-T-AuNP and (177)Lu-NT-AuNP was measured in clonogenic assays using BC cells with widely different EGFR densities: MDA-MB-468 (10(6) receptors/cell), MDA-MB-231 (10(5) receptors/cell), and MCF-7 cells (10(4) receptors/cell). Radiation absorbed doses to the cell nucleus of MDA-MB-468 cells were estimated based on subcellular distribution. Darkfield and fluorescence microscopy as well as radioligand binding assays revealed that (177)Lu-T-AuNP were specifically bound by BC cells dependent on their EGFR density whereas the binding and internalization of (177)Lu-NT-AuNP was significantly lower. The affinity of binding of (177)Lu-T-AuNP to MDA-MB-468 cells was reduced by 2-fold compared to (123)I-labeled panitumumab (KD = 1.3 ± 0.2 nM vs 0.7 ± 0.4 nM, respectively). The cytotoxicity of (177)Lu-T-AuNP was dependent on the amount of radioactivity incubated with BC cells, their EGFR density and the radiosensitivity of the cells. The clonogenic survival (CS) of MDA-MB-468 cells overexpressing EGFR was reduced to <0.001% at the highest amount of (177)Lu-T-AuNP tested (4.5 MBq; 6 × 10(11) AuNP per 2.5 × 10(4)-1.2 × 10(5) cells). (177)Lu-T-AuNP were less effective for killing MDA-MB-231 cells or MCF-7 cells with moderate or low EGFR density (CS = 33.8 ± 1.6% and 25.8 ± 1.2%, respectively). Because the β-particles emitted by (177)Lu have a 2 mm range, (177)Lu-NT-AuNP were also cytotoxic to BC cells due to a cross-fire effect but (177)Lu-T-AuNP were significantly more potent for killing MDA-MB-468 cells overexpressing EGFR than (177)Lu-NT-AuNP at all amounts tested. The cross-fire effect of the β-particles emitted by (177)Lu may be valuable for eradicating BC cells in tumors that have low or moderate EGFR expression or cells that are not targeted by (177)Lu-T-AuNP as a consequence of heterogeneous intratumoral distribution. The radiation dose to the nucleus of a single MDA-MB-468 cell was 73.2 ± 6.7 Gy, whereas (177)Lu-NT-AuNP delivered 5.6 ± 0.6 Gy. We conclude that (177)Lu-T-AuNP is a promising novel radiation nanomedicine with potential application for treatment of TNBC, in which EGFR are often overexpressed.

RNA oxidation catalyzed by cytochrome c leads to its depurination and cross-linking, which may facilitate cytochrome c release from mitochondria

PubMed Central

Tanaka, Mikiei; Jaruga, Pawel; Küpfer, Pascal A.; Leumann, Christian J.; Dizdaroglu, Miral; Sonntag, William E.; Chock, P. Boon

2015-01-01

Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegen-erative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H2O2 or by the Fe(II)/ascorbate/H2O2 system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H2O2 relative to the Fe(II)/ascorbate/H2O2 system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H2O2 concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H2O2, it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA–cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress. PMID:22683603

Design and engineering of a man-made diffusive electron-transport protein

PubMed Central

Fry, Bryan A.; Solomon, Lee A.; Dutton, P. Leslie

2016-01-01

Maquettes are man-made cofactor-binding oxidoreductases designed from first principles with minimal reference to natural protein sequences. Here we focus on water-soluble maquettes designed and engineered to perform diffusive electron transport of the kind typically carried out by cytochromes, ferredoxins and flavodoxins and other small proteins in photosynthetic and respiratory energy conversion and oxido-reductive metabolism. Our designs were tested by analysis of electron transfer between heme maquettes and the well-known natural electron transporter, cytochrome c. Electron-transfer kinetics were measured from seconds to milliseconds by stopped-flow, while sub-millisecond resolution was achieved through laser photolysis of the carbon monoxide maquette heme complex. These measurements demonstrate electron transfer from the maquette to cytochrome c, reproducing the timescales and charge complementarity modulation observed in natural systems. The ionic strength dependence of inter-protein electron transfer from 9.7 × 106 M−1s−1 to 1.2 × 109 M−1s−1 follows a simple Debye-Hückel model for attraction between +8 net charged oxidized cytochrome c and −19 net charged heme maquette, with no indication of significant protein dipole moment steering. Successfully recreating essential components of energy conversion and downstream metabolism in man-made proteins holds promise for in vivo clinical intervention and for the production of fuel or other industrial products. PMID:26423266

The relationship between black carbon concentration and black smoke: A more general approach

NASA Astrophysics Data System (ADS)

Heal, Mathew R.; Quincey, Paul

2012-07-01

The black carbon (BC) component of ambient particulate matter is an important marker for combustion sources and for its impact on human health and radiative forcing. Extensive data archives exist for the black smoke metric, the historic measure of ambient particle darkness. An expression presented in earlier publications (Quincey, 2007; Quincey et al., 2011) for estimating BC concentrations from traditional black smoke measurements is shown to have limitations that can be addressed by using a more systematic approach to the issue of corrections for increasing darkening of the filter. The form of the more general relationship is shown to be an off-axis parabola rather than the on-axis parabola of the earlier work. Existing data from co-located black smoke and aethalometer measurements at 5 UK sites are reanalysed in this context. At very low concentrations of dark particles (British Black Smoke index < ˜10 μg m-3) a simple linear relationship BC (/μg m-3) ≈ 0.27·BSIBRITISH will suffice. A parabolic relationship, [BC/μgm]=√{5.2-1.1+1.5×BSI+62-13+19}-7.9-0.9+1.1, quantitatively similar to the previously published relationship will be more reliable for BSIBRITISH values up to 20-25 μg m-3. The full set of data available was fitted empirically to the off-axis parabola over the range 0-80 μg m-3 as the quadratic: [BC/μg m-3] = (0.27 ± 0.03) · BSIBRITISH - (4.0 ± 0.2) × 10-4(BSIBRITISH)2, but this curve is highly dependent on the variations between the individual data sets. Adding the extra complexity of the full off-axis parabolic relationship is unlikely to be justified in practical situations. All expressions apply also to the OECD definition of black smoke with the substitution BSIBRITISH = 0.85·BSIOECD. However, in common with the previous approach, they apply only to black smoke values obtained from standard black smoke samplers with 25 mm diameter filters and ˜2 m3 day-1 volumetric flow rate, and presume a value 16.6 m2 g-1 for the specific absorption of BC in ambient particulate matter measured by aethalometry. Fitting uncertainties correspond to imprecision in estimated BC of ±5%, ±12% and ±18% at BSIBRITISH of 5, 20 and 80 μg m-3, respectively. Spatial and temporal variation in particle ensemble optical properties contributes to uncertainty in BC quantification.

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

PubMed

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

2014-07-11

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

DsRNA-free transmissible hypovirulence associated with formation of intra-hyphal hyphae in Botrytis cinerea.

PubMed

Cao, Jian Bo; Zhou, Yu; Zhang, Lei; Zhang, Jing; Yang, Long; Qin, Li Hong; Jiang, Dao Hong; Li, Guo Qing; Huang, Hung-Chang

2011-07-01

A spontaneous mutant CanBc-3HV and its parental strain CanBc-3 of Botrytis cinerea were investigated in terms of pathogenicity, colony morphology, hypovirulence transmissibility, presence of double-stranded RNA (dsRNA), and formation of intra-hyphal hyphae (IH). Results showed that inoculation of CanBc-3HV on detached leaves of Brassica napus did not produce any visible necrotic lesions (20°C, 72h), whereas inoculation of CanBc-3 caused necrotic leaf lesions. Compared to CanBc-3, CanBc-3HV grew slowly, formed numerous mycelial sectors, sporulated sporadically and failed to produce sclerotia on potato dextrose agar (PDA) (20°C, 15d). Hypovirulence and the abnormal cultural characteristics of CanBc-3HV were transmissible from CanBc-3HV to CanBc-3 in pair cultures on PDA. However, the transmission was unsuccessful from CanBc-3HV to another virulent strain CanBc-2 of B. cinerea. These results suggest that transmission of the hypovirulence and the abnormal cultural characteristics of CanBc-3HV are strain-specific. No dsRNA was detected in mycelia of either CanBc-3HV or CanBc-3, implying that the hypovirulence of CanBc-3HV is caused by a transmissible element (TE) of non-RNA mycoviral origin. Formation of IH through self-infection was observed in CanBc-3HV, CanBc-3T1 (a hypovirulent derivative of CanBc-3 trans-infected by TE in CanBc-3HV), but was not observed in CanBc-3, suggesting that IH formation is associated with the hypovirulence of CanBc-3HV. To our knowledge, this is the first report of dsRNA-free transmissible hypovirulence associated with IH formation in B. cinerea. Copyright © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Arginine functionalized bacterial cellulose nanofibers containing gel as an effective wound dressing; in vitro and in vivo evaluation.

PubMed

Feizabadi, Farideh; Minaiyan, Mohsan; Taheri, Azade

2018-02-19

Nanofibers such as bacterial cellulose nanofibers (BC-NFs) have gained increasing attention for use in wound dressings. Topical application of arginine can stimulate wound healing significantly. In order to promote the wound healing process, arginine functionalized BC-NFs containing gel (Arg-BC-NFs gel) was prepared by the electrostatic attachment of arginine on the surface of BC-NFs. The effect of pH was evaluated on the amount of the attached arginine on the BC-NFs surface. The attachment of arginine on BC-NFs surface was investigated by FTIR spectroscopy. The morphology of Arg-BC-NFs was evaluated using FESEM. The viscosity and spreadability of Arg-BC-NFs and the release of arginine from Arg-BC-NFs were evaluated. The effectiveness of Arg-BC-NFs gel was assessed in a full thickness wound model in rats. Re-epithelization, collagen deposition and neovascularization were investigated in the wound tissues using histological and immunohistochemical analysis. FTIR spectra and the zeta potential of BC-NFs confirmed the surface modification of BC-NFs by arginine. FESEM images showed the nanofibrous structure of Arg-BC-NFs. The release of arginine from Arg-BC-NFs gel was in a sustained release manner for 24 h. The appropriate viscosity and spreadability of Arg-BC-NFs gel confirmed its easy topical application. In vivo studies revealed that Arg-BC-NFs gel promoted wound closure at a faster rate than BC-NFs gel and arginine solution. Moreover, faster and more organized re-epithelialization, angiogenesis and collagen deposition were achieved in Arg-BC-NFs gel treated group in comparison to other groups. Arg-BC-NFs gel can be introduced as an effective wound dressing for acute wounds. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Characterization of MtoD from Sideroxydans lithotrophicus: a cytochrome c electron shuttle used in lithoautotrophic growth

DOE PAGES

Beckwith, Christopher R.; Edwards, Marcus J.; Lawes, Matthew; ...

2015-04-28

The autotrophic Sideroxydans lithotrophicus ES-1 can grow by coupling the oxidation of ferrous iron to the reduction of oxygen. Soluble ferrous iron is oxidized at the surface of the cell by an MtoAB porin-cytochrome complex that functions as an electron conduit through the outer membrane. Electrons are then transported to the cytoplasmic membrane where they are used to generate proton motive force (PMF) (for ATP synthesis) and NADH for autotrophic processes such as carbon fixation. As part of the mtoAB gene cluster, S. lithotrophicus also contains the gene mtoD that is proposed to encode a cytochrome c protein. We isolatedmore » mtoD from a Shewanella oneidensis expression system where the mtoD gene was expressed on a pBAD plasmid vector. Biochemical, biophysical, and crystallographic characterization of the purified MtoD revealed it as an 11 kDa monomeric protein containing a single heme. Sequence and structural alignment indicated that MtoD belonged to the class-1 cytochrome c family and had a similar fold to ferricytochrome c552 family, however the MtoD heme is bis-histidine coordinated and is substantially more exposed than the hemes of other family members. The reduction potential of the MtoD heme at pH 7 was +155 mV vs. Standard Hydrogen Electrode, which is approximately 100 mV lower than that of mitochondrial cytochrome c. Consideration of the properties of MtoD in the context of the potential respiratory partners identified from the genome suggests that MtoD could associate to multiple electron transfer partners as the primary periplasmic electron shuttle.« less

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

PubMed Central

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

2006-01-01

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

Analysis of transpacific transport of black carbon during HIPPO-3: implications for black carbon aging

NASA Astrophysics Data System (ADS)

Shen, Z.; Liu, J.; Horowitz, L. W.; Henze, D. K.; Fan, S.; Levy, H., II; Mauzerall, D. L.; Lin, J.-T.; Tao, S.

2014-06-01

Long-range transport of black carbon (BC) is a growing concern as a result of the efficiency of BC in warming the climate and its adverse impact on human health. We study transpacific transport of BC during HIPPO-3 using a combination of inverse modeling and sensitivity analysis. We use the GEOS-Chem chemical transport model and its adjoint to constrain Asian BC emissions and estimate the source of BC over the North Pacific. We find that different sources of BC dominate the transport to the North Pacific during the southbound (29 March 2010) and northbound (13 April 2010) measurements in HIPPO-3. While biomass burning in Southeast Asia (SE) contributes about 60% of BC in March, more than 90% of BC comes from fossil fuel and biofuel combustion in East Asia (EA) during the April mission. GEOS-Chem simulations generally resolve the spatial and temporal variation of BC concentrations over the North Pacific, but are unable to reproduce the low and high tails of the observed BC distribution. We find that the optimized BC emissions derived from inverse modeling fail to improve model simulations significantly. This failure indicates that uncertainties in BC removal as well as transport, rather than in emissions, account for the major biases in GEOS-Chem simulations of BC over the North Pacific. The aging process, transforming BC from hydrophobic into hydrophilic form, is one of the key factors controlling wet scavenging and remote concentrations of BC. Sensitivity tests on BC aging (ignoring uncertainties of other factors controlling BC long range transport) suggest that in order to fit HIPPO-3 observations, the aging timescale of anthropogenic BC from EA may be several hours (faster than assumed in most global models), while the aging process of biomass burning BC from SE may occur much slower, with a timescale of a few days. To evaluate the effects of BC aging and wet deposition on transpacific transport of BC, we develop an idealized model of BC transport. We find that the mid-latitude air masses sampled during HIPPO-3 may have experienced a series of precipitation events, particularly near the EA and SE source region. Transpacific transport of BC is sensitive to BC aging when the aging rate is fast; this sensitivity peaks when the aging timescale is in the range of 1-1.5 d. Our findings indicate that BC aging close to the source must be simulated accurately at a process level in order to simulate better the global abundance and climate forcing of BC.

Enhanced Solar Energy Absorption by Internally-mixed Black Carbon in Snow Grains

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

Flanner, M. G.; Liu, Xiaohong; Zhou, Cheng

2012-05-30

Here we explore light absorption by snowpack containing black carbon (BC) particles residing within ice grains. Basic considerations of particle volumes and BC/snow mass concentrations show that there are generally 0:05-109 BC particles for each ice grain. This suggests that internal BC is likely distributed as multiple inclusions within ice grains, and thus the dynamic effective medium approximation (DEMA) (Chylek and Srivastava, 1983) is a more appropriate optical representation for BC/ice composites than coated-sphere or standard mixing approximations. DEMA calculations show that the 460 nm absorption cross-section of BC/ice composites, normalized to the mass of BC, is typically enhanced bymore » factors of 1.8-2.1 relative to interstitial BC. BC effective radius is the dominant cause of variation in this enhancement, compared with ice grain size and BC volume fraction. We apply two atmospheric aerosol models that simulate interstitial and within-hydrometeor BC lifecycles. Although only {approx}2% of the atmospheric BC burden is cloud-borne, 71-83% of the BC deposited to global snow and sea-ice surfaces occurs within hydrometeors. Key processes responsible for within-snow BC deposition are development of hydrophilic coatings on BC, activation of liquid droplets, and subsequent snow formation through riming or ice nucleation by other species and aggregation/accretion of ice particles. Applying deposition fields from these aerosol models in offline snow and sea-ice simulations, we calculate that 32-73% of BC in global surface snow resides within ice grains. This fraction is smaller than the within-hydrometeor deposition fraction because meltwater flux preferentially removes internal BC, while sublimation and freezing within snowpack expose internal BC. Incorporating the DEMA into a global climate model, we simulate increases in BC/snow radiative forcing of 43-86%, relative to scenarios that apply external optical properties to all BC. We show that snow metamorphism driven by diffusive vapor transfer likely proceeds too slowly to alter the mass of internal BC while it is radiatively active, but neglected processes like wind pumping and convection may play much larger roles. These results suggest that a large portion of BC in surface snowpack may reside within ice grains and increase BC/snow radiative forcing, although measurements to evaluate this are lacking. Finally, previous studies of BC/snow forcing that neglected this absorption enhancement are not necessarily biased low, because of application of absorption-enhancing sulfate coatings to hydrophilic BC, neglect of coincident absorption by dust in snow, and implicit treatment of cloud-borne BC resulting in longer-range transport.« less

Cytochromes P450

PubMed Central

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

2011-01-01

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

Changes in Fire-Derived Soil Black Carbon Storage in a Sub-humid Woodland

NASA Astrophysics Data System (ADS)

White, J. D.; Yao, J.; Murray, D. B.; Hockaday, W. C.

2014-12-01

Fire-derived black carbon (BC) in soil, including charcoal, represents a potentially important fraction of terrestrial carbon cycling due to its presumed long persistence in soil. Interpretation of site BC retention is important for assessing feedbacks to ecosystem processes including nutrient and water cycling. However, interaction between vegetation disturbance, BC formation, and off site transport may exist that complicate interpretation of BC addition to soils from wildfire or prescribed burns directly. To investigate the relationship between disturbance and site retention on soil BC, we determined BC concentrations for a woodland in central Texas, USA, from study plots in hilly terrain with a fire scar dendrochronology spanning 100 years. BC values were determined from 13C nuclear magnetic resonance (NMR) spectroscopy. Estimated values showed mean BC concentration of 2.73 ± 3.06 g BC kg-1 (0.91 ± 0.51 kg BC m-2) for sites with fire occurrence within the last 40 years compared with BC values of1.21 ± 1.70 g BC kg-1 soil (0.18 ± 0.14 kg BC m-2) for sites with fire 40 - 100 years ago. Sites with no tree ring evidence of fire during the last 100 years had the lowest mean soil BC concentration of 0.05 ± 0.11 g BC kg-1 (0.02 ± 0.03 kg BC m-2). Molecular proxies of stability (lignin/N) and decomposition (Alkyl C/O-Alky C) showed no differences across the sites, indicating that low potential for BC mineralization. Modeled soil erosion and time since fire from fire scar data showed that soil BC concentrations were inversely correlated. A modified the ecosystem process model, Biome-BGC, was also used simulate the effects of fire disturbance with different severities and seasonality on C cycling related to the BC production, effect on soil water availability, and off-site transport. Results showed that BC impacts on ecosystem processes, including net ecosystem exchange and leaf area development, were predominantly related to fire frequency. Site BC loss rates were affected by initial slope-affected erosion, fire severity, vegetation type, and rate of vegetation recovery. The simulation results showed that fire types, such as high severity, was generally associated with low site BC retention related to low vertical transfer of BC into soils, buoyancy of BC particles, and surface runoff from unvegetated soils.

Enhanced leaf photosynthesis as a target to increase grain yield: insights from transgenic rice lines with variable Rieske FeS protein content in the cytochrome b6 /f complex.

PubMed

Yamori, Wataru; Kondo, Eri; Sugiura, Daisuke; Terashima, Ichiro; Suzuki, Yuji; Makino, Amane

2016-01-01

Although photosynthesis is the most important source for biomass and grain yield, a lack of correlation between photosynthesis and plant yield among different genotypes of various crop species has been frequently observed. Such observations contribute to the ongoing debate whether enhancing leaf photosynthesis can improve yield potential. Here, transgenic rice plants that contain variable amounts of the Rieske FeS protein in the cytochrome (cyt) b6 /f complex between 10 and 100% of wild-type levels have been used to investigate the effect of reductions of these proteins on photosynthesis, plant growth and yield. Reductions of the cyt b6 /f complex did not affect the electron transport rates through photosystem I but decreased electron transport rates through photosystem II, leading to concomitant decreases in CO2 assimilation rates. There was a strong control of plant growth and grain yield by the rate of leaf photosynthesis, leading to the conclusion that enhancing photosynthesis at the single-leaf level would be a useful target for improving crop productivity and yield both via conventional breeding and biotechnology. The data here also suggest that changing photosynthetic electron transport rates via manipulation of the cyt b6 /f complex could be a potential target for enhancing photosynthetic capacity in higher plants. © 2015 John Wiley & Sons Ltd.

Topography of tyrosine residues and their involvement in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes

PubMed Central

Kapralov, Alexandr A.; Yanamala, Naveena; Tyurina, Yulia Y.; Castro, Laura; Arias, Alejandro Samhan; Vladimirov, Yuri A.; Maeda, Akihiro; Weitz, Andrew A.; Peterson, Jim; Mylnikov, Danila; Demicheli, Verónica; Tortora, Verónica; Klein-Seetharaman, Judith; Radi, Rafael; Kagan, Valerian E.

2011-01-01

Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective towards peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues – via the generation of tyrosyl radicals (Tyr•) - are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H2O2-induced Tyr radicals in electron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H2O2-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance (NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex. PMID:21550335

Elevated extracellular [K+] inhibits death-receptor- and chemical-mediated apoptosis prior to caspase activation and cytochrome c release.

PubMed Central

Thompson, G J; Langlais, C; Cain, K; Conley, E C; Cohen, G M

2001-01-01

Efflux of intracellular K(+) and cell shrinkage are features of apoptosis in many experimental systems, and a regulatory role has been proposed for cytoplasmic [K(+)] in initiating apoptosis. We have investigated this in both death-receptor-mediated and chemical-induced apoptosis. Using Jurkat T cells pre-loaded with the K(+) ion surrogate (86)Rb(+), we have demonstrated an efflux of intracellular K(+) during apoptosis that was concomitant with, but did not precede, other apoptotic changes, including phosphatidylserine externalization, mitochondrial depolarization and cell shrinkage. To further clarify the role of K(+) ions in apoptosis, cytoprotection by elevated extracellular [K(+)] was studied. Induction of apoptosis by diverse death-receptor and chemical stimuli in two cell lines was inhibited prior to phosphatidylserine externalization, mitochondrial depolarization, cytochrome c release and caspase activation. Using a cell-free system, we have demonstrated a novel mechanism by which increasing [K(+)] inhibited caspase activation. In control dATP-activated lysates, Apaf-1 oligomerized to a biologically active caspase processing approximately 700 kDa complex and an inactive approximately 1.4 MDa complex. Increasing [K(+)] inhibited caspase activation by preventing formation of the approximately 700 kDa complex, but not of the inactive complex. Thus intracellular and extracellular [K(+)] markedly affect caspase activation and the initiation of apoptosis induced by both death-receptor ligation and chemical stress. PMID:11415444

A stromal region of cytochrome b6f subunit IV is involved in the activation of the Stt7 kinase in Chlamydomonas

PubMed Central

Zito, Francesca; Blangy, Stéphanie; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles

2017-01-01

The cytochrome (cyt) b6f complex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cyt b6f activates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplast petD gene, coding for subunit IV of the cyt b6f complex, and complementation of a ΔpetD host strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cyt b6f subunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cyt b6f and recombinant Stt7 kinase domain show that cyt b6f enhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cyt b6f complex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane. PMID:29078388

Do contemporary (1980-2015) emissions determine the elemental carbon deposition trend at Holtedahlfonna glacier, Svalbard?

NASA Astrophysics Data System (ADS)

Ruppel, Meri M.; Soares, Joana; Gallet, Jean-Charles; Isaksson, Elisabeth; Martma, Tõnu; Svensson, Jonas; Kohler, Jack; Pedersen, Christina A.; Manninen, Sirkku; Korhola, Atte; Ström, Johan

2017-10-01

The climate impact of black carbon (BC) is notably amplified in the Arctic by its deposition, which causes albedo decrease and subsequent earlier snow and ice spring melt. To comprehensively assess the climate impact of BC in the Arctic, information on both atmospheric BC concentrations and deposition is essential. Currently, Arctic BC deposition data are very scarce, while atmospheric BC concentrations have been shown to generally decrease since the 1990s. However, a 300-year Svalbard ice core showed a distinct increase in EC (elemental carbon, proxy for BC) deposition from 1970 to 2004 contradicting atmospheric measurements and modelling studies. Here, our objective was to decipher whether this increase has continued in the 21st century and to investigate the drivers of the observed EC deposition trends. For this, a shallow firn core was collected from the same Svalbard glacier, and a regional-to-meso-scale chemical transport model (SILAM) was run from 1980 to 2015. The ice and firn core data indicate peaking EC deposition values at the end of the 1990s and lower values thereafter. The modelled BC deposition results generally support the observed glacier EC variations. However, the ice and firn core results clearly deviate from both measured and modelled atmospheric BC concentration trends, and the modelled BC deposition trend shows variations seemingly independent from BC emission or atmospheric BC concentration trends. Furthermore, according to the model ca. 99 % BC mass is wet-deposited at this Svalbard glacier, indicating that meteorological processes such as precipitation and scavenging efficiency have most likely a stronger influence on the BC deposition trend than BC emission or atmospheric concentration trends. BC emission source sectors contribute differently to the modelled atmospheric BC concentrations and BC deposition, which further supports our conclusion that different processes affect atmospheric BC concentration and deposition trends. Consequently, Arctic BC deposition trends should not directly be inferred based on atmospheric BC measurements, and more observational BC deposition data are required to assess the climate impact of BC in Arctic snow.

NEW EVIDENCE FOR MORPHOLOGICAL AND GENETIC VARIATION IN THE COSMOPOLITAN COCCOLITHOPHORE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) FROM THE COX1b-ATP4 GENES(1).

PubMed

Hagino, Kyoko; Bendif, El Mahdi; Young, Jeremy R; Kogame, Kazuhiro; Probert, Ian; Takano, Yoshihito; Horiguchi, Takeo; de Vargas, Colomban; Okada, Hisatake

2011-10-01

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is a cosmopolitan coccolithophore occurring from tropical to subpolar waters and exhibiting variations in morphology of coccoliths possibly related to environmental conditions. We examined morphological characters of coccoliths and partial mitochondrial sequences of the cytochrome oxidase 1b (cox1b) through adenosine triphosphate synthase 4 (atp4) genes of 39 clonal E. huxleyi strains from the Atlantic and Pacific Oceans, Mediterranean Sea, and their adjacent seas. Based on the morphological study of culture strains by SEM, Type O, a new morphotype characterized by coccoliths with an open central area, was separated from existing morphotypes A, B, B/C, C, R, and var. corona, characterized by coccoliths with central area elements. Molecular phylogenetic studies revealed that E. huxleyi consists of at least two mitochondrial sequence groups with different temperature preferences/tolerances: a cool-water group occurring in subarctic North Atlantic and Pacific and a warm-water group occurring in the subtropical Atlantic and Pacific and in the Mediterranean Sea. © 2011 Phycological Society of America.

Structure-Guided Design of Peptides as Tools to Probe the Protein-Protein Interaction between Cullin-2 and Elongin BC Substrate Adaptor in Cullin RING E3 Ubiquitin Ligases.

PubMed

Cardote, Teresa A F; Ciulli, Alessio

2017-09-21

Cullin RING E3 ubiquitin ligases (CRLs) are large dynamic multi-subunit complexes that control the fate of many proteins in cells. CRLs are attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. Herein we describe a structure-guided biophysical approach to probe the protein-protein interaction (PPI) between the Cullin-2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel-Lindau complex (CRL2 VHL ) using peptides. Two peptides were shown to bind at the targeted binding site on Elongin C, named the "EloC site", with micromolar dissociation constants, providing a starting point for future optimization. Our results suggest ligandability of the EloC binding site to short linear peptides, unveiling the opportunity and challenges to develop small molecules that have the potential to target selectively the Cul2-adaptor PPI within CRLs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insight into Oxide-Bridged Heterobimetallic Al/Zr Olefin Polymerization Catalysts.

PubMed

Boulho, Cédric; Zijlstra, Harmen S; Hofmann, Alexander; Budzelaar, Peter H M; Harder, Sjoerd

2016-11-21

Reaction of (TBBP)AlMe⋅THF with [Cp* 2 Zr(Me)OH] gave [(TBBP)Al(THF)-O-Zr(Me)Cp* 2 ] (TBBP=3,3',5,5'-tetra-tBu-2,2'-biphenolato). Reaction of [DIPPnacnacAl(Me)-O-Zr(Me)Cp 2 ] with [PhMe 2 NH] + [B(C 6 F 5 ) 4 ] - gave a cationic Al/Zr complex that could be structurally characterized as its THF adduct [(DIPPnacnac)Al(Me)-O-Zr(THF)Cp 2 ] + [B(C 6 F 5 ) 4 ] - (DIPPnacnac=HC[(Me)C=N(2,6-iPr 2 -C 6 H 3 )] 2 ). The first complex polymerizes ethene in the presence of an alkylaluminum scavenger but in the absence of methylalumoxane (MAO). The adduct cation is inactive under these conditions. Theoretical calculations show very high energy barriers (ΔG=40-47 kcal mol -1 ) for ethene insertion with a bridged AlOZr catalyst. This is due to an unfavorable six-membered-ring transition state, in which the methyl group bridges the metal and ethene with an obtuse metal-Me-C angle that prevents synchronized bond-breaking and making. A more-likely pathway is dissociation of the Al-O-Zr complex into an aluminate and the active polymerization catalyst [Cp* 2 ZrMe] + . © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Measured Wavelength-Dependent Absorption Enhancement of Internally Mixed Black Carbon with Absorbing and Nonabsorbing Materials.

PubMed

You, Rian; Radney, James G; Zachariah, Michael R; Zangmeister, Christopher D

2016-08-02

Optical absorption spectra of laboratory generated aerosols consisting of black carbon (BC) internally mixed with nonabsorbing materials (ammonium sulfate, AS, and sodium chloride, NaCl) and BC with a weakly absorbing brown carbon surrogate derived from humic acid (HA) were measured across the visible to near-IR (550 to 840 nm). Spectra were measured in situ using a photoacoustic spectrometer and step-scanning a supercontinuum laser source with a tunable wavelength and bandwidth filter. BC had a mass-specific absorption cross section (MAC) of 7.89 ± 0.25 m(2) g(-1) at λ = 550 nm and an absorption Ångström exponent (AAE) of 1.03 ± 0.09 (2σ). For internally mixed BC, the ratio of BC mass to the total mass of the mixture was chosen as 0.13 to mimic particles observed in the terrestrial atmosphere. The manner in which BC mixed with each material was determined from transmission electron microscopy (TEM). AS/BC and HA/BC particles were fully internally mixed, and the BC was both internally and externally mixed for NaCl/BC particles. The AS/BC, NaCl/BC, and HA/BC particles had AAEs of 1.43 ± 0.05, 1.34 ± 0.06, and 1.91 ± 0.05, respectively. The observed absorption enhancement of mixed BC relative to the pure BC was wavelength dependent for AS/BC and decreased from 1.5 at λ = 550 nm with increasing wavelength while the NaCl/BC enhancement was essentially wavelength independent. For HA/BC, the enhancement ranged from 2 to 3 and was strongly wavelength dependent. Removal of the HA absorption contribution to enhancement revealed that the enhancement was ≈1.5 and independent of wavelength.

Plasma and Electro-energetic Physics

DTIC Science & Technology

2012-03-07

Dynamical Equations (with complex surfaces ): Relativistic Lorentz Force Law for relativistic momentum p and velocity u: tDcJcH tBcE   /)/1()/4...0.1-1 s • 3D, high-fidelity, parallel modeling of high energy density fields and particles in complex geometry with some surface effects...cathodes (500 µm separation) Tang, AFRL/RD 12 DISTRIBUTION A: Approved for public release; distribution is unlimited. ICEPIC simulations Equipotential

Four unexpected lanthanide coordination polymers involving in situ reaction of solvent N, N-Dimethylformamide

NASA Astrophysics Data System (ADS)

Jin, Jun-Cheng; Tong, Wen-Quan; Fu, Ai-Yun; Xie, Cheng-Gen; Chang, Wen-Gui; Wu, Ju; Xu, Guang-Nian; Zhang, Ya-Nan; Li, Jun; Li, Yong; Yang, Peng-Qi

2015-05-01

Four unexpected 2D lanthanide coordination polymers have been synthesized through in situ reactions of DMF solvent under solvothermal conditions. The isostructural complexes 1-3 contain four types of 21 helical chains. While the Nd(III) ions are bridged through μ2-HIDC2- and oxalate to form a 2D sheet along the bc plane without helical character in 4. Therefore, complex 1 exhibits bright red solid-state phosphorescence upon exposure to UV radiation at room temperature.

Resolving Size Distribution of Black Carbon Internally Mixed With Snow: Impact on Snow Optical Properties and Albedo

NASA Astrophysics Data System (ADS)

He, Cenlin; Liou, Kuo-Nan; Takano, Yoshi

2018-03-01

We develop a stochastic aerosol-snow albedo model that explicitly resolves size distribution of aerosols internally mixed with various snow grains. We use the model to quantify black carbon (BC) size effects on snow albedo and optical properties for BC-snow internal mixing. Results show that BC-induced snow single-scattering coalbedo enhancement and albedo reduction decrease by a factor of 2-3 with increasing BC effective radii from 0.05 to 0.25 μm, while polydisperse BC results in up to 40% smaller visible single-scattering coalbedo enhancement and albedo reduction compared to monodisperse BC with equivalent effective radii. We further develop parameterizations for BC size effects for application to climate models. Compared with a realistic polydisperse assumption and observed shifts to larger BC sizes in snow, respectively, assuming monodisperse BC and typical atmospheric BC effective radii could lead to overestimates of 24% and 40% in BC-snow albedo forcing averaged over different BC and snow conditions.

Evidence for prehistoric origins of Egyptian mummification in late Neolithic burials.

PubMed

Jones, Jana; Higham, Thomas F G; Oldfield, Ron; O'Connor, Terry P; Buckley, Stephen A

2014-01-01

Traditional theories on ancient Egyptian mummification postulate that in the prehistoric period (i.e. the Neolithic and Chalcolithic periods, 5th and 4th millennia B.C.) bodies were naturally desiccated through the action of the hot, dry desert sand. Although molding of the body with resin-impregnated linen is believed to be an early Pharaonic forerunner to more complex processes, scientific evidence for the early use of resins in artificial mummification has until now been limited to isolated occurrences during the late Old Kingdom (c. 2200 B.C.), their use becoming more apparent during the Middle Kingdom (c. 2000-1600 BC). We examined linen wrappings from bodies in securely provenanced tombs (pit graves) in the earliest recorded ancient Egyptian cemeteries at Mostagedda in the Badari region (Upper Egypt). Our investigations of these prehistoric funerary wrappings using a combination of gas chromatography-mass spectrometry (GC-MS) and thermal desorption/pyrolysis (TD/Py)-GC-MS have identified a pine resin, an aromatic plant extract, a plant gum/sugar, a natural petroleum source, and a plant oil/animal fat in directly AMS-dated funerary wrappings. Predating the earliest scientific evidence by more than a millennium, these embalming agents constitute complex, processed recipes of the same natural products, in similar proportions, as those utilized at the zenith of Pharaonic mummification some 3,000 years later. The antibacterial properties of some of these ingredients and the localized soft-tissue preservation that they would have afforded lead us to conclude that these represent the very beginnings of experimentation that would evolve into the famous mummification practice of the Pharaonic period.

Evidence for Prehistoric Origins of Egyptian Mummification in Late Neolithic Burials

PubMed Central

Jones, Jana; Higham, Thomas F. G.; Oldfield, Ron; O'Connor, Terry P.; Buckley, Stephen A.

2014-01-01

Traditional theories on ancient Egyptian mummification postulate that in the prehistoric period (i.e. the Neolithic and Chalcolithic periods, 5th and 4th millennia B.C.) bodies were naturally desiccated through the action of the hot, dry desert sand. Although molding of the body with resin-impregnated linen is believed to be an early Pharaonic forerunner to more complex processes, scientific evidence for the early use of resins in artificial mummification has until now been limited to isolated occurrences during the late Old Kingdom (c. 2200 B.C.), their use becoming more apparent during the Middle Kingdom (c. 2000-1600 BC). We examined linen wrappings from bodies in securely provenanced tombs (pit graves) in the earliest recorded ancient Egyptian cemeteries at Mostagedda in the Badari region (Upper Egypt). Our investigations of these prehistoric funerary wrappings using a combination of gas chromatography-mass spectrometry (GC-MS) and thermal desorption/pyrolysis (TD/Py)-GC-MS have identified a pine resin, an aromatic plant extract, a plant gum/sugar, a natural petroleum source, and a plant oil/animal fat in directly AMS-dated funerary wrappings. Predating the earliest scientific evidence by more than a millennium, these embalming agents constitute complex, processed recipes of the same natural products, in similar proportions, as those utilized at the zenith of Pharaonic mummification some 3,000 years later. The antibacterial properties of some of these ingredients and the localized soft-tissue preservation that they would have afforded lead us to conclude that these represent the very beginnings of experimentation that would evolve into the famous mummification practice of the Pharaonic period. PMID:25118605

Roles of Neuroglobin Binding to Mitochondrial Complex III Subunit Cytochrome c1 in Oxygen-Glucose Deprivation-Induced Neurotoxicity in Primary Neurons.

PubMed

Yu, Zhanyang; Zhang, Yu; Liu, Ning; Yuan, Jing; Lin, Li; Zhuge, Qichuan; Xiao, Jian; Wang, Xiaoying

2016-07-01

Neuroglobin (Ngb) is a tissue globin specifically expressed in brain neurons. Recent studies by our laboratory and others have demonstrated that Ngb is protective against stroke and related neurological disorders, but the mechanisms remain poorly understood. We previously identified cytochrome c1 (Cyc1) as an Ngb-interacting molecule by yeast two-hybrid screening. Cyc1 is a subunit of mitochondria complex III, which is a component of mitochondrial respiratory chain and a major source of reactive oxygen species (ROS) production under both physiological and pathological conditions. In this study, we for the first time defined Ngb-Cyc1 binding, and investigated its roles in oxygen-glucose deprivation (OGD)/reoxygenation-induced neurotoxicity and ROS production in primary neurons. Immunocytochemistry and co-immunoprecipitation validated Ngb-Cyc1 binding, which was significantly increased by OGD and Ngb overexpression. We found 4 h OGD with/without 4 h reoxygenation significantly increased complex III activity, but this activity elevation was significantly attenuated in three groups of neurons: Ngb overexpression, specific complex III inhibitor stigmatellin, or stigmatellin plus Ngb overexpression, whereas there was no significant differences between these three groups, suggesting Ngb-Cyc1 binding may function in suppressing OGD-mediated complex III activity elevation. Importantly, these three groups of neurons also showed significant decreases in OGD-induced superoxide anion generation and neurotoxicity. These results suggest that Ngb can bind to mitochondrial complex III subunit Cyc1, leading to suppression of OGD-mediated complex III activity and subsequent ROS production elevation, and eventually reduction of OGD-induced neurotoxicity. This molecular signaling cascade may be at least part of the mechanisms of Ngb neuroprotection against OGD-induced neurotoxicity.

[Molecular epidemiology and transmission of HIV-1 infection in Zhejiang province, 2015].

PubMed

Yang, J Z; Chen, W J; Zhang, W J; He, L; Zhang, J F; Pan, X H

2017-11-10

Objective: To understand the distribution of HIV-1 subtype diversity and its transmission characteristics in Zhejiang province. Methods: A total of 302 newly diagnosed HIV-1 positive patients were selected through stratified random sampling in Zhejiang in 2015. HIV-1 pol genes were sequenced successfully with reverse transcription PCR/nested PCR and phylogenetic analysis was conducted for 276 patients. Then a molecular epidemiologic study was performed combined with field epidemiological investigation. Results: Of 276 sequence samples analyzed, 122 CRF07_BC strains (44.2%), 103 CRF01_AE strains (37.3%), 17 CRF08_BC strains (6.1%), 9 B strains (3.2%), 6 CRF55_01B strains (2.2%), 5 C strains (1.8%), 1 CRF59_01B strain (0.4%), 1 CRF67_01B strain (0.4%), 1 A1 strain (0.4%), and 11 URFs strains (4.0%) were identified. Phylogenetic analysis revealed 16 clusters with only 15.1% (34/225) sequences involved among CRF07_BC and CRF01_AE strains. The clustered cases in MSM were higher than that in populations with other transmission routes. And clusters existed between the populations with different transmission routes. Conclusion: The major strains of HIV-1 in Zhejiang are CRF07_BC and CRF01_AE. The HIV subtypes showed more complexity in Zhejiang. It is necessary to strengthen the surveillance for HIV subtypes, carry out classified management and conduct effective prevention and control in the population at high risk.

KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer.

PubMed

Alshareeda, A T; Negm, O H; Green, A R; Nolan, C C; Tighe, P; Albarakati, N; Sultana, R; Madhusudan, S; Ellis, I O; Rakha, E A

2015-06-09

It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC. A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry. KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables. This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

Comparison of the protein-protein interfaces in the p53-DNA crystal structures: towards elucidation of the biological interface.

PubMed

Ma, Buyong; Pan, Yongping; Gunasekaran, K; Venkataraghavan, R Babu; Levine, Arnold J; Nussinov, Ruth

2005-03-15

p53, the tumor suppressor protein, functions as a dimer of dimers. However, how the tetramer binds to the DNA is still an open question. In the crystal structure, three copies of the p53 monomers (containing chains A, B, and C) were crystallized with the DNA-consensus element. Although the structure provides crucial data on the p53-DNA contacts, the active oligomeric state is unclear because the two dimeric (A-B and B-C) interfaces present in the crystal cannot both exist in the tetramer. Here, we address the question of which of these two dimeric interfaces may be more biologically relevant. We analyze the sequence and structural properties of the p53-p53 dimeric interfaces and carry out extensive molecular dynamics simulations of the crystal structures of the human and mouse p53 dimers. We find that the A-B interface residues are more conserved than those of the B-C. Molecular dynamics simulations show that the A-B interface can provide a stable DNA-binding motif in the dimeric state, unlike B-C. Our results indicate that the interface between chains A-B in the p53-DNA complex constitutes a better candidate for a stable biological interface, whereas the B-C interface is more likely to be due to crystal packing. Thus, they have significant implications toward our understanding of DNA binding by p53 as well as p53-mediated interactions with other proteins.

Monumental heritage exposure to urban black carbon pollution

NASA Astrophysics Data System (ADS)

Patrón, D.; Lyamani, H.; Titos, G.; Casquero-Vera, J. A.; Cardell, C.; Močnik, G.; Alados-Arboledas, L.; Olmo, F. J.

2017-12-01

In this study, aerosol light-absorption measurements obtained at three sites during a winter campaign were used to analyse and identify the major sources of Black Carbon (BC) particles in and around the Alhambra monument, a UNESCO World Heritage Site that receives over 2 million visitors per year. The Conditional Bivariate Probability Function and the Aethalometer model were employed to identify the main sources of BC particles and to estimate the contributions of biomass burning and fossil fuel emissions to the total Equivalent Black Carbon (EBC) concentrations over the monumental complex. Unexpected high levels of EBC were found at the Alhambra, comparable to those measured in relatively polluted European urban areas during winter. EBC concentrations above 3.0 μg/m3, which are associated with unacceptable levels of soiling and negative public reactions, were observed at Alhambra monument on 13 days from 12 October 2015 to 29 February 2016, which can pose a risk to its long-term conservation and may cause negative social and economic impacts. It was found that road traffic emissions from the nearby urban area and access road to the Alhambra were the main sources of BC particles over the monument. However, biomass burning emissions were found to have very small impact on EBC concentrations at the Alhambra. The highest EBC concentrations were observed during an extended stagnant episode associated with persistent high-pressure systems, reflecting the large impact that can have these synoptic conditions on BC over the Alhambra.

Black carbon aerosol size in snow.

PubMed

Schwarz, J P; Gao, R S; Perring, A E; Spackman, J R; Fahey, D W

2013-01-01

The effect of anthropogenic black carbon (BC) aerosol on snow is of enduring interest due to its consequences for climate forcing. Until now, too little attention has been focused on BC's size in snow, an important parameter affecting BC light absorption in snow. Here we present first observations of this parameter, revealing that BC can be shifted to larger sizes in snow than are typically seen in the atmosphere, in part due to the processes associated with BC removal from the atmosphere. Mie theory analysis indicates a corresponding reduction in BC absorption in snow of 40%, making BC size in snow the dominant source of uncertainty in BC's absorption properties for calculations of BC's snow albedo climate forcing. The shift reduces estimated BC global mean snow forcing by 30%, and has scientific implications for our understanding of snow albedo and the processing of atmospheric BC aerosol in snowfall.

[Prostate cancer diagnostic by saturation randomized biopsy versus rigid targeted biopsy].

PubMed

Defontaines, J; Salomon, L; Champy, C; Cholley, I; Chiaradia, M; de la Taille, A

2017-12-01

Optimal diagram teaming up randomized biopsy (BR) to targeted biopsy (BC) is still missing for the diagnostic of prostate cancer (CP). This study compares diagram of 6, 12 or 18 BR with or without BC rigid. Between January 2014 and May 2016, 120 patients had prostate biopsy BR and BC. Each patient had 18 BR and BC. Results compared sextant (6 BR), standard (12 BR) and saturation (18 BR) protocol with or without the adding of BC for the detection of CP. Rectal examination was normal, mean PSA at 8.99ng/mL and mean volume at 54cm 3 . It was first round for 48% of patients. Forty-four cancers were found by the group 18 BR+BC (control). The detection rate was respectively, for 6, 12 and 18 BR of 61%, 82% and 91%. The add of BC increased this detection of +27% for 6 BR+BC, +13% for 12 BR+BC and +9% for 18 BR+BC. BC found 70% of all CP. Nine percent of CP were missed by BR only. Significant CP (Gleason≥7) diagnostic was the same for 12 BR+BC and 18 BR+BC. The add of BC to BR increase the detection of CP by 10%. Twelve BR+BC is the optimal diagram for the diagnostic of CP finding 95% of CP and 97% of significant CP. 4. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Long-term landscape evolution of the Basal Complexes of Fuerteventura and La Gomera Islands, Canary Archipelago

NASA Astrophysics Data System (ADS)

Mansour, Sherif; Glasmacher, Ulrich A.; Albinger, Marie; Stoeckli, Daniel F.

2014-05-01

The Canarias archipelago consists from seven volcanic islands located at the northwestern African margin. Among them only Fuerteventura and La Gomera islands show distinctive wide exposures of the basal complex (BC) that is characteristic with complex geological history. The basal complex was exposed on the western part of Fuerteventura and northwestern sector of La Gomera because of giant landslide(s) which have removed most of the shield stage volcanic rocks (Ancochea et al., 1996; 2006; Stillman, 1999). Generally, landslides are a common feature in the earlier constructive stages of the entire archipelago and many other volcanic islands (McGuire, 1996). Integration of low temperature thermochronological data, and time-Temperature (t-T) numerical modelling have proven to be a powerful tool for reconstructing the thermal and tectonic history, defining and quantifying long-term landscape evolution in variety of geological settings. Therefore, zircon and apatite fission-track techniques and t-T paths modelling were applied to 36 samples representing the main rock units of the BC on both islands. Fuerteventura BC has experienced two very rapid cooling/exhumation events. While, La Gomera BC shows one long-lived very fast cooling/exhumation event. Interestingly, these very rapid cooling/exhumation events are synchronous with these major landslides. There are many reasons for the major landslides on such a volcanic island (see e.g. McGuire, 1996)., But, the most sufficient triggers for these huge mass wasting/landslides events on Fuerteventura and La Gomera are recommended to be the continuous igneous intrusions and dikes which have the potential to decrease the edifice stability, igneous extrusions which add new materials at the surface leading to over-steeping and overloading (McGuire, 1996), and major climatic changes of the Middle Miocene Climatic Optimum (Herold et al., 2011). References Ancochea, E., Brändle, J.L., Cubas, C.R.,Hernán, F.,Huertas, M.J., 1996. Volcanic complexes in the eastern ridge of the Canary Islands: the Miocene activity of the Island of Fuerteventura. Journal of Volcanology and Geothermal Research 70, 183-204. Ancochea, E., Hernán, F., Huertas, M.J., Brändle, J.L., Herrera, R., 2006. A new chronostratigraphical and evolutionary model for La Gomera: implications for the overall evolution of the Canarian Archipelago. Journal of Volcanology and Geothermal Research 157, 271-293. Herold, N., Huber, M., Greenwood, D.R., Müller, R.D., Seton, M., 2011. Early to Middle Miocene monsoon climate in Australia. Geology 39, 3-6. McGuire, W.J., 1996. Volcano instability: a review of contemporary themes. In: McGuire, W.J., Jones, A.P., Neuberg, J. (Eds.), Volcano Instability on the Earth and Terrestrial Planets. Geological Society of London, Special Publication 110, 1-23. Stillman, C.J., 1999. Giant Miocene landslides and the evolution of Fuerteventura, Canary Islands. Journal of Volcanology and Geothermal Research 94, 89-104.

Characterization of carbonaceous aerosols during the MINOS campaign in Crete, July August 2001: a multi-analytical approach

NASA Astrophysics Data System (ADS)

Sciare, J.; Cachier, H.; Oikonomou, K.; Ausset, P.; Sarda-Estève, R.; Mihalopoulos, N.

2003-10-01

During the major part of the Mediterranean Intensive Oxidant Study (MINOS) campaign (summer 2001, Crete Isl.), the Marine Boundary Layer (MBL) air was influenced by long range transport of biomass burning from the northern and western part of the Black Sea. During this campaign, carbonaceous aerosols were collected on quartz filters at a Free Tropospheric (FT) site, and at a MBL site together with size-resolved distribution of aerosols. Three Evolution Gas Analysis (EGA) protocols have been tested in order to better characterize the collected aged biomass burning smoke: A 2-step thermal method (Cachier et al., 1989) and a thermo-optical technique using two different temperature programs. The later temperature programs are those used for IMPROVE (Interagency Monitoring of Protected Visual Environments) and NIOSH 5040 (National Institute of Occupational Safety and Health). Artifacts were observed using the NIOSH temperature program and identified as interactions between carbon and dust deposited on the filter matrix at high temperature (T>550ºC) under the pure helium step of the analysis. During the MINOS campaign, Black Carbon (BC) and Organic Carbon (OC) mass concentrations were on average respectively 1.19±0.56 and 3.62±1.08 mgC/m3 for the IMPROVE temperature program, and 1.09±0.36 and 3.75±1.24 mgC/m3 for the thermal method. Though these values compare well on average and the agreement between the Total Carbon (TC) measurements sample to sample was excellent (slope=1.00, r2=0.93, n=56), important discrepancies were observed in determining BC concentrations from these two methods (average error of 33±22%). BC from the IMPROVE temperature program compared well with non-sea-salt potassium (nss-K) pointing out an optical sensitivity to biomass burning. On the other hand, BC from the thermal method showed a better agreement with non-sea-salt sulfate (nss-SO4), considered as a tracer for fossil fuel combustion during the MINOS campaign. The coupling between these two methods for determining BC brings here new insights on the origin of carbonaceous aerosols in a complex mixture of different sources. It brings also to our attention that important deviations in BC levels are observed using three widely used EGA's techniques and most probably none of the EGA tested here are well adapted to fully characterize this aerosol mixture. Spherical, smooth and silico-aluminated fly-ash observed by an Analytical Scanning Electron Microscope (ASEM) confirm the influence of coal combustion on the carbonaceous aerosol load throughout the campaign. A rough calculation based on a BC/nss-SO4 mass ratio suggests that biomass burning could be responsible for half of the BC concentration recorded during the MINOS campaign. From the plot of BC as a function of TC, two linear correlations were observed corresponding to 2 times series (before and after 12 August). Such good correlations suggest, from a first look, that both BC and OC have similar origin and atmospheric transport. On the other hand, the plot of BC as a function of TC obtained from the 2-step thermal method applied to DEKATI Low Pressure Cascade Impactor samples does not show a similar correlation and points out a non conservative distribution of this ratio with 2 super micron modes enriched in OC, correlated with sea salt aerosols and probably originating from gas-to-particle conversion.

Characterization of carbonaceous aerosols during the MINOS campaign in Crete, July-August 2001: a multi-analytical approach

NASA Astrophysics Data System (ADS)

Sciare, J.; Cachier, H.; Oikonomou, K.; Ausset, P.; Sarda-Estève, R.; Mihalopoulos, N.

2003-07-01

During the major part of the Mediterranean Intensive Oxidant Study (MINOS) campaign (summer 2001, Crete Isl.), the Marine Boundary Layer (MBL) air was influenced by long range transport of biomass burning from the northern and western part of the Black Sea. During this campaign, carbonaceous aerosols were collected on quartz filters at a Free Tropospheric (FT) site, and at a MBL site together with size-resolved distribution of aerosols. Three Evolution Gas Analysis (EGA) protocols have been tested in order to better characterize the collected aged biomass burning smoke: A 2-step thermal method (Cachier et al., 1989) and a thermo-optical technique using two different temperature programs. The later temperature programs are those used for IMPROVE (Interagency Monitoring of Protected Visual Environments) and NIOSH 5040 (National Institute of Occupational Safety and Health). Artifacts were observed using the NIOSH temperature program and identified as interactions between carbon and dust deposited on the filter matrix at high temperature (T=550°C) under the pure helium step of the analysis. During the MINOS campaign, Black Carbon (BC) and Organic Carbon (OC) concentrations were on average respectively 1.19±0.56 and 3.62±1.08 μgC/m3 for the IMPROVE temperature program, and 1.09±0.36 and 3.75±1.24 μgC/m3 for the thermal method. Though these values compare well on average and the agreement between the Total Carbon (TC) measurements sample to sample was excellent (slope = 1.00, r2=0.93, n=56), important discrepancies were observed in determining BC concentrations from these two methods (average error of 33±22%). BC from the IMPROVE temperature program compared well with non-sea-salt potassium (nss-K) pointing out an optical sensitivity to biomass burning. On the other hand, BC from the thermal method showed a better agreement with non-sea-salt sulfate (nss-SO4), considered as a tracer for fossil fuel combustion during the MINOS campaign. The coupling between these two methods for determining BC brings here new insights on the origin of carbonaceous aerosols in a complex mixture of different sources. It brings also to our attention that important deviations in BC levels are observed using three widely used EGA techniques and most probably none of the EGA tested here are well adapted to fully characterize this aerosol mixture. Spherical, smooth and silico-aluminated fly-ash observed by Analytical Scanning Electron Microscope (ASEM) confirm the influence of coal combustion on the carbonaceous aerosol load throughout the campaign. A raw calculation based on BC/nss-SO4 mass ratio suggests that biomass burning could be responsible for half of the BC concentration recorded during the MINOS campaign. From the plot of BC as a function of TC, two linear correlations were observed corresponding to 2 times series (before and after 12 August). Such good correlations suggest, from a first look, that both BC and OC have similar origin and atmospheric transport. On the other hand, the plot of BC as a function of TC obtained from the 2-step thermal method applied to DEKATI Low Pressure Cascade Impactor samples does not show a similar correlation and points out a non conservative distribution of this ratio with 2 super micron modes enriched in OC, correlated with sea salt aerosols and probably originating from gas-to-particle conversion.

Effect of in-situ aged and fresh biochar on soil hydraulic conditions and microbial C use under drought conditions.

PubMed

Paetsch, Lydia; Mueller, Carsten W; Kögel-Knabner, Ingrid; von Lützow, Margit; Girardin, Cyril; Rumpel, Cornelia

2018-05-01

Biochar (BC) amendments may be suitable to increase the ecosystems resistance to drought due to their positive effects on soil water retention and availability. We investigated the effect of BC in situ ageing on water availability and microbial parameters of a grassland soil. We used soil containing 13 C labeled BC and determined its water holding capacity, microbial biomass and activity during a 3 months incubation under optimum and drought conditions. Our incubation experiment comprised three treatments: soil without BC (Control), soil containing aged BC (BC aged ) and soil containing fresh BC (BC fresh ), under optimum soil water (pF 1.8) and drought conditions (pF 3.5). Under optimum water as well as drought conditions, soils containing BC showed higher soil organic carbon (SOC) mineralization as compared to control soil. Moreover, BC effects on the soil water regime increase upon in situ aging. Native SOC mineralization increased most for soils containing BC aged . The BC aged led to improved C use under drought as compared to the other treatments. We conclude that BC addition to soils can ameliorate their water regime, especially under drought conditions. This beneficial effect of BC increases upon its aging, which also improved native substrate availability.

Energetics of the Brazil Current in the Rio Grande Cone region

NASA Astrophysics Data System (ADS)

Brum, André Lopes; Azevedo, José Luiz Lima de; Oliveira, Leopoldo Rota de; Calil, Paulo Henrique Rezende

2017-10-01

The energetics of the Brazil Current (BC) in the region of the Rio Grande Cone (RGC, 30-35.5°S), a topographic rise in the southwest portion of the Brazilian continental margin, are analyzed using 16 years of numerical data from the Ocean General Circulation Model (OGCM) for the Earth Simulator (OFES). The main focus of this study is the eddy-mean flow interactions of the BC and the local energy budgets in the study region. The kinetic and potential energy balance equations are derived for mean and eddy flows, and the resulting terms are presented and discussed. The eddy-mean flow interactions exhibit complex spatial distributions, and the intensities of the energy budgets decrease with increasing depth. However, only the mean potential energy (MPE) budget decreases southward. Eddy kinetic energy (EKE) and eddy potential energy (EPE) exhibit similar horizontal distribution patterns. Additionally, the baroclinic and barotropic conversion rates increase downstream of the bump, where the eddy energy field exhibits along-stream variability that increases southward. Barotropic conversion is more intense between 50 and 200 m, where mean kinetic energy (MKE) and EKE are concentrated, and it exhibits a horizontal cross-stream variation pattern, with mean-to-eddy energy conversion observed on the offshore side of the BC. This result indicates that the turbulence associated with the stream jet increases as the BC moves away from the coast, with the conversion term acting to stabilize the flow. Baroclinic conversion exhibits a high intensity below 300 m (where MPE and EPE display peaks), and it has a greater influence on the eddy-mean flow interaction than does the barotropic conversion. The RGC directly affects the local dynamics of the BC by increasing the eddy field as soon as the BC reaches the bump. The energy diagrams illustrate a stream characterized by evolving barotropic and baroclinic instability processes throughout the water column. This result indicates an intrinsically unstable jet in the study region. Moreover, baroclinic instability is the main source of EKE in the RGC region.

Stabilization of nanoscale zero-valent iron (nZVI) with modified biochar for Cr(VI) removal from aqueous solution.

PubMed

Dong, Haoran; Deng, Junmin; Xie, Yankai; Zhang, Cong; Jiang, Zhao; Cheng, Yujun; Hou, Kunjie; Zeng, Guangming

2017-06-15

Three types of modified biochar (BC) were produced respectively with acid (HCl) treatment (HCl-BC), base (KOH) treatment (KOH-BC) and oxidation (H 2 O 2 ) treatment (H 2 O 2 -BC) of raw biochar. Both the raw biochar and modified biochars supported zero valent iron nanopartilces (nZVI) (i.e. nZVI@BC, nZVI@HCl-BC, nZVI@KOH-BC and nZVI@H 2 O 2 -BC) were synthesized and their capacities for Cr(VI) removal were compared. The results showed that the nZVI@HCl-BC exhibited the best performance and the underlying mechanisms were discussed. The surface elemental distribution maps of the nZVI@HCl-BC after reaction with Cr(VI) showed that Fe, Cr and O elements were deposited on the surface of HCl-BC evenly, indicating that the formed Cr(III)/Fe(III) could settle on the surface of HCl-BC uniformly rather than coated only on the nZVI surface. This reveals that the supporter HCl-BC could also play a role in alleviating the passivation of nZVI. Besides, the effects of mass ratio (nZVI/HCl-BC), pH, and initial Cr(VI) concentration on Cr(VI) removal were examined. At lower mass of HCl-BC, nZVI aggregation cannot be fully inhibited on the surface of HCl-BC, whereas excessive biochar can block the active sites of nZVI. Additionally, it was found that Cr(VI) removal by nZVI@HCl-BC was dependent on both pH and initial Cr(VI) concentration. Copyright © 2017 Elsevier B.V. All rights reserved.

Numerical Investigation on Absorption Enhancement of Black Carbon Aerosols Partially Coated With Nonabsorbing Organics

NASA Astrophysics Data System (ADS)

Zhang, Xiaolin; Mao, Mao; Yin, Yan; Wang, Bin

2018-01-01

This study numerically evaluates the effects of aerosol microphysics, including coated volume fraction of black carbon (BC), shell/core ratio, and size distribution, on the absorption enhancement (Eab) of polydisperse BC aggregates partially coated by organics, which is calculated by the exact multiple-sphere T-matrix method. The coated volume fraction of BC plays a substantial role in determining the absorption enhancement of partially coated BC aggregates, which typically have an Eab in the range of 1.0-2.0 with a larger value for larger coated volume fraction of BC as the shell/core ratio, BC geometry, and size distribution are fixed. The shell/core ratio, BC geometry, and size distribution have little impact on the Eab of coated BC with small coated volume fraction of BC, while they become significant for large coated volume fraction of BC. The Eab of partially coated BC particles can be slightly less than 1.0 for the large BC in the accumulation mode exhibiting large shell/core ratio and small coated volume fraction of BC, indicating that the absorption shows even slight decrease relative to uncoated BC particles. For partially coated BC aggregates in the accumulation and coarse modes, the refractive index uncertainties of BC result in the Eab differences of less than 9% and 2%, respectively, while those of organics can induce larger variations with the maximum differences up to 22% and 18%, respectively. Our study indicates that accounting for particle coating microphysics, particularly the coated volume fraction of BC, can potentially help to understand the differences in observations of largely variable absorption enhancements over various regions.

Mitochondrial modulators in experimental Huntington's disease: reversal of mitochondrial dysfunctions and cognitive deficits.

PubMed

Mehrotra, Arpit; Kanwal, Abhinav; Banerjee, Sanjay Kumar; Sandhir, Rajat

2015-06-01

Huntington's disease (HD) is a chronic neurodegenerative condition involving impaired mitochondrial functions. The present study evaluates the therapeutic potential of combined administration of mitochondrial modulators: alpha-lipoic acid and acetyl-l-carnitine on mitochondrial dysfunctions in 3-NP-induced HD. Our results reveal 3-NP administration resulted in compromise of mitochondrial functions in terms of: (1) impaired activity of mitochondrial respiratory chain enzymes, altered cytochrome levels, reduced histochemical staining of complex-II and IV, reduced in-gel activity of complex-I to V, and reduced mRNA expression of respiratory chain complexes; (2) enhanced mitochondrial oxidative stress indicated by increased malondialdehyde, protein carbonyls, reactive oxygen species and nitrite levels, along with decreased Mn-superoxide dismutase and catalase activity; (3) mitochondrial structural changes measured by mitochondrial swelling, reduced mitochondrial membrane potential and ultra-structure changes; (4) increased cytosolic cytochrome c levels, caspase-3 and -9 activity along with altered expression of apoptotic proteins (AIF, Bim, Bad, and Bax); and (5) impaired cognitive functions assessed using Morris water maze and Y-maze. Combination of mitochondrial modulators (alpha-lipoic acid + acetyl-l-carnitine) on the other hand ameliorated 3-NP-induced mitochondrial dysfunctions, oxidative stress, histologic alterations, and behavioral deficits, suggesting their therapeutic efficacy in the management of HD. Copyright © 2015 Elsevier Inc. All rights reserved.

Oxidative Stress in Cardiac Mitochondria Caused by Copper Deficiency May Be Insufficient to Damage Mitochondrial Proteins

USDA-ARS?s Scientific Manuscript database

Copper (Cu) deficiency may promote the generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain through inhibition of cytochrome c oxidase (CCO) and increased reduction of respiratory complexes upstream from CCO. In the present study, respiration, H2O2 production and...

[Black carbon content and distribution in different particle size fractions of forest soils in the middle part of Great Xing'an Mountains, China.

PubMed

Xu, Jia Hui; Gao, Lei; Cui, Xiao Yang

2017-10-01

Soil black carbon (BC) is considered to be the main component of passive C pool because of its inherent biochemical recalcitrance. In this paper, soil BC in the middle part of Great Xing'an Mountains was quantified, the distribution of BC in different particle size fractions was analyzed, and BC stabilization mechanism and its important role in soil C pool were discussed. The results showed that BC expressed obvious accumulation in surface soil, accounting for about 68.7% in the whole horizon (64 cm), and then decreased with the increasing soil depth, however, BC/OC showed an opposite pattern. Climate conditions redistributed BC in study area, and the soil under cooler and moister conditions would sequester more BC. BC proportion in different particle size fractions was in the order of clay>silt>fine sand>coarse sand. Although BC content in clay was the highest and was enhanced with increasing soil depth, BC/OC in clay did not show a marked change. Thus, the rise of BC/OC was attributed to the preservation of BC particles in the fine sand and silt fractions. Biochemical recalcitrance was the main stabilization mechanism for surface BC, and with the increasing soil depth, the chemical protection from clay mineral gradually played a predominant role. BC not only was the essential component of soil stable carbon pool, but also took up a sizable proportion in particulate organic carbon pool. Therefore, the storage of soil stable carbon and the potential of soil carbon sequestration would be enhanced owing to the existence of BC.

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

PubMed Central

Pakrasi, H B; Williams, J G; Arntzen, C J

1988-01-01

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII. Images PMID:3130246

Iron(IV)hydroxide pK(a) and the role of thiolate ligation in C-H bond activation by cytochrome P450.

PubMed

Yosca, Timothy H; Rittle, Jonathan; Krest, Courtney M; Onderko, Elizabeth L; Silakov, Alexey; Calixto, Julio C; Behan, Rachel K; Green, Michael T

2013-11-15

Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pK(a) (where K(a) is the acid dissociation constant) of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious auto-oxidation of tyrosine and tryptophan residues in the enzyme's framework. Here, we report on the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥90% yield. Using rapid mixing technologies in conjunction with Mössbauer, ultraviolet/visible, and x-ray absorption spectroscopies, we determine a pK(a) value for this compound of 11.9. Marcus theory analysis indicates that this elevated pK(a) results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

Axial iron coordination and spin state change in a heme c upon electrostatic protein-SAM interaction.

PubMed

Di Rocco, Giulia; Ranieri, Antonio; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Bonifacio, Alois; Sergo, Valter; Borsari, Marco; Sola, Marco

2013-08-28

A bacterial di-heme cytochrome c binds electrostatically to a gold electrode surface coated with a negatively charged COOH-terminated SAM adopting a sort of 'perpendicular' orientation. Cyclic voltammetry, Resonance Raman and SERRS spectroscopies indicate that the high-potential C-terminal heme center proximal to the SAM's surface undergoes an adsorption-induced swapping of one axial His ligand with a water molecule, which is probably lost in the reduced form, and a low- to high-spin transition. This coordination change for a bis-His ligated heme center upon an electrostatically-driven molecular recognition is as yet unprecedented, as well as the resulting increase in reduction potential. We discuss it in comparison with the known methionine ligand lability in monoheme cytochromes c occurring upon interaction with charged molecular patches. One possible implication of this finding in biological ET is that mobile redox partners do not behave as rigid and invariant bodies, but in the ET complex are subjected to molecular changes and structural fluctuations that affect in a complex way the thermodynamics and the kinetics of the process.

The effect of artichoke (Cynara scolymus L.) extract on respiratory chain system activity in rat liver mitochondria.

PubMed

Juzyszyn, Z; Czerny, B; Myśliwiec, Z; Pawlik, A; Droździk, M

2010-06-01

The effect of artichoke extract on mitochondrial respiratory chain (MRC) activity in isolated rat liver mitochondria (including reaction kinetics) was studied. The effect of the extract on the activity of isolated cytochrome oxidase was also studied. Extract in the range of 0.68-2.72 microg/ml demonstrated potent and concentration-dependent inhibitory activity. Concentrations > or =5.4 microg/ml entirely inhibited MRC activity. The succinate oxidase system (MRC complexes II-IV) was the most potently inhibited, its activity at an extract concentration of 1.36 microg/ml being reduced by 63.3% compared with the control (p < 0.05). The results suggest a complex inhibitory mechanism of the extract. Inhibition of the succinate oxidase system was competitive (K(i) = 0.23 microg/ml), whereas isolated cytochrome oxidase was inhibited noncompetitively (K(i) = 126 microg/ml). The results of this study suggest that the salubrious effects of artichoke extracts may rely in part on the effects of their active compounds on the activity of the mitochondrial respiratory chain system.

Structural and immunochemical relatedness suggests a conserved pathogenicity motif for secondary cell wall polysaccharides in Bacillus anthracis and infection-associated Bacillus cereus

PubMed Central

Saile, Elke; Klee, Silke R.; Hoffmaster, Alex; Kannenberg, Elmar L.

2017-01-01

Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying “anthrax-like” symptoms in Cameroon (Bc CA) and Côte d’Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10–20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains. PMID:28832613

Structural and immunochemical relatedness suggests a conserved pathogenicity motif for secondary cell wall polysaccharides in Bacillus anthracis and infection-associated Bacillus cereus.

PubMed

Kamal, Nazia; Ganguly, Jhuma; Saile, Elke; Klee, Silke R; Hoffmaster, Alex; Carlson, Russell W; Forsberg, Lennart S; Kannenberg, Elmar L; Quinn, Conrad P

2017-01-01

Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying "anthrax-like" symptoms in Cameroon (Bc CA) and Côte d'Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10-20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains.

Measurement of Atmospheric Black Carbon Concentrations, [BC]atm, in the Arctic Region from ~1700 to 2013

NASA Astrophysics Data System (ADS)

Husain, L.; Sarkar, S.; Jyethi, D. S.; Ruppel, M.; Dutkiewicz, V. A.

2015-12-01

Atmospheric black carbon (BC) aerosols play a key role in Earth's climate through direct and indirect effects. Due to a lack of long-term BC data, climate models are used to estimate BC based on fuel inventories, which have large uncertainties. Hence, long term BC data is needed to verify global models. We report here the first measurements of atmospheric BC concentrations, [BC]atm, from ~1700 to 2013 using sediments from Finnish lakes, Saanajarvi (SJ)(690 44' N, 200 52' E), and Vuoskojarvi (VJ)(69044'N, 26057'E). The cores were collected from the deepest parts of the lakes using a HTH gravity corer, sliced in 0.25 cm sections; freeze dried, and ages determined using 210Pb dating method. The BC was chemically separated, and [BC] determined by the thermal optical method. The [BC] varied from 50 to 1140µg/gdry weight in SJ; and 20 to 130µg/gdry weight in VJ. Husain et al.,(JGR, vol 113, D13102,doi:10.1029/2007JD009398, 2008) showed that the atmospheric deposition of BC into lake sediments depends on the characteristic of individual lakes, BC washout ratios, precipitation intensity, and sedimentation rates. The deposition rate, K, for a lake is defined by, [BC]sed = K[BC]atm where [BC]sed, is the concentration of BC in the sediment. We have measured [BC]atm from 1970 to 2010 in Kevo, Finland, where VJ and SJ are located. The [BC]atm from Kevo, and [BC]sed from VJ, and SJ were used to determine K for each of the lake. Owing to the availability of the long term atmospheric BC data from 1970 to 2010 multiple measurements of K were made, and provided a high measure of precision. The mean values of K for VJ, and SJ were 226 ± 60, and 830 ± 290 (m3air/ gdry weight). The K values were used to determine [BC]atm for the years before 1970. The [BC]atm from 2013 to 2006 was 82ng/m3. It increased slowly reaching a peak value of about 947 ± 322 ng/m3.The concentrations decreased subsequently to 244 ± 83ng/m3 in 1920, and changed little ~ 1774.The lowest concentration, 77 ± 26 ng/m3, was observed ~ 1700. The high atmospheric concentrations beyond 1850, perhaps, suggest a proportionally larger component from wood burning for heating. We plan to estimate [BC]atm using OsloCTM3 model, and inventory of fossil fuel, and biomass burning, and compare it with our measurements.

A review on black carbon emissions, worldwide and in China.

PubMed

Ni, Mingjiang; Huang, Jianxin; Lu, Shengyong; Li, Xiaodong; Yan, Jianhua; Cen, Kefa

2014-07-01

Black carbon (BC) produced from open burning (OB) and controlled combustion (CC) is a range of carbonaceous products of incomplete combustion of biomass and fossil fuel, and is deemed as one of the major contributors to impact global environment and human health. BC has a strong relationship with POPs, in waste combustion, BC promotes the formation of POPs, and then the transport of POPs in the environment is highly influenced by BC. However less is known about BC formation, measurement and emissions estimation especially in developing countries such as China. Different forms of BC are produced both in CC and OB. BC emission characteristics and combustion parameters which determine BC emissions from CC and OB are discussed. Recent studies showed a lack of common methodology and the resulting data for describing the mechanisms related to BC formation during combustion processes. Because BC is a continuum carbonaceous combustion product, different sampling and measuring methods are used for measuring their emissions with great quantitative uncertainty. We discuss the commonly used BC sampling and measuring methods along with the causes for uncertainty and measures to minimizing the uncertainty. Then, we discuss the estimations of BC emission factors and emission inventory for CC and OB sources. The total emissions of BC from CC and OB in China are also estimated and compared with previous BC emission inventories in this review and we find the inventories tend to be overestimated. As China becomes the largest contributor to global BC emissions, studies for characterizing BC emissions from OB and CC sources are absent in China. Finally, we comment on the current state of BC emission research and identify major deficiencies that need to overcome. Moreover, the advancement in research tools, measuring technique in particular, as discussed in this review is critical for researchers in developing countries to improve their capability to study BC emissions for addressing the growing climate change and public health concerns. Copyright © 2014 Elsevier Ltd. All rights reserved.

Loss of protohaem IX farnesyltransferase in mature dentate granule cells impairs short-term facilitation at mossy fibre to CA3 pyramidal cell synapses.

PubMed

Booker, Sam A; Campbell, Graham R; Mysiak, Karolina S; Brophy, Peter J; Kind, Peter C; Mahad, Don J; Wyllie, David J A

2017-03-15

Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity. Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low-frequency dentate to CA3 glutamatergic synaptic transmission. High-frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase-deficient mice. Intact presynaptic mitochondrial function is critical for the short-term dynamics of mossy fibre to CA3 synaptic function. Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole-cell patch-clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV-deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy-fibre synapse because the amplitude, input-output relationship and 50 ms paired-pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short-term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired-pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses with CA3 pyramidal cells via large mossy-fibre boutons, but rather to all synapses formed by dentate granule cells. Therefore, presynaptic mitochondrial function is critical for the short-term dynamics of synapse function, which may contribute to the cognitive deficits observed in pathological mitochondrial dysfunction. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Ferro- and antiferro-magnetism in (Np, Pu)BC

NASA Astrophysics Data System (ADS)

Klimczuk, T.; Shick, A. B.; Kozub, A. L.; Griveau, J.-C.; Colineau, E.; Falmbigl, M.; Wastin, F.; Rogl, P.

2015-04-01

Two new transuranium metal boron carbides, NpBC and PuBC, have been synthesized. Rietveld refinements of powder XRD patterns of {Np,Pu}BC confirmed in both cases isotypism with the structure type of UBC. Temperature dependent magnetic susceptibility data reveal antiferromagnetic ordering for PuBC below TN = 44 K, whereas ferromagnetic ordering was found for NpBC below TC = 61 K. Heat capacity measurements prove the bulk character of the observed magnetic transition for both compounds. The total energy electronic band structure calculations support formation of the ferromagnetic ground state for NpBC and the antiferromagnetic ground state for PuBC.

An Investigation of Black Carbon Degradation Potential in a Forest Soil Environment

NASA Astrophysics Data System (ADS)

William, H. C.; Lee, E.; Grannas, A.; Hatcher, P. G.

2003-12-01

Except for emission processes, there is currently little understanding of the mechanisms driving the degradation and biogeochemical cycling of black carbon (BC). Considering current estimates of the global BC pool (>2,500x1015gC), and its annual emission rates (55-205x1012 gC/year), BC represents roughly 16% of Earth's actively cycling organic carbon. Without significant chemical and biological degradation pathways, all of the actively cycling carbon on earth would have accumulated as charcoal in <100,000 years. This investigation show that charcoals recovered from experimental forest fires are altered significantly by microbial colonization, and mineral complexation during exposure to soil processes. Charcoal surface morphology and elemental composition were characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy, and BET surface area measurements. The influence of 90 years aging upon carbon functionality was probed by solid-state 13C NMR spectroscopy. The prevalence of fungal mycorhizae in these forest soil charcoals also motivated an investigation of black carbon degradation via extracellular enzymes and acids known be exuded by mycelia. Degradation is quantified by carbon loss, and soluble products are examined by mass spectrometry.

Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release.

PubMed

Düssmann, Heiko; Perez-Alvarez, Sergio; Anilkumar, Ujval; Papkovsky, Dmitri B; Prehn, Jochen Hm

2017-06-01

The detection of intracellular molecular oxygen (O 2 ) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O 2 -independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O 2 . Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O 2 accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at 'physiological' tissue O 2 levels (5% O 2 ). Multiplexing also allowed us to monitor intracellular O 2 during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O 2 despite a decrease in mitochondrial membrane potential.

Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release

PubMed Central

Düssmann, Heiko; Perez-Alvarez, Sergio; Anilkumar, Ujval; Papkovsky, Dmitri B; Prehn, Jochen HM

2017-01-01

The detection of intracellular molecular oxygen (O2) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6–pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O2-independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O2. Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O2 accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at ‘physiological’ tissue O2 levels (5% O2). Multiplexing also allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O2 despite a decrease in mitochondrial membrane potential. PMID:28569778

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

PubMed

Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

1995-04-04

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

Towards Soil and Sediment Inventories of Black Carbon

NASA Astrophysics Data System (ADS)

Masiello, C. A.

2008-12-01

A body of literature on black carbon (BC) concentrations in soils and sediments is rapidly accumulating, but as of yet, there are no global or regional inventories of BC in either reservoir. Soil and sediment BC inventories are badly needed for a range of fields. For example, in oceanography a global sediment BC inventory is crucial in understanding the role of biomass burning in the development of stable marine carbon reservoirs, including dissolved organic carbon and sedimentary organic carbon. Again in the marine environment, BC likely strongly impacts the fate and transport of anthropogenic pollutants: regional inventories of BC in sediments will help develop better environmental remediation strategies. In terrestrial systems well-constrained natural BC soil inventories would help refine ecological, agricultural, and soil biogeochemical studies. BC is highly sorptive of nutrients including nitrogen and phosphorous. The presence of BC in ecosystems almost certainly alters N and P cycling; however, without soil BC inventories, we cannot know where BC has a significant impact. BC's nutrient sorptivity and water-holding capacity make it an important component of agricultural soils, and some researchers have proposed artificially increasing soil BC inventories to improve soil fertility. Natural soil BC concentrations in some regions are quite high, but without a baseline inventory, it is challenging to predict when agricultural amendment will significantly exceed natural conditions. And finally, because BC is one of the most stable fractions of organic carbon in soils, understanding its concentration and regional distribution will help us track the dynamics of soil organic matter response to changing environmental conditions. Developing effective regional and global BC inventories is challenging both because of data sparsity and methodological intercomparison issues. In this presentation I will describe a roadmap to generating these valuable inventories.

Rational design of a high-strength bone scaffold platform based on in situ hybridization of bacterial cellulose/nano-hydroxyapatite framework and silk fibroin reinforcing phase.

PubMed

Jiang, Pei; Ran, Jiabing; Yan, Pan; Zheng, Lingyue; Shen, Xinyu; Tong, Hua

2018-02-01

Bacterial cellulose/hydroxyapatite (BC/HAp) composite had favourable bioaffinity but its poor mechanical strength limited its widespread applications in bone tissue engineering (BTE). Silk fibroin, which possesses special crystalline structure, has been widely used as organic reinforcing material, and different SFs have different amino acid sequences, which exhibit different bioaffinity and mechanical properties. In this regard, bacterial cellulose-Antheraea yamamai silk fibroin/hydroxyapatite (BC-AYSF/HAp), bacterial cellulose-Bombyx mori silk fibroin/hydroxyapatite (BC-BMSF/HAp), and BC/HAp nano-composites were synthesized via a novel in situ hybridization method. Compared with BC/HAp and BC-BMSF/HAp, the BC-AYSF/HAp exhibited better interpenetration, which may benefit for the transportation of nutrients and wastes, the adhesion of cells as well. Additionally, the BC-AYSF/HAp also presented superior thermal stability than the other two composites revealed by differential thermal analysis (DTA) and thermogravimetric analysis (TGA). Compression testing indicated that the mechanical strength of BC-BMSF/HAp was greatly reinforced compared with BC/HAp and was even a little higher than that of BC-AYSF/HAp. Tensile testing showed that BC-AYSF/HAp possesses extraordinary mechanical properties with a higher elastic modulus at low strain and higher fracture strength simultaneously than the other two composites. In vitro cell culture exhibited that MC3T3-E1 cells on the BC-AYSF/HAp membrane took on higher proliferative potential than those on the BC-BMSF/HAp membrane. These results suggested that compared with BC-BMSF/HAp, the BC-AYSF/HAp composite was more appropriate as an ideal bone scaffold platform or biomedical membrane to be used in BTE.

A potential large and persistent black carbon forcing over Northern Pacific inferred from satellite observations.

PubMed

Li, Zhongshu; Liu, Junfeng; Mauzerall, Denise L; Li, Xiaoyuan; Fan, Songmiao; Horowitz, Larry W; He, Cenlin; Yi, Kan; Tao, Shu

2017-03-07

Black carbon (BC) aerosol strongly absorbs solar radiation, which warms climate. However, accurate estimation of BC's climate effect is limited by the uncertainties of its spatiotemporal distribution, especially over remote oceanic areas. The HIAPER Pole-to-Pole Observation (HIPPO) program from 2009 to 2011 intercepted multiple snapshots of BC profiles over Pacific in various seasons, and revealed a 2 to 5 times overestimate of BC by current global models. In this study, we compared the measurements from aircraft campaigns and satellites, and found a robust association between BC concentrations and satellite-retrieved CO, tropospheric NO 2 , and aerosol optical depth (AOD) (R 2  > 0.8). This establishes a basis to construct a satellite-based column BC approximation (sBC*) over remote oceans. The inferred sBC* shows that Asian outflows in spring bring much more BC aerosols to the mid-Pacific than those occurring in other seasons. In addition, inter-annual variability of sBC* is seen over the Northern Pacific, with abundances varying consistently with the springtime Pacific/North American (PNA) index. Our sBC* dataset infers a widespread overestimation of BC loadings and BC Direct Radiative Forcing by current models over North Pacific, which further suggests that large uncertainties exist on aerosol-climate interactions over other remote oceanic areas beyond Pacific.

How shorter black carbon lifetime alters its climate effect.

PubMed

Hodnebrog, Øivind; Myhre, Gunnar; Samset, Bjørn H

2014-09-25

Black carbon (BC), unlike most aerosol types, absorbs solar radiation. However, the quantification of its climate impact is uncertain and presently under debate. Recently, attention has been drawn both to a likely underestimation of global BC emissions in climate models, and an overestimation of BC at high altitudes. Here we show that doubling present day BC emissions in a model simulation, while reducing BC lifetime based on observational evidence, leaves the direct aerosol effect of BC virtually unchanged. Increased emissions, together with increased wet removal that reduces the lifetime, yields modelled BC vertical profiles that are in strongly improved agreement with recent aircraft observations. Furthermore, we explore the consequences of an altered BC profile in a global circulation model, and show that both the vertical profile of BC and rapid climate adjustments need to be taken into account in order to assess the total climate impact of BC.

South Asian aerosols in perspective: Preface to the special issue

NASA Astrophysics Data System (ADS)

Moorthy, K. Krishna; Satheesh, S. K.; Sarin, M. M.; Panday, Arnico K.

2016-01-01

The south Asian region is one the world's most populous and fast-developing regions. The more than 1.7 billion population (˜24% of the world population) with highly diverse living habits, fast growing industrial and transport sectors, large and increasing demand for power, diverse fuel use for domestic and industrial purposes, and equally diverse geographical features make this region a large cauldron of emissions and atmospheric processes. It is being increasingly recognized to be among the global hotspots of aerosols and anthropogenic trace gases. The complex geography of this region adds considerable amount of natural aerosols (sea spray, windblown desert dust, pollen, etc) into the atmosphere, which mix with the man-made ones, making the aerosol environment one of the most complex in the world. The large spatial diversity of the sources coupled with the varying atmospheric dynamics, driven by the contrasting monsoons and the topography, make South Asia's aerosol and pollution very difficult to characterize, to model and to plan effective mitigation measures, despite the fairly good knowledge on their implications to radiative and climate forcing, health effects and environmental degradation. In the recent years, there have been several reports on the impact of aerosols (more importantly black carbon - BC) on the regional and global climate system including Asian monsoon, with the caveats of long-term impacts on the livelihoods of tens of millions of people in this region; though specifics of these are not yet unequivocally established. While tropospheric perturbations would produce strong regional signatures, their global impacts still remain marginally above the uncertainty levels (IPCC, 2013). There have been several recent investigations showing that deposition of aerosol black carbon (BC) on snow can reduce the snow albedo, leading to enhanced absorption of solar radiation and hence faster melting rates of glaciers. Though several investigators have reported that enhanced warming due to atmospheric BC at higher levels is responsible for the faster melting rates of snowfields and of white glaciers (ones that are not debris-covered); some studies have revealed that the snow albedo forcing overwhelms and is more important for older snow (Nair et al., 2013). Evidence on the record of BC and dust deposition in the Himalayan region is only beginning to emerge (Ming et al., 2008), based on ice-core studies. The full implications of deposition of absorbing aerosols (BC, brown carbon (BrC), and dust) on the Himalayan ice-pack and glaciers remain to be understood.

Polymerase chain reaction identification of three members of the Anopheles sundaicus (Diptera: Culicidae) complex, malaria vectors in Southeast Asia.

PubMed

Dusfour, Isabelle; Blondeau, Johanna; Harbach, Ralph E; Vythilingham, Indra; Baimai, Visut; Trung, Ho D; Sochanta, Tho; Bangs, Michael J; Manguin, Sylvie

2007-09-01

Anopheles sundaicus s.l., a major malaria vector taxon, occurs primarily along coastal areas and on islands in Southeast Asia. Our previous studies using cytochrome oxidase I, cytochrome-b, and internal transcribed spacer 2 markers discriminated three allopatric species: An. sundaicus s.s. in northern Borneo, An. epiroticus in Southeast Asia, and An. sundaicus E on Sumatra and Java, Indonesia. Morphological comparisons of three developmental stages did not reveal unique diagnostic characters that could reliably distinguish the three species. Therefore, we developed a multiplex polymerase chain reaction (PCR) assay based on two mitochondrial DNA markers to unambiguously identify them. This PCR was tested on 374 specimens from 24 different geographical populations, expanding our knowledge of the distribution of these species.

Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Amino, H; Tomitsuka, E; Kita, K

1999-08-04

We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.

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

PubMed

Lipka, J J; Waskell, L A

1989-01-01

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

Analysis of kinetoplast cytochrome b gene of 16 Leishmania isolates from different foci of China: different species of Leishmania in China and their phylogenetic inference

PubMed Central

2013-01-01

Background Leishmania species belong to the family Trypanosomatidae and cause leishmaniasis, a geographically widespread disease that infects humans and other vertebrates. This disease remains endemic in China. Due to the large geographic area and complex ecological environment, the taxonomic position and phylogenetic relationship of Chinese Leishmania isolates remain uncertain. A recent internal transcribed spacer 1 and cytochrome oxidase II phylogeny of Chinese Leishmania isolates has challenged some aspects of their traditional taxonomy as well as cladistics hypotheses of their phylogeny. The current study was designed to provide further disease background and sequence analysis. Methods We systematically analyzed 50 cytochrome b (cyt b) gene sequences of 19 isolates (16 from China, 3 from other countries) sequenced after polymerase chain reaction (PCR) using a special primer for cyt b as well as 31 sequences downloaded from GenBank. After alignment, the data were analyzed using the maximum parsimony, Bayesian and netwok methods. Results Sequences of six haplotypes representing 10 Chinese isolates formed a monophyletic group and clustered with Leishmania tarentolae. The isolates GS1, GS7, XJ771 of this study from China clustered with other isolates of Leishmania donovani complex. The isolate JS1 was a sister to Leishmania tropica, which represented an L. tropica complex instead of clustering with L. donovani complex or with the other 10 Chinese isolates. The isolates KXG-2 and GS-GER20 formed a monophyletic group with Leishmania turanica from central Asia. In the different phylogenetic trees, all of the Chinese isolates occurred in at least four groups regardless of geographic distribution. Conclusions The undescribed Leishmania species of China, which are clearly causative agents of canine leishmaniasis and human visceral leishmaniasis and are related to Sauroleishmania, may have evolved from a common ancestral parasite that came from the Americas and may have split off earlier than the other old world Leishmania. Our results also suggest the following: the isolates GS7, GS1 and XJ771 occur as part of the L. donovani complex; the JS1 isolate is L. tropica; and the isolate GS-GER20 identified as Leishmania gerbilli is close to KXG-2 which is L. turanica. PMID:23383990

Toward a mechanistic understanding of the effect of biochar addition on soil water retention

NASA Astrophysics Data System (ADS)

Yi, S.; Chang, N.; Guo, M.; Imhoff, P. T.

2014-12-01

Biochar (BC) is a carbon-rich product produced by thermal degradation of biomass in an oxygen-free environment, whose application to sediment is said to improve water retention. However, BC produced from different feedstocks and pyrolyzed at different temperatures have distinct properties, which may alter water retention in ways difficult to predict a priori. Our goal is to develop a mechanistic understanding of BC addition on water retention by examining the impact of BC from two feedstocks, poultry litter (PL) and hardwood (HW), on the soil-water retention curves (SWRC) of a uniform sand and a sandy loam (SL). For experiments with sand, BC and sand were sieved to the same particle size (~ 0.547 mm) to minimize effects of BC addition on particle size distribution. Experiments with SL contained the same sieved BC. PL and HW bicohars were added at 2 and 7% (w/w), and water retention was measured from 0 to -4.38 × 106 cm-H2O. Both BCs increased porosities for sand and SL, up to 39 and 13% for sand and SL, respectively, with 7% HW BC addition. The primary cause for these increases was the internal porosity of BC particles. While the matric potential for air-entry was unchanged with BC addition, BC amendment increased water retention for sand and SL in the capillary region (0 to -15,000 cm-H2O) by an average of 26 and 33 % for 7% PL and HW BC in sand, respectively, but only 7 and 14 % for 7% PL and HW BC in SL. The most dramatic influence of BC amendment on water retention occurred in the adsorption region (< -15,000 cm-H2O), where water retention increased by a factor of 11 and 22 for 7% PL and HW BC in sand, respectively, but by 140 and 190 % for 7% PL and HW BC in SL, respectively. The impact of BC on water retention in these sediments is explained primarily by the additional surface area and internal porosity of PL and HW BC particles. van Genuchten (VG) models were fitted to the water retention data. For SL where the impact of BC addition on water retention was less significant, a unimodal model fit water retention data well for unamended and BC-amended media: the addition of BC did not create a new class of small pores that could not be described with a unimodal VG model. While a unimodal model fitted the sand data well, a bimodal model was required for all BC-amended sand. The influence of BC type, mass fraction and sediment on water retention models will be discussed.