MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'BJ Energy' source. Sun, 21 Mar 2010 16:57:40 +0100 http://www.medworm.com/index.php?rid=3382057&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100122 A human homologue of the iron-sulfur cluster assembly protein IscA (hIscA1) has been cloned and expressed in Escherichia coli cells. The UV-visible absorption and EPR (electron paramagnetic resonance) measurements reveal that hIscA1 purified from E. coli cells contains a mononuclear iron center and that the iron binding in hIscA1 expressed in E. coli cells can be further modulated by the iron content in the cell growth medium. Additional studies show that purified hIscA1 binds iron with an iron association constant of approx. 2.0 x 1019 M-1, and that the iron-bound hIscA1 is able to provide the iron for the iron-sulfur cluster assembly in a proposed scaffold protein IscU of E. coli in vitro. The complementation experiments indicate that hIscA1 can partially substitute for IscA in restoring... BJ Energy journals http://www.medworm.com/rss/comments.php?id=3382057 Fri, 19 Mar 2010 00:00:00 +0100 3382057 http://www.medworm.com/index.php?rid=3345882&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20100164 Excessive generation of nitric oxide (.NO) in neuroinflammation, excitotoxicity, and during age-related neurodegenerative disorders entails the localized and concerted increase of nitric oxide synthase(s) expression in glial cells and neurons. The aim of this study was to assess the biological significance of the impact of .NO on the cell’s thiol status with emphasis on S-glutathionylation of targeted proteins. Exposure of primary cortical neurons or astrocytes to increasing flow rates of .NO (0.061 – 0.25 µM/s) resulted in (a) a decrease of glutathione (GSH) in neurons accompanied by formation of S-nitrosoglutathione (GSNO) and glutathione disulfide (GSSG); neurons were far more sensitive to .NO exposure than astrocytes; (b) a dose-dependent oxidation of the cell&#x20... BJ Energy journals http://www.medworm.com/rss/comments.php?id=3345882 Tue, 09 Mar 2010 00:00:00 +0100 3345882 http://www.medworm.com/index.php?rid=3301490&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091603 Bacillus megaterium flavocytochrome P450 BM3 (BM3, CYP102A1) is a biotechnologically important cytochrome P450/P450 reductase fusion enzyme. Mutants I401E, F261E and L86E were engineered near the heme 5-methyl group, to explore ability of the glutamate carboxylates to form ester linkages to the methyl group, as observed for eukaryotic CYP4 relatives. Although no covalent linkage was detected, mutants displayed marked alterations in substrate/inhibitor affinity, with L86E and I401E mutants having lower Kd values for arachidonic acid and lauric acid than wild-type (WT) BM3. All mutations induced positive shifts in heme Fe(III)/Fe(II) potential, with substrate-free I401E (-219 mV) being <170 mV more positive than WT BM3. The elevated potential stimulated FMN-to-heme electron transfer ~2-fo... BJ Energy journals http://www.medworm.com/rss/comments.php?id=3301490 Wed, 24 Feb 2010 00:00:00 +0100 3301490 http://www.medworm.com/index.php?rid=3222728&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091412 In this study we report on the identification and characterization of three novel chloroplast-targeted J-domain proteins, CDJ3-5, which in addition to their J-domains contain bacterial-type ferredoxin domains. In the databases we could identify homologs of CDJ3-5 in green algae, moss and higher plants, but not in cyanobacteria. Phylogenetic analyses allowed distinguishing two clades containing CDJ3/4 and CDJ5 that must have diverged early in an ancestor of the green lineage and have further diversified later on. Molecular and biochemical analysis of CDJ3 and CDJ4 from Chlamydomonas reinhardtii revealed that both are weakly expressed proteins that appear to be localized to the stroma and to thylakoid membranes, respectively. The low transcript levels of the CDJ3 and CDJ4 genes declined even... BJ Energy journals http://www.medworm.com/rss/comments.php?id=3222728 Fri, 29 Jan 2010 00:00:00 +0100 3222728 http://www.medworm.com/index.php?rid=3207799&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091373 Kidney function declines with advancing age and mitochondria have been implicated. We have examined the integrated function of mitochondria isolated from kidneys of 6 and 24 month Fischer 344 rats. Oxidative phosphorylation (OXPHOS) of intact mitochondria and cytochrome c oxidase activity in permeabilized mitochondria were determined with polarographic assays. The activities of the electron transport chain (ETC) complexes and the cytochrome content in solubilized mitochondria were measured using spectrophotometric methods. The respiratory complexes were evaluated with blue-native gel electrophoresis. Mitochondrial preparations were evaluated by immunoblotting for cytochrome c, Smac/Diablo, and the voltage dependant ion channel (VDAC). Mitochondrial morphology was examined by electron micro... BJ Energy journals http://www.medworm.com/rss/comments.php?id=3207799 Tue, 26 Jan 2010 00:00:00 +0100 3207799 http://www.medworm.com/index.php?rid=3110597&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091417 The bacterial hemoglobin (VHb) from Vitreoscilla displays several unusual properties that are unique among the globin family. When the VHb gene (vgb) is expressed from its natural promoter in either Vitreoscilla or E. coli, the level of VHb increases more than fifty fold under hypoxic conditions and decreases significantly during oxidative stress suggesting similar functioning of the vgb promoter in both organisms. Expression of VHb in E. coli induced antioxidant genes, katG and sodA, and conferred significant protection from oxidative stress. In contrast, when vgb was expressed in an oxyR mutant of E. coli, VHb levels increased and the strain showed high sensitivity to oxidative stress without induction of antioxidant genes; this indicates the involvement of OxyR in mediating the protecti... BJ Energy journals http://www.medworm.com/rss/comments.php?id=3110597 Tue, 22 Dec 2009 00:00:00 +0100 3110597 http://www.medworm.com/index.php?rid=3011640&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091515 In this paper we characterise the properties of the potent MCT1 inhibitor, AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858 binding sites (Et) on MCT1 and the transporter’s turnover number (Kcat). Derived values were 2.3 ± 1.4 nM, 1.29 ± 0.09 nmoles per ml packed cells and 12.2 ± 1.1 s-1 respectively. When expressed in Xenopus laevis oocytes MCT1 and MCT2 were potently inhibited by AR-C155858 whilst MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chi... BJ Energy journals http://www.medworm.com/rss/comments.php?id=3011640 Fri, 20 Nov 2009 00:00:00 +0100 3011640 http://www.medworm.com/index.php?rid=3007418&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091339 We previously showed that the Na+/H+ antiporter of Helicobacter pylori (HPNhaA) forms an oligomer in a native membrane of Escherichia coli, and conformational changes of oligomer occur between monomers of the oligomer during ion transport. In the current study, we use Blue Native PAGE to show that HPNhaA forms a dimer. Cysteine-scanning mutagenesis of residues 55 to 61 in a putative β-sheet region of loop1 and subsequent functional analyses revealed that the Q58C mutation resulted in an intermolecular disulfide bond. G56C, I59C, and G60C were found to be cross-linked by bifunctional cross-linkers. Furthermore, the Q58E mutant did not form a dimer, possibly due to electrostatic repulsion between monomers. These results imply that Gln58 and the flanking sequence in the p... BJ Energy journals http://www.medworm.com/rss/comments.php?id=3007418 Thu, 19 Nov 2009 00:00:00 +0100 3007418 http://www.medworm.com/index.php?rid=2998948&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091293 Prototypical electrophiles such as the lipid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) are well recognized for their therapeutic potential. Electrophiles modify signaling proteins in both the cytosol and mitochondrion which results in diverse cellular responses including cytoprotective effects and, at high doses, cell death. These findings led us to the hypothesis that targeting electrophiles to specific compartments in the cell can be used to fine-tune their biological effects. To examine this, we synthesized a novel mitochondrially-targeted analog of 15d-PGJ2 (mito-15d-PGJ2) and tested its effects on redox cell signaling. Mito-15d-PGJ2 caused profound defects in mitochondrial bioenergetics and mitochondrial membrane depolarization when compared to 15d-PGJ2. We also found that mi... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2998948 Mon, 16 Nov 2009 00:00:00 +0100 2998948 http://www.medworm.com/index.php?rid=2930384&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090775 Heme oxygenases (HOs) catalyze the oxidative cleavage of heme to biliverdin, iron and carbon monoxide. In plants, the product of the reaction is biliverdin IXα, the precursor of the phytochrome chromophore and is thus essential for proper photomorphogenesis. Arabidopsis thaliana contains one major biochemically characterized HO (HY1) and three additional putative HOs (HO2, HO3 and HO4). All four proteins are encoded in the nucleus but contain chloroplast translocation sequences at their N-termini. The transit peptides of all four proteins are sufficient for chloroplast translocalisation as shown by GFP reporter gene fusions. Overall, all four proteins can be divided into two subfamilies: HO1 and HO2. Here we show that all members of the HO1 subfamily (HY1, HO3, and HO4) are active m... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2930384 Tue, 27 Oct 2009 00:00:00 +0100 2930384 http://www.medworm.com/index.php?rid=2898123&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090902 Functional alterations in mitochondria such as overproduction of reactive oxygen species and overloading of calcium, with subsequent change in the membrane potential, are traditionally regarded as pro-apoptotic conditions. Although such events occur in the early phases of liver regeneration after 2/3 partial hepatectomy, hepatocytes do not undergo apoptosis but continue to proliferate until the mass of the liver is restored. The aim of this study was to establish whether tyrosine phosphorylation, an emerging mechanism of regulation of mitochondrial function, participates in the response to liver injury following PH and is involved in contrasting mitochondrial pro-apoptotic signalling. Mitochondrial tyrosine phosphorylation, negligible in the quiescent liver, was detected in the early phase... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2898123 Thu, 15 Oct 2009 23:00:00 +0100 2898123 http://www.medworm.com/index.php?rid=2841947&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091247 The ε subunit, a small subunit located in the F1 domain of ATP synthase and comprised of two distinct domains, an N-terminal β-sandwich structure and a C-terminal α-helical region, serves as an intrinsic inhibitor of ATP hydrolysis activity. This inhibitory function is especially important in photosynthetic organisms as the enzyme cannot synthesize ATP in the dark but may catalyze futile ATP hydrolysis reactions. To understand the structure-function relationship of this subunit in F1 from photosynthetic organisms, we solved the NMR structure of the ε subunit of ATP synthase obtained from thermophilic cyanobacteria Thermosynechococcus elongatus BP-1, and examined the flexibility of the C-terminal domains using molecular dynamics simulations. In addition, we rev... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2841947 Mon, 28 Sep 2009 23:00:00 +0100 2841947 http://www.medworm.com/index.php?rid=2785389&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20091158 We studied free fatty acid-induced uncoupling of heterologously expressed rat UCP1 in yeast mitochondria as well as UCP1 in rat BAT mitochondria. The proton conductance curves and the relationship between ubiquinone reduction level and membrane potential were determined in non-phosphorylating BAT and yeast mitochondria. The ADP/O method was applied to determine the ADP phosphorylation rate and the relationship between ubiquinone reduction level and respiration rate in yeast mitochondria. Our studies of membranous ubiquinone reduction level in mitochondria demonstrate that activation of UCP1 leads to a purine nucleotide-sensitive decrease in the ubiquinone redox state. Results obtained for non-phosphorylating and phosphorylating mitochondria, as the endogenous ubiquinone redox state was gra... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2785389 Thu, 10 Sep 2009 23:00:00 +0100 2785389 http://www.medworm.com/index.php?rid=2781383&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090934 Mitochondria play a critical role in mediating the cellular response to oxidants formed during acute and chronic cardiac dysfunction. It is widely assumed that, as cells are subject to stress, mitochondria are capable of drawing upon a “reserve capacity” which is available to serve the increased energy demands for maintenance of organ function, cellular repair, or detoxification of reactive species. This hypothesis further implies that impairment or depletion of this putative reserve capacity ultimately leads to excessive protein damage and cell death. However, it has been difficult to fully evaluate this hypothesis since much of our information about the response of the mitochondrion to oxidative stress derives from studies on mitochondria isolated from their cellular contex... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2781383 Wed, 09 Sep 2009 23:00:00 +0100 2781383 http://www.medworm.com/index.php?rid=2777783&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090991 In the present study, we describe the existence of a novel potassium channel in the plant (potato tuber) mitochondrial inner membrane. We found that substances known to modulate large-conductance calcium-activated potassium channel activity influenced the bioenergetics of potato tuber mitochondria. In isolated mitochondria, Ca2+ and NS1619 (a potassium channel opener) were found to depolarize the mitochondrial membrane potential and to stimulate resting respiration. These effects were blocked by iberiotoxin (a potassium channel inhibitor) in a potassium-dependent manner. Additionally, the electrophysiological properties of the large-conductance potassium channel present in the potato tuber inner mitochondrial membrane are described in a reconstituted system, using planar lipid bilay... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2777783 Tue, 08 Sep 2009 23:00:00 +0100 2777783 http://www.medworm.com/index.php?rid=2701852&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090666 The free radical theory of ageing proposes that reactive oxygen species (ROS) are major driving forces of ageing, and are also critically involved in cellular senescence. Besides the mitochondrial respiratory chain, alternative sources of ROS have been described, which might contribute to cellular senescence. NADPH oxidases are well-known sources of superoxide, which contribute to the antimicrobial capabilities of macrophages, a process involving the prototypical member of the family referred to as Nox2. However, in the recent years non-phagocytic homologues of Nox2 have been identified, which are involved in processes other than the host defence. Superoxide anions produced by these enzymes are believed to play a major role in signalling by MAP kinases and stress-activated kinases, but cou... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2701852 Thu, 13 Aug 2009 23:00:00 +0100 2701852 http://www.medworm.com/index.php?rid=2666767&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090716 Myoglobin (Mb) is a haemoprotein present in cardiac, skeletal and smooth muscle and is primarily responsible for the storage and “facilitated transfer” of molecular oxygen from the cell membrane to mitochondria. Also, Mb plays a role in regulating nitric oxide (•NO) homeostasis through (i) binding •NO (Mb-NO complex), (ii) oxidation of •NO to nitrate, and (iii) formation of vasoactive S-nitroso-Mb [Rayner BS, Wu B-J, Raftery M, Stocker R, Witting PK. J. Biol. Chem. 2005;280, 9985-9993]. Pathological •NO concentrations affect mitochondrial function and decrease cell viability through inducing apoptosis. Treatment of cultured rat vascular smooth muscle cells (VSMC) with cumulative doses (0.1, 1 or 10 μM) of •NO from the donors diethylam... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2666767 Mon, 03 Aug 2009 23:00:00 +0100 2666767 http://www.medworm.com/index.php?rid=2666766&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090594 In this study, we determined the effect of nitration of Tyr345 and Tyr368 in β-subunit of the F1-ATPase using site-directed mutagenesis. Nitration of β-subunit, achieved by using tetranitromethane (TNM), resulted in 66% ATPase activity loss. This treatment resulted in the modification of several Asn, Met, and Tyr. However, nitrated Tyr and ATPase inactivation were decreased in reconstituted F1 with Y368F (54%), Y345F (28%) and Y345,368F (1%) β-subunits indicating a clear link between nitration at these positions and activity loss, regardless of the presence of other modifications. Kinetic studies indicated that an F1 with one nitrated Tyr (345 or 368) or two Tyr368 was sufficient to grant inactivation. Tyr368 was 4-times more reactive to nitration due to its lower pKa.... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2666766 Mon, 03 Aug 2009 23:00:00 +0100 2666766 http://www.medworm.com/index.php?rid=2521661&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082427 In this study, we used isotopic tracing to demonstrate that both TTP and dCTP are solely synthesized by phosphorylation of thymidine and deoxycytidine, respectively, with no evidence for synthesis from other precursors. We also showed that UTP and CTP are synthesized by phosphorylation of uridine and cytidine, respectively, with no detectable role for the de novo pyrimidine synthesis pathway. Lastly, we demonstrated that AZT decreased the TTP pool by 50% in 30 min of perfusion while having no effect on other dNTPs. In summary, this work demonstrated that adult rat heart has a limited mechanism for dCTP and TTP synthesis and thus these pools may be more sensitive than replicating cells to drugs such as AZT that affect the salvage pathway. (Source: BJ Energy) BJ Energy journals http://www.medworm.com/rss/comments.php?id=2521661 Thu, 25 Jun 2009 23:00:00 +0100 2521661 http://www.medworm.com/index.php?rid=2503401&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082220 We present a model where small anti-cooperative redox interactions occur between haem a-CuA-CuB (steady state potential ranges: CuA 212-258 mV; haem a: 254-281 mV; CuB 227- 272 mV). Contrary to static equilibrium measurements, in the catalytic steady state there are no high potential redox centres (> 300 mV). We find that the overall reaction is correctly described by the classical model in which the Michaelis intermediate is a ferrocytochrome c – enzyme complex. However, the oxidation of ferrocytochrome c in this complex is not the sole rate-determining step. Turnover is instead dependent upon electron transfer from haem a to haem a3, but the haem a potential closely matches cytochrome c at all times. (Source: BJ Energy) BJ Energy journals http://www.medworm.com/rss/comments.php?id=2503401 Tue, 16 Jun 2009 23:00:00 +0100 2503401 http://www.medworm.com/index.php?rid=2503402&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090176 Oxidative stress has been implicated as a cause of various diseases such as anemia. We found that Cu,Zn-superoxide dismutase gene (SOD1)-deficiency causes anemia, and the production of autoantibodies against red blood cells (RBCs) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the human SOD1 (hSOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1−/− background (SOD1−/−;hSOD1tg/+). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1−/−;hSOD1tg/+ mice were approximately equivalent to those of SOD1+/+ mice. The produ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2503402 Tue, 09 Jun 2009 23:00:00 +0100 2503402 http://www.medworm.com/index.php?rid=2446196&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082410 We showed previously that Na+,K+-ATPase interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular weight (320-400 kDa) during molecular chromatography. We also found that microtubules assembled in vitro are able to bind Na+,K+-ATPase when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated Na+,K+-ATPase. Furthermore, we determined that the fifth cytoplasmic domain of Na+,K+-ATPase (CD5) is capable of interacting with acetylated tubulin. Taken together, our results are consistent with the idea that Na+... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2446196 Fri, 29 May 2009 04:00:00 +0100 2446196 http://www.medworm.com/index.php?rid=2432065&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090492 NADH:ubiquinone oxidoreductase (complex I) is an entry point for electrons into the respiratory chain in many eukaryotes. It couples NADH oxidation and ubiquinone reduction to proton translocation across the mitochondrial inner membrane. Because complex I deficiencies occur in a wide range of neuromuscular diseases, including Parkinson’s disease, there is a clear need for model eukaryotic systems to facilitate structural, functional and mutational studies. Here, we describe the purification and characterization of the complexes I from two yeast species, Pichia pastoris and Pichia angusta. They are obligate aerobes which grow to very high cell densities on simple media, as yeast-like, spheroidal cells. Both Pichia enzymes catalyze inhibitor-sensitive NADH:ubiquinone oxidoreduction, d... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2432065 Fri, 22 May 2009 04:00:00 +0100 2432065 http://www.medworm.com/index.php?rid=2379970&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090039 Thioesterase superfamily member 2 (Them2) is a 140 aa protein of unknown biological function that comprises a single hotdog fold thioesterase domain. Based on its putative association with mitochondria, accentuated expression in oxidative tissues and interaction with StarD2, a regulator of fatty acid metabolism, we explored whether Them2 functions as a physiologically relevant fatty acyl-CoA thioesterase. In solution, Them2 formed a stable homotetramer, which denatured in a single transition at 59.3 °C. Them2 exhibited thioesterase activity for medium- and long-chain acyl-CoAs, with KM values that decreased exponentially as a function of increasing acyl chain length. Steady state kinetic parameters for Them2 were characteristic of long chain mammalian acyl-CoA thioesterases, with mi... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2379970 Thu, 30 Apr 2009 04:00:00 +0100 2379970 http://www.medworm.com/index.php?rid=2363590&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090286 Because L-ascorbic acid (AA) scavenges various types of free radicals to form monodehydroascorbic acid (MDAA) and dehydroascorbic acid (DAA), its regeneration from the oxidized metabolites is critically important for humans and other animals that lack the ability to synthesize this antioxidant. To study the dynamic aspects of AA metabolism in the circulation, a long acting ascorbate oxidase (AOase) derivative was synthesized by covalently linking 2,4-bis[o-methoxy(polyethyleneglycol)]–6–chloro –S-triazine (PEG) to the enzyme. Fairly low concentrations of the modified enzyme (PEG-AOase) rapidly decreased AA levels in isolated fresh plasma and blood samples with concomitant increase in their levels of MDAA and DAA. In contrast, relatively high doses of PEG-AOase were req... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2363590 Thu, 23 Apr 2009 04:00:00 +0100 2363590 http://www.medworm.com/index.php?rid=2346641&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090309 Tissue damage resulting from the extracellular production of hypochlorous acid (HOCl) by the myeloperoxidase (MPO)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically-useful MPO inhibitors. Nitroxides are well-established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase-mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by M... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2346641 Mon, 20 Apr 2009 04:00:00 +0100 2346641 http://www.medworm.com/index.php?rid=2323275&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090242 In this study, western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidised at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 µM in the mitochondrial matrix of Jurkat T-lymphoma cells. While the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs other structural features must contribute to the innate resilience of Prx 3 to hyper... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2323275 Wed, 08 Apr 2009 04:00:00 +0100 2323275 http://www.medworm.com/index.php?rid=2323277&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090214 Cytochrome c Oxidase (CcO) is a multisubunit bigenomic protein complex which catalyzes the last step of the mitochondrial electron transport chain. The nuclear encoded subunits are thought to have roles either in the regulation or in the structural stability of the enzyme. Subunit Vb is a peripheral nuclear-encoded subunit of mammalian CcO that is dramatically reduced under hypoxia. Although subunit Vb contains different ligand binding sites and undergoes phosphorylation, its precise function remains unknown. In the present study we generated a cell line from RAW 264.7 murine macrophages, that contains 80% reduced subunit Vb. Functional analysis of these cells showed loss of CcO activity, disrupted membrane potential and lower ability to generate ATP. Resolution of complexes on Blue native... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2323277 Wed, 01 Apr 2009 04:00:00 +0100 2323277 http://www.medworm.com/index.php?rid=2283094&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081932 In conclusion, this study highlights that CPT1A is a prime target to increase hepatic LCFA ß-oxidation and that acting directly on the degree of its malonyl-CoA sensitivity may be a relevant strategy to prevent and/or correct hepatic steatosis. (Source: BJ Energy) BJ Energy journals http://www.medworm.com/rss/comments.php?id=2283094 Fri, 20 Mar 2009 04:00:00 +0100 2283094 http://www.medworm.com/index.php?rid=2283095&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082137 The continuous recycling of heme iron following phagocytosis and catabolism of senescent and damaged red blood cells by macrophages is a crucial process in the maintenance of systemic iron homeostasis. However, little is known about macrophage iron handling in hemolytic states resulting from a deficiency in antioxidant defenses. Our observations indicate that recently described [Iuchi Y, Okada F, Onuma K, Onoda T, Asao H, Kobayashi M, Fuji J. Biochem. J. (2007) 402, 219-227] the chronic but moderate regenerative hemolytic anemia of old superoxide dismutase 1 (SOD1) knockout mice is associated with red blood cells modifications and sensitivity to both intra- and extravascular hemolysis. We have then characterized the molecular pathways of iron turnover in the liver of SOD1-deficient mice. D... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2283095 Wed, 18 Mar 2009 04:00:00 +0100 2283095 http://www.medworm.com/index.php?rid=2242771&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090151 The biosynthesis of the tetrapyrrole framework has been investigated in the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough by characterization of the enzymes required for the transformation of 5-aminolevulinic acid into sirohydrochlorin. Porphobilinogen synthase (HemB) was found to be a zinc-dependent enzyme that exists in its native state as a homohexamer. Porphobilinogen deaminase (HemC) was shown to contain the dipyrromethane cofactor. Uroporphyrinogen III synthase is found fused with a uroporphyrinogen III methyltransferase (HemD-CobA). Both activities could be demonstrated in this amalgamated protein and the individual enzyme activities were separated by dissecting the relevant gene to allow the production of two distinct proteins. A gene annotated on the genome as a... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2242771 Fri, 06 Mar 2009 05:00:00 +0100 2242771 http://www.medworm.com/index.php?rid=2212543&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20090076 Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is a homotetramer, which is allosterically activated by the binding of fumarate. The fumarate binding site is located at the dimer interface of the NAD(P)-ME. In this paper, we decipher the functional role of the residue Lys57, which resides at the fumarate binding site and dimer interface and thus may be involved in the allosteric regulation and subunit-subunit interaction of the enzyme. In the present study, Lys57 is replaced with Ala, Cys, Ser and Arg. Site-directed mutagenesis and kinetic analysis strongly suggest that Lys57 (K57) is important for the fumarate-induced activation and quaternary structural organization of the enzyme. K57 mutant enzymes demonstrate a reduction of Km and an elevation of kcat following ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2212543 Tue, 24 Feb 2009 05:00:00 +0100 2212543 http://www.medworm.com/index.php?rid=2202354&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082397 Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PEPC protein kinase (PPCK) in plant acclimation to nutritional Pi deprivation. However, little is known about the genetic origin or phosphorylation status of native PEPCs from Pi-deficient (–Pi) plants. Transfer of Arabidopsis suspension cells or seedlings to –Pi growth media resulted in: (i) the marked transcriptional upregulation of genes encoding the PEPC isozyme AtPPC1, and PPCK isozymes AtPPCK1 and AtPPCK2, (ii) >2-fold increases in PEPC specific activity and amount of an immunoreactive 107 kDa PEPC polypeptide (p107), and (iii) in vivo p107 phosphorylation as revealed by i... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2202354 Thu, 19 Feb 2009 05:00:00 +0100 2202354 http://www.medworm.com/index.php?rid=2157779&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081299 The cholesteryl ester transfer protein (CETP) is a plasma protein synthesized in several tissues, mainly liver, which reduces plasma HDL-cholesterol and increases the risk of atherosclerosis. The effect of CETP levels on postprandial intravascular metabolism of triglycerides (TG) is an often-overlooked aspect of the relationship between CETP and lipoprotein metabolism. Here, we tested the hypothesis that CETP delays the plasma clearance of TG rich lipoprotein by comparing human CETP expressing transgenic (Tg) and non-transgenic (Non-Tg) mice. After an oral fat load, the postprandial triglyceridemia curve was markedly increased in CETP-Tg compared to Non-Tg mice (280±30 vs. 190±20 mg/dL.6h, respectively, p< 0.02). No differences in intestinal fat absorption and VLDL secreti... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2157779 Wed, 04 Feb 2009 05:00:00 +0100 2157779 http://www.medworm.com/index.php?rid=2138849&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081731 In conclusion: 1. The transamination of branched-chain 2-oxoacids is more important to the stimulation of insulin secretion than their catabolism. 2. Transamination decreases islet GABA concentrations by promoting GABA metabolism. 3. Inhibition of 2-oxoglutarate dehydrogenase by branched-chain 2-oxoacids may increase metabolic flux in the “GABA-shunt” at the expense of reduced TCA cycle flux. (Source: BJ Energy) BJ Energy journals http://www.medworm.com/rss/comments.php?id=2138849 Wed, 28 Jan 2009 05:00:00 +0100 2138849 http://www.medworm.com/index.php?rid=2126403&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081350 Heme oxygenase 1 (HO-1) is an essential antioxidant enzyme in the cell that exerts its effects through removal of pro-oxidant heme groups and the formation of antioxidant molecules and carbon monoxide. Decreased HO-1 in the vasculature is associated with increased development of atherosclerosis and also leads to extensive damage to the mitochondrion. These findings have led us to the hypothesis that mitochondria may play an important role in induction of HO-1 in endothelial cells. To investigate this, the effects of mitochondrial parameters such as membrane potential, protein thiols, and reactive oxygen species formation on induction of HO-1 by the electrophilic cyclopentenone 15-deoxy-prostaglandin J2 (15d-PGJ2) were assessed. Interestingly, the mitochondrially-targeted thiol reactive com... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2126403 Fri, 23 Jan 2009 05:00:00 +0100 2126403 http://www.medworm.com/index.php?rid=2117196&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082419 In low nanomolar concentrations, nitric oxide (NO) functions as a transmitter in brain and other tissues whereas near-micromolar NO concentrations are associated with toxicity and cell death. Control of the NO concentration, therefore, is critical for proper brain function but, while its synthesis pathway is well-characterised, the major route of breakdown of NO in brain is unclear. Previous observations indicate that brain cells actively consume NO at a high rate. The mechanism of this consumption was pursued in the present study. NO consumption by a preparation of central glial cells was abolished by cell lysis and recovered by addition of NADPH. NADPH-dependent consumption of NO localised to cell membranes and was inhibited by proteinase K, indicating the involvement of a membrane-bound... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2117196 Tue, 20 Jan 2009 05:00:00 +0100 2117196 http://www.medworm.com/index.php?rid=2117195&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082279 Heme oxygenase (HO) catalyses the degradation of heme to biliverdin, CO and ferrous iron via three successive oxygenation reactions, i.e. heme to α-hydroxyheme, α-hydroxyheme to α-verdoheme, and α-verdoheme to ferric biliverdin-iron chelate. Here, we determined the crystal structure of ferrous α-verdoheme-rat HO-1 complex at 2.2 Å resolution. The overall structure of the verdoheme complex was similar to that of the heme complex. H2O or OH- was coordinated to the verdoheme iron as a distal ligand. A hydrogen bond network consisting of water molecules and several amino acid residues was observed at the distal side of verdoheme. Such a hydrogen bond network was conserved in the structures of rat HO-1 complexes with heme and with the ferric biliverdin-... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2117195 Tue, 20 Jan 2009 05:00:00 +0100 2117195 http://www.medworm.com/index.php?rid=2098582&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082159 Although carnitine palmitoyltransferase-I (CPT-I) is generally regarded to present a major rate-controlling site in mitochondrial β-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall long-chain fatty acid (LCFA) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm. Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2098582 Tue, 13 Jan 2009 05:00:00 +0100 2098582 http://www.medworm.com/index.php?rid=2087827&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082003 Peroxiredoxin 5 (Prx5) is a distinct isoform of the peroxiredoxin gene family. The antioxidative and anti-apoptotic functions of Prx5 have been extensively demonstrated in cell culture experiments. Here, we provide the first functional analysis of Prx5 in a multi-cellular organism, Drosophila melanogaster. Like its mammalian, yeast or human counterparts, Drosophila peroxiredoxin 5 (dPrx5) is expressed in several cellular compartments, including cytosol, nucleus and the mitochondrion. Global over-expression of dPrx5 in flies increased resistance to oxidative stress and extended their life span by up to 30% under normal conditions. The dprx5 (-/-) null flies were comparatively more susceptible to oxidative stress, had higher incidence of apoptosis, and a shortened life span. TUNEL analysis r... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2087827 Wed, 07 Jan 2009 05:00:00 +0100 2087827 http://www.medworm.com/index.php?rid=2042407&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081999 c-type cytochromes are normally characterised by covalent attachment of the iron cofactor heme to protein through two thioether bonds between the vinyl groups of heme and the thiols of a Cys-Xxx-Xxx-Cys-His motif. Heme attachment is an enzyme catalysed, post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b562 containing a CXXCH heme-binding motif with the E. coli cytochrome c maturation (Ccm) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, without the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having heme inverted and attached through only one thioether bond. Here we use further variants of cytochrome b562 to investigate the substrate spe... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2042407 Wed, 17 Dec 2008 05:00:00 +0100 2042407 http://www.medworm.com/index.php?rid=2042406&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20082028 The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox (a.k.a. Nox2), which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox, and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox–p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its tandemly-arranged two SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. Here we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADP... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2042406 Wed, 17 Dec 2008 05:00:00 +0100 2042406 http://www.medworm.com/index.php?rid=2007852&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081981 The epimastigote stage of Trypanosoma cruzi undergoes programmed cell death (PCD) when exposed to fresh human serum (FHS). Although it has been known for over 30 years that complement is responsible for FHS-induced death, the link between complement activation and triggering of PCD has not been established. We have previously shown that mitochondrion participates in the orchestration of PCD in this model. Several changes in mitochondrial function were described, and in particular it was shown that mitochondrion-derived superoxide (O2•-) is necessary for PCD. In the present work, we established mitochondrial Ca2+ overload as the link between complement deposition and the observed changes in mitochondrial physiology and the triggering of PCD. We showed that complement activation ended... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2007852 Wed, 03 Dec 2008 05:00:00 +0100 2007852 http://www.medworm.com/index.php?rid=2007853&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080987 Nitrite (NO2-) recycling to nitric oxide (NO) is catalyzed by a number of enzymes and induces a protective vasodilatory effect under hypoxia/ ischemia. In the present work, we tested the in vitro ability of the three NOS isoforms to release NO from nitrite under anoxia using electrochemical detection, chemiluminescence and absorption spectroscopy. The release of free NO from anoxic nitrite solutions at 15µM was specific to the endothelial isoform eNOS and did not occur with neuronal nNOS and inducible iNOS. Unlike xanthine oxidase, eNOS reductase domain did not recycle nitrite to NO and WT eNOS did not reduce nitrate. Our data suggest that structural—and by inference—dynamic differences between nNOS and eNOS in the distal heme domains account for eNOS being the only is... BJ Energy journals http://www.medworm.com/rss/comments.php?id=2007853 Mon, 01 Dec 2008 05:00:00 +0100 2007853 http://www.medworm.com/index.php?rid=1970636&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081422 Cell surface localization of the glucose transporter, Glut1, is a cytokine-controlled process essential to support the metabolism and survival of hematopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. Here we show a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence through deletion or point mutation sharply decreased surface Glut1 levels and led to rapid targeting of internalized Glut1 to lysosomes for proteolysis, particularly in growth factor-deprived cells. The PDZ domain protein, GIPC, bound to Glut1 in part via the Glut1 C-terminal PDZ binding motif and we fo... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1970636 Wed, 19 Nov 2008 05:00:00 +0100 1970636 http://www.medworm.com/index.php?rid=1948372&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081478 Phytopathogenic fungi like Magnaporthe grisea are unique in having two catalase-peroxidase (KatG) paralogs located either intracellularly (KatG1) or extracellularly (KatG2). The coding genes have recently been shown to derive from a lateral gene transfer from a (proteo)bacterial genome followed by gene duplication and diversification. Here we demonstrate that Magnaporthe grisea KatG1 is expressed constitutively in rice blast fungus. It is the first eukaryotic catalase-peroxidase to be expressed heterologously in E. coli in high amount and purity with almost 100% haem occupancy. Recombinant MagKatG1 is an acidic, mainly homodimeric oxidoreductase with a predominant five-coordinated high-spin haem b. At 25 °C and pH 7.0, the reduction potential of the Fe(III)/Fe(II) couple is found to... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1948372 Mon, 10 Nov 2008 05:00:00 +0100 1948372 http://www.medworm.com/index.php?rid=1918603&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081161 β-Diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1 protomer adopts a single-domain β-barrel fold characteristic of the Cupin superfamily of proteins and features a mononuclear non-heme Fe2+ centre where a triad of histidines, His-62, His-64, and His-104, coordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-heme Fe2+, we replaced each histidine individually by Glu and Asn and compared binding of Fe2+ and four non-native, catalytically inactive metals to purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titrat... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1918603 Thu, 30 Oct 2008 04:00:00 +0100 1918603 http://www.medworm.com/index.php?rid=1896833&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080805 In this study it is reported that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 µM for 30 minutes) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 µM 3-BrPA, but this concentration caused more than 70% inhibition of glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose free medium. On the other hand, when cells were ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1896833 Wed, 22 Oct 2008 04:00:00 +0100 1896833 http://www.medworm.com/index.php?rid=1896832&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081423 Although the nitric oxide (NO)-mediated modification of iron-sulfur proteins has been well documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. Here, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli dihydroxyacid dehydratase (IlvD) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with the electron paramagnetic resonance (EPR) spectroscopy and the enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound dinitrosyl iron complex (DNIC) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reac... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1896832 Wed, 22 Oct 2008 04:00:00 +0100 1896832 http://www.medworm.com/index.php?rid=1896831&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081066 The ferric uptake regulator (Fur) is a prokaryotic transcriptional regulator that controls a large number of genes mainly related to iron metabolism. Frequently, several Fur homologues with different physiological roles are found in the same organism. The genome of the filamentous cyanobacterium Anabaena (Nostoc) sp. PCC 7120 codes for three different fur genes. FurA is an essential protein involved in iron homeostasis that also modulates dinitrogen fixation. FurA interacts with heme, impairing its DNA-binding ability. To explore functional differences between Fur homologues in Anabaena, factors affecting their regulation, as well as some biochemical characteristics have been investigated. While incubation of FurB with heme severely hinders it ability to interact with DNA, binding of heme ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1896831 Wed, 22 Oct 2008 04:00:00 +0100 1896831 http://www.medworm.com/index.php?rid=1884297&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081262 The SCaMCs constitute a subfamily of mitochondrial carriers responsible of the ATP-Mg/Pi exchange with at least three paralogues in vertebrates. SCaMCs members are proteins with two functional domains the C-terminal transporter domain and the N-terminal one which harbours Ca2+-binding EF-hands and facing the intermembrane space. Here, we have characterised a shortened fourth paralog, SCaMC-3-Like (also named as Slc25a41), which lacks the Ca2+-binding N-extension. SCaMC-3-Like orthologues are found exclusively in mammals showing around 60% identity to the carboxyl-half of SCaMC-3, its closest paralog. In mammalian genomes, SCaMC-3 and SCaMC-3-Like genes are adjacent on the same chromosome, forming a head-to-tail tandem array and show identical exon-intron boundaries indicating that SCaMC-3-... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1884297 Fri, 17 Oct 2008 04:00:00 +0100 1884297 http://www.medworm.com/index.php?rid=1854103&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081257 Enzymatic activation of phenylacetic acid (PAA) to phenylacetyl-CoA is an important step in the biosynthesis of the β-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and phenoxyacetic acid (POA) act as acyl donor in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a phenylacetate-CoA ligase, was cloned in Escherichia coli as a maltose binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA to phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with kcat/Km values of 0.23 ± 0.06 and 7.8 &#x0... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1854103 Mon, 06 Oct 2008 04:00:00 +0100 1854103 http://www.medworm.com/index.php?rid=1854102&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081713 CopR of Lactococcus lactis is a copper-responsive repressor involved in copper homeostasis. It controls the expression of a total of eleven genes, the CopR regulon, in a copper-dependent manner. In the absence of copper, CopR binds to the promoters of the CopR regulon. Copper releases CopR from the promoters, allowing transcription of the downstream genes to proceed. CopR binds through its N terminal domain to a 'cop box' of consensus TACAxxTGTA, which is conserved in Firmicutes. We here solved the NMR solution structure of the N-terminal DNA binding domain of CopR. The protein fold has a winged helix structure resembling that of the BlaI repressor which regulates antibiotic resistance in Bacillus licheniformis. CopR differs from other copper-responsive repressors and the present structure... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1854102 Mon, 06 Oct 2008 04:00:00 +0100 1854102 http://www.medworm.com/index.php?rid=1838490&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081426 The canonical Wnt signaling pathway acts by slowing the rate of ubiquitin mediated β-catenin degradation. This results in the accumulation and subsequent nuclear translocation of β-catenin, which induces expression of a number of genes involved in growth, differentiation and metabolism. The mechanisms regulating the Wnt signaling pathway in physiological contexts are still not fully understood. Here we provide evidence that changes in glucose levels within the physiological range can acutely regulate the levels of β-catenin in two macrophages cell lines (J774.2 and RAW264 cells). In particular we find that glucose induces these effects by promoting an autocrine activation of Wnt signaling that is mediated the hexosamine pathway and changes in N-linked glycosylation of ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1838490 Tue, 30 Sep 2008 04:00:00 +0100 1838490 http://www.medworm.com/index.php?rid=1830592&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081345 We have developed a simple native-like surface-tethered membrane system to investigate the activity of cytochrome bo3 (cbo3), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo3 substrate ubiquinol-10 (UQ-10). Tethered membranes are formed by self assembly from vesicles onto gold electrodes functionalised with cholesterol derivatives. Cytochrome bo3 activity was monitored using cyclic voltammetry with electron transfer to cbo3 mediated by UQ-10. The apparent KM for oxygen with this system is 1.1±0.4 μM, in good agreement with literature values for whole cell experiments and for purified cbo3. Increasing the concentration of lipophilic UQ-10 in the ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1830592 Fri, 26 Sep 2008 04:00:00 +0100 1830592 http://www.medworm.com/index.php?rid=1795450&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081082 This study provides evidence for SREBP-1c as a principal target for PUFA regulation of CiC transcription. In H4IIE cells overexpression of nSREBP-1c overrides arachidonic acid (C20:4, n-6) suppression but does not prevent the repression by docosahexaenoic acid (C22:6, n-3). ChIP assay in H4IIE cells showed that docosahexaenoic acid affects the binding of NF-Y, Sp1 and SREBP-1 to PUFA response region of CiC promoter whereas arachidonic acid alters only the binding of SREBP-1. Our data show that PUFA inhibition of hepatic Cic gene transcription is mediated not only by the nuclear level of SREBP-1c, but also might involve a reduction in Sp1 and NF-Y DNA binding, suggesting differential mechanisms in the Cic gene regulation by different PUFA. (Source: BJ Energy) BJ Energy journals http://www.medworm.com/rss/comments.php?id=1795450 Tue, 16 Sep 2008 04:00:00 +0100 1795450 http://www.medworm.com/index.php?rid=1783792&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081240 The insulin-sensitizing hormone, adiponectin, belongs to the expanding C1q/TNF family of proteins. We recently identified a family of adiponectin paralogs designated as C1q/TNF-related protein (CTRP)-1 to 7, and here describe CTRP10. Here, we show that CTRP1, CTRP2, CTRP3, CTRP5, and CTRP7 transcripts are expressed predominantly by adipose tissue. In contrast, placenta and eye expressed the highest levels of CTRP6 and CTRP10 transcripts, respectively. Expression levels of CTRP1, CTRP2, CTRP3, CTRP6, and CTRP7 transcripts are upregulated in 8-week-old obese (ob/ob) mice relative to lean controls. Treatment of mice with a PPAR-γ agonist, rosiglitazone, increased the expression of CTRP1 while decreased CTRP6 transcript levels. All CTRPs are secreted glycoproteins when expressed in mamm... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1783792 Thu, 11 Sep 2008 04:00:00 +0100 1783792 http://www.medworm.com/index.php?rid=1776518&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080740 The liver is responsible for key metabolic functions, including control of normal homeostasis in response to diet and xenobiotic metabolism/detoxification. We have previously shown that inactivation of the hepatic cytochrome P450 system through conditional deletion of P450 oxidoreductase (POR) induces hepatic steatosis, liver growth and P450 expression. We have exploited a new conditional model of POR deletion to investigate the mechanism underlying these changes. We demonstrate that P450 induction, liver growth and hepatic triglyceride homeostasis are intimately linked and provide evidence that the observed phenotypes result from hepatic accumulation of unsaturated fatty acids, which mediate these phenotypes by activation of the nuclear receptor CAR and, to a lesser degree, PXR. To our kn... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1776518 Tue, 09 Sep 2008 04:00:00 +0100 1776518 http://www.medworm.com/index.php?rid=1765562&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080762 In this study, we showed that KLF5 acts as a key regulator controlling the expression of fatty acid synthase (FASN) through the interaction with sterol regulatory element-binding protein-1 (SREBP-1) in androgen-dependent LNCaP prostate cancer cell line. The mRNA level of KLF5 increased when cells were treated with a synthetic androgen, R1881. Furthermore, KLF5 bound to SREBP-1 and enhanced the SREBP-1-mediated increase in the FASN promoter activity. The data also demonstrated that the expression of KLF5 in LNCaP prostate cancer cells enhanced FASN expression, whereas silencing of KLF5 by small interfering RNA down-regulated FASN expression. The proximal promoter region and the first intron of the FASN gene contain multiple CACCC elements that mediate the transcriptional regulation of the g... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1765562 Fri, 05 Sep 2008 04:00:00 +0100 1765562 http://www.medworm.com/index.php?rid=1756210&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080466 Mycobacterium tuberculosis flavoprotein reductase A (FprA) is a NAD(P)H- and FAD-binding reductase that is structurally/evolutionarily related to adrenodoxin reductase. Structural analysis implicates arginines 199 and 200 in interactions with the NADP(H) 2’-phosphate group. R199A, R200A and R199A/R200A mutants were characterized to explore the roles of these basic residues. All mutations abolished neutral FAD semiquinone stabilization in NADPH-reduced enzyme, owing to weakened NADPH affinity. Instead, FAD hydroquinone was formed in all mutants, and each displayed substantially enhanced auto-oxidation rates (20-40 fold) compared to NADPH-reduced wild-type FprA. Steady-state ferricyanide reduction studies revealed diminished NADPH affinity (higher Km values), but lower NADH Km values.... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1756210 Wed, 03 Sep 2008 04:00:00 +0100 1756210 http://www.medworm.com/index.php?rid=1752014&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20070722 The understanding of the mechanism, oxidant(s) involved, and how/what protein radicals are produced during the reaction of wild type Cu, Zn-superoxide dismutase (SOD1) with H2O2 and their fate is incomplete, but a better understanding of the role of this reaction is needed. We used immuno-spin trapping and mass spectrometry analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2 using human serum albumin (HSA) and mouse brain homogenate (mBH) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and carbonate radical anion-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via CO3•- from that initiated by copper released from t... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1752014 Tue, 02 Sep 2008 04:00:00 +0100 1752014 http://www.medworm.com/index.php?rid=1735753&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080847 Mice homozygous for a defect in the PTCD2 (pentatricopeptide domain protein 2) gene were generated in order to study the role of this protein in mitochondrial RNA metabolism. These mice displayed specific but variable reduction of ubiquinol-cytochrome c reductase complex activity in mitochondria of heart, liver and skeletal muscle due to a decrease in the expression of mitochondrial DNA encoded cytochrome b, the catalytic core of the complex. This reduction in mitochondrial function has a profound effect on the myocardium with replacement of ventricular cardiomyocytes by fibro-fatty tissue. Northern blotting showed a reduction in the mRNA for mitochondrial DNA encoded proteins cytochrome b (cytb) and ND5 (NADH dehydrogenase subunit 5) and an elevation in a combined pre-processed ND5-CYTB t... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1735753 Wed, 27 Aug 2008 04:00:00 +0100 1735753 http://www.medworm.com/index.php?rid=1724391&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20081133 Bacillus megaterium flavocytochrome P450 BM3 is a catalytically self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPH-cytochrome P450 reductase and P450 domains. Selected mutations at residue 264 in the heme (P450) domain of the enzyme lead to novel amino acid 6th (distal) coordination ligands to the heme iron. The catalytic, spectroscopic and thermodynamic properties of the A264M, A264Q and A264C variants were determined in both the intact flavocytochromes and heme domains of P450 BM3. Crystal structures of the mutant heme domains demonstrate axial ligation of P450 heme iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel heme iron ligand sets in the A264M/Q variants. In contrast, the crystal structure of the A264C variant reveals no d... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1724391 Fri, 22 Aug 2008 04:00:00 +0100 1724391 http://www.medworm.com/index.php?rid=1697850&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080690 Systems biology approaches, such as kinetic modelling, could provide valuable insights into how thioredoxins, glutaredoxins and peroxiredoxins (here collectively called redoxins), and the systems that reduce these molecules are regulated. However, it is not clear whether redoxins should be described as redox couples (with redox potentials) or as enzymes (with Michaelis-Menten parameters) in such approaches. We show that in complete redoxin systems, redoxin substrate saturation and other purported enzymatic behaviours result from limitations in the redoxin redox cycles in these systems. Michaelis-Menten parameters are therefore inappropriate descriptors of redoxin activity; data from redoxin kinetic experiments should rather be interpreted in terms of the complete system of reactions under ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1697850 Tue, 12 Aug 2008 04:00:00 +0100 1697850 http://www.medworm.com/index.php?rid=1672496&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080701 Nitroalkene derivatives of fatty acids act as adaptive, anti-inflammatory signaling mediators, based on their high affinity PPARγ ligand activity and electrophilic reactivity with proteins, including transcription factors. While free or esterified lipid nitroalkene derivatives have been detected in human plasma and urine, their generation by inflammatory stimuli has not been reported. Herein, we show increased nitration of cholesteryl-linoleate by activated murine J774.1 macrophages, yielding the mononitrated nitroalkene: cholesteryl-nitrolinoleate (CLNO2). CLNO2 levels were found to increase ~20-fold 24 hr after macrophage activation with E. coli lipopolysaccharide plus interferon-γ; this response was concurrent with an increase in the expression of inducible nitric oxide sy... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1672496 Fri, 01 Aug 2008 04:00:00 +0100 1672496 http://www.medworm.com/index.php?rid=1650272&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20070941 In this study, the products of the reaction of HOSCN with proteins has been studied. With albumin, thiols are oxidised preferentially forming unstable sulphenyl thiocyanate derivatives, as evidenced by the reversible incorporation of 14C from HOS14CN. On consumption of the HSA free thiol group, the formation of stable 14C-containing products and oxidation of Trp residues are observed. Oxidation of Trp residues is observed on reaction of HOSCN with other proteins (including myoglobin, lysozyme and trypsin inhibitor), but not free Trp, or Trp-containing peptides. Peptide mass mapping studies with HOSCN-treated myoglobin, showed the addition of 2 oxygen atoms on either Trp7 or Trp14 with equimolar or less oxidant, and the addition of a further 2 oxygen atoms to the other Trp with higher oxida... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1650272 Thu, 24 Jul 2008 04:00:00 +0100 1650272 http://www.medworm.com/index.php?rid=1623333&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080765 This study investigated transcriptional regulation of T-cad in endothelial cells (EC). Conditions of oxidative stress (serum-deprivation or presence of H2O2) elevate T-cad mRNA and protein levels in EC. Reporter gene analysis using serially deleted T-cad promoter stretches ranging from -99bps to -2304bps located the minimal promoter region of T-cad within -285bps from the translation start site. Reporter activity in EC transfected with -285bps construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. Electrophoretic mobility shift assay revealed specific nucleoprotein complex particular to -156 to -203bps which increased using nuclear extracts from oxidatively-stressed EC, suggesting the presence of redox-sensitive binding element(s)... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1623333 Tue, 15 Jul 2008 04:00:00 +0100 1623333 http://www.medworm.com/index.php?rid=1599692&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080369 Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) require a post-translational phosphopantetheinylation to become active. This reaction is catalysed by the 4'-phosphopantetheinyl transferase (PPTase). The ppt gene of Penicillium chrysogenum, encoding a protein that shares 50% homology with the stand-alone large PPTases, has been cloned. This gene is present as a single copy in the genome of the wild type and high penicillin producing strains (containing multiple copies of the penicillin gene cluster). Amplification of the ppt gene produced increases in isopenicillin N and benzylpenicillin biosynthesis. A PPTase defective mutant (Wis54-PPT—) was obtained. It required lysine and lacked pigment and penicillin production, but it still synthesized normal levels of ... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599692 Wed, 18 Jun 2008 04:00:00 +0100 1599692 http://www.medworm.com/index.php?rid=1599691&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080618 Non-enzymic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In this work, we analyze the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the plasma membrane calcium pump (PMCA) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remain unmodified after glycation of the protein. Further, phosphatidyleth... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599691 Wed, 18 Jun 2008 04:00:00 +0100 1599691 http://www.medworm.com/index.php?rid=1599693&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080300 In the plasma membrane fraction from Caco2, it was shown that active Nox1 endogenously co-localized with its regulatory components p22phox, NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI or NADP+. The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with that (15.1 pmol/mg) in the membranes from unstimulated Caco2, but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis exhibited approximately 36 pmol FAD and 43 pmol heme per mg membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/sec/mol heme. When the constitutively active fo... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599693 Mon, 02 Jun 2008 04:00:00 +0100 1599693 http://www.medworm.com/index.php?rid=1599695&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080280 We used 31P MR spectroscopy measurements of energetic intermediates (ATP, Pi, PCr) in combination with the analytical tools of metabolic control analysis to study in vivo energy metabolism in contracting skeletal muscle of anesthetized rats over a broad range of workload. According to our recent Modular Control Analysis (MoCA) used to describe regulatory mechanisms in beating heart, we defined the energetic system of muscle contraction as two modules (PCr-Producer and PCr-Consumer) connected by the energetic intermediates. Hypoxia and electrical stimulation were used in this in vivo study as reasonably selective modulations of Producer and Consumer respectively. As quantified by elasticity coefficients, the sensitivities of each module to PCr determine the control of steady state contract... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599695 Thu, 22 May 2008 04:00:00 +0100 1599695 http://www.medworm.com/index.php?rid=1599694&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080722 This study identifies the TAL deficiency as a modulator of mitochondrial homeostasis, Ca2+ fluxing and apoptosis. (Source: BJ Energy) BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599694 Thu, 22 May 2008 04:00:00 +0100 1599694 http://www.medworm.com/index.php?rid=1599697&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071701 To identify the functional unit of Ca2+-ATPase in the sarcoplasmic reticulum, we assessed Ca2+ transport activities occurring on sarcoplasmic reticulum membranes with different combinations of active and inactive Ca2+-ATPase molecules. We prepared heterodimers, comprising a native Ca2+-ATPase molecule and a Ca2+-ATPase molecule inactivated by fluorescein-5’-isothiocyanate labeling, by fusing vesicles loaded with each type of Ca2+-ATPase. The heterodimers exhibited neither Ca2+ transport nor ATP hydrolysis, suggesting that Ca2+ transport by the Ca2+-ATPase requires an interaction between functional Ca2+-ATPase monomers. This finding implies that the functional unit of the Ca2+-ATPase is a dimer. (Source: BJ Energy) BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599697 Mon, 12 May 2008 04:00:00 +0100 1599697 http://www.medworm.com/index.php?rid=1599696&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080344 To investigate cation adaptation and homeostasis in Aspergillus nidulans, two transcription factor-encoding genes have been characterised. The A. nidulans orthologue crzA of the Saccharomyces cerevisiae CRZ1 gene, encoding a transcription factor mediating gene regulation by Ca2+, has been identified and deleted. The crzA deletion phenotype includes extreme sensitivity to alkaline pH, Ca2+ toxicity and aberrant morphology connected with alterations of cell wall-related phenotypes such as reduced expression of a chitin synthase gene, chsB. A fully functional C-terminally GFP-tagged form of the CrzA protein is apparently excluded from nuclei in the absence of added Ca2+ but rapidly accumulates in nuclei upon exposure to Ca2+. In addition, the previously identified sltA gene, having no identif... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599696 Mon, 12 May 2008 04:00:00 +0100 1599696 http://www.medworm.com/index.php?rid=1599698&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071637 In this study, we have investigated the function of SHP as a putative repressor of HNF-6. Using transient transfection assays, we have shown that SHP represses the transcriptional activity of HNF-6. Confocal microscopy revealed that both SHP and HNF-6 colocalize in the nuclei of cells. SHP physically interacted with HNF-6 in protein-protein association assays in vitro. Electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays demonstrated that SHP inhibits the DNA binding activity of HNF-6 to an HNF-6-response element consensus sequence, and the HNF-6 target region of the endogenous glucose 6-phosphatase (G6Pase) promoter, respectively. Northern blot analysis of HNF-6 target genes in cells infected with adenoviral vectors for SHP and SHP small inhibitory... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599698 Wed, 07 May 2008 04:00:00 +0100 1599698 http://www.medworm.com/index.php?rid=1599699&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20080475 In this study, several Wolinella succinogenes mutants that produce variants of NrfH, the membrane anchor of the cytochrome c nitrite reductase complex, were constructed and characterized in order to improve the understanding of the putative menaquinol binding site, the maturation and function of the four covalently bound haem c groups and the importance of the N-terminal transmembrane segment. Based on amino acid sequence alignments, a homology model for W. succinogenes NrfH was constructed that underlines the overall conservation of tertiary structure in spite of a low sequence homology. The results support the proposed architecture of the menaquinol binding site in D. vulgaris NrfH, demonstrate that each histidine residue arranged in one of the four CX2CH haem c binding motifs is essenti... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599699 Fri, 25 Apr 2008 04:00:00 +0100 1599699 http://www.medworm.com/index.php?rid=1599700&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071615 Cisplatin accumulates in mitochondria being this organelle a major target for this drug in cancer cells. Thus, alterations in mitochondrial function have been implicated in cancer cell resistance to chemotherapeutic agents. Moreover, cisplatin toxic side effects seem to be associated with mitochondrial injury in vivo and in vitro. In order to clarify the potential effect of cisplatin in mitochondrial DNA maintenance and expression, we have analyzed rat liver mitochondrial DNA and RNA synthesis as well as their stability under the influence of in vivo treatment or in vitro exposure to cisplatin. We show that cisplatin causes a direct and significant impairment of mtDNA and mtRNA synthesis and decreased steady state levels of mtRNAs in isolated mitochondria. Furthermore, in vivo treatment of... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599700 Tue, 22 Apr 2008 04:00:00 +0100 1599700 http://www.medworm.com/index.php?rid=1599701&cid=s_37617_60_f&fid=37617&url=http%3A%2F%2Fwww.biochemj.org%2Fbj%2Fimps%2Frefer.htm%3FMSID%3DBJ20071298 Mycobacterium tuberculosis (Mtb) flavoprotein reductase A (FprA) is a NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports catalytic function of Mtb cytochrome P450s. Tryptophan 359, proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp359 is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define Trp359's role. W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone... BJ Energy journals http://www.medworm.com/rss/comments.php?id=1599701 Thu, 20 Mar 2008 04:00:00 +0100 1599701