BBA - General Subjects (v.1840, #11)

Electron flow into cytochrome c coupled with reactive oxygen species from the electron transport chain converts cytochrome c to a cardiolipin peroxidase: role during ischemia–reperfusion by Hema S. Aluri; David C. Simpson; Jeremy C. Allegood; Ying Hu; Karol Szczepanek; Scott Gronert; Qun Chen; Edward J. Lesnefsky (3199-3207).
Cytochrome c (Cyt c) is a mobile component of the electron transport chain (ETC.) which contains a tightly coordinated heme iron. In pathologic settings, a key ligand of the cyt c's heme iron, methionine (Met80), is oxidized allowing cyt c to participate in reactions as a peroxidase with cardiolipin as a target. Myocardial ischemia (ISC) results in ETC. blockade and increased production of reactive oxygen species (ROS). We hypothesized that during ischemia–reperfusion (ISC-REP); ROS generation coupled with electron flow into cyt c would oxidize Met80 and contribute to mitochondrial-mediated ETC. damage.Mitochondria were incubated with specific substrates and inhibitors to test the contributions of ROS and electron flow into cyt c. Subsequently, cyt c and cardiolipin were analyzed. To test the pathophysiologic relevance, mouse hearts that underwent ISC-REP were tested for methionine oxidation in cyt c.The combination of substrate/inhibitor showed that ROS production and electron flux through cyt c are essential for the oxidation of methionine residues that lead to cardiolipin depletion. The content of cyt c methionine oxidation increases following ISC-REP in the intact heart.Increase in intra-mitochondrial ROS coupled with electron flow into cyt c, oxidizes cyt c followed by depletion of cardiolipin. ISC-REP increases methionine oxidation, supporting that cyt c peroxidase activity can form in the intact heart.This study identifies a new site in the ETC. that is damaged during cardiac ISC-REP. Generation of a neoperoxidase activity of cyt c favors the formation of a defective ETC. that activates signaling for cell death.
Keywords: Cytochrome c peroxidase; Methionine sulfoxide; Cardiolipin; Mitochondria; ROS; Ischemia–reperfusion;

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP+ reductase by Anabel Soldano; Huili Yao; Mario Rivera; Eduardo A. Ceccarelli; Daniela L. Catalano-Dupuy (3208-3217).
Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans.A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and 1H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners.A plastidic type, high efficiently ferredoxin-NADP+ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis.Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP+ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans.Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.
Keywords: Leptospira interrogans; Heme oxygenase; Ferredoxin-NADP+ reductase; Iron; Electron transfer;

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin by Horia Nicolae Roman; Nedjma B. Zitouni; Linda Kachmar; Andrea Benedetti; Apolinary Sobieszek; Anne-Marie Lauzon (3218-3225).
Studies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin.To measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (Funb ) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin. Funb from unregulated actin (0.10 ± 0.01 pN) was significantly increased in the presence of CaD (0.17 ± 0.02 pN), tropomyosin (0.17 ± 0.02 pN) or both regulatory proteins (0.18 ± 0.02 pN). ERK phosphorylation of CaD significantly reduced the Funb (0.06 ± 0.01 pN). Inspection of the traces of the Funb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment.CaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels.This study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.
Keywords: Caldesmon; Myosin; Latch-state; Phosphorylation; In vitro motility assay laser trap; Tropomyosin;

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2μ2 by SeongJae Jang; Katsuki Ohtani; Atsushi Fukuoh; Kenichiro Mori; Takayuki Yoshizaki; Noritoshi Kitamoto; YounUck Kim; Yasuhiko Suzuki; Nobutaka Wakamiya (3226-3237).
Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated.We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in μ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out.We identified μ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2μ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules.Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2μ2.This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.
Keywords: Scavenger Receptors; Endocytosis; Adaptor complex; Collectin; Tyrosine motif;

Participation of thioredoxin in the V(V)-reduction reaction by Vanabin2 by Tatsuya Ueki; Masayuki Uwagaki; Sohei Yamamoto; Hitoshi Michibata (3238-3245).
It is well-understood that ascidians accumulate high levels of vanadium, a reduced form of V(III), in an extremely acidic vacuole in their blood cells. Vanabins are small cysteine-rich proteins that have been identified only from vanadium-rich ascidians. A previous study revealed that Vanabin2 can act as a V(V)-reductase in the glutathione cascade. AsTrx1, a thioredoxin gene, was cloned from the vanadium-rich ascidian, Ascidia sydneiensis samea, by PCR. AsTrx1 and Vanabin2 were prepared as recombinant proteins, and V(V)-reduction by Vanabin2 was assessed by ESR and ion-exchange column chromatography. Site-directed mutagenesis was performed to examine the direct involvement of cysteine residues. Tissue expression of AsTrx1 was also examined by RT-PCR.When reduced AsTrx1 and Vanabin2 were combined, Vanabin2 adopted an SS/SH intermediate structure while V(V) was reduced to V(IV). The loss of cysteine residues in either Vanabin2 or AsTrx1 caused a significant loss of reductase activity. V app and K app values for Vanabin2-catalyzed V(V)-reduction in the thioredoxin cascade were 0.066 mol-V(IV)/min/mol-Vanabin2 and 0.19 mM, respectively. The K app value was 2.7-fold lower than that observed in the glutathione cascade. The AsTrx1 gene was expressed at a very high level in blood cells, in which Vanabins 1–4 were co-expressed. AsTrx1 may contribute to a significant part of the redox cascade for V(V)-reduction by Vanabin2 in the cytoplasm of vanadocytes, but prevails only at low V(V) concentrations.This study is the first to report the reduction of V(V) in the thioredoxin cascade.Display Omitted
Keywords: Ascidian; Vanadium; Reductase; Metal; Redox;

Selenoproteins and selenium status in bone physiology and pathology by Zhichao Zhang; Jinsong Zhang; Jianru Xiao (3246-3256).
Emerging evidence supports the view that selenoproteins are essential for maintaining bone health.The current state of knowledge concerning selenoproteins and Se status in bone physiology and pathology is summarized.Antioxidant selenoproteins including glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), as a whole, play a pivotal role in maintaining bone homeostasis and protecting against bone loss. GPx1, a major antioxidant enzyme in osteoclasts, is up-regulated by estrogen, an endogenous inhibitor of osteoclastogenesis. TrxR1 is an immediate early gene in response to 1α,25-dihydroxyvitamin D3, an osteoblastic differentiation agent. The combination of 1α,25-dihydroxyvitamin D3 and Se generates a synergistic elevation of TrxR activity in Se-deficient osteoblasts. Of particular concern, pleiotropic TrxR1 is implicated in promoting NFκB activation. Coincidentally, TrxR inhibitors such as curcumin and gold compounds exhibit potent osteoclastogenesis inhibitory activity. Studies in patients with the mutations of selenocysteine insertion sequence-binding protein 2, a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins have clearly established a causal link of selenoproteins in bone development. Se transport to bone relies on selenoprotein P. Plasma selenoprotein P concentrations have been found to be positively correlated with bone mineral density in elderly women.A full understanding of the role and function of selenoproteins and Se status on bone physiology and pathology may lead to effectively prevent against or modify bone diseases by using Se.
Keywords: Selenium; Bone; Selenoprotein; Osteoblast; Osteoclast;

NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer by S. Mahindhoratep; H. Ait Bouda; Nelly El Shafey; D. Scherman; A. Kichler; Ch. Pichon; P. Midoux; N. Mignet; M.F. Bureau (3257-3263).
When activated, NF-κB can promote the nuclear import and transcription of DNA possessing NF-κB consensus sequences. Here, we investigated whether NF-κB is involved in the plasmid electrotransfer process.Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-κB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBα were studied as a marker of NF-κB activation.Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-κB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IκBα was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IκBα and thus of free NF-κB in the absence of any stimulation.pDNA electrotransfer can increase transgene expression independently of NF-κB.The insertion of NF-κB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-κB in muscle might be sufficient to enhance the activity of pDNA bearing NF-κB consensus sequences.
Keywords: NF-κB; IκBα; Electroporation; Muscle; Transgene expression; Transcription;

Microbial resistance to antibiotics has triggered the development of nanoscale materials as an alternative strategy. To stabilize these particles an inert support is needed.Porous nanomullite developed by sol–gel route is loaded with copper and silver nanoparticle by simple adsorption method. These nanocomposites are characterized using XRD, FTIR, TEM, SEM, EDAX and UV–visible spectrophotometer. Antibacterial activity of these nanocomposites against Gram positive and Gram negative bacteria are performed by bactericidal kinetics, flow cytometry and MTT assay. The underlying mechanisms behind the antimicrobial property and cell death are also investigated by EPR spectroscopy, intracellular ROS measurement and β-galactosidase assay. The cytocompatibility of the nanocomposites is investigated by cell viability (MTT), proliferation (Alamar blue) and wound healing assay of mammalian fibroblast cell line.Nanocomposites show a fairly uniform distribution of metal nanoparticle within mullite matrix. They show excellent antibacterial activity. Metal ions/nanoparticle is found to be released from the materials (CM and SM). Treated cells manifested high intracellular oxidative stress and β-galactosidase activity in the growth medium. The effect of nanocomposites on mammalian cell line depends on exposure time and concentration. The scratch assay shows normal cell migration with respect to control.The fabricated nanoparticles possess diverse antimicrobial mechanism and exhibit good cytocompatibility along with wound healing characteristics in mouse fibroblast cell line (L929).The newly synthesized materials are promising candidates for the development of antimicrobial ceramic coatings for biomedical devices and therapeutic applications.
Keywords: Mullite; Metal nanoparticle; Antibacterial activity; Cyto-compatibility;

Assembly of phagocyte NADPH oxidase: A concerted binding process? by Gilda Karimi; Chantal Houée Levin; Marie Claire Dagher; Laura Baciou; Tania Bizouarn (3277-3283).
The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b 558 and four cytosolic proteins (p67phox, p47phox, p40phox and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase.A wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47phox–p67phox complex.The data presented here are consistent with the absence of a catalytic role of the p47phox–p67phox interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b 558 allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b 558. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core.The activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.
Keywords: NADPH oxidase (Nox); Protein translocation; Cell free system; Arachidonic acid activation; Neutrophil;