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Cardiac ischemia and reperfusion (IR) injury induces excessive emission of deleterious reactive O 2 and N 2 species (ROS/RNS), including the non-radical oxidant peroxynitrite (ONOO−) that can cause mitochondria dysfunction and cell death. In this study, we explored whether IR injury in isolated hearts induces tyrosine nitration of adenine nucleotide translocase (ANT) and alters its interaction with the voltage-dependent anion channel 1 (VDAC1). We found that IR injury induced tyrosine nitration of ANT and that exposure of isolated cardiac mitochondria to ONOO− induced ANT tyrosine, Y81, nitration. The exposure of isolated cardiac mitochondria to ONOO− also led ANT to form high molecular weight proteins and dissociation of ANT from VDAC1. We found that IR injury in isolated hearts, hypoxic injury in H9c2 cells, and ONOO− treatment of H9c2 cells and isolated mitochondria, each decreased mitochondrial bound-hexokinase II (HK II), which suggests that ONOO− caused HK II to dissociate from mitochondria. Moreover, we found that mitochondria exposed to ONOO− induced VDAC1 oligomerization which may decrease its binding with HK II. We have reported that ONOO− produced during cardiac IR injury induced tyrosine nitration of VDAC1, which resulted in conformational changes of the protein and increased channel conductance associated with compromised cardiac function on reperfusion. Thus, our results imply that ONOO− produced during IR injury and hypoxic stress impeded HK II association with VDAC1. ONOO− exposure nitrated mitochondrial proteins and also led to cytochrome c (cyt c) release from mitochondria. In addition, in isolated mitochondria exposed to ONOO− or obtained after IR, there was significant compromise in mitochondrial respiration and delayed repolarization of membrane potential during oxidative (ADP) phosphorylation. Taken together, ONOO− produced during cardiac IR injury can nitrate tyrosine residues of two key mitochondrial membrane proteins involved in bioenergetics and energy transfer to contribute to mitochondrial and cellular dysfunction. • Peroxynitrite (ONOO−) induces tyrosine nitration of ANT and VDAC1. • ONOO− cause dissociation of ANT from VDAC1, and HK II from VDAC1 • Protein dissociation enhances cytochrome c release from mitochondria. • ONOO− impairs mitochondrial bioenergetics, and compromises cardiac function after IR. [ABSTRACT FROM AUTHOR]

We provide evidence for location and function of a small conductance, Ca 2+ -activated K + (SK Ca ) channel isoform 3 (SK3) in mitochondria (m) of guinea pig, rat and human ventricular myocytes. SK Ca agonists protected isolated hearts and mitochondria against ischemia/reperfusion (IR) injury; SK Ca antagonists worsened IR injury. Intravenous infusion of a SK Ca channel agonist/antagonist, respectively, in intact rats was effective in reducing/enhancing regional infarct size induced by coronary artery occlusion. Localization of SK3 in mitochondria was evidenced by Western blot of inner mitochondrial membrane, immunocytochemical staining of cardiomyocytes, and immunogold labeling of isolated mitochondria. We identified a SK3 splice variant in guinea pig (SK3.1, aka SK3a) and human ventricular cells (SK3.2) by amplifying mRNA, and show mitochondrial expression in mouse atrial tumor cells (HL-1) by transfection with full length and truncated SK3.1 protein. We found that the N-terminus is not required for mitochondrial trafficking but the C-terminus beyond the Ca 2+ calmodulin binding domain is required for Ca 2+ sensing to induce mK + influx and/or promote mitochondrial localization. In isolated guinea pig mitochondria and in SK3 overexpressed HL-1 cells, mK + influx was driven by adding CaCl 2 . Moreover, there was a greater fall in membrane potential (ΔΨ m ), and enhanced cell death with simulated cell injury after silencing SK3.1 with siRNA. Although SK Ca channel opening protects the heart and mitochondria against IR injury, the mechanism for favorable bioenergetics effects resulting from SK Ca channel opening remains unclear. SK Ca channels could play an essential role in restraining cardiac mitochondria from inducing oxidative stress-induced injury resulting from mCa 2+ overload. [ABSTRACT FROM AUTHOR]

Polybrominated diphenyl ethers (PBDEs), as typical persistent organic pollutants (POPs) have been world widely detected in environmental media and biological tissues. Compared with the extensive study on direct photolysis of PBDEs in the ultraviolet region (UV), indirect photodegradation of PBDEs under visible light triggered by the co-existing substances in the environment has been largely ignored. Herein, the indirect photolysis and debromination of organic brominated pollutants decabromodiphenyl ether (BDE209) induced by humic acid (HA) have achieved both in homogeneous and heterogeneous system under visible light irradiation. It shows a new transformation pathway of PBDEs in environment that plays an important role on their ultimate environmental fate. Different from direct photolysis of PBDEs in UV light, the indirect photolysis of PBDEs by HA under visible light irradiation experiences the electrons transfer from HA to PBDEs induced by a halogen-bond between HA and PBDEs. HA contributes to the activation of C-Br bond via formation of halogen bond, and promotes electron transfer under visible light irradiation, leading to meta- C-Br cleavage preferentially. To evaluate the effects of various functional groups in HA, eleven low-molecular-weight organic compounds (LMWOs) containing reactive oxygen terminal are selected to study the indirect photolysis of BDE209. Combined with the density functional theory (DFT) calculation results, it is shown that carboxyl group is proposed to be the key functional group capable of providing oxygen terminals to form halogen bonds. This work provides novel and valuable insights into the photolysis and debromination of PBDEs with co-existing HA in the environment under sunlight. [Display omitted] • The indirect visible light photolysis of BDE209 by HA is first realized. • The activation of C-Br bond is due to the halogen-oxygen bond between BDE209 and HA. • Carboxyl group in HA is proposed to be the key form halogen bonds with BDE209. [ABSTRACT FROM AUTHOR]

[Display omitted] • C-Br bond is activated on oxygen vacancies enriched Bi 2 WO 6. • Effective debromination is achieved on Bi 2 WO 6 under visible light irradiation. • Debromination on Bi 2 WO 6 follows a position-selective multi-electrons reduction mechanism. C Br bond activation is a critical step in the degradation of organic halogenated pollutants. Herein, by introducing oxygen vacancies with different concentration gradients into Bi 2 WO 6 , the CBr bond in decabromodiphenyl ether (BDE209) is activated, so that BDE209 is successfully dehalogenated under visible light. The study shows that the degradation rate of BDE209 on Bi 2 WO 6 is determined by both interface defects and crystallinity. Good crystallinity is conducive to the generation and separation of photogenerated electrons and holes, and an appropriate defect concentration can promote the C-Br activation of BDE209. In addition, through the tracking analysis of the degradation products of BDE209, the content of the meta -debromination products of BDE209 on Bi 2 WO 6 is significantly higher than that of the ortho- and para- debromination products, indicating that the reduction reaction is a position selective multi-electron mechanism. The study shows that oxygen vacancy assisted debromination is an excellent strategy for photocatalytic treatment of halogenated flame retardant pollutants. [ABSTRACT FROM AUTHOR]

Abstract: Hepatitis C virus (HCV) has a high rate of replication and lacks RNA-proofreading capabilities, thereby leading to variant or mutant viruses circulating within the host as a quasispecies. Previous work in our laboratory identified viral variants that emerged in a class-II immunodominant epitope NS3358-375 of the non-structural-3 (NS3) protein region of HCV, the sequence of which is based on genotype 1A, the most prevalent genotype in the United States. Further work suggested that positive immune selection pressure was driving viral variation. Paradoxically, viral variants account for only a small percentage of the circulating virus in human beings and in chimpanzees, suggesting that passive evasion is not the only means of escape by HCV. This observation suggests a unique pathogenesis for HCV as it persists in the host. In the current study, we hypothesize that viral variants are acting as altered peptide ligands (APLs). To test this hypothesis, we used cloned T cells specific for NS3358-375 peptide, which demonstrated attenuated T-cell and interferon-γ (IFN-γ) responses to individual variant peptides, when compared with the NS3358-375 stimulated T-cell clones. Furthermore, such variants could act as APLs, based on their ability to antagonize the IFN-γ proliferative responses of clones specific for NS3358-375. In addition, major histocompatibility complex (MHC) class II tetramer staining demonstrated that variant peptide–MHC complexes were able to specifically bind to NS3358-375 T-cell clones and that both the variant and NS3358-375 tetramers were able to bind to the same CD4+ T cells. Taken together, the results suggest that viral variants may act as APL to effectively blunt the T-cell response to an important HCV epitope. [Copyright &y& Elsevier]

Abstract: The mechanisms of action of gallium nitrate, an antineoplastic drug, are only partly understood. Using a DNA microarray to examine genes induced by gallium nitrate in CCRF-CEM cells, we found that gallium increased metallothionein-2A (MT2A) and heme oxygenase-1 (HO-1) gene expression and altered the levels of other stress-related genes. MT2A and HO-1 were increased after 6 and 16 h of incubation with gallium nitrate. An increase in oxidative stress, evidenced by a decrease in cellular GSH and GSH/GSSG ratio, and an increase in dichlorodihydrofluorescein (DCF) fluorescence, was seen after 1–4 h of incubation of cells with gallium nitrate. DCF fluorescence was blocked by the mitochondria-targeted antioxidant mitoquinone. N-Acetyl-L-cysteine blocked gallium-induced MT2A and HO-1 expression and increased gallium''s cytotoxicity. Studies with a zinc-specific fluoroprobe suggested that gallium produced an expansion of an intracellular labile zinc pool, suggesting an action of gallium on zinc homeostasis. Gallium nitrate increased the phosphorylation of p38 mitogen-activated protein kinase and activated Nrf-2, a regulator of HO-1 gene transcription. Gallium-induced Nrf-2 activation and HO-1 expression were diminished by a p38 MAP kinase inhibitor. We conclude that gallium nitrate induces cellular oxidative stress as an early event which then triggers the expression of HO-1 and MT2A through different pathways. [Copyright &y& Elsevier]

Abstract: The premise of this work is that within a given hepatitis C virus (HCV) protein there exists an array of Th1 and Th2 epitopes, each of which can provide synergistic (positive or negative) effects upon other epitopes by intramolecular, cytokine-mediated immunoregulation of helper T-cell responses. To address this question, we constructed minigene plasmids pHCVTh1, pHCVTh1X3 and pHCVThR, and HCV NS3 full-length plasmid pHCVNS3. 293T cells were transfected with these plasmids and cell lysates from the transfected cells were used to stimulate PBMC from a patient with chronic HCV infection. IL-2 and IFN-γ in the supernatant of the cultured PBMC were tested and proliferation of the PBMC was measured. The results demonstrate that interactions exist among helper T-cell epitopes; the synergistic effects of suppressive Th2 epitopes upon Th1 epitopes will inhibit the responses induced by Th1 epitopes, which may contribute to chronic infection by HCV; synergistic effects among Th1 epitopes induce higher levels of IFN-γ, which may suggest a new strategy for HCV vaccine development. Further, stimulation of an HCV NS3 specific clone with cell lysates from 293T cells transfected with different constructs shows that the HCV NS3 clone could respond to all suggesting that the epitope-specific suppression may be due to an imbalance of Type 1 and Type 2 cytokines or regulatory T-cells. [Copyright &y& Elsevier]

Abstract: Hepatitis C virus (HCV) causes non-A, non-B hepatitis and infects an estimated 170 million people worldwide. The treatment for HCV infection is often unsuccessful with high costs and many side-effects. There is a great need for alternative therapies including preventive and therapeutic vaccination for HCV infection. The experiments in this study were carried out to elucidate whether endogenously expressed antigen can be presented to helper T-cells restricted by class II molecules and to determine whether responses to plasmid-derived antigen resemble those that we have reported for recombinant antigens or synthetic peptides. To address these issues, a multi-epitope minigene was expressed in 293T-cells and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells (BLCL). The transfected BLCLs were employed as APCs to stimulate epitope-specific T-cell hybridomas (THC). The results demonstrated that the endogenously expressed minigene antigens could be processed and presented to T-cell hybridomas by HLA matched BLCL. Five out of seven incorporated epitopes were recognized. Blockade of HLA DR could abolish the release of IL-2, which demonstrated that the endogenously expressed minigene antigens were presented by MHC class II molecules. The presentation of endogenously expressed antigens was much more efficient than that of exogenous antigens, at least in the present study. The findings obtained here have important significance for the development of an HCV DNA vaccine. [Copyright &y& Elsevier]

Investigates the genes related to the transformation of immortalized human fetal fibroblast cell by means of differential display messenger RNA (mRNA). Formation of clone on agar after irradiation; Number of DNA fragments; Analysis of mRNA expression.

We hypothesized that NO• is generated in isolated cardiac mitochondria as the source for ONOO− production during oxidative stress. We monitored generation of ONOO− from guinea pig isolated cardiac mitochondria subjected to excess Ca2+ uptake before adding succinate and determined if ONOO− production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl 2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O 2 •−. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO− to form dityrosine (diTyr) in proportion to the ONOO− present. We found that exposing mitochondria to excess CaCl 2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H 2 O 2) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa2+ and succinate, increased mitochondrial ONOO− production via NO• and O 2 •−. Changes in mitochondrial ONOO− production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO− production, PTIO decreased production of ONOO−, and TEMPOL decreased ONOO− levels by converting more O 2 •− to H 2 O 2. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO− into the buffer is dependent both on O 2 •− released from mitochondria and NO• derived from a mtCa2+-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible. Unlabelled Image • Cardiac isolated mitochondria produce and release ONOO−, a product of O 2 •− and NO•. • O 2 •− is generated by complex I during oxidative stress induced by excess CaCl 2 and succinate. • NO• is generated within or near isolated mitochondria. • During oxidative stress mitochondrial ONOO− levels are modulated by O 2 •− and NO•. • Mitochondrial NOS is likely a variant of a non-inducible iNOS, but a nNOS may also contribute. [ABSTRACT FROM AUTHOR]

• Amylase activity were lower after drought treatment than during flood cultivation. • The expression levels of related amylase genes in dry cultivation were different during grain filling. • Key period for quality formation of dry cultivation was from 8DAF to 15DAF. • At 8–15DAF, accumulation of metabolites in dry cultivation rice was higher than controls. • At 40DAF, metabolite consumption of dry cultivation rice was faster than control. The differences between dry- and flood-cultivated rice and the reason behind low-quality dry-cultivated rice were clarified. The physiological traits, starch synthase activity, and grain metabolomics of 'Longdao 18′ were measured and analyzed at four growth stages. The brown, milled, and whole-milled rice rates and AGPase , SSS , and SBE activity were lower after drought treatment than during flood cultivation, while the chalkiness, chalky grain rate, amylose (16.57–20.999%), protein (7.99–12.09%), and GBSS activity were higher. Related enzymatic gene expression showed significant differences. Metabolic results showed pyruvate, glycine, and methionine upregulation at 8DAF and higher citric, pyruvic, and α-ketoglutaric acid content at 15DAF. Therefore, 8DAF-15DAF represented the crucial quality formation period for dry-cultivated rice. At 8DAF, the respiratory pathways used amino acids as signaling molecules and alternative substrates to adapt to energy shortages, arid environments and rapid protein accumulation and synthesis. Excessive amylose synthesis at 15DAF accelerated reproductive growth, promoting rapid premature aging. [ABSTRACT FROM AUTHOR]

Abstract: Ranolazine, an anti-anginal drug, is a late Na+ channel current blocker that is also believed to attenuate fatty acid oxidation and mitochondrial respiratory complex I activity, especially during ischemia. In this study, we investigated if ranolazine''s protective effect against cardiac ischemia/reperfusion (IR) injury is mediated at the mitochondrial level and specifically if respiratory complex I (NADH Ubiquinone oxidoreductase) function is protected. We treated isolated and perfused guinea pig hearts with ranolazine just before 30min ischemia and then isolated cardiac mitochondria at the end of 30min ischemia and/or 30min ischemia followed by 10min reperfusion. We utilized spectrophotometric and histochemical techniques to assay complex I activity, Western blot analysis for complex I subunit NDUFA9, electron paramagnetic resonance for activity of complex I Fe–S clusters, enzyme linked immuno sorbent assay (ELISA) for determination of protein acetylation, native gel histochemical staining for respiratory supercomplex assemblies, and high pressure liquid chromatography for cardiolipin integrity; cardiac function was measured during IR. Ranolazine treated hearts showed higher complex I activity and greater detectable complex I protein levels compared to untreated IR hearts. Ranolazine treatment also led to more normalized electron transfer via Fe–S centers, supercomplex assembly and cardiolipin integrity. These improvements in complex I structure and function with ranolazine were associated with improved cardiac function after IR. However, these protective effects of ranolazine are not mediated by a direct action on mitochondria, but rather indirectly via cytosolic mechanisms that lead to less oxidation and better structural integrity of complex I. [Copyright &y& Elsevier]