The differential NF-kB modulation by S-adenosyl-L-methionine,N-acetylcysteine and quercetin on the promotion stage of chemical hepatocarcinogenesis

S-adenosylmethionine (SAM), N-acetylcysteine (NAC) and quercetin exhibit a chemoprotective effect. Likely this effect is mediated by counteracting, oxidative stress and NF-kB activation. To test this hypothesis F344 rats were subjected to hepatocarcinogenesis with or without antioxidants. NAC decreased foci in number and area, SAM and quercetin decreased area. Lipid-peroxidation was decreased by antioxidants, but only SAM increased glutathione. SAM, in its regulation from IKK downwards, abolished the NF-kB activation. NAC decreased IKK and IkB-a phosphorylation, and Rel-A/p65 and NF-kB binding, though the last two were affected with less intensity compared to the NF-kB inhibitor. Quercetin decreased Rel-A/p65, without modifying upstream signalling. Although all antioxidants inhibited oxidative stress as shown by reduction of lipid peroxidation, not all exerted the same effect on NF-kB signalling pathway and only SAM increased GSH. The mechanisms exerted by SAM in the reduction of foci makes this compound a potential liver cancer therapeutic agent.     

ESR spin trapping studies of free radicals generated from nitrofuran derivative analogues of nifurtimox by electrochemical and Trypanosoma cruzi reduction

Electron spin resonance (ESR) spectra of radicals obtained from two analogues of the antiprotozoal drug nifurtimox by electrolytic and Trypanosoma cruzi reduction were analyzed. The electrochemistry of these compounds was studied using cyclic voltammetry. STO 3-21G ab initio and INDO molecular orbital calculations were performed to obtain the optimized geometries and spin distribution, respectively. The antioxidant effect of glutathione on the nitroheterocycle radical was evaluated. DMPO spin trapping was used to investigate the possible formation of free radicals in the trypanosome microsomal system. Nitro1 and Nitro2 nitrofuran analogues showed better antiparasitic activity than nifurtimox. Nitro2 produced oxygen redox cycling in T. cruzi epimastigotes. The ESR signal intensities were consistent with the trapping of either the hydroxyl radical or the Nitro2 analogue radicals. These results are in agreement with the biological observation that Nitro2 showed anti-Chagas activity by an oxidative stress mechanism.     

The Redox State of Glutathione,Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

Control of Redox Balance by the Stringent Response Regulatory Protein Promotes Antioxidant Defenses of Salmonella

We report herein a critical role for the stringent response regulatory DnaK suppressor protein (DksA) in the coordination of antioxidant defenses. DksA helps fine-tune the expression of glutathione biosynthetic genes and discrete steps in the pentose phosphate pathway and tricarboxylic acid cycle that are associated with the generation of reducing power. Control of NAD(P)H/NAD(P)⁺ redox balance by DksA fuels downstream antioxidant enzymatic systems in nutritionally starving Salmonella. Conditional expression of the glucose-6-phosphate dehydrogenase-encoding gene zwf, shown here to be under DksA control, increases both the NADPH pool and antioxidant defenses of dksA mutant Salmonella. The DksA-mediated coordination of redox balance boosts the antioxidant defenses of stationary phase bacteria. Not only does DksA increase resistance of Salmonella against hydrogen peroxide (H₂O₂), but it also promotes fitness of this intracellular pathogen when exposed to oxyradicals produced by the NADPH phagocyte oxidase in an acute model of infection. Given the role of DksA in the adjustment of gene expression in most bacteria undergoing nutritional deprivation, our findings raise the possibility that the control of central metabolic pathways by this regulatory protein maintains redox homeostasis essential for antioxidant defenses in phylogenetically diverse bacterial species.     

Effect of the Redox State of the Red Blood Cell Components on the Inactivation of Glutathione Peroxidase by Divicine

The redox state of red blood cell components was found to have profound effects on the specific inactivation of erythrocyte glutathione (GSH) peroxidase by divicine, a hydroquinone imine molecule of fava beans likely to be responsible, through redox cycling, of the oxidative damage of red blood cells ultimately resulting in the hemolysis of favism. Oxidation of hemoglobin is a necessary step for the inactivation to take place, apparently as a H₂O₂-MetHb adduct. On the other hand, the presence of either reduced NADP or glutathione enhances the inactivating effect although NADPH inhibits the oxidation of hemoglobin, and this suggests a catalytic role for MetHb in the inactivation process.     

Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli

Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H₂O₂ stimulation.     

Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment

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乙肝病毒DNA疫苗的构建及其诱导小鼠的免疫应答

构建含adr亚型HBV表面抗原基因的核酸疫苗 ,考察人白细胞介素II基因及重组白细胞介素II的免疫佐剂作用。用含有人白细胞介素II基因的真核表达质粒及基因重组白细胞介素II蛋白作为佐剂 ,将编码乙型肝炎病毒表面抗原的重组真核表达质粒 pVAX/HBS免疫BALB/C小鼠 (试验组 ) ,同时设置注射质粒pVAX的阴性对照组 ,并分别于第 2 ,4周后加强免疫各 1次。试验组在第 4周时开始有HBsAb产生 ,阴性对照组未测到HbsAb ,试验组和对照组均未检测到HBsAg。乙肝病毒DNA疫苗能引起小鼠特异性体液免疫应答 ,白细胞介素II的真核表达质粒的佐剂作用不明显 ,基因重组白细胞介素II蛋白具有提高小鼠对乙肝病毒核酸疫苗免疫应答水平的佐剂活性。     

The Heme Oxygenase 1 Inducer (CoPP) Protects Human Cardiac Stem Cells against Apoptosis through Activation of the Extracellular Signal-regulated Kinase (ERK)/NRF2 Signaling Pathway and Cytokine Release

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H₂O₂, concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells'' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.     

The Role of Heme Binding by DNA-protective Protein from Starved Cells (Dps) in the Tolerance of Porphyromonas gingivalis to Heme Toxicity

The widely expressed DNA-protective protein from starved-cells (Dps) family proteins are considered major contributors to prokaryotic resistance to stress. We show here that Porphyromonas gingivalis Dps (PgDps), previously described as an iron-storage and DNA-binding protein, also mediates heme sequestration. We determined that heme binds strongly to PgDps with an apparent K_d of 3.7 × 10⁻⁸ m and is coordinated by a single surface-located cysteine at the fifth axial ligand position. Heme and iron sequestered in separate sites by PgDps provide protection of DNA from H₂O₂-mediated free radical damage and were found to be important for growth of P. gingivalis under excess heme as the only iron source. Conservation of the heme-coordinating cysteine among Dps isoforms from the Bacteroidales order suggests that this function may be a common feature within these anaerobic bacteria.     

Effects of the acid polysaccharide fraction isolated from a cultivated Cordyceps sinensis on macrophages in vitro

The acid polysaccharide fraction (APSF) extracted from the mycelia of cultivated Cordyceps sinensis is water-soluble polysaccharide. In this study we evaluated the modulating effects of APSF on murine macrophage cell line RAW264.7. Phagocytotic assay by neutral red and FITC-dextran internalization showed that APSF stimulated the phagocytosis of macrophages. The nitrite levels in the culture supernatant determined using Griess reagent revealed the elevation of NO production after treatment with APSF. RT-PCR and immunocytochemistry assay indicated that APSF promoted both the mRNA and protein expressions of inducible nitric oxide synthase (iNOS). Furthermore, Western blotting demonstrated that NF-κB levels in nucleuses increased after APSF treatment, suggesting that APSF probably stimulated macrophage activities by activating the IκB-NF-κB pathway.     

Structural Basis for the Recognition of Oxidized Phospholipids in Oxidized Low Density Lipoproteins by Class B Scavenger Receptors CD36 and SR-BI

Specific oxidized phospholipids (oxPC_CD36) accumulate in vivo at sites of oxidative stress and serve as high affinity ligands for scavenger receptors class B (CD36 and SR-BI). Recognition of oxPC_CD36 by scavenger receptors plays a role in several pathophysiological processes. The structural basis for the recognition of oxPC_CD36 by CD36 and SR-BI is poorly understood. A characteristic feature of oxPC_CD36 is an sn-2 acyl group that incorporates a terminal γ-hydroxy (or oxo)-α,β-unsaturated carbonyl. In the present study, a series of model oxidized phospholipids were designed, synthesized, and tested for their ability to serve as ligands for CD36 and SR-BI. We demonstrated that intact the sn-1 hydrophobic chain, the sn-3 hydrophilic phosphocholine or phosphatidic acid group, and the polar sn-2 tail are absolutely essential for high affinity binding. We further found that a terminal negatively charged carboxylate at the sn-2 position suffices to generate high binding affinity to class B scavenger receptors. In addition, factors such as polarity, rigidity, optimal chain length of sn-2, and sn-3 positions and negative charge at the sn-3 position of phospholipids further modulate the binding affinity. We conclude that all three positions of oxidized phospholipids are essential for the effective recognition by scavenger receptors class B. Furthermore, the structure of residues in these positions controls the affinity of the binding. The present studies suggest that, in addition to oxPC_CD36, other oxidized phospholipids observed in vivo may represent novel ligands for scavenger receptors class B.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-alpha gene expression by reducing phosphorylation of ERK1/2 and p38 MAPK in macrophages

We previously reported that excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expression and production of tumour necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide. The present study investigated the suppressive mechanisms of tumour necrosis factor-alpha mRNA by excretory/secretory products in lipopolysaccharide-stimulated murine macrophages. Electrophoretic mobility shift assay and supershift assay revealed that neither nuclear translocation of nuclear factor-kappa B nor conformation of the p50/p65 nuclear factor-kappa B subunits was affected by the treatment of excretory/secretory products in lipopolysaccharide-stimulated macrophages. Inhibition of extracellular signal-regulated protein kinase 1/2 with PD98059 or p38 mitogen-activated protein kinase with SB203580 partially reduced tumour necrosis factor-alpha mRNA expression, and a combination of the two inhibitors additionally suppressed the level of tumour necrosis factor-alpha mRNA, revealing that both pathways are crucial for full induction of the gene. Northern blot analysis showed that excretory/secretory products additionally suppressed tumour necrosis factor-alpha mRNA expression in cells treated with PD98059 or SB208530 and, in turn, we found that excretory/secretory products reduced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated macrophages by Western blot analysis. This is the first report demonstrating that excretory/secretory products from parasites suppress tumour necrosis factor-alpha mRNA expression by reducing phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase without any effect on nuclear factor-kappa B activity in macrophages stimulated with lipopolysaccharide. We hypothesise that excretory/secretory products may enable this parasite to survive within the host.