C-peptide inhibits leukocyte-endothelium interaction in the microcirculation during acute endothelial dysfunction.

C-peptide is a cleavage product that comes from processing proinsulin to insulin that induces nitric oxide (NO) -mediated vasodilation. NO modulates leukocyte-endothelium interaction. We hypothesized that C-peptide might inhibit leukocyte-endothelium interaction via increased release of endothelial NO. Using intravital microscopy of the rat mesentery, we measured leukocyte-endothelium interactions after administration of C-peptide to the rat. Superfusion of the rat mesentery with either thrombin or L-NAME consistently and significantly increased the number of rolling, adhering, and transmigrated leukocytes. C-peptide significantly attenuated either thrombin- or L-NAME-induced leukocyte-endothelium interactions in rat mesenteric venules. A control scrambled sequence of C-peptide characterized by the same amino acid composition in a randomized sequence failed to inhibit leukocyte-endothelium interactions. These effects of C-peptide were associated with decreased surface expression of the cell adhesion molecules P-selectin and ICAM-1 on the microvascular endothelium. Endothelial nitric oxide synthase (eNOS) mRNA levels were increased in rats injected with C-peptide. This enhanced eNOS expression was associated with a marked increase in basal NO release from the aorta of C-peptide-treated rats. We conclude that C-peptide is a potent inhibitor of leukocyte-endothelium interaction and that this effect is specifically related to inhibition of endothelial cell adhesion molecules via maintenance of NO release from the vascular endothelium.     

Attenuation of experimental autoimmune myositis by blocking ICOS-ICOS ligand interaction

Polymyositis (PM) is an acquired, systemic, connective tissue disease characterized by the proximal muscle weakness and infiltration of mononuclear cells into the affected muscles. To understand its etiology and immunopathogenesis, appropriate animal model is required. It has been demonstrated that immunization with native human skeletal C protein induces severe and reproducible experimental autoimmune myositis (EAM) in Lewis rats, and that the muscle inflammatory lesions in the EAM mimic those of human PM. In the present study, we prepared recombinant skeletal C protein fragment and succeeded in inducing as severe EAM as that by native C protein. We found ICOS expression on muscle fiber-infiltrating T cells in the EAM rats, but not in normal rats. Treatment with anti-ICOS mAb reduced incidence and severity of myositis; decreased the number of muscle-infiltrating CD11b/c+, TCR+, and CD8a+ cells; and inhibited the expression of IL-1alpha and CCL2 in the hamstring muscles of the EAM rats. However, the treatment neither inhibited serum anti-C protein IgG level, C protein-induced proliferation of lymph node (LN) cells, or LN T cells, nor production of IFN-gamma by C protein-stimulated LN cells in EAM rats. These data indicate that analysis of C protein-induced EAM provides not only insights into pathogenesis of PM, but also useful information regarding development of effective immunotherapy against the disease. ICOS-ICOS ligand interaction would be a novel therapeutic target for PM.     

Ozone-induced inactivation of antioxidant enzymes

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.     

Regulation of antioxidant enzymes.

Free radicals generated by a partial reduction of O2 pose a serious hazard to tissues and vital organs, especially membrane lipids, connective tissues, and the nucleic acids of cells. For protection, enzymes have evolved that specifically attack O2-, hydrogen, and organic peroxides, and repair any damage incurred to DNA. With few exceptions, antioxidant enzymes are found in all aerobic and aerotolerant anaerobic organisms. Logic assumes that a basal level of antioxidant enzyme activity is maintained at all times. This may be true. Yet cells must have ways to amplify antioxidant enzyme activity to counter sudden increases in oxygen metabolites. The full details of that regulation are slowly coming to light. Bacteria possess a series of elaborate and interacting genes that can sense specific increases in intracellular H2O2 and O2-. In higher organisms, hormones and metal ion cofactors impose pre- and posttranslational control over the genetic expression of antioxidant enzymes. Furthermore, aging, cellular differentiation, and organ specificity must also be factored into the final equation in higher organisms. This review will discuss some of the more recent findings relevant to antioxidant enzyme regulation in bacteria and higher organisms.     

Attenuation of chronic neuroinflammation by a nitric oxide-releasing derivative of the antioxidant ferulic acid

Chronic neuroinflammation and oxidative stress contribute to the neurodegeneration associated with Alzheimer's disease and represent targets for therapy. Ferulic acid is a natural compound that expresses antioxidant and anti-inflammatory activities. Nitric oxide is also a key modulator of inflammatory responses. Grafting a nitric oxide-releasing moiety onto anti-inflammatory drugs results in enhanced anti-inflammatory activity. We compared the effectiveness of ferulic acid with a novel nitric oxide-releasing derivative of ferulic acid in an animal model of chronic neuroinflammation that reproduces many interesting features of Alzheimer's disease. Lipopolysaccharide was infused into the 4th ventricle of young rats for 14 days. Various doses of ferulic acid or its nitric oxide-releasing derivative were administered daily. Both drugs produced a dose-dependent reduction in microglia activation within the temporal lobe. However, the nitric oxide-releasing ferulic acid derivative was significantly more potent. If we delayed the initiation of therapy for 14 days, we found no reduction in microglial activation. In addition, both drugs demonstrated antioxidant and hydroxyl radical scavenging abilities in in vitro studies. Overall, our results predict that a treatment using nitric oxide-releasing ferulic acid may attenuate the processes that drive the pathology associated with Alzheimer's disease if the treatment is initiated before the neuroinflammatory processes can develop.     

Augmentation of antioxidant enzymes in vascular endothelium

The endothelium is a key site of injury from reactive oxygen species that can potentially be protected by the antioxidant enzymes superoxide dismutase and catalase. Large proteins, such as superoxide dismutase and catalase, do not readily penetrate cell membranes, which limits their efficacy in protecting cells from cellular reactions involving both intracellularly and extracellularly generated reactive oxygen species. Two methods are described that promote enzyme delivery to cultured endothelial cells and confer increased resistance to oxidative stress. The first method is to entrap the antioxidant enzymes within liposomes, which then become incorporated by endothelial cells and can increase enzyme specific activities by as much as 44-fold within 2 h. The second method involves covalent conjugation of polyethylene glycol (PEG) to superoxide dismutase and catalase, a technique that increases circulatory half-life and reduces protein immunogenicity. Conjugation of PEG to superoxide dismutase and catalase increased cellular-specific activities of these enzymes in cultured endothelial cells (but at a slower rate than for liposome entrapped enzymes) and rendered these cells more resistant to oxidative stress. Both liposome-mediated delivery and PEG conjugation offer an additional benefit over native superoxide dismutase and catalase because they can increase cellular antioxidant activities in a manner that can provide protection from both intracellular and extracellular superoxide and hydrogen peroxide.     

The role of antioxidant enzymes in photoprotection

The enzymatic component of the antioxidant system is discussed as one of the defensive mechanisms providing protection against excessive light absorption in plants. We present an analysis of attempts to improve stress tolerance by means of the creation of transgenic plants with elevated antioxidant enzyme activities and conclude that the effect of such transgenic manipulation strongly depends on the manner in which the stress is imposed. The following factors may diminish the differences in photosynthetic performance between transgenic plants and wild type under field conditions: effective functioning of the thermal dissipation mechanisms providing a primary line of defense against excessive light, long-term adjustments of the antioxidant system and other photoprotective mechanisms, the relatively low level of control over electron transport exerted by the Water–Water cycle, especially under warm conditions, and a decrease in the content of the transgenic product during leaf aging.     

Rapid energy exchange of enzymes with solvent water by dipolar interaction

One important problem for the function of proteins, especially enzymes, concerns the exchange of energy with the surrounding medium. In this paper, we study the interaction of vibrational degrees of freedom with the fluctuating water dipole moments. The rates of activation or deactivation attain a maximum at slow frequency vibrations near the water dispersion frequencies, i.e. in the gigahertz region. For medium proteins with molecular weights of ~10⁴ a.m.u., the rates are estimated to be of the order of magnitude of $\text{k}{}_{\mathrm{B}}\text{T}\text{h}$, the frequency factor of the transition state theory. We discuss the connection between energy exchange and reaction rates and show that a rapid energy exchange is at least a necessary condition for enzymatic catalysis.     

Characterization of the interaction between surfactants and enzymes by fluorescence probe

In order to investigate the mechanism of the different stimulatory effects of the biosurfactant rhamnolipid and the chemical surfactant Tween 80 on enzymatic hydrolysis of lignocellulose, the interaction between surfactants and enzymes was analyzed by the fluorescence probe method using pyrene as probe. Based on the evolution law of pyrene fluorescence spectroscopy in the “surfactants-enzymes” systems, the interaction relationship between surfactants and enzymes was analyzed and discussed in this paper. The results show that enzyme molecules bind with rhamnolipid molecules, participate in the formation of rhamnolipid micelles, and increase the inner hydrophobic polarity of micelles, but do not change the properties of rhamnolipid micelles above the CMC (Critical Micelle Concentration). Nevertheless, for Tween 80, enzyme molecules also participate in the forming of micelles, however, they exhibit a stronger interaction with enzymes above the CMC. Both rhamnolipid and Tween 80 bind more strongly with xylanase than cellulase. Considering also previous experimental results, it can be concluded that the interaction between surfactants and enzymes improve enzyme stability and activity, and, therefore, the efficiency of enzymatic hydrolysis of lignocellulose is enhanced. The findings further provide theoretical knowledge about the mechanism of the stimulative effects of surfactants on enzymatic hydrolysis of lignocellulose.     

Immunolocalization of antioxidant enzymes in adult hamster kidney

Summary Immunoperoxidase and immunogold techniques were used to localize the following antioxidant enzyme systems in the adult hamster kidney at the light and ultrastructural levels: superoxide dismutases, catalases, peroxidases and glutathione S-transferases. Each cell type in the kidney showed specific patterns of labelling of these enzymes. For example, proximal and distal tubular and transitional epithelial cells showed significant staining for all of these enzymes, while glomerular cells and cells of the thin loop of Henle did not show significant staining at the light microscope level. In addition, high levels of glutathione peroxidase were found in smooth muscle cells of renal arteries. At the ultrastructural level, each enzyme was found in a specific subcellular location. Manganese superoxide dismutase was found in mitochondria, catalase was localized in peroxisomes, while copper, zinc superoxide dismutase and glutathione S-transferase (liver and placental forms) were found in both the nucleus and cytoplasm. Glutathione peroxidase was found to have a broad intracellular distribution, with localization in mitochondria, peroxisomes, nucleus, and cytoplasm. Microvilli of tubular cells were labelled by antibodies to catalase, copper, zinc superoxide dismutase, glutathione peroxidase, and glutathione S-transferases. Cell types that were negative by light microscopy immunoperoxidase studies showed definite labelling with immunogold post-embedding ultrastructural techniques (glomerular cells and cells of the loop of Henle), demonstrating the greater sensitivity of the latter technique. These observations demonstrate that there are large variations in the levels of antioxidant enzymes in different cell types, and that even within a distinct cell type, the levels of these enzymes vary in different subcellular locations. Our results demonstrate for the first time the overall antioxidant enzyme status of individual kidney cell types, thereby explaining why different cell types have differing susceptibilities to oxidant stress. Possible physiological and pathological consequences of these findings are discussed.     

Interaction of 1-hydroxyethyl radical with antioxidant enzymes

There is considerable interest in the role of the 1-hydroxyethyl radical (HER) in the toxic effects of ethanol. The goal of this study was to evaluate the effects of HER on classical antioxidant enzymes. The interaction of acetaldehyde with hydroxylamine-o-sulfonic acid has been shown to produce 1, 1'-dihydroxyazoethane (DHAE); this compound appears to be highly unstable, and its decomposition leads to the generation of HER. Addition of DHAE into a solution of PBN led to the appearance of the typical EPR spectra of PBN/HER adduct. No PBN/HER spin adduct was detected when DHAE was incubated with 0.1 M PBN in the presence of GSH. In the absence of PBN, DHAE oxidized ascorbic acid to semidehydroascorbyl radical, presumably via an ascorbate-dependent one-electron reduction of HER back to ethanol. Catalase was progressively inactivated by exposure to DHAE-generated HER in a time and HER concentration-dependent manner. Ascorbic acid and PBN gave full protection to catalase against HER-dependent inactivation. The antioxidants 2-tert-butyl-4-methylphenol, propylgallate, and alpha-tocopherol-protected catalase against inactivation by 84, 88, and 39%, respectively. Other antioxidant enzymes were also sensitive to exposure to HER. Glutathione reductase, glutathione peroxidase, and superoxide dismutase were inactivated by 46, 36, and 39%, respectively, by HER. The results reported here plus previous results showing HER interacts with GSH, ascorbate, and alpha-tocopherol suggest that prolonged generation of HER in cells from animals chronically exposed to ethanol may lower the antioxidant defense status, thereby contributing to mechanisms by which ethanol produces a state of oxidative stress and produces toxicity.     

Expression of antioxidant enzymes in human inflammatory cells

Because antioxidant enzymesmay have an important role in the oxidant resistance ofinflammatory cells, we investigated the mRNA levels and specificactivities of manganese and copper-zinc superoxide dismutases (Mn SODand Cu,Zn SOD), catalase (Cat), and glutathione peroxidase, as well asthe concentrations of glutathione (GSH) in human neutrophils,monocytes, monocyte-derived macrophages, and alveolar macrophages.Levels of GSH and glutathione peroxidase activity in monocytes werethree times higher than in neutrophils, whereas the mRNA of Cat was50-fold and its specific activity 4-fold higher in neutrophils.Although Mn SOD mRNA levels were higher in neutrophils, enzymeactivities, as well as those of Cu,Zn SOD, were similar in allphagocytic cells. Neutrophils lost their viability, assessed by adeninenucleotide depletion, within 24 h ex vivo and more rapidly if GSH wasdepleted. However, neutrophils were the most resistant cell type toexogenousH₂O₂.In conclusion, high Cat activity of neutrophils appears to explaintheir high resistance against exogenousH₂O₂,whereas low GSH content and GSH-related enzymes seem to account for thepoor survival of human neutrophils.

     

Leaf senescence and activities of the antioxidant enzymes

Senescence is a genetically regulated process that involves decomposition of cellular structures and distribution of the products of this degradation to other plant parts. Reactions involving reactive oxygen species are the intrinsic features of these processes and their role in senescence is suggested. The malfunction of protection against destruction induced by reactive oxygen species could be the starting point of senescence. This article reviews biochemical changes during senescence in relation to reactive oxygen species and changes in antioxidant protection.     

Spontaneous hypomorphic mutations in antioxidant enzymes of mice

An antioxidant enzymatic system is pivotal for aerobic animals to minimize the damage induced by reactive oxygen species. Spontaneous mutant animals with altered antioxidant enzyme activity should be useful for the study of the function of these enzymes in vivo. We examined the nucleotide sequences of the genes for the major antioxidant enzymes, including catalase (Cat), superoxide dismutase (Sod1, Sod2, Sod3), glutathione peroxidase (Gpx1, Gpx2, Gpx3, Gpx4, Gpx5), and glutathione reductase (Gsr) in 10 inbred mouse strains. Nonsynonymous nucleotide polymorphisms were identified in all genes, except for Gpx1, Gpx3, and Gpx4. Notably, the SJL/J mouse strain possessed unique nucleotide substitutions in the Gsr and Sod2 genes, which led to Asp39Ala and Val138Met amino acid substitutions in GSR and SOD2, respectively. The specific activity of GSR of SJL/J mice was reduced to 65% of that of NZB/N mice. In vivo activity, however, was higher in SJL/J, due to upregulated expression of the enzyme. The SOD2 activity in SJL/J mice was reduced to half that of other mouse strains. Consistent with this reduction, oxidative damage in the mitochondria was increased as demonstrated by a decrease of total glutathione and an increase in the levels of protein oxidation. These spontaneous hypomorphic alleles would be valuable in the study of free radical biology.     

miR-17* suppresses tumorigenicity of prostate cancer by inhibiting mitochondrial antioxidant enzymes

Aberrant micro RNA (miRNA) expression has been implicated in the pathogenesis of cancer. Recent studies have shown that the miR-17-92 cluster is overexpressed in many types of cancer. The oncogenic function of mature miRNAs encoded by the miR-17-92 cluster has been identified from the 5' arm of six precursors. However, the function of the miRNAs produced from the 3' arm of these precursors remains unknown. The present study demonstrates that miR-17* is able to suppress critical primary mitochondrial antioxidant enzymes, such as manganese superoxide dismutase (MnSOD), glutathione peroxidase-2 (GPX2) and thioredoxin reductase-2 (TrxR2). Transfection of miR-17* into prostate cancer PC-3 cells significantly reduces levels of the three antioxidant proteins and activity of the luciferase reporter under the control of miR-17* binding sequences located in the 3'-untranslated regions of the three target genes. Disulfiram (DSF), a dithiolcarbomate drug shown to have an anticancer effect, induces the level of mature miR-17* and cell death in PCa cells, which can be attenuated by transfection of antisense miR-17*. Increasing miR-17* level in PC-3 cells by a Tet-on based conditional expression system markedly suppresses its tumorigencity. These results suggest that miR-17* may suppress tumorigenicity of prostate cancer through inhibition of mitochondrial antioxidant enzymes.     

Inducing gene expression of cardiac antioxidant enzymes by dietary phenolic acids in rats

An increase in oxidative stress is suggested to be intimately involved in the pathogenesis of heart failure. Phenolic acids are widespread in plant foods; they contain important biological and pharmacological properties. This study evaluated the role of phenolic acids on the expression of antioxidant enzymes in the heart of male Sprague-Dawley rats. Gallic acid, ferulic acid and p-coumaric acid at a dosage of 100 mg kg(-1) body weight significantly increased the activities of cardiac superoxide dismutase, glutathione peroxidase (GPx) and catalase (CAT) as compared with control rats (P<.05). The changes in cardiac CuZnSOD, GPx and CAT mRNA levels induced by phenolic acids were similar to those noted in the enzyme activity levels. A significant (P<.05) increase in the GSH/GSSG ratio was observed in the heart of phenolic acid-treated rats. The heart homogenates obtained from rats that were administered phenolic acids displayed significant (P<.05) increases in capacity for oxygen radical absorbance compared with control rats. Immunoblot analysis revealed the increased cardiac total level of Nrf2 in phenolic acid-treated rats. Interestingly, phenolic acid-mediated antioxidant enzyme expression was accompanied by up-regulation of heme oxygenase-1. This study demonstrates that antioxidant enzymes in rat cardiac tissue can be significantly induced by phenolic acids following oral administration.     

Antibiotic interaction with proteolytic enzymes]

The effect of penicillin, tetracycline, aminoglucozide antibiotics and streptomycin on BAEE-esterase activity of trypsin was studied. It was found that benzylpenicillin in amounts of 50, 100 and 300 mg, ampicillin in an amount of 25 mg, methicillin in an amount of 12 mg and tetracycline in an amount of 2.5 mg as calculated per 1 mg of trypsin had no effect in vitro on the esterase activity of the enzyme. Neomycin, kanamycin and streptomycin in amounts of 5, 10, 100 or 300 mg per 1 mg of trypsin catalyzed splitting of BAEE by trypsin. When the antibiotics were added to the bile, its esterase activity increased. Preliminary intramuscular administration of trypsin and kanamycin to the rats had no effect on the ampicillin levels in the blood serum and brain and did not affect the permeability of the hemato-encephalic barrier as compared to the use of trypsin alone.