At last the organ grinder...

Abstract

A prominent and early feature of the retinopathy of diabetes mellitus is a diffuse increase in vascular permeability. As the disease develops, the development of frank macular oedema may result in vision loss. That reactive oxygen species production is likely to be elevated in the retina, and that certain regions of the retina are enriched in substrates for lipid peroxidation, may create an environment susceptible to oxidative damage. This may be more so in the diabetic retina, where hyperglycaemia may lead to elevated oxidant production by a number of mechanisms, including the production of oxidants by vascular endothelium and leukocytes. There is substantial evidence from animal and clinical studies for both impaired antioxidant defences and increased oxidative damage in the retinae of diabetic subjects that have been, in the case of animal studies, reversible with antioxidant supplementation. Whether oxidative damage has a causative role in the pathology of diabetic retinopathy, and thus whether antioxidants can prevent or correct any retinal damage, has not been established, nor has the specific nature of any damaging species been characterised.     

Role of metal-catalyzed autoxidation in Maillard reaction damage to proteins in vivo

Summary

Administration of 0.1 mmol/kg of diquat to Fischer-344 rats causes acute hepatic necrosis by mechanisms that appear to involve increased generation of reactive oxygen species, but the critical targets of the proposed oxidations have not been identified. In the present study the effects of diquat-induced redox stresses on hepatic protein thiol status were determined by derivatization of subcellular fractions with monobromobimane and separation of the fluorescent derivatives by SDS-PAGE. No differences in hepatic thiol status were seen in animals 2 or 6 h after diquat, relative to saline-treated controls, despite documentation of injury by elevated plasma transaminase activities. Hepatic DNA fragmentation was increased in diquat-treated animals (24.9±5.1 vs 6.7±0.3% (controls) at 2 h; 57.2±4.1 vs 4.6±0.3% (controls) at 6 h, P<0.001). However, 8-hydroxydeoxyguanosine (8-OHdG) contents in hepatic DNA were not increased by diquat (35.3±6.2 μmol 8-OHdG/mol deoxyguanosine (dG)) over saline-treated controls (28.3±2.6). Plasma NH₃ concentrations increased in diquat-treated rats from 49 μM in controls to 170 μM 6 h after treatment with diquat. Hepatic activities of glutamine synthetase (GS) were lower in diquat-treated rats (39.7±13.0 mU/mg protein) than in controls (65.8±13.4, P<0.001), but activities of carbamyl phosphate synthetase-I (CPS-I), were not decreased significantly. The oxidation of proteins to forms reactive with 2,4-dinitrophenylhydrazine (DNPH) was investigated in subcellular fractions by Western blot analyses with a monoclonal antibody to DNP-derivatized bovine serum albumin (BSA). N-terminal sequencing of bands exhibiting reactivity with anti-DNP-BSA antibodies indicated protein carbonyl formation in malate dehydrogenase, protein disulfide isomerase, and glutathione transferase. The functional consequences of oxidation of these proteins are not known but the observation of protein carbonyl formation and no measurable loss of protein thiol content are consistent with iron chelate-mediated oxidation in the transformation critical to expression of tissue damage. The time course data are consistent with DNA fragmentation as a mechanism contributing to the development of cell injury, but the absence of increases in 8-OHdG indicates that direct oxidation of DNA may not be responsible.     

24-Hour variation in gene expression of redox pathway enzymes in rat hypothalamus: effect of melatonin treatment

Abstract

The 24-h changes in medial basal hypothalamic (MBH) gene expression of redox pathway enzymes nitric oxide synthase (NOS)-1 and NOS-2, heme oxygenase (HO)-1 and HO-2, Cu/Zn- and Mn-superoxide dismutases (SOD) and catalase were examined in adult male Wistar rats kept under an alternating regimen of light/dark. Half of the animals received melatonin (～60 μg/day) in the drinking water. After 1 month, rats were killed at six different time intervals, throughout a 24-h cycle. MBH mRNA levels were measured by real-time PCR analysis. In controls, gene expression of NOS-2 and HO-2 peaked at the early light phase while that of HO-1 showed a maximum at the middle of the dark phase. None of MBH mRNAs encoding NOS-1, Cu/Zn-SOD, Mn-SOD and catalase exhibited significant 24-h variations in control rats. Melatonin administration decreased significantly mRNAs for NOS-1, NOS-2, HO-1 and HO-2 as well as changed their 24-h profile. Melatonin augmented gene expression of the antioxidant enzymes Cu/Zn-SOD, Mn-SOD or catalase at certain time intervals only. The results are compatible with the view that the principal indirect (i.e. gene expression of redox pathway enzymes) effect of melatonin on redox pathway in the hypothalamus is mainly exerted via down-regulation of pro-oxidant enzyme mRNAs.     

Level of nitrated proteins in the plasma,platelets and liver of patients with liver cirrhosis

Abstract

Over-expression of nitric oxide synthase (NOS) and nitric oxide (NO) formation are associated with the pathogenesis of liver cirrhosis. NO-related stress alters the functions of biomolecules, especially proteins, probably as a result of nitration. The aim of this study was to assess the level of protein nitration and its correlation with the severity of the disease. Liver cirrhosis patients with different grades of severity (grades A, B, and C according to the Child–Pugh classification) were enrolled in this study. Nitroprotein content, arginine, citrulline, NO in terms of total nitrite, nitrosothiol (RSNO) and protein carbonyls were measured in blood. Immunohistochemical detection of nitroprotein was carried out in liver sections of cirrhosis patients. A significant elevation in the levels of serum and platelet arginine, arginase, citrulline, plasma, and platelet nitroproteins, RSNO, total nitrite, protein carbonyls and also a significant amount of nitrated proteins by immunohistochemical detection in tissue were observed in cirrhosis patients. The alterations were highly significant in grade C patients with bleeding complications when compared to those of grade B and A patients. In platelets, both cytosolic and cytoskeletal proteins were found to be nitrated significantly. The level of nitrite seems to have positive correlation with the level of nitroproteins in different grades of cirrhosis. The level of nitroproteins in plasma, platelets and liver tissue can be correlated with the severity of liver cirrhosis.     

Tezosentan increases nitric oxide signaling via enhanced hydrogen peroxide generation in lambs with surgically induced acute increases in pulmonary blood flow

We have previously shown that acute increases in pulmonary blood flow (PBF) are limited by a compensatory increase in pulmonary vascular resistance (PVR) via an endothelin‐1 (ET‐1) dependent decrease in nitric oxide synthase (NOS) activity. The mechanisms underlying the reduction in NO signaling are unresolved. Thus, the purpose of this study was to elucidate mechanisms of this ET‐1–NO interaction. Pulmonary arterial endothelial cells were acutely exposed to shear stress in the presence or absence of tezosentan, a combined ET_A/ET_B receptor antagonist. Shear increased NO_x, eNOS phospho‐Ser1177, and H₂O₂ and decreased catalase activity; tezosentan enhanced, while ET‐1 attenuated all of these changes. In addition, ET‐1 increased eNOS phospho‐Thr495 levels. In lambs, 4 h of increased PBF decreased H₂O₂, eNOS phospho‐Ser1177, and NO_X levels, and increased eNOS phospho‐Thr495, phospho‐catalase, and catalase activity. These changes were reversed by tezosentan. PEG‐catalase reversed the positive effects of tezosentan on NO signaling. In all groups, opening the shunt resulted in a rapid increase in PBF by 30 min. In vehicle‐ and tezosentan/PEG‐catalase lambs, PBF did not change further over the 4 h study period. PVR fell by 30 min in vehicle‐ and tezosentan‐treated lambs, and by 60 min in tezosentan/PEG‐catalase‐treated lambs. In vehicle‐ and tezosentan/PEG‐catalase lambs, PVR did not change further over the 4 h study period. In tezosentan‐treated lambs, PBF continued to increase and LPVR to decrease over the 4 h study period. We conclude that acute increases in PBF are limited by an ET‐1 dependent decrease in NO production via alterations in catalase activity, H₂O₂ levels, and eNOS phosphorylation. J. Cell. Biochem. 114: 435–447, 2013. © 2012 Wiley Periodicals, Inc.