Oxidized proteins and their contribution to redox homeostasis.

Proteins are major target for radicals and other oxidants when these are formed in both intra- and extracellular environments in vivo. Formation of lesions on proteins may be highly sensitive protein-based biomarkers for oxidative damage in mammalian systems. Oxidized proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases. ROS scavenging activities of intact proteins are weaker than those of misfolded proteins or equivalent concentrations of their constituent amino acids. Protein oxidation and enhanced proteolytic degradation, therefore, have been suggested to cause a net increase in ROS scavenging capacity. However, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxidized proteins, may contribute to the observed accumulation and damaging actions of oxidized proteins during ageing and in pathologies such as diabetes, arteriosclerosis and neurodegenerative diseases. Protein oxidation may play a controlling role in cellular remodelling and cell growth. There is some evidence that antioxidant supplementation may protect against protein oxidation, but additional controlled studies of antioxidant intake to evaluate the significance of dietary/pharmacological antioxidants in preventing physiological/pathological oxidative changes are needed.

Therapeutic efficacy of ozone in patients with diabetic foot.

Oxidative stress is suggested to have an important role in the development of complications in diabetes. Because ozone therapy can activate the antioxidant system, influencing the level of glycemia and some markers of endothelial cell damage, the aim of this study was to investigate the therapeutic efficacy of ozone in the treatment of patients with type 2 diabetes and diabetic feet and to compare ozone with antibiotic therapy. A randomized controlled clinical trial was performed with 101 patients divided into two groups: one (n = 52) treated with ozone (local and rectal insufflation of the gas) and the other (n = 49) treated with topical and systemic antibiotics. The efficacy of the treatments was evaluated by comparing the glycemic index, the area and perimeter of the lesions and biochemical markers of oxidative stress and endothelial damage in both groups after 20 days of treatment. Ozone treatment improved glycemic control, prevented oxidative stress, normalized levels of organic peroxides, and activated superoxide dismutase. The pharmacodynamic effect of ozone in the treatment of patients with neuroinfectious diabetic foot can be ascribed to the possibility of it being a superoxide scavenger. Superoxide is considered a link between the four metabolic routes associated with diabetes pathology and its complications. Furthermore, the healing of the lesions improved, resulting in fewer amputations than in control group. There were no side effects. These results show that medical ozone treatment could be an alternative therapy in the treatment of diabetes and its complications.

Effect of increase of dietary micronutrient intake on oxidative stress indicators in HIV/AIDS patients.

Several recent studies in human immunodeficiency virus (HIV) patients have identified micronutrient deficiencies as affecting progression to acquired immunodeficiency syndrome (AIDS) and death. Although the mechanisms are not known, micronutrient deficiencies may exacerbate the oxidative stress induced by HIV. In addition, infection and its evolution likely lead to an increased requirement for nutritional micronutrients, especially antioxidants. To evaluate this, 40 relatively healthy, institutionalized HIV-infected individuals were recruited for assessment before or three months after fresh fruit and vegetable supply were increased due to seasonal supply. Seven-day dietary records were recorded at the beginning (December) and end of the three-month study period (March). Oxidative stress indices and CD4+, CD38+/CD8+, and CD95+ T-lymphocyte subsets were also measured at these times. No significant differences were found in calorie or protein intake across the study period, but vitamin A, C, and E intakes all increased. A number of redox indicators were modified (increase: total antioxidant status, glutathione peroxidase, and glutathione; and decrease: superoxide dismutase) during the study period. However, no change in malondialdehyde, hydroperoxides, or DNA damage was noted but a significant reduction in CD38+/CD8+ relative count was seen. Within the context and limitations of this study, the increase of dietary fruits and vegetables intake for three months had some beneficial effects on nutrition, systemic redox balance, and immune parameters in HIV-infected persons.

Protective effect of gossypitrin on carbon tetrachloride-induced in vivo hepatotoxicity.

Carbon tetrachloride (CCl4) is a known environmental biohazard, which induces lipid peroxidation (LPO) and oxidative damage in rat liver. In this study, the hepatoprotective effect of Gossypitrin, a flavonoid extracted from Hibiscus elatus S.W, was investigated against the CCl4-induced in vivo hepatotoxicity. The levels of malondialdehyde (MDA) were assayed as an index of LPO and the levels of catalase (CAT) activity as a biomarker of oxidative damage. Leakage of aspartate aminotransferase (ALT) and lactate dehydrogenase (LDH), liver weight/body weight ratio as well as morphological parameters were used as signs of hepatotoxicity. CCl4 (1 ml/kg), intraperitoneally injected into rats, caused increased MDA production and CAT activity, and also a significant ALT and LDH leakage as compared to levels of these constituents in the control group. Changes in morphology, including steatosis, cells forming balloon cells and necrosis were evaluated in the hepatotoxin-induced damage. Treatment of rats with Gossypitrin (3.98, 5.97 and 8.95 mg/kg) 2 h before and 2 h after CCl4 injection, protected hepatocytes against cell injury induced by CCl4 and its efficacy as an antioxidant was similar to vitamin E (used as a reference antioxidant). These results are consistent with the conclusion that the toxicity of CCl4 is due to LPO and the generation of reactive oxygen species (ROS), and that Gossypitrin's protective effects relate to its direct radical scavenging ability and other antioxidative processes induced by its structure.

Protective effect of Mangifera indica L. extract (Vimang) on the injury associated with hepatic ischaemia reperfusion.

The effect of Mangifera indica L. extract (Vimang) on treatment of injury associated with hepatic ischaemia/reperfusion was tested. Vimang protects from the oxidative damage induced by oxygen-based free radicals as shown in several in vitro test systems conducted. The ability of Vimang to reduce liver damage was investigated in rats undergoing right-lobe blood fl ow occlusion for 45 min followed by 45 min of reperfusion. The ischaemia/reperfusion model leads to an increase of transaminase (ALT and AST), membrane lipid peroxidation, tissue neutrophil in filtration, DNA fragmentation, loss of protein -SH groups, cytosolic Ca2+ overload and a decrease of catalase activity. Oral administration of Vimang (50, 110 and 250 mg/kg, b.w.) 7 days before reperfusion, reduced transaminase levels and DNA fragmentation in a dose dependent manner (p < 0.05). Vimang also restored the cytosolic Ca2+ levels and inhibited polymorphonuclear migration at a dose of 250 mg/kg b.w., improved the oxidation of total and non protein sulfhydryl groups and prevented modification in catalase activity, uric acid and lipid peroxidation markers (p < 0.05). These data suggest that Vimang could be a useful new natural drug for preventing oxidative damage during hepatic injury associated with free radical generation.

Neuroprotective efficacy of nimesulide against hippocampal neuronal damage following transient forebrain ischemia.

Cyclooxygenase-2 is involved in the inflammatory component of the ischemic cascade, playing an important role in the delayed progression of the brain damage. The present study evaluated the pharmacological effects of the selective cyclooxygenase-2 inhibitor nimesulide on delayed neuronal death of hippocampal CA1 neurons following transient global cerebral ischemia in gerbils. Administration of therapeutically relevant doses of nimesulide (3, 6 and 12 mg/kg; i.p.) 30 min before ischemia and at 6, 12, 24, 48 and 72 h after ischemia significantly (P<0.01) reduced hippocampal neuronal damage. Treatment with a single dose of nimesulide given 30 min before ischemia also resulted in a significant increase in the number of healthy neurons in the hippocampal CA1 sector 7 days after ischemia. Of interest is the finding that nimesulide rescued CA1 pyramidal neurons from ischemic death even when treatment was delayed until 24 h after ischemia (34+/-9% protection). Neuroprotective effect of nimesulide is still evident 30 days after the ischemic episode, providing the first experimental evidence that cyclooxygenase-2 inhibitors confer a long-lasting neuroprotection. Oral administration of nimesulide was also able to significantly reduce brain damage, suggesting that protective effects are independent of the route of administration. The present study confirms the ability of cyclooxygenase-2 inhibitors to reduce brain damage induced by cerebral ischemia and indicates that nimesulide can provide protection when administered for up to 24 h post-ischemia.

Anti-inflammatory activity and lipid peroxidation inhibition of iridoid lamiide isolated from Bouchea fluminensis (Vell.) Mold. (Verbenaceae).

Anti-inflammatory activity of an ethanolic extract from Bouchea fluminensis leaves was demonstrated. From de ethanolic extract, the active compound was isolated and characterized as the iridoid lamiide. The activity of lamiide on rat-brain phospholipid peroxidation showed a powerful effect (IC(50)=0.92+/-0.01 mM) and an anti-inflammatory activity in carrageenin-induced paw edema test (ED(50)=62.3+/-7 mg/kg weight).

Selection of a WEHI-3B leukemia cell subclone resistant to inhibition by cholera toxin.

The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms. After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT. In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation. To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT. This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells). Stimulation ofWEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside. Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHl-3B/CT/REV. These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation). Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation.

Similar protective effect of ischaemic and ozone oxidative preconditionings in liver ischaemia/reperfusion injury.

Many studies indicate that oxygen free-radical formation after reoxygenation of liver may initiate the cascade of hepatocellular injury. It has been demonstrated that controlled ozone administration may promote an oxidative preconditioning or adaptation to oxidative stress, preventing the damage induced by reactive oxygen species (ROS) and protecting against liver ischaemia-reperfusion (I/R) injury. On the basis of those results we postulated that ozone treatment in our experimental conditions has biochemical parameters similar to the ischaemic preconditioning (IscheP) mechanism. Four groups of rats were classified as follows: (1) sham-operated animals subjected to anaesthesia and laparotomy, plus surgical manipulation; (2) I/R animals were subjected to 90 min of right-lobe hepatic ischaemia, followed by 90 min of reperfusion; (3) IscheP, previous to the I/R period (as in group 2): animals were subjected to 10 min of ischaemia and 10 min of reperfusion; (4) ozone oxidative preconditioning (OzoneOP), previous to the I/R period (as in group 2): animals were treated with ozone by rectal insufflation 1 mg kg (-1). The rats received 15 ozone treatments, one per day, of 5-5.5 ml at the ozone concentration of 50 microg ml (-1). The following parameters were measured: serum transaminases (AST, ALT) and 5'-nucleotidase (5 '-NT), with morphological determinations, as indicators or hepatocellular injury; total sulfhydryl groups, calcium levels and calpain activity as mediators which take part in xanthine deshydrogenase (XDH) conversion to xanthine oxidase (XO) (reversible and irreversible forms, respectively); XO activities and malondialdehyde + 4-hydroyalkenals as indicators of increased oxidative stress. AST, ALT levels were attenuated in the IscheP (130 +/- 11.4 and 75 +/- 5.7 U l (-1)) with regard to the I/R group (200 +/- 22 and 117 +/- 21.7 U l (-1)) while the OzoneOP maintained both of the enzyme activities ( 89.5 +/- 12.6 and 43.7 +/- 10 U l (-1)) without statistical differences (P< 0.05) in comparison with the sham-operated ( 63.95 +/- 11 and 19.48 +/- 3.2 U l (-1)). Protective effects of both the preconditioning settings on the preservation of total sylfhydryl groups (IscheP: 6.28 +/- 0.07, OzoneOP: 6.34 +/- 0.07 micromol mg prot (-1)), calcium concentrations (IscheP: 0.18 +/- 0.09, OzoneOP: 0.20 +/- 0.06 micromol mg prot (-1)), and calpain activity (IscheP: 1.04 +/- 0.58, OzoneOP: 1.41 +/- 0.79 U mg prot (-1)) were observed. Both of the preconditionings attenuated the increase of total XO associated to I/R injury. Generation of malondialdehyde + 4 hydroxyalkenals was prevented by IscheP and OzoneOP without statistical differences between the two protective procedures. These results provide evidence that both of the preconditioning settings share similar biochemical mechanisms of protection in the parameters which were measured. Although there were no differences from a biochemical point of view between Ischaemic and OzoneOPs, the histological results showed a more effective protection of OzoneOP than IscheP in our experimental conditions.