Gregorio Martínez

Assessment of the Relative Contribution of COX-1 and COX-2 Isoforms to Ischemia-induced Oxidative Damage and Neurodegeneration Following Transient Global Cerebral Ischemia

We investigated the relative contribution of COX‐1 and/or COX‐2 to oxidative damage, prostaglandin E2 (PGE2) production and hippocampal CA1 neuronal loss in a model of 5 min transient global cerebral ischemia in gerbils. Our results revealed a biphasic and significant increase in PGE2 levels after 2 and 24–48 h of reperfusion. The late increase in PGE2 levels (24 h) was more potently reduced by the highly selective COX‐2 inhibitor rofecoxib (20 mg/kg) relative to the COX‐1 inhibitor valeryl salicylate (20 mg/kg). The delayed rise in COX catalytic activity preceded the onset of histopathological changes in the CA1 subfield of the hippocampus. Post‐ischemia treatment with rofecoxib (starting 6 h after restoration of blood flow) significantly reduced measures of oxidative damage (glutathione depletion and lipid peroxidation) seen at 48 h after the initial ischemic episode, indicating that the late increase in COX‐2 activity is involved in the delayed occurrence of oxidative damage in the hippocampus after global ischemia. Interestingly, either selective inhibition of COX‐2 with rofecoxib or inhibition of COX‐1 with valeryl salicylate significantly increased the number of healthy neurons in the hippocampal CA1 sector even when the treatment began 6 h after ischemia. These results provide the first evidence that both COX isoforms are involved in the progression of neuronal damage following global cerebral ischemia, and have important implications for the potential therapeutic use of COX inhibitors in cerebral ischemia.

Contribution to characterization of oxidative stress in HIV/AIDS patients

Infection by human immunodeficiency virus (HIV) causes persistent chronic inflammation. Viral Tat protein plays a role in the intracellular increase of reactive oxygen species (ROS) thus increasing apoptotic index, mostly the one mediated by FAS/CD95, and depleting CD4+ T lymphocytes. The aim of this study was to investigate whether there is a relationship between an extensive array of redox status indices (glutathione (GSH), malondialdehyde (MDA), peroxidation potential, total antioxidant status, glutathione peroxidase (GPx), superoxide dismutase (SOD), total hydroperoxide (TH), DNA fragmentation) and relative CD4, CD95, CD38/CD8 T lymphocyte counts in HIV/AIDS patients compared to healthy subjects. Blood samples from 85 HIV/AIDS patients and 40 healthy subjects were tested by spectrophotometric techniques in order to measure oxidative stress indices, and by flow cytometry to quantify T cell subsets. Patients were divided in two groups according to CDC 1993 guidelines. CD95 and CD38 increase paralleled the severity of HIV infection. Both a reduction of GSH levels and an increase in MDA and TH levels were detected in the plasma of HIV+ patients. These patients also showed an increase of DNA fragmentation in lymphocytes as well as a significant (P<0.05) reduction of GPx and an increase in SOD activity in erythrocytes. Relatively to the control group, HIV-infected patients had significantly differences in global indices of total antioxidant status. These results corroborate that substantial oxidative stress occurs during HIV infection. To our knowledge this study is the first relating oxidative stress indices with both CD38/CD8 and CD95 lymphocytes subsets.

Time course of oxidative damage in different brain regions following transient cerebral ischemia in gerbils.

The time course of oxidative damage in different brain regions was investigated in the gerbil model of transient cerebral ischemia. Animals were subjected to both common carotid arteries occlusion for 5 min. After the end of ischemia and at different reperfusion times (2, 6, 12, 24, 48, 72, 96 h and 7 days), markers of lipid peroxidation, reduced and oxidized glutathione levels, glutathione peroxidase, glutathione reductase, manganese-dependent superoxide dismutase (MnSOD) and copper/zinc containing SOD (Cu/ZnSOD) activities were measured in hippocampus, cortex and striatum. Oxidative damage in hippocampus was maximal at late stages after ischemia (48-96 h) coincident with a significant impairment in glutathione homeostasis. MnSOD increased in hippocampus at 24, 48 and 72 h after ischemia, coincident with the marked reduction in the activity of glutathione-related enzymes. The late disturbance in oxidant-antioxidant balance corresponds with the time course of delayed neuronal loss in the hippocampal CA1 sector. Cerebral cortex showed early changes in oxidative damage with no significant impairment in antioxidant capacity. Striatal lipid peroxidation significantly increased as early as 2 h after ischemia and persisted until 48 h with respect to the sham-operated group. These results contribute significant information on the timing and factors that influence free radical formation following ischemic brain injury, an essential step in determining effective antioxidant intervention.

Evaluation of the in vitro antioxidant activity of Mangifera indica L. extract (Vimang)

An extract of Mangifera indica L. (Vimang) was tested in vitro for its antioxidant activity using commonly accepted assays. It showed a powerful scavenger activity of hydroxyl radicals and hypochlorous acid and acted as an iron chelator. The extract also showed a significant inhibitory effect on the peroxidation of rat‐brain phospholipid and inhibited DNA damage by bleomycin or copper‐phenanthroline systems. Copyright

Nimesulide limits kainate-induced oxidative damage in the rat hippocampus

Kainate induces a marked expression of cyclooxygenase-2 after its systemic administration. Because cyclooxygenase-2 activity is associated to the production of reactive oxygen species, we investigated the effects of nimesulide, a selective cyclooxygenase-2 inhibitor, on kainate-induced in vivo oxidative damage in the rat hippocampus. A clinically relevant dose of nimesulide (6 mg/kg, i.p. ) was administered three times following kainate application (9 mg/kg, i.p.). After 24 h of kainate administration, the drastic decrease in hippocampal glutathione content and the significant increase in lipid peroxidation were attenuated in nimesulide-treated rats, suggesting that the induction of cyclooxygenase-2 is involved in kainate-mediated free radicals formation.

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.

Effects of Hypericum extract on the acetylcholine release: a loose patch clamp approach

The St. Johns Wort (Hypericum perforatum) extract (Hp) represents one of the most useful natural therapeutic agents in the treatment of moderate and mild depression. The antidepressant effects of Hp are different, by a molecular mechanism point of view, when compared to those of other antidepressant drugs and, we think, a further pharmacological characterization is needed. It is suggested that the neurochemical effects of Hp could be bind either to its activity on the uptake of some mediators in the central nervous system or to the inhibition of some enzymatic activity at the receptor level. The present study carried out with the loose patch clamp (LPC) in the mouse neuromuscular junction, indicates a potentiation of the acetylcholine (ACh) action at the mouse neuromuscular junction. The spontaneous release of ACh was unaffected by Hp indicating that neither presynaptic nor postsynaptic function are modified by Hp. Indeed, both the frequency and the amplitude of the miniature end-plate currents (mepcs) were unmodified by Hp. Furthermore, the mepcs decay time (tau), i.e. the apparent cholinergic channel life time, was significantly increased after Hp treatment. The other parameter affected was the amplitude of the evoked end-plate currents (epcs) which was constantly and in a dose dependent manner increased by Hp. These findings suggest a possible action of Hp on the acetylcholinesterase (AChE) in terms of a reduction of the degradation rate of ACh.

Effects of a brassinosteroid analogue to mosquito larvae.

Many plant compounds are able to modulate herbivore growth and reproduction by directly interacting with steroid hormones systems. In insects, several classes of phytochemicals, including brassinosteroids and related substances, interfere with molting and reproduction. The effects of the molting-hormone antagonist pesticide DI-31, a brassinosteroid analogue, on Aedes aegypti mosquito larvae were tested in two different exposure scenarios. After static exposure of first-instar larvae, the calculated NOEC, LOEC, and LC(50) values referenced to 19 d were 0.03, 0.036, and 0.04 mg/mL, respectively. Semistatic exposure of fourth-instar larvae revealed them to be slightly less susceptible than the younger larvae (NOEC 0.03 mg/mL, LOEC 0.036 mg/mL, LC(50) 0.049 mg/mL referenced to 19 days). In both cases mortality was immediate and larval development was retarded. This study suggests that A. aegypti could be a useful model for the detection of hormonally active substances such as DI-31.

Improved Pharmacological Properties for Superoxide Dismutase Modified with Carboxymethycellulose

Superoxide dismutase (SOD) was chemically modified with carboxymethylcellulose through two different synthetic procedures: Reductive alkylation with the periodate-oxidized polymer (SOD-CMCox), and the formation of amide linkages through a carbodiimide catalyzed reaction (SODCMCedac). The SOD-CMCox and SOD-CMCedac conjugates contained about 1.8–1.2mol of polymer per mol of protein, and retained 68–78% of the initial catalytic activity, respectively. The glycosidated enzymes were more resistant to inactivation with H2O2 and their plasma half-life times were prolonged to 34.7h – 6.6h when compared with 4.8min for native SOD. The anti-inflammatory activity of the enzyme was 2–2.4 times increased after conjugation with the polymer.