The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes

Tolerance against oxidative stress generated by high light intensities or the catalase inhibitor aminotriazole (AT) was induced in intact tobacco plants by spraying them with hydrogen peroxide (H₂O₂). Stress tolerance was concomitant with an enhanced antioxidant status as reflected by higher activity and/or protein levels of catalase, ascorbate peroxidase, guaiacol peroxidases, and glutathione peroxidase, as well as an increased glutathione pool. The induced stress tolerance was dependent on the dose of H₂O₂ applied. Moderate doses of H₂O₂ enhanced the antioxidant status and induced stress tolerance, while higher concentrations caused oxidative stress and symptoms resembling a hypersensitive response. In stress-tolerant plants, induction of catalase was 1.5-fold, that of ascorbate peroxidase and glutathione peroxidase was 2-fold, and that of guaiacol peroxidases was approximately 3-fold. Stress resistance was monitored by measuring levels of malondialdehyde, an indicator of lipid peroxidation. The levels of malondialdehyde in all H₂O₂-treated plants exposed to subsequent high light or AT stress were similar to those of unstressed plants, whereas lipid peroxidation in H₂O₂-untreated plants stressed with either high light or AT was 1.5- or 2-fold higher, respectively. Although all stress factors caused increases in the levels of reduced glutathione, its levels were much higher in all H₂O₂-pretreated plants. Moreover, significant accumulation of oxidized glutathione was observed only in plants that were not pretreated with H₂O₂. Extending the AT stress period from 1 to 7 days resulted in death of tobacco plants that were not pretreated with H₂O₂, while all H₂O₂-pretreated plants remained little affected by the prolonged treatment. Thus, activation of the plant antioxidant system by H₂O₂ plays an important role in the induced tolerance against oxidative stress. Received 11 December 2001; received after revision 25 January 2002; accepted 4 February 2002     

Inhibition of lipid peroxidation by diterpenoid fromPodocarpus nagi

A diterpenoid, totarol (1), fromPodocarpus nagi was evaluated as an antioxidant. This diterpenoid inhibited autoxidation of linoleic acid. Mitochondrial and microsomal lipid peroxidation induced by Fe(III)-ADP/NADH or Fe(III)-ADP/NADPH were also inhibited. Nagilactone E (2), a norditerpene lactone isolated from the same source, had no antioxidative activity. Furthermore, totarol protected red cells against oxidative hemolysis. This diterpene was shown to be effective in protecting biological systems against oxidative stresses.     

Effects of myocardial ischemia and reperfusion on mitochondrial function and susceptibility to oxidative stress

We investigated the effects of ischemia duration on the functional response of mitochondria to reperfusion and its relationship with changes in mitochondrial susceptibility to oxidative stress. Mitochondria were isolated from hearts perfused by the Langendorff technique immediately after different periods of global ischemia or reperfusion following such ischemia periods. Rates of O₂ consumption and H₂O₂ release with complex I- and complex II-linked substrates, lipid peroxidation, overall antioxidant capacity, capacity to remove H₂O₂, and susceptibility to oxidative stress were determined. The effects of ischemia on some parameters were time dependent so that the changes were greater after 45 than after 20 min of ischemia, or were significantly different to the nonischemic control only after 45 min of ischemia. Thus, succinate-supported state 3 respiration exhibited a significant decrease after 20 min of ischemia and a greater decrease after 45 min, while pyruvate malate-supported respiration showed a significant decrease only after 45 min of ischemia, indicating an ischemia-induced early inhibition of complex II and a late inhibition of complex I. Furthermore, both succinate and pyruvate malate-supported H₂O₂ release showed significant increases only after 45 min of ischemia. Similarly, whole antioxidant capacity significantly increased and susceptibility to oxidants significantly decreased after 45 min of ischemia. Such changes were likely due to the accumulation of reducing equivalents, which are able to remove peroxides and maintain thiols in a reduced state. This condition, which protects mitochondria against oxidants, increases mitochondrial production of oxyradicals and oxidative damage during reperfusion. This could explain the smaller functional recovery of the tissue and the further decline of the mitochondrial function after reperfusion following the longer period of oxygen deprivation. Received 18 May 2001; received after revision 17 July 2001; accepted 24 July 2001     

Differential effect of silybin on the Fe2+-ADP and t-butyl hydroperoxide-induced microsomal lipid peroxidation

Summary We have observed a differential effect of silybin dihemisuccinate on rat liver microsonal oxygen consumption and on lipid peroxidation induced by NADPH-Fe²⁺-ADP and t-butyl hydroperoxide. These results are ascribed to the antioxidant properties of the flavonoid. The differences observed in the effect of the catalysts may be a consequence of the different capacity of silybin to act as a scavenger of free radicals formed by NADPH-Fe²⁺-ADP or t-butyl hydroperoxide.This research was supported in part by grant B-2019-8412 from Dirección de Investigación y Bibliotecas, Universidad de Chile.     

Effect of chronic ethanol treatment on peroxisomal acyl-CoA oxidase activity and lipid peroxidation in rat liver and heart

Summary Chronic ethanol administration was shown to increase catalase and acyl-CoA oxidase activities in rat myocardium but did not alter the activity of liver peroxisomal enzymes. As a result of alcohol consumption a 2–3-fold increase in the level of lipid peroxidation was observed in the heart tissue while in the liver the induction was much less pronounced.     

Effect of physical exercise on the specific activity of carbonic anhydrase isozyme in human erythrocytes

Summary Significant decreases in the levels of both carbonic anhydrase type B and total esterase activity of human erythrocytes were observed after physical exercise (bicycle ergometer, 150 W for 30 min). Since carbonic anhydrase B-dependent esterase activity likewise decreased, the decrease in the total esterase activity would be caused by the decrease of carbonic anhydrase B activity. The specific activity of carbonic anhydrase B tended to decrease after the exercise. On the other hand no such effects were noted for carbonic anhydrase type C.     

Effect of various stressors on the levels of lipid peroxide,antioxidants and Na+, K+-ATPase actrivity in rat brain

The level of malondialdehyde (MDA), an index of lipid peroxidation, and the antioxidants superoxide dismutase (SOD) and glutathione (GSH), as well as the activity of Na⁺, K⁺-ATPase, were assessed in whole rat brain after immobilization, anemic hypoxia (NaNO₂) and 72 h starvation. The effect of these stressors on plasma glucose and corticosterone levels was also observed. Hypoxia and starvation stimulated the lipidj peroxide formation in braini as indicated by an increase in the level of MDA, being higher after starvation than hypoxia. Brain SOD activity was also increased in response to hypoxia and starvation while GSH content was only diminished ini hypoxia. However, neither MDA nor antioxidants were affected by immobilization. On the other hand, the activity of brain Na⁺, K⁺-ATPase was significantly increased by immobilization and hypoxia but decreased in starvation. A similar pattern of change was also observed in plasma glucose and corticosterone levels in response to these stressors. These results elucidate differences in the biochemical response of animals towards various types of stress, with increased lipid peroxide formation in hypoxia and starvation.     

Effect of splenectomy on the in-vitro migration inhibition response to sheep erythrocytes in the lizard,Calotes versicolor

Summary Splenectomy completely erased the PFC response to SRBC in lizards. In contrast, it has very little effect on the degree of MI to all doses ranging from 10⁴ to 10⁹, excepting the lowest dose, 10³ SRBC.Supported by PL-480 (NIH) research grant, No. 01-077.Grateful to Madurai University for the award of Studentship during the tenure of the work and to Dr P. W. Askenase for the gift of capillaries used in this study.     

Relationship between the pentose phosphate shunt and methemoglobin reductase activity in human erythrocytes: Effect of aging on methemoglobin reductase activity

Summary The increase in methemoglobin reductase activity in human erythrocytes upon incubation with inosine, phosphate, pyruvate occurs only in the presence of methylene blue. No difference in activity of the methemoglobin reductases was observed between enzyme extracts of fresh cells and aged cells.     

Oxidative stress in the moderately halophilic bacteriumDeleya halophila: Effect of NaCl concentration

The sensitivity ofDeleya halophila to oxidative stress caused by hydrogen peroxide (H₂O₂) was found to vary, depending on the NaCl concentration of the growth medium. Pretreatment of the bacteria at a low concentration of H₂O₂ (50 M) protected the cells against the lethal effects of higher levels (1–2 mM) of H₂O₂. Exposure ofD. halophila cells to 50 M H₂O₂ resulted in the induction of several proteins (hydrogen peroxide-inducible proteins, hips). However, the kinetics of induction, the extent of induction and the number of hips appear to be influenced by the salt concentration of the growth medium. Five of the hips exhibited apparent molecular masses identical to those of five heat shock proteins (hsps).     

Relationship between the pentose phosphate shunt and methemoglobin reductase activity in human erythrocytes: Effect of aging on methemoglobin reductase activity.

The increase in methemoglobin reductase activity in human erythrocytes upon incubation with inosine, phosphate, pyruvate occurs only in the presence of methylene blue. No difference in activity of the methemoglobin reductases was observed between enzyme extracts of fresh cells and aged cells.     

Oxidative modifications of low-density lipoproteins (LDL) by the human endothelial cell line EA.hy 926

Modifications of LDL by the EA.hy 926 cell line were compared to those generated by human umbilical vein endothelial cells (HUVEC). Thiobarbituric acid reactive substances (TBARS) index values (TBARS sample/TBARS cell-free control ratio) were 2.64±0.18 (m±SE, n=11) and 3.12±0.24 (n=11), for HUVEC and EA.hy 926, respectively. The percentage of the most electronegative modified LDL fraction (fraction C), assessed by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC), represented 14±3% (n=34) and 22±13% (n=10) of total modified LDL in HUVEC and EA.hy 926, respectively. LDL modified by both cell lines showed increased agarose electrophoretic mobility and apo B100 fragmentation on SDS-PAGE. None of the results were significantly different between the two cell lines. Superoxide anion production was 0.12±0.04 (n=11) and 0.07±0.01 nmol/min/mg cell protein (n=11) in HUVEC and EA.hy 926, respectively. Cell-specific effects on LDL were abrogated in cysteine-free medium. Moreover, cell-modified LDL were similarly degraded by J774 macrophage-like cells. We conclude that EA.hy 926 cells are a good model for investigating endothelial cell-induced modifications of LDL. Advantages include ready availability and less individual variability than with HUVEC.     

Visualisation of lectin binding sites on the surface of human platelets using lectins adsorbed to gold granules

Summary Washed human platelets have been incubated with the lectins WGA, ConA and RCA₁, adsorbed to differentsized gold particles. Plasma membrane receptors for each lectin were then located by scanning and transmission electron microscopy.Acknowledgments. We would like to thank Mlle Dominique Dupuis for technical assistance. This work was supported in part by grant No. CRL 78.5.128.1 from INSERM.     

Acute60Co-gamma irradiation of rats decreases the inhibitory effect of succinate on the lipid peroxidation of liver mitochondria

Summary Succinate inhibits NADPH-dependent lipid peroxidation of liver mitochondria. This effect of succinate decreased 12 h after whole-body⁶⁰Co-gamma irradiation, depending on the dose of irradiation.     

Effect of volatile anesthetics on the circular dichroism of bilirubin bound to human serum albumin

The characteristic circular dichroism of bilirubin bound to human serum albumin undergoes a remarkable sign inversion on addition of halothane, chloroform and other volatile anesthetics. This sign inversion, which is completely reversed by removal of the anesthetic, reflects a pronounced conformational change of the bound ligand; probably a complete inversion of chirality. The observation suggests that association of volatile anesthetics with proteins can markedly alter the internal topography of receptor sites and potentially influence the stereoselectivity of ligand binding.     

Effect of volatile anesthetics on the circular dichroism of bilirubin bound to human serum albumin.

The characteristic circular dichroism of bilirubin bound to human serum albumin undergoes a remarkable sign inversion on addition of halothane, chloroform and other volatile anesthetics. This sign inversion, which is completely reversed by removal of the anesthetic, reflects a pronounced conformational change of the bound ligand; probably a complete inversion of chirality. The observation suggests that association of volatile anesthetics with proteins can markedly alter the internal topography of receptor sites and potentially influence the stereoselectivity of ligand binding.