Expression analysis of genes involved in apoptosis, proliferation and endoplasmic reticulum stress in ionomycin/PMA treated Jurkat cells.

PURPOSE: Activation of T cells by direct stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) results in numerous downstream signals that activate pathways enabling T cells to proliferate and produce cytokines. Inducible T cell activation is regulated predominantly at the transcriptional level. Therefore, we were interested to analyze the transcriptional activity of the 19 genes involved in the regulation of several important cellular processes. METHODS: Quantitative real-time (RT) PCR analysis was performed using mRNA-specific primers and SybrGreen for relative mRNA expression levels of all the examined genes. RESULTS: Our results showed c-kit expression in Jurkat cells, further confirmed by sequencing of c-kit mRNAspecific PCR product. The expected increased expression of interleukin (IL)-2 mRNA, together with moderate Ki-67 upregulation, indicate the proliferation of PMA/Io treated Jurkat cells. Significant upregulation of nuclear factor (NF)-κB, JNK and the prosurvival Bcl-2 was followed by activation of only one protein kinase RNA-like endoplasmic reticulum kinase (PERK) out of 3 main endoplasmic reticulum (ER) stress subpathways (ATF6 and spliced XBP were downregulated). NF-κB and JNK activation, as well as ERK downregulation were reactive oxygen species (ROS)-independent, shown by the lack of activation of antioxidative enzymes (SOD, NOS, GSTP1, gGCS and GR). C-kit was downregulated in the absence of exogenous SCF (c-kit ligand). CONCLUSION: Based on these data it is concluded that the PMA/Io treatment of Jurkat cells induced increased expression of IL-2, followed by upregulation of prosurvival genes belonging to the Bcl-2 family. Neither c-kit nor the antioxidative system were activated, excluding their role in Jurkat T-cell activation in the absence of exogenous c-kit ligand SCF.

Antioxidant defense in mitochondria during diapause and postdiapause development of European corn borer (Ostrinia nubilalis, Hubn.).

Antioxidant enzymes (CAT, catalase; GPx, selenium nondependent glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; DHAR, dehydroascorbate reductase) were determined in the mitochondria of diapausing and non-diapausing larvae and pupae of both diapausing and non-diapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). CAT, GST, and DHAR activity in mitochondria of diapausing larvae were reduced compared to non-diapausing larvae. Pupae of diapaused-larvae possessed lower GST, but higher DHAR activities compared to pupae of non-diapaused individuals. Comparison between larvae and pupae revealed lower GPx activity in the mitochondria of pupae. CAT activity in the mitochondria of pupae was higher compared to diapausing larvae, but lower than in non-diapausing ones. Correlation and canonical discriminant analyses revealed different antioxidant enzyme compositions for a particular stage and developmental pattern. Our results show that antioxidant enzymes have a similar role in the regulation of energetics in mitochondria as that in diapause and metamorphosis.

Oxidative stress in diabetic pregnancy: SOD, CAT and GSH-Px activity and lipid peroxidation products.

Diabetic pregnancy is often complicated by a number of pathological conditions among which is increased oxidative stress. This study was conducted to investigate the parameters of oxidative stress in 90 patients divided into the three groups: pregnant women with Type 1 diabetes mellitus, healthy pregnant women and non-pregnant women. In pregnancy groups all parameters were followed in 1st, 2nd and 3rd trimester. Diabetic control was monitored by fasting blood glucose and glycosylated hemoglobin (HbA(1c)) and these values, as well as measured biochemical parameters (urea, creatinine, total cholesterol and uric acid), were appropriate throughout the study. The concentration of TBARS, as a measure of lipid peroxidation, and activity of antioxidant enzymes superoxide dismutase (Cu, Zn-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were investigated in hemolysate of erythrocytes. TBARS concentration increased significantly in pregnant women when compared with control group (non-pregnant women), as well as in pregnant diabetics compared with healthy pregnant women. The SOD activity was gradually increased in the group of normal pregnant women vs. non-pregnant group, but decreased significantly in the group of diabetic pregnant women. Catalase activity was significantly increased only in 3rd trimester diabetic pregnant women. Increased lipid peroxidation and reduced antioxidant status, despite good diabetic control, show that pregnant women are exposed to oxidative stress to a greater degree than controls.

Role of antioxidant defense during different stages of preadult life cycle in European corn borer (Ostrinia nubilalis, Hubn.): Diapause and metamorphosis.

Antioxidant enzymes, total glutathione (GSH), and ascorbic acid (ASA) were determined in whole body homogenates of nondiapausing larvae, diapausing larvae during the diapausing period (October, December, and February), and in pupae emerged from both diapausing and nondiapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). The activities of catalase, selenium nondependent glutathione peroxidase (GPx), and glutathione-S-transferase (GST), as well as the content of GSH and ASA, were found to vary throughout the larval diapause. Compared to diapausing larvae, nondiapausing larvae were higher in levels of catalase, GPx, GST, and dehydroascorbate reductase (DHAR) activity. GSH content was also increased. However, nondiapausing larvae contained less ASA than diapausing ones. Pupae had higher GPx and GST activity and an increased ASA content compared to larvae. The pupae emerged from nondiapausing larvae had higher GST, glutathione reductase (GR), and DHAR activities, but lower GPx activity and ASA content than those emerged from diapausing larvae. Correlation analysis revealed differences in the way the antioxidant level is equilibrated for a particular stage and developmental pattern. The results suggest that cellular antioxidants are involved in both the protection of cells and the regulation of redox levels during the pre-adult stages of Ostrinia nubilalis. Arch. Insect Biochem. Physiol. 55:79-89, 2004.

Selenium-dependent GSH-Px in erythrocytes of patients with hypertension treated with ACE inhibitors.

This study is aimed at examining whether essential arterial hypertension (HTN) or ACE inhibitors have any effect on erythrocyte selenium (Se)-dependent and Se-non-dependent glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity. Eleven patients with HTN (2 men and 9 women) and 9 healthy volunteers were included in this study after clinical examination and laboratory investigation. The activities of all three enzymes were determined and then the patients were assigned to receive ACE inhibitor therapy consisting of captopril, 25 to 50 mg daily, or enalapril, 10 to 40 mg daily. After 1 year, the determination of antioxidant enzymes was repeated. Our results showed that the initial values of Se-dependent GSH-Px in patients treated with ACE inhibitors were significantly lower (19.60 +/- 3.50 microM NADPH/min(-1)/mgHb(-1)) compared with the controls (28.64 +/- 4.93 microM NADPH/min(-1)/mgHb(-1); p < 0.001), whereas the activity of Se-non-dependent GSH-Px was significantly enhanced (13.55 +/- 1.46 microM NADPH/min(-1)/mgHb(-1); p < 0.001) compared with the control group (9.44 +/- 0.81 microM NADPH/min(-1)/mgHb(-1); p < 0.001). ACE inhibitors did not significantly change the activity of Se-dependent GSH-Px or Se-non-dependent GSH-Px. No significant alteration was observed in SOD activity.

Selenium-dependent and selenium-non-dependent glutathione peroxidase in patients with Balkan endemic nephropathy.

We studied the activity of erythrocyte selenium (Se)-dependent, Se-non-dependent glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in uremic patients (UP) in clinically healthy members from families affected with Balkan nephropathy (HMF/BEN) and in healthy volunteers from endemic settlements (control group). The SOD activity was not significantly different in the groups studied and the Se-non-dependent GSH-Px activity in HMF/BEN and UP was not different from the control group. However, the activity of Se-dependent GSH-Px in UP was lower compared with the control group, whereas the mean value of the Se-dependent GSH-Px activity in HMF/BEN was not significantly different when compared with the other two investigated groups.

Partial desiccation induced by sub-zero temperatures as a component of the survival strategy of the Arctic collembolan Onychiurus arcticus (Tullberg).

The mechanism by which the freeze susceptible Arctic collembolan Onychiurus arcticus survives winter temperatures of -25 degrees C in the field is not fully understood but exposure to sub-zero temperatures (e.g. -2.5 degrees C) is known to induce dehydration and lower the supercooling point (SCP). In this study, changes in the water status and certain biochemical parameters (measured in individual Collembola) during a 3-week exposure to decreasing temperatures from 0 to -5.5 degrees C were studied. Osmotically active and inactive body water contents were measured by differential scanning calorimetry (DSC), water soluble carbohydrates by high performances liquid chromatography (HPLC) and glycogen by enzymatic assays. The activity of trehalase and trehalose 6-phosphate synthase were also measured. During the experiment, total water content decreased from 70 to 40% of fresh weight, mostly by the loss of osmotically active water with only a small reduction in the osmotically inactive component. The SCP decreased from -7 to -17 degrees C. Analysis of the results shows that if O. arcticus is exposed to -7 degrees C in the presence of ice, all osmotically active water would be lost due to the vapour pressure gradient between the animals supercooled body fluids and the ice. Under these conditions the estimated SCP would reach a minimum of c. -27 degrees C, but the Collembola may never freeze as all the osmotically active water has been lost, the animal becoming almost anhydrobiotic. Trehalose concentration increased from 0.9 to 94.7&mgr;g mg(-1)fw while glycogen reserves declined from 160 to 7.7 nmol glucose equivalents mg(-1) protein. Trehalase activity declined as the temperature was reduced, while trehalose 6-phosphate activity peaked at 0 degrees C. By adopting a strategy of near anhydrobiosis induced by sub-zero temperatures, O. arcticus, which was previously thought to be poorly adapted to survive severe winter temperatures, is able to colonise high Arctic habitats.

The effect of selenium on antioxidant system in erythrocytes and liver of the carp (Cyprinus carpio L.)

The effect of selenium-supplemented diet (sodium selenate and selenium yeast) on antioxidant in erythrocytes and liver of the carp (Cyprinus carpio L.) fingerlings was studied. With this goal, the activities of glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-S-transferase (GST), as well as glutathione (GSH + GSSG) level, were determined. In the group supplemented with sodium selenate, no significant changes in the activity of the above enzymes were recorded in both the erythrocytes and in the liver, with the exception of GST activity that was significantly reduced in the plasma compared with the controls. Glutathione content was at the control level. In the group supplemented with selenium-yeast, the activities of GSH-Px, CAT, and SOD were significantly increased in erythrocytes, whereas GST activity and plasma content of GSH + GSSG were reduced compared with the controls. At the same time, the activities of hepatic SOD and GST were increased compared with the controls. These results demonstrate that organically bound selenium (selenium-yeast) acts more efficiently on antioxidant system of the carp fingerlings than inorganic selenium salts.