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The possible cardioprotective effects of preconditioning by ischaemia (IPC) or a low dose of H2O2 (HPC) prior to a high dose of H2O2 was investigated. Langendorff-perfused rat hearts (n = 10 in each group) were subjected to 10 min of 140 micromol/L H2O2 and 30 min recovery after either (1) control perfusion, (2) 20 micromol/L H2O2 for 10 min, recovery 10 min, or (3) 2 x 2 min global ischaemia and 5 min reperfusion. 140 micromol/L H2O2 increased left ventricular end-diastolic pressure from 0 to 68+/-8 mmHg in controls (mean+/-SEM), which was attenuated by IPC (46+/-9 mmHg, p<0.001) and HPC (18+/-4 mmHg, p < 0.001 compared to controls, p < 0.01 compared to IPC). HPC, but not IPC, improved coronary flow (p < 0.02) and left ventricular developed pressure (p < 0.001) during recovery. Troponin T release was similar in all groups. Tissue thiobarbituric acid reactive substances, antioxidant capacity, catalase, and glutathione peroxidase were not influenced by 140 micromol/L H2O2. H2O2 decreased the level of tissue glutathione. This reduction was augmented by HPC (p <0.02) and attenuated by IPC (p < 0.02). H2O2 increased superoxide dismutase (p < 0.04). The increase was attenuated by IPC (p < 0.05), but not influenced by HPC. HPC efficiently protected cardiac function in H2O2-induced cardiac injury, while IPC had only a small protective effect. The functional protection cannot be explained by reduction of irreversible injury, attenuation of lipid peroxidation, or modification of tissue antioxidant parameters. more...

Fluorescence in situ hybridization studies using non-breakpoint DNA probes were performed to detect the X;18 translocation on 4-microm sections of synovial sarcoma from paraffin blocks. This was done by using commercially available, large target unique sequence DNA probes for regions of the X chromosome short-arm and the 18 chromosome long-arm together with centromere probes for the alternate chromosomes. We determined that such probe combinations could detect the presence of the diagnostic X;18 translocation in interphase cells. Spatial association of dual color signals from the X centromere and the 18 unique sequence probe, as well as between an 18 centromere and the X unique sequence probe, was seen in a significantly higher percentage of synovial sarcoma cells (81.1% +/- 7.7%, confidence interval 95%) than in control nonsynovial soft tissue sarcomas (14.7% +/- 8.3%) and control peripheral blood lymphocytes (5.6% +/- 0.6%). The observed spatial association supports the use of this strategy to detect the X;18 translocation in synovial sarcoma and suggests that this technique could be applied in the diagnosis of other types of tumors with characteristic translocations when histopathological findings are inconclusive. This study is the first report describing the use of nonbreakpoint unique sequence probes for detecting translocations in tumors on paraffin-embedded slides. more...

Several recent experiments have shown that melatonin is an efficient antioxidant and free radical scavenger. In the present study the antioxidative effect of melatonin was compared with that of pinoline. Pinoline (6-methoxy-tetrahydro-beta-carboline) can be formed in the mammalian body under physiological conditions from 5-hydroxytryptamine or as a tricyclic metabolite of melatonin. Both melatonin and pinoline inhibited lipid peroxidation and showed comparable activity in a total antioxidant status test. Melatonin and pinoline concentration-dependently scavenged hydroxyl radicals with IC50 11.4+/-1.0 microM for melatonin and 62.3+/-3.8 microM for pinoline. These results support the importance of the indolic part of the molecule and the 5-methoxy group common to both compounds in terms of the ability of these molecules to quench the hydroxyl radicals. As pinoline has been shown to exert an antidepressant-like effect in behavioral experiments and has been reported to have a low toxicity, this compound should be further studied as a potential antidepressant with pronounced antioxidative effects. These results further support the importance of pineal gland in antioxidative protection. more...

The effects of long-term alimentary (drinking water) iron overload on the parameters of oxidative stress were evaluated. The study group (n = 35) from a village in southern Estonia was 37.1 +/- 13.3 years old, and the mean period of drinking water iron overload was 20.6 +/- 9.3 years. The serum iron content was significantly higher than normal. The total iron-binding capacity of serum tended to be lowered. There was no change in the transferrin content. The parameters of lipid peroxidation like conjugated dienes and thiobarbituric acid reactive substances showed also significant differences. In addition, the red blood cell glutathione content was also decreased. The total antioxidant capacity of serum was not changed. It can be concluded from our results that a long-term alimentary iron overload results in a positive serum iron balance, which, in turn, yields an increased oxidative stress. more...

The effect of the 24-amino-acid-long peptide, PD(1), on rat cerebral cortical Na+/K+ -exchanging ATPase (EC 3.6.1.37) has been studied. Incubation of the enzyme preparation (25 degrees C for 10-25 min) with the peptide (10(-7) - 10(-4) M) did not appreciably affect the activity of the enzyme, only 5-8% activation being registered. On the other hand, PD(1) completely eliminated the cooperative nature of Na+ -binding to Na+/K+ -exchanging ATPase (n(H) decreased from 1.4 to 0.9) and slightly (1.2-fold) decreased the affinity for Na+. ATP, a substrate of activity for Na+/K+ -exchanging ATPase, blocked the PD(1)-promoted effect on the cooperativity for Na+. Incubation of cerebral cortical membranes with 5 x 10(-4) M PD(1) revealed a shift (from 19.5 degrees C to 21.4 degrees C) of the typical break on the Arrhenius plot (15-37 degrees C). Prolonged incubation of enzyme preparation (25 degrees C for 1-2 h) with PD(1) (4.5 x 10(-4) - 0.7 x 10(-2) M) followed by centrifugation of the mixture at 53,000 g for 90 min, resulted in loss of the activity both in the supernatant and the sediment, while the protein content in the supernatant and the sediment remained unchanged. After a short incubation (25 degrees for 10 min) with PD(1) (1 x 10(-6) M), followed by centrifugation, the full activity of Na+/K+ -exchanging ATPase in the sediment was restored. These data suggest that peptitergent PD(1) does not solubilize the transmembrane protein Na+/K+ -exchanging ATPase, although it abolishes the cooperative effect of Na+. more...

Pinoline (6-methoxy-1,2,3,4-tetrahydro-beta-carboline) has been found in similar concentrations to those of melatonin in various mammalian tissues. The present study investigates the subcellular distribution of in vivo administered [3H]-pinoline in mouse tissues using light and electron microscopic autoradiography. The antioxidative capacity of pinoline was determined in vitro and compared with that of the structurally similar pineal hormone melatonin. Autoradiograms revealed that [3H]-pinoline was present in all tissues studied 30 min after administration, but in most tissues binding was nonspecific. Adrenal glands showed the highest specific binding for [3H]-pinoline 0.5-1 h after administration of the labelled compound. In all tissues a large amount (approximately 40%) of radioactivity was located in nuclei of cells. Specific nuclear binding was the highest in adrenal glands and cerebral cortex. Both pinoline and melatonin potently inhibited lipid peroxidation. Therefore, we suggest that pinoline may act in cells and nuclei as an antioxidant. Direct genomic effects of pinoline cannot be excluded. more...

Background: Despite therapy, arterial hypertension continues to be a risk factor of coronary artery disease (CAD). The role of oxidative stress, an important source of vascular injury, in the genesis of this increased risk also needs to be defined, because several antihypertensive drugs have demonstrated antioxidant effects. This study tested the existence of oxidative stress in young (< 40 years) untreated patients with uncomplicated essential hypertension (EH)., Methods: Lipid peroxidation (LP) products (diene conjugates, basal and Fe-stimulated levels of thiobarbituric acid reactive substances) were detected spectrophotometrically in serum together with markers of antioxidant status (serum antioxidative capacity (AOC), red blood cell (RBC) glutathione) in 32 patients with mild-to-moderate EH and in 26 matched normotensive controls., Results: All LP products were elevated (P < 0.01), while serum AOC was decreased (P < 0.001) and RBC glutathione increased (P < 0.05) in EH patients compared with controls. The presence of hypercholesterolaemia was not found to influence the differences in the measured parameters between hypertensive patients and controls significantly., Conclusions: The results indicate that oxidative stress occurs in young patients with uncomplicated EH. Therefore antihypertensive treatment; especially in patients whose vascular disease is still reversible, should provide antioxidant protection. more...

Oxidative stress and subsequent lipid peroxidation have been suggested as pathogenetically important for postischaemic reperfusion injury. We studied the time-course of oxidative stress in 14 adults undergoing cardiac surgery, evaluating serum levels of lipid peroxidation products--diene conjugates (DC) and basal and Fe-stimulated thiobarbituric acid reactive substances (TBARS, FeTEBARS)--as well as markers of blood antioxidant status--serum antioxidative capacity (AOC) and red blood cell glutathione (RBC-GSH) at 6 perioperative time-points. Arterial TBARS were significantly increased 15 minutes after start of cardiopulmonary bypass, 5 minutes after release of aortic cross-clamp and 15 minutes after cessation of bypass, compared with the preoperative levels (respective means 20.8, 38.5, 34.8 vs 7.5 nmol/g protein, p < 0.05). AOC had decreased at these times (means 21.3, 18.1, 23.2 vs 34.9%, p < 0.05). The TBARS changes correlated with AOC decrease (r = 0.30, p < 0.001). Changes in serum DC and RBC-GSH were not statistically significant. All lipid peroxidation parameters had returned to preoperative levels on the following morning, while antioxidative capacity remained suppressed (28.1%, p < 0.05). These data demonstrate a definite time-course of oxidative stress markers in arterial blood during open-heart surgery. more...

Several peptidic and non-peptidic factors can modulate Na,K-ATPase activity, among them mainly inhibitors of this enzyme, ouabain being the most effective. In a very few cases only, activation of Na,K-ATPase by endogenous factors has been recorded. We have investigated the effect on Na,K-ATPase of a novel regulatory peptide, PEC-60, recently isolated from porcine intestine. Various biological effects have been described for PEC-60 in different tissues, including brain. We have found that PEC-60 caused a dose-dependent activation of Na,K-ATPase from rat brain frontal cortex, whereas the carboxymethylated form of PEC-60 or other hormonal peptides had no effect. The maximal value of activity reaches up to 125% at close to micromolar concentrations of PEC-60 and the dependence can be described with a bell-shaped curve, indicating a complex mechanism for the interaction. The activation of the enzyme by PEC-60 is apparently related to Na(+)-dependent steps of the Na,K-ATPase system. The kinetic parameters for K(+)-phosphatase were unaffected. Moreover, the activating effect was enhanced by preincubation at low concentrations of ATP that transform the enzyme into the Na(+)-form. Due to the crucial physiological role of Na,K-ATPase, its activity has to be finely controlled and thus PEC-60 may be one of the endogenous factors that regulate this enzyme. more...

The role of lipid-protein interaction in the regulation of the brain Na-pump by different neurotropic agents (prostaglandin E2, middle molecules from blood plasma of uremic patients, neuropeptide galanin, the oligopeptide fraction from brain) was investigated. We established a definite correlation between the lipid status (the peroxidativity of the lipids, the phospholipids and cholesterol content) of the Na,K-ATPase enzyme preparation (plasma membrane fragments) and the influence of the neurotropic agents. Besides, after the treatment with delipidative agents (phospholipase A2, SDS) the inhibitory effects of these neurotropic agents, were diminished significantly. These facts do not contradict our previous suggestion that the lipid-protein interactions underlay the regulative action mechanism of the natural bioactive ligands. more...

A method is presented for obtaining the preparation of N+, K+-ATPase from the cattle brain. The specific activity of the preparation is 5 units (mu mole Pi per 1 min) per 1 mg of protein. A water-soluble derivate of carbodiimide is shown to inhibit reversibly both Na+, K+-ATPase and K+-phosphatase. ATP, Na+ and K+ manifest a protective effect against inhibition, and Na+ and K+ revealed a competition with the inhibitor for the enzyme. p-Chloromercuribenzoate inhibits irreversibly Na+, K+-ATPase and K+-phosphatase activities. The substrates ATP and p-nitrophenylphosphate protected these activities against inhibition. The phosphororganic compound O-n-butyl-S-(beta-ethyl-mercaptoethyl)-methyl thiophosphate has no significant effect on the Na+, K+-ATPase and K+-phosphatase activities. more...

Plasma levels of gastrin, adrenocorticotropin (ACTH) and somatotropin (STH) were determined 24 hours after pylorus ligation and after pylorus ligation and vagotomy by radioimmunoassay. Pylorus ligation increases the serum levels of these hormones, while vagotomy inhibits the production of these ones. We conclude that the antiulcerogenetic effect of surgical vagotomy is closely related with the changes in the level of these hormones. more...