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General Physiology and Biophysics

Volume 24, 2005, No. 1


  Thioredoxin – structural and functional complexity
P Štefanková 1), M Kollárová, I Barák

1)Petra Štefanková, Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-1, 842 15 Bratislava 4, Slovakia.

Thioredoxins are small globular proteins that proved to be excellent model for investigating the relationship between the structure of protein and their physico-chemical and functional properties. The results from the experiments on thioredoxins offer the basic for the development of the new paradigms in the field of chemistry, biophysics and biology of proteins, with special attention to redox reaction in living cells, protein stability and design. It is a good example of broad class of sulphur-containing redox proteins.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 3-11.

  Antidiabetic effect of Scoparia dulcis: effect on lipid peroxidation in streptozotocin diabetes
L Pari 1), M Latha

1)Leelavinothan Pari, Department of Biochemistry, Faculty of Science, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India.

Oxidative damage has been suggested to be a contributory factor in the development and complications of diabetes. The antioxidant effect of an aqueous extract of Scoparia dulcis, an indigenous plant used in Ayurvedic medicine in India was studied in rats with streptozotocin-induced diabetes. Oral administration of Scoparia dulcis plant extract (SPEt) (200 mg/kg body weight) for 3 weeks resulted in a significant reduction in blood glucose and an increase in plasma insulin. The aqueous extract also resulted in decreased free radical formation in tissues (liver and kidney) studied. The decrease in thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HPX) and increase in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and glutathione-S-transferase (GST) clearly show the antioxidant properties of SPEt in addition to its antidiabetic effect. The effect of SPEt at 200 mg/kg body weight was better than glibenclamide, a reference drug.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 13-26.

  Effect of ajmaline on transient outward current in rat ventricular myocytes
M Bébarová 1), P Matejovič, M Pásek, M Šimurdová, J Šimurda

1)Department of Physiology, Faculty of Medicine, Masaryk University, Komenského nám 2, 662 43 Brno, Czech Republic.

The mechanism of ajmaline-induced inhibition of the transient outward current (Ito) has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of Ito in a concentration-dependent, but frequency- and use-independent manner. In contrast to the single exponential time course of Ito-inactivation in control conditions (τi = 37.1 ± 2.7 ms), the apparent inactivation was fitted by a sum of two exponentials under the effect of ajmaline with concentration-dependent fast and slow components (τf = 11.7 ± 0.8 ms, τs = 57.6 ± 2.7 ms at 10 µmol/l) suggesting block development primarily in the open channel state. An improved expression enabling to calculate the association and dissociation rate constants from the concentration dependence of τf and τs was derived and resulted in kon = 4.57 × 106 ± 0.32 × 106 mol–1·l·s–1 and koff = 20.12 ± 5.99 s–1. The value of Kd = 4.4 µmol/l calculated as koff / kon was considerably lower than IC50 = 25.9 ± 2.9 µmol/l evaluated from the concentration dependence of the integrals of Ito. Simulations on a simple model combining Hodgkin–Huxley type gating kinetics and drug-channel interaction entirely in open channel state agreed well with the experimental data including the difference between the Kd and IC50. According to the model, the fraction of blocked channels increases upon depolarization and declines if depolarization is prolonged. The repolarizing step induces recovery from block with time constant of 52 ms. We conclude that in the rat right ventricular myocytes, ajmaline is an open channel blocker with fast recovery from the block at resting voltage.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 27-45.

  Effects of vitamin C on liver enzymes and biochemical parameters in rats anesthetized with halothane
A Karakilçik 1), A Hayat, N Aydilek, M Zerin, M Çay

1)A. Ziya Karakilçik, Department of Physiology, Faculty of Medicine, Harran University, 63300 Sanliurfa, Turkey.

Halothane is an important human and veterinary anesthetic, which produces free radicals during biotransformation. Occasionally, these free radicals may cause hepatic injury, especially in case of multiple halothane exposures over short periods. Vitamin C may protect cellular lipids and lipoproteins against oxidative damage by the free radicals. This study investigated the effects of vitamin C on liver enzymes and other biochemical parameters in rats anesthetized with halothane. One group of rats was used as a control, and saline (0.9% NaCl) was injected intraperitoneally into these animals as a placebo. The second group of rats was used as an anesthesia control group and was only anesthetized by halothane for 2 h. The third group was anesthetized by halothane and injected vitamin C intraperitoneally. Activities of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase enzymes were significantly increased (p < 0.05, p < 0.01, p < 0.05, respectively) by halothane anesthesia, but decreased (p < 0.05, p < 0.05, p < 0.05, respectively) with administration of vitamin C. Concentrations of triglycerides, cholesterol, total bilirubin and creatinine were statistically affected (p < 0.05, p < 0.01, p < 0.05, and p < 0.01, respectively) by injection of vitamin C. Values of erythrocyte counts, packet cell volumes, hemoglobin concentration, leukocyte counts, rates of neutrophils and lymphocytes were significantly affected (p < 0.01, p < 0.05, p < 0.05, p < 0.01, p < 0.001 and p < 0.01, respectively) by halothane anesthesia. The values of erythrocyte counts, leukocyte counts, neutrophil and lymphocyte rates were significantly decreased (p < 0.05, p < 0.05, p < 0.05, p < 0.01 and p < 0.01, respectively) with administration of vitamin C. Based upon these results, vitamin C may play an important role in the prevention of hepatic cellular injury inflicted by halothane anesthesia.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 47-55.

  Insulin facilitates the induction of the slow Na+ channels in immature Xenopus oocytes
G Charpentier 1)

1)Gilles Charpentier, Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.

Endogenous slow sodium channels have been described in the membrane of immature Xenopus laevis oocytes. The opening of these channels is a complex process comprising an induction phase leading the channels from a state of electrical inexcitability into a voltage-dependent state. The mechanism by which the depolarization of the membrane causes the induction is dependent upon an enzymatic cascade implying a phospholipase C (PLC) and a protein kinase C (PKC). The existence of different isoforms of PLC has been described in the oocytes, each isoform being activated by distinct membrane receptors upon ligand binding. The present work investigated the effects of insulin known to bind to membrane receptor tyrosine kinases and to activate PLC-γ isoforms. Our results in current and voltage clamp experiments showed that insulin facilitated the induction of the slow Na+ channels in a dose-dependent way. The current/voltage relationships indicated that the gating properties of the channels were not altered by the hormone. Lavendustins and tyrphostin, inhibitors of the epidermal growth factor signaling pathway, failed to block insulin effect as well as induction of the sodium channels. The results support the idea that some of the enzymes activated by insulin could also be involved in the acquisition of the channel voltage dependency and activated by sustained depolarization of the membrane.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 57-73.

  Fluoro-Jade B evidence of induced ischemic tolerance in the rat spinal cord ischemia: physiological, neurological and histopathological consequences
J Orendáčová 1), T Ondrejčák, K Kuchárová, D Čížková, S Jergová, B Mitrušková, E Račeková, I Vanický, J Maršala

1)Judita Orendáčová, Institute of Neurobiology, Center of Excellence, Slovak Academy of Sciences, Šoltésovej 4, 040 01 Košice, Slovakia.

Fluoro-Jade B, a marker of degenerating neurons, was used to label histopathological changes in the rat spinal cord after transient ischemia and ischemic preconditioning (IPC). To characterize postischemic neurodegenerations and consequent neurological changes, a particular attention was paid to the standardization of ischemic conditions in animals of both groups. 1. The control ischemic rats were submitted to a reversible occlusion of descending aorta by insertion and subsequent inflation of a 2F Fogarty catheter for 12 min. 2. In the IPC rats, an episode of short 3 min occlusion and 30 min reperfusion preceded the 12 min ischemia. Postischemic motor function testing (ambulation and stepping) was provided repeatedly for evaluation of neurological status 2 h and 24 h after surgery and at the end of postischemic survival, i.e. after 48 h. Fluoro-Jade B staining was used to demonstrate degenerated neurons. In the control rats, neurological consequences of histopathological changes in lumbosacral spinal cord, manifested as paraplegia, were present after 12 min ischemia. Thus, numbers of degenerated Fluoro-Jade B positive cells were visible in gray matter of the most injured L4–S2 spinal cord segments. Slight motor function impairment, consequential from significant decreasing in Fluoro-Jade B-positivity in the L4–S2 spinal cord segments of the IPC rats, was considered the pathomorpfological evidence that IPC induces spinal cord tolerance to ischemia. Our results are consistent with the previously published silver impregnation method for histopathological demonstration of ischemic degeneration.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 75-87.

  Deformability of multilamellar vesicles
M Richterová 1), V Lisý

1)Department of Biophysics, Institute of Physics, P. J. Šafárik University, Jesenná 5, 041 54 Košice, Slovakia.

Although a free unilamellar vesicle has zero or almost zero genuine surface tension, the multilamellar vesicle (“onion”) exhibits a nonzero effective surface tension σeff. The expression for σeff used in the literature is σeff ∼ sqrt[κB/d0], where B is the interaction modulus between the vesicle bilayers, d0 the repeating distance between the bilayers in the droplet, and κ their bending rigidity. In this paper we calculate the contributions to the effective surface tension of a lamellar droplet in the case when the layers interact with one another and when they are free. It is shown that the interaction contribution to the surface tension is small and σeff is determined mainly by κ, the radius of the droplet R0, and the number of the shape undulation modes lmax. A nonzero surface tension of the layers is also included in the calculation which is necessary when the vesicle membrane is stressed in the complex of other membranes.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 89-97.

  Binding of a 23 kD endonuclease to the rat liver nuclear matrix
N Grdović 1), M Vidaković, G Poznanović

1)Department of Molecular Biology, Institute for Biological Research, Despot Stephen Boulevard 142, 11060 Belgrade, Serbia and Montenegro.

In a previous paper we have described a 23 kD nuclear endonuclease (p23) that was mostly found to exist in a state of association with the isolated rat hepatocyte nuclear matrix. To investigate the nature of this interaction, the nuclear matrix was prepared using different procedures and examined for the presence/absence of the enzyme by activity gel analysis. Treatment of isolated nuclei with sodium tetrathionate (NaTT), a sulfhydryl-cross-linking agent, led to the complete recovery of p23 in the nuclear matrix, whereas incubation of nuclei with dithiothreitol (DTT), a sulfhydryl-reducing agent, led to its complete solubilization and resulting absence from the nuclear matrix. Exposure of the isolated nuclear matrix to DTT in high-ionic strength buffer, a procedure that promotes the solubilization of the internal nuclear matrix, caused the nearly complete solubilization of p23. It was concluded that disulfide bonds play an essential role in the association of p23 with the nuclear matrix and that p23 is mostly localized in the nuclear matrix interior.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 99-111.

  Effect of pharmacologically selective antidepressants on serotonin uptake in rat platelets
Z Fišar 1), M Anders, L Kališová

1)Zdeněk Fišar, Department of Psychiatry, 1st Faculty of Medicine, Charles University, Ke Karlovu 11, 120 00 Prague 2, Czech Republic.

We tested a hypothesis that a long-term administration of antidepressants acting through different primary biochemical mechanisms is associated with changes in the platelet serotonin (5-hydroxytryptamine, 5-HT) transport. Laboratory rats were administered norepinephrine reuptake inhibitors (desipramine, maprotiline), selective 5-HT reuptake inhibitor (citalopram), reversible monoamine oxidase inhibitor (moclobemide), and lithium (inositol monophosphatase inhibitor among others) during a 4-week period. Apparent kinetic parameters of platelet 5-HT transport were analyzed. Significant decrease in apparent Michaelis constant (KM) was found after the administration of all tested antidepressants except for desipramine. There was certain increase in maximal velocity (Vmax) values following the administration of desipramine, maprotiline, and citalopram; however, the all Vmax changes were not significant. Vmax/KM ratio representing limiting permeability at low extracellular concentrations of 5-HT was systematically increased in all the tested drugs, but significant changes were occurred only in maprotiline- and citalopram-treated rats. Adaptive changes in platelet 5-HT transport induced by citalopram were opposite to the acute inhibitory effect of this drug on 5-HT transporter activity. An increase in limiting membrane permeability for 5-HT could be included in the common adaptive effect of the long-term administration of antidepressants that differ in pharmacologic selectivity.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 113-128.

  Effects of stress and of amphetamine on passive avoidance conditioning in rats
L Trnečková 1), S Hynie, P Šída, Z Hliňák, I Krejčí, V Klenerová

1)Lenka Trnečková, Institute of Medical Biochemistry, First Medical Faculty, Charles University in Prague, Albertov 4, 128 00 Prague 2, Czech Republic.

This study examined the effects of immobilization stress combined with water immersion (ICS) and/or amphetamine (AM) on different memory phases in the passive avoidance task in rats. The performance of rats was evaluated in the retention tests 24 and 48 h after a single acquisition trial. ICS exposure lasting 1 h impaired retention of the learned avoidance response if applied 2 to 4 h before or immediately after training. The stressor did not affect retrieval if presented 5 or 2 h before the retention test. AM was used i.p. at the dose of 8 or 1 mg/kg. Neither 8 mg AM administered 4 h before nor 8 or 1 mg doses given after training did not impair the retention performance in unstressed rats. The 1 mg AM prevented the impairment of retention in animals exposed to the stressor 3 or 4 h before training but had no effect when the stronger impairment was induced by ICS 2 h before training. However, when given 1 h before retention testing, 1 mg AM attenuated even the severe impairment induced by the pre-training stressor exposure. Our results suggest that ICS impairs primarily the early phase of memory consolidation and a low dose of AM can prevent this effect.

General Physiology and Biophysics. Volume 24, 2005, No. 1: 129-142.