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General Physiology and Biophysics

Volume 29, 2010, No. 2


  Effects of one-day reperfusion after transient forebrain ischemia on circulatory system in the rat
Petra Kravcukova 1), Viera Danielisova, Miroslava Nemethova, Jozef Burda , Miroslav Gottlieb

1)Institute of Neurobiology SAS, Šoltesovej 4/6, 040 01 Košice, Slovakia.

Although ischemia/reperfusion injury remains incompletely understood, it appears that reactive oxygen species produced mainly during postischemic recirculation play a critical role. The present study examined the impact of forebrain ischemia and subsequent one‐day reperfusion on several blood parameters. We determined glutamate concentration in whole blood, measured Cu/Zn‐ and Mn‐SOD (superoxide dismutase) activity in blood cells as well as plasma, and investigated the prevalence of single and double strand breaks of lymphocyte DNA. The results of our experiment showed that the concentration of glutamic acid in whole blood was increased by about 25%. Antioxidant activity of total SOD and Cu/Zn‐SOD was reduced in blood cells and plasma. Mn‐SOD activity in blood cells was not affected by ischemic insult and one‐day reperfusion, but we detected its significantly lower activity in samples of plasma. We observed a weakly reduced level of double and a significantly elevated level of single strand breaks of lymphocyte DNA. In conclusion, one day of recovery after the ischemic attack failed to return peripheral circulatory system to physiological conditions. Reduced antioxidant capacity in the blood and an elevated level of excitotoxic amino acid glutamate may cause lymphocyte DNA damage, and probably contribute to insufficient postischemic recovery of brain tissue.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 113-121.

  The effect of pH on the activity of soluble peroxidase in needles of Serbian spruce (Picea omorika (Panč.) Purkinye): application of a mathematical model
Danijela Laketa 1), Jelena Bogdanović, Radivoje Prodanović, Aleksandar Kalauzi, Ksenija Radotić

1)Faculty of Biology, University of Belgrade, Studentski trg 3-5, 11000 Belgrade, Serbia.

We studied the dependence of peroxidase (POD) activity on pH in crude extract of Picea omorika (Panč.) Purkinye needles and in its acidic and basic fractions, obtained by ion exchange chromatography. Nonlinear regression was applied on the activity data with pH as the explaining variable, using the Levenberg‐Marquardt algorithm. Studying crude extract at three different temperatures, the shape of the simulated activity/pH dependences indicated an existence of two components, which was confirmed by mathematical modeling. The kinetic parameters Act0, KEH and KEOH of both components are presented. The curves and pH optima shifted under increasing temperatures towards lower pH values, which was verified after decomposition. Nonlinear regression detected the presence of two components for both fractions, and there is no considerable difference between their pH optima. Our results show for the first time that the sum of components, each described by the mathematical model employed, can be used to explain the complex pH‐related POD activity in the extract with two or more enzyme forms simultaneously active.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 122-128.

  Analysis of rat papillary muscle transverse deformation by laser diffraction
Adolfo Virgen-Ortiz 1), Alejandro Apolinar-Iribe, Martín Ríos-Martínez, José Marín, Jesús Muñiz

1)Departamento de Ciencias Químico Biológicas y Agropecuarias, División de Ciencias e Ingeniería, Unidad Regional Sur, Universidad de Sonora. Blvd. Lázaro Cárdenas No. 100, Colonia Francisco Villa, CP 85880, Navojoa, Sonora, México.

We use laser diffraction in the analysis of the transversal deformation that the papillary muscle of the female and male Wistar rat may undergo when is subjected to different tension (tension range, 0‐30 mN) in the longitudinal plane. Papillary muscles from the right ventricle were illuminated at normal incidence with a He‐Ne laser lasing at 594 nm at room temperature. The far‐field diffraction pattern projected to a screen was recorded with a digital camera for its analysis. The analysis of the stress‐strain curves from the two experimental groups shows that the papillary muscles from male rats exhibit a higher stiffness in the transversal axis compared to the female rats.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 129-133.

  Effect of selected flavones on cancer and endothelial cells
Martina Pilátová 1), Viktória Stupáková, Lenka Varinská, Marek Šarišský, Ladislav Mirossay, Andrej Miroššay, Peter Gál, Vladimír Kraus, Katarína Dianišková, Ján Mojžiš

1)Department of Pharmacology, Faculty of Medicine, P. J. Šafárik University, Košice, Slovakia.

In our study we used quercetin (3,3´,4´,5,7-pentahydroxyflavone) as the reference standard to compare antiproliferative and antiangiogenic effects of chrysin (5,7-dihydroxyflavone) and 3-hydroxyflavone. Our data indicates that chrysin and 3-hydroxyflavone showed significantly higher cytotoxic effect than reference standard quercetin. These tested agents significantly decreased cell survival with the efficacy of 65‐85% at the concentration 100 µmol/l for HUVEC, lung carcinoma and leukemic cells being the most sensitive. Cell cycle analysis indicates that quercetin and 3-hydroxyflavone might affect the cell cycle of Jurkat cells by a similar or the same mechanism of action which lead to G2/M arrest as well as to an increase in sub‐G0/G1 fraction. Treatment of Jurkat cells with chrysin resulted only increase in the fraction of cells with sub‐G0/G1 DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was confirmed by DNA fragmentation and by staining with annexin V. All three tested flavones inhibited endothelial cell migration after 24 h of incubation at a concentration 100 µmol/l. At a lower concentration (10 µmol/l) only quercetin significantly inhibited migration of endothelial cells. Furthermore, in our experiments decreased secretion of matrix metalloproteinases (MMP-2 and MMP-9) was observed after a 72 h treatment with quercetin. No decrease in secretion of MMP-2 and MMP-9 was seen after chrysin and 3-hydroxyflavone treatment. On the other hand, our results showed that none of three flavonoids blocked microcapillary tube formation. Further studies are necessary to investigate the mechanism of action and to find out the relationship between the structure, character and position of substituents of natural substances and their biological activities.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 134-143.

  Long‐term melatonin administration enhances the antioxidant potential of human plasma maintained after discontinuation of the treatment
Aleksandra Piechota 1), Stanis&lslash;awa Lipinska, Janusz Szemraj, Anna Gorąca

1)Experimental and Clinical Physiology, Department of Cardiovascular Physiology, Medical University, Mazowiecka 6/8, 92-215 &Lslash;ódź, Poland.

We investigated the effect of long‐term oral melatonin administration on the antioxidant capacity of plasma. The study was performed on healthy volunteers divided into two groups: the control group (without melatonin treatment) and the study group treated with 6 mg of melatonin per day for two weeks, 2 hours before bedtime. Blood samples were drawn: before melatonin administration, on the 7th and 14th day of melatonin treatment and on the 10th day after the last dose of melatonin. It was shown that oral administration of melatonin increases plasma antioxidant ferric reducing ability (FRAP assay) (p < 0.05) and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging (p < 0.01), and decreases thiobarbituric acid reactive substances (TBARS) (p < 0.05) and DNA damage (p < 0.001). This protective effect is maintained for at last 10 days after discontinuation of the treatment. The present work highlights that the antioxidant capacity of plasma was significantly higher on the 10th day after the discontinuation of melatonin treatment than on the 14th day of its administration. Our findings indicated that a long‐term oral melatonin administration maintained the increased antioxidant capacity of plasma and prevented oxidative damage to DNA after hormone administration was discontinued.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 144-150.

  Desmopressin stimulates bile secretion in anesthetized rats
Sepideh Ghazaee 1), Zoya Gorenko, Ludmila Karbovska, Stanislav Veselsky, Petro Yanchuk, Mykola Makarchuk

1)Department of Human and Animal Physiology, Faculty of Biology, National Taras Schevchenko University of Kyiv, Volodymyrska 64, 01601 Kyiv, Ukraine.

One of the synthetic analogues of antidiuretic hormone – desmopressin is used in patients with central diabetes insipidus and in those with coagulation disorders. However, its effects on bile secretion are not fully defined. We investigated the effect of desmopressin on bile formation and determined the role of V1a vasopressin receptors in the action of desmopressin on choleresis. Rats were injected intraportally with a bolus of desmopressin; the changes of bile flow, the content of free and conjugated cholates were compared with control animal group. Selective antagonist of V1a receptors was injected 10 minutes before desmopressin treatment and the findings were compared with the results after desmopressin injection alone. Desmopressin increased bile flow, secretion of total cholates like amino acids conjugated, while diminished free bile acids content. Secreted bile volume and conjugated bile acids content were reduced in V1a receptors antagonist+desmopressin‐treated rats. In contrast, free bile acids content was more than the results in desmopressin‐treated rats. Desmopressin at concentrations nearly equal to physiological concentrations of natural hormone in blood shows its choleretic effect. Antagonist of V1a vasopressin receptors modulates desmopressin action. This certifies the role of these receptors in the action of desmopressin on different processes of bile formation.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 151-159.

  Mouse and human mitochondrial nucleoid – detailed structure in relation to function
Jarmil Prachař 1)

1)Laboratory of Electron Microscopy, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovakia.

The independent mitochondrial genetic information is organized in so‐called mitochondrial nucleoids that, in vertebrates, typically contain 5‐7 genetic units. The total number of nucleoids per cell is several hundred in cultured cells. Mitochondrial nucleoids, similarly to the whole mitochondrial network, have recently been successfully and extensively visualized using fluorescent and confocal microscopy. In the present work, we show high‐resolution micrographs of mouse and human mitochondrial nucleoids obtained by transmission electron microscopy. Position in the mitochondria, size, general appearance and other properties of the human nucleoids appear the same as those of mouse nucleoids, and all observations are also in full agreement with the results obtained in different laboratories using different approaches. Most of nucleoids are located inside mitochondrial tubes. However, we show directly that certain part of the nucleoids close to inner membrane is bound to the complex of molecules that crosscut both, the inner and the outer mitochondrial membranes. Nucleoids in cells starving for serum are mostly more dense than those in dividing cells. We discuss the position, appearance and other properties of the nucleoids in relation to functional stage. Other electron‐dense structures inside mitochondria that could be erroneously considered to be mitochondrial nucleoids are also described.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 160-174.

  The binding affinity of carcinogenic N-nitrosodimethylamine and N-nitrosomethylaniline to cytochromes P450 2B4, 2E1 and 3A6 does not dictate the rate of their enzymatic N-demethylation
Miroslav Šulc 1), Petr Hodek, Marie Stiborová

1)Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 43 Prague 2, Czech Republic.

The interaction of carcinogenic N-nitrosodimethylamine (NDMA) and N-nitrosomethylaniline (NMA) with cytochromes P450 (CYP), CYP2B4, CYP2E1 and CYP3A6, and their metabolism by these enzymes reconstituted with NADPH-CYP reductase in liposomes were studied. Using the difference spectroscopy, the lowest values of spectral dissociation constants (KS) of the binary complex of NDMA and NMA with the enzyme were found for CYP2E1. Both N-nitrosamines bind to the heme iron atom as ligands. On the contrary, the binding of NDMA and NMA to CYP2B4 gives the type I spectra. NDMA is bound to CYP3A6 analogously as to CYP2B4, whereas no difference spectra were acquired with NMA and CYP3A6. All tested CYPs oxidize NDMA and NMA. CYP2E1 exhibits the lowest Km values, 7.5 and 5.0 µmol/l for NDMA and NMA, respectively, and for CYP3A6 we found 30.0 and 10.0 µmol/l for NDMA and NMA, respectively, while CYP2B4 exhibits the lowest affinity for both carcinogens. In spite of different binding affinities of NDMA and NMA, the values of Vmax for their oxidation were, however, similar for all tested CYPs. The results demonstrate that investigations utilizing several enzymatic approaches are necessary to properly evaluate the mechanism and efficiency of NDMA and NMA oxidation by CYPs in vitro.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 175-185.

  May modifications of human plasma proteins stimulated by homocysteine and its thiolactone induce changes of hemostatic function of plasma in vitro?
Beata Olas 1), Joanna Ko&lslash;odziejczyk, Joanna Malinowska

1)Department of General Biochemistry, Institute of Biochemistry, University of &Lslash;ódź, Banacha 12/16, 90-237 &Lslash;ódź, Poland.

Homocysteine (Hcys) may be implicated in different diseases, especially in cardiovascular illnesses. The most reactive form of Hcys is its cyclic thioester – homocysteine thiolactone (HTL), which is formed in plasma and represents up to 0.29% of plasma total Hcys. Recently, it has been observed that Hcys and HTL may modify plasma proteins, including albumin, hemoglobin or fibrinogen, but the role of this process is not yet well known. The aim of our study in vitro was to investigate the modifications of human plasma total proteins after incubation with the reduced form of Hcys in concentrations 10‐100 µmol/l, and HTL in concentrations 1‐0.1 µmol/l, which correspond to levels found in human plasma during hyperhomocysteinemia in vivo. The aim of our study was also to explain the effects of Hcys and HTL on coagulation activity of human plasma. We showed that in model system in vitro Hcys and HTL change the level of thiol, amino and carbonyl groups in plasma total proteins. Moreover, our studies reported that not only Hcys (10‐100 µmol/l), but also HTL (at lower concentrations than Hcys) modulates the coagulation properties of human plasma.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 186-193.

  Central effects of ghrelin on the adrenal cortex: a morphological and hormonal study
Verica Milošević 1), Darko Stevanović, Dejan Nešić, Branka Šošić-Jurjević, Vladimir Ajdžanović, Vesna Starčević, Walter Severs

1)Institute for Biological Research ", Siniša Stanković", University of Belgrade, 142 Despot Stefan Blvd., 11060 Belgrade, Serbia.

Ghrelin, a growth hormone secretagogue that exerts an important role in appetite and weight regulation, participates in the activation of the hypothalamo‐pituitary‐adrenal (HPA) axis. Male Wistar rats (5/group) received daily for 5 days, via an ICV (intracerebroventricular) cannula, 5 µl phosphate buffered saline with or without 1 µg of rat ghrelin. Two hours after the last injection, blood and adrenal glands were collected from decapitated rats for blood hormone analyses and histologic and morphometric processing. Ghrelin treatment resulted in increased (p < 0.05) body weight (13%), absolute whole adrenal gland weight (18%) and whole adrenal gland volume (20%). The absolute volumes of the entire adrenal cortex, ZG, ZF, and ZR also increased (p < 0.05) after ghrelin by 20%, 21%, 21% and 11%, respectively. Ghrelin-treated rats had elevated (p < 0.05) blood concentrations of ACTH, aldosterone and corticosterone (68%, 32% and 67%, respectively). The data clearly provide both morphological and hormonal status that ghrelin acts centrally to exert a global stimulatory effect on the adrenal cortex. Clarifying of the ghrelin precise role in the multiple networks affecting the stress hormone release, besides its well known energy and metabolic disbalance effects, remains a very important research goal.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 194-202.

  Mitochondrial function in heart and kidney of spontaneously hypertensive rats: influence of captopril treatment
Jana Mujkošová 1), Oľga Uličná, Iveta Waczulíková, Jana Vlkovičová, Oľga Vančová, Miroslav Ferko, Stefan Polak, Attila Ziegelhöffer

1)Institute for Heart Research, Centre of Excellence for Cardiovascular Research, Slovak Academy of Sciences, 840 05 Bratislava, Slovakia.

Effect of captopril treatment on capability of heart and kidney mitochondria to produce ATP was investigated in spontaneously hypertensive rats (SHR). Heart mitochondria from SHR responded to hypertension with tendency to compensate the elevated energy demands of cardiac cells by moderate increase in mitochondrial Mg2+‐ATPase activity, membrane fluidity (MF) and in majority of functional parameters of the mitochondria (p > 0.05). Significant increase exhibited only the oxygen consumption (QO2; p < 0.01‐0.001) and oxidative phosphorylation rate (OPR; p < 0.003) with glutamate + malate (GLUT+MAL) as substrates. Lowering the blood pressure (p < 0.02) captopril also eliminated the above compensatory response and impaired the oxidative ATP production by decreasing OPR (p < 0.001). Kidney mitochondria of SHR experienced serious disarrangement in parameters of oxidative ATP production: increase in Mg2+‐ATPase activity (p < 0.05) but, also scattered QO2 values (p < 0.03‐0.01) leading to decrease in OPR and the ADP:O (p < 0.05‐0.01) values with both GLUT+MAL and succinate as substrates. Captopril treatment does not alleviated but even worsened the above alterations. Mg2+‐ATPase became also decreased and the depression of ADP:O became aggravated (p < 0.0001).

General Physiology and Biophysics. Volume 29, 2010, No. 2: 203-207.

  The lipid peroxidation in breast cancer patients
Magdalena Kedzierska 1), Beata Olas, Barbara Wachowicz, Arkadiusz Jeziorski, Janusz Piekarski

1)Department of General Biochemistry, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland.

The aim of our study was to estimate oxidative stress (by using different biomarkers of lipid peroxidation – isoprostanes and thiobarbituric acid reactive substances (TBARS)) in patients with invasive breast cancer, patients with benign breast diseases and in a control group. We observed a statistically increased level of TBARS in plasma and isoprostanes in urine of patients with invasive breast cancer in comparison with a control group. The concentration of tested biomarkers in plasma or urine from patients with invasive breast cancer was also higher than in patients with benign breast diseases. Moreover, the levels of tested markers in patients with benign breast diseases and in a control group did not differ. Considering the data presented in this study, we suggest that free radicals induce peroxidation of unsaturated fatty acid in patients with breast cancer.

General Physiology and Biophysics. Volume 29, 2010, No. 2: 208-210.