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Zoznam článkov

General Physiology and Biophysics


Volume 30, 2011, No. 3

Content:


  Pro-oxidative effect of peroxynitrite regarding biological systems: a special focus on high-molar-mass hyaluronan degradation
Eva Hrabarova 1), Ivo Juranek, Ladislav Soltes

1)Department of Carbohydrate Enzymology, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic. eva.hrabarova@savba.sk.


Current understanding on the role of peroxynitrite in etiology and pathogenesis of some human diseases, such as cardio-vascular diseases, stroke, cancer, inflammation, neurodegenerative disorders, diabetes mellitus and diabetic complications has recently led to intensive investigation of peroxynitrite involvement in physiology and pathophysiology. Mechanism of cytotoxic effects of peroxynitrite involve its reactions with lipids, DNA/RNA, proteins, and polysaccharides, thus triggering cellular responses ranging from subtle changes of cell functioning to severe oxidative damage of the affected macromolecules leading to necrosis or apoptosis. The present work is aimed at providing a brief overview of i) peroxynitrite biosynthesis and reaction pathways in vivo, ii) its synthetic preparation in vitro, and iii) to reveal its potential damaging role in vivo, on actions studied via monitoring in vitro hyaluronan degradation. The complex biochemical behavior of peroxynitrite is determined by a number of variables, such as chemistry of the reaction itself, depending mostly on the involvement of conformational structures of different energy states, concentration of the species involved, content of reactive intermediates and trace transition metal ions, contribution of carbon dioxide, presence of trace organics, and by the reaction kinetics. Recently, in vitro studies of oxidative cleavage of hyaluronan have, in fact, been the subject of growing interest. Here we also describe our experimental set-up for studying peroxynitrite-mediated degradation of hyaluronan, a system, which may be suitable for testing prospective pharmacological substances.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 223-238.

 
  Investigation of ABC transporter from mycobacterial arabinogalactan biosynthetic cluster
Petronela Dianišková 1), Jana Korduláková, Henrieta Škovierová, Devinder Kaur, Mary Jackson, Patrick Brennan, Katarína Mikušová

1)Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia. mikusova@fns.uniba.sk.


Two genes from the "mycobacterial arabinogalactan biosynthetic cluster" spanning the region from Rv3779 to Rv3809c in the genome of Mycobacterium tuberculosis H37Rv were annotated as possible components of the ATP-binding cassette transporter. Rv3781 encodes a nucleotide-binding domain and Rv3783 determines production of a membrane-spanning domain. We have examined possible roles of these genes in mycobacterial cell wall biosynthesis through inactivation of their respective orthologs in Mycobacterium smegmatis mc2155, phenotypic characterization of the mutant strains via metabolic labeling with [U-14C]-glucose, cell-free reactions with UDP-[U-14C]-galactose monitoring galactan build-up and transcriptional analysis. Several lines of evidence suggest that this ABC transporter is involved in biosynthesis of arabinogalactan, although more investigation is needed to establish its precise role or the transported substrate.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 239-250.

 
  The β isoenzyme of Ca2+/calmodulin-dependent kinase type II as possible mediator of somatostatin functions in pituitary tumour cells
Davide Cervia 1)

1)IDepartment for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Viterbo, Italy. d.cervia@unitus.it.


Somatostatin or somatostatin release inhibiting factor (SRIF) analogues are indicated for the treatment of somatotropinomas that hypersecrete growth hormone (GH). Indeed, SRIF inhibits intracellular Ca2+ concentration ([Ca2+]i), thus allowing the inhibition of GH secretion. In the present study, our hypothesis was that Ca2+/calmodulin-dependent kinase type II (CaMKII), a multifunctional serine/threonine protein kinase, is part of those signalling mechanisms mediating SRIF functions. All four CaMKII isoenzymes (termed α, β, γ and δ) are expressed in rat somatotroph GC cells, although only CaMKIIβ is inhibited by SRIF at both mRNA and protein levels. Similarly to SRIF, the specific knockdown of CaMKIIβ by RNA interference induces a decrease of [Ca2+]i. The effects of SRIF and those of CaMKIIβ knockdown are non-additive. These results are confirmed by the pharmacological blockade of CAMKII. We also observed that, similarly to SRIF, the specific knockdown of CaMKIIβ induces a decrease of both GH content/secretion. These results raise the hypothesis that CaMKIIβ may mediate, at least in part, the SRIF-induced control of [Ca2+]i. In addition, CaMKIIβ seems to play a positive role in maintaining the exocytosis of GH. Our data provide a framework for better elucidating the pathophysiological role of SRIF transduction network in somatotropinomas.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 251-262.

 
  Modulations of behavioral consequences of minor cortical ischemic lesion by application of free radicals scavengers
Kateryna Deykun 1), Marie Pometlova, Barbora Schutova, Jan Mares

1)Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Charles University in Prague, 120 00 Prague, Czech Republic. k.deykun@gmail.com.


Functional and morphological consequences of ischemic lesions are partially related to the production of reactive oxygen species (ROS). The aim of the study was to create a unilateral photothrombic lesion with minimal morphological changes and minor sensorimotor and cognitive deficits and also to test whether the application of ROS scavengers after the end of induction of ischemia had improved the functional outcome. Adult Wistar male rats were randomly divided into five groups: naive control, sham operated animals, animals with induced ischemia, and two groups of animals with induced ischemia and subsequent ROS scavenger application –melatonin or tempol. The group subjected to ischemia showed a significant decline in performance in sensorimotor tests and the Morris water maze (MWM) test, compared to control animals. Tempol (50 mg/kg, i.p.) did not improve sensorimotor function and did not change spatial learning. Melatonin (100 mg/kg, i.p.), on the contrary, resulted in a significant improvement in animals’ performances. All the ischemia subjected animals had increased speed of swimming in the MWM test, compared to the control group. Our findings showed that subsequent application of ROS scavengers improve ischemia outcomes, with melatonin being more potent. Conversely, neither melatonin, nor tempol decreased swimming speed cased by ischemia.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 263-270.

 
  Interaction between sympathetic nervous system and renin angiotensin system on MMPs expression in juvenile rat aorta
Houcine Dab 1), Rafik Hachani, Wassim Hodroj, Mohsen Sakly, Giampiero Bricca, Kamel Kacem

1)Laboratoire de Pathologies Vasculaires, Unité de Physiologie Intégrée, Faculté des Sciences de Bizerte, Universite de Carthage, Tunisia. houcine.dab@fsb.rnu.tn.


The aim of our present study is to investigate the interaction between angiotensin II (ANG II) and sympathetic nervous system (SNS) on matrix metalloproteinase MMP-2 and MMP-9 expression and activity in juvenile rat aorta under normal conditions. Sympathectomy with guanethidine and blockade of the ANG II receptors (AT1R) by losartan were performed alone or in combination on new-born rats. mRNA, protein expression and activity of MMP-2 and MMP-9 were examined by Q-RT-PCR, immunoblotting and zymography, respectively. MMP-2 mRNA and protein amount were decreased after sympathectomy or AT1R blockade and an additive effect was observed after combined treatment. However, MMP-9 expression was reduced to the same level in the three treated groups. There were some detectable gelatinolytic activity of the MMPs in both control and treated rats. We concluded that ANG II stimulates directly and indirectly (via sympathostimulator pathway) the MMP-2 expression but seems unable to affect MMP-9 expression through direct pathway. Combined inhibition of SNS and ANG II were more efficient than a single inhibition in reducing MMP amounts in rat vessels.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 271-277.

 
  Antidiabetic and antioxidant potentials of Euphorbia hirta leaves extract studied in streptozotocin-induced experimental diabetes in rats
Sorimuthu Subramanian 1), Subramanian Bhuvaneshwari, Gopalan Prasath

1)Department of Biochemistry, University of Madras, Guindy campus, Chennai 600 025, India. subbus2020@yahoo.co.in.


IEuphorbia hirta, commonly known as asthma weed, is a popular folk remedy for the treatment of various ailments. Recent studies have indicated that plant has potent antioxidant properties. As part of an ongoing programme to validate the use of some reputed herbs in Indian traditional medicines, the present study was aimed to evaluate the antidiabetic and antioxidant potentials of E. hirta leaves in streptozotocin-induced experimental diabetes in rats. Oral administration of E. hirta leaves extract (300 mg/kg b.w./rat/day) for a period of 30 days indicated the antidiabetic nature of the leaves extract. Determination of the lipid peroxides, hydroperoxides, and both enzymatic and non-enzymatic antioxidants evidenced the antioxidant potential of the leaves extract. Assay of enzymes such as serum aspartate transaminase (AST), serum alanine transaminase (ALT) and serum alkaline phosphatase (ALP) revealed the non-toxic nature of E. hirta leaves. The hypoglycemic activity of the leaves extract was comparable with gliclazide, a standard reference drug.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 278-285.

 
  Study of membrane attachment and in vivo co-localization of TerB protein from uropathogenic Escherichia coli KL53
Olga Alekhina 1), Lenka Valkovicova, Jan Turna

1)Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia. turna@fns.uniba.sk.


The tellurite resistance operon has been found in a wide range of bacteria. We have previously identified the ter operon (terXYW and terZABCDEF) of the uropathogenic strain Escherichia coli KL53. In this study, we use an innovative approach to identify putative protein-protein interaction partners for one of the essential tellurite resistance proteins – TerB. We observe that N-terminus of TerB attaches to the periplasmic membrane, while the C-terminus is partly localized in the cytoplasm. Subsequently, by methods of in vivo cross-linking and mass-spectroscopic analysis, we have determined the proteins from both the membrane and cytoplasmic fractions, which can potentially interact with TerB.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 286-292.

 
  The effects of vitamin C and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart
Vladimir Jakovljevic 1), Dusica Djordjevic, Dragan Djuric

1)Department of Physiology, Faculty of Medicine, University of Kragujevac, Serbia. drvladakgbg@yahoo.com.


The aim of this study was to assess the effects of vitamin C (ascorbic acid) on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by Nω-nitro-L-arginine monomethyl ester (L-NAME) in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180–200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40–120 cm H2O). Coronary flow, nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were determined. The experiments were performed during control conditions and in presence of vitamin C (100 µM) alone or vitamin C (100 µM) + L-NAME (30 µM). Administration of vitamin C induced only increase of nitrite levels, while vitamin C + L-NAME induced significant decrease of coronary flow above autoregulatory range, i.e. especially at higher coronary perfusion pressure (CPP) values, accompanied with similar dynamic in nitrite outflow. Vitamin C + L-NAME also induced significant decrease in TBARS production. The results of our study show no significant effects of vitamin C administration either on ROS levels or on coronary flow in isolated rat heart.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 293-300.

 
  Biophoton emissions from cell cultures: biochemical evidence for the plasma membrane as the primary source
Blake Dotta 1), Carly Buckner, Dianne Cameron, Robert Lafrenie, Michael Persinger

1)Department of Biology, Laurentian University, Sudbury, Ontario, Canada. mpersinger@laurentian.ca.


Photon emissions were measured at ambient temperature (21°C) in complete darkness once per min from cultures of 106 cells during the 12 h following removal from 37°C. The energy of emission was about 10–20 J/s/cell. Of 8 different cell lines, B16-BL6 (mouse melanoma cells) demonstrated the most conspicuous emission profile. Acridine orange and ethidium bromide indicated the membranes were intact with no indication of (trypan blue) cell necrosis. Treatments with EGF and ionomycin produced rapid early (first 3 h) increases in energy emission while glutamine-free, sodium azide and wortmanin-treated cells showed a general diminishment 3 to 9 h later. The results suggested the most probable origin of the photon emission was the plasma cell membrane. Measures from cells synchronized at the M- and S-phase supported this inference.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 301-309.

 
  Functional changes in the septal GABAergic system of animals with a model of temporal lobe epilepsy
Anton Malkov 1), Irina Popova

1)Laboratory of System Organization of Neurons, Institute of Theoretical and Experimental Biophysics of RAS, Pushchino, 142290 Moscow region, Russia. malkovae@gmail.com.


The septal GABAergic system plays a central role in the regulation of activity and excitability of the hippocampus (the main locus of temporal lobe epilepsy, TLE), but the character of changes the septum undergoes in this pathology remains unknown. To address this issue we studied the influences on GABAergic receptors in septal slices from the brain of epileptic guinea pigs compared to a control. In the epileptic brain, the overall increase in the mean frequency of neuronal discharges and the rise in the number of bursting neurons were revealed. The inhibitory action of exogenously applied GABA on neuronal activity is sharply enhanced, whereas the efficacy of action of GABAA and GABAB receptor blockers decreases, indicating the alteration of intraseptal inhibitory processes in epilepsy. In epilepsy, GABA sharply increases the oscillatory activity of the part of pacemakers, and the opposite effect was observed in the control. In epileptic animals, the GABA receptor blockers did not affect burst neurons, indicating the disturbance of the tonic GABAergic control of the oscillatory activity. Thus, we demonstrated for the first time that the activity of septal neurons and their reactions to GABAergic substances in animals with TLE model changed sharply compared to healthy ones.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 310-320.

 
  The effect of chronic food restriction on immunopositive ACTH cells in peripubertal female rats
Verica Milošević 1), Vladimir Ajdžanović, Desanka Bogojević, Ivana Medigović, Svetlana Ivanović-Matić, Vesna Martinović , Ilijana Grigorov

1)Institute for Biological Research “Siniša Stanković”, University of Belgrade,Belgrade, Serbia. dimi@ibiss.bg.ac.rs.


In peripubertal female rats, we have previously found that 50% food restriction (FR) increases plasma IL-6, haptoglobin and both alanine transaminase (ALT) and alkaline phosphatase (AST) aminotransferases, indicating the existence of an inflammatory response. To study whether such FR influences the hypothalamic-pituitary-adrenal (HPA) axis, we examined by immunohistochemistry the morphofunctional features of pituitary adrenocorticotroppic (ACTH) cells. In FR rats the volume and volume density of ACTH cells as well as plasma ACTH levels were increased by 17.6%, 12.5% and 13.4%, respectively, in comparison with controls (p<0.05). We concluded that chronic FR is a systemic stressor in young females, capable to stimulate the HPA axis, probably as a result of IL-6 action.

General Physiology and Biophysics. Volume 30, 2011, No. 3: 321-324.