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General Physiology and Biophysics

Volume 32, 2013, No. 4


  Characterization of membrane-bound fatty acid desaturases
Tatiana Klempova 1), Daniel Mihalik, Milan Certik

1)Department of Biochemical Technology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinskeho 9, 812 37 Bratislava, Slovak Republic.

Membrane-bound desaturases play key role in metabolism of polyunsaturated fatty acids. Characterization of these enzymes and their genes is the first step in basic understanding of their proper functioning in living cells as well as in tailor-made preparation of highly-specific and highly-productive strains of microorganisms interesting for applied biotechnology. It is also the crucial step in creation of transgenic agricultural crops with enhanced content of individual polyunsaturated fatty acids. Properties and applications of identified membrane-bound desaturases genes and enzymes are discussed in this review.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 445-458.

  Genetic aspects of vitamin D receptor and metabolism in relation to the risk of multiple sclerosis
Lucia Krizova 1), Branislav Kollar, Daniela Jezova, Peter Turčáni

1)1st Department of Neurology, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic.

Recent findings suggest that polymorphisms in vitamin D pathway genes are candidates for association with multiple sclerosis susceptibility. It has been now well demonstrated that vitamin D has immunomodulatory functions that may be favorable for reduction of multiple sclerosis risk. Current research has been focused on identification of new variants of genes involved in vitamin D pathway, namely in vitamin D receptor and enzymes of vitamin D metabolism. These variants have been intensively studied as possible genetic predictors of both vitamin D levels and the risk of multiple sclerosis. Considering the findings available up-to-date, we may recognize two groups of genetic variants. The first group of genes was found to predict vitamin D levels but not the risk of multiple sclerosis. The second group of genetic variants is represented by promising genes predicting vitamin D levels as well as the risk of multiple sclerosis. A strong association with increased risk of the disease has been observed for a rare variant in the CYP27B1 gene encoding a vitamin D-activating enzyme. Observed interaction between genetic and epidemiological findings brings the rationale for supplementation trials of vitamin D. Although promising effects of vitamin D supplementation have emerged, the results obtained so far are inconclusive and the real therapeutic significance of vitamin D supplementation remains to be elucidated.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 459-466.

  Nuclear receptors – target molecules for isoflavones in cancer chemoprevention
Lucia Bialešová 1), Július Brtko, Vladimír Lenko, Dana Macejova

1)Laboratory of Molecular Endocrinology, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovak Republic.

Breast cancer is the most occurring type of cancer among women. In Slovakia, there are yearly diagnosed about 1900 new cases of this disease. Breast cancer treatment is very expensive, psychic stressful and in some cases ineffective. Therefore, it is essential to search for new and/or alternative methods for breast cancer treatment, since nuclear receptors are considered to be a central goal for maximizing treatment opportunities in breast cancer. Among natural ligands for estrogen receptors (ERα and ERβ), which are member of nuclear receptors superfamily, belongs also isoflavones. These natural compounds have similar structure to main female hormon-17β estradiol. A rich source of isoflavones is soy and its products. Three aglycones form of isoflavones (genistein, daidzein, glycitein) are predominantly found in soybean and red clover. Among other important isoflavones belongs also biochanin A and formononetin.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 467-478.

  Interactions of y+LAT1 and 4F2hc in the y+l amino acid transporter complex: consequences of lysinuric protein intolerance-causing mutations
Minna Toivonen 1), Maaria Tringham, Johanna Kurko, Perttu Terho, Olli Simell, Kaisa Heiskanen, Juha Mykkänen

1)Department of Medical Biochemistry and Genetics, Institute of Biomedicine, University of Turku, Turku, Finland.

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by recessive mutations in the SLC7A7 gene encoding y+L amino acid transporter 1 (y+LAT1), which combines with 4F2hc to generate an active transporter responsible for the system y+L amino acid transport. We have previously shown that the y+LAT1 proteins with point mutations are expressed in the plasma membrane, while those with frameshift mutations are retained in the cytoplasm. This finding has prompted us to study whether the difference in localization is due to the inability of the structurally altered mutant y+LAT1 proteins to heteromerize with 4F2hc. For this purpose, we utilized FACS technique to reveal fluorescence resonance energy transfer (FRET) in cells expressing wild type or LPI-mutant CFP-tagged y+LAT1 and YFP-tagged 4F2hc. The heteromerization of y+LAT1 and 4F2hc within the cell is not disrupted by any of the tested LPI mutations. In addition, the expression rate of the LPI mutant y+LAT1 proteins was significantly lower and cellular mortality was markedly increased than that of the wild type y+LAT1 in transfected samples. Our results indicate that the FACS-FRET method provides an alternative approach for screening of potential protein associations.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 479-488.

  Effects of the blood components on the AMPA and NMDA synaptic responses in brain slices in the onset of hemorrhagic stroke
Anatoly Mokrushin 1), Larisa Pavlinova

1)Laboratory of Regulation of Neuron Function, I. P. Pavlov Institute of Physiology of the Russian Academy of Sciences, St-Petersburg, Russia.

Blood-borne events play a major role in post bleeding disturbances of the neuronal network. However, very little is known about the early effects of blood plasma, leucocytes, and the red blood cells on the AMPA and NMDA-mediated synaptic responses in the onset of experimental intracranial hemorrhage (ICH). In this study, we used the technique of on-line monitoring of electrophysiological parameters referred to synaptic activity in piriform cortex of SHR rat slice. We exposed the olfactory cortex slices to diluted autologous blood or its components and compared with effects of ferric chloride. Whole blood exerted a total inhibition of synaptic activity in piriform cortex within first 5 min. Dilution of blood induced prolonged epileptic synaptic activation of NMDA receptors. Blood plasma and fraction of leucocytes induced hyperactivation of neurons transforming to epileptiform discharges. Fraction of red blood cells acted biphasic, an initial sharp activity of AMPA- and NMDA-mediated receptors replaced by a following total depression. Our slice-based models of experimental stroke revealed the mechanism of the earliest pathophysiologic events occur in brain tissue during bleeding that may be relevant to the human ICH.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 489-504.

  ABT-737 accelerates butyrate-induced death of HL-60 cells. Involvement of mitochondrial apoptosis pathway
Andrea Štefaniková 1), Katarína Kliková, Jozef Hatok, Peter Racay

1)Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic.

The aim of presented study was to determine effect of NaBu in combination with ABT-737 on cell survival of leukemic cell line HL-60. In addition, analysis of molecular mechanism of NaBu action with a focus on mitochondrial apoptosis was performed. Both NaBu and ABT-737 are inducing death of HL-60 cells with different kinetics. ABT-737-induced cell death is fast while NaBu-induced death preceded by cell cycle arrest in G2 phase is rather slow. Cell viability preceded by cell cycle arrest in G2 phase was significantly decreased after 48 hours of incubation with 2 and 5 mmol/l of NaBu while it was significantly decreased after 24 hours of incubation with 1 μmol/l of ABT-737 combined with 2 and 5 mmol/l of NaBu. Incubation of HL-60 cells with NaBu was associated with increased level of pro-apoptotic protein BIMEL and decreased levels of anti-apoptotic proteins of Bcl-2 family as well as GRP78 involved in ER stress signalling. It seems that ABT-737 accelerates NaBu-induced death of HL-60 cells due to mitochondrial apoptosis resulting from ABT-737-mediated inhibition of functions and NaBu-induced decrease of the levels of anti-apoptotic Bcl-2 family proteins as well as due to accelerated decrease of GRP78 observed after the treatment of cells with combination of NaBu and ABT-737. The effect of combination of both drugs on survival of HL-60 cells seems to be synergistic at high concentrations of NaBu (2 and 5 mmol/) while it is rather antagonistic at concentrations of NaBu less than 1 mmol/l. Finally, it might be assumed that NaBu is capable to induce cell death with mechanisms independent from mitochondrial apoptosis.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 505-516.

  Suppression of exaggerated neuronal oscillations by oxytocin in a rat model of Parkinson’s disease
Oytun Erbas 1), Fatih Oltulu, Dilek Taskiran

1)Department of Physiology, Ege University School of Medicine, 35100 Izmir, Turkey.

Increased oscillatory activity has been demonstrated in the basal ganglia of Parkinson’s disease (PD) patients. The aim of the present study was to evaluate the effects of oxytocin on local field potentials (LFPs) in a rotenone-induced rat model of PD. Adult male Sprague-Dawley rats were unilaterally injected with rotenone (3 µg/µl in DMSO) into the left substantia nigra pars compacta whereas vehicle group was received only DMSO. PD developed rats were then administered either oxytocin (160 µg/kg/day, i.p.) or saline for three weeks. Following treatment period, LFPs were recorded from the left striatum of freely moving rats and neuronal cell loss was evaluated by Nissl staining. We found significant increase in all frequency bands except delta in saline group when compared with vehicle (p < 0.0005), while treatment of oxytocin prevented these alterations in EEG recordings. Besides, histopathological evaluation of the striatal sections revealed a significant cell loss (p < 0.005), whereas administration of rats with oxytocin significantly lessened the neuronal death. These findings suggest that injury of dopaminergic neurons triggers exaggerated neuronal oscillations in the striatum and oxytocin may have some inhibitory effects on neuronal activity in PD.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 517-525.

  Attenuated vascular responsiveness to K+ channel openers in diabetes mellitus: the differential role of reactive oxygen species
Daniel Owu 1), Nelson Orie, Chukwuemeka Nwokocha, Morris Muzyamba, Lucie Clapp, Eme Osim

1)Department of Physiology, College of Medical Sciences, University of Calabar, Calabar, Nigeria.

The current study examined the responsiveness of blood vessels from diabetic rats to K+ channel openers and explored whether ROS might be involved in any changes. Responses were measured in aortic rings isolated from four weeks streptozotocin (65 mg/kg)-induced diabetic rats. Relaxation to levcromakalim (ATP-sensitive potassium channel KATP opener, 10-9-10-5 mol/l) and (+/-)-naringenin (large conductance calcium–activated channel BKCa opener, 10-8-10-3 mol/l) were recorded in phenylephrine (1 µmol/l) pre-contracted segments in the absence and presence of superoxide dismutase (SOD, 100 µmol/l) and apocynin (an antioxidant and inhibitor of NADPH oxidase, 100 µmol/l). Contractions to phenylephrine (10-9-10-5 mol/l) and relaxation to acetylcholine (ACh, 10-9-10-5 mol/l) were also recorded. Relaxation curves for levcromakalim, naringenin and ACh for the diabetic group were shifted to the right (p < 0.05) compared with the control. Contractions to phenylephrine were enhanced in the diabetic group (p < 0.01). SOD restored the ACh response but not those of K+ channel openers. On the other hand, apocynin restored the relaxation to naringenin but had no effect on both levcromakalim and ACh responses. The results suggest that both KATP and BKCa activities are attenuated in diabetes mellitus and that ROS appears to contribute only to the change in BKCa function.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 527-534.

  Quercetin attenuates oxidative stress in the blood plasma of rats bearing DMBA-induced mammary cancer and treated with a combination of doxorubicin and docetaxel
Sabina Tabaczar 1), Anna Pieniążek, Jan Czepas, Joanna Piasecka-Zelga, Krzysztof Gwoździński, Aneta Koceva-Chyła

1)Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Łódź, Poland.

The development of side-effects during doxorubicin-docetaxel (DOX-DTX) chemotherapy is considered as related to generation of oxidative stress by DOX. The addition of docetaxel potentiates this effect. Thus, antioxidants are assumed as a promising remedy for neutralizing deteriorating effects of reactive oxygen species (ROS) in pathological conditions and polyphenolic antioxidants are suitable candidates for such a therapeutic approach. We evaluated the ability of quercetin to attenuate oxidative stress developed during the process of DMBA carcinogenesis and DOX-DTX chemotherapy in the blood plasma of rats bearing mammary tumors. We have found that quercetin significantly improved the plasma nonenzymatic antioxidant capacity (NEAC) and reduced lipid peroxidation, which suggest the beneficial effect of flavonoid. The inclusion of quercetin to the DOX-DTX chemotherapy was also advantageous. A considerable decrease of carbonyls and lipid peroxidation products (TBARS) and improvement of the endogenous antioxidant defense system (an increase of NEAC, thiols and SOD activity) were observed compared to rats treated with DOX-DTX chemotherapy. These results suggest that quercetin could protect blood plasma constituents against oxidative damage evoked by DOX and DTX.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 535-543.

  The phenomenon of synaptic vesicle clustering as the prefusion state in the model system of exocytosis
Vitaliy Gumenyuk 1), Alexander Chunikhin, Nina Himmelreich, Irene Trikash

1)Department of Neurochemistry, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, 9 Leontovich str., 01601, Kiev, Ukraine.

Our findings concern to the synaptic vesicle interactions that were reconstructed in the cell-free system and are thought to represent the different states of exocytosis pathway. The combination of different technical approaches allowed to study the features of aggregation and calcium-dependent homotypic fusion of synaptic vesicles. Electron microscopy observations of synaptic vesicle fraction purified from the rat brain showed the appearance of large particles formed by aggregated synaptic vesicles in the presence of the nerve terminal cytosolic proteins only. This data were confirmed by dynamic light scattering measurements indicating an importance of the cytosolic proteins for the formation of synaptic vesicle clusters. The scanning confocal microscopy and imaginative exploitation of fluorescence probe R18 allowed to distinguish the process of synaptic vesicle clustering from the synaptic vesicle fusion. The stimulating effect of antiepileptic drug, ethosuximide and sodium valproate on the formation of synaptic vesicle aggregates has been revealed. Experiments with the removal of cholesterol showed that such modification of synaptic vesicle membranes did not change the ability of synaptic vesicles to form the clusters, reducing their Ca2+-triggered membrane fusion. Thus, our data have shown that aggregated state of synaptic vesicles represent an intermediate stage of the fusion pathway, where aggregation of synaptic vesicles is preceded by Ca2+-dependent membrane fusion.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 545-558.

  Comparative spectroscopic studies on liposomes containing chlorophyll a and chlorophyllide a
Sanja Milenkovic 1), Marcela Bărbînţă-Pătraşcu, Gabriel Baranga, Dejan Markovic, Laura Ţugulea

1)Faculty of Technology, University of Niš, Leskovac, Serbia.

Chlorophyll a (Chla) and chlorophyllyde a (Chlida) - a derivative of Chla, have been incorporated in the lipid bilayers of two types of liposomes, small unilamellar vesicles (SUV) and multilamelar vesicles (MLV). The objective of the present work was to compare the spectral behaviour of Chla and Chlida incorporated in the lipid bilayers and their sensing behaviour at molecular level. The VIS absorption and fluorescence emission presented differences depending on the type of liposomes and inserted pigment, reflecting the different localization of porphyrin macrocycle in the lipid moieties. The temperature dependence of emission anisotropy and fluorescence intensity, for both Chla and Chlida incorporated in DPPC SUV, revealed the presence of different lipid phases. The degree of incorporation of quercetin (QCT) in liposome membrane was studied by using Chla and Chlida as molecular sensors. The fluorescence polarisation data and the fluorescence quenching process provided arguments for the insertion of the QCT at the interface lipid/water, in the vicinity of lipid polar heads and porphyrin macrocycle. The phytyl chain of Chla penetrating in the hydrophobic core of the lipid bilayers is responsible for the observed differences among Chla and Chlida in sensing the lipid phase transition and the fluorescence quenching process induced by QCT.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 559-567.

  Physiological regulation of pro-inflammatory cytokines expression in rat cardiovascular tissues by sympathetic nervous system and angiotensin II
Houcine Dab 1), Rafik Hachani, Mohsen Sakly, Giampiero Bricca, Kamel Kacem

1)Unité de Physiologie Intégrée, Laboratoire de Pathologies Vasculaires, Université de Carthage, Faculté des Sciences de Bizerte, Tunisia.

Pro-inflammatory cytokines regulation by sympathetic nervous system (SNS) and angiotensin II (ANG II) was widely described in cardiovascular system, but the role of such neuro-humoral interaction needs further investigation in this context. We tested SNS-ANG II interaction on IL-6 and TNF-α mRNA expression in left ventricle and aorta from normotensive rats by sympathectomy with guanethidine and blockade of the ANG II AT1 receptors (AT1R) antagonist with losartan. mRNA synthesis of IL-6 and TNF-α were performed by Q-RT-PCR. In the left ventricle, IL-6 mRNA increased by 63% (p < 0.01) after sympathectomy, still unchanged after losartan treatment and decreased by 38% (p < 0.05) after combined treatment. TNF-α mRNA decreased by 44% (p < 0.01), only after combined treatment. In the aorta, IL-6 mRNA increased equally by 65% (p < 0.05) after sympathectomy or losartan treatment. TNF-α mRNA decreased by 28, 41, and 42% (p < 0.05) after sympathectomy, losartan and combined treatments, respectively. Our data suggest that ANG II stimulates directly (via AT1R) and indirectly (via SNS) IL-6 mRNA synthesis in left ventricle and aorta and TNF-α mRNA in left ventricle. ANG II seems unable to influence directly TNF-α mRNA synthesis in the aorta but can stimulate this cytokine via SNS. The results are relevant to prevent or reduce proinflammatory cytokines overexpression seen in cardiovascular diseases.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 569-575.

  Protective effects of Artemisia campestris upon fenthion-induced nephrotoxicity in adult rats and their progeny
Mediha Sefi 1), Afef Troudi, Fatma Hamida, Nejla Soudani, Tahia Boudawara, Najiba Zeghal

1)Animal Physiology Laboratory, UR/08-73, Department of Life Sciences, University of Sfax, Sciences Faculty of Sfax, Sfax, Tunisia.

The purpose of this study was to assess the possible protective effects of Artemisia campestris against fenthion-induced nephrotoxicity in adult rats and their progeny. Fenthion was administered orally at a dose of 551 ppm, which represented ¼ of LD50, for 21 consecutive days to pregnant and lactating rats. Oxidative stress was monitored in the kidney by measuring malondialdehyde (MDA), GSH levels, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase activities (GPx). Fenthion caused a significant induction of oxidative damage in kidney as evidenced by increased MDA levels from 5.32 ± 0.47 nmol/100 mg tissue to 11.72 ± 0.83 nmol/100 mg tissue for pups and from 5.18 ± 0.45 nmol/100 mg tissue to 10.84 ± 1.67 nmol/100 mg tissue for dams (p < 0.001). A significant increase (p < 0.001) in the activities of SOD, CAT and GPx was observed. Co-administration of Artemisia c. at a dose of 5% (w/w) in the diet of fenthion-treated rats showed a significant reno-protection against fenthion-induced cytotoxic effects. It could be concluded that Artemisia c. is promising as a protective agent against nephrotoxicity during the exposure to fenthion.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 577-588.

  Aquaporin, forward osmosis and biomimetic membranes
Nikolai Kocherginsky 1)

1)Biomime, Urbana, Illinois, USA.

Aquaporin attracted attention not only of physiologists and biophysicists, but also of chemical engineers. Here we critically analyze a paper describing aquaporin-based artificial membranes, suggested for forward osmosis-based water purification (Wang et al. 2012, Small 8, pp. 1185–1190). Related papers published later by the same group are also discussed. We indicate recently developed general approach to describe membrane transport, membrane permeability and selectivity, which is applicable for forward osmosis. In addition, we also mention our papers describing simple nitrocellulose-based membranes, which have selective aqueous channels without proteins, but successfully imitate many properties of biomembranes.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 589-594.

  Reply from Honglei Wang, Tai-Shung Chung, Yen Wah Tong, Wenyuan Xie and Fang He
Honglei Wang 1), Tai-Shung Chung, Yen Tong, Wenyuan Xie, Fang He

1)Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 119260, Singapore.

Kocherginsky presented a critical analysis of our article (published in Small 8, 2012, pp. 1185–1190) in his paper “Aquaporin, forward osmosis and biomimetic membranes” (in Gen. Physiol. Biophys. 4, 2013, pp. 589–594). However, after a detailed analysis of his arguments, it is easy to recognize that Kocherginsky did not understand the permeability of aquaporin-based biomimetic membranes nor the membrane structures in our article. It is clear that Kocherginsky’s interpretation of our article was based on wrong calculations and assumptions that are not justified. Herein, the main points mentioned in Kocherginsky’s paper are summarized and a point-by-point response detailed showing how the errors have arisen.

General Physiology and Biophysics. Volume 32, 2013, No. 4: 595-596.