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General Physiology and Biophysics


Volume 35, 2016, No. 3

Content:


  Glycosylation of α2δ1 subunit: a sweet talk with Cav1.2 channels
Joanna Lazniewska 1), Norbert Weiss

1)Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic. weiss@uochb.cas.cz.


Bosentan, an endothelin-1 (ET) receptor antagonist is an important drug for the effective management of patients with pulmonary arterial hypertension. Bosentan has a rather complicated pharmacokinetics in humans involving multiple physiological components that have a profound influence on its drug disposition. Bosentan is mainly metabolized by cytochrome P450 (CYP) 3A4 and 2C9 enzymes with the involvement of multiple transporters that control its hepatic uptake and biliary excretion. The involvement of phase 2 metabolism of bosentan is a key to have an enhanced biliary excretion of the drug-related products. While bosentan exhibits high protein binding restricting the drug from extensive distribution and significant urinary excretion, bosentan induces its own metabolism by an increased expression of CYP3A4 on repeated dosing. Due to the above properties, bosentan has the potential to display drug-drug interaction with the co-administered drugs, either being a perpetrator or a victim. The intent of this review is manifold: a) to summarize the physiological role of CYP enzymes and hepatic-biliary transporters; b) to discuss the mechanism(s) involved in the purported liver injury caused by bosentan; c) to tabulate the numerous clinical drug-drug interaction studies involving the physiological interplay with CYP and/or transporters; d) to provide some perspectives on dosing strategy of bosentan.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 239-242.

 
  Clinical drug-drug interactions of bosentan, a potent endothelial receptor antagonist, with various drugs: Physiological role of enzymes and transporters
Nuggehally Srinivas 1)

1)Suramus Bio, J. P. Nagar, I Phase Bangalore 560078, Karnataka, India. srini.suramus@yahoo.com.


Bosentan, an endothelin-1 (ET) receptor antagonist is an important drug for the effective management of patients with pulmonary arterial hypertension. Bosentan has a rather complicated pharmacokinetics in humans involving multiple physiological components that have a profound influence on its drug disposition. Bosentan is mainly metabolized by cytochrome P450 (CYP) 3A4 and 2C9 enzymes with the involvement of multiple transporters that control its hepatic uptake and biliary excretion. The involvement of phase 2 metabolism of bosentan is a key to have an enhanced biliary excretion of the drug-related products. While bosentan exhibits high protein binding restricting the drug from extensive distribution and significant urinary excretion, bosentan induces its own metabolism by an increased expression of CYP3A4 on repeated dosing. Due to the above properties, bosentan has the potential to display drug-drug interaction with the co-administered drugs, either being a perpetrator or a victim. The intent of this review is manifold: a) to summarize the physiological role of CYP enzymes and hepatic-biliary transporters; b) to discuss the mechanism(s) involved in the purported liver injury caused by bosentan; c) to tabulate the numerous clinical drug-drug interaction studies involving the physiological interplay with CYP and/or transporters; d) to provide some perspectives on dosing strategy of bosentan.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 243-258.

 
  Effects of Cd2+ on the epithelial Na+ channel (ENaC) investigated by experimental and modeling studies
Maria Mernea 1), Roxana Ulăreanu, Octavian Călborean, Sergiu Chira, Octavian Popescu, Dan Mihailescu, Dana Cucu

1)Department of Anatomy, Physiology and Biophysics, Faculty of Biology, University of Bucharest, Bucharest, Romania. dana.cucu@bio.unibuc.ro.


The function of the epithelial Na+ channel from the apical membrane of many Na+ transporting epithelia is modulated by various chemical compounds from the extracellular space, such as heavy metals, protons or chloride ions. We have studied the effect of extracellular Cd2+ on the function of the epithelial Na+ channel (ENaC) in heterologously expressed Xenopus laevis oocytes and Na+-transporting epithelia. We assayed channel function as the amiloride-sensitive sodium current (INa). Cd2+ rapidly and voltage-independently inhibited INa in oocytes expressing αβγ Xenopus ENaC (xENaC). The extracellular Cd2+ inhibited Na+ transport and showed no influence on ENaC trafficking, as revealed by concomitant measurements of the transepithelial current, conductance and capacitance in Na+-transporting epithelia. Instead, amiloride inhibition was noticeably diminished in the presence of Cd2+ on the apical membrane. Using molecular modeling approaches, we describe the amiloride binding sites in rat and xENaC structures, and we present four putative binding sites for Cd2+. These results indicate that ENaC functions as a sensor for external Cd2+.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 259-271.

 
  Ultrastructure of mitochondrial nucleoid and its surroundings
Jarmil Prachař 1)

1)Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovak Republic. jarmil.prachar@savba.sk.


Mitochondrial nucleoids (hereafter nucleoids) contain genetic information, mitochondrial DNA, prerequisite for mitochondrial functioning, particularly information required for mitochondrial electron transport. To understand nucleoid functioning, it is imperative to know its ultrastructure and dynamics in the context of the actual mitochondrial state. In this study, we document the internal structure, different positions of nucleoids inside the mitochondrial tube and their different morphology. The nucleoid cores appear in section as circular or slightly oval objects ranging from 50 to 100 nm in diameter. They are mainly located in the matrix between cristae inside the mitochondrial tube but they are also frequently found close to the inner mitochondrial surface. In tightly packed form, their interior exhibits sophisticated nucleoprotein regularity. The core surroundings form an electron-lucent thick layer which is probably partitioned into separate chambers. We suggest that the morphology of nucleoids mirrors the mode of energy production, glycolysis versus oxidative phosphorylation. The new high resolution transmission electron microscopy method enabled us to obtain morphological characteristics on yet unpublished level.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 273-286.

 
  New mechanisms of vesicles migration
Viviana Aursulesei 1), Decebal Vasincu, Daniel Timofte, Lucia Vrajitoriu, Irina Gatu, Dan Iacob, Vlad Ghizdovat, Calin Buzea, Maricel Agop

1)”Grigore T. Popa” University of Medicine and Pharmacy, Medical First Department, 16 University Str., Iaşi - 700115, Romania. m.agop@yahoo.com.


In multicellular organisms, both health and disease are defined by means of communication patterns involving the component cells. Despite the intricate networks of soluble mediators, cells are also programed to exchange complex messages pre-assembled as multimolecular cargo of membranous structures known as extracellular vesicles (EVs). Several biogenetic pathways produce EVs with different properties able to orchestrate neighboring cell reactions or to establish an environment ripe for spreading tumor cells. Such an effect is in fact an extension of similar physiological roles played by exosomes in guiding cell migration under nontumoral tissue remodeling and organogenesis. We start with a biological thought experiment equivalent to Bénard’s experiment, involving a fluid layer of EVs adherent to an extracellular matrix, in a haptotactic gradient, then, we build and present the first Lorenz model for EVs migration. Using Galerkin’s method of reducing a system of partial differential equations to a system of ordinary differential equations, a biological Lorenz system is developed. Such a physical frame distributing individual molecular or exosomal type cell-guiding cues in the extracellular matrix space could serve as a guide for tissue neoformation of the budding pattern in nontumoral or tumoral instances.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 287-298.

 
  The tolerability of dextran-coated iron oxide nanoparticles during in vivo observation of the rats
Cristina Popa 1), Alina Prodan, Carmen Ciobanu, Daniela Predoi

1)National Institute of Materials Physics, P.O. Box MG 07, 07725, Magurele, Romania. dpredoi@gmail.com.


Superparamagnetic iron oxide nanoparticles (SPION) have attracted a lot of interest due to their widespread biomedical and diagnostic applications. Coating the SPIONs with various surface layers can provide an interface between the core and the surrounding environment. The aim of this study was to examine the in vivo behaviour of dextran-coated iron oxide nanoparticles (D-IONPs) in aqueous suspensions. The SPIONs stabilized with dextran (D-IONPs) were synthesized in aqueous solutions by co-precipitation method. The average grain size deduced from transmission electron microscopy is 7.5 nm. The hematological parameters registered for the rats exposed to D-IONPs at 1 ml/kg have had values approximately equal to those examined for the control specimen. The architecture of liver and kidneys was not affected after one day of intraperitoneal injection of D-IONPs compared to the reference group. After 21 and 28 days respectively from the administration of the D-IONPs solution, the liver and kidneys from the injected rats showed a normal aspect without abnormalities compared to the rats uninjected. Our findings suggest that the administration of 1 ml/kg D-IONPs did not cause any toxicological effect since the parameters of renal and liver function were in the normal range as reported to the control group.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 299-310.

 
  Assessment of DNA damage in Ehlrich carcinoma after treatment with doxorubicin encapsulated in nanoscales thermosensitive liposomes in combination with localized hyperthermia
Monira Rageh 1), Medhat Shafaa, Mona Elhefnawy, Mohamed El-Nagdy

1)Biophysics Department, Faculty of Science, Cairo University, Cairo, Egypt. medhatwi@hotmail.com.


Nanoscales thermosensitive liposomes (TSL) composed of synthetic lipids (dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine), were used for doxorubicin encapsulation with 70% encapsulated efficiency. The liposomes were characterized by dynamic light scattering, transmission electron microscopy and turbidity method. Additionally, the liposomes exhibited a significant release of doxorubicin (Dox) by 60% within 5 min at 42°C. To assess the therapeutic efficacy of Dox in combination with hyperthermia, Dox free and encapsulated TSL were administered directly to Ehrlich tumor bearing mice at 1 mg/kg dose. Immediately after the drug administration, hyperthermia was applied to mention the temperature inside the tumor site at 42°C either for 5 min and 30 min. The results indicate a significant increase in the percent of apoptotic and necrotic cells in the treated group. Moreover, disrupts the integrity and the amount of intact DNA in tumor cells. In conclusion, Dox and hyperthermia may serve as a useful targeted drug delivery system for management of Ehrlich carcinoma.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 311-322.

 
  Fructus sophorae attenuates secretion of proinflammatory mediators and cytokines through the modulation of NF-κB and MAPK signaling pathways in LPS-stimulated RAW 264.7 macrophages
Yung Hyun Choi 1), Hye-Joo Kang

1)Department of Biochemistry, Dongeui University College of Korean Medicine, 52-57, Yangjeong-ro, Busanjin, Busan 614-052, Republic of Korea. choiyh@deu.ac.kr.


This study investigated the inhibitory effects and underlying mechanisms of fructus sophorae, the dried ripe fruit of Styphnolobium japonicum (L.) Schott, on the production of proinflammatory molecules in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The results indicated that pretreatment with noncytotoxic concentrations of fructus sophorae extract (FSE) significantly inhibited the release of the proinflammatory mediators nitric oxide (NO) and prostaglandin E2, which were associated with the downregulation of both mRNA and protein for inducible NO synthase and cyclooxygenase-2, respectively, in LPS-challenged RAW 264.7 cells. FSE also blocked the LPS-induced expression of the proinflammatory cytokines interleukin (IL)-6 and IL-1β. Furthermore, the results showed that FSE efficiently attenuated the LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB) and phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but not p38 MAPK. These results suggest that FSE exhibits anti-inflammatory activity by inhibiting proinflammatory mediators and cytokines through the inactivation of NF-κB, ERK and JNK, and it may offer therapeutic potential for treating inflammatory diseases accompanied with macrophage activation.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 323-331.

 
  Novel SCN1A variants in Dravet syndrome and evaluating a wide approach of patient selection
Milan Surovy 1), Andrea Soltysova, Miriam Kolnikova, Pavol Sykora, Denisa Ilencikova, Andrej Ficek, Jan Radvanszky, Ludevit Kadasi

1)Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina Bratislava, 842 15 Slovak Republic. milan.surovy015@gmail.com.


Voltage-gated sodium channels are essential for generation and propagation of the action potential mainly in nerve and muscle cells. Causative variants in SCN1A gene which codes the main, pore-forming subunit of the channel expressed in central nervous system are associated predominantly with Dravet syndrome (DS), as well as with generalized epilepsy with febrile seizures plus (GEFS+) making it one of the most significant epilepsy gene. Our goal was to determine whether SCN1A screening is relevant in patients with a broad range of epileptic syndromes. 52 patients diagnosed with DS, generalized epilepsy with GEFS+ or similar types of epileptic syndromes were included. Sequencing of the protein coding parts of the gene complemented with MLPA analysis was carried out. One already described nonsense variant, four novel protein truncating variants and a deletion encompassing the whole SCN1A gene were revealed, all in heterozygous state. All identified variants were found in DS patients with 85.7% sensitivity, thus supporting the role of profound SCN1A gene variants in etiology of DS phenotype. No causative variants were identified in any of non-DS epileptic patients in our cohort, suggesting a minor, but not irrelevant role for SCN1A in patients with other types of childhood epilepsy.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 333-342.

 
  Gender and chronological age affect erythrocyte membrane oxidative indices in citrate phosphate dextrose adenine-formula 1 (CPDA-1) blood bank storage condition
Hayriye Erman 1), Uğur Aksu, Ahmet Belce, Pınar Atukeren, Duygu Uzun, Tamer Cebe, Ahmet Kansu, Remisa Gelişgen, Ezel Uslu, Seval Aydın, Ufuk Çakatay

1)Department of Medical Biochemistry, Faculty of Medicine, Istanbul Medeniyet University, Goztepe Training and Research Hospital, Istanbul, Turkey. cakatay@yahoo.com.


It is well known that in vitro storage lesions lead to membrane dysfunction and decreased number of functional erythrocytes. As erythrocytes get older, in storage media as well as in peripheral circulation, they undergo a variety of biochemical changes. In our study, the erythrocytes with different age groups in citrate phosphate dextrose adenine-formula 1 (CPDA-1) storage solution were used in order to investigate the possible effect of gender factor on oxidative damage. Oxidative damage biomarkers in erythrocyte membranes such as ferric reducing antioxidant power, pro-oxidant-antioxidant balance, protein-bound advance glycation end products, and sialic acid were analyzed. Current study reveals that change in membrane redox status during blood-bank storage condition also depends on both gender depended homeostatic factors and the presence of CPDA-1. During the storage period in CPDA-1, erythrocytes from the male donors are mostly affected by free radical-mediated oxidative stress but erythrocytes obtained from females are severely affected by glyoxidative stress.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 343-351.

 
  Could nitric oxide be a mediator of action of oxytocin on myocardial injury in rats? (Biochemical, histological and immunohistochemical study)
Noha Hussien 1), Ayman Mousa

1)Department of Physiology, Benha Faculty of Medicine, Benha University, Cairo, Egypt. drnohaibrahim79@gmail.com.


Oxytocin (OT) was revisited recently as a hormone of cardiovascular system with several new functions in cardiovascular regulation. But less is known about its role in acute myocardial injury (MI). The aim of our study was to investigate the possible protective effect of OT on the biochemical, histological and immunohistochemical changes of MI induced by isoprenaline (ISO) in adult male albino rats and studying the possible role of nitric oxide (NO) in its action. Forty male albino rats were divided into 5 groups: control rats (Group I), acute MI rats (Group II), rats pretreated with OT prior to induction of MI (Group III), rats injected with a combination of OT and atosiban (ATO, OT receptor antagonist) prior to induction of MI (Group IV). In Group V, a combination of OT and nitric oxide synthase inhibitor (L-NAME) were injected to the rats prior to induction of MI. The heart wall in all groups were taken and processed for histological, immunohistochemical, morphometrical and biochemical studies. We concluded that OT has antioxidant, anti-inflammatory and anti-apoptotic effects on MI and its effects is mediated through NO.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 353-362.

 
  Methionine-choline deprivation alters liver and brain acetylcholinesterase activity in C57BL6 mice
Danijela Vučević 1), Ivana Cerović, Dusan Mladenovic, Milena Vesković, Ivana Stevanović, Bojan Jorgačević, Rada Ješić Vukićević, Tatjana Radosavljevic

1)Institute of Pathophysiology "Ljubodrag Buba Mihailović", Faculty of Medicine, University of Belgrade, Dr Subotića 9, 11000 Belgrade, Serbia. danibovuc@med.bg.ac.rs.


Choline and methionine are precursors of acetylcholine, whose hydrolysis is catalyzed by acetylcholinesterase (AChE). Considering the possibility of their common deficiency, we investigated the influence of methionine-choline deprivation on AChE activity in liver and various brain regions (hypothalamus, hippocampus, cerebral cortex and striatum) in mice fed with methionine-choline deficient (MCD) diet. Male C57BL/6 mice (n = 28) were randomly and equally divided into following groups: control group fed with standard diet for 6 weeks (C) and groups fed with MCD diet for 2 weeks (MCD2), 4 weeks (MCD4) and for 6 weeks (MCD6). After the diet, mice were sacrificied and AChE activity in liver and brain was determined spectrophotometrically. Hepatic AChE activity was higher in MCD2, MCD4 and MCD6 compared to control (p < 0.01), with most prominent increase in MCD6. AChE activity in hypothalamus was higher in MCD4 and MCD6 vs. control (p < 0.05 and p < 0.01, respectively), as well as in MCD6 compared to MCD4 (p < 0.01). In hippocampus, increase in AChE activity was shown in MCD6 compared to control (p < 0.01). In cortex and striatum, increase in AChE activity was noted in MCD6 compared to control (p < 0.05). Our findings indicate the increase of hepatic and brain AChE activity in mice caused by methionine-choline deprivation.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 363-370.

 
  Effect of noise stress on cardiovascular system in adult male albino rat: implication of stress hormones, endothelial dysfunction and oxidative stress
Mona Said 1), Ola El-Gohary

1)Physiology Department, Faculty of Medicine, Benha University, Benha, Qalubia, Egypt. dr.monaabdelazim@gmail.com.


Noise pollution has been realized as an environmental stressor associated with modern life style that affects our health without being consciously aware of it. The present study investigated the effect of acute, chronic intermittent and chronic continuous exposure to noise of intensity 80–100 dB on heart rate and mean systemic arterial blood pressure in rats and the possible underlying mechanisms. Noise stress causes significant increase in heart rate, mean systemic arterial blood pressure as well as significant increase in plasma levels of corticosterone, adrenaline, noradrenaline, endothelin-1, nitric oxide and malondialdehyde with significant decrease in superoxide dismutase and these values are significantly more worse in chronic continuous exposure to noise than acute or chronic intermittent exposure. These findings suggest that noise stress has many adverse effects on cardiovascular system via increasing plasma levels of stress hormones, oxidative stress and endothelial dysfunction. These findings have major implication in the management of adverse cardiovascular reactions of people subjected to daily noise stress.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 371-377.

 
  Adipogenesis and aldosterone: a study in lean tryptophan-depleted rats
Michal Pokusa 1), Natasa Hlavacova, Agnesa Csanova, Michael Franklin, Štefan Zórad, Daniela Jezova

1)Laboratory of Pharmacological Neuroendocrinology and Laboratory of Metabolic Regulations, Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovak Republic. michal.pokusa@savba.sk.


Next to epithelial tissues, mineralocorticoid receptors are also expressed in adipose tissue and are involved in the process of adipogenesis. Mineralocorticoid receptors in adipose tissue are likely to be activated mainly by glucocorticoids. The aim of the present study was to test the hypothesis that the processes related to adipogenesis are modified under the conditions associated with high circulating aldosterone. We have made advantage of a model of depression based on tryptophan depletion in which we have previously demonstrated that the elevation of serum aldosterone precedes that of corticosterone. Sixty adult female Sprague-Dawley rats were fed either a low tryptophan diet or control diet for 4 (elevation of aldosterone only), 7 and 14 days (broader neuroendocrine activation) respectively. Gene expression of several adipogenic factors, CD31, interleukin-6, adiponectin, resistin and leptin were evaluated. Levels of mRNAs coding for adipogenic, angiogenic and inflammatory factors in adipose tissue were elevated at 4 and 7 days of tryptophan depletion. Additionally, gene expression of aldosterone sensing 11-β-hydroxysteroid dehydrogenase 2 and mineralocorticoid receptors were elevated. All changes disappeared at 14 days of tryptophan depletion. Synchronously an increase of adipose tissue mass was observed. Although direct evidence is not provided, observed changes in gene expression may be related to the action of aldosterone on mineralocorticoid receptors. Our findings represent the first data on any changes in gene expression in adipose tissue in animal models of depression

General Physiology and Biophysics. Volume 35, 2016, No. 3: 379-386.

 
  A comparative study of protein patterns of human estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines
Dana Flodrova 1), Lucia Toporova, Dana Macejova, Marketa Lastovickova, Július Brtko, Janette Bobalova

1)Institute of Analytical Chemistry of the CAS, v. v. i., Veveří 97, 602 00 Brno, Czech Republic. flodrova@iach.cz.


In the present study, we analyzed the cell lysates of human tumour cell lines representing two major clinically different types of breast cancer. Our main goal was to show the differences between them on proteomic level. Gel electrophoresis followed by MALDI-TOF MS analysis was used for proteins determination. Exactly 98 proteins were unequivocally identified and 60 of them were expressed differentially between MDA-MB-231 and MCF-7 cell lines. Among the proteins reported here, some well-known breast cancer markers (e.g., annexin A1, annexin A2 and vimentin) were identified in the MDA-MB-231 cell line and thus we were able to distinguish both cell lines sufficiently.

General Physiology and Biophysics. Volume 35, 2016, No. 3: 387-392.