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General Physiology and Biophysics

Volume 36, 2017, No. 5


  Antioxidative effects of diallyl trisulfide on hydrogen peroxide-induced cytotoxicity through regulation of nuclear factor-E2-related factor-mediated thioredoxin reductase 1 expression in C2C12 skeletal muscle myoblast cells
Ji Sook Kang 1), Gi-Yiung Kim, Byung Woo Kim, Yung Hyun Choi

1)Anti-Aging Research Center and Blue-Bio Industry RIC, Dongeui University, 176 Eomgwangno Busanjin-gu, Busan 614-714, Republic of Korea.

Diallyl trisulfide (DATS) is one of the major sulfur-containing compounds in garlic oil. In this study, we analyzed the effects of DATS against hydrogen peroxide (H2O2)-induced oxidative stress in C2C12 myoblasts. DATS preconditioning significantly attenuated H2O2-induced growth inhibition and DNA damage, as well as apoptosis by decreasing the generation of ROS. Treatment with DATS alone effectively upregulated the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and thioredoxin reductase 1 (TrxR1), which was associated with the increased phosphorylation of Nrf2. However, the protective effects of DATS against H2O2-induced growth reduction and ROS accumulation were significantly abolished by auranofin, an inhibitor of TrxR activity. Moreover, DATS-mediated phosphorylation of Nrf2 and induction of TrxR1 were markedly reduced by genetic silencing of Nrf2. DATS treatment also induced the phosphorylation extracellular signal-regulating kinase (ERK), and analysis using specific inhibitors of cellular signaling pathways demonstrated that only ERK activation was involved in Nrf2 phosphorylation and TrxR1 induction. In addition, the cytoprotective potentials were abrogated in C2C12 cells pretreated with an ERK specific inhibitor. The results demonstrate that DATS protects against oxidative stress-induced DNA damage and apoptosis in C2C12 cells in part through the activation of Nrf2-mediated TrxR1 induction via the ERK signaling pathway.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 129-139.

  Comparative molecular dynamics study of dimeric and monomeric forms of HIV-1 protease in ligand bound and unbound state
Monikaben Padariya 1), Umesh Kalathiya

1)Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdansk University of Technology, Narutowicza St 11/12, 80-233 Gdansk, Poland.

Human immunodeficiency virus type 1 protease is a viral-encoded enzyme and it is essential for replication and assembly of the virus. Inactivation of HIV-1 protease causes production of immature, noninfectious viral particles and thus HIV-1 protease is an attractive target in anti-AIDS drug design. In our current work, we performed molecular dynamics (MD) calculations (500 ns) for two different ligands (COM5 – designed in our previous study, and Darunavir) and made effort to understand dynamics behaviour of our designed compound COM5. An apo form of HIV-1 protease as monomer and dimer form was also studied in order to analyze response of protein to the ligand. MD results suggest that presence of ligand in hinders the stability of HIV-1 protease and one monomer from dimer systems is dominant on other monomer in terms of interaction made with ligands. We were able to trace functional residues as well as continuous motion of opening and closing (clapping) of flap region in HIV-1 protease (apo form) during entire 1000 ns of MD simulation. COM5 showed almost similar behaviour towards HIV-1 protease enzyme as Darunavir and propose as promising lead compound for the development of new inhibitor for HIV-1 protease.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 141-154.

  Effects of heat and freeze on isolated erythrocyte submembrane skeletons
Ivan Ivanov 1), Boyana Paarvanova, Veselin Ivanov, Kathrin Smuda, Hans Bäumler, Radostina Georgieva

1)Department of Physics and Biophysics, Medical Faculty of Thracian University, Stara Zagora 6000, Bulgaria.

In this study we heated insoluble residues, obtained after Triton-X-100 (0.1 v/v%) extraction of erythrocyte ghost membranes (EGMs). Specific heat capacity, electric capacitance and resistance, and optical transmittance (280 nm) sustained sharp changes at 49°C (TA) and 66°C (TC), the known denaturation temperatures of spectrin and band 3, respectively. The change at TA was selectively inhibited by diamide (1 mM) and taurine mustard (1 mM) while its inducing temperature was selectively decreased by formamide in full concert with the assumed involvement of spectrin denaturation. In the residues of EGMs, pretreated with 4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid (DIDS), the change at TC was shifted from 66 to 78°C which indicated the involvement of band 3 denaturation. The freeze and rapid thaw of EGM residues resulted in a strong reduction of cooperativity of band 3 denaturation while the slow thaw completely eliminated the peak of this denaturation. These effects of freeze-thaw were prevented in residues obtained from DIDS-treated EGMs. The freeze-thaw of residues slightly affected spectrin denaturation at 49°C although an additional denaturation appeared at 55°C. The results indicate preserved molecular structure and dynamics of the membrane skeleton in Triton-X-100 extracts of EGMs. The freeze-thaw inflicted strong damage on band 3 and spectrin-actin skeleton of EGM extracts which is relevant to cryobiology, cryosurgery and cryopreservation of cells.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 155-165.

  Salicylic acid and ascorbic acid retrieve activity of antioxidative enzymes and structure of Caralluma tuberculata calli on PEG stress
Riaz Rehman 1), Muhammad Zia, Muhammad Chaudhary

1)Horticulture and Floriculture Institute, Government of Punjab, Rawalpindi, Pakistan.

Biochemical adaptations and morphological changes are cellular aptitude originated on biotic and abiotic stresses. Polyethylene glycol (PEG) induces drought stress in the nutrient solution. In the present investigation, Caralluma tuberculata calli is exposed to PEG and antioxidative molecules. By increasing the level of antioxidative enzymes (SOD, POD, CAT, APX, and GR), the PEG-stressed calli falls off upon exposure to non-enzymatic antioxidants (ascorbic acid and salicylic acid). Under PEG-stress, several cellular and sub-cellular changes such as alteration in plasma membrane thickness, change in nucleus shape, increase in nucleoli, deformation of thylakoid membranes, and increase in plastoglobuli are observed through electron microscopic images. From our results we conclude that application of PEG (a drought causative agent) leads to an increase in the level of antioxidative enzymes and also deformation of cellular organelles. However, application of ascorbic acid and salicylic acid eradicate drought effect induced by PEG.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 167-174.

  Glutathione is the main endogenous inhibitor of protein glycation
Sabina Galiniak 1), Grzegorz Bartosz, Izabela Sadowska-Bartosz

1)Department of Biochemistry and Cell Biology, Faculty of Biology and Agriculture, University of Rzeszów, Rzeszów, Poland.

Glycation is the cause of diabetic complications and contributes to the development of other diseases and aging. Numerous exogenous compounds have been tested for their anti-glycating activity. The aim of this study was to answer the question, which endogenous compounds at physiological concentrations can effectively prevent glycation. A set of endogenous compounds has been tested for the ability to protect albumin from glucose-induced glycation in vitro at a concentration of 1 mM and in a physiological concentration range. Only glutathione was found to protect significantly against glycation at physiological concentrations. Glutathione depletion increased the rate of hemoglobin glycation in erythrocytes incubated with high glucose concentrations. These results indicate that the level of glutathione is the main determinant of glycation of intracellular proteins.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 175-186.

  Novel seleno-hydantoin palladium(II) complex – antimigratory, cytotoxic and prooxidative potential on human colon HCT-116 and breast MDA-MB-231 cancer cells
Marko Živanović 1), Jelena Košarić, Biljana Šmit, Dragana Šeklić, Radoslav Pavlović, Snežana Marković

1)Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac, Serbia.

Selenium and palladium containing compounds separately exert multifunctional effects on cells. While selenium containing compounds usually exert antioxidative properties, palladium(II) containing compounds are cytotoxic and prooxidative. Here we investigated biological effects of bicyclic seleno-hydantoin cis-7a-ethyl-5-methyl-5-phenylselanylmethyl-tetrahydro-pyrrolo[1,2-c]imidazole-1,3-dione (Hid-Se), and its palladium(II) complex, trans-bis-(cis-7a-ethyl-5-methyl-5-phenylselanylmethyl-tetrahydro-pyrrolo[1,2-c]imidazole-1,3-dionato) palladium(II) chloride ((Hid-Se)2Pd) on human colon HCT-116 and breast MDA-MB-231 cancer cell lines. Hid-Se and (Hid-Se)2Pd showed prooxidative and cytotoxic character. In all performed experiments (Hid-Se)2Pd proved to be more active, i.e. this substance exerted greater prooxidative effect, cytotoxicity and influence on cell migration potential. Even though Hid-Se and (Hid-Se)2Pd enhanced migration of HCT-116 cells, very important feature of these substances is the strong antimigratory potential on metastatic MDA-MB-231 cells.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 187-196.

  Molecular crowding has no effect on the dilution thermodynamics of the biologically relevant cation mixtures
Daria Głogocka 1), Magdalena Przybyło, Marek Langner

1)Department of Biomedical Engineering, Faculty of Fundamental Problems of Technology, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.

The ionic composition of intracellular space is rigorously maintained in the expense of high-energy expenditure. It has been recently postulated that the cytoplasmic ionic composition is optimized so the energy cost of the fluctuations of calcium ion concentration is minimized. Specifically, thermodynamic arguments have been produced to show that the presence of potassium ions at concentrations higher than 100 mM reduce extend of the energy dissipation required for the dilution of calcium cations. No such effect has been measured when sodium ions were present in the solution or when the other divalent cation magnesium was diluted. The experimental observation has been interpreted as the indication of the formation of ionic clusters composed of calcium, chloride and potassium. In order to test the possibility that such clusters may be preserved in biological space, the thermodynamics of ionic mixtures dilution in solutions containing albumins and model lipid bilayers have been measured. Obtained thermograms clearly demonstrate that the energetics of calcium/potassium mixture is qualitatively different from calcium/sodium mixture indicating that the presence of the biologically relevant quantities of proteins and membrane hydrophilic surfaces do not interfere with the properties of the intracellular aqueous phase.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 197-204.

  The first Slovak Legius syndrome patient carrying the SPRED1 gene mutation
Martina Sekelska 1), Lenka Briatkova, Tomas Olcak, Anna Bolcekova, Denisa Ilencikova, Ludevit Kadasi, Andrea Zatkova

1)Department of Molecular Biology, Comenius University, Faculty of Natural Sciences, Mlynska dolina, Ilkovicova 6, 842 15 Bratislava, Slovakia.

Autosomal dominant disorder Legius syndrome (NF1- like syndrome) shows phenotype features that overlap with neurofibromatosis type 1 (NF1), such as CALMs, freckling, macrocephaly and learning disability. Mutation analysis provides an important tool in order to distinguish two entities that have different clinical implications. We analyzed SPRED1 gene by cDNA and/or gDNA sequencing in a cohort of 46 Slovak patients in whom previously NF1 mutation was excluded. In one case we identified a nonsense mutation c.46C>T (p.Arg16*) in exon 2 of SPRED1 gene, confirming diagnosis of Legius syndrome. This mutation was reported previously.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 205-210.

  Mitochondrial hyperpolarization and cytochrome-c release in microwave-exposed MCF-7 cells
Meric Esmekaya 1), Ayşe Canseven, Handan Kayhan, Mehmet Tuysuz , Bahriye Sirav, Nesrin Seyhan

1)Department of Biophysics, Faculty of Medicine, Gazi University, Ankara, Turkey.

This study examines the effects of a 2.1-GHz WCDMA-modulated microwave (MW) radiation on apoptotic activity and mitochondrial membrane potential (ΔΨm) in MCF-7 cells. The cells were exposed to the MW at a specific absorption rate (SAR) of 0.528 W/kg for 4 or 24 h. The antiproliferative effect of MW exposure was determined by the MTT test. Cytochrome-c and p53 levels were determined by an ELISA method. The relative ΔΨm was analysed by JC-1 staining using flow cytometer. Apoptotic rate of the cells was measured by Annexin-V-FITC staining. All assays were performed after certain time of incubations (15 min–4 h) following MW exposure. MW-exposed cells showed a significant decrease in viability when compared to unexposed cells. A significantly larger decrease was observed after longer exposure. The percentage of apoptotic cells, amount of cytochrome-c, and relative ΔΨm were significantly higher in MW-exposed cells. The percent of apoptotic cells and relative ΔΨm in 24 h MW-exposed group was significantly higher than those in 4 h MW-exposed group. However, no significant change was observed in p53 levels. These results demonstrated that exposure to 2.1-GHz WCDMA-modulated MW radiation caused hyperpolarization of mitochondria that in turn induced apoptosis in MCF-7 cells.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 211-218.

  Inhibition of Kupffer cell functions modulates arsenic intoxication in Wistar rats
Oluyomi Adeyemi 1), Ekong Meyakno, Musbau Akanji

1)Medicinal Biochemistry and Toxicology Laboratory, Department of Biological Sciences, Landmark University, Omu-Aran, Nigeria.

Study determined the influence of the inhibition of Kupffer cell functions by GdCl3 in arsenic intoxication. Twenty-four Wistar rats weighing between 150 and 160 g were randomly assigned into four groups. Group 1 received sodium arsenite (1.5 mg/kg b.w.) once a day, Group 2 received GdCl3 (2 mg/kg b.w.) once, 24 hours before commencing the arsenite (1.5 mg/kg b.w.) treatment. Group 3 received GdCl3 (2 mg/kg b.w.) once and subsequently given distilled water. Group 4 received distilled water only. The treatments were daily by oral gavage and lasted for 28 days. Animals were euthanized 24 hours after the last treatment. Arsenic exposure elevated the activities of rat plasma AST, ALT, ALP and γ-GT, indicative of liver injury. Arsenic exposure in rat lowered GSH concentration but potentiated inflammation and oxidative stress evidenced in the raised levels of MPO, NO and MDA. Rats with arsenic exposure were predisposed to atherosclerosis, lowering the HDL-C but elevated the LDL-C concentration. The histopathological assessment showed degenerating cellular lesion caused by arsenic. However, the inhibition of Kupffer cell functions by GdCl3 suppressed arsenic intoxication improving the liver function indices, oxidative stress status, lipid profile, neutrophilic inflammation and ultimately restored the cellular architecture. Data suggest that specific inhibition of Kupffer cells by GdCl3 protected against arsenic intoxication.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 219-227.

  Unequal size of ions in modified Wicke-Eigen model of electric double layer
Ekaterina Gongadze 1), Veronika Kralj-Iglič, Aleš Iglič

1)Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Tržaška cesta 25, SI-1000 Ljubljana, Slovenia.

Numerous electric double layer models have been developed through the years to capture the complex electrode/electrolyte interface. In the present study, the Wicke-Eigen model of electric double layer is generalized to incorporate the asymmetric size of cations and anions. Analytical expressions for the spatial distribution of ions and water dipoles are derived. Asymmetric and symmetric Wicke-Eigen models are analysed. Arguments are given in favour of changing the recently adopted name of so-called Bikerman model/equation to Wicke-Eigen model/equation.

General Physiology and Biophysics. Volume 36, 2017, No. 5: 229-234.