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General Physiology and Biophysics

Volume 33, 2014, No. 4


  The crystal structure of oxy hemoglobin from high oxygen affinity bird emu (Dromaius novaehollandiae)
Mohamed Mohamed Abubakkar 1), Kadhirvel Saraboji, Mon Nanjappa Ponnuswamy

1)Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India..

Hemoglobin is an honorary enzyme, a two-way respiratory carrier, transporting oxygen from the lungs to the tissues and facilitating the return transport of carbon dioxide. Hemoglobin has high affinity for oxygen and low affinity for carbon dioxide and other substances in the arterial circulation, whereas in the venous circulation these relative affinities are upturned. The oxygen affinity of hemoglobin increases with the fall in temperature and decreases with the increase in pH and 2, 3-bisphosphoglycerate; point mutations also affect the tetrameric arrangement and alter the oxygen affinity. Though several studies have revealed the specific reasons for the adaptation of increased oxygen affinity of avian hemoglobins at high-altitudes, further structural insights on hemoglobins from high oxygen affinity species are required to understand the detailed oxygen adaptation at the molecular level. Herein, we describe the structural investigation of hemoglobin from emu (Dromaius novaehollandiae), a high oxygen affinity bird. Hemoglobin from emu was purified using anion-exchange chromatography, crystallized and determined the structure in the oxy form at a resolution of 2.3 Å; the R-factor of the model was 19.2%. The structure was compared with other oxy hemoglobins of high oxygen affinity avian species; significant changes are noted at intra-subunit contacts which provide the clues for increased oxygen affinity of emu hemoglobin.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 373-382.

  Laser interferometric analysis of glucose and sucrose diffusion in agarose gel
Sławomir Wąsik 1), Michał Arabski, Kazimierz Dworecki, Joanna Janoska, Jacek Semaniak, Karol Szary, Andrzej Ślęzak

1)Institute of Physics, Jan Kochanowski University in Kielce, Świętokrzyska 15, 25-406 Kielce, Poland.

The paper presents the investigation results of glucose and sucrose diffusion in agarose gel studied with laser interferometry method and the results of fluorescence analysis of the macroscopic gel structure. The diffusion kinetics of these substances released from aqueous solutions of a molar concentration of 0.05 M into the agarose solutions of concentrations of 0.5% and 3% in two gravitational configurations of measuring system was analysed. In the first configuration the solute diffused according, whereas in the second one – opposite to the gravitational force. The diffusion was analysed in the time period between 120 and 2400 s with a time interval of Δt = 120 s. We observed that the convective instabilities were damped well by the agarose gel, which gives the possibility of the interferometric studies of the diffusive transport for other substances in different gravitational configurations of the system. The time characteristics of glucose and sucrose fluxes in both configurations of the system and the gravitational polarisation coefficient values were obtained. The substantial differences in fluxes of glucose and sucrose diffused according and opposite to the gravitational force were observed. Additionally, we observed the differences between the diffusive fluxes of these substances in both configurations in dependence on the gel solution concentration (which is associated with gel porosity dependent on its concentration) and the kind of diffused substance.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 383-391.

  Biotransformation and nitroglycerin-induced effects on antioxidative defense system in rat erythrocytes and reticulocytes
Snežana Marković 1), Nataša Đorđević, Milena Ćurčić, Andraš Štajn, Mihajlo Spasić

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

The effects of nitroglycerin (glyceryl trinitrate – GTN) are mediated by liberated nitric oxide (NO) and formed reactive nitrogen species, which induces oxidative stress during biotransformation in red blood cells (RBCs). The aim of this study was to evaluate effects of GTN on antioxidative defense system (AOS) in rat erythrocytes (without) and reticulocytes (with functional mitochondria). Rat erythrocyte and reticulocyte-rich RBC suspensions were aerobically incubated (2 h, 37°C) without (control) or in the presence of different concentrations of GTN (0.1–1.5 mM). After incubation, concentrations of non-enzymatic components of AOS, activities of antioxidative enzymes and oxidative pentose phosphate (OPP) pathway activity were followed in RBC suspensions. In rat reticulocytes, GTN decreased the activity of mitochondrial MnSOD and increased the activity of CuZnSOD. In rat RBCs, GTN induced increase of Vit E concentration (at high doses), but decreased glutathione content and activities of all glutathione-dependent antioxidative enzymes; the OPP pathway activity significantly increased. GTN biotransformation and induction of oxidative stress were followed by general disbalance of antioxidative capacities in both kinds of RBCs. We suggest that oxidative stress, MnSOD inhibition and depletion of glutathione pool in response to GTN treatment lead to decreased bioavailability of NO after GTN biotransformation in rat reticulocytes.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 393-401.

  Ionizing radiation as preconditioning against transient cerebral ischemia in rats
Natália Kokošová 1), Viera Danielisova, Beňadik Šmajda, Jozef Burda

1)Department of Animal Physiology, Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University, Šrobárova 2, 040 01 Košice, Slovak Republic.

Induction of ischemic tolerance (IT), the ability of an organism to survive an otherwise lethal ischemia, is the most effective known approach to preventing postischemic damage. IT can be induced by exposing animals to a broad range of stimuli. In this study we tried to induce IT of brain neurons using ionizing radiation (IR). A preconditioning (pre-C) dose of 10, 20, 30 or 50 Gy of gamma rays was used 2 days before an 8 min ischemia in adult male rats. Ischemia alone caused the degeneration of almost one half of neurons in CA1 region of hippocampus. However, a significant decrease of the number of degenerating neurons was observed after higher doses of radiation (30 and 50 Gy). Moreover, ischemia significantly impaired the spatial memory of rats as tested in Morris's water maze. In rats with a 50 Gy pre-C dose, the latency times were reduced to values close to the control level. Our study is the first to reveal that IR applied in sufficient doses can induce IT and thus allow pyramidal CA1 neurons to survive ischemia. In addition, we show that the beneficial effect of IR pre-C is proportional to the radiation dose.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 403-410.

  Spoke ring and anchorage of nuclear pore complex revealed by high resolution transmission electron microscopy
Jarmil Prachař 1)

1)Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 83391 Bratislava, Slovak Republic.

Using several different methods, the nuclear pore complex (NPC) was shown to be anchored in the nuclear envelope into the specific curved region, called pore membrane. Three transmembrane nucleoporins in the equatorial region of NPC contain hydrophobic stretches, which exhibit the ability to intersect the phospholipid bilayer. Using transmission electron microscopy, we observed three different evaluable morphological situations in the section through the NPC spoke ring (SR). We suppose that some sections are directed through one type of subunit that is responsible for anchoring. Other sections are directed through the second type of subunit that may provide pore membrane bending. Finally, the spoke ring is sectioned between aforementioned subunits where the pore membrane is best preserved. The proposed anchor is represented by the chains of protein complexes which replace phospholipid bilayer in a relatively large area. Second subunit, presumed bending module is represented by the bundles of chains copying the shape of the pore membrane from the side of the NPC. This work is based on very high resolution resulting in unique and complicated images of tangled and cut off protein chains, nevertheless, it provides insight into how some proteins interact with or replace the membrane.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 411-423.

  Vincristine-induced expression of P-glycoprotein in MOLM-13 and SKM-1 acute myeloid leukemia cell lines is associated with coexpression of nestin transcript
Denisa Imrichova 1), Martina Coculova, Lucia Messingerova, Zdena Sulová, Albert Breier

1)Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlarska 5, 83334, Bratislava Slovak Republic.

Nestin is a class 6 filament protein typically expressed in neural stem/progenitor cells. However, nestin expression has been observed in other tissues during mammalian embryogenesis. In human neural stem/progenitor cells, coexpression of nestin and P-glycoprotein (P-gp, ABCB1 member of the ABC transporter family) was detected. P-gp-mediated drug efflux is the most common molecular cause of multidrug resistance in neoplastic cells. Nestin expression has also been detected in various human solid tumours as well as in the corresponding established cell lines. Interestingly, expression of nestin in different leukemia cells has been recently reported. Here, we show that expression of P-gp is associated with the simultaneous expression of nestin in acute myeloid leukemia cell lines (MOLM-13 and SKM-1) under the selective pressure of vincristine, a substance that may induce P-gp expression in neoplastic cells.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 425-431.

  Gossypol enhances radiation induced autophagy in glioblastoma multiforme
Hoda Keshmiri-Neghab 1), Bahram Goliaei, Alireza Nikoofar

1)Laboratory of Biophysics and Molecular Biology, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran.

Malignant gliomas (glioblastoma multiforme) are the most aggressive of the primary brain tumors. Radiotherapy is an important tool for treatment of cancer but malignant gliomas are usually resistant to radiotherapy and other adjuvant therapies. Thus new drugs are needed to increase the efficiency of radiotherapy in order to improve the therapeutic outcome of tumor patients. Recent investigations showed that gossypol, natural polyphenolic compound produced by cotton plants, is a promising agent against solid tumors. The current study was defined to evaluate whether the combinatorial effect of radiation and gossypol would induce higher level of cell death on U-87 MG than single agent treatment and its possible mechanism of action. Clonogenic survival assay showed that ionizing radiation plus gossypol significantly inhibited clonogenic growth of irradiated cells as compared with either treatment alone. Acridine orange/etidium bromide staining confirmed that there was no significant increase in necrotic and apoptotic cells, but irradiated cells in combination with gossypol showed a significant increase in accumulation of acidic vesicular organelle. The results obtained herein indicated that gossypol is a promising drug that induced autophagic cell death in radioresistant malignant glioma.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 433-442.

  Sodium tungstate alleviates biomechanical properties of diabetic rat femur via modulation of oxidative stress
Baris Donmez 1), Nihal Ozturk, Mehmet Sarikanat, Nurettin Oguz, Ramazan Sari, Semir Ozdemir

1)Department of Nutrition and Dietetics, School of Health, Akdeniz University, 07070, Antalya, Turkey.

Diabetes mellitus leads to bone disorders such as osteopenia and osteoporosis that can increase fracture risk. On the other hand, sodium tungstate is an inorganic compound which exerts anti-diabetic activity in experimental studies due to its suggested insulin-mimetic or antioxidant activity. Therefore this study was designed to investigate the effect of tungstate on bone quality in diabetic rat femurs. The rats were divided into four groups: Control (C), tungstate-treated control (C+Tung), diabetes (STZ-D) and tungstate-treated diabetes (STZ-D+Tung). Diabetes mellitus was induced by single injection of streptozotocin (50 mg/kg). The treated rats received 150 mg/kg/day of sodium tungstate for 12 weeks. Sodium tungstate achieved a little (17%) but significant reduction on blood glucose levels, while it didn’t recover the reduced body weights of diabetic rats. In addition, impaired bone mechanical quality was reversed, despite the unchanged mineral density. Sodium tungstate administration significantly lowered the 2-thiobarbituric acid reactive substances and restored the activity of tissue antioxidant enzymes such as glutathione peroxidase, catalase and superoxide dismutase in diabetic rats. On the other hand, glutathione levels didn’t change in either case. These findings indicate that tungstate can improve the reduced mechanical quality of diabetic rat femurs due probably to reduction of reactive oxygen species and modulation of antioxidant enzymes as well as reduction in blood glucose levels.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 443-452.

  The effect of cupric and ferric ions on antioxidant properties of human serum albumi
Gholamreza Rezaei Behbehani 1), Katayon Gonbadi, Nasrin Eslami

1)Chemistry Department, Imam Khomeini International University, Qazvin, Iran.

The interaction of both ferric (Fe3+) and cupric (Cu2+) ions with human serum albumin (HSA) was assayed at a temperature of 27°C in aqueous solution using isothermal titration calorimetry. The association equilibrium constant and the molar enthalpy for one binding is 1.7×105 M–1 and –31.37 kJ•M–1, respectively. To obtain the binding parameters of metal ion-protein interaction over the whole range of Fe3+ concentrations, the extended solvation model was applied. The solvation parameters obtained from this model were attributed to the structural change of HSA. The binding parameters obtained from the extended solvation model indicate that the stability of HSA was decreased as a result of its binding with ferric ions, which cause dampening the antioxidant property of HSA. Cuperic ion increases the stability of HSA considerably, indicating that the antioxidant property of human serum albumin are increased as a result of its interaction with cupric ion.

General Physiology and Biophysics. Volume 33, 2014, No. 4: 453-456.