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General Physiology and Biophysics

Volume 27, 2008, No. 4


  Quantum dots versus organic fluorophores in fluorescent deep-tissue imaging – merits and demerits
Rumiana Bakalova 1), Zhivko Zhelev, Veselina Gadjeva

1)Department of Biophysics, Molecular Imaging Center, National Institute of Radiological Sciences, 4-91 Anagawa, Inage-kun, Chiba-shi, Chiba 263-8555, Japan.

The use of fluorescence in deep-tissue imaging is rapidly expanding in last several years. The progress in fluorescent molecular probes and fluorescent imaging techniques gives an opportunity to detect single cells and even molecular targets in live organisms. The highly sensitive and high-speed fluorescent molecular sensors and detection devices allow the application of fluorescence in functional imaging. With the development of novel bright fluorophores based on nanotechnologies and 3D fluorescence scanners with high spatial and temporal resolution, the fluorescent imaging has a potential to become an alternative of the other non-invasive imaging techniques as magnetic resonance imaging, positron-emission tomography, X-ray, computing tomography. The fluorescent imaging has also a potential to give a real map of human anatomy and physiology. The current review outlines the advantages of fluorescent nanoparticles over conventional organic dyes in deep-tissue imaging in vivo and defines the major requirements to the "perfect fluorophore". The analysis proceeds from the basic principles of fluorescence and major characteristics of fluorophores, light-tissue interactions, and major limitations of fluorescent deep-tissue imaging. The article is addressed to a broad readership – from specialists in this field to university students.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 231-242.

  Gastric relaxation induced by electrical and chemical stimulation of the area postrema in the rat
Masanao Kawachi 1), Nobuaki Hori, Mineo Takei, Tadashi Kurimoto, Norio Akaike, Yushi Ito

1)Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.

Area postrema (AP) is considered to be an important neural center for emesis in carnivores. However, it is also known that AP mediates motor responses induced by apomorphine in rats which do not have an emetic reflex. To shed more light on the possible role of AP in the control of gastric motility in physiological or pathophysiological conditions, we observed the effects of electrical or chemical (apomorphine) stimulation of AP neurons on intragastric pressure (IGP) or intragastric volume (IGV) in rat. We found that electrical stimulation (ES) reduces IGP, and this is sensitive to hexamethonium or L-NAME, and apomorphine also reduces IGP and increases IGV. In slice preparations, apomorphine (10 µmol/l) increased the frequency of spontaneous single unit discharges of AP neurons recorded extracellularly. We also succeeded retrograde labeling of AP neurons by DiI applied into the gastric corpus, for the first time. These observations indicate that rat stomach receives efferent neural input from AP and the excitation of AP neurons relaxes the stomach in rat, suggesting some functional roles of AP neurons in the regulation of gastric motility.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 243-252.

  Characterization and regulation of basal calcium influx in human peripheral blood lymphocytes
Boris Lakatoš 1), Jana Slováková, Karin Kaiserová, Jozef Orlický, Ľudovít Varečka

1)Department of Biochemistry and Microbiology, Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia.

The basal 45Ca2+ influx into resting human blood lymphocytes was measured. This process showed biphasic kinetics with first rapid phase followed by the second long-lasting and markedly slower phase. Further, it showed signs of saturability and reaches maximal values at 37°C and extracellular pH 7.2. The basal 45Ca2+ influx was stimulated by addition of submicromolar concentrations of 4β-phorbol-12-myristate-13-acetate, and this effect was abolished by protein kinase C (PKC) inhibitor Ro-31-8220. In the regulation of basal 45Ca2+ influx is probably only partially involved adenylate cyclase pathway as show results with intracellular c-AMP elevating agents (dB-c-AMP, 3-isobutyl-1-metylxantine and forskolin). Uncoupler 3,3',4',5-tetrachloro-salicylanilide (TCS) in micromolar concentrations stimulated basal 45Ca2+ influx and its effect was more significant in media with high extracellular concentration of K+.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 253-262.

  Long-term exercise training affects age-induced changes in HSP70 and apoptosis in rat heart
Farhad Soufi 1), Safar Farajnia, Naser Aslanabadi, Naser Ahmadiasl, Mohammadreza Alipour, Mohsen Alipour, Yousef Doustar, Jalal Abdolalizadeh, Farzam Sheikhzadeh

1)Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

The aim of this study was to test the effects of age and long-term exercise training on antioxidant, heat shock protein 70 (HSP70) expression and apoptosis by comparing the hearts of sedentary and trained rats. Training groups went under 3-, 6- and 9-months of regular exercise (25 m/min with a 0% slope, 60 min/day and 6 days/week). Level of glutathione increased with age in trained and sedentary control rats but level of this factor unchanged by training. Activity of mitochondrial superoxide dismutase (mtSOD) increased in heart homogenates of 6- and 9-months trained animals as compared with their sedentary. The rates of apoptosis were increased with age but level of apoptosis in 9-months trained group was significantly lower than corresponding sedentary. Levels of HSP70 expression were significantly decreased with age while long-term training induced marked increase in HSP70 expression levels. These results show that a long-term regular exercise affects age-induced changes in mtSOD, HSP70 and apoptosis as it increases mtSOD activities and HSP70 expression levels and elicits a marked reduction in apoptosis rate in rat myocardium. However, a shorter training program is not effective.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 263-270.

  A circular dichroism study of the stability of guanine quadruplexes of thrombin DNA aptamers at presence of K+ and Na+ ions
Slavomíra Poniková 1), Marian Antalik, Tibor Hianik

1)Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, 842 48 Bratislava, Slovakia.

The effect of K+ and Na+ on the properties of DNA aptamers that selective binds to fibrinogen (FIBRI) and heparin (HEPA) exosites of human α-thrombin was studied by circular dichroism (CD). The complexes of FIBRI-K+ were slightly more stable than HEPA-K+. However, lower stability was observed for HEPA-K+ at presence of Na+ in comparison with FIBRI-K+. The analysis of CD melting curves suggests differences in thermal stability of both aptamers at presence of K+. The melting temperatures (Tm) and changes in van’t Hoff enthalpy for HEPA-K+ complexes were lower in comparison with those for FIBRI-K+. With increasing HEPA concentration the Tm value increased, but Tm did not change with increasing FIBRI concentration. This suggests formation of HEPA aggregates, while FIBRI aptamers are in monomeric form.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 271-277.

  Loss of membrane cholesterol affects lysosomal osmotic stability
Shu-Jing Hao 1), Jun-Fang Hou, Nan Jiang, Guo-Jiang Zhang

1)Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, P. R. China.

Lysosomal destabilization is critical for the organelle and living cells. Using methyl-β-cyclodextrin (MβCD) to selectively deplete lysosomal membrane cholesterol, we investigated the effect of cholesterol on the organelle osmotic stability. The results show that loss of membrane cholesterol caused changes in the lysosomal osmotic properties. The lysosomes lost the ability to resist osmotic shock and became more sensitive to osmotic stress. As a result, the lysosomes lost membrane integrity rapidly. Microscope observation showed that the lysosomes were liable to swell in the hypotonic sucrose medium. It is presumably due to an enhancement of the lysosomal permeability to water caused by the loss of membrane cholesterol. The results indicate an important role of cholesterol in the maintenance of lysosomal stability.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 278-283.

  Transport of lithium across the lamprey (Lampetra fluviatilis) erythrocyte membrane
Gennadii Gusev 1), Natalia Agalakova, Tatiana Ivanova

1)Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr. 44, 194223 St. Petersburg, Russia.

Lithium, capable of replacing Na+ in various membrane transport processes, was used to investigate Na+ transport pathways across the lamprey erythrocytes membrane. The values of Li+ influxes have ranged from 8 to 24 mmol/l cells/h. Intracellular accumulation of Li+ was associated with loss of cellular Na+, the value of which was less than the value of Li+ influx. Both Li+ influx and Na+ efflux were partially inhibited by amiloride. The amiloride-sensitive Li+ influx was considerably stimulated by hyperosmotic cell shrinkage. The treatment of lamprey erythrocytes with blockers of protein phosphatases (fluoride and cantharidin) also resulted in a considerable increase in Li+ accumulation within the cells. No significant difference was observed between the values of Li+ and Na+ (22Na) influxes measured in red cells incubated simultaneously in isotonic LiCl and NaCl media (9.2 ± 2.1 and 7.8 ± 1.3 mmol/l cells/h, respectively). In hypo- and hypertonic media, however, the rate of Na+ influx in lamprey erythrocytes was approximately twice higher as compared to the rate of Li+ influx, what was determined by the difference in the amiloride-sensitive components. In acidified lamprey erythrocytes (intracellular pH 6.0) Li+ and Na+ influxes were considerably increased due to activation of amiloride-sensitive Na+/H+ (Li+/H+) exchange mechanism, although the activity of Na+/H+ exchange was much greater than that of Li+/H+ exchange. The data obtained confirm the hypothesis on the presence of two amiloride-sensitive systems of Na+ transport in the lamprey red blood cells.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 284-290.

  The effect of simvastatin on coenzyme Q and antioxidant/oxidant balance in diabetic-hypercholesterolaemic rats
Magdaléna Kuželová 1), Adriana Adameova, Zuzana Sumbalová, Ingrid Paulíková, Anna Harčárová, Pavel Švec, Jarmila Kucharská

1)Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojárov 10, 832 32 Bratislava, Slovakia.

The effect of simvastatin administered for 10 days on coenzyme Q and antioxidant/oxidant balance in a rat model of diabetes mellitus and hypercholesterolaemia was studied. In the diabetic-hypercholesterolaemic rats the signs of oxidative stress-decreased α-tocopherol/cholesterol in the plasma (p < 0.01) and α-tocopherol in liver (p < 0.001) together with increased lipid peroxidation in the liver (TBARS, p < 0.05) were found. Increased coenzyme Q9 concentrations in the plasma (p < 0.05) and liver (p < 0.01), coenzyme Q10 in the myocardium (p < 0.05) and in the liver (p < 0.01) may indicate adaptation to oxidative stress. Administration of simvastatin (10 mg/kg) to the diabetic-hypercholesterolaemic rats counteracted increased myocardial (coenzyme Q10, p < 0.05) and liver (total coenzyme Q9, p < 0.05) coenzyme Q concentrations but did not improve α-tocopherol depletion and increased formation of TBARS in the liver. Even though simvastatin treatment did not induce coenzyme Q deficiency in plasma, heart and liver of the diabetic-hypercholesterolaemic rats as compared to the control levels, it was not able to prevent completely the changes in antioxidant/oxidant balance induced by diabetes and hypercholesterolaemia. The results highlight the importance of studying the effect of statins on the coenzyme Q levels in the animal models of pathological conditions known to change the initial antioxidant defence system.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 291-298.

  Mutation analysis of the CFTR gene in Slovak cystic fibrosis patients by DHPLC and subsequent sequencing: identification of four novel mutations
Peter Kolesár 1), Gabriel Minarik, Marian Baldovic, Andrej Ficek, László Kovács, Ludevit Kadasi

1)Department of Molecular Biology, Faculty of Natural Science, Comenius University, Bratislava, Slovakia.

Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in Caucasians. Its incidence is approximately 1 : 2500 newborns. CF is caused by mutations in the transmembrane conductance regulator (CFTR) gene, which encodes an important chloride ion channel. The disease affects the respiratory, digestive and reproductive systems. To date more than 1550 mutations and polymorphisms have been identified throughout the CFTR gene, making the DNA diagnosis more difficult. Rapid accurate identification of CFTR gene mutations is important for confirming the clinical diagnosis, for cascade screening in families at risk for CF, for understanding the correlation between genotype and phenotype, and moreover it is also the only means for prenatal diagnosis. Individuals suspect of CF are in Slovakia presently screened for the presence of 30 common mutations, giving mutation detection rate only approximately 48%. To increase the detection rate we applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons. The fragments showing abnormal elution profiles were subsequently sequenced to characterize the DNA change. We have identified a total of 28 different mutations up to present not found in Slovak CF patients, and 17 different polymorphisms. Four mutations (G437D, H954P, H1375N, and 3120+33G>T) are novel, not yet found in any other CF patient all over the word.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 299-305.

  Cytotoxic effects of catechol to neuroblastoma N2a cells
Rute Lima 1), Lisandro Alvarez, Maria Costa, Silvia Costa, Jorge Clarêncio, Ramon El-Bachá

1)Laboratory of Neurochemistry and Cell Biology, Department of Biochemistry and Biophysics, Institute of Health Sciences, Federal University of Bahia, 40110-100 Salvador, Bahia, Brazil.

The mechanisms of catechol-induced cytotoxicity were studied in cultures of neuroblastoma N2a cells. The minimal cytotoxic concentration after 72 h was 20 µmol·l–1. The EC50 after 72 h was 38 µmol·l–1. There was not a correlation between the cytotoxicity and the formation of quinones in the medium. Catechol-induced cytotoxicity was increased significantly when superoxide dismutase (SOD) was added. The addition of catalase did not protect cells, but this enzyme reverted the deleterious effect of SOD. The experimental studies showed a detrimental effect of deferoxamine on catechol-induced cytotoxicity suggesting that cells need iron to maintain its metabolism. NF-κB inhibitors increased the cytotoxicity, suggesting that this factor is also important for cell viability. L-cysteine and N-acetyl-L-cysteine protected cells significantly in a dose-dependent manner. The use of monochlorobimane showed that catechol induced reduced glutathione (GSH) depletion after 24 h, prior to cell death. The mode of cell death was studied by flow cytometry after double staining with annexin V and propidium iodide. Catechol induced apoptosis after 72 h. Furthermore, catechol also induced nuclear fragmentation. These data showed that catechol-induced cytotoxicity to N2a cell was not directly a consequence of reactive oxygen species production. Rather, it was due to GSH depletion followed by the induction of apoptosis.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 306-314.

  Transport of non-electrolyte solutions through membrane with concentration polarization
S&lslash;awomir Grzegorczyn 1), Jolanta Jasik-Ślezak, Katarzyna Michalska-Ma&lslash;ecka, Andrzej Ślęzak

1)Department of Biophysics, Silesia Medical University, H. Jordan Str. 19, 41 808 Zabrze, Poland.

Mathematical model of the volume fluxes through neutral membrane with concentration boundary layers on both sides of this membrane is presented. This model, based on the Kedem-Katchalsky equations, describes the volume flux generated by osmotic and hydrostatic forces for non-homogeneous and non-electrolyte solutions. Nonlinear equation for volume flux was used for numerical calculation in linear regime of hydrodynamic stability. In the steady state of non-homogeneous solutions the dependence of volume flux on pressure difference is shifted with regard to this dependence for homogeneous solution, while the volume flux as a function of osmotic pressure between chambers is characterized by different angle of inclination for homogeneous and non-homogeneous solutions.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 315-321.

  Imaging compaction of single supercoiled DNA molecules by atomic force microscopy
Olga Limanskaya 1), Alex Limanskii

1)Department of New Infectious Diseases, Institute of Microbiology and Immunology, Pushkinskaya St. 14, 61057 Kharkov, Ukraine.

Supercoiled pGEMEX DNA, 3993 bp in length, was immobilized on different substrates (freshly cleaved mica, standard amino mica and modified amino mica with increased hydrophobicity and surface charge density compared with standard amino mica) and was visualized by atomic force microscopy (AFM) in air. Plectonemically supercoiled DNA (scDNA) molecules, as well as extremely compacted single molecules, were visualized on amino-modified mica, characterized by increased hydrophobicity and surface charge density. We show four-fold increase in DNA folding on the mica surface with high positive charge density. This result is consistent with a strongly enhanced molecular flexibility facilitated by shielding of the DNA phosphate charges. The formation of minitoroids with about a 50 nm diameter and molecules in spherical conformation was the final stage of single molecule compaction. A possible model of conformational transitions for scDNA in vitro in the absence of protein is proposed based on AFM image analysis. Compaction of the single scDNA molecules, up to minitoroids and spheroids, appears to be caused by screening of the negatively charged DNA phosphate groups. The high surface charge density from positively charged amino groups on mica, on which DNA molecules were immobilized, is an obvious candidate for the screening effect.

General Physiology and Biophysics. Volume 27, 2008, No. 4: 322-337.