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General Physiology and Biophysics


Volume 34, 2015, No. 2

Content:


  Stac gets the skeletal L-type calcium channel unstuck
Norbert Weiss 1)

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


Commentary to: Stac adaptor proteins regulate trafficking and function of muscle and neuronal L-type Ca2+ channels. (Proc. Natl. Acad. Sci. 2015, pp. 602–606)

General Physiology and Biophysics. Volume 34, 2015, No. 2: 101-103.

 
  Emerging evidence for specific neuronal functions of auxiliary calcium channel α2δ subunits
Stefanie Geisler 1), Clemens Schöpf, Gerald Obermair

1)Division of Physiology, Department of Physiology and Medical Physics, Medical University Innsbruck, 6020 Innsbruck, Austria. Gerald.Obermair@i-med.ac.at.


In nerve cells the ubiquitous second messenger calcium regulates a variety of vitally important functions including neurotransmitter release, gene regulation, and neuronal plasticity. The entry of calcium into cells is tightly regulated by voltage-gated calcium channels, which consist of a heteromultimeric complex of a pore forming α1, and the auxiliary β and α2δ subunits. Four genes (Cacna2d1-4) encode for the extracellular membrane-attached α2δ subunits (α2δ-1 to α2δ-4), out of which three isoforms (α2δ-1 to -3) are strongly expressed in the central nervous system. Over the years a wealth of studies has demonstrated the classical role of α2δ subunits in channel trafficking and calcium current modulation. Recent studies in specialized neuronal cell systems propose roles of α2δ subunits beyond the classical view and implicate α2δ subunits as important regulators of synapse formation. These findings are supported by the identification of novel human disease mutations associated with α2δ subunits and by the fact that α2δ subunits are the target of the anti-epileptic and anti-allodynic drugs gabapentin and pregabalin. Here we review the recently emerging evidence for specific as well as redundant neuronal roles of α2δ subunits and discuss the mechanisms for establishing and maintaining specificity.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 105-118.

 
  Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres
Ivan Husu 1), Melinda Magyar, Tibor Szabó, Béla Fiser, Enrique Gómez-Bengoa, László Nagy

1)Department of Chemistry, University of Rome „La Sapienza”, Rome, Italy..


Many herbicides employed in agriculture and also some antibiotics bind to a specific site of the reaction centre protein (RC) blocking the photosynthetic electron transport. Crystal structures showed that all these compounds bind at the secondary ubiquinone (QB) site albeit to slightly different places. Different herbicide molecules have different binding affinities (evaluated as inhibition constants, KI, and binding enthalpy values, ΔHbind). The action of inhibitors depends on the following parameters: (i) herbicide molecular structure; (ii) interactions between herbicide and quinone binding site; (iii) protein environment. In our investigations KI and ΔHbind were determined for several inhibitors. Bound herbicide structures were optimized and their intramolecular charge distributions were calculated. Experimental and calculated data were compared to those available from databank crystal structures. We can state that the herbicide inhibition efficiency depends on steric and electronic, i.e. geometry of binding with the protein and molecular charge distribution, respectively. Apolar bulky groups on N-7 atom of the inhibitor molecule (like t-buthyl in terbutryn) are preferable for establishing stronger interactions with QB site, while such substituents are not recommended on N-8. The N-4,7,8 nitrogen atoms maintain a larger electron density so that more effective H-bonds are formed between the inhibitor and the surrounding amino acids of the protein.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 119-133.

 
  Interaction of cysteine-rich cationic antimicrobial peptides with intact bacteria and model membranes
Krisztina Nagy 1), Kata Mikuláss, Attila Végh, Attila Kereszt, Éva Kondorosi, György Váró, Zsolt Szegletes

1)Institute of Biophysics, Biological Research Centre of Hungarian Academy of Sciences, Temesvári krt. 62., Szeged, Hungary. nagy.krisztina@brc.mta.hu.


Antimicrobial peptides are small proteins that exhibit a broad spectrum of antimicrobial activity. Their chemical structure allows them to interact (attach and insert) with membranes. The fine details about this interaction and their mode of action are not fully clarified yet. In order to better understand this mechanism, we have performed in situ atomic force microscopy studies using two types of nodule specific cysteine-rich NCR peptides on Escherichia coli bacteria and on natural purple membrane. On intact bacteria, both NCR247 and NCR335 caused increase in the surface roughness, indicating the damage of the bacterial cell envelope. In case of the tightly packed purple membrane, it is clear that the peptides prefer to disrupt the border of the disks indicating a strong lipid preference of the interaction. These results verify the concept that the first target of NCR peptides is probably the bacterial cell envelope, especially the lipid matrix.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 135-144.

 
  Pressure-induced wall thickness variations in multi-layered wall of a pollen tube and Fourier decomposition of growth oscillations
Mariusz Pietruszka 1), Aleksandra Haduch-Sendecka

1)University of Silesia, Faculty of Biology and Environment Protection, Laboratory of Plant Physiology, Katowice, Poland. mariusz.pietruszka@us.edu.pl.


The augmented growth equation introduced by Ortega is solved for the apical portion of the pollen tube as an oscillating volume, which we approach in the framework of a two-fluid model in which the two fluids represent the constant pressure and the fluctuating features of the system. Based on routine Fourier analysis, we calculate the energy spectrum of the oscillating pollen tube, and discuss the resonant frequency problem of growth rate oscillations. We also outline a descriptive model for cell wall thickness fluctuations associated with small, yet regular variations (~ 0.01 MPa) observed in turgor pressure. We propose that pressure changes must lead to the sliding of wall layers, indirectly resulting in a wave of polarization of interlayer bonds. We conclude that pollen tube wall thickness may oscillate due to local variations in cell wall properties and relaxation processes. These oscillations become evident because of low amplitude/high frequency pressure fluctuations δP being superimposed on turgor pressure P. We also show that experimentally determined turgor pressure oscillates in a strict periodical manner. A solitary frequency f0 ≈ 0.066 Hz of these (~ 0.01 MPa in magnitude) oscillations for lily pollen tubes was established by the discrete Fourier transform and Lorentz fit.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 145-156.

 
  CaV1.2 and CaV1.3 L-type calcium channels regulate the resting membrane potential but not the expression of calcium transporters in differentiated PC12 cells
Lucia Lichvárová 1), Ľubica Lacinová

1)Institute of Molecular Physiology and Genetics, Centre of Excellence for Cardiovascular Research, Slovak Academy of Sciences, Bratislava, Slovak Republic..


PC12 cells differentiated under the influence of the neuronal growth factor (NGF) serve as a model of both sympathetic neurons and chromaffin cells. NGF-induced differentiation critically depends on elevated intracellular calcium concentration. Main pathway for Ca2+ entry in excitable cells is represented by voltage-dependent calcium channels including L-type calcium channels (LTCC). We investigated role of CaV1.2 and CaV1.3 LTCC subtypes in NGF-differentiated PC12 cells. The expression of LTCC subtypes was downregulated by transfection of NGF-differentiated PC12 cells with siRNA for either CACNA1C or CACNA1D gene. Efficiency of gene silencing was verified by RT-PCR and by functional essay. The dominant LTCC subtype in PC12 cells was CaV1.2. Downregulation of either LTCC significantly hyperpolarized the resting membrane potential. Expression of mRNA for intracellular calcium transporters inositol trisphosphate receptor type 1, 2 and 3, ryanodine receptor type 1 and 2 and sarco/endoplasmic reticulum Ca2+ ATPase type 2 as well as plasma membrane transporters Na+-Ca2+ exchanger type 1 and 2 was not altered in the absence of either LTCC subtype. In conclusion, Ca2+ influx through CaV1.2 or to CaV1.3 channel subtypes contributes to maintenance of the resting membrane potentials of NGF-differentiated PC12 cells but is not required for regulation of expression of genes for calcium-transporting proteins.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 157-165.

 
  The effect of long-term administered CRAC channels blocker on the functions of respiratory epithelium in guinea pig allergic asthma model
Martina Sutovska 1), Michaela Kocmálová, Marta Joskova, Marián Adamkov, Soňa Fraňová

1)Department of Pharmacology, Jessenius Faculty of Medicine, Comenius University, Sklabinska Street 26, 036 01 Martin, Slovak Republic. sutovska@jfmed.uniba.sk.


Previously, therapeutic potency of CRAC channels blocker was evidenced as a significant decrease in airway smooth muscle hyperreactivity, antitussive and anti-inflammatory effects. The major role of the respiratory epithelium in asthma pathogenesis was highlighted only recently and CRAC channels were proposed as the most significant route of Ca2+ entry into epithelial cells. The aim of the study was to analyse the impact of long-term administered CRAC channels blocker on airway epithelium, e.g. cytokine production and ciliary beat frequency (CBF) using an animal model of allergic asthma. Ovalbumin-induced allergic airway inflammation of guinea pigs was followed by long-term (14 days lasted) therapy by CRAC blocker (3-fluoropyridine-4-carboxylic acid, FPCA). The influence of long-term therapy on cytokines (IL-4, IL-5 and IL-13) in BALF and in plasma, immunohistochemical staining of pulmonary tissue (c-Fos positivity) and CBF in vitro were used for analysis. Decrease in cytokine levels and in c-Fos positivity confirmed an anti-inflammatory effect of long-term administered FPCA. Cytokine levels in BALF and distribution of c-Fos positivity suggested that FPCA was a more potent inhibitor of respiratory epithelium secretory functions than budesonide. FPCA and budesonide reduced CBF only insignificantly. All findings supported CRAC channels as promising target in the new strategy of antiasthmatic treatment.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 167-176.

 
  Protective effect of allicin against glycidamide-induced toxicity in male and female mice
En-Ting Wang 1), Dong-yan Chen, Huang-you Liu, Hai-Yang Yan, Yuan Yuan

1)College of Quartermaster Technology, Jilin University, 130062 Changchun, China. yuanyuan1024@gmail.com.


Acrylamide is known to be a neurotoxic, genotoxic, and carcinogenic compound. Glycidamide has a close relationship to the toxic mechanism of acrylamide. In order to explore the toxic mechanism of acrylamide, we further discussed the effects of oral administration of allicin on glycidamide-induced toxicity by determining the hematological parameters like AST, ALT, LDH, BUN, creatinine, ROS, and 8-OHdG, and biochemical parameters such as MDA, MPO, SOD, GST and GSH in the kidney, liver, brain and lung of male and female mice for the first time. We found that the same dose of glycidamide had more toxic effects and damage effects to the mice compared to the previous study of acrylamide. It could markedly increase the level of AST, ALT, LDH, BUN, ROS, 8-OHdG, MDA, MPO while decrease the SOD, GST and GSH. However, our data showed the oral administered allicin with a concentration of 5, 10, and 20 mg/kg b.w./day could significantly decrease the damage indexes of AST, ALT, LDH, BUN, ROS, 8-OHdG, MDA, and MPO, while increase the antioxidant indicators of SOD, GST and GSH. Thus allicin could be used as an effective dietary supplement for the chemoprevention of glycidamide genotoxicity internally, and to prevent the tissue damage and toxicity induced by glycidamide.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 177-187.

 
  Cholinergic properties of new 7-methoxytacrine-donepezil derivatives
Vendula Sepsova 1), Jana Karasova, Gunnar Tobin, Daniel Jun, Jan Korabecny, Pavla Cabelova, Katerina Janska, Jan Krusek, Kristyna Skrenkova, Kamil Kuca, Ondrej Soukup

1)Depatment of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic. vendula.sepsova@unob.cz.


Organophosphorus nerve agents inhibit acetylcholinesterase (AChE) which causes the breakdown of the transmitter acetylcholine (ACh) in the synaptic cleft. Overstimulation of cholinergic receptors (muscarinic and nicotinic) by excessive amounts of ACh causes several health problems and may even cause death. Reversible AChE inhibitors play an important role in prophylaxis against nerve agents. The presented study investigated whether 7-methoxytacrine (7-MEOTA) and 7-MEOTA-donepezil derivatives can act as central and peripheral reversible AChE inhibitors and simultaneously antagonize muscarinic and nicotinic receptors. The possible mechanism of action was studied on cell cultures (patch clamp technique, calcium mobilization assay) and on isolated smooth muscle tissue (contraction study). Furthermore, the kinetics of the compounds were also examined. CNS availability was predicted by determining the passive blood-brain barrier penetration estimated via a modified PAMPA assay. In conclusion, this study provides promising evidence that the new synthesized 7-MEOTA-donepezil derivatives have the desired anticholinergic effect; they can inhibit AChE, and nicotinic and muscarinic receptors in the micromolar range. Furthermore, they seem to penetrate readily into the CNS. However, their real potency and benefit must be verified by in vivo experiments.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 189-200.

 
  Cytotoxic effect of extract from Dunaliella salina against SH-SY5Y neuroblastoma cells
Belkis Atasever-Arslan 1), Kaan Yilancioglu, Maide Bekaroglu, Emre Taskin, Eyup Altinoz, Selim Cetiner

1)Department of Molecular Biology and Genetics, Faculty of Engineering and Natural Sciences, Üsküdar University, Istanbul, Turkey. belkisatasever.arslan@uskudar.edu.tr.


Cytotoxic effects of essential oils extracted from Dunaliella salina on SH-SY5Y human neuroblastoma cells were investigated in this study. GC-MS analysis was used for determination of the composition of essential oils found in Dunaliella salina extract. All experimented concentrations of Dunaliella salina extract on SH-SY5Y human neuroblastoma cells were significantly more cytotoxic than the tested concentrations of the extract on ECV304 human endothelial cells used as a control. Fifthy compounds were detected in GC-MS analysis of the extract, and five major compounds were predominantly found as follows: octadecanoic acid, methyl ester (27.43%); hexadecanoic acid, methyl ester (Cas) methyl palmitate (24.82%); 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z)- (7.39%); octadecanoic acid (5.03%), pentadecanoic acid (3.60%). The cytotoxic activity of Dunaliella salina extract on SH-SY5Y human neuroblastoma cells might be due to high concentrations of octadecanoic acid and hexadecanoic acid. Furthermore, results indicate that the extract demonstrates some proliferative effect on ECV304 cells in a dose-dependent manner between 0.25 and 5 μg/ml. These results suggest that Dunaliella salina may have anticancer potential against human neuroblastoma cells.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 201-207.

 
  Molecular pharmacology of antihistamines in inhibition of oxidative burst of professional phagocytes
Radomír Nosáľ 1), Viera Jančinová, Katarína Drábiková, Tomáš Perečko

1)Department of Cellular Pharmacology, Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovak Republic. radomir.nosal@savba.sk.


Antihistamines of the H1 and H3/H4 groups interfere with oxidative burst of human professional phagocytes in vitro. In the concentration of 10 μM, H1 antihistamines of the 1st and 2nd generation inhibited oxidative burst of human neutrophils in the rank order of potency: dithiaden > loratadine > brompheniramine > chlorpheniramine > pheniramine. Of the H1 antihistamines, the most effective was dithiaden in suppressing oxidative burst of whole human blood and dose-dependently the chemiluminescence of isolated neutrophils at extra- and intracellular level. Inhibition of free oxygen radical generation in isolated neutrophils by dithiaden resulted from the inhibition of protein kinase C activation. The potentiation of recombinant caspase-3 by dithiaden is supportive of the antiinflammatory effect of dithiaden and suggestive of increasing the apoptosis of professional phagocytes. Of the H3/H4 antihistamines, the most effective was JNJ7777120 in decreasing chemiluminescence in whole blood and also at extra- and intracellular sites of isolated neutrophils. JNJ 10191584 and thioperamide were less effective and the latter significantly potentiated free oxygen radical generation intracellularly. The results demonstrated that, compared with the H3/H4 antihistamines investigated, H1 antihistamines were much more potent in inhibiting free oxygen radical generation in human professional phagocytes. This finding should be taken into account therapeutically.

General Physiology and Biophysics. Volume 34, 2015, No. 2: 209-216.