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71.
B Tencerová A Zahradníková J Gaburjáková M Gaburjáková 《The Journal of general physiology》2012,140(2):93-108
The synergic effect of luminal Ca(2+), cytosolic Ca(2+), and cytosolic adenosine triphosphate (ATP) on activation of cardiac ryanodine receptor (RYR2) channels was examined in planar lipid bilayers. The dose-response of RYR2 gating activity to ATP was characterized at a diastolic cytosolic Ca(2+) concentration of 100 nM over a range of luminal Ca(2+) concentrations and, vice versa, at a diastolic luminal Ca(2+) concentration of 1 mM over a range of cytosolic Ca(2+) concentrations. Low level of luminal Ca(2+) (1 mM) significantly increased the affinity of the RYR2 channel for ATP but without substantial activation of the channel. Higher levels of luminal Ca(2+) (8-53 mM) markedly amplified the effects of ATP on the RYR2 activity by selectively increasing the maximal RYR2 activation by ATP, without affecting the affinity of the channel to ATP. Near-diastolic cytosolic Ca(2+) levels (<500 nM) greatly amplified the effects of luminal Ca(2+). Fractional inhibition by cytosolic Mg(2+) was not affected by luminal Ca(2+). In models, the effects of luminal and cytosolic Ca(2+) could be explained by modulation of the allosteric effect of ATP on the RYR2 channel. Our results suggest that luminal Ca(2+) ions potentiate the RYR2 gating activity in the presence of ATP predominantly by binding to a luminal site with an apparent affinity in the millimolar range, over which local luminal Ca(2+) likely varies in cardiac myocytes. 相似文献
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73.
S Betanzos-Lara O Novakova RJ Deeth AM Pizarro GJ Clarkson B Liskova V Brabec PJ Sadler A Habtemariam 《Journal of biological inorganic chemistry》2012,17(7):1033-1051
The synthesis and characterization of complexes [(η(6)-arene)Ru(N,N')X][PF(6)], where arene is para-cymene (p-cym), biphenyl (bip), ethyl benzoate (etb), hexamethylbenzene (hmb), indane (ind) or 1,2,3,4-tetrahydronaphthalene (thn), N,N' is 2,2'-bipyrimidine (bpm) and X is Cl, Br or I, are reported, including the X-ray crystal structures of [(η(6)-p-cym)Ru(bpm)I][PF(6)], [(η(6)-bip)Ru(bpm)Cl][PF(6)], [(η(6)-bip)Ru(bpm)I][PF(6)] and [(η(6)-etb)Ru(bpm)Cl][PF(6)]. Complexes in which N,N' is 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione or 4,7-diphenyl-1,10-phenanthroline (bathophen) were studied for comparison. The Ru(II) arene complexes undergo ligand-exchange reactions in aqueous solution at 310?K; their half-lives for hydrolysis range from 14 to 715?min. Density functional theory calculations on [(η(6)-p-cym)Ru(bpm)Cl][PF(6)], [(η(6)-p-cym)Ru(bpm)Br][PF(6)], [(η(6)-p-cym)Ru(bpm)I][PF(6)], [(η(6)-bip)Ru(bpm)Cl][PF(6)], [(η(6)-bip)Ru(bpm)Br][PF(6)] and [(η(6)-bip)Ru(bpm)I][PF(6)] suggest that aquation occurs via an associative pathway and that the reaction is thermodynamically favourable when the leaving ligand is I?>?Br?≈?Cl. pK (a)* values for the aqua adducts of the complexes range from 6.9 to 7.32. A binding preference for 9-ethylguanine (9-EtG) compared with 9-ethyladenine (9-EtA) was observed for [(η(6)-p-cym)Ru(bpm)Cl][PF(6)], [(η(6)-hmb)Ru(bpm)Cl](+), [(η(6)-ind)Ru(bpm)Cl](+), [(η(6)-thn)Ru(bpm)Cl](+), [(η(6)-p-cym)Ru(phen)Cl](+) and [(η(6)-p-cym)Ru(bathophen)Cl](+) in aqueous solution at 310?K. The X-ray crystal structure of the guanine complex [(η(6)-p-cym)Ru(bpm)(9-EtG-N7)][PF(6)](2) shows multiple hydrogen bonding. Density functional theory calculations show that the 9-EtG adducts of all complexes are thermodynamically preferred compared with those of 9-EtA. However, the bmp complexes are inactive towards A2780 human ovarian cancer cells. Calf thymus DNA interactions for [(η(6)-p-cym)Ru(bpm)Cl][PF(6)] and [(η(6)-p-cym)Ru(phen)Cl][PF(6)] consist of weak coordinative, intercalative and monofunctional coordination. Binding to biomolecules such as glutathione may play a role in deactivating the bpm complexes. 相似文献
74.
An evaluation of field and noninvasive genetic methods for estimating Eurasian otter population size
Petra Hájková Barbora Zemanová Kevin Roche Bedřich Hájek 《Conservation Genetics》2009,10(6):1667-1681
Successful conservation and management of rare and elusive species requires reliable estimates of population size, but acquisition
of such data is often challenging. We compare the two most frequently used methods of assessing abundance of Eurasian otter
(Lutra lutra) populations, noninvasive genetic sampling (NGS) based on genotyping of faeces and field surveys using snow tracking. In
a 100-km2 oligotrophic otter habitat with linear water bodies, both methods yielded very similar estimates (10–12 individuals). However,
in a 100-km2 fishpond area, consisting of a complex network of rivers, fishponds, channels and marshes, genotyping of faeces revealed
the presence of a higher number of individuals (46–50 genotypes) than the snow survey (38 individuals). NGS data analysed
by capture-mark-recapture (CMR)-based software CAPWIRE provided even higher estimates, being twice the number assessed through
snow tracking (76–81 individuals, CI95% = 49–96 and 55–89). Our results suggest that the performance of both NGS and snow tracking is comparable in simple linear
habitats, but in complex habitats with very high otter density a combination of genetic and field methods, or CMR analysis
using genetic data, is recommended. We emphasise that to obtain reliable estimates using NGS it is necessary to follow strict
protocols for detection and elimination of genotyping errors. Based on a literature review and our experience, we suggest
improvements that may increase the success rate and efficiency of NGS for otters. 相似文献
75.
Hynek Strnad Lukáš Lacina Michal Kolář Zdeněk Čada Čestmír Vlček Barbora Dvořánková Jan Betka Jan Plzák Martin Chovanec Jana Šáchová Jaroslav Valach Markéta Urbanová Karel Smetana Jr 《Histochemistry and cell biology》2010,133(2):201-211
Epithelial–mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium, including the tumor progression and spread. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity toward normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor-associated fibroblast activity that influences normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4, respectively, to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in the medium conditioned by cancer-associated fibroblast. Presence of these growth factors was also detected in tumor samples. 相似文献
76.
Andrej?Godány Katarína?Majzlová Viera?Horváthová Barbora?Vidová ?tefan?Jane?ekEmail author 《Biologia》2010,65(3):408-415
The presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid
sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine
(present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ±
0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from
85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining
activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease
was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could
contribute to the thermostability of the α-amylase from T. hydrothermalis. 相似文献
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Linhová M Branská B Patáková P Lipovský J Fribert P Rychtera M Melzoch K 《Folia microbiologica》2012,57(4):307-311
We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone–butanol–ethanol
(ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein
diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish
two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and
cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains,
BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination
under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability.
Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination.
We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation. 相似文献
79.
Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis. 相似文献
80.
Peter Segla Dušan Mikloš Barbora Kaliňáková Martina Palicová Marian Valko Tadeusz Glowiak 《Inorganica chimica acta》2004,357(14):4172-4180
The synthesis and characterization of seven new solid complexes, [Cu(2-MeSnic)2 (phen)] (2-MeSnic = 2-methylthionicotinate, phen = 1,10-phenanthroline), [CuX2(bipy)(H2O)] (X = 2-MeSnic or nic (nicotinate), bipy = 2,2′-bipyridine), [Cu(isonic)2(bipy)(H2O)] · H2O (isonic = isonicotinate), [Cu(bipy)2(H2O)](2-MeSnic)2 · 3H2O, [Cu(phen)2(H2O)](isonic) 2 · 2H2O and [Cu(phen)2(H2O)](nic)2 · 3H2O, are reported. The composition and stereochemistry as well as the mode of ligand coordination have been determined by elemental analysis, IR, electronic and EPR spectra. The carboxyl group of the pyridinecarboxylate anions coordinates to the Cu(II) atom as an unidentate or as a chelating ligand. The EPR spectra of studied complexes are monomeric except for the spectrum of [Cu(2-MeSnic)2(bipy)(H2O)], which shows triplet state feature. Half-field transition, observed for [Cu(2-MeSnic)2(bipy)(H2O)], [Cu(bipy)2(H2O)](2-MeSnic)2 · 3H2O and [Cu(phen)2(H2O)](nic)2 · 3H2O, was used to estimate the interspin copper-copper distances. In all cases, the available evidence supports square-pyramidal environment about the copper(II) atom, which is confirmed by crystal and molecular structure of one of the products, namely [Cu(2-MeSnic)2(bipy)(H2O)]. The antimicrobial effects have been tested on various strains of bacteria, yeasts and filamentous fungi. 相似文献