We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDS-PAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDI-TOF MS) and sequenced by Liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified., J. Žurmanová, D. Maláčová, F. Půta, P. Novák, J. Říčný, T. Soukup., and Obsahuje bibliografii a bibliografické odkazy
Glutamate is a well-characterized excitatory neurotransmitter in the central nervous system (CNS). Recently, glutamate receptors (GluRs) were also found in peripheral tissues, including the heart. However, the function of GluRs in peripheral organs remains poorly understood. In the present study, we found that N-methyl-D-aspartate (NMDA) could increase intracellular calcium ([Ca2+]i) level in a dose-dependent manner in cultured neonatal rat cardiomyocytes. NMDA at 10-4 M increased the levels of reactive oxygen species (ROS), cytosolic cytochrome c (cyto c), and 17-kDa caspase-3, but depolarized mitochondrial membrane potential, leading to cardiomyocyte apoptosis. In addition, NMDA treatment induced an increase in ba x mRNA but a decrease in bcl-2 mRNA expression in the cardiomyocytes. The above effects of NMDA were bloc ked by the NMDA receptor antagonist (+)-5-methyl- 10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and by ROS scavengers glutathione (GSH) and N-acetylcystein (NAC). These results suggest that stimulation of NMDA receptor in the cardiomyocyte may lead to apoptosis via a Ca2+, ROS, and caspase-3 mediated pathway. These findings suggest that NMDA receptor may play an important role in myocardial pathogenesis., X. Gao, X. Xu, J. Pang, C. Zhang, J. M. Ding, X. Peng, Y. Liu, J.-M. Cao., and Obsahuje bibliografii a bibliografické odkazy
The aim of this work was to evaluate ontogeny of reactive nitrogen species (RNS) production by peripheral blood phagocytes in pig. Pig fetuses (55 and 92 days of gestation) and postnatal piglets (1, 3, 8, 17, 31 and 41 days after birth) were used. RNS production was measured by fluorescent probes diaminofluorescein-diacetate (DAF-FMDA) and dichloro-fluorescein-diacetate (H2DCFDA). Levels of nitration of cell proteins were established by immunofluorescent detection of nitrotyrosine. Levels of plasma nitrites/nitrates were detected spectrophotometrically by Griess reaction. Nitric oxide production measured by DAF-FMDA in neutrophils decreased during postnatal life. Spontaneous RNS measured by H2DCFDA decreased from 55th day of gestation to the 41st day of life. Phorbol-12-myristate-13-acetate activated production decreased during postnatal life. Production of NO measured by DAF-FMDA in macrophages decreased from the 1st to 41st day after birth. RNS production measured by H2DCFDA in monocytes did not show any significant changes during ontogeny. The level of nitrotyrosine significantly decreased from the 3rd to 17th day. Levels of plasma nitrites/nitrates gradually decreased from the 55th day of gestation to the 41st day after birth. A temporary increase in all parameters occurred after weaning, but without any significance. In conclusion, RNS production has a decreasing trend during ontogeny and is transiently upregulated after weaning., P. Zelníčková, M. Faldyna, J. Ondráček, H. Kovářů, F. Kovářů., and Obsahuje bibliografii a bibliografické odkazy
To understand the contribution of potassium (K+) channels, particularly α-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of α-dendrotoxin (α-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of α-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing α-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs., H. Haghdoust, M. Janahmadi, G. Behzadi., and Obsahuje bibliografii a bibliografické odkazy
Raloxifen is a selective estrogen receptor modulator which prevents bone loss in ovariectomized female mice in a fashion similar to estrogens. Since testosterone-deficient male mice also lose bone mass, we were interested in testing the effects of raloxifen on bones in intact and castrated male mice. Bone density was significantly reduced in castrated animals (1.36±0.04 g/ml) as compared to intact animals (1.42±0.03 g/ml) (p<0.01). When castrated mice with extraordinarily low concentrations of testosterone and with reduced weight of seminal vesicles were treated with raloxifen, the changes in bone density and bone minerals resulting from castration (1.36±0.04 g/ml) were entirely prevented (1.40±0.01 g/ml). Cortical bone was lost in orchidectomized mice, and this decrease in cortical thickness of the femur was prevented by raloxifen administration. Raloxifen in a dose used in humans for treatment of osteoporosis decreased the weight of seminal vesicles, an organ which is highly sensitive to the androgenic effect, decreased the concentration of testosterone (12.5±2.8 μmol/l) (p<0.01) but not to the same level as in the case of castrated animals (0.6±0.3 μmol/l), and did not have any effect on bone density or mineral content in intact mice. The results of the present study may thus be interpreted as supporting the hypothesis that raloxifen is an effective agent against the deleterious effects of castration-induced osteopenia in male mice and also support the hypothesis that estrogens may have physiological skeletal effects in male mice., P. D. Broulík, K. Broulíková., and Obsahuje bibliografii a bibliografické odkazy
Oxidative stress has been implicated to play a major role in aging and age-related diseases. In the present study, we investigated the effects of aging on the total antioxidant capacity, uric acid, lipid peroxidation, total sulfhydryl group content and damage to DNA in adult (6 months), old (15 months) and senescent (26 months) male Wistar rats. The antioxidant capacity, determined by phycoerythrin-based TRAP method (total peroxyl radical-trapping potential) was significantly decreased in the plasma and myocardium of old and senescent rats, whereas plasma level of uric acid was elevated in 26-month-old rats. Age-related decline in plasma and heart antioxidant capacity was accompanied by a significant loss in total sulfhydryl group content, increased lipid peroxidation and higher DNA damage in lymphocytes. Correlations between TRAP and oxidative damage to lipids, proteins and DNA suggest that the decline in antioxidant status may play an important role in age-related accumulation of cell damage caused by reactive oxygen species., M. Sivoňová, Z. Tatarková, Z. Ďuračková, D. Dobrota, J. Lehotský, T. Matáková, P. Kaplán., and Obsahuje bibliografii a bibliografické odkazy
C-type natriuretic peptides (CNP) play an inhibitory role in smooth muscle motility of the gastrointestinal tract, but the effect of CNP on delayed rectifier potassium currents is still unclear. This study was designed to investigate the effect of CNP on delayed rectifier potassium currents and its mechanism by using conventional whole-cell patch- clamp technique in guinea-pig gastric myocytes isolated by collagenase. CNP significantly inhibited delayed rectifier potassium currents [IK (V)] in dose-dependent manner, and CNP inhibited the peak current elicited by depolarized step pulse to 86.1±1.6 % (n=7, P<0.05), 78.4±2.6 % (n=10, P< 0.01) and 67.7±2.3 % (n=14, P<0.01), at concentrations of 0.01 μmol/l, 0.1 μmol/l and 1 μmol/l, respectively, at +60 mV. When the cells were preincubated with 0.1 μmol/l LY83583, a guanylate cyclase inhibitor, the 1 μmol/l CNP-induced inhibition of IK (V) was significantly impaired but when the cells were preincubated with 0.1 μmol/l zaprinast, a cGMP-sensitive phosphodiesterase inhibitor, the 0.01 μmol/l CNP-induced inhibition of IK (V) was significantly potentiated. 8-Br-cGMP, a membrane permeable cGMP analogue mimicked inhibitory effect of CNP on IK (V). CNP-induced inhibition of IK (V) was completely blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). The results suggest that CNP inhibites the delayed rectifier potassium currents via cGMP-PKG signal pathway in the gastric antral circular myocytes of the guinea-pig., H. Y. Xu, X. Huang, M. Yang, J.-B. Sun, L.-H. Piao, Y. Zhang, L. Gao, W.-X. Xu., and Obsahuje bibliografii a bibliografické odkazy
The aim of our study was to test the in fluence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 ºC or 42.5 ºC) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 ºC) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 ºC or 42.5 ºC) or standard temperature (control 37.5 ºC), post-incubated overnight (16-20 h) at 37.5 ºC and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 ºC did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 ºC. We stern-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 ºC) and embryos (exposed to 41.5 ºC). Following the elevation of temperature to 42.5 ºC embryo development was dramati cally compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis., A. V. Makarevich, L. Olexiková, P. Chrenek, E. Kubovičová, K. Fréharová, J. Pivko., and Obsahuje bibliografii a bibliografické odkazy