Mitochondria are involved in cellular functions that transcend the traditional role of these organelles as the energy factory of the cell. Their relative inaccessibility and the difficulties involved in attempts to study them in their natural environment - the cytosol - has delayed much of this understanding and they still have many secrets to yield. One of the relatively new fields in this respect is undoubtedly the analysis of mitochondrial membrane potential. The realization that its alteration may have important pathophysiological consequences has led to an increased interest in measuring this variable in a variety of biological settings, including cardiovascular diseases. Measurements of mitochondrial membrane potential tell us much about the role of mitochondria in normal cell function and in processes leading to cell death. However, we must be aware of the limitations of using isolated mitochondria, single cells and different fluorescent indicators., L. Škárka, B. Ošťádal., and Obsahuje bibliografii
We investigated how selected electromorphological parameters of myelinated axons influence the preservation of interspike intervals when the propagation of action potentials is corrupted by axonal intrinsic noise. Hereby we tried to determine how the intrinsic axonal noise influences the performance of axons serving as carriers for temporal coding. The strategy of this coding supposes that interspike intervals presented to higher order neurons would minimally be deprived of information included in interspike intervals at the axonal initial segment. Our experiments were conducted using a computer model of the myelinated axon constructed in a software environment GENESIS (GEneral NEural SImulation System). We varied the axonal diameter, myelin sheath thickness, axonal length, stimulation current and channel distribution to determine how these parameters influence the role of noise in spike propagation and hence in preserving the interspike intervals. Our results, expressed as the standard deviation of spike travel times, showed that by stimulating the axons with regular rectangular pulses the interspike intervals were preserved with a microsecond accuracy. Stimulating the axons with pulses imitating postsynaptic currents, greater changes of interspike intervals were found, but the influence of implemented noise on the jitter of interspike intervals was approximately the same., E. Kuriščák, S. Trojan, Z. Wünsch., and Obsahuje bibliografii
The pattern-reversal (P-VEPs) and the motion-onset (M-VEPs) of visual evoked potentials were modeled by means of three damped oscillators (O1, O2, O3) of identical construction. The O1, assumed to simulate the response of primary visual area (V1), was driven by the firing density of the lateral geniculate nuclei. O1 contributed mainly to the N75 and P100 peaks of the P-VEPs. The O2, driven by the O1 output, mimics the activity of V2, V3a, and MT. It contributed to the negative peak N145 of the P-VEPs or to the N160 in the M-VEPs. The O3 was suggested to model late slow processes probably of an attentive origin. The model parameters were set by optimization to follow the P-VEPs and M-VEPs obtained as a grand average of four young volunteers (PZ - A2 lead). The evoked potentials were described with normalized root mean square error lower than 13 %., J. Kremláček, M. Kuba, J. Holčík., and Obsahuje bibliografii
Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1x10-5 and 1x10-6 mol.l-1 was significantly potentiated by NEM (1x10-7-1x10-9 mol.l-1) and DMA (1x10-6 and 1x10-7 mol.l-1) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin., A. Miroššay, L. Mirossay, M. Šarišský, P. Papp, J. Mojžiš., and Obsahuje bibliografii
Summary The aim of the study was to characterize by molecular profiling two glomerular diseases: IgA nephropathy (IgAN) and focal segmental glomerulosclerosis (FSGS) and to identify potential molecular markers of IgAN and FSGS progression. The expressions of 90 immune-related genes were compared in biopsies of patients with IgAN (n=33), FSGS (n=17) and in controls (n=11) using RT-qPCR. To identify markers of disease progression, gene expression was compared between progressors and non-progressors in 1 year follow-up. The results were verified on validation cohort of patients with IgAN (n=8) and in controls (n=6) using laser-capture microdissection, that enables to analyze gene expression separately for glomeruli and interstitium. In comparison to controls, patients with both IgAN and FSGS, had lower expression of BAX (apoptotic molecule BCL2-associated protein) and HMOX-1 (heme oxygenase 1) and higher expression of SELP (selectin P). Furthermore, in IgAN higher expression of PTPRC (protein-tyrosine phosphatase, receptor-type C) and in FSGS higher expression of BCL2L1 (regulator of apoptosis BCL2-like 1) and IL18 compared to control was observed. Validation of differentially expressed genes between IgAN and controls on another cohort using laser-capture microdissection confirmed higher expression of PTPRC in glomeruli of patients with IgAN. The risk of progression in IgAN was associated with higher expression EDN1 (endothelin 1) (AUC=0.77) and FASLG (Fas ligand) (AUC=0.82) and lower expression of VEGF (vascular endothelial growth factor) (AUC=0.8) and in FSGS with lower expression of CCL19 (chemokine (C-C motif) ligand 19) (AUC=0.86). Higher expression of EDN1 and FASLG along with lower expression of VEGF in IgAN and lower expression of CCL19 in FSGS at the time of biopsy can help to identify patients at risk of future disease progression., I. Tycová, P. Hrubá, D. Maixnerová, E. Girmanová, P. Mrázová, L. Straňavová, R. Zachoval, M. Merta, J. Slatinská, M. Kollár, E. Honsová, V. Tesař, O. Viklický., and Seznam literatury
Using histochemical analysis (NADPH-diaphorase, Fluoro-Jade B dye and bis-benzimide 33342 Hoechst) we studied the influence of intraperitoneal administration of nicotine (NIC), kainic acid (KA) and combination of both these substances on hippocampal neurons and their changes. In experiments, 35-day-old male rats of the Wistar strain were used. Animals were pretreated with 1 mg /kg of nicotine 30 min prior to the kainic acid application (10 mg/kg). After two days, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blades of the dentate gyrus and in the hilus of the dentate gyrus. Fluoro-Jade B positive cells were examined in the same areas in order to elucidate a possible neurodegeneration. In animals exposed only to nicotine the number of NADPH-diaphorase positive neurons in the CA3 area of the hippocampus and in the hilus of the dentate gyrus was higher than in controls. In contrast, KA administration lowered the number of NADPH-diaphorase positive cells in all studied hippocampal areas and in both blades of the dentate gyrus. Massive cell degeneration was observed in CA1 and CA3 areas of the hippocampus and in the hilus of the dentate gyrus after kainic acid administration. Animals exposed to kainic acid and pretreated with nicotine exhibited degeneration to a lesser extent and the number of NADPH-diaphorase positive cells was higher compared to rats, which were exposed to kainic acid only., V. Riljak, M. Milotová, K. Jandová, J. Pokorný, M. Langmeier., and Obsahuje bibliografii a bibliografické odkazy
Growth of the A549 cell line in a perfusion system suitable for use in a magnetic resonance study has been characterized and shown to be stable physiologically and hence appropriate for serial observations. Several methods of monitoring cell growth were compared to assess the behavior of the cells in this system. Comparison between NMR metabolite data and cell growth via cell counting showed that 31P NMR signals accurately reported cell doubling time. In contrast to most NMR cell culture systems, viable cells can be recovered from the perfusion system after the NMR measurements for further biochemical studies. These data further suggest that this system will be useful for studying the physiology and biochemistry of exponentially growing cells for at least two days in NMR tube culture., E. G. Shankland, J. C. Livesey, R. W. Wiseman, K. A. Krohn., and Obsahuje bibliografii
Myofibrillar creatine kinase (CK) that buffers ATP during fluctuating muscle energy metabolism has been selected for studies of conformational changes underlying the cellular control of enzyme activity. The force field was computed for three energetic states, namely for the substrate-free CK molecule, for the molecule conjugated with the MgATP complex, and for the molecule conjugated with the pair of reactants MgATP-creatine. Without its substrates, the enzyme molecule assumes an inactive "open" form. Upon binding of the MgATP complex, the CK molecule takes up a reactive "closed" conformation. Subsequent binding of creatine yields a nonreactive "intermediary" conformation. Acid-base catalysis is considered to be the basic principle for the reversible transfer of the phosphoryl group between the substrates. The results indicate that the substrate-induced energy minimizing conformational changes do not represent a sufficient condition for CK activity and that some other essential component of physiological control at the cellular level is involved in the transition from the intermediary to the closed structure of the molecule., J. A. Mejsnar, B. Sopko, M. Gergor., and Obsahuje bibliografii
Renal medullary endothelin B receptors (ETB) mediate sodium excretion and blood pressure (BP) control. Several animal models of hypertension have impaired renal medullary ETB function. We found that 4-week high-caloric diet elevated systolic BP in Dahl salt-sensitive (Dahl S) rats (126±2 vs. 143±3 mm Hg, p<0.05). We hypothesized that renal medullary ETB function is dysfunctional in DS rats fed a high-caloric diet. We compared the diuretic and natriuretic response to intramedullary infusion of ETB agonist sarafotoxin 6c (S6c) in DS rats fed either a normal or high-caloric diet for 4 weeks. Urine was collected during intramedullary infusion of saline for baseline collection followed by intramedullary infusion of either saline or S6c. We first examined the ETB function in DS rats fed a normal diet. S6c increased urine flow (2.7±0.3 μl/min during baseline vs. 5.1±0.6 μl/min after S6c; p<0.05; n=5) and sodium excretion (0.28±0.05 vs. 0.81±0.17 μmol/min; p<0.05), suggesting that DS rats have renal medullary ETB function. However, DS rats fed a high-caloric diet displayed a significant increase in urine flow (2.7±0.4 vs. 4.2±0.4 μl/min, baseline vs. S6c infusion, respectively; p<0.05, n=6), but no significant change in sodium excretion in response to S6c (0.32±0.06 vs. 0.45±0.10 μmol/min). These data demonstrate that renal medullary ETB function is impaired in DS rats fed a high-caloric diet, which may be contributed to the elevation of blood pressure during high-caloric feeding in this model., W. Kittikulsuth, K. A. Hyndman, J. S. Pollock, D. M. Pollock., and Seznam literatury
Recently, the genetic cause of several syndromic forms of glycemia dysregulation has been described. One of them, MEHMO syndrome, is a rare X-linked syndrome recently linked to the EIF2S3 gene mutations. MEHMO is characterized by Mental retardation, Epilepsy, Hypogonadism/hypogenitalism, Microcephaly, and Obesity. Moreover, patients with MEHMO had also diabetes and endocrine phenotype, but detailed information is missing. We aimed to provide more details on the endocrine phenotype in two previously reported male probands with MEHMO carrying a frame-shift mutation (I465fs) in the EIF2S3 gene. Both probands had a neonatal hypoglycemia, early onset insulindependent diabetes, and hypopituitarism due to dysregulation and gradual decline of peptide hormone secretion. Based on the clinical course in our two probands and also in previously published patients, neonatal hypoglycemia followed by earlyonset diabetes and hypopituitarism may be a consistent part of the MEHMO phenotype., J. Staník, M. Škopková, D. Staníková, K. Brennerová, L. Barák, L. Tichá, J. Hornová, I. Klimeš, D. Gasperiková., and Seznam literatury