The ubiquitin-proteasome pathway fulfills major biological functions, but its physiologic tissue distribution and the interrelationship between pathway component activities and ubiquitin pools are unknown. Therefore, we analyzed free and conjugated ubiquitin, ubiquitin-protein ligation rates (UbPL) and chymotryptic- and tryptic-like proteasome peptidase activities in porcine skeletal muscle, heart, lung, liver, spleen and kidney (n=5 each). There were considerable differences between tissues (p<0.05 for all parameters). Lung and spleen showed high levels of free and conjugated ubiquitin and high UbPL. Proteasome activities were highest in kidney and heart. There were linear relationships between tryptic-like and chymotryptic-like proteasome peptidase activities (r2 = 0.624, p<0.001) and between free and conjugated ubiquitin tissue levels (r2 = 0.623, p<0.001). Tissue levels of free and conjugated ubiquitin correlated linear with UbPL (p<0.005), but they were not correlated with proteasome peptidase activities. The results suggest that tissue ubiquitin pools are tightly regulated and indicate a constant proportion of conjugated ubiquitin. They further support the hypothesis that ubiquitin-protein ligase systems, and probably deubiquitylating enzymes, are key regulators of ubiquitin homeostasis. The detected differences are suggestive of tissue-specific roles of ubiquitin-proteasome pathway components. Besides the known importance of the ubiquitin proteasome pathway in heart, kidney and the immune system, the results suggest the lung as another organ in which ubiquitin proteasome pathway components may also significantly contribute to disease processes., M. B. Patel, M. Majetschak., and Obsahuje bibliografii a bibliografické odkazy
The 24-hour urinary excretion of 6-β-hydroxycortisol (6β-OHC) and the urinary ratio of 6β-hydroxycortisol/cortisol (6β-OHC/UFC) have been proposed as noninvasive probes for human cytochrome P450 3A4 isoform (CYP3A4). In this study, we evaluated within- and between-day variability of 6β-OHC excretion and 6β-OHC/UFC ratio in nine Caucasian men with cardiac disease. Each study participant was asked to collect 24-hour urine specimens during four consecutive days in five standardized time intervals. Concentrations of UFC and 6β-OHC were determined by immunoassay and the high-performance liquid chromatographic (HPLC) method, respectively. The HPLC method was accurate and precise, as indicated by the recovery rate of 96.5-103.3 % and less than 5.2 % and 6.3 % of the coefficient of variation for within-run and between-run assay, respectively. In patients, diurnal variations in UFC and 6β-OHC excretion were parallel. Consequently, 6β-OHC/UFC ratio remained stable during the day. Both, 6β-OHC excretion and 6β-OHC/UFC ratio showed significant relationship between 24-hour value and values measured in corresponding collection periods with best correlations obtained from night interval (22.00-06.00, r = 0.86-0.91). These results indicated that urinary 6β-OHC excretion and 6β-OHC/UFC ratio measured in overnight/morning urine could precisely reflect 24-hour values even in severely ill patients. In addition, a simple and sensitive HPLC method was described for determination of 6β-OHC in urine., S. Mičuda, L. Šišpera, M. Hodač, P. Pařízek, L. Fuksa, E. Brčáková, J. Cerman, J. Cermanová, J. Martínková., and Obsahuje bibliografii a bibliografické odkazy
Perinatal cerebral hypoxia represents a major cause of obstetric complications and the resulting transient oxygen deficiency might belong to early risk factors for schizophrenia. The aim of this study was to evaluate possible long-term behavioral changes induced by one hour of continuous bilateral common carotid artery occlusion in 12-day-old male rats. Post-ischemic behavioral disturbances were evaluated in social (play) behavior on postnatal day 22 (PND 22), open field test (PND 35 and 50) and prepulse inhibition of the acoustic startle reflex (PND 50). Transient ischemia in neonatal rats was not significantly altered in social dyadic interactions evaluated in pre-weaning pups, but resulted in enhanced locomotor activity in pubertal rats (PND 35) and impaired prepulse inhibition of the startle reflex in post- pubertal males (PND 50). These behavioral alterations suggest that perinatal hypoxic/ischemic insults may represent a risk factor for later manifestation of specific features relevant to schizophrenia in predisposed individuals., F. Tejkalová, M. Kaiser, J. Klaschka, F. Šťastný., and Obsahuje bibliografii a bibliografické odkazy
Based on the biological significance of the ubiquitin-proteasome pathway (UPP) and its potential role during sepsis, burns and ischemia-reperfusion injury, we hypothesized that the systemic response to traumatic shock (TS) is accompanied by tissue-specific UPP alterations. Therefore, we studied tissue ubiquitin pools, chymotryptic- and tryptic-like proteasome peptidase activities and ubiquitin-protein ligation (UbPL) rates in skeletal muscle, heart, lung, liver, spleen and kidney using a clinically relevant porcine model (bilateral femur fracture/hemorrhage followed by fluid resuscitation). TS induced a systemic reduction of tissue- specific high molecular mass ubiquitin-protein conjugates (>50 kDa). Free ubiquitin was unaffected. The dynamic organ patterns of ubiquitin pools paralleled the typical physiological response to TS and resuscitation. Reduction of ubiquitin-protein conjugates was most pronounced in heart and lung (p<0.05 vs. control) and accompanied by significant increases in proteasome peptidase and UbPL activities in these organs. Unlike all other tissues, spleen proteasome peptidase and UbPL activities were significantly reduced 10 h after TS. These findings support the concept that the UPP could play an important role in regulation of cell functions during the early whole-body response to TS. The UPP might be a therapeutic target to improve the metabolic care after TS, particularly in the heart, lung, and spleen., M. B. Patel, S. A. Earle, M. Majetschak., and Obsahuje bibliografii a bibliografické odkazy
Exposure to hyperbaric oxygen leads to increased amount of reactive oxygen species (ROS) that are derived from various sources. After the discovery that ROS can function as signaling molecules, the idea of ROS being hazardous to biological tissues has been challenged. The aim of this study was to examine the changes in oxidative stress parameters in diabetics undergoing hyperbaric oxygen therapy (HBOT) due to foot ulcers. Twenty patients, who received HBOT for diabetic foot ulcers, were included in the study. Blood samples were taken before HBOT and 30 min after exit from the chamber, on the day of the first and the 15th HBOT sessions. They were used for the determinations of malondialdehyde (MDA), 8-isoprostane and advanced oxidation protein products (AOPPs). 8-Isoprostane and AOPP levels were not altered significantly after the first HBOT session, while both were increased on the fifteenth day (p<0.05). MDA was significantly increased only after the first HBOT session, and remained unchanged on the fifteenth day (within-day variations). Plasma AOPP levels were lowered significantly after fifteen consecutive HBOT sessions (between-day variations). Decreased AOPP levels suggest that increased oxygenation of tissues due to HBO therapy may activate some endogenous factors that prevent hazardous effects of the disease itself., F. Gürdöl, M. Cimşit, Y. Öner-İyidoğan, Ş. Körpinar, S. Yalçinkaya, H. Koçak., and Obsahuje bibliografii a bibliografické odkazy
Adenosine is secreted from adipocytes, binds to adenosine A1 receptor and modulates various functions of these cells. In the present study, the effects of an adenosine A1 receptor antagonist (DPCPX; 0.01, 0.1 and 1 μM) on lipogenesis, glucose transport, lipolysis and the antilipolytic action of insulin were tested in rat adipocytes. DPCPX had a very weak effect on lipogenesis and did not significantly affect glucose uptake. In adipocytes incubated with 1 μM DPCPX, lipolysis increased. This effect was blunted by insulin and by a direct inhibitor of protein kinase A. Moreover, 0.1 μM DPCPX substantially enhanced the lipolytic response to epinephrine and increased cAMP in adipocytes. However, DPCPX was ineffective when lipolysis was stimulated by direct activation of protein kinase A. Adipocyte exposure to epinephrine and insulin with or without 0.1 μM DPCPX demonstrated that this antagonist increased the release of glycerol. However, despite the presence of DPCPX, insulin was able to reduce lipolysis. It is concluded that DPCPX had a weak effect on lipogenesis, whereas lipolysis was significantly affected. The partial antagonism of adenosine A1 receptor increased lipolysis in cells incubated with epinephrine alone and epinephrine with insulin due to the synergistic action of 0.1 μM DPCPX and epinephrine., T. Szkudelski, K. Szkudelska, L. Nogowski., and Obsahuje seznam literatury
L-malate, a tricarboxylic acid cycle (TCA ) intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production and may be involved in the beneficial effects of improving physical stamina. In the present study, we investigated the effects of L-malate on the performance of forced swimming time and blood biochemical parameters related to fatigue – blood urea nitrogen (BUN), glucose (Glc), creatine kinase (CK) , total protein (TP) and lactic acid (LA). To investigate the effects of L-malate on the malate-aspartate shuttle and energy metabolism in mice, the activities of enzymes related to the malate-aspartate shuttle were measured. L-malate was orally administered to mice continuously for 30 days using a feeding atraumatic needle. The swimming time was increased by 26.1 % and 28.5 %, respectively, in the 0.210 g/kg and 0.630 g/kg L-malate-treated group compared with the control group. There were no differences in the concentrations of Glc, BUN and TP between the L-malate-treated groups and the control groups. However, the levels of CK were significantly decreased in the L-malate-treated groups. The results predict a potential benefit of L-malate for improving physical stamina and minimizing muscle damage during swimming exercise. The activities of cytosolic and mitochondrial malate dehydrogenase were significantly elevated in the L-malate-treated group compared with the control group. These enzymatic activities may be useful indicators for evaluating changes affecting the malate-aspartate shuttle and energy metabolism in the liver of mice., J. L. Wu, Q. P. Wu, J. M. Huang, R. Chen, M. Cai, J. B. Tan., and Obsahuje bibliografii a bibliografické odkazy
The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP - 1, 2. 5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST-1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS prod uction was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation., Tomáš Roušar ... [et al.]., and Obsahuje seznam literatury
In this study we have evaluated the effect of maximal incremental cycling exercise (IE) on the systemic release of prostacyclin (PGI2), assessed as plasma 6-keto-PGF1α concentration in young healthy men. Eleven physically active - untrained men (mean ± S.D.) aged 22.7 ± 2.1 years; body mass 76.3 ± 9.1 kg; BMI 23.30 ± 2.18 kg · m-2; maximal oxygen uptake (VO2max) 46.5 ± 3.9 ml · kg-1 · min-1, performed an IE test until exhaustion. Plasma concentrations of 6-keto-PGF1α, lactate, and cytokines were measured in venous blood samples taken prior to the exercise and at the exhaustion. The net exercise-induced increase in 6-keto-PGF1α concentration, expressed as the difference between the end-exercise minus pre-exercise concentration positively correlated with VO2max (r=0.78, p=0.004) as well as with the net VO2 increase at exhaustion (r=0.81, p=0.003), but not with other respiratory, cardiac, metabolic or inflammatory parameters of the exercise (minute ventilation, heart rate, plasma lactate, IL-6 or TNF-α concentrations). The exercise-induced increase in 6-keto-PGF1α concentration was significantly higher (p=0.008) in a group of subjects (n=5) with the highest VO2max when compared to the group of subjects with the lowest VO2max, in which no increase in 6-keto-PGF1α concentration was found. In conclusion, we demonstrated, to our knowledge for the first time, that exercise-induced release of PGI2 in young healthy men correlates with VO2max, suggesting that vascular capacity to release PGI2 in response to physical exercise represents an important factor characterizing exercise tolerance. Moreover, we postulate that the impairment of exercise-induced release of PGI2 leads to the increased cardiovascular hazard of vigorous exercise., J. A. Zoladz ... [et al.]., and Obsahuje seznam literatury
Two forms of gonadotropin-releasing hormone (GnRH), GnRH-I and GnRH-II, are commonly present in mammals. The main hormone controlling reproduction is GnRH-I acting through its receptor (GnRHR-I), whereas the function of GnRH-II is unknown. In primates, it has been suggested that GnRH-II is a specific agonist for the structurally distinct GnRHR-II. Here we compared effects of GnRH-I and GnRH-II on intracellular calcium and gonadotropin hormone release in neonatal rat gonadotrophs in vitro and the dependence of agonist actions on cyclic nucleotide levels. Both agonists elevated intracellular calcium and stimulated gonadotropin secretion in a concentration-dependent manner, with comparable peak amplitudes, but GnRH-I was three times more potent than GnRH-II. Antide, a specific GnRHR-I antagonist, completely blocked the action of both agonists on gonadotropin release. Inhibition of adenylyl cyclase activity by melatonin and MDL significantly attenuated GnRH-I- and GnRH-II-induced calcium signaling and gonadotropin release, whereas inhibition of soluble guanylyl cyclase activity was ineffective. GnRH-II also generated calcium oscillations in a fraction of gonadotrophs not expressing melatonin receptors. These results indicate that GnRH-I and GnRH-II act on the same GnRHR to stimulate gonadotropin release through intracellular calcium and cyclic nucleotide signaling, and that GnRH-II is less potent agonist for this receptor in neonatal rat gonadotrophs., A. Balík ... [et al.]., and Obsahuje seznam literatury