We studied cadmium toxicity in murine hepatocytes in vitro. Cadmium effects on intracellular free Ca2+ concentration ([Ca2+]i) were assayed, using a laser scanning confocal microscope with a fluorescent probe, Fluo-3/AM. The results showed that administration of cadmium chloride (CdCl2, 5, 10, 25 μM) resulted in a dose-dependent decrease of hepatocyte viability and an elevated aspartate aminotransfe rase (AST) activity in the culture medium (p<0.05 for 25 μM CdCl2 vs. control). Significant increases of lactate dehydrogenase (LDH) activities in 10 and 25 μM CdCl2-exposed groups were observed (p<0.05 and p<0.01, respectively). A greatly decreased albumin content and a more malondialdehyde (MDA) formation also occurred after CdCl2 treatment. The Ca2+ concentrations in the culture medium of CdCl2-exposed hepatocytes were significantly decreased, while [Ca2+]i appeared to be significantly elevated (p<0.05 or p<0.01 vs. control). We found that in Ca2+-containing hydroxyethyl piperazine ethanesulfonic acid-buffered salt solution (HBSS) only, CdCl2 elicited [Ca2+]i increases, which comprised an initially slow ascent and a strong elevated phase. However, in Ca2+-containing HBSS with addition of 2-aminoethoxydiphenyl borane (2-APB), CdCl2 caused a mild [Ca 2+] i elevation in the absence of an initial rise phase. Removal of extracellular Ca2+ showed that CdCl2 induced an initially slow [Ca2+]i rise alone without being followed by a markedly elevated phase, but in a Ca2+-free HBSS with addition of 2-APB, CdCl2 failed to elicit the [Ca2+]i elevation. These results suggest that abnormal Ca2+ homeostasis due to cadmium may be an important mechanism of the development of the toxic effect in murine hepatocytes. [Ca2+]i elevation in acutely cadmium-exposed hepatocytes is closely related to the extracellular Ca2+ entry and an excessive release of Ca2+ from intracellular stores., S. S. Wang, L. Chen, S. K. Xia., and Obsahuje bibliografii a bibliografické odkazy
Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i ) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca2+ pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent with a NTPDase identity. The presence of a NTPDase was demonstrated by immunohistochemistry in rat SMG. Immunoreactivity was stronger in ductal cells than in mucous and serous acini. Although this enzyme was observed in the plasma membrane, colocalization with the ER marker calnexin revealed a specific subcellular localization in this organelle of all three types of cell. The putative function of this NTPDase activity in salivary glands is discussed., M. A. Ostuni ...[et al.]., and Obsahuje seznam literatury
During shock, prognosis of a patient depends largely on intestinal barrier function. The potency of gut epithelium to represent an obstacle to toxins is determined by the blood supply. All established methods of mucosal function determination necessitate the functional involvement of bloodstream. Microdialysis allows monitoring of extracellular substances in the gut submucosa, but its potential use for gut barrier integrity assessment is unknown. Twelve rats underwent perfusion of the descending colon either with 20 % ethanol or control medium (vehicle). Both media contained equal amounts of a radioactive tracer substance (51Cr-EDTA). Mucosal permeability for 51Cr-EDTA was assessed by microdialysate to luminal perfusate activity ratios. Sampling was performed using the colon submucosal microdialysis technique. The group subjected to ethanol treatment had profound macro- and microscopical alterations in perfused colonic segment associated with a significant increase in tracer permeability during ethanol exposure (2.354±0.298 % for ethanol as opposed to 0.209±0.102 % for control group, p<0.01), which remained elevated for 60 min after cessation of ethanol administration (3.352±0.188 % for ethanol compared to 0.140± 0.0838 % for the control group, p<0.001). Submucosal microdialysis with radioactive tracer substance can be considered a feasible and advantageous alternative of gut barrier function estimation. Parallel monitoring of local tissue chemistry with this method remains a challenge in the future., N. Cibiček, H. Živná, Z. Zadák, J. Kulíř, E. Čermáková, V. Palička., and Obsahuje bibliografii a bibliografické odkazy
a1_Focal cerebral contusion can be dynamic and expansive. It has been proved that subsequent expansive contusion is caused by brain parenchyma damage, especially BBB damage. We investigated a group of patients with traumatic brain injury. The patients (n=18) were divided in to group I (n=7) of patients submitted to neurosurgery due to expansive contusion, and group II (n=11) of patients without surgery. Serum concentrations of NSE and S-100B protein were measured by electrochemiluminescence immunoassay, interleukin-6 (IL-6) was measured by chemiluminescent sequential immunometric assay and matrix metalloproteinases (MMP-9, MMP-2) were measured by immunoassays. Cortical biopsy specimens of brain were investigated by electron microscopy in patients with trauma brain injury submitted to neurosurgery. Biochemical investigation from first day up to third day after traumatic brain injury proved increased values of IL-6 (302.2±119.9 vs. 59.6±11.9 ng/l, p<0.02) and S-100B protein (3.064±1.064 vs. 0.649±0.182 μ g/l, p<0.05) in patients with expansive lesion compared to patients without expansive contusion. Significantly higher levels of MMP-9 (150.4±28.46 vs. 74.11±13.16 ng/l, p<0.05) and of MMP-2 (814.5±126.3 vs. 523.1±25.28 ng/l, p<0.05) were found during first 3 days after admission in group I compared to group II. MMP-9 has also elevated in group II from lower values after admission (74.11±13.16 ng/l) up to high levels on the 10th day of hospitalization (225.1±49.35 ng/l )., a2_Ultrastructural investigation of endothelial cells and surrounded tissue revealed perivascular hemorrhage, increased pinocytic activity of endothelial cells, and cytotoxic edema of astroglial cells. Multivesical bodies were disclosed inside the endothelial cells. Higher levels of serum protein S-100B and IL-6 correlated with ultrastructural changes of endothelial cells, and with inflammatory response following TBI, respectively., D. Vajtr ... [et al.]., and Obsahuje seznam literatury
Under normal conditions, antioxidants at the corneal surface are balanced with the production of reactive oxygen species without any toxic effects. Danger from oxidative stress appears when natural antioxidants are overwhelmed leading to antioxidant/prooxidant imbalance. The aim of the present study was to examine the activities of enzymes contributing to the antioxidant/prooxidant balance in normal corneal epithelium of various mammals. The enzyme activities of antioxidant superoxide dismutase and glutathione peroxidase, as well as prooxidant xanthine oxidoreductase/xanthine oxidase were examined using biochemical methods. Results show that superoxide dismutase activity is high in rabbits and guinea pigs, whereas in pigs the activity is low and in cows it is nearly absent. In contrast, glutathione peroxidase activity is high in cows, pigs and rabbits, whereas in guinea pigs the activity is low. As far as prooxidant enzymes are concerned, elevated xanthine oxidoreductase/xanthine oxidase activities were found in rabbits, lower activities in guinea pigs, very low activity in cows and no activity in pigs. In conclusion, the above results demonstrate inter-species variations in activities of enzymes participating in antioxidant/prooxidant balance in the corneal epithelium. It is suggested that the levels of antioxidant and prooxidant enzymes studied in the corneal epithelium might be associated with the diurnal or nocturnal activity of animals. UV rays decompose hydrogen peroxide to damaging hydroxyl radicals and perhaps for this reason large animals with diurnal activity (cow, pig) require more effective peroxide removal (high glutathione peroxidase activity) together with the suppression of peroxide production (low superoxide dismutase activity, low xanthine oxidoreductase activity)., J. Kovačeva, J. Pláteník, M. Vejražka, S. Štípek, T. Ardan, Č. Čejka, A. Midelfart, J. Čejková., and Obsahuje bibliografii a bibliografické odkazy
Acetylcholinesterase (AChE) inhibitors represent standard treatment of Alzheimer´s disease. Cholesterol plays an important role in Alzheimer´s disease development. Because cholesterol synthesis may be inhibited by statins or bisphosphonates, we hypothesized that these drugs might possibly have an influence on cholinesterases. Moreover, we also evaluated if the cholesterol-lowering agents that cross the blood-brain barrier (e.g. simvastatin) should be more effective than those which do not (e.g. atorvastatin). Four groups of rats were orally administered simvastatin, atorvastatin, alendronate or vehicle for seven days. Thereafter, blood samples were taken and the basal ganglia, septum, frontal cortex, and hippocampus were isolated from brains for measurement of acetylcholinesterase activity. In the blood, activities of neither acetyl- nor butyrylcholinesterase were influenced by any of the applied drugs. In the brain, no significant changes in AChE activity were observed after administration of atorvastatin. Both simvastatin and alendronate significantly suppressed the activity of AChE in the frontal cortex. In conclusion, our results confirmed the hypothesis that cholesterol-modifying drugs modulate AChE activity and it is more reasonable to use a blood-brain barrier penetrating drug., Ľ. Cibičková, V. Palička, N. Cibiček, E. Čermáková, S. Mičuda, L. Bartošová, D. Jun., and Obsahuje bibliografii a bibliografické odkazy
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