Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia-reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.
- MeSH
- acetylcholin metabolismus MeSH
- acetylcholinesterasa analýza metabolismus MeSH
- butyrylcholinesterasa analýza metabolismus MeSH
- GPI-vázané proteiny analýza metabolismus MeSH
- izoenzymy analýza metabolismus MeSH
- kolagen analýza metabolismus MeSH
- krysa rodu rattus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- membránové proteiny analýza metabolismus MeSH
- myokard enzymologie MeSH
- potkani Wistar MeSH
- proteiny nervové tkáně analýza metabolismus MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Histochemická diagnostika Hirschsprungovy nemoci byla na našem pracovišti zavedena v 70. letech minulého století, v roce 2015 jsme nově přidali imunohistochemický průkaz kalretininu na řezech z parafinových bloků. Od té doby jsme kombinací obou metod diagnostikovali morbus Hirschsprung u 13 pacientů, u 34 jsme jej vyloučili. Výhodou vyšetření exprese kalretininu je snadná interpretace a vysoká spolehlivost nezávislá na věku, genetickém pozadí a rozsahu choroby. Počet nediagnostických vzorků byl mimořádně nízký (3,8 %). Histochemie je pomocnou metodou, která koriguje nejasné nálezy a stále se osvědčuje při diagnostice krátkého a ultrakrátkého segmentu. Hranice aganglionárního úseku, které jsme před operací stanovili pomocí etážových odběrů s kalretininem, velmi dobře korelovaly s nálezem v definitivním resekátu. Proti tomu histochemická detekce vedla častěji k podhodnocení, protože reaktivita acetylcholinesterázy se bez ohledu na délku postiženého segmentu orálním směrem vytrácí.
Histochemical diagnosis of Hirschsprung´s disease at our institution was introduced in the 1970s, calretinin imunohistochemistry on formalin fixed tissue was newly added in 2015. Employing both methods we were able to confirm Hirschsprung´s disease in 13 patients and exclude it in 34 patients since then. Calretinin seems highly reliable and easy to evaluate, it is not influenced by patient´s age, associated genetic features or the length of agangliosis. The number of inadequate samples was very low (3.8%). Histochemistry is useful as an adjunct tool to correct equivocal findings of calretinin staining and to facilitate diagnosis of short and ultra-short Hirschsprung´s disease. Serial biopsies from distal rectum and adjacent large bowel were obtained to assess the length of agangliosis preoperatively. The results of calretinin imunohistochemistry correlated very well with the findings in the colectomy specimens. In contrast, the length of affected bowel detected by histochemistry was often underestimated because acetylcholinesterase activity always diminishes orally irrespective of the length of aganglionic portion.
- MeSH
- acetylcholinesterasa analýza MeSH
- biopsie MeSH
- dítě MeSH
- Hirschsprungova nemoc * diagnóza chirurgie patologie MeSH
- imunohistochemie MeSH
- kalbindin 2 * analýza MeSH
- kojenec MeSH
- kolonoskopie MeSH
- lidé MeSH
- mladiství MeSH
- novorozenec MeSH
- rektum anatomie a histologie MeSH
- vrozené vady MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
Acetylcholinesterase reactivators (oximes) are compounds used for antidotal treatment in case of organophosphorus poisoning. The dissociation constants (pK(a1)) of ten standard or promising acetylcholinesterase reactivators were determined by ultraviolet absorption spectrometry. Two methods of spectra measurement (UV-vis spectrometry, FIA/UV-vis) were applied and compared. The soft and hard models for calculation of pK(a1) values were performed. The pK(a1) values were recommended in the range 7.00-8.35, where at least 10% of oximate anion is available for organophosphate reactivation. All tested oximes were found to have pK(a1) in this range. The FIA/UV-vis method provided rapid sample throughput, low sample consumption, high sensitivity and precision compared to standard UV-vis method. The hard calculation model was proposed as more accurate for pK(a1) calculation.
- MeSH
- acetylcholinesterasa analýza chemie MeSH
- chemie farmaceutická metody normy MeSH
- oximy analýza chemie MeSH
- reaktivátory cholinesterázy analýza chemie MeSH
- spektrofotometrie ultrafialová metody normy MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
CART (cocaine- and amphetamine-regulated transcript) peptide is a neuropeptide with a powerful central anorexigenic effect. Specific CART peptide binding sites, most likely CART peptide receptors, have been found in PC12 cells. This study further characterizes the CART peptide binding sites in PC12 cells. After differentiation to a neuronal phenotype with nerve growth factor, the number of CART peptide binding sites in PC12 cells tripled. Following dexamethasone treatment, which transforms PC12 cells into chromaffin-like cells, the number of CART peptide binding sites substantially decreased. CART peptide did not affect the differentiation or acetylcholinesterase activity of PC12 cells, indicating that CART peptide does not participate in differentiation or neuronal activity. CART peptide increased the phosphorylation of SAPK/JNK (stress-activated protein kinase/c-Jun-amino-terminal kinase) and subsequent c-Jun protein expression. These effects were reversed by SP600125, a specific JNK-kinase inhibitor. CART peptide did not significantly affect ERK (extracellular signal-regulated kinase), CREB (cAMP responsive element binding protein), or p38 phosphorylation and c-Fos protein expression. Central administration of CART peptide into mice also resulted in increased c-Jun positive cells in dorsomedial hypothalamic nucleus and nucleus of the solitary tract, areas involved in food intake regulation. Activation of c-Jun by CART peptide might indicate a possible role of CART peptide in managing stress conditions rather than a role in cell proliferation or differentiation as well as the more complex and/or specific regulation ways by transcription factors in some nuclei involved in food intake regulation. The characteristics of stress that CART peptide potentially mediates should be further studied.
- MeSH
- acetylcholinesterasa analýza MeSH
- buňky PC12 MeSH
- hypothalamus účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nucleus solitarius účinky léků metabolismus MeSH
- proteiny nervové tkáně metabolismus farmakologie MeSH
- receptory peptidů metabolismus MeSH
- signální transdukce fyziologie MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Poisoning from chemical warfare agents (CWAs) such as sarin is associated with neuronal degeneration. This damage is thought to result from glutamatergic excitotoxicity such as seen following kainic acid induced seizures. In order to search for novel neuroprotectants it is necessary to select good mouse models for susceptibility to nerve agent-induced seizures and the resulting neurodegeneration. The mouse strains tested (C57BL/6, ICR, DBA/2, SW, and FVB/N, Harlan Laboratory) had widely different sensitivity to sarin as shown by differences in the dose required resulting in 50% mortality, LD50. Differences also were observed among the strains in Fluoro-Jade C staining with the C57BL/6 and DBA having little to no staining when euthanized at 7 days whereas the other strains did. Differences in acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity were found among the strains as well. The ICR strain was excluded from the FOB and weight data due to difficulty getting a consistent LD50. Weight loss and FOB scores were similar for all strains. All strains had inhibited AChE activity after sarin exposure and exhibited inhibition of CNS BuChE after sarin exposure but only ICR and SW reached significance.
- MeSH
- acetylcholinesterasa analýza krev MeSH
- butyrylcholinesterasa analýza krev MeSH
- chemické bojové látky otrava MeSH
- cholinesterasové inhibitory aplikace a dávkování otrava MeSH
- chování zvířat účinky léků MeSH
- degenerace nervu * chemicky indukované patologie MeSH
- druhová specificita MeSH
- hmotnostní úbytek účinky léků MeSH
- inbrední kmeny myší * MeSH
- LD50 MeSH
- myši inbrední C57BL MeSH
- myši inbrední DBA MeSH
- myši inbrední ICR MeSH
- myši MeSH
- neurony patologie účinky léků MeSH
- otrava organofosfáty MeSH
- pohybová aktivita účinky léků MeSH
- sarin * aplikace a dávkování otrava MeSH
- tělesná hmotnost účinky léků MeSH
- záchvaty chemicky indukované patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Flow injection analysis (FIA) is an analytical method where the reaction mixture is injected into flow of liquid. The reaction product is monitored by a suitable detector such as ultraviolet/visible (UV/VIS) spectrophotometric or electrochemical detector. Mass spectrometric detectors (MS) are coming to be a standard equipment of analytical laboratories in the present time. This work is focused on application of FIA-MS instrumentation for monitoring of Ellman's reaction where both reactants (acetylthiocholine and 5,5’-dithiobis-2-nitrobenzoic acid, DTNB) and the reaction product (5-mercapto-2-nitrobenzoic acid) are monitored. This reaction is usually used for monitoring of acetylcholinesterase and butyrylcholinesterase. Due to its simplicity, the developed method is generally applicable for monitoring of enzymatic reactions of cholinesterases. The main advantage of this method is high selectivity and reduction of influence of compounds, which are reacting with DTNB, resulting in a color product of Ellman's reaction.
Illegal poisoning of wildlife and domestic animals is a worldwide issue. The carbamates primarily used as pesticides are often misused for such a purpose. In this study, 181 birds, mammals and baits were analysed over the period 2004-2009 for possible intoxication by carbamates. Intoxication by carbamate carbofuran was diagnosed in 89 cases, and in another 19 cases (nine Wild Boars and 10 Bisons) intoxication with another carbamate-methomyl - was proven. Incidental ingestion of the marten bait was the main cause of intoxication. Although the distribution of carbofuran was prohibited in 2007, no decline in the number of intoxicated animals in the following two years was detected. New cases of intoxication by carbofuran are anticipated in the future until all remaining stock is expended.
- MeSH
- acetylcholinesterasa analýza MeSH
- cholinesterasové inhibitory otrava škodlivé účinky toxicita MeSH
- chromatografie plynová MeSH
- financování organizované MeSH
- gastrointestinální obsah chemie MeSH
- histologické techniky MeSH
- játra chemie MeSH
- karbamáty otrava MeSH
- karbofuran otrava MeSH
- methomyl otrava MeSH
- organofosforové sloučeniny otrava toxicita MeSH
- otrava epidemiologie veterinární MeSH
- pesticidy otrava toxicita MeSH
- příčina smrti MeSH
- ptáci MeSH
- retrospektivní studie MeSH
- rezidua pesticidů analýza MeSH
- savci MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Organophosphates present serious fulmination in several aspects of human life. Detection of organophosphates is frequently based on following acetylcholinesterase (AChE) inhibition. Although limit of detection and sensitivity for AChE-based assays seem to be intriguing, the identification of organophosphates is not currently efficient in this way. We introduce an improvement of AChE-based assay by reactivators using a selective come-back of AChE activity after previous inhibition. We have chosen four organophosphates: paraoxon-ethyl, paraoxon-methyl, trichlorfon, methamidophos as representative pesticides and the three most available reactivators: HI-6, obidoxime, pralidoxime. Reactivation was realized in the 96-wells photometric microplates and activity of human recombinant AChE was followed by reaction of Ellman's reagent with one of enzyme digestion product: thiocholine. Distinguishing of reactivation efficacy was judged by the independent two population t-test. The most significant identification was based on methamidophos inhibited AChE reactivation by HI-6 or pralidoxime and paraoxon-ethyl inhibited AChE by obidoxime; moreover, identification of trichlorfon and paraoxon-methyl was possible, too. Practical impact of described method is discussed.
