ATP sulfurylase (ATPS) catalyzes the first step of sulfur assimilation in photosynthetic organisms. An ATPS type A is mostly present in freshwater cyanobacteria, with four conserved cysteine residues. Oceanic cyanobacteria and most eukaryotic algae instead, possess an ATPS-B containing seven to ten cysteines; five of them are conserved, but only one in the same position as ATPS-A. We investigated the role of cysteines on the regulation of the different algal enzymes. We found that the activity of ATPS-B from four different microorganisms was enhanced when reduced and decreased when oxidized. The LC-MS/MS analysis of the ATPS-B from the marine diatom Thalassiosira pseudonana showed that the residue Cys-247 was presumably involved in the redox regulation. The absence of this residue in the ATPS-A of the freshwater cyanobacterium Synechocystis sp. instead, was consistent with its lack of regulation. Some other conserved cysteine residues in the ATPS from T. pseduonana and not in Synechocystis sp.were accessible to redox agents and possibly play a role in the enzyme regulation. Furthermore, the fact that oceanic cyanobacteria have ATPS-B structurally and functionally closer to that from most of eukaryotic algae than to the ATPS-A from other cyanobacteria suggests that life in the sea or freshwater may have driven the evolution of ATPS.
- MeSH
- chromatografie kapalinová MeSH
- cystein metabolismus MeSH
- dithiothreitol farmakologie MeSH
- mikrořasy enzymologie MeSH
- molekulární modely MeSH
- oxidace-redukce účinky léků MeSH
- peptidy chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- sulfurylasa chemie metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
Elevated levels of pteridines can indicate the activation of cellular immune system by certain diseases. No work dealing with the simultaneous determination of urinary neopterin, biopterin and their reduced forms has been published. Therefore, a new SPE-UHPLC-FD method for the analysis of these compounds has been developed. The main emphasis was put on the stability of dihydroforms during the sample processing and storage. As a stabilizing agent, dithiothreitol, at various concentrations, and various pH values (3.8-9.8) of working solutions were tested. Chromatographic separation was performed under HILIC isocratic conditions on BEH Amide column. The method was linear for the calibration standard solutions in the range of 10-10,000 ng/ml (dihydroforms) and 0.5-1000 ng/ml (oxidized forms), and for real samples in the range of 25-1000 ng/ml (dihydroforms) and 1-100 ng/ml (oxidized forms). The development of a new SPE sample preparation method was carried out on different types of sorbents (based on a mixed-mode cation exchange, porous graphitic carbon and a polymer comprising hydrophilic and hydrophobic components). Final validation was performed on a MCAX SPE column. Method accuracy ranged from 76.9 to 121.9%. The intra- and inter-day precision did not exceed 10.7%. The method provided high sensitivity for the use in routine clinical measurements of urine (LLOQ 1 ng/ml for oxidized forms and 25 ng/ml for dihydroforms). Average concentrations of biopterin, neopterin, and dihydrobiopterin found in urine of healthy persons were related to the mol of creatinine (66.8, 142.3, and 257.3 μmol/mol of creatinine, respectively) which corresponded to the literature data. The concentration of dihydroneopterin obtained using our method was 98.8 μmol/mol of creatinine.
It has been shown that drug resistance is extremely common in hepatocellular carcinoma (HCC) and is one of the major problems in HCC chemotherapy. However, the detailed mechanisms remain largely unknown. We have previously shown that endoplasmic reticulum (ER) stress is involved in the tumorigenesis of HCC. Here, we demonstrated that the unfolded protein response (UPR) inhibits cisplatin-induced HCC cell apoptosis. In HCC cells, cisplatin treatment triggers the UPR, which subsequently inhibits cisplatin-induced apoptosis. Importantly, mild ER stress precondition suppresses the sensitivity of HCC cells to cisplatin-induced apoptosis through autophagy regulation. Furthermore, heat-shock protein 27 (Hsp27) is involved in the cytoprotective role of the UPR in cisplatin-induced apoptosis. We also demonstrated that Hsp27 inhibits cisplatin- induced HCC cell death through autophagy activation. Taken together, our results indicate that the UPR inhibits cisplatin-induced apoptosis in HCC cells, at least in part, by Hsp27-mediated autophagy activation.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza fyziologie účinky léků MeSH
- autofagie fyziologie účinky léků MeSH
- cisplatina farmakologie metabolismus MeSH
- dithiothreitol farmakologie MeSH
- endoplazmatické retikulum fyziologie účinky léků MeSH
- hepatocelulární karcinom patologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory jater patologie MeSH
- proteiny tepelného šoku HSP27 genetika metabolismus MeSH
- reakce na tepelný šok MeSH
- signální dráha UPR fyziologie MeSH
- tunikamycin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
The potential pro-survival role of phosphatidylinositol 3-kinase (PI3K)/Akt during endoplasmic reticulum stress has been well-characterized. However, the detailed mechanisms remain largely unknown. Here, we showed that PI3K/Akt inhibition promoted endoplasmic reticulum stress-induced apoptosis in a glucose-regulated protein 78 (GRP78)-dependent manner. During endoplasmic reticulum stress, high levels of Akt phosphorylation were sustained for at least 18 h in HEK293 cells. Importantly, PI3K/Akt enhanced GRP78 accumulation through increasing its stability following endoplasmic reticulum stress. Furthermore, Akt1, but not Akt2 or Akt3, was involved in GRP78 stability regulation. These results suggest that PI3K/Akt inhibits endoplasmic reticulum stress-induced apoptosis in HEK293 cells, at least in part, by promoting GRP78 protein stability.
- MeSH
- apoptóza fyziologie účinky léků MeSH
- buněčné linie MeSH
- buňky NIH 3T3 MeSH
- dithiothreitol farmakologie MeSH
- endoplazmatické retikulum metabolismus účinky léků MeSH
- fosfatidylinositol-3-kinasy genetika metabolismus MeSH
- fyziologický stres MeSH
- inhibitory enzymů farmakologie MeSH
- lidé MeSH
- myši MeSH
- proteiny tepelného šoku genetika metabolismus MeSH
- protoonkogenní proteiny c-akt genetika metabolismus MeSH
- thapsigargin farmakologie MeSH
- transkripční faktor CHOP metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
We have previously reported that the reducing agent dithiothreitol (DTT) strongly increases thermally induced activity of the transient receptor potential vanilloid receptor-1 (TRPV1) channel. Here, we show that exposure to oxidizing agents also enhances the heat-induced activation of TRPV1. The actions of sulfhydryl modifiers on heat-evoked whole-cell membrane currents were examined in TRPV1-transfected human embryonic kidney 293T cells. The sensitizing effects of the membrane-permeable oxidizing agents diamide (1 mM), chloramine-T (1 mM), and the copper-o-complex (100:400 microM) were not reversed by washout, consistent with the stable nature of covalently modified sulfhydryl groups. In contrast, the membrane-impermeable cysteine-specific oxidant 5,5'-dithio-bis-(2-nitrobenzoic acid) (0.5 mM) was ineffective. The alkylating agent N-ethylmaleimide (1 mM) strongly and irreversibly affected heat-evoked responses in a manner that depended on DTT pretreatment. Extracellular application of the membrane-impermeable reducing agent glutathione (10 mM) mimicked the effects of 10 mM DTT in potentiating the heat-induced and voltage-induced membrane currents. Using site-directed mutagenesis, we identified Cys621 as the residue responsible for the extracellular modulation of TRPV1 by reducing agents. These data suggest that the vanilloid receptor is targeted by redox-active substances that directly modulate channel activity at sites located extracellularly as well as within the cytoplasmic domains. The results obtained demonstrate that an optimal redox state is crucial for the proper functioning of the TRPV1 channel and both its reduced and oxidized states can result in an increase in responsiveness to thermal stimuli.
- MeSH
- buněčné linie MeSH
- diamid farmakologie MeSH
- dithiothreitol farmakologie MeSH
- ethylmaleimid farmakologie MeSH
- financování organizované MeSH
- kapsaicin farmakologie MeSH
- kationtové kanály TRPV fyziologie genetika MeSH
- krysa rodu rattus MeSH
- kyselina dithionitrobenzoová farmakologie MeSH
- lidé MeSH
- membránové potenciály účinky léků MeSH
- metoda terčíkového zámku MeSH
- missense mutace genetika MeSH
- mutace genetika MeSH
- mutantní proteiny fyziologie genetika MeSH
- oxidancia farmakologie MeSH
- peroxid vodíku farmakologie MeSH
- redukční činidla farmakologie MeSH
- sulfhydrylová reagencia farmakologie MeSH
- transfekce MeSH
- vysoká teplota MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
Adenylyl cyclase (AC) in brain cortex from young (12-day-old) rats exhibits markedly higher activity than in adult (90-day-old) animals. In order to find some possibly different regulatory features of AC in these two age groups, here we modulated AC activity by dithiothreitol (DTT), Fe(2+), ascorbic acid and suramin. We did not detect any substantial difference between the effects of all these tested agents on AC activity in cerebrocortical membranes from young and adult rats, and the enzyme activity was always about two-fold higher in the former preparations. Nevertheless, several interesting findings have come out of these investigations. Whereas forskolin- and Mn(2+)-stimulated AC activity was significantly enhanced by the addition of DTT, increased concentrations of Fe(2+) ions or ascorbic acid substantially suppressed the enzyme activity. Lipid peroxidation induced by suitable combinations of DTT/Fe(2+) or by ascorbic acid did not influence AC activity. We have also observed that PKC- or protein tyrosine kinase-mediated phosphorylation apparently does not play any significant role in different activity of AC determined in cerebrocortical preparations from young and adult rats. Our experiments analysing the presumed modulatory role of suramin revealed that this pharmacologically important drug may act as a direct inhibitor of AC. The enzyme activity was diminished to the same extent by suramin in membranes from both tested age groups. Our present data show that AC is regulated similarly in brain cortex from both young and adult rats, but its overall activity is much lower in adulthood.
- MeSH
- adenylátcyklasy antagonisté a inhibitory chemie metabolismus MeSH
- buněčná membrána metabolismus MeSH
- časové faktory MeSH
- dithiothreitol farmakologie MeSH
- financování organizované MeSH
- fluoridy farmakologie MeSH
- fosforylace MeSH
- guanosin 5'-O-(3-thiotrifosfát) metabolismus MeSH
- kolforsin farmakologie MeSH
- krysa rodu rattus MeSH
- kyselina askorbová farmakologie chemie MeSH
- mangan farmakologie MeSH
- mozek enzymologie metabolismus MeSH
- mozková kůra enzymologie metabolismus MeSH
- peroxidace lipidů MeSH
- potkani Wistar MeSH
- proteinkinasa C metabolismus MeSH
- sloučeniny hliníku farmakologie MeSH
- stárnutí MeSH
- suramin farmakologie chemie metabolismus MeSH
- tyrosinkinasy metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- železo chemie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
