Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).
- MeSH
- bakteriální proteiny izolace a purifikace toxicita MeSH
- cytotoxiny izolace a purifikace toxicita MeSH
- detergenty chemie MeSH
- erytrocyty účinky léků MeSH
- Escherichia coli metabolismus MeSH
- hemolýza MeSH
- hemolyziny izolace a purifikace toxicita MeSH
- lidé MeSH
- lipopolysacharidy analýza MeSH
- močovina chemie MeSH
- nádorové buněčné linie MeSH
- oktoxynol chemie MeSH
- ovce MeSH
- THP-1 buňky MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Chemical restraint of wild animals is practiced to accomplish intended procedures such as capture, clinical examination, collection of diagnostic samples, treatment and/or transport. Extra-label use of animal medicinal drugs is often necessary in wildlife because most approved therapeutics do not list wild species on the labelling. Here, we used cellular in vitro models, a cutting-edge tool of biomedical research, to examine cytotoxicity of anaesthetic agents in fallow deer and extrapolate these data for anaesthetic risks in wildlife. METHODS: We examined the cytotoxic effects of ketamine, xylazine, and ketamine-xylazine, i.e. the Hellabrunn mixture, on liver-, heart- and kidney-derived cell cultures prepared from a fallow deer (Dama dama) specimen. In line with preliminary studies we exposed cells to 10 µM, 50 µM, 100 µM, 1 mM, and 10 mM ketamine or xylazine. The combination of ketamine-xylazine was dosed at 0.025+0.02 mg/ml, 0.05+0.04 mg/ml, 0.75+0.06 mg/ml, 0.1+0.08 mg/ml, and 0.125+0.1 mg/ml per one well containing 10 000 cells. The quantification of cytotoxicity was based on lactate dehydrogenase activity released from damaged cells. RESULTS: Liver-derived cells show higher sensitivity to the cytotoxic effects of both ketamine and xylazine administered as single drugs when compared with cells cultured from the heart and kidney. The Hellabrunn mixture induced significantly higher cytotoxicity for kidney-derived cells ranging from 16.78% to 35.6%. Single and combined exposures to ketamine and xylazine resulted only in high-dose cytotoxicity in the heart-derived cells. CONCLUSIONS: Our results indicate that immobilization drugs significantly differ in their cytotoxic effects on cells derived from various organs of the fallow deer.
- MeSH
- analgetika toxicita MeSH
- cytotoxiny toxicita MeSH
- játra cytologie metabolismus MeSH
- ketamin analogy a deriváty toxicita MeSH
- ledviny cytologie metabolismus MeSH
- srdce účinky léků MeSH
- testy toxicity metody MeSH
- vysoká zvěř * MeSH
- xylazin analogy a deriváty toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
2'-Deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2'-deoxythymidine 5'-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.
- MeSH
- buněčné linie MeSH
- cytotoxiny metabolismus toxicita MeSH
- deoxyuridin analogy a deriváty metabolismus toxicita MeSH
- DNA biosyntéza genetika metabolismus MeSH
- inhibitory enzymů metabolismus toxicita MeSH
- intracelulární prostor účinky léků metabolismus MeSH
- lidé MeSH
- poškození DNA * MeSH
- proliferace buněk účinky léků MeSH
- replikace DNA účinky léků MeSH
- S fáze účinky léků MeSH
- tetrahydrofoláty biosyntéza MeSH
- thymidin metabolismus farmakologie MeSH
- thymidinmonofosfát metabolismus MeSH
- thymidylátsynthasa antagonisté a inhibitory MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Oxime HI-6 is an efficient reactivator of the acetylcholinesterase inhibited by organophosphorous nerve agents. In this study we have estimated cytotoxicity of HI-6 by the colony forming assay and genotoxicity by the comet assay on human and rodent cell lines. IC50 of HI-6 assessed by the colony forming capacity was 3.59 mM for HeLa cells and 5.18 mM for a mouse cell line L929. Small difference in cytotoxicity was found among other cell lines tested: IC50 was 1.61 mM for human A549 cells, 1.14 mM for UROtse line, 1.96 mM and 1.71 mM for Chinese hamster cells AA8 and UV-20, respectively. The A549 cell viability measured with the MTT test was 5 times decreased comparing 2 and 24 hours of HI-6 oxime treatment. The 5 mM HI-6 concentration reduced the viability within 2 hours to 95% only, however, it induced a significant number of DNA breaks in mouse cells L929, and also in human UROtse and HepG2 cells. 1-β-D-arabinofuranosylcytosine (10(-4) M) and hydroxyurea (10(-2) M), supplemented to the cultivation medium, did not cause any significant accumulation of DNA breaks during treatment, which indicated that the nucleotide excision repair was not acting on the induced DNA damage.
- MeSH
- analýza kolonii tvořících jednotek MeSH
- antidota toxicita MeSH
- buněčné linie účinky léků fyziologie MeSH
- cytotoxiny toxicita MeSH
- druhová specificita MeSH
- křečci praví MeSH
- krysa rodu rattus MeSH
- LD50 MeSH
- lidé MeSH
- mutageny toxicita MeSH
- myši MeSH
- oximy toxicita MeSH
- poškození DNA fyziologie MeSH
- pyridinové sloučeniny toxicita MeSH
- reaktivátory cholinesterázy toxicita MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The biological activity of cyanobacteria and their chemical components have been widely studied due to their blooms in eutrophic waters worldwide. The primary goal of this study was to determine if individual cyanobacterial species and mixtures of cyanobacteria collected from the environment contain compounds with the potential for interaction with signaling pathways of the aryl hydrocarbon receptor (AhR), androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR) and retinoid acid receptor (RAR). Cytotoxicity and specific toxic potencies of products of freshwater cyanobacteria were determined by use of in vitro reporter gene trans-activation assays. The testing included samples prepared from five selected single cyanobacterial species cultivated in laboratory and five complex cyanobacterial biomasses collected from blooms in surface waters in the Czech Republic. The results demonstrate estrogenic potencies of extracts of cyanobacterial biomasses. Among the laboratory single species, the extract of Planktothrix agardhii (intracellular metabolites) had a potency of estrogenic equivalents (EEQ) of 3.8 ng 17β-estradiol/g dw. The estimates of EEQs of samples prepared from complex cyanobacterial biomasses collected from freshwaters in the Czech Republic ranged from 19 to 2200 ng 17β-estradiol/g dw. Several samples prepared from the environmental cyanobacterial biomasses potentiated the androgenic potency of dihydrotestosterone. There was no dioxin-like, glucocorticoid or anti/retinoic activity observed for any of the extracts studied. Extracts of natural complex cyanobacterial biomasses exhibited greater and more frequent presence of compounds with specific modes of action, mainly estrogenic, and also greater cytotoxicity than extracts of single cyanobacterial species. The demonstrated estrogenic potency of the compounds present in complex cyanobacterial biomasses is of environmental relevance, and could potentially contribute to endocrine disruptive effects in aquatic ecosystems in case of great bloom densities.
- MeSH
- androgenní receptory metabolismus MeSH
- bakteriální toxiny toxicita MeSH
- biomasa MeSH
- biotest MeSH
- buněčné linie MeSH
- cytotoxiny toxicita MeSH
- endokrinní disruptory toxicita MeSH
- estrogeny toxicita MeSH
- komplexní směsi toxicita MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mikrocystiny toxicita MeSH
- nádorové buněčné linie MeSH
- receptory aromatických uhlovodíků metabolismus MeSH
- receptory glukokortikoidů metabolismus MeSH
- receptory kyseliny retinové metabolismus MeSH
- receptory pro estrogeny metabolismus MeSH
- signální transdukce účinky léků MeSH
- sinice chemie MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Using exhaustive chromatographic separation we have isolated (-)-tigloyl-deangeloyl-gomisin F as a novel dibenzocyclooctadiene lignan from schisandra chinensis. With the help of HPLC, we further isolated (+)-schisandrin, (+)-deoxyschisandrin, (+)-γ-schisandrin, (-)-gomisin J, (+)-gomisin A, (-)-gomisin N, (-)-tigloyl-gomisin P, (-)-wuweizisu C, (-)-gomisin D, rubrisandrin A, (-)-gomisin G, (+)-gomisin K (3) and (-)-schisantherin C. A full NMR description of (-)-schisantherin C was carried out with the aim to confirm previous reports of its structure. Compounds isolated were identified on the basis of UV, IR, (1)H- and (13)C-NMR and MS. The cytotoxicity of lignans was tested for the BY-2 cell line alone and as a synergistic effect with the cytotoxic agent camptothecin. Lignans showed various toxicity and synergistic and antagonistic effects on camptothecin-induced cytotoxicity. Cytotoxicity against colon cancer cell line LoVo was also tested.
- MeSH
- apoptóza účinky léků MeSH
- buněčné linie MeSH
- chemická frakcionace MeSH
- cytotoxiny chemie izolace a purifikace toxicita MeSH
- lidé MeSH
- lignany chemie izolace a purifikace toxicita MeSH
- nádorové buněčné linie MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- proliferace buněk účinky léků MeSH
- rostlinné extrakty chemie MeSH
- Schisandra chemie MeSH
- tabák účinky léků MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A number of cytotoxicity assays are currently available, each of them using specific approach to detect different aspects of cell viability, such as cell integrity, proliferation and metabolic functions. In this study we compared the potential of five commonly employed cytotoxicity assays (WST-1, XTT, MTT, Brilliant blue and Neutral red assay) to detect antiproliferative effects of three selenium compounds, sodium selenite, seleno-L-methionine (SeMet) and Se-(Methyl)selenocysteine (SeMCys) on three colorectal cancer cell lines in vitro. Cells were exposed to the selected selenium compounds in the concentration range of 0-256 microM during 48 h. WST-1 and XTT failed to detect cytotoxic effect, with the exception of the highest concentration of selenium compounds tested. Conversely, the metabolic activity of selenium treated cells measured by WST-1 and XTT significantly increased in comparison to untreated controls. MTT, Neutral red and Brilliant blue assays were more sensitive and yielded mutually comparable results, with significant decrease of measured parameters in a concentration-dependent manner. To a smaller extent, the results were affected by the different chemical nature of the selenium compounds tested as well as by the biological properties of individual cell lines.
- MeSH
- antikarcinogenní látky terapeutické užití toxicita MeSH
- cystein analogy a deriváty terapeutické užití toxicita MeSH
- cytotoxiny terapeutické užití toxicita MeSH
- lidé MeSH
- methionin analogy a deriváty terapeutické užití toxicita MeSH
- nádorové buněčné linie MeSH
- nádory tračníku farmakoterapie metabolismus prevence a kontrola MeSH
- organoselenové sloučeniny terapeutické užití toxicita MeSH
- proliferace buněk účinky léků MeSH
- seleničitan sodný terapeutické užití toxicita MeSH
- testy toxicity metody MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Kardiotoxicita je jedním z možných nežádoucích úcinku onkologické lécby. K diagnostice kardiotoxicity byla doporucena rada metod. V našich podmínkách se rutinne používá elektrokardiografie a echokardiografie. Clánek podává prehled o možnostech elektrokardiografie v diagnostice kardiotoxicity onkologické lécby a obsahuje vlastní zkušenosti autoru s touto problematikou.
Cardiac toxicity is one of the possible adverse events of oncology treatment. Various methods have been recommended for diagnostics of cardiac toxicity. In our conditions, electrocardiography and echocardiography are routinely used. The article gives a review of the potential of electrocardiography in diagnostics of cardiac toxicity of oncology treatment and contains the authors' own experience with this problem.
- MeSH
- antracykliny škodlivé účinky terapeutické užití toxicita MeSH
- cytostatické látky škodlivé účinky toxicita MeSH
- cytotoxiny škodlivé účinky toxicita MeSH
- elektrokardiografie metody účinky léků využití MeSH
- financování organizované využití MeSH
- kardiomyopatie diagnóza etiologie patofyziologie MeSH
- nádory farmakoterapie MeSH
- srdeční arytmie diagnóza etiologie MeSH
- syndrom dlouhého QT MeSH
The preparation of various 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils with alkyl chain lengths C(1)-C(12) is described. The synthesis is based on the preparation of 5-[chloro-(4-nitro-phenyl)-methyl]-uracil and subsequent substitution of chlorine with appropriate alcohols. The resulting ethers were tested for their cytotoxic activity in vitro against five cancer cell lines. The compounds were less active in lung resistance protein expressing cell lines, suggesting the involvement of this multidrug resistant protein in control of the biological activity. Cytotoxic substances induced rapid inhibition of DNA and modulation of RNA synthesis followed by induction of apoptosis. The data indicate that the biological activity of 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils depends on the alkyl chain length.
- MeSH
- alkoholy chemická syntéza toxicita MeSH
- antitumorózní látky chemická syntéza toxicita MeSH
- buňky K562 MeSH
- cytotoxiny chemická syntéza toxicita MeSH
- financování organizované MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- stereoizomerie MeSH
- uracil analogy a deriváty chemická syntéza toxicita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
