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Biomedical subjects

K Horáková

Publications and source records attributed to K Horáková.

At least 19 recordsLinked to original sources
PubMed2006

Direct detection of bacterial faecal indicators in water samples using PCR.

The presence of enteric pathogens in water resources represents a serious risk for public health. Therefore, their precise detection, and especially detection of E. coli, which is obviously regarded as the main indicator of faecal contamination of water, is an essential step in ensuring bacterial safety of water. Numerous PCR protocols for detection of E. coli have been published to date. They are usually based on amplification of regions derived from lacZ (beta-D-galactosidase) and uidA (beta-D-glucuronidase) gene sequences. However, these methods are not universal enough for precise detection of all E. coli strains found in water samples. We developed a novel triplex PCR method for detection of E. coli in which cyd gene coding for cytochrome bd complex was co-amplified along with lacZ and uidA genes. Our triplex PCR approach significantly increases the specificity and reliability of E. coli detection in water samples. This approach allowed us to distinguish Shigella flexneri from E. coli. In addition, we were able to detect even non-coliform Klebsiella and Raoutella spp., some of which can also cause infections to humans.

Base Sequence↗
PubMed2004

A comparison of the traditional method of counting viable cells and a quick microplate method for monitoring the growth characteristics of Listeria monocytogenes.

AIMS: To determine: (i) the growth parameters (specific growth rate, lag time, asymptotic amount of growth, generation time and time for maximum growth rate) of Listeria monocytogenes in different broths by standard cultivation methods and (ii) whether a microplate method in conjunction with a standard nondedicated plate reader could be adapted to routine assay. METHODS AND RESULTS: Growth curves were determined from cell numbers in a standard tube method at 2 h intervals by serial dilution and plating, and in a microplate method by absorbance measurements. Growth curves were fitted with a modified Gompertz function. CONCLUSIONS: The microplate method was similar to the standard cultivation methods in accuracy, required less chemical reagents, and considerably reduced the time required for analyses. This work also illustrates that growth characteristics of bacteria are not necessarily constant, and depend on the methodology used. SIGNIFICANCE AND IMPACT OF THE STUDY: It is not the intended purpose of this paper to present all the data for the media tested but instead to illustrate the success of the microplate method for studying growth kinetics compared to a standard cultivation method and system precision. The method will be of considerable benefit to laboratories unable to afford dedicated workstations.

Bacteriological Techniques↗
PubMed2001

Detection of drug-induced, superoxide-mediated cell damage and its prevention by antioxidants.

The mode of the cytotoxic activity of three benzo(c)fluorene derivatives was characterized. The observed morphological changes of lysosomes or variations of mitochondrial activity are assumed to be the consequence of cell protection against oxidative damage and/or the part of the damage process. To establish the relationship between the quantity of superoxide (O2*-) generated and the degree of damage resulting from O2*-, a simple system based on measurement of 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) reductase activity in the presence of superoxide dismutase (SOD) was used. The functionality of the chosen battery of in vitro tests was proved using several known superoxide inducers: cyclosporin A (CsA) and benzo(a)pyrene (BP), as well as noninducers: citrinin (CT) and cycloheximide (CH). From the results followed that the cell growth tests are much better indices of toxicity than the other tests. The model system for the evaluation of the protective capacity of antioxidants against superoxide-induced cytotoxicity included simultaneous exposure of HeLa cells to cytotoxic drugs and to quercetin (Qe), an antioxidant of plant origin. The complete abolishment of the inhibition of cell proliferation and clonogenic survival was concluded to be due to the protective effect of the antioxidant. These observations correlated with the decrease of superoxide content as estimated by the INT-reductase assay in the presence of SOD using the same model system, as well as with the increase of intracellular SOD content and its activity.

Antioxidants↗
PubMed2001

Antibacterial and mutagenic activities of new isothiocyanate derivatives.

Nine newly synthesized isothiocyanate derivatives were demonstrated to posses antibacterial and genotoxic activities in vitro. 4-Hydroxybutyl isothiocyanate exhibited a broad antibacterial effect, with MIC values of 762 mumol/L for Staphylococcus aureus and Escherichia coli. Ethyl 4-methylsulfoxidobutanoate had the highest antibacterial activity in Gram-positive bacteria, the MIC value being 425 mumol/L for S. aureus. The highest tested concentrations of ethyl 4-isothiocyanatobutanoate and 4-hydroxybutyl isothiocyanate produced a bacteriocidal effect in Gram-positive bacteria. The compounds showed no mutagenic effects on Salmonella typhimurium tester strains TA 98 and TA 100, either in the absence or in the presence of a metabolically active microsomal S9 fraction from rat liver using standard Ames test.

Animals↗
PubMed1999

Stobadine inhibits lysosomal enzyme release in vivo and in vitro.

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.

Acetylglucosaminidase↗
PubMed1999

The use of cell culture systems for the assessment of general cellular toxicity and to detect the nature and location of free radical damage.

In the process of developing compounds to counteract the damaging effects of free radicals in biological systems it is important to determine the type and the intracellular location of specific toxic events. For that purpose cultured cells are used, because a properly designed cell culture system allows to asses specific damage at the cellular and subcellular level and can contribute to an evaluation of the biochemistry of free radical damage. A wide range of changes in cellular activities resulting from oxidative injury in vitro have been demonstrated. Data from many laboratories indicate that cell culture system coupled with appropriate analytical techniques can be used to explore cellular and biochemical details of damage induced by free radicals. The type of reactive oxygen species used to generate the radicals, the rate of radical production, and the location of action of the toxic species must be taken into account to understand the biochemistry of the system. An effort is made to analyse the considerable progress made in the development of appropriate in vitro models and end-points for use in testing and characterising the nature and location of free radical cytotoxicity.

Animals↗
PubMed1999

Comparison of isoproterenol-induced changes in lysosomal enzyme activity in vivo and in vitro.

Isoproterenol was used as a drug which, when administered in high doses, is able to induce lysosomal enzyme activity changes in in vivo conditions. We correlated lysosomal enzyme activity in the absence and presence of isoproterenol, obtained in whole animals and in HeLa and HepG2 cells in tissue culture. In vivo experiments: male Wistar rats (270-300 g) were treated subcutaneously with isoproterenol in various doses. Effect of isoproterenol on lysosomal enzyme activity was assayed in the heart after differential centrifugation. In vitro experiments: Isoproterenol in concentrations 0.1-100 microg/ml was added to HeLa and HepG2 cells and the activity of lysosomal enzyme was measured in the cell homogenate. In the sedimentable and nonsedimentable fractions of the rat myocardium, the isoproterenol-induced changes in the activity of lysosomal enzyme were time-and dose-dependent. In HeLa cells, isoproterenol administration caused a dose-dependent increase of lysosomal enzyme activity, while in HepG2 cells the activity remained unchanged. Thus the isoproterenol-induced changes in lysosomal enzyme activity in the rat myocardium were comparable with the results found in vitro in HeLa cells.

Acetylglucosaminidase↗
PubMed1998

Disodium cromoglycate is neither cytotoxic nor genotoxic in mammalian cells in vitro.

The cytotoxic and genotoxic effects of disodium cromoglycate on mammalian cells were investigated. We used two types of hamster cells, a primary culture of Syrian hamster embryo cells and the cell line V79-4. Cytotoxicity was studied by using proliferation and plating efficiency assays. No toxic effect of disodium cromoglycate on hamster cells in the tested concentration range (2.4-48 microM) was observed. The growth of treated V79 cells was slightly stimulated during proliferation compared with control cells. This stimulation was not observed in the stationary phase of growth. Plating experiments confirmed that disodium cromoglycate has no cytotoxic effect on V79 cells. The genotoxicity of disodium cromoglycate (up to concentration 96 microM) was studied in both cell types using two assays, the DNA synthesis inhibition test and alkaline DNA unwinding with hydroxyapatite chromatography. In V79 cells, disodium cromoglycate had no effect on DNA synthesis. In Syrian hamster embryo cells disodium cromoglycate acts as a mild metabolic inhibitor of DNA synthesis. This effect is reversible. No single-strand breaks were found after treatment with disodium cromoglycate in either cell type. These results confirm that disodium cromoglycate is without cytotoxic effect (up to 48 microM) and, furthermore, lacks genotoxicity at least up to 96 microM.

Animals↗
PubMed1993

Expression of disodium cromoglygate 'protective' effects observed during V79 cell proliferation.

Disodium cromoglycate (DSCG) in equimolar concentration of 0.56 mumol/l preincubated with an asynchronous cell population decreases the inhibition of proliferation and results in a 100% elimination of inhibition of colony formation induced by benfluron (BF). DSCG protects the cells from unbalanced growth and unbalanced metabolism (e.g., prevention of increase or decrease in protein cell content, glycolytic activity and in amino acid metabolism) which are both the integrating parts of BF cytotoxic reaction. The protective effect of DSCG is manifested also on synchronous cell populations, particularly in G2 phase (26%). DSCG also stabilizes cell membrane by preventing the alterations of its permeability caused by the cytolytic concentrations of benfluron (at a concentration of 1.05 mumol/l and more).

Amino Acids↗
PubMed1993

Ethyl 4-isothiocyanatobutanoate--antiproliferative activity in vitro and in vivo.

A new isothiocyanate (ITC) derivate ethyl 4-isothiocyanatobutanoate (E-4IB) induces an immediate dose-dependent inhibitory action on the division of HeLa cells in the concentration range 1.0-0.1 mg/l. Concomitant with the decrease in cell proliferation which follows E-4IB treatment the protein:cell number ratio increases and DNA accumulates. Cells which have lost their ability to divide do not stop their glucose metabolism and only partly stop their glutamine metabolism. The increased content of DNA suggests that cells synthesize DNA without entering into mitosis and that dying cells are in late S or G2 phases prior to death. E-4IB produces a significant growth inhibition of transplanted sarcoma cells B77-RF in rats (at 28 mg/kg). A 57% regression in tumor volume was observed for at least 30 days following the completion of the in vivo treatment. These findings support the presumption that E-4IB is a potential anti-cancer drug. However, further studies are needed for the optimization of its in vivo activity.

Animals↗
PubMed1992

The relation between the synergistic effect of cytostatics and metabolism of V 79B cells in vitro.

A single-dose simultaneous application of methotrexate (MTX; 0.002/microgram ml-1) and cisplatin (cis-Pt; 0.0002/microgram ml-1) had a permanent synergistic effect on both synchronized and asynchronous cell populations of V 79B cells. Successive combination of the drugs was manifested synergistically when MTX was applied first. The synchronized cell population was more sensitive to the cytostatics than the asynchronous population. Treatment with MTX alone, or the combination of MTX-cis-Pt, as well as their successive combination with the first drug being cis-Pt, caused gluconeogenesis.

Animals↗
PubMed1991

Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin.

Monoclonal antibody against light chains of human cardiac myosin (MLC) was labelled with horseradish peroxidase. The conjugation was performed by two different methods with glutaraldehyde and periodate respectively. The binding activities of the conjugates were tested by enzyme linked immunosorbent assay (ELISA) on the microtitration plates with immobilized MLC (1-1000 ng per well). A comparison of both methods revealed their universal suitability for the preparation of conjugates as well as their applicability. The use of conjugates shortens the time needed and improves the ELISA method for MLC estimation. Specific advantages of the glutaraldehyde and the periodate method concern diverse details.

Antibodies, Monoclonal↗
PubMed1989

Study of metabolism and growth limitation of human leukemia cell lines.

The relationship between proliferation and metabolism of four leukemia cell lines (BALL-1, JOK-1, Jurkat, and MOLT-4) in batch culture was studied. The maximum cell density (1.5-2.3 x 10(6) cells/ml) without change of medium was observed on days 6-8 of cultivation. At the same time, the original concentration of glucose in the medium (10 mmol/l) fell to 3.5-4 mmol/l. While BALL-1 and MOLT-4 cells, on day 4 of cultivation, converted 82% of glucose into lactate, on day 7 this value was 50%, or there was no lactate production (MOLT-4), respectively. On the other hand, the values of the coefficient of glycolysis showed that Jurkat and JOK cells converted also other compounds into lactate. Part of the utilized glutamine was employed by all four cell lines: 1. as a precursor of glutamic acid, and 2. as a source of energy. BALL-1 and JOK-1 cells converted part of arginine into ornithine. At the time when the proliferation of the cells ceased, the level of ammonia reached a toxic concentration of 2.0-3.6 mmol/l. Since these cell lines utilized only a part of carbon and nitrogen sources in the medium, it was suggested that the final cell density was limited by a growth inhibitor (i.e. ammonia) and not by a lack of nutrients.

Amino Acids↗
PubMed1988

The study of cytotoxic effect of benfluron.

The efficiency of benfluron (B-F) was measured by determining the inhibition of cell proliferation in HeLa and V/79 cells. B-F induced an acute cytotoxic reaction in dependency on the concentration applied. A biphasic unbalanced growth was an integral part of this reaction. In connection with the decrease in cell proliferation which followed B-F treatment at micromolar concentrations, the protein:cell volume ratios increased while at nanomolar concentrations decreased. These variations must be taken into account when expressing and interpreting the results of cell metabolism. The glucose and amino acid metabolism of treated V/79 cells differed markedly when expressed as a function of cell number or as function of protein content per cell.

Animals↗