Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the ε-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted "super-substrates" with catalytic constants of up to 15,000,000 M-1s-1 for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD+ has IC50 values below 200 pM.

• MeSH
• Fluorescent Dyes chemistry   pharmacology   MeSH
• Photochemical Processes MeSH
• Histone Deacetylases chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Molecular Structure MeSH
• Positron-Emission Tomography * MeSH
• Sirtuins antagonists & inhibitors   chemistry   metabolism   MeSH
• Thioamides chemistry   pharmacology   MeSH
• Electron Transport MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We developed a one-step direct assay for the determination of histone deacylase (HDAC) activity by substituting the carbonyl oxygen of the acyl moiety with sulfur, resulting in thioacylated lysine side chains. This modification is recognized by class I HDACs with different efficiencies ranging from not accepted for HDAC1 to kinetic constants similar to that of the parent oxo substrate for HDAC8. Class II HDACs can hydrolyze thioacylated substrates with approximately 5-10-fold reduced kcat values, which resembles the effect of thioamide substitution in metallo-protease substrates. Class IV HDAC11 accepts thiomyristoyl modification less efficiently with an ∼5-fold reduced specificity constant. On the basis of the unique spectroscopic properties of thioamide bonds (strong absorption in spectral range of 260-280 nm and efficient fluorescence quenching), HDAC-mediated cleavage of thioamides could be followed by ultraviolet-visible and fluorescence spectroscopy in a continuous manner. The HDAC activity assay is compatible with microtiter plate-based screening formats up to 1536-well plates with Z' factors of >0.75 and signal-to-noise ratios of >50. Using thioacylated lysine residues in p53-derived peptides, we optimized substrates for HDAC8 with a catalytic efficiency of >250000 M-1 s-1, which are more than 100-fold more effective than most of the known substrates. We determined inhibition constants of several inhibitors for human HDACs using thioacylated peptidic substrates and found good correlation with the values from the literature. On the other hand, we could introduce N-methylated, N-acylated lysine residues as inhibitors for HDACs with an IC50 value of 1 μM for an N-methylated, N-myristoylated peptide derivative and human HDAC11.

• MeSH
• Biocatalysis MeSH
• Histone Deacetylases chemistry   genetics   metabolism   MeSH
• Histone Deacetylase Inhibitors chemistry   metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Lysine chemistry   metabolism   MeSH
• Thioamides chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Lectins are a group of ubiquitous proteins which specifically recognize and reversibly bind sugar moieties of glycoprotein and glycolipid constituents on cell surfaces. The mutagenesis approach is often employed to characterize lectin binding properties. As lectins are not enzymes, it is not easy to perform a rapid specificity screening of mutants using chromogenic substrates. It is necessary to use different binding assays such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), enzyme-linked lectin assays (ELLA), or glycan arrays for their characterization. These methods often require fluorescently labeled proteins (MST), highly purified proteins (SPR) or high protein concentrations (ITC). Mutant proteins may often exhibit problematic behaviour, such as poor solubility or low stability. Lectin-based cell agglutination is a simple and low-cost technique which can overcome most of these problems. In this work, a modified method of the agglutination of human erythrocytes and yeast cells with microscopy detection was successfully used for a specificity study of the newly prepared mutant lectin RS-IIL_A22S, which experimentally completed studies on sugar preferences of lectins in the PA-IIL family. Results showed that the sensitivity of this method is comparable with ITC, is able to determine subtle differences in lectin specificity, and works directly in cell lysates. The agglutination method with microscopy detection was validated by comparison of the results with results obtained by agglutination assay in standard 96-well microtiter plate format. In contrast to this assay, the microscopic method can clearly distinguish between hemagglutination and hemolysis. Therefore, this method is suitable for examination of lectins with known hemolytic activity as well as mutant or uncharacterized lectins, which could damage red blood cells. This is due to the experimental arrangement, which includes very short sample incubation time in combination with microscopic detection of agglutinates, that are easily observed by a small portable microscope.

• MeSH
• Agglutination drug effects   MeSH
• Agglutination Tests * methods   MeSH
• Bacterial Proteins pharmacology   MeSH
• Erythrocytes cytology   drug effects   MeSH
• Hemagglutination drug effects   MeSH
• Hemolysis drug effects   MeSH
• Yeasts cytology   drug effects   MeSH
• Lectins pharmacology   MeSH
• Humans MeSH
• Microscopy MeSH
• Surface Plasmon Resonance MeSH
• Escherichia coli Proteins pharmacology   MeSH
• Saccharomyces cerevisiae cytology   drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

With aim to develop effective proof-of-concept approach which can be used in a development of new preparations for the inhalation therapy, we designed a new screening method for simple and rapid simultaneous determination of antibacterial potential of plant volatiles in the liquid and the vapour phase at different concentrations. In addition, EVA (ethylene vinyl acetate) capmat™ as vapour barrier cover was used as reliable modification of thiazolyl blue tetrazolium bromide (MTT) assay for cytotoxicity testing of volatiles on microtiter plates. Antibacterial activity of carvacrol, cinnamaldehyde, eugenol, 8-hydroxyquinoline, thymol and thymoquinone was determined against Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae using new broth microdilution volatilization method. The cytotoxicity of these compounds was evaluated using MTT test in lung fibroblast cells MRC-5. The most effective antibacterial agents were 8-hydroxyquinoline and thymoquinone with the lowest minimum inhibitory concentrations (MICs) ranging from 2 to 128μg/mL, but they also possessed the highest toxicity in lung cell lines with half maximal inhibitory concentration (IC50) values 0.86-2.95μg/mL. The lowest cytotoxicity effect was identified for eugenol with IC50 295.71μg/mL, however this compound produced only weak antibacterial potency with MICs 512-1024μg/mL. The results demonstrate validity of our novel broth microdilution volatilization method, which allows cost and labour effective high-throughput antimicrobial screening of volatile agents without need of special apparatus. In our opinion, this assay can also potentially be used for development of various medicinal, agricultural, and food applications that are based on volatile antimicrobials.

• MeSH
• Acrolein analogs & derivatives   chemistry   MeSH
• Anti-Bacterial Agents chemistry   MeSH
• Benzoquinones chemistry   MeSH
• Cell Line MeSH
• Eugenol chemistry   MeSH
• Phytochemicals chemistry   MeSH
• Haemophilus influenzae drug effects   MeSH
• Humans MeSH
• Microbial Sensitivity Tests methods   MeSH
• Monoterpenes chemistry   MeSH
• Oxyquinoline chemistry   MeSH
• Staphylococcus aureus drug effects   MeSH
• Streptococcus pneumoniae drug effects   MeSH
• Volatile Organic Compounds chemistry   MeSH
• Tetrazolium Salts MeSH
• Thiazoles MeSH
• Thymol chemistry   MeSH
• Volatilization * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The analytical performance and evaluation of a kit-based ELISA for the determination of acrylamide in fried potato and corn chip samples are described. The sample homogenate is subjected to clean-up using SPE, followed by analyte derivatization and ELISA detection. Accuracy, precision and linearity of the ELISA procedure have been validated using spiked samples. Analytical recovery ranged from 91.8% to 96.0% with coefficients of variation below 15%. Good linearity over a wide range of dilution and minimal assay drift was observed within a microtiter plate. IC50 value of the calibration curve was 110 ng/mL, with the limit of detection about 5 ng/mL and dynamic range from 10 to 1000 ng/mL. The high specificity of the ELISA was demonstrated by cross-reactivity study using 11 potential cross-reactants. A good correlation between the results obtained from the ELISA and GC-MS within the concentration range 120-1500 μg/kg was found in the chip samples (r=0.992, n=120). The data demonstrate that the evaluated and validated ELISA has a potential utility in a quick, simple and reliable acrylamide screening analysis for the medium- and large-sized food companies, as well as for residue laboratories and the food industry dealing with improving the chemical safety of foods available to the consumer.

• MeSH
• Acrylamide analysis   MeSH
• Food Analysis methods   MeSH
• Enzyme-Linked Immunosorbent Assay methods   MeSH
• Food Contamination analysis   MeSH
• Zea mays chemistry   MeSH
• Gas Chromatography-Mass Spectrometry MeSH
• Reproducibility of Results MeSH
• Solanum tuberosum chemistry   MeSH
• Cooking methods   MeSH
• Hot Temperature MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

A microtiter plate-based assay was developed for the automatic monitoring of degradation profile of the yellow-coloured nitrophenolic compounds. The method enables to reduce the intervals between measurements of substrate concentration to minutes and to overcome the problem of discontinuity of sampling typical for conventional methods. The concentrations of nitrophenolic compounds were calculated from the absorbance values determined automatically by BIOSCREEN C. Verification of the method was based on the comparison of results with the conventional HPLC method results. The values of the rate and saturation constants were comparable for both the microtiter plate-based assay and the conventional HPLC method. The automatic method described here seems to be efficient for the screening degradation studies, which requires the treatment of quantity of samples.

• MeSH
• Arthrobacter isolation & purification   metabolism   growth & development   MeSH
• Bacteriological Techniques methods   instrumentation   MeSH
• Biodegradation, Environmental MeSH
• Financing, Organized MeSH
• Catechols metabolism   MeSH
• Kinetics MeSH
• Culture Media MeSH
• Molecular Sequence Data MeSH
• Nitrophenols metabolism   MeSH
• Soil Microbiology MeSH
• Rhodococcus genetics   isolation & purification   classification   metabolism   MeSH
• Sequence Analysis, DNA MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Evaluation Study MeSH