hymecromone and 4-methylumbelliferyl-heptanoate

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an alpha-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this alpha-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH.

Topics: Acylation; Amino Acid Sequence; Animals; Binding Sites; Butyrates; Catalysis; Cell Line; Chlorocebus aethiops; COS Cells; Cysteine; Gene Deletion; Gene Expression; Humans; Hymecromone; Iodoacetamide; Lipase; Mercaptoethanol; Mutagenesis, Site-Directed; Mutation; Nitrophenols; Phenylalanine; Point Mutation; Protein Folding; Recombinant Proteins; Sequence Homology, Amino Acid; Spodoptera; Substrate Specificity; Transfection

A fluorimetric method using 4-methylumbelliferyl heptanoate (MUH) has been developed for detecting cell-mediated cytotoxicity and cell proliferation. The assay is based on the hydrolysis of the fluorochrome (MUH) by intracellular esterases of viable cells resulting in the production of highly fluorescent 4-methylumbelliferone that can be measured in a microplate fluorimeter. Because of a similarity to the principle of the widely used colorimetric MTT assay, a comparison was made between the two assays when measuring cell proliferation and LAK cell cytotoxicity to different target cell types. The results have shown that the MUH assay represents a method for evaluating both cell-mediated cytotoxicity and cell proliferation which is completely comparable to the MTT method. The rapidity of the new cytotoxicity assay, 5 h in contrast to 9 h for the MTT assay, its applicability to both adherently and nonadherently growing target cells and its high accuracy due to the avoidance of centrifugation steps make this method a serious contender for replacing conventional radioactive techniques.

Topics: Animals; Cell Division; Cytotoxicity Tests, Immunologic; Esterases; Female; Fluorescent Dyes; Hymecromone; In Vitro Techniques; Killer Cells, Lymphokine-Activated; Mice; Mice, Inbred AKR; Mice, Inbred BALB C; Spectrometry, Fluorescence

In order to measure cell mediated cytotoxicity to adherent growing cell lines in vitro more rapidly and conveniently, a fluorometric microassay was developed and results were compared with those obtained by the 51Cr release assay. The fluorometric method is based on the hydrolysis of the fluorochrome 4-methylumbelliferyl heptanoate (MUH) by intracellular esterases of viable cells. Melanoma cell monolayers were incubated with lymphokine activated killer (LAK) cells for 4 h at various effector: target (E:T) cell ratios (E:T = 16, 8, 4, 2:1). Thereafter surviving adherent melanoma cells were stained with MUH for 30 min and fluorescence was measured directly in a 96 well plate reader. For the calculation of LAK cell cytotoxicity fluorescence values were corrected for the number of nonspecifically detached tumor cells during the washes and the number of nonspecifically adherent LAK cells. Using identical target and effector cell preparations both assays showed a nearly proportional increase of percentage cytotoxicity with rising numbers of lymphocytes. Compared with the 51Cr release assay, however, higher cytotoxicity values were obtained with the fluorometric MUH microassay: 57% with MUH versus 26% with 51Cr and 39% versus 14% for cell lines StML-11 and SKMel-28, respectively (E:T ratio = 16:1). The higher cytotoxicity rates obtained with the fluorometric MUH microassay were not due to the additional 30 min staining with MUH or due to nonspecific hydrolysis of MUH by extracellular esterases released from damaged cells, as could be shown by a series of experiments. In conclusion, a simple and rapid fluorometric microassay has been developed showing reliable reproducibility and a higher sensitivity compared with the 51Cr release assay for the determination of cellular cytotoxicity to adherent growing cell lines, avoiding hazardous radioactive labels.

Topics: Cell Adhesion; Chromium Radioisotopes; Cytotoxicity, Immunologic; Fluoroimmunoassay; Humans; Hymecromone; Immunity, Cellular; In Vitro Techniques; Killer Cells, Lymphokine-Activated; Melanoma; Tumor Cells, Cultured

In the present study we describe a simple technique for the determination of keratinocyte proliferation in vitro, based on the hydrolysis of a fluorogenic substrate by cell esterases. Normal and transformed human keratinocytes were grown in microtiter plates and were incubated with 4-methylumbelliferyl heptanoate after 3, 5, and 7 days. The fluorescence was quantified using an automatic fluorescence detection unit. The fluorescence showed a strong correlation with the cell number at various growth phases. In addition, the method reliably detected the growth inhibitory effect of recombinant interferon gamma on human keratinocytes. The fluorometric assay is a simple, fast and reliable method to assess cell number in keratinocyte cultures.

Topics: Cell Count; Cell Division; Cell Line, Transformed; Epidermal Cells; Humans; Hymecromone; Infant, Newborn; Interferon-gamma; Keratins; Male; Recombinant Proteins; Reference Values; Spectrometry, Fluorescence

The production of an enzyme, 4-methylumbelliferyl heptanoate hydrolase, in Escherichia coli exposed to enriched and nonenriched seawater was studied. In all media, except for seawater with no or very small amounts of organic material and seawater enriched with peptone, 4-methylumbelliferyl heptanoate hydrolase activity increased by 2 to 3 orders of magnitude within 2 days. Increased enzyme activity was assumed to be related to cells not undergoing lysis but adapting to conditions of nutrient limitation.

Topics: Colony Count, Microbial; Culture Media; Escherichia coli; Hydrolases; Hymecromone; Seawater; Umbelliferones; Water Microbiology

Three fluorogenic methylumbelliferone (MU) substrates were evaluated for rapid detection of total and fecal coliform bacteria (TC and FC) in drinking water. 4-MU-beta-D-galactoside, MU-heptanoate, and MU-glucuronide were used to determine enzyme activity as a surrogate measure of coliform concentration. Coliforms occurring in river water and in potable water artificially contaminated with raw sewage were tested. The initial rate of hydrolysis (delta F) of MU-beta-D-galactoside showed promise as an indicator of TC and FC within 15 min. delta F of MU-glucuronide was insufficient in the 15-min assay, and combinations of the MU substrates did not enhance delta F. A direct membrane filter method incorporating MU-beta-D-galactoside into an agar medium allowed the detection of as few as 1 FC per 100 ml within 6 h.

Topics: Colony Count, Microbial; Enterobacteriaceae; Fluorescent Dyes; Galactosides; Glycosides; Hydrolysis; Hymecromone; Sewage; Umbelliferones; Water Microbiology

A microplate method for assessing cell growth and viability based on the hydrolysis of fluorogenic substrates by cell esterases has been investigated. Living cells incubated with fluorescein diacetate or 4-methylumbelliferyl heptanoate generate a fluorescent product which is proportional to the number of cells. This can be used as a simple and economical readout for various bioassays such as, for example, the assessment of IL-2 and IL-3 on factor-dependent cell lines, and on antigen- and mitogen-stimulated proliferation of lymphocytes. This fluorometric assay has similar sensitivity to the measurement of [3H]thymidine uptake and greater sensitivity than standard colorimetric assays. Incubation with MUH for a period of 30-60 min at 22 degrees C is adequate.

Topics: Biological Assay; Cell Line; Esterases; Fluorescein; Fluoresceins; Fluorescent Dyes; Hymecromone; Immunologic Techniques; Interleukin-2; Interleukin-3; Kinetics; Lymphocyte Activation; Lymphokines; Spectrometry, Fluorescence; Structure-Activity Relationship; Umbelliferones