A modified capillary assay for chemotaxis

Summary A modification is described of the capillary assay for chemotaxis. It employs a 96-well dilution plate and its cover. Capillary tubes are inserted through the cover and are supported by small rubber collars. The method is faster and less tedious and gives more precise results than earlier methods.     

A quantitative assay for ciliate chemotaxis

A quantitative bioassay for ciliate chemotaxis based on the capillary principle is described using Tetrahymena thermophila as test organism. The attractant-containing assay tube designed for the bioassay attracts up to 4 X 10(4) cells in 2 h which makes electronic cell counting of the chemotactic response feasible. The attractants used are solutions of proteose peptone and yeast extract which also are growth media for this organism.     

A simplified assay for phosphoenolpyruvate.

A new method for analysis of phosphoenolpyruvate has been developed. The assay is based upon the stoichiometric conversion of ADP to ATP by the enzyme pyruvate kinase in the presence of variable amounts of PEP, and subsequent measurement of the ATP with a luciferin-luciferase preparation.     

A high-throughput capillary assay for bacterial chemotaxis

We present a high-throughput capillary assay in order to characterize the chemotactic response of the E. coli bacterium. We measure the number of organisms attracted into an array of 96 capillary tubes containing the attractant L-aspartate. The effect of bacterial concentration on the chemotactic response is reported. Such high-throughput assay can be used to characterize bacterial chemotaxis function of a wide range of biochemical parameters.     

A simplified assay for phospholipase C.

A new assay for phospholipase C activity that uses alkaline phosphatase to convert phosphorylcholine to inorganic phosphate is described. The determination of inorganic phosphate is performed in the presence of phosphatidylcholine and protein after the addition of sodium dodecyl sulfate. Phospholipase C activity determined by this coupled enzyme assay agrees well with data obtained by extracting and measuring phosphoryl[¹⁴C]choline produced from phosphatidyl[methyl-¹⁴C]choline. The assay is sensitive to 1 nmol of phosphate, requires no removal of protein or phospholipid, and will work with a variety of phospholipid substrates. The assay is faster and more sensitive than previously published procedures. Stimulation of phospholipase C from Clostridium perfringens by ammonium sulfate is also reported.     

Paradoxical effects of adenosine on neutrophil chemotaxis

Chemotaxis of rabbit neutrophils is most sensitive to inhibition by 3-deazaadenosine, followed by 3-deaza-(+/-)aristeromycin, 3-deaza-(+/-)aristeromycinylhomocysteine, 3-deazaadenosylhomocysteine, and adenosylhomocysteine, in that order. Although adenosine by itself had no effect on the chemotaxis of neutrophils, it essentially abolished the inhibitory effects of 3-deaza-adenosine on chemotaxis and the reduction of nitroblue tetrazolium. Paradoxically, adenosine enhanced the inhibition of chemotaxis by 3-deazaadenosylhomocysteine slightly and that of 3-deaza-(+/-)aristeromycin significantly. Adenosine alone unexpectedly inhibited phospholipid methylation to the same extent as 3-deazaadenosine, and reduced protein carboxymethylation to a lesser degree. The inhibition of these two methylation reactions by 3-deazaadenosine was, however, not substantially altered in the presence of adenosine. Drastic changes in the ratio of adenosylmethionine/nucleosidylhomocysteine were observed in the presence of adenosine, 3-deazaadenosine, 3-deaza-(+/-)aristeromycin, or of adenosine in combination with each of the latter compounds. There was no significant effect on the binding of chemotactic peptide to receptors, or on the ratio of ATP/ADP in cells treated by the analogs. These results suggest that the inhibition of methylation reactions per se is not enough to account for the inhibition of both chemotaxis and the reduction of nitroblue tetrazolium by neutrophils.     

Effect of dimethylthiourea on the neutrophil myeloperoxidase pathway

The sulfur-centered compound dimethylthiourea (DMTU) affords antioxidant protection in animal models of acute lung injury, an effect that has been attributed to its OH. scavenging properties. Although DMTU can also react with H2O2 in certain experimental systems, the effect of DMTU on the neutrophil myeloperoxidase (MPO) pathway has not been studied. DMTU (1-10 mM) completely blocked stable oxidants and hypochlorous acid formation by phorbol myristate acetate- and zymosan-stimulated neutrophils. DMTU also provided complete inhibition when incubated with cell-free supernatants after the formation of the MPO products. DMTU prevented the oxidative inactivation of alpha 1-antitrypsin by neutrophil-stable oxidants. Evidence that DMTU was oxidized by the MPO products was obtained by titration of oxidized DMTU with reduced glutathione. Surprisingly, supernatants from cells incubated with DMTU (10 mM) consumed two- to threefold higher amounts of reduced glutathione than supernatants from cells incubated with taurine (15 mM). Metabolic studies with stimulated neutrophils and experiments with the MPO enzyme system in a cell-free system suggested that DMTU acts by scavenging the products of the MPO pathway rather than by blocking H2O2 production in the intact cell. These findings demonstrate that DMTU blocks the neutrophil MPO pathway in addition to its known ability to scavenge other reactive O2 species. The capacity of DMTU to scavenge MPO products may explain some of its protective effects in acute lung injury.     

A fluorometric assay for the quantitation of aminosugars.

A method is presented for the quantitative determination of aminosugars in glycoproteins. Glycoproteins are acid hydrolyzed and reacted with the fluorogenic reagent 1-dimethylaminonaphthalene-5-sulfonyl chloride. Following cellulose thin layer electrophoresis in pyridine/acetic acid (pH 4.4), 5 h at 500 V, appropriate areas of the plate are scraped and the dansyl hexosamines are eluted with 95% ethanol. Aliquots are then applied to a silica gel tlc plate and subjected to ascending thin layer chromatography in cyclohexane/ethylacetate/ethanol (6/4/3). After spraying the plate with triethanolamine/isopropanol ($\text{1}\text{4}$), fluorescent intensities are measured by in situ scanning. The aminosugar content of the glycoprotein is determined from a curve generated from a series of standards run concurrently on each plate. The method clearly resolves glucosamine from galactosamine, and is sensitive for the detection of aminosugars in the subnanomole range.     

An improved chamber for direct visualisation of chemotaxis

There has been a growing appreciation over the last decade that chemotaxis plays an important role in cancer migration, invasion and metastasis. Research into the field of cancer cell chemotaxis is still in its infancy and traditional investigative tools have been developed with other cell types and purposes in mind. Direct visualisation chambers are considered the gold standard for investigating the behaviour of cells migrating in a chemotactic gradient. We therefore drew up a list of key attributes that a chemotaxis chamber should have for investigating cancer cell chemotaxis. These include (1) compatibility with thin cover slips for optimal optical properties and to allow use of high numerical aperture (NA) oil immersion objectives; (2) gradients that are relatively stable for at least 24 hours due to the slow migration of cancer cells; (3) gradients of different steepnesses in a single experiment, with defined, consistent directions to avoid the need for complicated analysis; and (4) simple handling and disposability for use with medical samples. Here we describe and characterise the Insall chamber, a novel direct visualisation chamber. We use it to show GFP-lifeact transfected MV3 melanoma cells chemotaxing using a 60x high NA oil immersion objective, which cannot usually be done with other chemotaxis chambers. Linear gradients gave very efficient chemotaxis, contradicting earlier results suggesting that only polynomial gradients were effective. In conclusion, the chamber satisfies our design criteria, most importantly allowing high NA oil immersion microscopy to track chemotaxing cancer cells in detail over 24 hours.     

A sensitive and selective assay for chloramine production by myeloperoxidase

We describe a new assay for the chlorination activity of myeloperoxidase and detection of chloramines. Chloramines were detected by using iodide to catalyze the oxidation of either 3,3',5,5'-tetramethylbenzidine (TMB) or dihydrorhodamine to form strongly absorbing or fluorescent products, respectively. With TMB as little as 1 muM taurine chloramine could be detected. The sensitivity of the dihydrorhodamine assay was about 10-fold greater. The chlorination activity of myeloperoxidase was measured by trapping hypochlorous acid with taurine and subsequently using iodide to promote the oxidation reactions of the accumulated taurine chloramine. A similar approach was used to detect hypochlorous acid production by stimulated human neutrophils. Iodide-dependent catalysis distinguished N-chloramines from N-bromamines. This allows for discrimination between heme peroxidases that generate either hypochlorous acid or hypobromous acid. The assay has distinct advantages over existing assays for myeloperoxidase with regard to sensitivity, specificity, and its ease and versatility of use.     

A simplified assay method for galactosyl ceramide beta-galactosidase

In a previously described method for determining the activity of cerebroside galactosidase, the enzyme preparation was incubated with an emulsion of cerebroside which had been labeled in the galactose moiety. The liberated galactose was separated from the emulsion by liquid-liquid partitioning, but the presence of detergent necessitated the use of careful agitation and a backwash in order to reduce the contamination of the aqueous layer with excess emulsified substrate. This problem is eliminated by adding a large amount of lipid to reduce the emulsifying power of the detergent. It may be that other lipid hydrolase assays, based on the same principle, would benefit by this approach. Some additional improvements in the assay system are described.     

A new simplified assay for larval migration inhibition

, , , and 1992. A new simplified assay for larval migration inhibition. International Journal for Parasitology 22: 1183–1185. A simple method is described for the in vitro detection of substances that impair the motility of third-stage larvae of gastro-intestinal nematodes. The test is based on the ability of larvae to freely migrate through selected mesh sizes of nylon sieves and the reduced ability of larvae to migrate after preincubation with, and in the presence of, substances that inhibit or reduce larval motility.     

Isolation and properties of human neutrophil myeloperoxidase

Human leukocyte myeloperoxidase has been purified to homogeneity by a three-step procedure which includes dialysis of a granule extract against low-salt buffer. Sephadex G-75 chromatography, and carboxymethylcellulose chromatography. The final product was homogeneous when examined by acid polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrifugation. The molecular weight determined by the latter procedure was 118000. With or without reduction of the protein by 2-mercaptoethanol, subunits were formed which migrated as a single band after sodium dodecyl sulfate gel electrophoresis. With reduction, the molecular weight of the apparently identical subunits was 59000, and 42000 without reduction. Other general properties of human leukocyte myeloperoxidase, including amino acid composition, amino terminal sequence analysis, and absorption spectra, are also reported. Myeloperoxidase, in the presence of hydrogen peroxide and chloride ion, and no other substrate, autoinactivates. After completion of the inactivation reaction, several oxidizable amino acids in the enzyme are modified, and the absorption peak at 430 nm disappears. The presence of a substrate of the myeloperoxidase system (alpha-1-proteinase inhibitor), or of high concentration of chloride ion, completely protects the enzyme from autoinactivation.     

Quantitative assay for algal chemotaxis.

A quantitative capillary assay is described for measuring chemoreception in the neritic and littoral unicellular alga Dunaliella tertiolecta. Lucite chemotaxis plates were used in the assay with 3-microliter capillaries. A Coulter Counter was employed to determine algal cell numbers. D. tertiolecta is attracted to ammonium ion with a maximum positive response at 10(-3) M. Inclusion of calcium and L-methionine in the chemotaxis medium stimulates algal chemoreception, although neither chemical is essential for motility. Attraction of the chlorophyte to ammonium is dependent on time of incubation, cell density, and pH. The optimum pH for attraction was found to be 6.25.     

Quantitative assay for algal chemotaxis.

A quantitative capillary assay is described for measuring chemoreception in the neritic and littoral unicellular alga Dunaliella tertiolecta. Lucite chemotaxis plates were used in the assay with 3-microliter capillaries. A Coulter Counter was employed to determine algal cell numbers. D. tertiolecta is attracted to ammonium ion with a maximum positive response at 10(-3) M. Inclusion of calcium and L-methionine in the chemotaxis medium stimulates algal chemoreception, although neither chemical is essential for motility. Attraction of the chlorophyte to ammonium is dependent on time of incubation, cell density, and pH. The optimum pH for attraction was found to be 6.25.     

Nicotinanilides as inhibitors of neutrophil chemotaxis

A series of 4-fluoronicotinanilides was synthesized and shown to be novel, potent, and selective inhibitors against GRO-alpha-driven human neutrophil chemotaxis. Compounds of this class may be useful for the treatment of inflammatory, autoimmune, and allergic disorders.