Chemiluminescent picture of diphenyleneiodonium-inhibited NADPH oxidase: a bimodal process and its logistic-exponential model-based description.

A chemiluminescence (CL) study of diphenyleneiodonium-inhibited NADPH oxidase was performed on a cellular system containing neutrophils stimulated by phorbol myristate acetate, indicating a complex bimodal structure of CL processes corresponding to different stages of the inhibition. The complex structure of these processes was described by a superposition of two logistic-exponential (LE) models, characterizing these processes as bimodal ones. To determine the mechanistic foundation of the LE model-described processes, a generalized form of the second-order dynamic system of CL reactions, the solution to which corresponds to the LE model, was constructed. The diphenyleneiodonium effects on neutrophil NADPH oxidase were separated from the total bimodal CL of the whole measurement system by the use of difference CL processes. These difference processes were also found to be bimodal; thus, inhibitor-induced reduction of CL could be described by a second-order dynamic system. The rate constants and initial concentrations in this dynamic system were determined by the least squares method applied to numerical solutions approximating the difference processes. Using interrelations between the parameters of the dynamic system, cooperative effects in the inhibitor reactions with NADPH oxidase were found and described quantitatively. Other evidences of cooperativity were obtained from integral characteristics of the CL reduction process, i.e. dose-response and progress curves, determined by numerical integration of the LE models constituting the superposition. On this basis, it was also possible to detect a specific binding of the inhibitor to the enzyme. Finally, putative reaction mechanisms suggested by the model obtained were considered and compared with those known at present.

Comparison of different procedures for the lipid extraction from HL-60 cells: a MALDI-TOF mass spectrometric study.

A human leukaemia cell line--HL-60--can be differentiated into neutrophils or macrophages and both differentiation processes are accompanied by changes of the lipid composition. Various methods were described for the extraction of lipids from cellular systems, but only two of them were applied to the HL-60 cell line so far. In this study we compared five selected extraction methods for the lipid extraction from HL-60 cells with regard to their qualitative analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS): chloroform/methanol at volume ratios 2:1 and 1:2, isopropanol/ chloroform, isopropanol/hexane and butanol. In addition, the cholesterol and phospholipid concentrations in organic extracts were measured by colorimetric assays. Results can be summarized as follows: For the analysis of polar phospholipids obtained from HL-60 cells by MALDI-TOF MS, a chlorofom/methanol (1:2) or isopropanol/chloroform mixture or butanol can be applied as extraction systems On the other hand, if one would like to analyze changes in triacylglycerols, then chloroform/methanol (2:1) would be the method of choice.

Fluoro-modified chemotactic peptides: fMLF analogues.

A small library of peptide analogues of the chemotactic tripeptide For-Met-Leu-Phe-NH2 modified by substitution of Leu at position 2 by three different fluorinated amino acids varying in content of fluorine, length of the fluorinated side chain, and alkylation degree at the alpha-carbon atom was synthesized. The influence of the fluorine substitution on the biological activity was investigated by measuring the oxidative activity of neutrophils using a luminol-dependent chemiluminescence assay.

Involvement of phosphatidic acid in both degranulation and oxidative activity in fMet-Leu-Phe stimulated polymorphonuclear leukocytes.

BACKGROUND/AIM: Polymorphonuclear leukocytes (neutrophils) release a variety of toxic agents--proteins and reactive oxygen species (ROS)--that are used to inactivate foreign microorganisms in the non-specific immune response. This study was undertaken to compare intracellular signalling pathways that lead to the ROS production as well as degranulation of azurophilic granules of human fMet-Leu-Phe/cytochalasin B stimulated neutrophils. METHODS: Luminol-amplified chemiluminescence was used for monitoring the oxidative activity of human neutrophils in the presence of various inhibitors. The elastase activity was assessed in the neutrophil supernatant as a marker for degranulation of azurophilic granules. RESULTS: Tested inhibitors of enzymes of signalling cascades showed the same effect on the ROS production and on the activity of elastase released from neutrophils. The only difference was obtained with staurosporine: it inhibited the chemiluminescence response, but increased the elastase release. CONCLUSION: Early signalling pathways leading to the ROS production and the degranulation are ubiquitous in human neutrophils. They are branching most probably at the point of the phosphatidic acid production by phospholipase D. A protein kinase activated by this lipid second messenger might play a central regulatory role in human neutrophils.