Accelerating effect of glutathione on hydroxylation of phenylalanine by stimulated polymorphonuclear leukocytes

Sulfhydryl compounds significantly accelerated the hydroxylation of phenylalanine by stimulated polymorphonuclear leukocytes. The reduced form of glutathione (G-SH) was most effective. The hydroxylation reaction in the presence of G-SH was largely prevented by superoxide dismutase and hydroxyl radical scavengers. The results suggest that a much faster production of hydroxyl radical may occur in a reaction mixture containing both G-SH and stimulated polymorphonuclear leukocytes than in that containing stimulated polymorphonuclear leukocytes alone.     

Study on the cupric phenanthroline-induced beta-glucuronidase release in saponin-permeabilized polymorphonuclear leukocytes.

Saponin-permeabilized polymorphonuclear leukocytes (PMNs) released beta-glucuronidase, a lysosomal enzyme, dose-dependently in response to cupric phenanthroline (CuPh), a mild oxidant, which catalyzes the formation of disulfide bridges. The beta-glucuronidase release induced by CuPh was inhibited by ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Both dithiothreitol (DTT) and N-(6-aminohexyl)-5-chloro-naphthalene sulfonamide (W-7) also inhibited the beta-glucuronidase release induced by CuPh. CuPh elicited a decrease in protein-bound free sulfhydryls simultaneously, and this decrease was not restored by EGTA treatment. CuPh inhibited Ca2+ uptake into Ca2+ store sites, and promoted a Ca2+ efflux from Ca2+ store sites. It also inhibited Ca(2+)-adenosine triphosphatase (ATPase) activity in permeable PMNs. DTT, a sulfhydryl reducing agent, suppressed both the beta-glucuronidase release and the Ca2+ uptake in CuPh-treated permeable PMNs. On the other hand, chloromercuriphenylsulfonic acid (CMPS), a sulfhydryl modifier, decreased the amount of free sulfhydryls in protein and released beta-glucuronidase in permeable PMNs dose-dependently, but EGTA did not inhibit either reaction. Neither CuPh nor CMPS released beta-glucuronidase from intact PMNs. These results indicate that both CuPh and CMPS act on intra-PMN target molecules to exert their influence, but the involved mechanisms are different in nature. Alteration in calcium movement is responsible for the beta-glucuronidase release in the CuPh-treated permeable PMNs.     

Activation by cathepsin G of latent gelatinase secreted from rat polymorphonuclear leukocytes

Rat polymorphonuclear leukocytes secrete a latent gelatinase with a molecular weight of about 96 kilodaltons (kDa). Activation of the latent 96-kDa gelatinase by cathepsin G was studied by using sodium dodecyl sulfate-substrate polyacrylamide gel electrophoresis. Cathepsin G activated the 96-kDa gelatinase by converting it to two lower molecular-weight species of 76 and 61 kDa, which were slightly different from the gelatinase species generated by treatment with 4-aminophenylmercuric acetate, an activator of latent gelatinase.     

Lipid peroxidation of the erythrocyte membrane caused by stimulated polymorphonuclear leukocytes in the presence of ferritin

Lipid peroxidation of erythrocyte membrane was caused by phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMN) in the presence of ferritin. PMN themselves were not peroxidized. A lag period was observed before the start of the peroxidation reaction. In contrast, ferritin iron was continuously released by PMA-stimulated PMN, suggesting that accumulation of free iron in the reaction system was important for proceeding of the peroxidation reaction. Superoxide dismutase, catalase, hydroxyl radical scavengers and an iron chelator, diethylenetriaminepenta-acetic acid, inhibited the lipid peroxidation, indicating that the lipid peroxidation is initiated by a hydroxyl radical generated from the interaction of H2O2 with ferrous iron released from ferritin.     

Chemiluminescence response and endothelial cell damage following lipopolysaccharide priming of polymorphonuclear leukocytes

Conclusions Neutrophils can be directly stimulated by endothelial cell suspensions, an observation that has also been made by experiments with nylon fiber, as an artificial model for PMNL/EC interactions [5]. As shown by experiments with endothelial cell suspensions a further amplification of this process following LPS-priming up to 10 ng/ml blood has been demonstrated.Unstimulated neutrophils do not lead to an endothelial cell damage, as also could be demonstrated by electron micrographs. An increased ¹¹¹In release occurred following neutrophil stimulation between 10 and 20 ng LPS/ml blood, but cannot be explained by an increased oxygen radical production of neutrophils. Therefore, these data suggest, that other neutrophil derived products rather than oxygen radicals contribute to an endothelial cell damage in this system.

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Chemiluminescenz-Antwort und Endothelzellschaden nach Lipopolysaccharid-Stimulation polymorphkerniger Leukocyten

     

The measurement of chemiluminescence,aggregation, and 5-hydroxy-6,8,11,14-eicosatetraenoic acid production of n-formyl-methioninyl-leucyl-phenylalanine-stimulated human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) which had been treated with nordihydroguaiaretic acid (NDGA) or indomethacin were stimulated with varying doses of n-formyl-methioninyl-leucyl-phenylalanine (FMLP). Aggregation and chemiluminescence (CL) were simulta-neously measured. Supernatant solutions were obtained and saved from cells stimulated with 10^?6M FMLP. These solutions were assayed for 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) using radioimmunoassay (RIA) techniques and used as a measure of 5-lipoxygenase (LO) activity. It was found that 10^?5M NDGA inhibited CL from PMNs stimulated with FMLP. However, NDGA had much less effect on aggregation of these cells. Indomethacin had little effect on PMN aggregation and less effect on CL than NDGA following FMLP stimulation. Indomethacin (10^?5M) did inhibit CL stimulated with FMLP at FMLP doses of 10^?7 and 10^?6M but had no effect of CL at FMLP doses of 10^?5M. Finally, RIA for 5-HETE showed that the effects of NDGA and indomethacin on 5-HETE production could be correlated with their effects on CL responses of cells stimulated with 10^?6M FMLP. These results support two conclusions. First, PMN-CL and LO activity may be correlated. Second, PMN-CL is not necessarily associated with PMN aggregation. These observations may indicate that LO products are not necessary for PMN aggregation to occur.     

Biotransformation of phorbol by human intestinal bacteria

Anaerobic incubation of phorbol (1) from Croton tiglium with human intestinal bacteria afforded five metabolites: isophorbol (2), deoxyphorbol (3), 4beta,9alpha,20-trihydroxy-13,15-seco-1,6,15-tigliatriene-3,13-dione (4), 4beta,9alpha,20-trihydroxy-15,16,17-trinor-1,6-tigliadiene-3,13-dione (5) and 4beta,9a,20-trihydroxy-14(13-->12)-abeo-12alphaH-1,6-tigliadiene-3,13-dione (6). All these metabolites (2-6) were identified and characterized by spectroscopic means, including two-dimensional (2D)-NMR. Nine defined strains from the human intestine showed an ability to transform 1 to these metabolites.     

Effect of liquiritin on human intestinal bacteria growth: metabolism and modulation

Licorice is one of the oldest and most frequently used prescribed traditional Chimese medicines. However, the route and metabolites of liquiritin by human intestinal microflora are not well understood and its metabolites may accumulate to exert physiological effects. Therefore, our objective was to screen the ability of the bacteria to metabolize liquiritin and assess the effect of this compound on the intestinal bacteria. Finally, six strains, comprising Bacteroides sp. 22 and sp.57, Veillonella sp. 31 and sp.48, Bacillus sp. 46 and Clostridium sp. 51, were isolated and their abilities to convert liquiritin studied. A total of five metabolites were identified using ultra performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry in human incubated solution. The results indicated that hydrolysis, hydrogenation, methylation, deoxygenation and acetylation were the major routes of metabolism of liquiritin. On the other hand, effect of liquiritin on different strains of intestinal bacteria growth was detected using an Emax precision microplate reader. Growth of certain pathogenic bacteria, such as Enterobacter, Enterococcus, Clostridium and Bacteroides, was significantly repressed by liquiritin, while growth of commensal probiotics such as Lactobacillus and Bifidobacterium was less severely affected. Our observation provided further evidence for the importance of intestinal bacteria in the metabolism, and the potential activity of liquiritin in human health and disease. Copyright © 2014 John Wiley & Sons, Ltd.     

Proteome analysis of rat polymorphonuclear leukocytes: a two-dimensional electrophoresis/mass spectrometry approach

The development of a two-dimensional (2-D) map of rat polymorphonuclear (PMN) leukocytes is here reported for the first time. The map is built up by utilizing a wide immobilized pH gradient (IPG), pH 3-10, in the first dimension and also a narrower IPG pH 4.5-8.5 gradient. In addition, the map is constructed by adopting the most recent protocols in 2-D mapping, which call for reduction and alkylation of the sample prior to the start of any electrophoretic step, including the IPG dimension. Fifty-two major protein spots have been so far identified by utilizing both matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray quadrupole (Q)-TOF mass spectrometry. A large number of house-keeping and cytoskeleton proteins were detected, together with proteins which are specific to PMN organelles or related to PMN functions such as phagocytosis and chemotaxis. The results obtained demonstrate the possibility of obtaining a single 2-D gel based proteomic map of PMN with representative proteins from different cellular compartments, also including membrane components, allowing the study of PMN protein expression on a proteome-wide scale. The aim of this project is to build an extensive database of such proteins, to be utilized for future studies where the expression of PMN proteins is used as a disease- or drug treatment marker.     

Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia

Fluorescence spectroscopy has the potential to improve the in vivo detection of intraepithelial neoplasias; however, the presence of inflammation can sometimes result in misclassifications. Inflammation is a common and important pathologic condition of epithelial tissues that can exist alone or in combination with neoplasia. It has not only been associated with the presence of cancer but also with the initiation of cancer by damage induced due to the oxidative activity of inflammatory cells. Microscopic examination of cervical biopsies has shown increased numbers of polymorphonuclear and mononuclear leukocytes in inflamed tissues mostly confined to the stroma. The purpose of this study was to characterize the fluorescence properties of human polymorpho- and mononuclear leukocytes and compare their fluorescence to that of cervical cancer cells. Human neutrophils were purified from peripheral blood and their fluorescence characterized over an excitation range of 250-550 nm. There are four notable excitation emission maxima: the tryptophan peak at 290 nm excitation, 330 nm emission; the NAD(P)H peak at 350 nm excitation, 450 nm emission, the FAD peak at 450 nm excitation, 530 nm emission and an unidentified peak at 500 nm excitation, 530 nm emission. Treatment of these peripheral blood neutrophils with 40 nM phorbol myristate acetate or with the chemotactic peptide formyl-Met-Leu Phe (1 M) demonstrated a significant increase in NAD(P)H fluorescence. Isolated mononuclear cells have similar emission peaks for tryptophan and NAD(P)H and a small broad peak at 450 nm excitation, 530 nm emission suggestive of FAD. Comparison of the fluorescence from leukocytes to epithelial cancer cell fluorescence has demonstrated the presence of these fluorophores in different quantities per cell. The most notable difference is the high level of tryptophan in cervical epithelial cancer cells, thus offering the potential for discrimination of inflammation.