NADPH-dependent reduction of 2,6-dichlorophenol-indophenol by the phagocytic vesicles of pig polymorphonuclear leucocytes.
Ontology highlight
ABSTRACT:
SUBMITTER: Wakeyama H
PROVIDER: S-EPMC1154259 | biostudies-other | 1983 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The present study was aimed at determining the metabolic profile of PMNs in obese subjects, and to explore its potential relationship with insulin resistance (IR). To achieve this goal, a pilot clinical study was performed using PMNs from 17 patients with obesity and IR, and 17 lean controls without IR, which was validated in an additional smaller cohort (consisting of 10 patients and 10 controls). PMNs were isolated from peripheral blood and nuclear magnetic resonance was used to perform the metabolomic analysis. A total of 48 metabolites were quantified. The main metabolic change found in PMNs was a significant increase in 2-aminoisobutyric acid with a direct correlation with HOMA-IR (p<0.001), BMI (p<0.000001) and waist circumference (p<0.000001). By contrast, a decrease of 3-hydroxyisovalerate was observed with an inverse correlation with HOMA-IR (p = 0.001), BMI (p = 0.001) and waist circumference (p = 0.0001). Notably, the metabolic profile in plasma was different than that obtained in PMNs. In summary, our results suggest that the change in 3-hydroxyisovalerate and 2-aminoisobutyric is the key metabolic fingerprint in PMNs of obese subjects with IR. In addition, our methodology could be an easy and reliable tool for monitoring the effect of treatments in the setting of precision medicine.
| S-EPMC6044522 | biostudies-literature
Project description:A phagocytic vesicle fraction with high NADPH-dependent superoxide-forming activity was obtained in large quantity from pig blood polymorphonuclear leucocytes, phagocytosing oil droplets in the presence of cyanide. The activity of the homogenate of the phagocytosing cells was 40 times that of the resting cells, and 70% of the activity in the homogenate was recovered in the phagocytic vesicle fraction. Essentially all of the superoxide-forming activity was extracted by repeated extraction with a mixture containing deoxycholate and Tween 20. The extract had a superoxide-forming activity of 1 mumol/min per mg of protein with NADPH, and one-fifth of this with NADH, Km values being similar to those of the vesicle fraction (40 microM for NADPH and 400 microM for NADH). A stoichiometric relationship of 1:2 for NADPH oxidation and superoxide formation was obtained, in agreement with the reaction NADPH +2O2 leads to NADP+ + 2O2 -. + H+. The activity of the extract was enhanced 2-fold by the addition of FAD, suggesting that the flavin is a component of the enzyme system. The Km value for FAD was 0.077 microM. The activities in both vesicle fraction and extract were labile even on refrigeration, but could be kept for several months at -70 degrees C.
| S-EPMC1158526 | biostudies-other
Project description:1. Two cyclic AMP-independent casein/glycogen synthase kinases were purified from pig polymorphonuclear leucocytes by chromatography on phosphocellulose followed by affinity chromatography on casein-Sepharose 4B or gel filtration on Bio-Gel A-1.5m. When the affinity step was used, the specific activities were 86 and 43units/mg of protein for casein kinase 1 and 2, respectively, whereas these values were 94 and 90units/mg of protein when the gel-filtration step was used. 2. These kinases differ as follows: (a) the molecular weight of casein kinase 1 (38000) is very much lower than that of casein kinase 2 (185000); (b) the K(m) for casein (0.46mg/ml) and K(a) for Mg(2+) (0.3mm) of casein kinase 1 are lower than those of casein kinase 2 (0.90mg/ml and 1.7mm respectively); (c) KCl stimulates the phosphorylation of casein by casein kinase 1, whereas it inhibits phosvitin phosphorylation by this enzyme; on the contrary, the effect of KCl on casein kinase 2 is very similar with either casein or phosvitin as substrate; (d) although both kinases phosphorylate rabbit muscle glycogen synthase I, the ratio of glycogen synthase to casein phosphorylation by casein kinase 1 is about 4-fold greater than that by casein kinase 2. Furthermore, (32)P incorporation into glycogen synthase promoted by casein kinase 1 (3.6mol of (32)P/mol of 85000-dalton subunit) is twice that observed with casein kinase 2 (1.8mol of (32)P/mol of 85000-dalton subunit). Such a phosphorylation results in a decrease in the glucose 6-phosphate-independence ratio of glycogen synthase to 10-15 with casein kinase 1 and to 35-45 with casein kinase 2. 3. The activity of both kinases is neither stimulated by cyclic AMP, Ca(2+) and calmodulin nor inhibited by cyclic AMP-dependent protein kinase inhibitor protein. 4. No phosphorylation kinase activity was observed with casein kinase 1 and 2 at either pH6.8 or 8.2 in the presence of Ca(2+). 5. Activities of both kinases on casein and glycogen synthase decreased in parallel when incubated at 50 degrees C.
| S-EPMC1162674 | biostudies-other
Project description:ObjectivesPolymorphonuclear leucocytes (PMNs) constitute the first line of host defence and are crucial in maintaining periodontal health. Their survival and function are modulated by mesenchymal stromal cells (MSCs) from different origin. Gingival MSCs (GMSCs) play an important role in maintaining oral health and in the initial inflammatory response. The present study aimed to investigate the effects of GMSCs on PMNs apoptosis and reactive oxygen species (ROS) production.MethodsPMNs were either directly incubated with untreated, interleukin (IL)-1β- or tumour necrosis factor (TNF)-α-treated GMSCs or stimulated with their conditioned media. Resulting ROS production was evaluated by dichlorofluorescin diacetate staining, whereas PMNs apoptosis was assessed by Annexin V staining, followed by flow cytometry analysis.ResultsWhile conditioned media of untreated and TNF-α-treated GMSCs did not affect apoptosis of PMNs, it was significantly delayed by conditioned media of GMSCs treated with IL-1β. In direct co-culture, GMSCs exerted anti-apoptotic effects on PMNs independently of the previous stimulation. However, the strongest impact was observed by IL-1β-treated GMSCs. ROS production of PMNs was not influenced by GMSCs or their conditioned media.ConclusionThis study demonstrates for the first time the immunomodulatory properties of GMSCs towards PMNs, revealing that IL-1β enhances anti-apoptotic effects of GMSCs.
| S-EPMC9290793 | biostudies-literature
Project description:1. Homogenates of guinea-pig polymorphonuclear leucocytes were separated by differential centrifugation into six particulate fractions and a soluble fraction. 2. The distributions in these fractions of protein, DNA, succinate dehydrogenase, beta-glucuronidase, peroxidase, alkaline phosphatase, acid phosphatase (against p-nitrophenyl phosphate and beta-glycerophosphate), cathepsin, and catalase were compared. 3. Almost all of the DNA sedimented in the first two pellets, indicating that the nuclei were relatively intact. 4. The four hydrolases and peroxidase showed different distribution patterns, although these activities were previously reported to be localized mainly in the single ;granule' fraction isolated from leucocytes. 5. The particles containing peroxidase, acid phosphatase and alkaline phosphatase all exhibited latency. Maximum activity for each enzyme was obtained at roughly similar concentrations of Triton X-100. 6. The acid phosphatase of these cells was distributed between two populations of particles that differed in both sedimentation characteristics and density. The acid phosphatase(s) of the two populations showed slightly different substrate specificities. This bimodal distribution was not an artifact of the procedure used to elicit the cells. 7. Catalase was recovered almost entirely in the soluble fraction and showed no latency in freshly prepared homogenates. No urate oxidase was detected. 8. We conclude that the ;granule' fraction of the polymorphonuclear leucocyte, as isolated by previous workers, contains at least three, probably more, populations of particles with different enzyme contents, and that these cells probably do not contain peroxisomes.
| S-EPMC1185347 | biostudies-other
Project description:An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.
| S-EPMC1144305 | biostudies-other
Project description:Symptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40-70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase- and RecA-deficient Gc, which are more sensitive to ROS in vitro, had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.
| S-EPMC2771623 | biostudies-literature
Project description:Plasma membranes prepared from guinea-pig peritoneal polymorphonuclear leucocytes showed an immune-complex-binding activity that corresponded well with the activity in intact cells. The characteristics of this activity were reversible binding, dependence on the Fc portion of antigen-complexed IgG (immunoglobulin G), competition with aggregated IgG and independence from energy metabolism. These results support the conclusion that the binding activity found in the isolated plasma membranes is an Fc-receptor activity of guinea-pig peritoneal polymorphonuclear leucocytes. The activity showed Kd = 6.7 x 10(-8) M-IgG and maximum binding of 17 pmol of IgG/mg of membrane protein when measured with an immune complex of alpha-amylase and homologous guinea-pig anti-(alpha-amylase) IgG. Inhibition of the Fc-receptor activity by a series of various salts indicated the contribution of the hydrophobic interaction to the binding. Inhibitory effects of salts or metal-chelating reagents on the Fc-receptor activity were also observed on superoxide generation by these cells induced by the immune complex, suggesting a role of the Fc receptor as the immune-complex-binding site responsible for the initiation of superoxide generation.
| S-EPMC1158400 | biostudies-other
Project description:The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.
| S-EPMC1138384 | biostudies-other
Project description:The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O2. The activity of the oxidase at the plasma membrane can be measured as electron current (I(e)), and the voltage dependence of I(e) was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from -120 to 200 mV, were applied to obtain 'steady-state' current-voltage relationships (I-V). The amplitude of I(e) recorded at -40 mV was minimal at 8 microM NADPH and saturated above 1 mM, with half-maximal activity (K(m)) observed at approx. 110 microM NADPH. Comparison of I-V values obtained at different NADPH concentrations revealed that the voltage-dependence of I(e) is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, I(e) was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (-60 to 60 mV), the I-V curve strongly rectifying only below -100 mV. At lower NADPH concentrations the I-V curve was progressively shifted to more positive potentials and I(e) became voltage-independent also within the physiological range. Consequently, the K(m) of the oxidase decreased by approx. 40% (from 100 to 60 microM) when the membrane potential increased from -60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the I(e)-V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of I(e) reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase.
| S-EPMC1138955 | biostudies-literature