scholarly journals Surface events in polymorphonuclear leucocyte activation: insights from a hydrophobic membrane antigen that triggers the respiratory burst

1987 ◽  
Vol 88 (5) ◽  
pp. 591-601
Author(s):  
H. Rosen ◽  
S. Gordon
Keyword(s):  
Guinea Pig ◽  
Respiratory Burst ◽  
Morphological Characteristics ◽  
Apparent Molecular Weight ◽  
Polymorphonuclear Leucocyte ◽  
Polymorphonuclear Neutrophils ◽  
Direct Immunofluorescence ◽  
Hydrophobic Membrane ◽  
Myelomonocytic Cells ◽  
Triton X

The rat monoclonal antibodies (Mab) 1A10.4 and IG4 were raised to a solubilized NADPH-oxidase preparation from guinea-pig polymorphonuclear neutrophils (PMN). They bind to a surface antigen restricted to guinea-pig myelomonocytic cells and on binding to the PMN surface can stimulate the respiratory burst (RB) and degranulation. This response was specific to these Mab and was not found with several other Mab restricted to myelomonocytic cells but with other antigenic epitopes. In order to understand the role of this molecule in stimulating the RB, we have characterized the antigen (Ag) by a variety of techniques. It is a hydrophobic membrane molecule with an apparent molecular weight of 8–10(x10(3] and a pI of 6.2. The Ag has been partially purified by extraction in organic solvents and high-pressure liquid chromatography on silica and is probably a proteolipid. Studies on Mab-dependent triggering of PMN secretion indicated that cross-linking of the cell surface was critical. We therefore used direct immunofluorescence under triggering and non-triggering conditions to show that stimulation of the RB by Mab correlated with redistribution of the surface Ag into patches. This patching was associated with aggregation of surface Ag and transfer of Ag from a Triton X-100-extractable to a Triton X-100-inextractable membrane domain. These movements of surface Ag, which included both patching and capping resulting from aggregation of a hydrophobic membrane component, occurred at 4 degrees C and were insensitive to inhibition by cytoskeletal inhibitors. These specific probes that control triggering of the RB have permitted the dissection of PMN stimulation into discrete membrane events by correlating the biochemical and morphological characteristics of a new PMN surface Ag as it stimulates exocytosis.

Glycogen in guinea pig neutrophils: Ultrastructural study of neutrophils at the intradermal site of BCG injection

1983 ◽  
Vol 41 ◽  
pp. 726-727
Author(s):  
Corazon D. Bucana
Keyword(s):  
Bone Marrow ◽  
Guinea Pig ◽  
Electron Density ◽  
Peritoneal Cavity ◽  
Ultrastructural Study ◽  
Guinea Pigs ◽  
Random Distribution ◽  
Glycogen Content ◽  
Polymorphonuclear Neutrophils ◽  
Ultrastructural Studies

In the circulating blood of man and guinea pigs, glycogen occurs primarily in polymorphonuclear neutrophils and platelets. The amount of glycogen in neutrophils increases with time after the cells leave the bone marrow, and the distribution of glycogen in neutrophils changes from an apparently random distribution to large clumps when these cells move out of the circulation to the site of inflammation in the peritoneal cavity. The objective of this study was to further investigate changes in glycogen content and distribution in neutrophils. I chose an intradermal site because it allows study of neutrophils at various stages of extravasation.Initially, osmium ferrocyanide and osmium ferricyanide were used to fix glycogen in the neutrophils for ultrastructural studies. My findings confirmed previous reports that showed that glycogen is well preserved by both these fixatives and that osmium ferricyanide protects glycogen from solubilization by uranyl acetate.I found that osmium ferrocyanide similarly protected glycogen. My studies showed, however, that the electron density of mitochondria and other cytoplasmic organelles was lower in samples fixed with osmium ferrocyanide than in samples fixed with osmium ferricyanide.

scholarly journals Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

1991 ◽  
Vol 280 (3) ◽  
pp. 745-751 ◽  
Author(s):  
N M Hooper ◽  
A Bashir
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Aqueous Phase ◽  
Hydrophobic Membrane ◽  
Rich Phase ◽  
Glycosyl Phosphatidylinositol ◽  
Ionic Detergent ◽  
Membrane Spanning ◽  
Triton X ◽  
Insoluble Pellet

Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.

Ketamine inhibits polymorphonuclear leucocyte CD11b expression and respiratory burst activity in endotoxemic rats

2007 ◽  
Vol 56 (4) ◽  
pp. 149-153 ◽  
Author(s):  
Z. Q. Zhou ◽  
Y. Q. Yu ◽  
S. W. Feng ◽  
M. Yu ◽  
H. J. Liu ◽  
...  
Keyword(s):  
Respiratory Burst ◽  
Polymorphonuclear Leucocyte ◽  
Burst Activity ◽  
Respiratory Burst Activity ◽  
Cd11b Expression

Cleavage of human von Willebrand factor by platelet calcium-activated protease

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 352-356 ◽  
Author(s):  
TJ Kunicki ◽  
RR Montgomery ◽  
J Schullek
Keyword(s):  
Von Willebrand Factor ◽  
Sodium Dodecylsulfate ◽  
Apparent Molecular Weight ◽  
Aminocaproic Acid ◽  
Crossed Immunoelectrophoresis ◽  
Nonionic Detergent ◽  
Epsilon Aminocaproic Acid ◽  
Triton X ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In human platelet lysates prepared by addition of nonionic detergent (Triton X-100) or by sonication, the multimer composition and electrophoretic mobility of platelet von Willebrand factor (vWF) were consistently modified under conditions that would favor activation of the endogenous calcium-activated, sulfhydryl-dependent neutral protease (CAP). By sodium dodecylsulfate-agarose gel electrophoresis, native platelet vWF contained some multimers that were larger than those characteristic of plasma vWF. Modified platelet vWF contained a multimer population equivalent to or smaller than that of plasma vWF plus an additional fast-migrating band. In crossed immunoelectrophoresis (CIE), modified platelet vWF was characterized by a more anodic distribution and the appearance of a distinct, cross- reactive, anodic component previously designated VIIIR:Ag fragment. In the presence of calcium, radiolabeled purified plasma vWF was also degraded by the protease in question, with a decrease in the apparent molecular weight of the reduced monomer from 230,000 to 205,000. The VIIR:Ag fragment isolated from the same degraded plasma vWF by preparative CIE was shown to be composed of an identical mol wt 205,000 subunit. Because cleavage of plasma or platelet vWF was inhibited by prior addition of leupeptin, EDTA, ethylene glycol bis (beta-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), or N-ethylmaleimide (agents known to inhibit platelet CAP) but was unaffected by numerous other protease inhibitors, including soybean trypsin inhibitor, benzamidine, hirudin, phenylmethylsulfonyl fluoride, aprotonin, or epsilon-aminocaproic acid (none of which inhibits platelet CAP), we conclude that proteolysis of vWF observed in this study is a direct effect of CAP and is not mediated by way of secondary proteases.

scholarly journals PMN heterogeneity: long-term stability of fluorescent membrane potential responses to the chemoattractant N-formyl-methionyl-leucyl- phenylalanine in healthy adults and correlation with respiratory burst activity

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 611-618 ◽  
Author(s):  
MP Fletcher ◽  
BE Seligmann
Keyword(s):  
Membrane Potential ◽  
Respiratory Burst ◽  
Healthy Adults ◽  
Polymorphonuclear Neutrophils ◽  
Cyanine Dye ◽  
Potential Response ◽  
Long Term Stability ◽  
Nbt Reduction ◽  
Wbc Count ◽  
Immature Cells

Abstract Polymorphonuclear neutrophils (PMNs) were isolated from 24 healthy adults 20 to 61 years of age and the proportion of cells that demonstrated depolarization responses to the synthetic chemotaxin N- formyl-methionyl-leucyl-phenylalanine (FMLP) were enumerated using the lipophilic fluorescent cyanine dye 3,3′-di-pentyl-oxacarbocyanine [di-O- C(5)(3)] and flow cytometry. The membrane potential responses were correlated to the cells' respiratory burst capabilities as measured by nitroblue tetrazolium (NBT) reduction and/or superoxide production in response to FMLP; 40.2% +/- 15.1% (mean +/- SD) of cells depolarized to FMLP. The mean SE for duplicate determinations 1 hour apart was 3.8% (range 0.4% to 13.6%, n = 15). There was no correlation between the percentage of depolarizing PMNs and the yield of cells, the percentage of immature cells, or the circulating WBC count. There was no difference in the average age of men (34.9 +/- 9.9 years, n = 11) v women (33.8 +/- 8.5, n = 13) (mean +/- SD) studied, or in the percentage of depolarizing PMNs when men and women were compared (42.2 +/- 10.6% v 43.1 +/- 13.3%, respectively). However, there was a significant increase in the percentage of depolarizing PMNs with increasing age of women (r = .61, P less than .025) but not men (r = .03, P greater than .05). Analysis of variance revealed significantly greater person-to-person variability in the membrane potential response than in day-to-day variability in the same person (P less than .0005). The percentage of depolarizing PMNs in response to FMLP was significantly correlated with the percentage of NBT-positive cells from both purified PMNs and from whole blood (r = .849, P less than .0005, r = .857, P less than .05, respectively), and with the amount of superoxide produced, expressed as a percentage of that amount produced by cytochalasin B (cyto-B)-pretreated cells (r = .565, P less than .01). The data indicate that PMNs from healthy adults demonstrate a heterogeneous membrane potential response to the chemotaxin FMLP that correlates with the cells' oxidative responsiveness and that intersubject differences can be detected. In addition, the proportion of responsive PMNs increases with increasing age in women.

Effects of non-ionic and anionic detergents on lipid-associated tissue ferritin.

1988 ◽  
Vol 34 (1) ◽  
pp. 152-154
Author(s):  
B E Cham ◽  
P Roeser ◽  
A Nikles
Keyword(s):  
Guinea Pig ◽  
Liver Homogenate ◽  
Diisopropyl Ether ◽  
Detergent Concentration ◽  
Pig Liver ◽  
Ionic Detergents ◽  
Ionic Detergent ◽  
Anionic Detergents ◽  
Triton X ◽  
Guinea Pig Liver

Abstract Lipid-associated ferritin from homogenates of guinea pig liver is released from its conjugate(s) by incubation with the non-ionic detergents Triton X-100 and Nonidet P-40 but not by incubation with the anionic detergent deoxycholate. The amount of lipid-associated ferritin released from its conjugate(s) depends on the concentration of the non-ionic detergents. At a final non-ionic detergent concentration of about 20 g/L, all lipid-associated ferritin is released from its conjugate(s) in a liver homogenate. The amount released is identical with the amount of the lipid-associated ferritin obtained by extraction of the same liver homogenate with a mixture of butanol and diisopropyl ether.

scholarly journals The metabolism of cyclic nucleotides in the guinea-pig pancreas. Adenylate cyclase and guanylate cyclase

1980 ◽  
Vol 186 (2) ◽  
pp. 499-505 ◽  
Author(s):  
M Lemon ◽  
P Methven ◽  
K Bhoola
Keyword(s):  
Adenylate Cyclase ◽  
Guinea Pig ◽  
Guanylate Cyclase ◽  
Cyclic Nucleotides ◽  
Cyclase Activity ◽  
Dependent Manner ◽  
Guanylate Cyclase Activity ◽  
Triton X ◽  
Dose Dependent ◽  
Significant Activation

Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.

Effect of the Mucoactive Drug Nacystelyn on the Respiratory Burst of Human Blood Polymorphonuclear Neutrophils

1997 ◽  
Vol 10 (5-6) ◽  
pp. 287-292 ◽  
Author(s):  
A.M Nagy ◽  
F Vanderbist ◽  
N Parij ◽  
P Maes ◽  
P Fondu ◽  
...  
Keyword(s):  
Human Blood ◽  
Respiratory Burst ◽  
Polymorphonuclear Neutrophils

Sevoflurane and Isoflurane Protect the Reperfused Guinea Pig Heart by Reducing Postischemic Adhesion of Polymorphonuclear Neutrophils 

1999 ◽  
Vol 91 (2) ◽  
pp. 521-530 ◽  
Author(s):  
Bernhard Heindl ◽  
Florian M. Reichle ◽  
Stefan Zahler ◽  
Peter F. Conzen ◽  
Bernhard F. Becker
Keyword(s):  
Guinea Pig ◽  
Cardiac Function ◽  
Minimum Alveolar Concentration ◽  
Volatile Anesthetics ◽  
Underlying Mechanism ◽  
Polymorphonuclear Neutrophils ◽  
Guinea Pig Heart ◽  
Cd11b Expression ◽  
Heart Work ◽  
The Difference

Background Polymorphonuclear neutrophils (PMNs) contribute to reperfusion injury. Because volatile anesthetics can reduce PMN adhesion in the reperfused, nonworking heart, the authors analyzed whether this action of volatile anesthetics affects cardiac performance after ischemia and reperfusion and further clarified the underlying mechanism. Methods Isolated guinea pig hearts perfused with crystalloid buffer and performing pressure-volume work were used. Hearts were subjected to 15 min global ischemia and 20 min reperfusion. In the intervention groups an intracoronary bolus of 3 x 10(6) PMNs was applied in the second min of reperfusion, either in the absence or presence of 0.5 or 1 minimum alveolar concentration sevoflurane or isoflurane. The number of sequestered PMNs was calculated from the difference between coronary input and output (coronary effluent) of PMNs. Performance of external heart work, determined pre- and postischemically, served as criterion for recovery of myocardial function. Additionally, the expression of the integrin CD11b on the cell surface of PMN was measured before and after coronary passage. Results Injection of PMN in the reperfusion phase, but not under nonischemic conditions, reduced recovery of external heart work significantly (from 55+/-7% to 19+/-11%). Addition of sevoflurane or isoflurane in concentrations of 0.5 and 1 minimum alveolar concentration to the perfusate reduced postischemic PMN adhesion from 36+/-8% to basal values (20+/-7%) and prevented decline of cardiac function. CD11b expression on PMNs increased significantly during postischemic coronary passage under control conditions. Again, both anesthetics in both concentrations inhibited that activation. Conclusions Volatile anesthetics reduce PMN adhesion in the reperfused coronary system and thereby preserve cardiac function. Reduced expression of the adhesion molecule CD11b on PMNs in the presence of sevoflurane or isoflurane is, at least in part, responsible for the cardioprotective effect.

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