scholarly journals Neutrophils from patients after burn injury express a deficiency of the oxidase components p47-phox and p67-phox

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4321-4329 ◽  
Author(s):  
J Rosenthal ◽  
GW Thurman ◽  
N Cusack ◽  
VM Peterson ◽  
HL Malech ◽  
...  
Keyword(s):  
Respiratory Burst ◽  
Oxidase Activity ◽  
Thermal Injury ◽  
Burn Injury ◽  
Sodium Dodecyl ◽  
Burn Patients ◽  
Biochemical Basis ◽  
Free System ◽  
The Status ◽  
Oxidase Enzyme

Infection is a major cause of morbidity and mortality in patients after thermal injury. This predisposition to infections is related, in part, to abnormal polymorphonuclear leukocyte (PMN) function and a diminished respiratory burst. To evaluate the biochemical basis for the defective respiratory burst after major burns, the status of the oxidase enzyme system and its components was investigated. PMNs were isolated from 24 patients with 12% to 62% burns. Oxidase activity of intact PMNs, measured as superoxide anion (O2-) generation or oxygen consumption, was decreased in burn compared with healthy controls. Subcellular fractions from patient PMNs generated less O2- in the sodium dodecyl sulfate cell-free system, and this was related to a diminished contribution by cytosol but not by plasma membrane. Subsequently, cytosol was separated with CM-Sepharose, yielding two fractions; one contained the p47-phox and p67-phox (47/67 mix) and the other contained the remaining cytosolic components (run through [RT]). Although the contribution to oxidase activity made by RT from patient cytosol was similar to that of control, the activity of p47/67 mix from PMNs of burn patients was deficient. Quantitative assays using an immunoautoradiographic technique showed a consistent, but significant decrease in both p47-phox and p67-phox. The addition of purified or human recombinant p47-phox but not p67-phox corrected the diminished oxidase activity of cytosol from burn patients. Thus, decreased respiratory burst activity found in PMNs from individuals with thermal injury was associated with a specific, quantitative deficiency of p47- phox.

scholarly journals Neutrophils from patients after burn injury express a deficiency of the oxidase components p47-phox and p67-phox

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4321-4329 ◽  
Author(s):  
J Rosenthal ◽  
GW Thurman ◽  
N Cusack ◽  
VM Peterson ◽  
HL Malech ◽  
...  
Keyword(s):  
Respiratory Burst ◽  
Oxidase Activity ◽  
Thermal Injury ◽  
Burn Injury ◽  
Sodium Dodecyl ◽  
Burn Patients ◽  
Biochemical Basis ◽  
Free System ◽  
The Status ◽  
Oxidase Enzyme

Abstract Infection is a major cause of morbidity and mortality in patients after thermal injury. This predisposition to infections is related, in part, to abnormal polymorphonuclear leukocyte (PMN) function and a diminished respiratory burst. To evaluate the biochemical basis for the defective respiratory burst after major burns, the status of the oxidase enzyme system and its components was investigated. PMNs were isolated from 24 patients with 12% to 62% burns. Oxidase activity of intact PMNs, measured as superoxide anion (O2-) generation or oxygen consumption, was decreased in burn compared with healthy controls. Subcellular fractions from patient PMNs generated less O2- in the sodium dodecyl sulfate cell-free system, and this was related to a diminished contribution by cytosol but not by plasma membrane. Subsequently, cytosol was separated with CM-Sepharose, yielding two fractions; one contained the p47-phox and p67-phox (47/67 mix) and the other contained the remaining cytosolic components (run through [RT]). Although the contribution to oxidase activity made by RT from patient cytosol was similar to that of control, the activity of p47/67 mix from PMNs of burn patients was deficient. Quantitative assays using an immunoautoradiographic technique showed a consistent, but significant decrease in both p47-phox and p67-phox. The addition of purified or human recombinant p47-phox but not p67-phox corrected the diminished oxidase activity of cytosol from burn patients. Thus, decreased respiratory burst activity found in PMNs from individuals with thermal injury was associated with a specific, quantitative deficiency of p47- phox.

Knockdown of Neutrophil p29 Peroxiredoxin (Prx) in Transgenic K562 Cells Expressing Phox Proteins Results in Diminished Oxidase Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1643-1643
Author(s):  
Daniel R. Ambruso ◽  
Gail Thurman
Keyword(s):  
Western Blot ◽  
Respiratory Burst ◽  
Oxidase Activity ◽  
Enzyme System ◽  
K562 Cells ◽  
Free System ◽  
Transfected Cells ◽  
Cell Free System ◽  
Cell Counts ◽  
Oxidase Enzyme

Abstract Introduction: Production of reactive oxygen species through a respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of the components of the NADPH oxidase enzyme system (p47phox, p67phox, p40phox, gp91phox, p22phox and Rac) and expression of the activity of the system to produce superoxide anion (O2 −). We recently identified a neutrophil protein with an approximate MW of 29 kDa which binds to the p67phox, is classified as a peroxiredoxin (Prx) and translocates to the plasma membrane during stimulation of the neutrophil. Additional studies demonstrate that this protein (p29 Prx) increases production of O2 − in a cell-free system of oxidase activity in a specific, stoichiometric manner and that the cysteine residues of p29 Prx, at amino acid positions 47 and 91, are required for this activity. The current studies demonstrate the role of p29 Prx in oxidase activity using the technique of small interfering RNA (siRNA) to degrade specific mRNA and decrease the expression of the protein. Methods: siRNA probes for p29 Prx were constructed based on standard constraints for unique 19 nucleotide binding sites along with other sequence selection criteria. Six probes were constructed based on the cDNA sequence of p29 Prx; one resulted in significant knockdown of p29 Prx. Inactive siRNA fluorescently labeled was obtained commercially. K562 cells, stably transfected with the p67phox, p47phox, gp91phox and low affinity fMLP receptor, were cultured under standard conditions and expressed p29 Prx message by RT-PCR and protein by Western blot. Transgenic K562 cells were transfected with siRNA or GFP labeled control siRNA with Nucleofector technology. Cell counts and viability were determined by standard techniques. For Western blots, proteins from cell lysates were separated on 10% SDS-PAGE and blotted onto nitrocellulose, and specific proteins were detected with polyclonal antibodies to p29 Prx, actin or p67phox, p47phox, gp91phox, and p22phox by chemiluminescent technique. Results: After transfection with the active siRNA for p29 Prx, inactive or labeled siRNA, the K562 cells were harvested at 24, 48, and 72 hours. A knockdown by one of the 6 siRNAs resulted in decreased levels of p29 Prx by Western blot. Optimum knockdown was achieved by transfection of 6 μg siRNA and the decrease in p29 Prx observed at 24 hours but was optimum after 48 hours. Under these conditions, a decrease in p29 Prx by 50–60% detected by Western blot was achieved with no differences in levels of actin or any of the phox proteins. The viability of control cells and siRNA transfected cells was not different. Cells transfected with siRNA for p29 Prx which demonstrated a knockdown of this protein exhibited decreased respiratory burst (O2 −) in response to fMLP (measured by chemiluminescence) or PMA (cytochrome c reduction) compared to control porated or transfected cells. Conclusion: These results correlate with in vitro studies of recombinant p29 Prx in the SDS cell-free system of oxidase activation. Decreased levels of p29 Prx result in decreased oxidase activity on transgenic K562 cells. p29 Prx may be important for the expression of the oxidase enzyme system through its antioxidant or signaling activity.

scholarly journals Proteinuria after Burn Injury

1981 ◽  
Vol 18 (6) ◽  
pp. 353-360 ◽  
Author(s):  
Peter G Shakespeare ◽  
Edward J Coombes ◽  
Joan Hambleton ◽  
Diane Furness
Keyword(s):  
Total Protein ◽  
Burn Injury ◽  
Serum Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Burn Patients ◽  
Tubular Proteinuria ◽  
Glomerular Lesion ◽  
Protein Excretion ◽  
Urine Pool

Qualitative and quantitative aspects of the excretion of protein after burn injury have been investigated. The excretion of total protein and of albumin have been measured nephelometrically while the excretion of proteins of both serum and non-serum origin have been measured by immunoelectrophoresis using antiserum against serum proteins or against a pool of urine from burned patients. Comparison of the patterns of proteins observed in urine using these two different antisera showed the presence of at least three major proteins that did not originate from the plasma. The excretion of two of these three proteins followed closely the pattern of total protein excretion which reached a maximum at five to seven days after injury. The excretion of albumin was greatest in the first 48 hours after injury. Examination of the original burn patients' urine antigen by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that this urine pool contained greater amounts of lower molecular weight proteins than were observed in a pool of normal urines. The observations suggest that a biphasic renal pathophysiology is observed after burn injury. Initially, there develops a mild transient glomerular lesion which progresses to a different state which shows at least some of the features of a tubular proteinuria.

Vestigial respiratory burst activity in wound macrophages

1999 ◽  
Vol 276 (6) ◽  
pp. R1587-R1594 ◽  
Author(s):  
Christopher C. Nessel ◽  
William L. Henry ◽  
Balduino Mastrofrancesco ◽  
Jonathan S. Reichner ◽  
Jorge E. Albina
Keyword(s):  
Nadph Oxidase ◽  
Respiratory Burst ◽  
Oxidase Activity ◽  
Reactive Oxygen ◽  
Human Neutrophils ◽  
Reactive Oxygen Intermediates ◽  
Oxygen Intermediates ◽  
Free System ◽  
Nadph Oxidase Activity ◽  
Text Production

Macrophages from experimental wounds in rats were tested for their capacity to generate reactive oxygen intermediates. Measurements of superoxide and H2O2release, [Formula: see text]-dependent lucigenin chemiluminescence, oxygen consumption, hexose monophosphate shunt flux, and NADPH oxidase activity in cell lysates indicated, at best, the presence of a vestigial respiratory burst response in these cells. The inability of wound cells to release[Formula: see text] was not rekindled by priming with endotoxin or interferon-γ in vivo or in vitro. NADPH oxidase activity in a cell-free system demonstrated that wound macrophage membranes, but not their cytosols, were capable of sustaining maximal rates of [Formula: see text] production when mixed with their corresponding counterparts from human neutrophils. Immune detection experiments showed wound macrophages to be particularly deficient in the cytosolic component of the NADPH oxidase p47- phox. Addition of recombinant p47- phox to the human neutrophil-cell membrane/wound macrophage cytosol cell-free oxidase assay, however, failed to support[Formula: see text] production. Present findings indicate an unexpected deficit of wound macrophages in their capacity to generate reactive oxygen intermediates.

scholarly journals Early abnormal fibrinolysis and mortality in patients with thermal injury: a prospective cohort study

BJS Open ◽  
2021 ◽  
Vol 5 (2) ◽  
Author(s):  
A E Pusateri ◽  
T D Le ◽  
J W Keyloun ◽  
L T Moffatt ◽  
T Orfeo ◽  
...  
Keyword(s):  
Cohort Study ◽  
Prospective Cohort Study ◽  
Prospective Cohort ◽  
Odds Ratio ◽  
Thermal Injury ◽  
Burn Injury ◽  
Total Body Surface Area ◽  
Burn Patients ◽  
Clot Strength ◽  
Increased Risk

Abstract Introduction Abnormal fibrinolysis early after injury has been associated with increased mortality in trauma patients, but no studies have addressed patients with burn injury. This prospective cohort study aimed to characterize fibrinolytic phenotypes in burn patients and to see if they were associated with mortality. Methods Patients presenting to a regional burn centre within 4 h of thermal injury were included. Blood was collected for sequential viscoelastic measurements using thromboelastography (RapidTEG™) over 12 h. The percentage decrease in clot strength 30 min after the time of maximal clot strength (LY30) was used to categorize patients into hypofibrinolytic/fibrinolytic shutdown (SD), physiological (PHYS) and hyperfibrinolytic (HF) phenotypes. Injury characteristics, demographics and outcomes were compared. Results Of 115 included patients, just over two thirds were male. Overall median age was 40 (i.q.r. 28–57) years and median total body surface area (TBSA) burn was 13 (i.q.r. 6–30) per cent. Some 42 (36.5 per cent) patients had severe burns affecting over 20 per cent TBSA. Overall mortality was 18.3 per cent. At admission 60.0 per cent were PHYS, 30.4 per cent were SD and 9.6 per cent HF. HF was associated with increased risk of mortality on admission (odds ratio 12.61 (95 per cent c.i. 1.12 to 142.57); P = 0.041) but not later during the admission when its incidence also decreased. Admission SD was not associated with mortality, but incidence increased and by 4 h and beyond, SD was associated with increased mortality, compared with PHYS (odds ratio 8.27 (95 per cent c.i. 1.16 to 58.95); P = 0.034). Discussion Early abnormal fibrinolytic function is associated with mortality in burn patients.

scholarly journals Vitamin D status and its influence on outcomes following major burn injury and critical illness

2018 ◽  
Vol 6 ◽  
Author(s):  
Khaled Al-Tarrah ◽  
Martin Hewison ◽  
Naiem Moiemen ◽  
Janet M. Lord
Keyword(s):  
Vitamin D ◽  
Critical Illness ◽  
Vitamin D Deficiency ◽  
Critically Ill ◽  
Critically Ill Patients ◽  
Thermal Injury ◽  
Burn Injury ◽  
Vitamin D Supplementation ◽  
Burn Patients ◽  
Vitamin D Status

Abstract Vitamin D deficiency is common among the general population. It is also observed in up to 76% of critically ill patients. Despite the high prevalence of hypovitaminosis D in critical illness, vitamin D is often overlooked by medical staff as the clinical implications and consequences of vitamin D deficiency in acute contexts remain to be fully understood. Vitamin D has a broad range of pleotropic effects on various processes and systems including the immune-inflammatory response. 1α,25-dihydroxyvitamin D (1,25(OH)2D), has been shown to promote a tolerogenic immune response limiting deleterious inflammatory effects, modulation of the innate immune system, and enhancement of anti-microbial peptides. Vitamin D deficiency is frequently observed in critically ill patients and has been related to extrinsic causes (i.e., limited sunlight exposure), magnitude of injury/illness, or the treatment started by medical doctors including fluid resuscitation. Low levels of vitamin D in critically ill patients have been associated with sepsis, organ failure, and mortality. Despite this, there are subpopulations of critical illness, such as burn patients, where the literature regarding vitamin D status and its influence on outcomes remain insufficient. Thermal injury results in damage to both burned and non-burned tissues, as well as induces an exaggerated and persistent immune-inflammatory and hypermetabolic response. In this review, we propose potential mechanisms in which burn injury affects the vitamin D status and summarizes current literature investigating the influence of vitamin D status on outcomes. In addition, we reviewed the literature and trials investigating vitamin D supplementation in critically ill patients and discuss the therapeutic potential of vitamin D supplementation in burn and critically ill patients. We also highlight current limitations of studies that have investigated vitamin D status and supplementation in critical illness. Thermal injury influences vitamin D status. More studies investigating vitamin D depletion in burn patients and its influence on prognosis, via standardized methodology, are required to reach definitive conclusions and influence clinical practice.

Antioxidant Effects of Neutrophil p29 Peroxiredoxin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3087-3087
Author(s):  
Daniel R. Ambruso ◽  
Gail Thurman ◽  
Carolina Gonzalez-Aller
Keyword(s):  
Western Blot ◽  
Respiratory Burst ◽  
Fluorescent Protein ◽  
Enzyme System ◽  
Oxidant Stress ◽  
Western Blots ◽  
Free System ◽  
Cell Lysates ◽  
Cell Counts ◽  
Oxidase Enzyme

Abstract Production of reactive oxygen species through a respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of the components of the NADPH oxidase enzyme system (p47phox, p67phox, p40phox, gp91phox, p22phox and Rac) and expression of the activity of the system to produce superoxide anion (O2 −). We recently identified a neutrophil protein with an approximate MW of 29 kDa which binds to the p67phox, is classified as a peroxiredoxin (Prx) and translocates to the plasma membrane during stimulation of the neutrophil. Additional studies demonstrate that this protein (p29 Prx) increases production of O2 − in a cell-free system of oxidase activity in a specific, stoichiometric manner and that the cysteine residues of p29 Prx, at amino acid positions 47 and 91, are required for this activity. The current studies demonstrate the antioxidant activity of p29 Prx using the technique of small interfering RNA (siRNA) to degrade specific mRNA and decrease the expression of the protein. siRNA probes for p29 Prx were constructed based on standard constraints for unique 19 nucleotide binding sites along with other sequence selection criteria. Nine probes were constructed based on the cDNA sequence of p29 Prx; one resulted in significant knockdown of p29 Prx. A vector for green fluorescent protein was obtained commercially. U293 cell line was cultured under standard conditions and expressed p29 Prx message by RT-PCR and protein by Western blot. U293 cells were transfected with siRNA or GFP with calcium chloride. Cell counts and viability were also determined by standard techniques. For Western blots, proteins from cell lysates were separated on 10% SDS-PAGE and blotted onto nitrocellulose, and specific proteins were detected with polyclonal antibodies to p29 Prx, lamin or actin by chemiluminescent technique. U293 cells were transfected with the siRNA for p29 Prx and the vector for GFPb and the cells were harvested at 24, 48, and 72 hours. A knockdown by one of the 6 siRNAs resulted in decreased levels of p29 Prx by Western blot. Optimum knockdown was achieved by transfection of 1 μg siRNA and the decrease in p29 Prx observed at 24 hours but was maximum at 48 and 72 hours. Greater than 90% transfection was achieved documented by green fluorescence of the cells. Under these conditions, a decrease in p29 Prx >90% detected by Western blot was achieved with no differences in levels of actin or lamin in the cell lysates. In separate experiments, U293 cells were transfected with siRNA for p29 Prx and the vector for GFP and exposed to an oxidant stress (paraquot, 100 μM). The level of p29 Prx, actin, and lamin; cell counts; and viability were determined. Cells transfected with siRNA had lower p29 Prx (<10%) but not actin or lamin. The viability of control cells and siRNA transfected cells after 48 hours was no different (91% and 94%, respectively). Paraquot decreased the viability of nontransfected cells (68%), and knockdown of p29 Prx in the presence of paraquot resulted in poorer viability (49%). The number of cells harvested was slightly decreased in the paraquot treated groups but not in the untreated control or siRNA groups. These results suggest an antioxidant effect of p29 Prx in the cell and support its role in protecting the oxidase enzyme system in the neutrophil.

Cysteine Residues on Neutrophil p29 Peroxiredoxin Are Required for Enhanced Oxidase Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 654-654
Author(s):  
Daniel R. Ambruso ◽  
Gail Thurman ◽  
Carolina Gonzalez-Aller
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Recombinant Proteins ◽  
Respiratory Burst ◽  
Oxidase Activity ◽  
Cysteine Residues ◽  
Dependent Manner ◽  
Oxidase Enzyme ◽  
O2 Production

Abstract Introduction: Production of reactive oxygen species (ROS) through the respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of an active NADPH oxidase enzyme system. This system has several components including gp91phox and p22phox comprising the membrane associated cytochrome b558 and the cytosolic components p47phox, p67phox, p40phox and rac2. Recently, we identified a novel protein with a molecular weight of 29 kDa which binds p67phox by immunoprecipitation and yeast two-hybrid analysis (Leavey et al J Biol Chem2002;277:45181–7). This protein is classified as peroxiredoxin (Prx) by its amino acid sequence and enzyme activity. Prxs oxidize H2O2 through conserved cysteines. The neutrophil p29 Prx has a cysteine at amino acid residue 47 flanked by a consensus sequence shared by other 1-cys Prx proteins and a second cysteine at residue 91. We have also shown that neutrophil p29 Prx translocates to the cell membrane during oxidase activation and enhances oxidase activity in a cell-free assay with recombinant phox cytosolic components and neutrophil plasma membrane. The current studies were undertaken to evaluate requirements of cysteine residue(s) for the effect on the oxidase. Methods: cDNA for p29 Prx was cloned into pBlueBacHis vector and a recombinant protein was expressed in a baculovirus expression system as previously described. Two additional recombinant proteins with ser substituted for cys at residue 47 and 91 were generated using the PCR overlap technique and also expressed in Sf9 cells. The wild type (WT), C47S, and C91S recombinant proteins were purified by affinity chromatography. The effect of these proteins on oxidase activity was determined in a SDS cell-free system of oxidase activity using plasma membrane (1 μg), p47phox (100 nM), p67phox (100 nM), a constitutively active Rac and 1 mM NADPH. O2− production was measured as SOD inhibitable cytochrome c reduction. Results: O2− Production (nmol/min) Concentration (μM) WT C47S C91S * Numbers are mean ± SEM, n=3 ** Significantly different from no addition, p<0.05 0 7.15 ± 0.46* 6.38 ± 0.81 7.11 ± 0.10 0.55 11.86 ± 1.33** 5.42 ± 0.73 6.57 ± 0.54 0.83 14.58 ± 2.09** 7.32 ± 1.02 7.29 ± 6.70 1.10 17.66 ± 2.50** 7.41 ± 1.19 7.64 ± 0.76 1.65 19.93 ± 2.82** 8.37 ± 1.37 8.14 ± 1.23 WT p29 Prx increased oxidase activity in a concentration dependent manner. Neither the C47S nor C91S mutants altered oxidase activity to a significant degree. Additional studies varying concentration of NADPH at several concentrations of p29 Prx demonstrated that p29 Prx increased the Vmax without changing the km for NADPH. At a fixed concentration of NADPH, the increase in rate of O2− generation was dependent upon p29 Prx concentration demonstrating saturable kinetics with a km for p29 Prx of 1.72 ± 0.65 μM and a Vmax (increase in velocity) of 15.4 ± 3.0 nmol/min (mean ± SEM for four separate studies). Conclusion: Neutrophil p29 Prx enhances oxidase enzyme activity. The enhancement is specific, exhibiting a saturable effect dependent on the concentration of p29 Prx. However, replacement of either conserved or non-conserved cysteine residues with serines resulted in an inactive protein. Cysteine residues which are required for Prx activity are also critical to the effects of neutrophil p29 Prx on the oxidase.

FEATURES OF THE CYTOKINE PROFILE IN BURN PATIENTS

2020 ◽  
Vol 17 (2) ◽  
pp. 161-174
Author(s):  
P.A. Selivanov ◽  
◽  
N.V. Bychkova ◽  
N.V. Chinenova ◽  
N.M. Kalinina ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Burn Injury ◽  
Skin Flap ◽  
Fatal Outcome ◽  
Burn Patients ◽  
Healthy Individuals ◽  
Laboratory Examination ◽  
Pronounced Increase ◽  
The Status ◽  
Anti Inflammatory

Introduction. Currently, more and more attention is paid to burn injury as one of the most significant medical and social problems. Treatment of burn injury is performed by autotransplantation of the skin flap. The efficiency of using this method is from 70 to 90%. A significant reason for the failure of skin flap transplantation in burn disease is the lack of objective methods for assessing the patient's condition on the eve of free autodermoplasty surgery. Assessment of the levels of both Pro-and anti-inflammatory cytokines in burned patients is not included in the algorithm of examination and monitoring of the status of these patients. The aim of the study: to study the levels of pro — and anti-inflammatory cytokines to expand the algorithm of laboratory examination in preparing patients for free autodermoplasty.Materials and methods. The levels of IL-6, IL-8, and IL-10 in the peripheral blood of 50 patients with burn injury compared to healthy individuals were studied by enzyme immunoassay. Results. The serum content of all studied cytokines was significantly increased upon admission to the hospital in all burn patients compared to healthy individuals. A particularly pronounced increase was observed in patients with a fatal outcome. The results of autodermoplasty correlated with the severity of hyperproduction of proinflammatory cytokines on the eve of surgery. Conclusion. A justification for the addition of the algorithm for laboratory examination of burned patients by determining cytokines-interleukins 6, 8 and 10. Monitoring the level of cytokines in burn injury is appropriate for objectifying the choice of the timing of surgery, which will help reduce the mortality rate and increase the effectiveness of free autodermoplasty.

scholarly journals Respiratory burst oxidase activation can be dissociated from phosphatidylinositol bisphosphate degradation in a cell-free system from human neutrophils

Blood ◽  
1989 ◽  
Vol 73 (1) ◽  
pp. 296-300
Author(s):  
AE Traynor ◽  
PJ Scott ◽  
AL Harris ◽  
JA Badwey ◽  
LA Sklar ◽  
...  
Keyword(s):  
Respiratory Burst ◽  
Second Messengers ◽  
Sodium Dodecyl ◽  
Human Neutrophils ◽  
Free System ◽  
Phosphatidylinositol Bisphosphate ◽  
Membrane Particles ◽  
A Cell ◽  
Phosphatidylinositol 4 ◽  
Respiratory Burst Oxidase

Activation of the respiratory burst oxidase in cell-free preparations from 32P-labeled neutrophils was compared with changes in levels of radioactively labeled phosphoinositides in the same preparations. With membrane particles, treatment with sodium dodecyl sulfate (SDS) in the presence of cytosol led to activation of the oxidase without an alteration in levels of labeled phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol 4-phosphate (PIP). Conversely, solubilization of the membrane particles with deoxycholate resulted in loss of nearly 98% of the radioactive PIP2 without activation of the oxidase. In this solubilized preparation, the oxidase could subsequently be fully activated by SDS in the presence of cytosol, even though the labeled PIP2 was almost totally depleted. Two PIP2-derived second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, as well as the protein kinase C activator phorbol myristate acetate (PMA), failed to activate the oxidase. These results suggest that in a cell- free preparation from human neutrophils, detergent-mediated activation of the respiratory burst oxidase is independent of changes in the levels of phosphoinositides or phosphoinositide-derived second messengers.

Sign in / Sign up

Export Citation Format

Share Document