Search PubMed⌕ Search

Biomedical subjects

N Borregaard

Publications and source records attributed to N Borregaard.

At least 127 records · Page 7Linked to original sources
PubMed1987

Prevention of tissue damage: inhibition of myeloperoxidase mediated inactivation of alpha 1-proteinase inhibitor by N-acetyl cysteine, glutathione, and methionine.

The ability of the sulphur compounds, N-acetyl cysteine, Methionine, and Glutathione to prevent inactivation of alpha 1-proteinase inhibitor by Myeloperoxidase-H2O2-Cl--system was investigated in vitro with purified components. The Myeloperoxidase system, or its main product HOCl by itself, readily abrogated the ability of alpha 1-proteinase inhibitor to inhibit elastase. This inactivation of alpha 1-proteinase inhibitor was effectively prevented by micromolar concentrations of N-acetyl cysteine, Methionine and reduced Glutathione, whereas oxidized Glutathione was much less effective. These results indicate that the sulphydryl compounds work as scavengers of the products of the Myeloperoxidase system, and might be useful in inflammatory disorders, to prevent tissue damage inflicted by this system.

Acetylcysteine↗
PubMed1987

Development of the phagocytic and cidal capacity during maturation of myeloid cells: studies on cells from patients with chronic myelogenous leukaemia.

Myeloid cells from peripheral blood of patients with chronic myelogenous leukaemia were isolated and fractionated by density gradient centrifugation using Lymphoprep gradient followed by discontinuous Percoll gradients. Six fractions were obtained, each enriched in one of the morphologically identifiable types of myeloid cells from myeloblasts to polymorphonuclear neutrophils. Each of these cell types were functionally and biochemically characterized. The development of the capacities for phagocytosis and killing of yeast cells and the ability to generate a respiratory burst of phagocytosis correlated closely with the content of cytochrome b and vitamin B12-binding protein, a marker of specific granules. These results support the notion that the specific granules provide the developing neutrophil with components which are essential for its microbicidal activity.

Blood Bactericidal Activity↗
PubMed1987

Stimulated mobilization of monocyte Mac-1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface.

Monocytes were stimulated to increase their cell surface quantity of leukocyte adhesion proteins p150,95 and Mac-1 by the chemoattractant formyl-methionyl-leucyl-phenylalanine, or other mediators such as platelet-derived growth factor, tumor necrosis factor, C5a, and leukotriene B4. Dose-response curves indicated variations in the sensitivity of monocytes and granulocytes to these mediators. These increases were independent of protein synthesis and half-maximal at 2 min. Human alveolar and murine peritoneal macrophages, cells that had previously diapedised, could not be induced to upregulate Mac-1 or p150,95. Detergent permeabilization studies in monocytes indicated that these proteins were stored in internal latent pools, which were reduced upon stimulation. Electron microscopy utilizing rabbit antiserum against p150,95 revealed these proteins on the plasma membrane, and in intracellular vesicles and peroxidase negative granules. Together with other functional studies, these findings suggest that the mobilization of Mac-1 and p150,95 from an intracellular compartment to the plasma membrane regulates the monocyte's ability to adhere and diapedese.

Animals↗
PubMed1987

Lactic acid concentration and acid-base balance in cerebrospinal fluid: observations of diagnostic importance in non-Hodgkin lymphoma.

Measurements of lactic acid concentration and gas analysis were performed in lumbar cerebrospinal fluid from 36 patients without malignant central nervous system involvement and four patients with meningeal dissemination of non-Hodgkin lymphoma. The upper lactic acid concentration in controls of 2.48 mmol/l was exceeded in all four patients, also in cases with low blast counts and normal protein and glucose content. The pH, pCO2, pO2 and standard bicarbonate concentration in spinal fluid of patients with meningeal dissemination in non-Hodgkin lymphoma did not show significant differences compared with other patients and controls. Determination of the lactic acid concentration in cerebrospinal fluid add information, relevant to the diagnosis of meningeal involvement in non-Hodgkin lymphoma.

Acid-Base Equilibrium↗
PubMed1985

Anomalous neutrophil granule distribution in a patient with lactoferrin deficiency: pertinence to the respiratory burst.

Neutrophils from a patient with lactoferrin deficiency were examined and the quantity and subcellular localization of protein markers were determined on Percoll density gradients. Distribution of azurophilic and specific granule markers was abnormal in that azurophilic granules were lighter than normal and appeared in the fraction of the gradient where normally the specific granules sediment. The specific granule membrane markers, cytochrome b-235 and its associated flavoprotein, were abnormally distributed in the gamma fraction, the site of the plasma membrane marker alkaline phosphatase. Thus, the b-cytochrome-flavoprotein complex had either been incorporated into the plasma membrane or was still present in the membranes of granules that were abnormally light and cosedimented with the plasma membranes. This is of particular interest in regard to the patient's respiratory burst oxidase function, since the b-cytochrome/flavoprotein complex normally translocates from the specific granules to the plasma membrane to constitute the active respiratory burst oxidase. The functional consequences of this abnormal distribution are discussed, as is the importance of characterizing both intragranular enzymatic markers and granule membrane proteins to define granular disorders.

Alkaline Phosphatase↗
PubMed1985

The respiratory burst of phagocytosis: biochemistry and subcellular localization.

Using nitrogen cavitation and Percoll density gradient centrifugation for subcellular centrifugation of human neutrophils, approximately 90% of the low potential b-cytochrome, unique for phagocytes, as well as 50% of the flavoproteins in normal neutrophils were found in a granule fraction which co-sedimented with the specific granules. Upon stimulation of the intact cells with phorbol myristate acetate, both the b-cytochrome and the flavoprotein translocated from this granule fraction to the fractions which contained the plasma membranes and the NADPH oxidase activity. In neutrophils from two patients with chronic granulomatous disease, both the b-cytochrome and the flavoprotein of the granules were absent, but flavoprotein was present in normal amounts in the membrane and cytosol fractions. Taken together, these findings suggest that the specific granules, or granules co-sedimenting with the specific granules, are important stores for the components of the NADPH oxidase, which is responsible for the respiratory burst. Analysis of the stoichiometry of CO2 generation, H+ secretion and O2 consumption by stimulated neutrophils indicated that the hexose monophosphate shunt is the source of both protons and electrons for the NADPH oxidase activity, as well as of the extra protons secreted during the respiratory burst.

Carbon Dioxide↗
PubMed1985

Neutrophils autoinactivate secretory products by myeloperoxidase-catalyzed oxidation.

The neutrophil response to inflammatory stimuli involves the formation of reactive oxygen species and secretion of granule enzymes. In studying secretion of vitamin B12 binding protein by human neutrophils, we noted a major decrease in total recoverable activity from the extracellular fluid plus the stimulated cells (54% of resting cells). Recovery of B12 binding protein from neutrophils exposed to phorbol myristate acetate or opsonized zymosan was significantly enhanced on addition of heme enzyme inhibitors (azide, cyanide) or catalase or when halide-free medium was used. The changes in B12 binding protein recovery were attributable entirely to increases in extracellular fluid levels, and cell pellet content was unaffected. These data indicate extracellular destruction of functional B12 binding protein by the halide-dependent heme enzyme myeloperoxidase and H2O2. Kinetic studies demonstrated rapid secretion of B12 binding protein in the first two to five minutes, followed by its inactivation over the next 20 to 30 minutes. A cell-free extract of vitamin B12 binding protein was readily inactivated on exposure to purified myeloperoxidase, H2O2, and a halide. These findings document a functional interaction among products of the neutrophil specific granules (B12 binding protein), azurophil granules (myeloperoxidase), and metabolic burst (H2O2). They provide an interesting model for the modulation of the inflammatory response by oxidation of secretory products of neutrophils.

Catalysis↗
PubMed1984

Development of the superoxide-generating system during differentiation of the HL-60 human promyelocytic leukemia cell line.

Utilizing the induced differentiation of HL-60 promyelocytic leukemia cells as a model of myeloid maturation, we examined the development of the superoxide-generating system, focusing on NADPH oxidase activity, membrane depolarization, and cytochrome b content. NADPH oxidase activity, measured as NADPH-dependent superoxide production, increased with both spontaneous and N,N-dimethylformamide-induced differentiation. Activity in particulate fractions from induced HL-60 cells and human peripheral blood polymorphonuclear leukocytes was proportional to their relative rates of superoxide production, but activity from uninduced cells was surprisingly high: one-third that from induced cells, despite only 7% their rate of superoxide generation. NADPH oxidase activities in phagocytic vesicles from induced HL-60 cells and polymorphonuclear leukocytes were equal, indicating the equivalence of the enzyme system in active portions of their cell membranes. Separation by centrifugal elutriation of the HL-60 cell population into fractions of varying maturity confirmed the relationship of NADPH oxidase activity to advancing differentiation in both dimethylformamide-induced and spontaneously maturing cells. Membrane potential change, an early event related to activation of the oxidase, was followed by 3,3'-dipropylthiodicarbocyanine dye fluorescence. The depolarization response increased dramatically in both magnitude and initial rate of change during differentiation. The cells' cytochrome b content increased 3-fold with induction of differentiation, in proportion to the change in NADPH oxidase activity.

Cell Differentiation↗
PubMed1984

Subcellular localization of the human neutrophil NADPH oxidase. b-Cytochrome and associated flavoprotein.

Human neutrophils were fractionated by nitrogen cavitation and Percoll density centrifugation, and the subcellular localization of FAD-flavoprotein, b-cytochrome, NADH-cytochrome b5 reductase, and NADPH-dependent cytochrome c reductase were determined in normal cells, cells from two patients with chronic granulomatous disease (CGD), and normal cells that had been stimulated with phorbol myristate acetate. In normal cells, a FAD-flavoprotein is found in a 1:2 molar ratio, with cytochrome b in the fractions containing the specific granules. Triton X-114 phase distribution indicates that the b-cytochrome but not the b-cytochrome-associated flavoprotein is an integral membrane protein. 80% of this flavoprotein, as well as all the b-cytochrome, was absent in these fractions from 2 CGD patients, although these patients had normal quantities of FAD in the fractions containing plasma membranes and cytosol. During stimulation the b-cytochrome-associated flavoprotein of the granules translocates with the b-cytochrome to the plasma membrane where NADPH oxidase is localized. Definition of the role of these NADPH oxidase constituents may provide a molecular description of the normal neutrophil respiratory burst and the molecular defect(s) in CGD.

Cell Fractionation↗
PubMed1984

Bactericidal mechanisms of the human neutrophil. An integrated biochemical and morphological model.

The bactericidal activity of the human neutrophil is dependent on a coordinated series of events by which the bacteria become confined to a vacuole. Fusion of the azurophil and specific granules with the phagocytic vacuole results in secretion of BPI, the primary oxygen independent bactericidal protein, and of myeloperoxidase into the phagolysosome. Simultaneously, an electron transport chain, the NADPH oxidase, is activated in the membrane of the phagolysosome, resulting in generation of H2O2, which together with myeloperoxidase and Cl- forms a highly bactericidal agent. Digestion of the killed bacteria is subsequently effectuated by proteases and lipases of the neutrophil granules. The neutrophil thus has several highly efficient bactericidal systems that overlap to a certain degree, thereby giving the neutrophil an overcapacity to kill. This is appreciated in the defence against microorganisms, but is increasingly being recognized as a cause of perturbation of serum protease anti-protease homeostasis that may cause major tissue destruction. The recent achievements in the understanding of neutrophil function will hopefully permit better control to be exerted over this potent cell.

Antimicrobial Cationic Peptides↗
PubMed1984

Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.

Phagocytosis by neutrophils is accompanied by a burst in O2 consumption and activation of the hexose monophosphate shunt (HMPS). Proton secretion equal to the amount of O2 consumed is an additional feature of the respiratory burst, but its source has not been identified, nor has the source of all electrons donated to O2 in the respiratory burst. We chemically quantitated total CO2 generation in human neutrophils and found that proton secretion elicited by phagocytosis was accompanied by a stoichiometric increase in CO2 generation. Addition of carbonic anhydrase and its inhibitors had no effect on either the quantities of CO2 measured or the quantities of protons secreted. Therefore, the CO2 generated in the respiratory burst of stimulated neutrophils is hydrated to form H2CO3, which then dissociates, accounting for the observed proton secretion. Furthermore, the CO2 generated corresponds to the O2 consumed with a respiratory quotient of nearly 1. We conclude on the basis of this and previous studies that the HMPS activity is the source of both the electrons for the NADPH oxidase and of protons secreted in association with the respiratory burst.

Carbonic Anhydrases↗
PubMed1983

Absence of cytochrome b-245 in chronic granulomatous disease. A multicenter European evaluation of its incidence and relevance.

The heme-containing protein cytochrome b-245 has been proposed as a primary component of the microbicidal oxidase system of phagocytes that normally generates superoxide-free radicals but when defective is associated with chronic granulomatous disease. We measured this cytochrome in granulocytes from 27 patients with chronic granulomatous disease and from 64 members of their families. It was undetectable in all 19 of the men in whom the defect appeared to be located on the X chromosome. Female relatives who were heterozygous carriers had reduced concentrations of the cytochrome and variable proportions of cells that were unable to generate superoxide; these two characteristics were closely related (r = 0.93 in the 16 mothers and 0.85 in all 24 carriers, P less than 0.001). In contrast, in all eight patients (seven women) with a probable autosomal recessive inheritance the cytochrome was present but nonfunctional. The properties tested, including midpoint potential, carbon monoxide binding, and organelle distribution, were normal, but the cytochrome did not undergo reduction on cellular stimulation. Thus, absence or malfunction of the cytochrome b-245 may be the causal molecular defect in chronic granulomatous disease, implicating it in the microbicidal oxidase system.

Adolescent↗
PubMed1983

Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation.

We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b-cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil.

Calcimycin↗
PubMed1983

A variant form of X-linked chronic granulomatous disease with normal nitroblue tetrazolium slide test and cytochrome b.

Chronic granulomatous disease was diagnosed in a boy who suffered from severe generalized infections. Family investigations revealed the inheritance of the disease to be X-linked. However, unlike other cases of X-linked chronic granulomatous disease, the membrane oxidase of the neutrophils from this patient was not totally defective and sufficient activity was left to result in a normal phorbol myristate acetate-stimulated nitroblue tetrazolium slide test. Also, unlike the usual findings in X-linked chronic granulomatous disease, cytochrome b was present in normal amounts in the neutrophils from this patient. The cytochrome was normal, judged from its midpoint potential of -245 mV and its ability to bind CO. It is thus apparent that X-linked chronic granulomatous disease may result from at least two different defects and that the phorbol myristate acetate stimulated nitroblue tetrazolium slide test fails to detect some cases.

Adolescent↗
PubMed1983

Early changes in cyclic AMP and calcium efflux during phagocytosis by neutrophils from normals and patients with chronic granulomatous disease.

During phagocytosis of serum-treated zymosan particles (STZ) human neutrophils respond within 5 sec by a two-fold increase in cAMP concomitantly with an increase in 45Ca-efflux from intracellular stores. The changes in cyclic AMP and calcium efflux during phagocytosis were essentially the same whether the cells were preincubated in the presence of 5 mM glucose or without glucose. However, the phagocytic capacity, and oxygen consumption and degranulation during phagocytosis were reduced about 20%-25% in the absence of glucose. Addition of 2-deoxyglucose and iodoacetamide, which results in depletion of cellular ATP, abolished the cAMP increments during phagocytosis and profoundly inhibited calcium efflux. At the same time, the phagocytic capacity and STZ-induced oxygen consumption and degranulation were severely impaired. The mitochondrial inhibitors, cyanide, azide, and antimycin A, in the presence of glucose, did not affect the cellular ATP levels. Neither were cAMP increments and calcium effluxes during phagocytosis affected by these drugs, and likewise these did not affect phagocytosis or the post-phagocytic events. Neutrophils from two patients with X-linked chronic granulomatous disease (CGD) and one patient with the autosomal recessive form elicited normal increases in cAMP levels and calcium effluxes after addition of STZ despite no increase in oxygen consumption showing that these early metabolic events are not affected by the lack of oxidase function.

Adenosine Triphosphate↗