The respiratory chain of Azotobacter vinelandii. III. The effect of cyanide in the presence of substrates.

(1) Cyanide (100 microM) causes a rapid disappearance of the band (648 nm) of oxidized cytochrome d in particles of Azotobacter vinelandii oxidizing NADH. The rate of disappearance of the band can be related to the rate of inhibition of the oxygen consumption. (2) The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide in the presence of substrates deviate from first-order, indicating that at least two conformations of the enzyme are involved. (3) The rate of binding of cyanide to cytohrrome d increases the larger the rate of turnover of the oxidase. From this it is concluded that cyanide binds preferentially to the enzymically active oxidized conformation of cytochrome d. (4) The instantaneous inhibition of the oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) by cyanide is related neither to the binding of cyanide to cytochrome d as determined spectrophotometrically, nor to the rate of inhibition of the oxidation of NADH. This indicates that different oxidases are involved in the oxidation of NADH and of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), in line with the conclusion of Jones and Redfearn (1967) Biochim. Biophys. Acta 143, 340-353. (5) From experiments in which the change in redox states of cytochrome b1 and c-551 were related to the rate of binding of cyanide to cytochrome d, it is concluded that the cytochromes b1 and d are located in the main pathway to oxygen, whereas cytochrome c-551 functions in the branch via cytochrome o. (6) After prolonged incubation of particles with cyanide in the presence of NADH a residual activity (5%) was found in which a b-type cytochrome is involved. This shows the existence of a third but minor pathway to oxygen.

Activation of the cAMP-dependent signaling pathway downregulates the expression of interleukin-3 and granulocyte-macrophage colony-stimulating factor in activated human T lymphocytes.

Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signaling pathways. We have examined the modulating effects of the cAMP-dependent signaling pathway on the expression of interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in activated human T lymphocytes. 2'-O-dibutyryl-cAMP (db-cAMP), prostaglandin E2 (PGE2), isoproterenol (ISO), and isobutyl-methyl-xantin (IBMX) costimulated with concanavalin A (Con A) or Con A plus the phorbolester phorbol myristate acetate (PMA) inhibited IL-3 and GM-CSF mRNA accumulation compared to the effects of Con A or Con A plus PMA alone. Nuclear run-on experiments revealed that the inhibitory effect of db-cAMP could partially be ascribed to a five-fold reduction in transcription rate of both the IL-3 and GM-CSF gene in the presence of Con A or Con A plus PMA. mRNA stability studies demonstrated that PMA increased the stability of both transcripts. db-cAMP did not affect the stability of IL-3 and GM-CSF mRNAs in Con A activated cells. In contrast, in Con A plus PMA activated cells, db-cAMP significantly reduced the half-life of both transcripts: IL-3 >240 minutes vs. 90 minutes and GM-CSF 90 minutes vs. 60 minutes. Finally, in accordance with the mRNA data, db-cAMP, PGE2, and ISO reduced the secretion of IL-3 and GM-CSF protein in Con A and Con A plus PMA activated cells. In conclusion, these data demonstrate that the protein kinase A (PKA)-dependent signaling pathway is an important regulatory mechanism in controlling IL-3 and GM-CSF gene expression in activated human T lymphocytes.

A single centrifugation step method for the simultaneous separation of different leukocytes with special reference to basophilic leukocytes.

A simple method of separating mononuclear leukocytes, basophils, neutrophils and an eosinophil fraction from human blood is described, using discontinuous Percoll gradient centrifugation. Mononuclear cells were found in the upper layer (band 1) at a density of less than or equal to 1.072. The second band with a density of less than or equal to 1.082 contained most of the basophilic leukocytes (av. 70% and 8-20% of the differential cell count) and a minor part of the mononuclear cells (av. 8%). In the third band, density less than or equal to 1.1, 82-91% of the polymorphonuclear cells (PMN) were found, consisting mainly of neutrophils. 35-70% of the eosinophilic PMN cells are located in band four (density greater than 1.1). The function of the basophil-enriched fraction was tested by degranulation with specific antigen.

Induction of low density and up-regulation of CD11b expression of neutrophils and eosinophils by dextran sedimentation and centrifugation.

Neutrophils and eosinophils circulating in an activated state are of low density. However, purification procedures such as dextran sedimentation and centrifugation may influence the density and function of cells. In the present study we have evaluated the effect of dextran sedimentation and subsequent centrifugation on the density and CD11b expression of neutrophils and eosinophils. Direct density separation of whole blood resulted in 17.7 +/- 9.0% low density neutrophils (< 1.090 g/ml) and 8.7 +/- 3.5% low density eosinophils (< 1.093 g/ml). Dextran sedimentation at room temperature prior to density separation yielded 57.8 +/- 14.7% low density neutrophils and 43.2 +/- 8.0% of low density eosinophils. Additional centrifugation after dextran sedimentation resulted in an increase of these numbers to 91.7 +/- 3.1 and 69.8 +/- 11.7% respectively. The density shifts were found with hypertonic as well as isotonic Percoll. Furthermore, it was shown that dextran sedimentation resulted in an increased CD11b expression on neutrophils as well as eosinophils. During subsequent washing by centrifugation, a further increase in CD11b expression was observed together with lactoferrin release. The effects of dextran sedimentation on density and CD11b expression were independent of extracellular calcium. These results indicate that dextran sedimentation induces the release of specific granule compartments with subsequent expression of CD11b, resulting in changes in granulocyte density.

Cyclosporine, FK506, mycophenolate mofetil, and prednisolone differentially modulate cytokine gene expression in human airway-derived epithelial cells.

BACKGROUND: The immunosuppressive effects of cyclosporine (CsA), tacrolimus (FK506), mycophenolate mofetil (MMF), and prednisolone in cells from the immunological compartment are well documented. In contrast, limited information is available with respect to the effects of these immunosuppressive drugs on airway-epithelial cells, although these cells may contribute to the development of obliterative bronchiolitis (OB) through the production of interleukin (IL)-6 and IL-8. METHODS: We studied the production of IL-6 and IL-8 proteins by airway-derived epithelial cell lines and primary epithelial cell cultures obtained from lung brushings. Transcriptional mechanisms were detected by transient transfections. RESULTS: We demonstrate that CsA dose dependently induces the production of the proinflammatory cytokines IL-6 and IL-8 in both cell lines and primary epithelial cells. FK506 and MMF were also able to upregulate IL-8, although the effect was less dramatic than observed for CsA. Low concentrations of prednisolone (0.01 and 0.001 microg/ml) enhanced IL-6 and IL-8 secretion, whereas concentrations > or =0.01 microg/ml significantly diminished IL-6 secretion. Furthermore, we showed that CsA and prednisolone mediate their effects at the transcriptional level. CONCLUSIONS: The data provide evidence that relevant concentrations of CsA and MMF in vivo may enhance the inflammatory processes in the lower airways of patients after lung transplantation.

Potentiation of adenylyl cyclase in human peripheral blood mononuclear cells by cell-activating stimuli.

The isoprenaline-induced production of cAMP in human peripheral blood mononuclear cells (PBMC) was potentiated significantly by incubating PBMC with isoprenaline in the presence of phytohaemagglutinin (PHA), Concanavalin A (Con A) or A23187. This potentiation, that proved to be dependent on the concentration of PHA, Con A or A23187, increased the maximal response but did not cause a change in the potency of isoprenaline. Potentiation could not be induced by the phorbol ester phorbol-myristate acetate, suggesting that protein kinase C-dependent pathways are not likely to be involved in potentiation of adenylyl cyclase. Potentiation could be inhibited by chelating extracellular Ca2+ with EGTA and also by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamine, an inhibitor of calmodulin. Potentiation could not be induced by preincubation of PBMC with PHA, suggesting that transient biochemical changes are involved. It was concluded from these results that potentiation in PBMC probably involves the activation of Ca2+/calmodulin-dependent adenylyl cyclase subtypes. Potentiation of the adenylyl cyclase activity could be an important physiological mechanism in vivo preventing cells from becoming "over stimulated".

Phorbol 12-myristate 13-acetate induces beta-adrenergic receptor uncoupling and non-specific desensitization of adenylate cyclase in human mononuclear leukocytes.

Tumour-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate beta-adrenergic receptor responses in various cell types, presumably by the activation of protein kinase C. In the present investigation we assessed the effect of PMA on the beta-adrenergic receptor-adenylate cyclase system of human mononuclear leukocytes (MNL). It was found that incubation of MNL with PMA resulted in a time- and concentration-dependent desensitization of isoproterenol-induced adenylate cyclase activity. However, the effect of PMA was not restricted to the beta-adrenergic receptor system, since basal adenylate cyclase activity and histamine-, prostaglandin E1-, 5'-guanylylimidodiphosphate (GppNHp)-, and NaF-stimulated values were also reduced. By contrast, no effect was found on the forskolin-induced adenylate cyclase activity. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate had no effect on adenylate cyclase at all, suggesting that the observed PMA effect was specifically mediated by activation of protein kinase C. The reduced beta-adrenergic response induced by PMA was not associated with a reduced beta-adrenergic receptor number, indicating uncoupling of this receptor from adenylate cyclase. Isoproterenol competition curves for 3H-dihydroalprenolol binding to membranes from untreated and PMA-treated cells demonstrated that the uncoupling was due to a reduced ability of the agonist to promote formation of the guanine nucleotide-sensitive high affinity state of the receptor. The results indicate that PMA may cause desensitization of catecholamine-responsive adenylate cyclase in MNL, and that the major locus of alteration is the guanine nucleotide regulatory protein.

Allergen-induced recruitment of inflammatory cells in lavage 3 and 24 h after challenge in allergic asthmatic lungs.

To determine whether a link exists between the recruitment of inflammatory cells in the airways on a bronchial and bronchoalveolar level and the development of allergen-induced increase in bronchial hyperresponsiveness after allergen challenge, we used bronchial lavage and bronchoalveolar lavage to assess the airway responses to allergen. Twelve symptomatic atopic asthmatics were studied. In all patients bronchial and bronchoalveolar lavage was performed before, 3 h, and 24 h after allergen challenge. Monoclonal antibodies were used directed against T cells (CD3, CD4, CD8) and the eosinophil cationic protein (EG2). Eight patients showed a dual asthmatic response; four patients showed only an early asthmatic reaction after allergen challenge. Clear differences were found between bronchial and bronchoalveolar lavage. Activated eosinophils (EG2) were significantly increased both at 3 h (p = 0.01) and 24 h (p = 0.005). The number of activated eosinophils was significantly higher in the dual responders. A correlation was observed between the severity of the late asthmatic reaction (LAR) and the number of epithelial cells in the bronchial recovery at 3 h, but not at 24 h, in patients who clinically developed a LAR. No significant changes in the number of CD3, CD4, and CD8 cells 3 and 24 h after the challenge both in the bronchial and bronchoalveolar recovery were observed. We conclude that the number of activated eosinophils in bronchial lavage is associated with the development of the LAR and allergen-induced increase in bronchial hyperresponsiveness.

Human allogeneic CD2+ lymphocytes activate airway-derived epithelial cells to produce interleukin-6 and interleukin-8. Possible role for the epithelium in chronic allograft rejection.

BACKGROUND: The adhesion of lymphocytes to the epithelium and the release of proinflammatory cytokines are important features observed during acute and chronic allograft rejection. Development of chronic rejection in lung-transplantation patients is preceded by high levels of interleukin (IL)-6 and IL-8 protein in the bronchoalveolar lavage. Therefore, we studied the expression of IL-6 and IL-8 in cocultures of epithelial cells and allogeneic lymphocytes. METHODS: IL-6 and IL-8 protein levels were determined in supernatants of the airway-derived epithelial cell line A549 and in primary epithelial cells obtained from lung-brushings after coculturing with autologous and allogeneic lymphocytes. Transcriptional mechanisms were detected by transient transfections. RESULTS: Coculture-supernatants of epithelial cells and allogeneic CD2+ lymphocytes show high levels of IL-6 and IL-8 protein due to transcriptional activation of the respective genes in epithelial cells. Highest productions were measured when the epithelial-cell:lymphocyte ratio was 1:10. Highly purified CD4+ and/or CD8+ cells were unable to induce the same response as observed with the total lymphocyte-population. Depletion of CD4+ and/or CD8+ had no effect on the IL-6 and IL-8 production induced by the total CD2+ lymphocyte-population. However, depletion of CD56+ cells diminished the lymphocyte-induced IL-6 and IL-8 production by > 75%. CONCLUSION: These data show that allogeneic CD2+ lymphocytes are able to activate lung-derived epithelial cells, resulting in the release of proinflammatory cytokines, which have a prominent role in chronic allograft rejection observed in lung-transplantation patients.

Purification of specific precipitinogen and extraction of endotoxin from Haemophilus influenzae.

After purifying a Haemophilus influenzae precipitinogen from endotoxic activity by means of ultracentrifugation, column chromatography (Sepharose 6B) and ion exchange chromatography (DEAE Sephadex A25) a fraction was obtained which still contained a specific precipitinogen that was virtually free of endotoxin. Furthermore, during the chromatographic procedures fractions with a high and a low molecular weight endotoxic activity were found. The limulus lysate test was more sensitive in the high molecular weight fractions and the LD50 in mice in the low molecular weight fractions with endotoxic activity.

Muscarinic receptors in human airway smooth muscle are coupled to phosphoinositide metabolism.

In the present study we investigated whether muscarinic receptors in human airway smooth muscle are coupled to phosphoinositide metabolism as a possible transduction mechanism of contraction. Using isolated bronchial smooth muscle preparations, we found that the muscarinic agonist methacholine caused a time- and concentration-dependent accumulation of inositol phosphates in the presence of lithium, an effect which could be inhibited by atropine. Apart from its physiological significance, this finding may have great relevance for the biochemical investigation of cholinergic hyperresponsiveness in the airways of asthmatic patients.

Characterization of (-)-[3H]dihydroalprenolol binding to intact and broken cell preparations of human peripheral blood lymphocytes.

In this study we compared characteristics of (-)-[3H]dihydroalprenolol ([3H]DHA) binding sites in crude membrane preparations of human peripheral blood lymphocytes with those of intact, viable cells. A valid determination of specific beta-adrenergic receptor binding in both preparations was obtained by defining non-specific [3H]DHA binding with 10(-6) M l- or dl-propranolol or 10(-3) M l-isoproterenol. Higher concentrations of propranolol were used in prior reports on lymphocyte membranes. We showed that these concentrations may inhibit non-specific binding, causing non-saturability and inhomogeneity of the estimated 'specific' binding. In the intact cell preparations, inclusion of 10(-4) M phentolamine was necessary to reduce the high degree of non-specific binding. By contrast, phentolamine (10(-4) M) showed no effect on the [3H]DHA binding to membrane preparations. At 37 degrees C the [3H]DHA binding to beta-adrenergic receptor sites in both intact and broken cell preparations was rapid and reversible. The sites were stereoselective, as l-propranolol was about two orders of magnitude more potent to inhibit [3H]DHA binding than was the d-isomer. In both preparations, agonists competed for specific binding with a rank order of potency isoproterenol greater than epinephrine greater than norepinephrine, which indicated a beta 2-type of adrenergic receptor. The specific [3H]DHA binding was saturable and Scatchard analysis revealed comparable numbers of homogeneous, non-cooperative binding sites (approximately 1250 receptors/cell in the membrane preparations and 1700 receptors/cell in the intact cells). In spite of these similarities the membrane sites showed a lower affinity for the antagonists [3H]DHA and propranolol than did the intact cell sites, whereas their affinity for the agonists was increased. These differences indicate that the membrane system might be less suited to provide physiologically significant information about the beta-adrenergic receptor system.

Evidence for a direct relationship between phosphoinositide metabolism and airway smooth muscle contraction induced by muscarinic agonists.

The relationship between bovine tracheal muscle contraction and phosphoinositide metabolism was studied with the muscarinic agonists, methacholine, oxotremorine, and McN-A-343. Analysis of the dose-response curves for contraction and inositol phosphates accumulation with these agonists demonstrated a direct relationship between the two parameters, with a considerable reserve of inositol phosphate production for the full contractile agonists, methacholine and oxotremorine, and no reserve for the partial agonist, McN-A-343.

Determination of N tau-methylhistamine in plasma and urine by isotope dilution mass fragmentography.

The determination in N tau-methylhistamine by gas chromatography-mass spectrometry is described using bis-heptafluorobutyryl derivatives and deuterium labelled N tau-methylhistamine as internal standard. A description of the synthesis of the internal standard, N tau-trideuteromethylhistamine, is given. Normal concentrations for plasma were 1.4 +/- 0.4 microgram/l (mean +/- 1 S.D., n = 10). The normal values for urine ranged from 60 to 280 microgram/24 h (n = 20). Five asthmatic patients showed above-normal plasma concentrations.

Determination of N tau-methylimidazoleacetic acid (a histamine metabolite) in urine by gas chromatography using nitrogen-phosphorus detection.

N tau-Methylimidazoleacetic acid, the quantitatively most important metabolite of histamine, was isolated from urine by ion exchange chromatography. After esterification with 2-propanol and extraction, N tao-methylimidazoleacetic acid was analyzed by capillary gas chromatography with nitrogen-phosphorus detection, using N tao-ethylimidazoleacetic acid as internal standard. The synthesis of this internal standard is described. In contrast to the methods hitherto described, this method is appropriate for use in clinical chemical laboratories. Normal 24-h excretion ranged from 8.3 to 18.5 mumol (n = 20). Five patients with mastocytosis, a patient with chronic myelocytic leukemia and a patient after an anaphylactoid reaction on acetylsalicylic acid showed highly elevated values.

No long-lasting or intermittent mast cell activation in acute coronary syndromes.

BACKGROUND: Unstable coronary syndromes, such as acute myocardial infarction and unstable angina pectoris are mostly due to rupture of an atherosclerotic plaque. Recently mast cells were found to participate actively in the inflammatory process of atherosclerosis by excreting proteolytic and pro-inflammatory substances with the ability to cause plaque instability and rupture. Mast cell activity can be determined by measuring serum levels of tryptase, as has been demonstrated in patients with anaphylaxis and mastcytosis. HYPOTHESIS: Acute coronary events (acute myocardial infarction and unstable angina pectoris) are associated with increased mast cell activity, reflected by elevated serum tryptase levels. METHODS: Serum levels of tryptase were determined in the following three groups of patients: 13 patients with acute myocardial infarction, 10 patients with unstable angina pectoris, and 14 patients without ischaemic cardiovascular disease who were used as controls. Patients with known IgE mediated allergic diseases and/or anti-histaminical drugs were excluded. RESULTS: The groups were comparable for sex, blood pressure, smoking and cholesterol levels. The controls tended to be younger (P=0.05). Levels of tryptase did not differ between patients with acute myocardial infarction (7.9+/-4.6 microg/l), unstable angina pectoris (6.0+/-2.1 microg/l) or controls (6.9+/-4.1 microg/l), nor could a relation with levels of C-reactive protein be demonstrated. CONCLUSION: Serum levels of tryptase are not elevated in patients with acute coronary syndromes. This implies that increased mast cell activity, if any, in unstable coronary syndromes is not reflected systemically. Other, more specific methods will be needed to determine the activity of the mast cell in vivo.

Eosinophil derived neurotoxin (EDN) levels in commercial human urinary preparations of glycoprotein hormones.

Eosinophil derived neurotoxin (EDN) is a ubiquitous human ribonuclease, occurring not only in eosinophils, but also in many tissues and body fluids. It may be a contaminant of commercial human urinary preparations of chorionic gonadotropin (hCG) and other glycoprotein hormones. Here we describe the use of a fast commercial assay to quantify this contaminant and demonstrate that the content varies much between different commercial glycoprotein hormone preparations. As this ribonuclease may have a cytotoxic activity on certain cells, it is useful to be able to determine its quantity in a fast and reliable way in these preparations.