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Biomedical subjects

D G Dearborn

Publications and source records attributed to D G Dearborn.

At least 37 records · Page 2Linked to original sources
PubMed1988

Phosphoinositide content of erythrocyte membranes in cystic fibrosis.

Phosphoinositide content was measured in erythrocyte membranes from 11 patients with cystic fibrosis (CF) and from 12 control subjects to determine whether altered levels of phosphatidylinositol-4-phosphate (Ptdlns4P) or phosphatidylinositol-4,5-bisphosphate (Ptdlns(4,5)P2) are responsible for the decrease in Ca2+-adenosine triphosphatase (Ca2+-ATPase) activity in this disorder. Isolated membranes were extracted with an acidified chloroform-methanol solvent system. The recovered lipids were separated by one-dimensional thin-layer chromatography and quantified with a colorimetric assay for phosphorus. The results are expressed in molar percent, moles of phosphoinositide times 100 divided by the total number of moles of phospholipid per membrane. The means +/- SEM of Ptdlns(4,5)P2, Ptdlns4P, and phosphatidylinositol (Ptdlns) in CF membranes (1.07 +/- 0.18, 1.02 +/- 0.22, and 2.32 +/- 0.36 molar percent, respectively) were indistinguishable from controls (0.91 +/- 0.14, 0.85 +/- 0.12, and 2.21 +/- 0.32 molar percent, respectively) (P greater than 0.20 for all three pairs). The accuracy of quantitative recovery throughout the procedure was determined by adding a radioactive internal standard, L-3-phosphatidyl[2-3H]inositol to 10 membrane preparations. Although quantitative recoveries, as determined by percent radioactivity recovered, varied from 54% to 92%, mean Ptdlns(4,5)P2, Ptdlns4P, and Ptdlns levels appropriately corrected from tracer loss were still indistinguishable between the two groups. We conclude that absolute phosphoinositide levels are not altered in cystic fibrosis erythrocyte membranes and that the differences in Ca2+-ATPase activity cannot be explained on this basis.

Cystic Fibrosis↗
PubMed1987

Studies on the mechanism of T cell inhibition by the Pseudomonas aeruginosa phenazine pigment pyocyanine.

Pseudomonas aeruginosa and its products have been shown to inhibit mitogen-induced human lymphocyte blastogenesis as measured by [3H]TdR uptake. The phenazine pigment pyocyanine has been identified as one of the inhibitors present in cellfree culture supernatants. To determine the mechanism of the inhibitory action of pyocyanine, we studied its effect on the early stages of T cell activation. Pyocyanine inhibited lymphocyte stimulation induced by specific antigens, the lectin concanavalin A and the calcium ionophore, ionomycin, suggesting that its inhibitory effect is not dependent on interference with the T cell antigen receptor complex itself. Using quin-2, we showed that pyocyanine did not interfere with the mitogen-induced increase in cytosolic-free Ca2+. We also showed that pyocyanine did not interfere with the function of calmodulin stimulated Ca2+-Mg2+ ATPase activity, indicating that the mechanism of action of pyocyanine differs from that of the structurally related phenothiazine compounds. Analysis of IL 2 production and IL 2 receptor expression clearly showed that pyocyanine inhibits the production of this essential lymphokine as well as the expression of IL 2 receptors on the T cell membrane. This inhibition is dose dependent and not due to cellular toxicity. There was parallel inhibition of growth in cell volume as well as [3H]TdR uptake. Thus, our results demonstrate that pyocyanine inhibits T cell proliferation by decreasing the production of the critical lymphokine IL 2 and by decreasing the expression of the IL 2 receptor. Local suppression of lymphocyte stimulation by phenazine pigments such as pyocyanine may interfere with cellular immune responses that may be necessary for eradication of chronic infection with P. aeruginosa.

Adult↗
PubMed1987

In vitro effect of synthetic pyocyanine on neutrophil superoxide production.

Pyocyanine, a low-molecular-weight phenazine pigment produced by Pseudomonas aeruginosa, has previously been shown to strongly inhibit human lymphocyte blastogenesis. We now report that synthetic pyocyanine can also affect the generation of superoxide by human peripheral blood polymorphonuclear leukocytes (PMNs) in a dose-dependent manner. Superoxide production by PMNs stimulated with phorbol myristate acetate (PMA) was measured in the presence and absence of pyocyanine, phenazine, and trifluoperazine, a phenothiazine of similar chemical structure to the phenazine pigments. Pyocyanine at 50 microM inhibited superoxide production to 28.9 +/- 2.8% of PMA control values, whereas at the lower concentration of 1 microM, the production of superoxide was significantly enhanced (203 +/- 31.7% of PMA control values). Phenazine, the tricyclic parent compound of pyocyanine, had only a minor effect. Trifluoperazine had a marked inhibitory effect on superoxide generation at concentrations above 1 microM. None of the compounds induced superoxide generation in the absence of PMA. Pyocyanine at all concentrations, unlike phenothiazines, had very little effect on the release of neutrophil granule enzymes. The effect of P. aeruginosa phenazine pigments on polymorphonuclear phagocytes is of significance, since inhibition of host PMN function at sites of infection could result in ineffective bacterial killing, whereas enhanced PMN function could lead to greater tissue damage. These two possibilities are not mutually exclusive and may coexist depending on local pyocyanine concentrations.

Adult↗
PubMed1986

MR imaging in infants with airway obstruction: preliminary observations.

Magnetic resonance (MR) imaging of the trachea and main bronchi was performed in seven infants (aged 3-15 months) with symptoms of airway obstruction. Diagnoses made clinically, radiologically, and by transnasal flexible fiberoptic endoscopy included vascular ring (one patient), tracheal compression by the innominate artery (five patients), and tracheomalacia (one patient). MR images in sagittal and axial sections clearly demonstrated tracheal compression at the level of the innominate artery in three infants and tracheal narrowing due to a vascular ring in one patient. The other three patients had airway narrowing apparently unrelated to aberrant vascular structures on MR images. The appearance of the airways on MR images corresponded closely to endoscopic observations. It is concluded that MR imaging is capable of demonstrating airway obstruction in infants and delineating any relationship to major mediastinal blood vessels.

Aorta, Thoracic↗
PubMed1986

Regional distribution of macroscopic lung disease in cystic fibrosis.

We used macroscopic morphometry and multivariate analysis (Hotelling's T2) to quantitate the extent of chronic lung disease and its regional distribution in formalin-inflated right lungs obtained at autopsy from 17 young adults with cystic fibrosis (CF) and 5 without CF as control subjects. Lungs were point-counted, by lobe, for 6 anatomic variables: parenchyma, nonparenchyma, bronchi, vessels, cysts, and emphysema, and the results were expressed for each as volume proportion (Vv). Considering all 6 variables simultaneously, CF lungs differed significantly from control lungs (p = 0.003). For individual variables, Vv bronchus was significantly elevated and Vv parenchyma was significantly reduced in the CF group. Within the CF lungs, there was a significant difference between upper and lower lobes when all variables were considered together (p = 0.001). For individual variables, Vv parenchyma and Vv vessels were significantly less, and Vv bronchus was significantly greater in the upper compared with the lower lobe. There was no difference in the upper versus lower lobe Vv for any variable in the control group. These results indicate that lung disease and remodeling are irregularly distributed in CF and that the upper lobe is disproportionately severely involved.

Adolescent↗
PubMed1985

Erythrocyte cytosolic free Ca2+ and plasma membrane Ca2+-ATPase activity in cystic fibrosis.

The properties of the Ca2+, Mg2+-ATPase of erythrocyte membranes from patients with cystic fibrosis (CF) were extensively compared to that of healthy controls. Following removal of an endogenous membrane inhibitor of the ATPase, activation of the enzyme by Ca2+, calmodulin, limited tryptic digestion or oleic acid, as well as inhibition by trifluoperazine, were studied. The only properties found to be significantly different (CF cells vs controls) were calmodulin-stimulated peak activity (90 vs 101, P less than 0.02) and trypsin-activated peak activity (92 vs 102, P less than 0.02). No significant difference could be measured in the steady-state Ca2+-dependent phosphorylation of CF and control erythrocyte membranes indicating similar numbers of enzyme molecules per cell. The functional state of Ca2+ homeostasis in intact erythrocytes was investigated by measuring the resting cytosolic free Ca2+ levels using quin-2. Both CF and control erythrocytes maintained cytosolic free Ca2+ between 20 to 30 nM. Addition of 50 uM trifluoperazine resulted in an increase in erythrocyte cytosolic free Ca2+ to about 50 nM in both CF and control cells. Estimates of erythrocyte membrane permeability using the steady-state uptake of 45Ca into intact erythrocytes revealed no differences between CF and control cells. These results confirm that there is a small decrease in the calmodulin-stimulated activity of the erythrocyte Ca2+, Mg2+-ATPase in CF. However, this deficit is apparently not large enough to impair the ability of the CF erythrocyte to maintain normal resting levels of cytosolic free Ca2+.

Calcium↗
PubMed1985

Pulmonary air cysts in cystic fibrosis: relation of pathologic features to radiologic findings and history of pneumothorax.

One lung obtained from each of 21 consecutive autopsies in adolescents and young adults with cystic fibrosis was studied prospectively by macroscopic morphometry and light microscopy to determine the prevalence, morphology, and radiographic appearance of subpleural air cysts, which potentially contribute to spontaneous pneumothorax. In 15 lungs, 41 cysts of three anatomic types were identified: bronchiectatic cysts (23), interstitial cysts (13), and emphysematous bullae (5). All cysts were significantly more numerous in the upper lobe. Bronchiectatic cysts had the largest mean diameter, occupied from less than 1 per cent to 47.7 per cent of upper lobe volume in nine patients, and produced large multiloculated hyperlucencies on chest radiographs in five cases. All six lungs with prior pneumothorax contained at least one cyst, but no significant difference was found in the type or proportion of lung volume occupied by cysts between lungs with and without pneumothorax. Patients with large cysts had significantly lower chest radiograph scores, but there was no correlation between the proportion of lung volume occupied by cysts and patient age or duration of either symptomatic lung disease or colonization by bacteria. On chest radiographs only bronchiectatic cysts with conglomerate diameters of greater than 3 cm were visible. Smaller lesions could not be separated from ring shadows produced by bronchiectasis.

Adult↗
PubMed1985

Biochemical and pathologic evidence for proteolytic destruction of lung connective tissue in cystic fibrosis.

The risk for proteolysis of lung connective tissue was evaluated in patients with cystic fibrosis (CF) with chronic, severe lung infections by measuring uninhibited elastase activity in sputum samples and urinary excretion of desmosines (cross-linking amino acids in elastin). Of the 16 patients included in the study, 11 were infected with Pseudomonas aeruginosa, 2 with Pseudomonas cepacia, and 2 with both P. aeruginosa and P. cepacia. Uninhibited elastase activity (0.34 to 20.2 micrograms elastin degraded/mg protein/30 min) was detected in the sputum samples from each of 13 patients tested. Serine elastase activity was detected in the sputum of each of 12 patients, and metalloelastase (P. aeruginosa elastase and possibly macrophage elastase) activity was detected in the sputum of 11 of 12 patients tested. Male patients with CF excreted significantly more elastin cross-links (desmosines) in their urine than did control male subjects (3.6 +/- 1.7 micrograms/kg/24 h versus 1.5 +/- 0.6 micrograms/kg/24 h; p less than 0.01), and there was a significant correlation (p less than 0.05) between urine desmosine excretion and the severity of lung disease in the patients with CF as indicated by chest roentgenogram score. In 3 autopsied patients, abnormal elastin fibers were seen by light microscopy in all lung compartments. Fragmented and exfoliated elastin, evidence of active elastolysis, was noted in bronchial ulcers and abscesses. The results of this study suggest that proteolytic destruction of lung connective tissue is an ongoing process in the chronically infected CF lung and that this proteolysis contributes to the pathologic changes observed in airways and alveolar parenchyma.

Adolescent↗
PubMed1985

Immune complexes and complement abnormalities in patients with cystic fibrosis. Increased mortality associated with circulating immune complexes and decreased function of the alternative complement pathway.

Serum samples from 139 patients with cystic fibrosis (CF) were tested for complement abnormalities and circulating immune complexes (CIC). We found no consistent changes in whole complement activity. However, we found CIC in 29% of these patients and decreased activity of the alternative complement pathway (ACP) in 36%. During 5 yr of observation, mortality was much higher in patients whose sera contained CIC (p less than 0.001) or decreased ACP activity (p less than 0.01). Of patients with both abnormalities, 31% died; however, no deaths occurred in patients with normal ACP activity and negative tests for CIC (p less than 0.001). During a subsequent 2.5-yr period, 55% of patients greater than or equal to 21 yr old with both findings died. In contrast, no deaths occurred in older patients lacking this combination (p = 0.0062). Circulating immune complexes but not decreased ACP activity were an independent risk factor for death. Our findings support the hypothesis that humoral immune mechanisms may contribute to morbidity and mortality in CF.

Adolescent↗
PubMed1984

Cytosolic free calcium concentration and intracellular calcium distribution in lymphocytes from cystic fibrosis patients.

Lymphocytes prepared from normal individuals and patients with cystic fibrosis (CF) were compared with regard to intracellular Ca2+ concentration, distribution, and handling. No difference between control and CF was found in the concentration of cytosolic free Ca2+ (98 +/- 5 vs 102 +/- 7 nM), and no difference was observed in the kinetics with which control and CF cells restored cytoplasmic Ca2+ toward normal following a perturbation induced by cold-exposure. However, total intracellular Ca2+ is about 25% higher in CF lymphocytes than in control. Of this excess Ca2+, about 50% appears to be sequestered in mitochondria. This suggests that some difference in Ca2+ handling does exist, but the significance of this in cystic fibrosis remains to be determined.

Calcium↗
PubMed1984

Inhibition of the elastase of Pseudomonas aeruginosa by N alpha-phosphoryl dipeptides and kinetics of spontaneous hydrolysis of the inhibitors.

The rates of hydrolysis of N-[(alpha-L-rhamnopyranosyloxy)phospho]-L-leucyl-L-tryptophan (phosphoramidon), N alpha-phosphoryl-L-leucyl-L-tryptophan (PO3LeuTrp), N alpha-phosphoryl-L-leucyl-L-phenylalanine (PO3LeuPhe), and N alpha-phosphoryl-L-leucyl-L-phenylalaninamide (PO3LeuPheNH2) were followed by proton nuclear magnetic resonance spectroscopy. The rates of hydrolysis (kobsd) of PO3LeuTrp, PO3LeuPhe, and PO3LeuPheNH2 were all first order in phosphorylamide concentration over the pH range studied (3.8-9.5). The values for kobsd at pH 7.3 and 37 degrees C are as follows: PO3LeuTrp, 0.35 h-1; PO3LeuPhe, 0.63 h-1; PO3LeuPheNH2, 0.73 h-1. The values for kobsd do not significantly change between pH 5 and pH 8 but dramatically decreased with increasing pH. The hydrolysis of PO3LeuPhe and PO3LeuPheNH2 above a pH of approximately 5 was positively correlated with the concentration of monoanionic species (NHRPO3H)1-, and the values for the first-order rate constants for the respective monoanionic species were calculated to be 0.66 +/- 0.03 h-1 and 1.07 +/- 0.10 h-1. Phosphoramidon was not found to hydrolyze after 6 days at 37 degrees C at a pH of 4.6 and 7.7, while the phosphorylamide PO3LeuTrp, synthesized by the removal of L-rhamnose from phosphoramidon by base hydrolysis, was found to rapidly hydrolyze under these conditions. Solvolysis in aqueous methanol of PO3LeuPhe and PO3LeuPheNH2 indicates that the hydrolysis reaction is bimolecular, proceeding by way of direct attack of solvent (H2O, CH3OH) on phosphorus.(ABSTRACT TRUNCATED AT 250 WORDS)

Dipeptides↗
PubMed1984

Carbon-13 NMR studies of native and modified ovine submaxillary mucin.

Natural abundance 13C NMR spectroscopy has been used to study the solution structure and dynamics of the ovine submaxillary mucin (OSM). Results at both 45.3 and 67.9 MHz show the extremely viscous mucin to possess sufficient internal segmental flexibility to allow high-resolution 13C NMR studies. Essentially all of the resonances in the spectra have been assigned to individual carbons of the carbohydrate disaccharide side chain alpha- NeuNAc2 ----6 alpha-Gal-NAc-Ser/Thr and to the protonated carbons of the major peptide residues. Spin-lattice relaxation times and nuclear Overhauser enhancements reveal that the internal mobility of the mucin is unaffected by large changes in molecular weight and hence bulk viscosity. On the basis of the relaxation measurements the peptide and carbohydrate side chain mobilities increase stepwise from the glycosylated peptide residue alpha-carbons to the terminal sialic acid (NeuNAc) side-chain C9 carbon. Removal of the terminal sialic acid C8 and C9 side-chain carbons as well as the complete removal of the NeuNAc residue does not alter the dynamics of the peptide core. However, the removal of carbons C8 and C9 from the NeuNAc residue produces an increase in its ring mobility or conformational flexibility. Complete removal of sialic acid produces an increase in the mobility or flexibility of the GalNAc ring and reduces the chemical shift sensitivity of the GalNAc ring carbons to the different serine and threonine linkages. The pKa value for the sialic acid carboxyl group in the intact mucin is 2.0, while it increases to 2.4 after the removal of the NeuNAc C8 and C9 side-chain carbons. This change in pKa confirms the intramolecular hydrogen bond interaction of the C8 hydroxyl with the C2 carboxyl group in the alpha-NeuNAc residue as previously suggested by Jennings and Bhattacharjee [ Jennings , H.J., & Bhattacharjee , A.K. (1977) Carbohydr . Res. 55, 105-112]. The relaxation time values and temperature dependence of the chemical shift of the NeuNAc C7 carbon suggest that this group is also involved in an intramolecular interaction. Overall the 13C NMR results indicate that the relatively simple mucous glycoprotein, OSM, is a highly extended and internally flexible molecule which in solution possesses little secondary structure.

Animals↗
PubMed1984

Calcium-ATPase activity in cystic fibrosis erythrocyte membranes: decreased activity in patients with pancreatic insufficiency.

The activity of Ca-ATPase (Ca2+,Mg2+-ATPase, ATP phosphohydrolase, EC 3.6.1.3) was measured in erythrocyte membrane preparations from 37 cystic fibrosis patients, 27 with pancreatic insufficiency and 10 with pancreatic sufficiency, and from 24 healthy controls. The mean maximal calcium-stimulated specific activities, in the absence and presence of purified calmodulin, of the pancreatic sufficient patients (34.3 +/- 4.2 and 75.9 +/- 6.9 nmol/min/mg) was indistinguishable from that of controls (35.8 +/- 2.6 and 84.3 +/- 4.7 nmol/min/mg), while both activities of patients with pancreatic insufficiency were significantly decreased (28.9 +/- 1.3, p less than 0.02; 65.2 +/- 3.0, p less than 0.001) compared to the control group. Similarly, the mean erythrocyte membrane (Na + K)ATPase activity was decreased only for those patients with a history of steatorrhea and who clinically required pancreatic enzyme therapy and had low immunoreactive trypsin levels (10.6 +/- 0.8 versus control, 13.4 +/- 1.1, and pancreatic sufficient patients, 13.3 +/- 1.4 nmol/min/mg; p less than 0.025). No correlation was found between any of the ATPase activities and the clinical scores of the patients, suggesting the lack of significant contribution of general clinical status to the activities of those cation transporters.

Adolescent↗
PubMed1983

The resistance to tryptic hydrolysis of peptide bonds adjacent to N epsilon,N-dimethyllysyl residues.

A peptide from sperm whale myoglobin, residues 132-153, and a chromogenic substrate, H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide diacetate, were selected to investigate the susceptibility of peptide bonds adjacent to N epsilon,N-dimethyllysyl residues to tryptic hydrolysis. The peptides were exhaustively methylated using formaldehyde and sodium cyanoborohydride (N. Jentoft and D. G. Dearborn (1979) J. Biol. Chem. 254, 4359-4365). Unmodified and methylated peptides were digested with trypsin or submaxillary protease, an enzyme that catalyzes the hydrolysis of only arginyl bonds in proteins. Trypsin catalyzed the hydrolysis of the methylated apomyoglobin peptide only at the single arginyl residue and not at any of the four N epsilon,N-dimethyllysyl residues. Trypsin also failed to catalyze the hydrolysis of reductively methylated H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide. Even a 17-fold molar excess of the methylated substrate did not appear to alter the rate of tryptic hydrolysis of the unmodified substrate. These results are discussed with regard to the interactions of substrates within the specificity site of trypsin.

Animals↗
PubMed1983

In vitro inhibition of lymphocyte proliferation by Pseudomonas aeruginosa phenazine pigments.

Human lymphocyte proliferation is inhibited in vitro in the presence of killed Pseudomonas aeruginosa or cell-free P. aeruginosa culture supernatants. A comparison of culture supernatants obtained under similar conditions from Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Pseudomonas cepacia strains demonstrated that all P. aeruginosa supernatants were strongly inhibitory, whereas supernatants from other bacteria were mildly inhibitory or not inhibitory at all. These P. aeruginosa inhibitors prevent proliferative responses of resting cells upon mitogen activation and decrease [3H]thymidine uptake when added to human lymphocytes undergoing active proliferation in culture. The inhibitory effect is reversible and not due to cytotoxicity. Most of the inhibitory activity present in crude supernatants was detected in ultrafiltrates of molecular weights below 2,000. Purified P. aeruginosa pyocyanine, a low-molecular-weight phenazine pigment present in culture supernatant, was strongly inhibitory for lymphocyte proliferation. Extraction of pyocyanine and phenazine pigments from inhibitory P. aeruginosa supernatants eliminated their inhibitory activity. Inhibitors were recovered from reverse-phase chromatographic cartridges by both chloroform and methanol elution, indicating that pyocyanine and other phenazine pigments present in P. aeruginosa supernatants are responsible for the inhibition of lymphocyte proliferation. In addition to the identification of phenazine pigments as lymphocyte proliferation inhibitors, several criteria ruled out major contributions of P. aeruginosa polysaccharide, exotoxin A, and proteases to this phenomenon. P. aeruginosa strains selected for very low protease production or for very low exotoxin A production produced supernatants as inhibitory for lymphocyte proliferation as supernatants obtained from clinical P. aeruginosa isolates. Purified P. aeruginosa lipopolysaccharide and protease preparations failed to induce reversible lymphocyte proliferation inhibition. Finally, heat inactivation of P. aeruginosa supernatants at 100 degrees C for 60 min inactivates exotoxin A and proteases but produced only a moderate decrease of the inhibitory activity for lymphocyte proliferation.

Cell Division↗