Complement receptor expression on neutrophils at an inflammatory site, the Pseudomonas-infected lung in cystic fibrosis.

Activation of human neutrophils (PMN) is accompanied by rapid upregulation of CR1, the C3b receptor, and CR3, the iC3b receptor, which also serves as the PMN's major adherence protein. This is necessary for migration and phagocytosis, but the extent of expression of these proteins on PMN at inflammatory sites has not been determined. We used monoclonal antibodies and flow cytometry to assess CR1 and CR3 expression on PMN in bronchoalveolar lavage (BAL) fluid of cystic fibrosis (CF) patients chronically infected with pseudomonas and in sterile joint fluid of arthritis patients. Resting peripheral blood PMN from these patients and normals expressed similar low levels of CR1 and CR3, and the patients' PMN increased CR1 and CR3 expression normally when stimulated in vitro. CR3 expression on CF BAL PMN was 90 +/- 12% of that on the same patient's blood cells stimulated in vitro with FMLP. In contrast, CR1 expression on BAL PMN was only 27 +/- 8% of that on stimulated blood cells. Similar results were obtained for joint PMN. This pattern could be reproduced in vitro by treating FMLP-stimulated blood cells with BAL supernatants or with pseudomonas or PMN elastase. The serine protease inhibitors, PMSF and alpha 1-antitrypsin prevented the lavage supernatant from reducing CR1 expression, while metalloprotease inhibitors had no effect. Treatment of PMN with elastase in vitro decrease their ability to kill opsonized Pseudomonas aeruginosa. These results suggest that PMN at inflammatory sites have maximally upregulated expression of their complement receptors, but that CR1 is then cleaved by proteolysis in situ. Although not related to the basic defect in CF, this may interfere with efficient phagocytosis and contribute to the CF patient's inability to eradicate chronic lung infection.

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+.

A recombinant peptide model of the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator: comparison of wild-type and delta F508 mutant forms.

A series of recombinant peptides, each including the sequence proposed to be the first nucleotide-binding fold of cystic fibrosis transmembrane conductance regulator (CFTR), has been produced in an attempt to find a model peptide that would autologously fold into a soluble structure with native-like properties. The peptide NBDIF, which contains the 267-amino acid sequence of CFTR from 384 to 650, meets these requirements. The peptide was produced with a high expression bacterial plasmid pRSET, purified from inclusion bodies following solubilization with 6 M guanidine-HCl and refolded from 8 M urea. Competitive displacement of trinitrophenol-ATP by nucleotides reveals binding of ATP and related nucleotides with KDs in the low micromolar range; the KD for ATP gamma S is 1.0 +/- 0.4 microM and for ADP 8.8 +/- 3.1 microM. The native-like character of the model peptide's structure is further supported by the findings that the KD for the ATP analog, 5'-adenylimidodiphosphate, is fourfold lower than the KD for the methylene analog, 5'-adenylmethylenediphosphonate, and that ATP binding slows the trypsin proteolysis of NBDIF. The CD spectra of NBDIF and the parallel peptide containing the most common cystic fibrosis mutation, deletion of Phe 508, are essentially indistinguishable, both spectra indicating 28% alpha-helix and 23% beta-sheet, with insignificant differences in the amounts of beta-turns and random structure. Extensive investigation using multiple conditions with highly purified preparations of the model peptides demonstrates that they do not support ATP hydrolysis. These large recombinant peptides offer practical models for the investigation of the first nucleotide-binding domain of CFTR.

Sequence homologies between nucleotide binding regions of CFTR and G-proteins suggest structural and functional similarities.

Sequence homology between the alpha-subunits of G-proteins and other GTP-binding proteins and certain regions within the nucleotide binding domains (NBDs) of cystic fibrosis transmembrane conductance regulator (CFTR) indicates that these protein structures may be similar. A sequence alignment of the NBDs of CFTR and NBDs from other membrane transporters, forms the basis of a structural model. This model predicts that one of the conserved sequences GGQR, within which a number of CF mutations occur, forms part of the nucleotide binding pocket and serves as an ON/OFF conformational switch as observed in GTP binding proteins. Furthermore, based on subtle sequence differences between the first and second NBDs of CFTR and from structure-activity data, we suggest that the nucleotide binding site environments of the two NBDs are different.

Overview of investigations into pulmonary hemorrhage among infants in Cleveland, Ohio.

Idiopathic pulmonary hemorrhage was diagnosed in 37 infants in the Cleveland, Ohio, area between 1993 and 1998. This rare disorder has been related to 12 deaths, including 7 originally thought to be sudden infant death syndrome. Thirty of the infants were African American, all of whom lived in a limited geographic area of eastern metropolitan Cleveland, an area of older housing stock. An investigation led by the Centers for Disease Control and Prevention has found an association with household exposure to a toxigenic mold, Stachybotrys chartarum, and other fungi. The rapidly growing lungs of young infants appear to be especially vulnerable to the toxins made by toxigenic molds. Environmental tobacco smoke was frequently present in the infants' homes and may be a trigger precipitating the acute bleeding. Stachybotrys, although not thought to be a common mold, is known to have a wide geographic distribution. An additional 101 cases of acute, idiopathic pulmonary hemorrhage have been reported in infants in the United States over the past 5 years. In this overview, the investigations are summarized, the clinical profile is described, the toxicity of S. chartarum is discussed, and pathophysiologic concepts are presented.

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.

Acute pulmonary hemorrhage in infants associated with exposure to Stachybotrys atra and other fungi.

BACKGROUND: A geographic cluster of 10 cases of pulmonary hemorrhage and hemosiderosis in infants occurred in Cleveland, Ohio, between January 1993 and December 1994. STUDY DESIGN: This community-based case-control study tested the hypothesis that the 10 infants with pulmonary hemorrhage and hemosiderosis were more likely to live in homes where Stachybotrys atra was present than were 30 age- and ZIP code-matched control infants. We investigated the infants' home environments using bioaerosol sampling methods, with specific attention to S atra. Air and surface samples were collected from the room where the infant was reported to have spent the most time. RESULTS: Mean colony counts for all fungi averaged 29 227 colony-forming units (CFU)/m3 in homes of patients and 707 CFU/m3 in homes of controls. The mean concentration of S atra in the air was 43 CFU/m3 in homes of patients and 4 CFU/m3 in homes of controls. Viable S atra was detected in filter cassette samples of the air in the homes of 5 of 9 patients and 4 of 27 controls. The matched odds ratio for a change of 10 units in the mean concentration of S atra in the air was 9.83 (95% confidence interval, 1.08-3 X 10(6)). The mean concentration of S atra on surfaces was 20 X 10(6) CFU/g and 0.007 x 10(6) CFU/g in homes of patients and controls, respectively. CONCLUSION: Infants with pulmonary hemorrhage and hemosiderosis were more likely than controls to live in homes with toxigenic S atra and other fungi in the indoor air.

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.

Pulmonary hemorrhage in infants and children.

Following a brief presentation of important clinical concepts regarding pulmonary hemorrhage in infants and children, recent reports on secondary and immune-related disorders causing acute pulmonary hemorrhage are reviewed. Idiopathic pulmonary hemosiderosis is updated noting the compilation of Japanese cases and current treatments. The recent increase in idiopathic pulmonary hemorrhage and hemosiderosis in young infants, particularly in Cleveland, is discussed along with management suggestions and an apparent relationship to some sudden infant death syndrome cases.

Reversible thermal conformation changes in human serum low-density lipoprotein.

Human serum beta(1) (LDL(2)) lipoprotein was preparatively divided into three subfractions on the basis of density, and the protein conformation was investigated by optical rotatory dispersion and circular dichroism. The protein conformation was found to vary substantially as a function of lipid composition and temperature in a gradual and reversible manner. Surprisingly, the reversible temperature range includes physiological temperature. From the character of the spectra, it appears that alpha-helix, disordered, and beta conformations can be present in the protein moiety. The presence of these conformations and their distribution appears to depend upon both lipid content and temperature. The importance of this conformational flexibility in its relation to the role of the lipoprotein in lipid transport is discussed.

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.

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.

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.

Expression in Escherichia coli of cytoplasmic portions of the cystic fibrosis transmembrane conductance regulator: apparent bacterial toxicity of peptides containing R-domain sequences.

Large peptide segments (148-479 amino acids) of the cystic fibrosis transmembrane conductance regulator, which are projected to represent cytoplasmic portions of this large membrane protein, were expressed in Escherichia coli using two T7 RNA polymerase vectors, pET11a and pRSET. Five of the nine peptides were readily expressed at high levels (15-35 mg/liter) and one at an intermediate level (10 mg/liter), but three could not be expressed at >1.5 mg/liter regardless of efforts to further optimize the system. Preinduction testing of these latter plasmids failed to demonstrate any plasmid instability, while bacterial survival was drastically curtailed upon induction, beyond that observed with the other plasmids. Peptides containing the second half of exon 13 (residues 700-830; R domain) appear to be especially toxic to the expressing bacteria. Peptides including this hydrophilic segment may be inhibiting the bacterial permeases which are known to he homologous to other portions of CFTR.

Characterization of a human cryoglobulin complex: a crystalline adduct of a monoclonal immunoglobulin G and albumin.

An unusual human cryoglobulin complex was characterized as a two-component system containing monoclonal immunoglobulin G (IgG) and serum albumin in a 1:2 mole ratio. This complex appeared to be an antibody-antigen complex, since the mole ratio was appropriate and the isolated Fab of the IgG associated with the albumin. The cryoglobulin apparently arose as a result of specific association and/or aggregation of the IgG albumin adduct, since the cryoglobulin complex formed a crystalline precipitate. The IgG and albumin were separated and characterized with respect to immunological cross-reactivities, sedimentation velocities, isoelectric properties, and amino acid composition. The extent of precipitation of the cryoglobulin complex was maximal at pH 7.8, was decreased by added ions including citrate, ethylenediaminetetraacetic acid, NaCl, and CaCl2, and was decreased by increasing temperature. Both the nature of the cold-precipitable complex and the solubility properties differed from those described for other cryoglobulins.

Lipophosphonoglycan of the plasma membrane of A canthamoeba castellanii. Inositol and phytosphingosine content and general structural features.

Lipophosphonoglycan, a major component of the plasma membrane of Acanthamoeba castellanii, has now been shown to contain 8% inositol and 13% C25- and C24-phytosphingosines in addition to the previously identified content of neutral sugars (26%), amino sugars (3%), aminophosphonates (10%), acidhydrolyzable phosphate (3%), and long chain fatty acids (14%). The fatty acids and phytosphingosines are in ceramide groups. Lipophosphonoglycan can be separated by dodecyl sulfate-polyacrylamide electrophoresis into two major components that are similar in composition except for different oligosaccharide groups. A tentative structural model incorporating these features is proposed in which each of the two components of lipophosphonoglycan is conceived as an oligomeric inositol-containing glycosphingolipid.

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.

[13C]Methylated ribonuclease A. 13C NMR studies of the interaction of lysine 41 with active site ligands.

A unique resonance in the 13C NMR spectrum of [13C]methylated ribonuclease A has been assigned to a N epsilon, N-dimethylated active site residue, lysine 41. The chemical shift of this resonance was studied over the pH range 3 to 11, and the titration curve showed two inflection points, at pH 5.7 and 9.0. The higher pKa, designated pKa1, was assigned to the ionization of the lysyl residue itself while the pKa of 5.7, designated pKa2, was assigned on the basis of its pKa to the ionization of a histidyl residue which is somehow coupled to lysine 41. Both pKa values are measurably perturbed by the binding of active site ligands including nucleotides, nucleosides, phosphate, and sulfate. In most cases, the alterations in pKa values induced by the ligands were larger for pKa2. The ligand-induced perturbations in pKa2 generally paralleled those reported for histidine 12, another active site residue (Griffin, J. H., Schechter, A. N., and Cohen, J. S. (1973) Ann. N. Y. Acad. Sci. 222, 693-708). The sensitivity of the N epsilon, N-dimethylated lysine 41 resonance to the histidyl ionization may result from a conformational change in the active site region of ribonuclease which is coupled to the histidyl ionization. This coupling between lysine 41 and another ribonuclease residue, which has not been documented previously, offers new insight into the interrelationship between residues in the active site of this well characterized enzyme.