A quantitative analysis of C3 binding to O-antigen capsule, lipopolysaccharide, and outer membrane protein of E. coli 0111B4

The binding of serum C3 to the O-antigen capsule (OAg Cap), lipopolysaccharide (LPS), and outer membrane proteins (OMP) of Escherichia coli 0111B4 was examined. Bacteria were intrinsically labeled with [3H] or [14C]galactose (*gal) in the OAg Cap and LPS moieties or with [14C]leucine (*leu) to label proteins. Organisms were then incubated in serum containing differentially labeled C3, the above fractions were separated, and the proportion of each binding to a column containing anti-C3 was measured. The OAg Cap fraction bound 72 to 82% of the C3, which bound to E. coli 0111B4 during incubation in absorbed 10% pooled normal human serum (10% PNHS) or absorbed 40% C8-deficient serum (C8D). This distribution did not change when the organism was presensitized with immune IgG before serum incubation. A total of 2.93% +/- 0.48 of OAg Cap and 0.52% +/- 0.16 of LPS *gal bound specifically to Sepharose-containing antibodies to C3 (A:C3-Seph) after incubation in 10% PNHS; these values increased to 10.1% +/- 4.5 and 1.8% +/- 0.3, respectively, when C3 deposition was increased fourfold by incubation in 40% C8D. When encapsulated E. coli 0111B4 was incubated in 10% PNHS containing biotinylated C3, specific attachment of OAg Cap *gal to avidin-Sepharose was demonstrated in 1% sodium dodecyl sulfate (SDS), and complete release of bound *gal but not C3 occurred with 1 M NH2OH. When a mutant of E. coli 0111B4 lacking OAg Cap was incubated in 40% C8D, the outer membrane (OM) bound 85% of C3. Five percent of OM *gal from the unencapsulated organism bound to A:C3-Seph in 0.05% SDS, indicating that the fraction of LPS molecules with bound C3 increased threefold in the absence of OAg Cap. OAg Cap does not contain protein, and no net specific binding of *leu from OAg Cap fractions to A:C3 was detectable; 2.4 to 3.6% of OM *leu bound to A:C3-Seph. Immunoprecipitation of 82.9% of OAg Cap *gal with antisera that were directed to E. coli 0111B4 was associated with co-precipitation of 69.5% of C3 in the capsular fraction. Therefore, the majority of C3 bound to E. coli 0111B4 was covalently attached to OAg Cap and LPS. As corroboration of experiments with whole bacteria, purified OAg Cap and purified LPS consumed C3 when incubated in serum in the fluid phase. These results are the first to evaluate the acceptor site for C3 deposition on a Gram-negative organism incubated in serum, and show that LPS, OAg Cap, and OMP are all major acceptor sites for C3 in nonimmune serum.     

Relationship between intermediate filaments and microfilaments in cultured fibroblasts: evidence for common foci during cell spreading

Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers. Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF. These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.     

A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture

A rapid method for the direct conjugation of affinity-purified antibodies with fluorescein (termed DCAPA) is described. This procedure involves the immobilization of antibodies as antigen-antibody complexes on nitrocellulose blots, and subsequently the bound antibodies are reacted with fluorescein isothiocyanate. An enriched sample of smooth muscle tropomysin transferred to nitrocellulose paper by the Western blotting procedure has been used as the affinity medium for purification of specific tropomyosin antibody from whole rabbit antiserum. Direct conjugation of the antibody with fluorescein was carried out following the binding of antibody to antigen. Direct conjugation and affinity purification of antibodies directed against tropomyosin was accomplished in 2-3 d using an enriched tropomyosin sample and whole antiserum directed against tropomyosin. The immunofluorescence images obtained with this procedure exhibit distinct advantages with regard to background fluorescence and overall specificity of antibody binding. The usefulness of this direct conjugation method in various experimental protocols is discussed.     

Leak Current Rectification in Myxicola Giant Axons : Constant field and constant conductance components

Early leak current, i.e. for times similar to the time to peak of the transient current was measured in Myxicola giant axons in the presence of tetrodotoxin. The leak current-voltage relation rectifies, showing more current for strong depolarizing pulses than expected from symmetry around the holding potential. A satisfactory practical approximation for most leak corrections is constant resting conductance. The leak current-voltage curve rectifies less than expected from the constant field equation. These curves cannot be reconstructed by summing the constant field currents for sodium and potassium using a P_Na/P_K ratio obtained in the usual way, from zero current constant field fits to resting membrane potential data. Nor can they be reconstructed by summing the constant field current for potassium with that for any other single ion. They can be reconstructed, however, by summing the constant field current for potassium with a constant conductance component. It is concluded that the leak current and the resting membrane potential, therefore, are determined by multiple ionic components, at least three and possibly many. Arguments are presented suggesting that ion permeability ratios obtained in the usual way, by fitting the constant field equation to resting membrane potential data should be viewed with skepticism.     

SPORADIC ACUTE GLOMERULONEPHRITIS IN ADULTS

Nineteen adults who had acute glomerulonephritis were reviewed with respect to the clinical course and long-term follow-up. The age range was from 17 to 55 years. Only one patient died during the acute episode. In 11 cases, onset occurred between November and January and 15 of the patients had a known respiratory tract infection three to 30 days before the onset. The most important symptoms noted were weight gain, edema, dyspnea, oliguria and red or smoky urine. The most prominent physical signs were elevated blood pressure, edema, abnormalities in the chest and fever of over 100° F. Fifteen patients showed roentgen evidence of pulmonary vascular congestion, pleural effusion, cardiomegaly, pneumonia or a combination of these abnormalities. All the patients had proteinuria and red blood cells in the urine, and half of them had red blood cell casts. Azotemia, when present, subsided in 9.4 days. The average diastolic pressure was 105 mm. of mercury and the mean fall was 26 mm. in 23.5 days. At six months, nine of the 13 patients still being observed continued to show proteinuria or microscopic hematuria (seven showed both). A late follow-up of ten patients showed one to have significant hypertension and one to have early functional impairment and inconstant proteinuria. In these cases the average blood pressure was 140/91 mm. as compared with 119/74 mm. at the time of discharge.Sporadic glomerulonephritis in adults presents essentially the same pattern as it does in children. Urinary abnormalities may persist for months or even years, and neither the present series nor those reported by others clearly reveal the ultimate prognosis.