scholarly journals The Unitary Nature of "Complete" and "Incomplete" Pathologic Cold Hemagglutinins

Blood ◽  
1962 ◽  
Vol 19 (3) ◽  
pp. 379-398 ◽  
Author(s):  
JOHN P. LEDDY ◽  
NORMA C. TRABOLD ◽  
JOHN H. VAUGHAN ◽  
SCOTT N. SWISHER
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cold Agglutinin ◽  
Complement Components ◽  
Cold Agglutinins ◽  
Zone Electrophoresis ◽  
Human Sera ◽  
Normal Human ◽  
Physicochemical Methods ◽  
Reduced Activity

Abstract Several human pathologic sera containing high titered cold agglutinins were studied to determine whether the serologic activity ascribed to an "incomplete cold antibody" could be separated from the "complete" cold agglutinin activity. Separation was not achieved by physicochemical methods, including zone electrophoresis, density gradient ultracentrifugation, and anion exchange chromatography. Both activities were susceptible to destruction by mercaptans. Neither activity could be differentially absorbed from the sera. Using "Bombay" and I-negative ("i") red cells, a difference in specificity of the two activities for the H or I antigen of human erythrocytes could not be demonstrated. The simplest interpretation of these findings is that there is only one antibody involved, the cold agglutinin, and that the serologic manifestation usually attributed to an additional "incomplete cold antibody", i.e. the production of a positive antiglobulin reaction of the "non-γ-globulin" type, results from an interaction of complement components with the cold agglutinin-erythrocyte complex. Three of these cold agglutinating sera were unreactive with I-negative erythrocytes, in keeping with the reported anti-I specificity of these antibodies. A fourth serum retained moderate, though greatly reduced, activity against these cells, and the interpretation of this finding is discussed. The anti-H specificity of the incomplete cold antibodies in normal human sera was confirmed by their failure to sensitize "Bombay" erythrocytes. This was in sharp contrast to the excellent reactivity of the pathologic sera with these cells, demonstrating that pathologic cold agglutinins are unrelated to the incomplete cold antibodies present in most normal sera.

scholarly journals REAGINIC ACTIVITY ASSOCIATED WITH IGG IMMUNOGLOBULIN

1966 ◽  
Vol 123 (5) ◽  
pp. 845-858 ◽  
Author(s):  
Robert T. Reid ◽  
Percy Minden ◽  
Richard S. Farr
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Anion Exchange ◽  
Bovine Serum ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Unique Property ◽  
Serum Fractions ◽  
Human Sera

Five human sera with reaginic activity to a number of allergens were fractionated using anion exchange chromatography. In each serum, fractions which contained detectable IgG and no detectable IgA had capacity to fix to skin and subsequently elicit a P-K reaction. Four of these sera had reaginic activity about equally distributed between fractions containing only IgG and fractions containing mixtures of IgG and IgA. A fifth serum contained reaginic activity to crystalline bovine serum albumin (BSA) and most of the activity was associated with the fraction which contained only IgG. This serum was extensively studied using a variety of techniques and it was confirmed that most of the reagin to BSA in this serum was in those fractions containing only IgG. Since reaginic activity can no longer be considered a unique property of IgA the implications of finding antibody with reaginic qualities in immunoglobulins other than IgA are discussed.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

scholarly journals Characterization of mucins from cultured normal human tracheobronchial epithelial cells

2000 ◽  
Vol 278 (6) ◽  
pp. L1118-L1128 ◽  
Author(s):  
David J. Thornton ◽  
Thomas Gray ◽  
Paul Nettesheim ◽  
Marj Howard ◽  
Ja Seok Koo ◽  
...  
Keyword(s):  
Charge Density ◽  
Gradient Centrifugation ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Mrna Levels ◽  
Early Passage ◽  
Respiratory Mucin ◽  
Normal Human ◽  
Human Airways

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5Bas the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH2-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.

Are cardiovascular and sympathoadrenal effects of human “new pressor protein” preparations attributable to human coagulation β-FXIIa?

2004 ◽  
Vol 286 (3) ◽  
pp. H837-H846 ◽  
Author(s):  
Peter C. Papageorgiou ◽  
Ali Pourdjabbar ◽  
Akis A. Amfilochiadis ◽  
Eleftherios P. Diamandis ◽  
Frans Boomsma ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Coagulation Cascade ◽  
Plasma Catecholamine ◽  
Response Curves ◽  
Normal Human ◽  
New Pressor Protein

“New pressor protein” (NPP) derived from normal human plasma is an extra renal enzyme that shares strong sequence homology with human coagulation β-FXIIa. Under our bioassay conditions, human NPP (10–20 μl plasma equivalent/∼300 g rat iv) can raise the systolic blood pressure (SBP) by 40–50 mmHg, the diastolic blood pressure (DBP) by 15–20 mmHg, and the heart rate (HR) by 70–90 beats/min. Plasma epinephrine (of adrenal medullary origin) and norepinephrine rise by about 50- and 10-fold, respectively. Because β-FXIIa is not normally associated with pressor properties, we endeavored to substantiate that the hypertensive effects of impure NPP preparations used in our experiments are attributable to their content of β-FXIIa. We carried out comparisons with highly purified (>90%) commercial human β-FXIIa and found that by gel filtration (Sephadex G-100 and G-75), NPP bioactivity appeared in the ∼30-kDa elution zone, consistent with the molecular mass of β-FXIIa. Retention time using fast-protein liquid chromatography anion exchange chromatography was identical. Molecular mass and comigration were confirmed by SDS-PAGE gel electrophoresis, and the recovered ∼30-kDa protein bands yielded β-FXIIa fragments identified by mass spectrometry. Matched doses of the NPP preparations produced dose-response curves very similar to those elicited by β-FXIIa with respect to increments of SBP, DBP, and HR, whereas plasma catecholamine increments were generally comparable. We propose that β-FXIIa is substantially, if not exclusively, responsible for the observed effects of our NPP preparations and that this points to a novel axis connecting the FXII coagulation cascade and the sympathoadrenal gland to other cardiovascular regulatory mechanisms.

scholarly journals Structural analysis of the N-glycans from human immunoglobulin A1: comparison of normal human serum immunoglobulin A1 with that isolated from patients with rheumatoid arthritis

1994 ◽  
Vol 299 (1) ◽  
pp. 261-275 ◽  
Author(s):  
M C Field ◽  
S Amatayakul-Chantler ◽  
T W Rademacher ◽  
P M Rudd ◽  
R A Dwek
Keyword(s):  
Rheumatoid Arthritis ◽  
Human Serum ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Normal Human Serum ◽  
Complex Type ◽  
Gamma Chain ◽  
One Dimensional ◽  
Normal Individuals ◽  
Normal Human

The primary structures of the N-linked oligosaccharides from normal human serum IgA1 were determined by a combination of sequential exoglycosidase digestion, Bio-Gel P-4 chromatography, anion-exchange chromatography and one-dimensional n.m.r. spectroscopy. Three major N-linked disialylated biantennary-complex-type structures were found (55%). The remaining N-linked oligosaccharides consisted of at least nine further structures, some of which (7%) were of the triantennary type and included disialylated triantennary oligosaccharides with outer-arm fucose substitution [Fuc alpha 1-3(4)]. Compared with IgG, the N-glycan structures on IgA are more completely processed: the outer arms have a higher proportion of galactose and sialic acid, and only trace levels of incompletely galactosylated oligosaccharides, commonly found on IgG, were detected. Analysis of the sialylated O-glycans revealed that 64% were [NeuAc2 alpha 3(6)]2Gal beta 3GalNAc and 9% were [NeuAc2 alpha 3(6)]-Gal beta 4GlcNAc beta 6[NeuAc2 alpha 3(6)Gal beta 3]GalNAc, and 27% were monosialylated. The N-linked glycosylation of both serum IgA1 and IgG isolated from a group of six normal individuals was compared with that from ten patients with rheumatoid arthritis (RA). In contrast with the hypogalactosylation found in IgG from diseased sera, there was no evidence of an equivalent decrease in the galactosylation of the IgA1 oligosaccharides. In addition, the N-glycosylation of IgA1 was remarkably consistent within the group of normal individuals. These data suggest that incomplete galactosylation of N-linked glycans and its augmentation in RA does not extend to IgA1 and that the RA-associated galactosyltransferase deficiency may be restricted to cells producing gamma-chain.

scholarly journals Evaluation of a Capillary Electrophoresis Method for Routine Determination of Hemoglobins A2 and F

1999 ◽  
Vol 45 (2) ◽  
pp. 237-243 ◽  
Author(s):  
Frédéric Cotton ◽  
Changying Lin ◽  
Bernard Fontaine ◽  
Béatrice Gulbis ◽  
Jacques Janssens ◽  
...  
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Hemoglobin F ◽  
Low Concentrations ◽  
Zone Electrophoresis ◽  
Electrophoresis Method ◽  
Capillary Zone ◽  
Hb S

Abstract Hemoglobin A2 (Hb A2) and hemoglobin F (Hb F) are important analytes in the diagnosis and follow up of Hb diseases. We evaluated a new capillary zone electrophoresis (CZE) kit for Hb A2 and Hb F measurements. The imprecision ranged from 3% to 6% for Hb A2 and Hb F at physiological and pathological concentrations. The method compared well with cation-exchange HPLC for Hb A2 and Hb F and with anion-exchange chromatography in microcolumns (MAEC), for Hb A2. Nevertheless, higher results were obtained [Hb A2 CZE (%) = 1.233 Hb A2 HPLC − 0.2; Hb A2 CZE (%) = 1.190 Hb A2 MAEC + 0.1; Hb FCZE (%) = 1.118 Hb FHPLC + 0.4], and new reference values had to be determined (Hb A2 2.7–3.8%; Hb F <1.2%). Quantification of Hb A2 was not influenced by Hb S. Measurement of Hb F was accurate and precise except at low concentrations in Hb AS patients. This new CZE kit is rapid, precise, and reliable, and seems appropriate for use in clinical laboratories.

scholarly journals The variability of hemolysis in the cold agglutinin syndrome

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 409-416 ◽  
Author(s):  
WF Rosse ◽  
JP Adams
Keyword(s):  
Complement Activation ◽  
Cold Agglutinin ◽  
Complement Components ◽  
Cold Agglutinins ◽  
Thermal Amplitude

Abstract The amount of lysis effected by cold agglutinins is directly related to the ability of the antibody to initiate complement activation. This ability is modified by the concentration of antibody, its thermal amplitude (the highest temperature at which the antibody will react with the cell), the degree to which antibody fixation is modified by the presence of complement components (particularly C3) on the membrane, and the degree to which antibody, once fixed, is able to fix the components of complement. In vitro measurement of these factors correlates with the rate of hemolysis in vivo.

scholarly journals The variability of hemolysis in the cold agglutinin syndrome

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 409-416 ◽  
Author(s):  
WF Rosse ◽  
JP Adams
Keyword(s):  
Complement Activation ◽  
Cold Agglutinin ◽  
Complement Components ◽  
Cold Agglutinins ◽  
Thermal Amplitude

The amount of lysis effected by cold agglutinins is directly related to the ability of the antibody to initiate complement activation. This ability is modified by the concentration of antibody, its thermal amplitude (the highest temperature at which the antibody will react with the cell), the degree to which antibody fixation is modified by the presence of complement components (particularly C3) on the membrane, and the degree to which antibody, once fixed, is able to fix the components of complement. In vitro measurement of these factors correlates with the rate of hemolysis in vivo.

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