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A Kulczycki

Publications and source records attributed to A Kulczycki.

53 records · Page 3Linked to original sources
PubMed1980

Improved cell surface radioiodination of macrophages.

Rabbit alveolar macrophages at high cell concentrations are inefficiently radiolabeled by the usual lactoperoxidase-catalyzed iodination method. Soluble macromolecules secreted by macrophages were found to inhibit the radioiodination of macrophages and other cells. A modified procedure is described which minimizes the presence of such inhibitory material and thereby considerably improves the radioiodination efficiency for both alveolar macrophages and the macrophage-like P388D1 cell line.

Animals↗
PubMed1980

Purification of Fc gamma receptor from rabbit alveolar macrophages that retains ligand-binding activity.

The Fc gamma receptor of rabbit alveolar macrophages was purified by affinity chromatography by using rabbit gamma-globulin (Rab gamma G) coupled to Sepharose. Macrophage preparations were efficiently labeled with 125I by using a modified lactoperoxidase method. After incubation of NP-40 cell lysates with Rab gamma G-Sepharose, elution at 4 degrees C with 0.5 N acetic acid containing 1% NP-40 and rapid neutralization allowed recovery of active Fc gamma receptor. Purified Fc gamma receptor retained its ligand-binding activity, since approximately 41 to 72% of labeled material specifically rebound to Rab gamma G-Sepharose. Active receptor also rebound to human IgG- and rat IgG-sepharose. Active Fc gamma receptor did not bind to Sepharose coupled to rabbit Fab, rabbit F(ab)'2 or human F(ab)'2 fragments, nor to Sepharose coupled to chicken IgG. Analysis of Fc gamma receptor by SDS polyacrylamide gels demonstrated a broad peak of radioactivity in the apparent m.w. range of 50,000 to 70,000 in 5.6% acrylamide gels and 35,000 to 55,000 in 9% gels. Labeled receptor with similar structural characteristics and ligand-binding activity was also obtained from highly purified adherent cell populations and from macrophages biosynthetically-labeled with [14C]glucosamine in culture.

Animals↗
PubMed1979

Characterization of the IgE receptor isolated from human basophils.

The IgE receptor of human basophils was purified by using simple and repetitive affinity chromatography on human IgE-Sepharose. Basophils were partially purified from peripheral blood of patients with chronic myelogenous or basophilic leukemia. Cells were labeled with 125I by using the lactoperoxidase method and were solubilized with nonionic detergent. Elution of IgE-Sepharose with 0.5 N acetic acid, 1% NP-40 allowed recovery of active IgE receptor. Analysis of human IgE receptor by SDS polyacrylamide gel electrophoresis with 10% gels demonstrated one major radioactive peak with an apparent m.w. of 58,000 to 68,000, somewhat larger than rat IgE receptor. The purified human IgE receptor was active since approximately 10 to 42% of labeled receptor could specifically rebind to insolubilized human IgE. Rebinding was blocked by nanomolar concentrations of soluble human IgE or rat IgE but not by human or rat IgG, heat-inactivated human IgE, or heat-aggregated human IgG; thus it appears that rat IgE receptor. The relative abilities of active rat IgE and active human IgE to inhibit human IgE receptor rebinding could not be precisely determined because of the limitations in assessing the proportion of human IgE that retains receptor-binding activity.

Animals↗
PubMed1977

Studies of the mechanism of tunicamycin in hibition of IgA and IgE secretion by plasma cells.

Tunicamycin, an antibiotic which blocks the formation of N-acetylglucosamine-lipid intermediates, thereby preventing glycosylation of glycoproteins, inhibits the secretion of IgA and IgE by MOPC 315 mouse plasma cells and IR162 rat plasma cells, respectively. At 0.5 microng of tunicamycin per ml, D-[14C]glucosamine incorporation into newly synthesized immunoglobulin was inhibited greater than 90% while the overall rate of protein synthesized was much less inhibited (40% in the case of MOPC 315 cells and 13% in the case of IR162 cells). This dose of tunicamycin produced an 85% inhibition of IgA secretion by the MOPC 315 cells and a complete inhibition of intact IgE secretion by the IR162 plasma cells. In contrast, tunicamycin had little effect on the secretion of normally nonglycosylated lambda light chains or on cell-free protein synthesis, demonstrating that tunicamycin is not a general inhibitor of protein synthesis or a non-specific inhibitor of protein secretion. No enhancement of intracellular degradation of nonglycosylated immunoglobulin could be demonstrated. Electron microscopy of tunicamycin-treated MOPC 315 cells revealed marked dilatations of the rough endoplasmic reticulum, and direct immunofluorescence indicated that the dilated rought endoplasmic reticulum contained IgA. These data indicate that glycosylation of newly synthesized IgA and IgE may be necessary for normal secretion to occur.

Acetylglucosamine↗
PubMed1977

Release of slow reacting substance (SRS) from rat basophilic leukemia (RBL-1) cells.

When rat basophilic leukemia (RBL-1) cells were exposed to the ionophore A23187, a substance was released that produced a prolonged contraction of guinea pig ileum resembling that seen with slow reacting substances (SRSs) from various sources. The response was temperature, dose, and the time dependent with no activity being demonstrated in unstimulated cells. Several lines of evidence indicated that the RBL-1 product was markedly similar or identical to SRSs obtained from non-neoplastic tissues: 1) appropriate behavior in seven different chromatographic systems, 2) an appropriate profile of activity on various smooth muscle preparations, 3) an ability of low concentrations of the selective SRS inhibitor FPL 55712 to block the guinea pig ileal response, 4) failure of chymotrypsin to destroy activity, 5) loss of the activity after incubation with arylsulfatase, and 6) an ability to release activity from cells preincubated with indomethacin. Since RBL-1 cells can be grown in considerable guantity and under optimal conditions an average of 1500 SRS units/10(7) cells can be obtained, these cells should be useful as a biosynthetic source in further attempts to purify and characterize the SRS molecule.

Animals↗
PubMed1976

Modulation of cyclic AMP in purified rat mast cells. III. Studies on the effects of concanavalin A and anti-IgE on cyclic AMP concentrations during histamine release.

Changes in rat mast cell cyclic adenosine 3',5' monophosphate (cAMP) concentrations during stimulation of histamine release by concanavalin A (con A) and anti-IgE were studied. Con A caused an increase in cAMP with a mean peak level at 20 sec of 232% of control (range 164% to 365%). Con A-stimulated cells demonstrated falls toward control levels after 20 sec, but generally remained above control for at least 5 min. By 10 min cAMP had returned to control values. The con A effect on cAMP occurred in the absence of phosphatidyl serine but was markedly inhibited by 5 mM alpha-methyl-D-mannose. Anti-IgE induced a less marked increase in cAMP (157% of control, range 110% to 540% of control) which reached a peak at 20 sec. Two monospecific goat anti-rat myeloma IgE antisera induced similar changes in cAMP whereas normal goat IgG had no effect. These peak values were followed by a rapid decrease in cAMP. Within 2 min the cAMP content of anti-IgE stimulated cells had fallen to levels well below control and remained below control levels from 45 sec to over 15 min. Histamine release in both systems began after the peak cAMP levels, during the period of rapid destruction of cAMP.

Animals↗
PubMed1976

The rat basophilic leukemia cell receptor for IgE. I. Characterization as a glycoprotein.

Rat basophilic leukemia cells were labeled either enzymically with 125I or biosynthetically by culture in the presence of 14C-glucosamine or 3H-amino-acids and then extracted with NP-40. IgE-anti-IgE precipitates insolubilized a radiolabeled macromolecule from these extracts largely or entirely absent in control IgG-anti-IgG percipitates. When specific precipitates were boiled in sodium dodecyl sulfate (SDS) and analyzed by polyacrylamide gel electrophoresis in the presence of SDS, most of the 14C or 125I radioactivity was in the area corresponding to an apparent m.w of 60,000 to 70,000 in 5.9% gels. In 10% and 12% gels, faster mobility was demonstrated indicating an atypical electrophoretic behavior often associated with glycoproteins and a presumptive m.w. of 50,000 or less. Since only IgE-containing precipitates localized label in this region and since such precipitates from cells saturated with IgE prior to surface iodination failed to show this band, the labeled macromolecule appears to be the IgE receptor itself. Analysis of the acid hydrolysates of precipitated 14C radioactivity demonstrated that label was entirely in hexosamines and sialic acid. 125I and 14C labels in the recepotr region were eliminated almost completely with pepsin and pronase and to a lesser extent with trypsin.

Animals↗
PubMed1975

Ionophore A-23187 induced histamine release from rat mast cells and rat basophil leukemia (RBL-1) cells.

Ionophore A-23187 releases histamine from normal mast cells apparently by promoting Ca++ influx (Foreman et al, Nature 245: 249, 1973). In our hands at concentrations of greater than 0.2 mug/ml release occurs in 1 to 2 min, is blocked by metabolic inhibitors, and is unaccompanied by cytotoxicity (trypan-blue uptake, lactic dehydrogenase (LDH) release). At higher doses (0.5 mug/ml) histamine release is followed by significant cytotoxicity, but again Ca++ is required. In parallel studies, we examined cultured rat basophilic leukemia (RBL-1) cells. These cells, which apparently have normal surface receptors for IgE, contained approximately 700 ng histamine/10(6) cells but did not release histamine when IgE-mediated release was looked for. They do not respond to doses of ionophore which would be expected to give non-cytotoxic histamine release. At higher doses histamine release is preceded by progressive LDH release: LDH release is 75% complete at 5 min whereas 10 min are required for 75% maximal histamine release. This reaction requires Ca++ and is temperature dependent but is not inhibited by metabolic poisons (2-deoxyglucose, dinitrophenol, CN-). These studies suggest that either Ca++ does not enter into these cells normally or that one or more mechanisms which are ordinarily triggered by the changes in Ca++ flow are unresponsive in the RBL-1 cells. These studies also underline the importance of ruling out cytotoxicity in ionophore-induced phenomena.

Animals↗
PubMed1974

The interaction of IgE with rat basophilic leukemia cells. II. Quantitative aspects of the binding reaction.

Previous studies indicated that cultured rat basophilic leukemia cells have surface receptors which bind IgE with high specificity. In this paper we describe some quantitative aspects of the phenomenon. The reaction mechanism appears to consist of a simple reversible binding reaction with k(1) = 9.6 x 10(4) M(-1) sec(-1) and k(-1) 1.6 x 10(-5) sec(-1) at 37 degrees C. The calculated K(A) is therefore 6 x 10(9) M(-1). The activation energy of binding was found to be 7.8 kcal/mol. The number of binding sites/cell varied between 3 x 10(5) to over 1 x 10(6). The binding was insensitive to pH's between 6-8 but at pH 3.0 complete dissociation of bound IgE occurred in approximately 1 min at 0 degrees C leaving the receptors for IgE intact. Ca(++) plus EDTA and Mg(++) plus EDTA produce a fairly marked reduction in binding capacity though these reagents alone produce much smaller effects.

Animals↗
PubMed1974

The interaction of IgE with rat basophilic leukemia cells. I. Evidence for specific binding of IgE.

A rat basophilic leukemia cell line originally described by Eccleston et al. (3) has been successfully transplanted into eight Wistar strains and adapted to suspension cell culture without noticeable morphological changes. The cells deplete the reaginic activity of mouse and rat immune sera to an extent roughly equivalent to that reported for normal rat mast cells. Studies utilizing radioiodinated antilight-chain antibodies and radioiodinated partially purified rat IgE indicate that the cells bind IgE to their surface membrane with high specificity. Heating or mildly reducing the rat IgE destroyed its binding activity. The binding is unaffected by the presence or absence of Ca(++) and Mg(++), and is markedly inhibited by reaginic but not by normal rat or mouse serums. The affinity of these cells for human IgE, if present at all, is substantially lower than for rat IgE.

Animals↗