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Biomedical subjects

Y Bushkin

Publications and source records attributed to Y Bushkin.

29 records · Page 2Linked to original sources
PubMed1986

Stimulation and expansion of a human T-cell subpopulation by a monoclonal antibody to T-cell receptor molecule.

A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG1,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In long-term cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an alpha-subunit (45-48 kD) and a beta-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pI from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.

Antibodies, Monoclonal↗
PubMed1985

Structural characteristics of Tla products.

Biochemical study of thymus leukemia antigen (TL) from thymocytes of various Tla genotypes and from leukemia cells revealed features that, given present evidence, are peculiar to TL among class I products of the H-2:Qa:Tla region of chromosome 17. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of TL from thymocytes of all TL+ mouse strains, precipitated by anti-TL antiserum or monoclonal antibodies, showed two closely migrating bands of equal intensity in the heavy (H) chain position (45-50,000 mol wt). Comparison of these two bands by two-dimensional isoelectric focusing (2D IEF)-SDS-PAGE and 2D chymotryptic peptide mapping showed no differences indicative of protein dissimilarity. Thus, the two components of the H chain doublet may differ only in a feature of glycosylation that does not affect charge. The two leukemias studied gave only a single band in the H chain position. On 2D peptide mapping and 2D IEF-SDS-PAGE, the patterns for TL of Tlaa and Tlae thymocytes, which are closely related serologically, were broadly similar, but clearly different from the pattern typical of Tlac and Tlad thymocytes. 2D peptide maps of TL from Tlaa thymocytes and Tlaa leukemia cells did not differ. Leukemia cells of Tlab origin (thymocytes TL-) gave 2D peptide and 2D IEF-SDS-PAGE patterns of a third type. With the exception of Tlaa, thymocytes of TL+ mice yielded additional TL products of higher molecular weight than the TL H chain.

Animals↗
PubMed1985

Biochemical characterization of a p43,12 complex: comparison with human and murine class I molecules.

A monoclonal antibody designated as C21 reacting with a p43,12 complex was developed against human thymocytes. It stained predominantly the early hematopoietic cells of the lymphoid lineage and also thymocytes, peripheral B-cells and activated T- and B-cells similarly to OKT10. The heavy chain of this antigen was a glycoprotein of Mr 43,000 (p43). Sequential immunoprecipitation with C21 and OKT10 antibodies indicated that they both reacted with an identical heavy-chain molecule. This observation was further documented by two-dimensional analysis. Monoclonal antibody C21 was used to probe a p43,12 complex further. Structural polymorphism of the p43 heavy chain isolated from T- and B-cells of different individuals was not detected by chymotryptic peptide mapping, although molecules from these cell types possessed a different charge on two-dimensional gels. An unusual observation was made regarding this complex on MOLT4 cells. The light chain co-precipitated from these cells was 12,000 daltons and had a pI distinct from that of beta 2-microglobulin but similar to the pI of the beta t molecule. Comparison between chymotryptic peptide maps of the p43 heavy chain and those of the human and murine class I molecules such as HLA, T6, H-2K, Qa-2 and TL revealed no apparent homology. We have shown, however, that the peptide backbone of p43, as studied by both tunicamycin treatment of cells and endoglycosidase F digestion of immunoprecipitates, was identical in size to that of murine Qa-1. These results suggest that the p43 antigen may be homologous to murine Qa-1 or another class I antigen encoded in the murine TL:Qa region.

Amino Acid Sequence↗
PubMed1984

T cell antiidiotypic antibodies reveal differences between two human leukemias.

Two different human T cell leukemias were compared, using antiidiotype-like murine monoclonal antibodies. In each case these antibodies immunoprecipitated disulfide-linked heterodimer molecules from their respective leukemic cells. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the two idiotype-bearing molecules a major difference in molecular weight was observed, which could be attributed to a similar difference in size of the heavily iodinated chain of either heterodimer. The lightly iodinated chains of both molecules co-migrated at 43 Kd, but appeared to have different isoelectric points on two-dimensional gel analysis. The possibility that these two different heterodimers correspond to different classes of the putative T cell receptor for antigen is discussed. Assays of proliferation of the leukemic cells using Sepharose-bound antiidiotype-like monoclonal antibody showed that one of the leukemic cell types proliferated readily in response to its antiidiotypic antibody. This proliferation was not associated with measurable production of IL-2 and appeared to be a direct effect of the antiidiotypic antibody, which may mimic antigen in its interaction with the T cell receptor for antigen. The other leukemic cell type did not respond to Sepharose-bound antiidiotypic antibody and was generally unresponsive to lymphokines and mitogens. It is possible that the two leukemic cell types represent different stages of T cell differentiation.

Animals↗
PubMed1984

Biochemical characterization of the human T6 antigen: a comparison between T6 and murine TL.

The human T6 antigen was studied by two monoclonal antibodies: OKT6 and Leu-6. A third monoclonal antibody, C56 (developed in our laboratory), was found to have similar properties to those of OKT6. On SDS-PAGE, all three antibodies precipitated a 48,000-12,000-dalton heterodimer. Two-dimensional gel electrophoresis and chymotryptic peptide map analysis revealed that these antibodies precipitated in identical 48,000-dalton heavy chain which was distinguishable from the HLA-A,B,C heavy chains. The single 12,000-dalton light chain precipitated with OKT6 antibody was shown to be distinct from beta 2-microglobulin by its pI. The two light chains precipitated with Leu-6 antibody were resolved by charge into beta 2-microglobulin and the more basic 12,000-dalton peptide identical to that precipitated with OKT6. In addition to beta 2-microglobulin, the latter component (presumably beta t) was also found in the light-chain fraction precipitated from the thymocytes with a monoclonal antibody recognizing the framework of HLA-A,B,C heavy chains. Using chymotryptic peptide mapping, no polymorphism was detected among the heavy chains of the T6 antigen isolated from thymocytes of four individuals. All three monoclonal antibodies failed to precipitate murine TL from ASL1 leukemia cell lysates. Similarly, none of the six monoclonal and two conventional anti-TL antibodies reacted with T6. Although a high degree of homology was found by peptide map analysis among the TL molecules encoded by the Tlaa, Tlad and Tlae alleles, a comparison between their peptide maps and that of T6 revealed no similarity. Despite previous suggestions that T6 is homologous to murine TL, the present biochemical studies do not support this hypothesis.

Animals↗
PubMed1984

Preparation and characterization of monoclonal antibodies directed at epitopes of human IFN-gamma.

Five monoclonal antibodies (A7, B24, I14, L12, and M2) recognizing different epitopes of the human natural IFN-gamma were prepared by immunizing BALB/c mice with a highly purified human natural IFN-gamma preparation (10(7) U/mg). All five antibodies had high IFN-gamma-binding activity but exhibited differential IFN-gamma-neutralizing activities. Furthermore, none of them neutralized the antiviral activity exhibited by either IFN-alpha or IFN-beta preparations, indicating thus their specificity for IFN-gamma. The A7, L12, M2, and I14 monoclonal antibodies, but not the B24, blocked the augmentation of natural killer cytotoxicity, mediated by peripheral blood monocyte-depleted lymphocytes, by Escherichia coli-derived IFN-gamma or natural IFN-gamma but not by IFN-alpha 2. All five monoclonal antibodies precipitated an identical molecular complex containing two major protein components with molecular weights of 20,000 (20 kD) and 25,000 (25 kD) and two minor components with molecular weights of 17,000 (17 kD) and 45,000 (45 kD). Treatment of the immunoprecipitated IFN-gamma molecule with endoglycosylase F led to a stepwise removal of the carbohydrate portions on both the 25 and 20 kD chains, which resulted in the appearance of both 16 kD and 18 kD chains. The hereby reported monoclonal anti-IFN-gamma antibodies will prove useful as probes for purification and for rapid assay of human IFN-gamma molecule.

Animals↗
PubMed1984

Monoclonal antibodies to human sarcoma and connective tissue differentiation antigens.

The use of monoclonal antibodies to distinguish human sarcoma from carcinoma cells has been explored. Spleen cells from a BALB/c mouse immunized with a human malignant fibrohistiocytoma were fused with cells of the mouse P3U1 plasmacytoma cell line. Antibodies were then screened for reactivity against human sarcoma and carcinoma cells growing in culture. This work has yielded 2 immunoglobulin G monoclonal antibodies VIE4 and VIF3 which, respectively, reacted with 85% (17 of 20) and 90% (18 of 20) of sarcoma lines tested but with none of eight carcinoma cell line preparations. Reactivity against normal fibroblasts was also demonstrated. By immunofluorescence, the antigens detected by the two antibodies appear to have distinctive intracellular distributions. Immunoprecipitation with VIF3 has shown that it is detecting a protein with a molecular weight of 70,000. When tested against pathological frozen tissue sections, VIF3 reacted with four of 11 and VIE4 with three of 11 human sarcomas but with none of ten carcinomas tested. VIF3 occasionally bound to normal adult connective tissues, whereas no such reactivity was seen with VIE4. These antibodies appear to be directed to fibroblastic markers associated with sarcomas and connective tissue differentiation antigens.

Animals↗
PubMed1982

The biochemical genetics of TL antigens on mouse thymocytes.

Molecules bearing thymus leukemia (TL) alloantigen were isolated by immunoprecipitation from detergent-solubilized thymocyte lysates. Antisera used included monoclonal antibodies (anti-TL.m1, anti-TL.m2, anti-TL.m3), monospecific anti-TL.5 alloantisera and multispecific anti-TL.1,2,3,5 antiserum. Apparently, each of these reagents immunoprecipitates the same single 45,000 molecular weight Tla gene product as shown by identity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing and by sequential precipitation studies. Allelic TL molecules, coded by the Tlaa and Tlad genes, were shown to be distinguishable by SDS-PAGE, and tryptic peptide mapping experiments. Both allelic TL molecules could be isolated from thymocytes of (Tlaa x Tlad)F1 mice. These results suggest that, at least for the Tlaa-Tlad allelic differences, the polymorphism and antigenicity of TL is determined by variation in amino acid composition.

Animals↗
PubMed1982

Membrane-bound and secreted IgA contain structurally different alpha-chains.

Three different forms of alpha-chains are synthesized by BF0.3 and 615.2, two cloned cell lines derived from the murine B lymphoma 1.29. The three forms of alpha-chains differ in size, pI, cellular location, and rate of turnover. They were identified by means of lactoperoxidase-catalyzed radioiodination, internal 14C or 35S labeling, and immunofluorescence techniques as membrane-bound(alpha m), secreted (alpha s), and intracellular (alpha ic) proteins. Comparison of immunoglobulin products of the two lymphoma lines with those of a hybridoma cell line, Id 150, which secretes IgA of the 1.29 idiotype but lacks membrane IgA, confirmed the assignments of alpha m, alpha s, and alpha ic. Results of biosynthetic labeling of BF0.3, 615.2, and Id 150 in the presence and absence of tunicamycin suggest that the difference in m.w. and charge observed between alpha m and alpha s can be attributed to differences in primary amino acid structure rather than different degrees of glycosylation.

Animals↗
PubMed1981

Further biochemical data on Qa-2.

The Qa-2 differentiation alloantigen is coded by a gene situated between the D and Tla loci of the murine major histocompatibility complex (H-2). Qa-2-bearing protein was isolated by immunoprecipitation and found to be composed of subunits of 40 000 and 12 000 daltons by SDS polyacrylamide gel electrophoresis (PAGE). The 12 000 dalton material was identified as beta 2-microglobulin (beta 2M) by its molecular weight (SDS PAGE), charge (isoelectric focusing), antigenicity (reactivity with xenogenic anti-beta 2M), and genetics. The 40 000 dalton mol. wt. of Qa-2 heavy chains is 5 000 daltons less than that of D and K molecules (45 000 daltons). The quantity of Qa-2 isolated by immunoprecipitation was found to vary in strain-specific fashion and as much as a 15-fold difference was observed.

Animals↗