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R E Cone

Publications and source records attributed to R E Cone.

At least 91 records · Page 5Linked to original sources
PubMed1979

Differential radiolabelling of lymphocyte membrane alloantigens and immunoglobulins: variation of H2O2 concentration during lactoperoxidase catalyzed cell surface radio-iodination.

The effect of H2O2 concentration on lactoperoxidase catalyzed cell surface radio-iodination and subsequent isolation of Murine splenic lymphocyte Ia, H-2K and Lyb-3 surface antigens and membrane immunoglobulins was studied. For most membrane polypeptides analyzed 0.3 mM H2O2 proved to be optimal for the recovery of radiolabelled antigens from detergent lysates of labelled cells by immunoprecipitation. Marked variations among surface antigens and membrane immunoglobulin polypeptide chains were observed for the iodination and recovery of these proteins above and below the optimal peroxide concentration. The results suggest that cell surface radio-iodination conditions should be standardized to the requirements of the particular membrane protein being studied. The differential iodination and recovery of discrete membrane components above and below optimal conditions may prove useful in the analysis of surface membrane protein structure and membrane association.

Animals↗
PubMed1979

Antigen-binding T cells: dose response and kinetic studies on the development of different subsets.

We have described a number of the parameters involved in the in vitro induction of specific SRBC-binding T cells (T rosette-forming cells, T-RFC). Although T-RFC precursors pass through nylon, most of the induced cells do not; nor do detectable numbers of Ly 1+2, 3- cells bind antigen with sufficient stability to form rosettes. The ratio of Ly 2,3:Ly 1,2,3 T-RFC varies with time after immunization and with the dose of antigen used for stimulation. Relatively high or low doses of antigen selectively induce Ly 1,2,3 T-RFC. Ly 2,3 T-RFC, when they appear, follow Ly 1,2,3 T-RFC. Pretreatment of T cells with anti-Ly sera before RFC induction prevents formation by Ly2+ T-RFC. Since anti-Ly 1 treatment blocks RFC formation and since Ly 1,2,3, T-RFC always precede the appearance of Ly 2,3, T-RFC, our results suggest that some Ly 1+ cells (Ly 123 at least, but perhaps also Ly 1) may act as inducers, precursors, and/or amplifiers for Ly 2,3 RFC as they appear to do for Ly 2,3 suppressor and killer cells. Thus, our results confirm and extend the observed similarities between T-RFC and other Ly 2+ cells such as killer and suppressor cells as well as their differences from Ly 1+ helper cells.

Animals↗
PubMed1979

Turnover and shedding of Ia antigens by murine spleen cells in culture.

Lactoperoxidase-catalyzed radioiodination was used to examine the metabolic fate of surface Ia antigens on murine spleen cells in culture. Ia antigens, detected predominately on splenic B lymphocytes, were lost from the cultured cells with biphasic kinetics: a 4 to 6 hr rapid phase, t 1/2 = 5 hr followed by slow release through 20 hr, t 1/2 = 30 hr. The rapid loss of Ia antigens observed was abolished by both harsh iodination conditions and nonphysiologic incubation conditions. The rapid decline in Ia activity was shown to be due to shedding of intact Ia antigens from the cell and to predominant release of IA subregion-coded proteins. Release of Ia antigens from the cell was accomplished by replacement at the cell surface, and thus reflected net membrane Ia turnover. Ia shedding was shown to be extremely temperature dependent, reflecting both a comparatively high activation enthalpy and entropy requirement for turnover.

Animals↗
PubMed1978

Molecular identification of a surface structure on B cells (Lyb-3) and its relationship to B cell triggering.

Lactoperoxidase-catalyzed radioiodination of cell surface proteins and immunochemical procedures are used to identify murine splenic lymphocyte membrane components bound by anti-Lyb-3 serum. This antiserum defines membrane components (Lyb-3) on a subpopulation of murine B cells that may function as a receptor for T cell signals. SDS-PAGE analysis of surface-labeled membrane components bound by anti-Lyb-3 serum demonstrated a single molecular species of 68,000 d. The polypeptides recognized by anti-Lyb-3 are not composed of disulfide-linked subunits and bear no antigenic relationship with known membrane immunoglobulins (IgM or IgD). Absorption of anti-Lyb-3 serum with the 68,000 d polypeptides removed the ability of anti-Lyb-3 serum to augment the in vivo immune response of mice to low doses of sheep erythrocytes. The latter provides formal proof that the 68,000 d polypeptide bound by anti-Lyb-3 serum is the target on the B cell membrane for the immunoenhancing activity of the antiserum.

Animals↗
PubMed1978

Membrane proteins of the P388D1 macrophage cell line: isolation of membrane polypeptides that bind to the Fc portion of aggregated IgG.

Lactoperoxidase-catalyzed cell surface radioiodination was employed to radiolabel membrane polypeptides of a murine macrophage cell line P388D1. Optimal conditions for radioiodination of P388D1 cells were determined and were found to differ from conditions used to label lymphoid cells. SDS-polyacrylamide gel electrophoresis of detergent soluble membrane polypeptides revealed that 9 to 10 molecular species from 1 X 10(5) to 0.15 X 10(5) daltons were labeled. Radioiodinated, Triton X-100 extracted P388D1 membrane polypeptides were subjected to affinity chromatography on aggregated IgG:Sepharose columns. Elution of the bound polypeptides and analysis by SDS-polyacrylamide gel electrophoresis revealed polypeptides with an apparent molecular size of 8 X 10(4), which possess binding affinity for the Fc portion of aggregated IgG. The 8 X 10(4) dalton membrane polypeptides do not readily aggregate, are resistant to degradation, are not composed of disulfide-linked subunits, and do not appear to contain much carbohydrate. Cellular binding characteristics paralleled the binding of soluble receptor for sieved fractions of aggregated IgG suggesting that these polypeptides may be responsible for the in situ binding of aggregated IgG to P388D1 cells.

Animals↗
PubMed1977

Nylon adherent antigen-specific rosette-forming T cells.

Shortly after intravenous immunization of mice with heterologous erythrocytes (RBC) antigen-specific Thy 1+ cells which form rosettes with the immunizing RBC (thymic-derived lymphocytes-forming rosettes [T-RFC]) appear in the spleen. These T-RFC are much less stable than Thy 1- RFC (non-thymic-derived [B-RFC]) although most if not all of both classes of RFC adhere to nylon. T-RFC are induced with low doses of antigen (which fail to induce B-RFC) and are inhibited by higher antigen doses which are optimal for induction of B-RFC. Pretreatment of mice with cyclophosphamide prevents the high dose inhibition of T-RFC. Although there are many parallels between the production of T-RFC and delayed-type hypersensitivity (DTH) it is unlikely that the T-RFC are essential for DTH reactions since DTH can be transferred with cells which pass through nylon, and such cells are almost totally depleted of T-RFC. Thus immunization can lead to the production of large numbers of antigen-specific T-RFC whose functional role in the immune response is unknown. However, the characteristics of the T-RFC suggest that they may play an important role in amplification of suppressor cell activity.

Animals↗
PubMed1976

Immunoglobulin turnover in B lymphocyte subpopulations.

"In vitro" turnover of leucine-labeled and of radioiodinated IgM has been studied with cells from various lymphoid organs of nude mice, i.e. lymph nodes, thoracic duct, spleen and bone marrow, as well as with subpopulations of B cells from spleen and bone marrow separated by free flow electrophoresis. Three types of IgM-producing lymphocytes could be distinguished by their turnover rates of IgM, by the size of the released IgM and by the capacity of the IgM molecules to be labeled by the lactoperoxidase-catalyzed radioiodination reaction and/or by incorporation of radioactive leucine. Type I cells release 7-8 S IgM rapidly (t1/2 = 1-3h); the released IGM is leucine-labeled and radioiodinated. Type II cells release 7-8 S IgM slowly (t1/2 =10-30); the released IgM is leucine labeled and radioiodinated. Type III cells release 19 S IgM rapidly (t1/2 =2-4 h); the released IgM is leucine labeled, but not radioiodinated. Lymph nodes and thoracic duct contain predominantly type II cells, bone marrow contains type I and II cells, spleen contains type I,II and III cells. It is suggested that type III cells are Ig-secreting plaque-forming plasma cells, type II cells are small, resting "memory" B cells, and type I cells may be newly formed antigen-inexperienced B cells.

Animals↗
PubMed1975

Turnover of radioiodinated and of leucine-labeled immunoglobulin M in murine splenic lymphocytes.

Lactoperoxidase-catalyzed radioiodination of cell surface proteins and biosynthetic incorporation of tritiated leucine were used to study the size and turnover of cell-associated immunoglobulins in splenic lymphocytes of different size from BALB/c, C3H, and nu/nu mice. Small spleen cells, separated from large cells by velocity sedimentation, showed a single slow rate of turnover (t1/2 = 10-30 h) of surface radioiodinated or leucine-labeled IgM released as the monomeric 7-8 S subunit of IgM. Mitogen-activated large B cells and nonstimulated large spleen cells, separated by velocity sedimentation, released leucine-labeled IgM in an initial rapid phase (t1/2 = 2-4 h) as 19 S IgM, and later released 7-8 S IgM slowly (t1/2 = 10-30 h). The iodination reaction changed neither the biphasic mode of turnover, nor, even at higher concentration of H2O2, the size of the actively secreted 19 S IgM. Radioiodination of mitogen-activated large cells or of unstimulated large cells labeled only the slowly released 7-8 S IgM, not the actively secreted 19 S IgM. Large cells of nonstimulated spleen, however, released radioiodinated as well as leucine-labeled IgM also rapidly (t1/2 = 1-3 h). These rapidly released IgM molecules were found to be 7-8 S IgM subunits. Our results indicate that these turnover rate and size differences of radioiodinated and of leucine-labeled IgM released from murine spleen cells are in part resulting from the differences in the methodology of labeling and are in other parts due to different small and large splenic lymphocytes releasing IgM of different size at different rates.

Animals↗
PubMed1974

Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes. Selective isolation of immunoglobulin and the theta-antigen by non-ionic detergents.

Accessible surface proteins of thymus-derived lymphocytes (T-cells) of normal CBA mice and bone-marrow-derived lymphocytes (B-cells) of congenitally athymic nu/nu mice were analysed. The surfaces of lymphocytes were radioiodinated by using the enzyme lactoperoxidase (EC 1.11.1.7), then solubilized either in acid-urea or in the non-ionic detergent Nonidet P-40. These lysates were then precipitated with antisera specific to either immunoglobulin or the theta-alloantigen in order to assess the presence of these surface markers. Comparable amounts of radioactivity in proteins specifically precipitable as immunoglobulin were obtained from T-lymphocytes and B-lymphocytes when the cells were disrupted by acid-urea. This immunoglobulin had mol. wt. approx. 180000 and was composed of light chains and mu-type heavy chains. When radioiodinated lymphocytes were solubilized with Nonidet P-40, 3-4% of radioiodinated high-molecular-weight protein of B-cells consisted of immunoglobulin, a result similar to that found with acid-urea extraction. However, with the detergent extraction, only 0.1% of T-cell surface protein was precipitable by anti-globulin reagents. The theta-alloantigen was isolated from CBA T-cells both by acid-urea and by detergent lysis. This protein possessed a mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate which was consistent with a mol. wt. of 60000. An identical component was isolated from the theta-positive thymoma WEHI 105. The theta-antigen was not isolated from B-cells by either of the extraction procedures used. These results provide further evidence that the surface membranes of normal T-cells and B-cells differ in physicochemical properties. In particular, various surface components possess differential solubilities in non-ionic or organic solvents. This observation provides an explanation for discrepant results that have appeared in the literature concerning the isolation of immunoglobulin from T-lymphocytes.

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