Patricia J. McConahey

[11] Radioiodination of proteins by the use of the chloramine-T method

Publisher Summary This chapter describes the radioiodination of proteins by the use of the chloramine-T method. The incorporation of radioiodine into protein molecules provides increased dimensions to the study of their interactions and subsequent fates. This incorporation involves the covalent linkage of radioiodine in the tyrosyl and to a lesser degree histidine residue of the protein molecule. Information gained through the use of radioiodinated proteins can be used with confidence if it is established that minimal, if any, alteration occurs in the reactivity of the radioiodinated protein versus that observed in its native form. Chloramine-T radioiodination of proteins involves both the oxidation of radioiodide to its reactive state, H 2 OI + , and the oxidation of sulfhydryl groups of the protein molecules. There must be sufficient chloramine-T available to complete both the reactions. Therefore, proteins, with high reducing capacity, may be poorly radioiodinated under the standard conditions. For example, keyhole limpet hemocyanin under standard conditions is minimally radioiodinated. However, by increasing the chloramine-T to 100/μg per milligram of protein, an average 50% incorporation of radioiodine is achieved with no apparent denaturation. Proteins that cannot be radioiodinated successfully with standard conditions should be repeated at increased chloramine-T to the protein ratios.

A comparative immunologic analysis of several murine strains with autoimmune manifestations

Abstract The genetic autoimmune disease (lupus-like) in mice of several strains (NZB, NZB × W, MRL 1 , MRL n , BXSB) has been analyzed comprehensively. Comparison of the histoimmunopathologic, serologic, and cellular characteristics of these mice reveals several pathogenic common denominators but no obvious etiologic factors in common. In all kinds of autoimmune mice studied, the lupus-like syndrome consists of a fatal immune complex glomerulonephritis, thymic atrophy, and lymphoid hyperplasia that varies in degree. The lymphoid hyperplasia is most prominent in MRL 1 mice and it is of T-cell origin. MRL 1 mice, in addition, may exhibit arteritis and arthritis. Serologically, all SLE mice have abnormally elevated serum Ig levels, antinuclear antibodies, anti-ds- and ssDNA antibodies, and anti-hapten antibodies. Some of the MRL 1 mice, in addition to the above antibodies, develop antibodies to Sm ( MRL 1 and MRL n ) and antiglobulins ( MRL 1 ). Natural thymocytotoxic antibodies and anti-erythrocyte antibodies are predominantly found in NZB and NZB × W mice. With advanced age and disease all lupus mice have depressed complement levels and circulating immune complexes. Some of these complexes in all lupus mice are composed of retroviral gp70-anti-gp70 antibodies, whereas MRL 1 mice, in addition, contain circulating IgG-IgG RF complexes. Characterizing the various recognized lymphoid cell surface markers, one notes that each strain of lupus mice has some variation from the normal distribution, but in no two of these strains are the abnormalities the same. The B cells of these mice seem advanced in maturity, whereas the developmental isotype diversity is normal in all of them. NZB and NZB × W mice are characterized by a nonspecific early B-cell hyperactivity but it is not known if this activity is responsible for their subsequent specific autoantibody production. The proliferating T cells of older MRL 1 mice seem to exert excessive helper activity on syngeneic B cells. Both antigen-specific and antigen-nonspecific Con A-induced suppression are within normal limits in all lupus mice. Although transfer studies have shown that the basic defect is associated with the lymphoid system, from our work and that of others it appears that the abnormalities in these mice are polygenic and different from one strain to another. Thus, murine lupus as a syndrome can be the result of many different abnormalities which eventuate in B-cell hyperactivity and involvement of a number of the available endogenous antigens in a systemic immune complex disease which is responsible for the “characteristic” clinical picture of systemic lupus erythematosus.

Quantitative Aspects of Plasma Membrane-Associated Immunoglobulin in Clones of Diploid Human Lymphocytes

The amount of κ and λ chains and Fc fragment associated with the plasma membrane and in the entire cell has been measured for eight lines of human lymphocytes and 21 clones derived from two diploid lines. There was considerable variation in the nature and amount of membrane-associated immunoglobulin and total immunoglobulin among different cell lines and clones. Cells with different phenotypes for membrane-associated immunoglobulin were utilized to show that it is not simply absorbed from the culture medium onto the plasma membrane. The methods are useful for selecting variants which lack or have altered membrane-associated immunoglobulin.

Type C Virus Expression in Lymphoma-Paralysis-Prone Wild Mice

Abstract Wild mice trapped near Lake Casitas (LC) in southern California showed a high prevalence of infectious type C virus in the liver, spleen, and thymus within the first few weeks of life. By young adulthood about 80% of LC mice (including their genital tissues) were infected. Virus isolates from these mice cause lymphoma and lower limb paralysis under both natural and experimental conditions. Mice destined to develop paralysis showed higher levels of serum gs antigen early in life, whereas mice destined to develop lymphoma or remain free of these diseases could not be distinguished by this test. The individual variation in virus expression suggested that differences in virus type or in the immune or other host defense mechanisms greatly influenced susceptibility or resistance to indigenous type C virus-caused disease in LC wild mice.