Biomedical subjects
Publications and source records attributed to M E Percy.
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Human thymus-leukemia-associated antigen (HThy-L), a saline-soluble antigen, was previously detected by immunodiffusion (but not on the cell surface) in significant quantity in extracts of normal thymocytes, cells from cultured T-cell lines, and erythrocyte-rosette-positive leukemia blasts. Two species of HThy-L were identified and isolated from normal human thymus tissue after extraction in tris buffer, ammonium sulfate fractionation, acid precipitation of inactive fractions, DEAE-cellulose (DE-52) chromatography, Sephadex G-100 gel filtration, and carboxymethyl-cellulose (CM-52) chromatography; On Sephadex G-100, both HThy-L species had a similar molecular weight (40,000--50,000), but they eluted in different positions on DE-52 and CM-52. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that each of the 2 HThy-L species contained 2 components with molecular weights of approximately 43,000 and 23,000. Further purification of HThy-L on Sephadex G-50 showed that the 43,000-dalton component possessed HThy-L activity.
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Despite the absence of B lymphocytes, peripheral blood lymphocytes (PBL) from four of five patients with congenital agammaglobulinemia (cAgamma) generated a specific hemolytic plaque-forming cell (HcPFC) response in vitro to sheep red blood cells and ovalbumin. The kinetics, antigenic, and cellular requirements were similar to normals, but significantly less HcPFC were found in patient cultures. Normal but not patient HcPFC-precursor cells were inactivated by treatment with anti-mu antisera whereas generated HcPFC in both controls and patients were sensitive to treatment with anti-mu. Pokeweed mitogen (PWM) and dextran sulfate (DXS) enhanced the HcPFC-response of normal PBL; cAgamma-cells were unresponsive to DxS and, in the presence of PWM, the development of HcPFC was inhibited. These findings indicate the presence of B lymphocyte precursors in the majority of patients with cAgamma investigated.
Cultures of peripheral blood lymphocytes (PBL) in which specific hemolytic plaque-forming cells (HcPFC) had been induced were labeled with 14C-amino acids. Antigen-specific products in the culture supernatants were characterized by using indirect immune precipitation in conjunction with specific immunoabsorbents and/or gel filtration followed by SDS-polyacrylamide gel electrophoresis. After 5 days of culture with antigen (sheep red blood cells or ovalbumin) newly synthesized IgM and specific IgM antibody were demonstrated in culture supernatants from normal donors and from four out of five patients with congenital agammaglobulinemia (cAgamma). Secreted products bound specifically to antigen and pretreatment of labeled supernatants with anti-mu and anti-L chain antisera, but not with anti-gamma antiserum, prevented binding. Typical mu- and L chains constituted only a proportion of the anigen-binding peptides recognized by the anti-mu reagents. Induction of IgM antibody synthesis was dependent on the presence of antigen and was correlated with the generation of HcPFC. No major differences between the antigen-induced products of cAgamma and normal PBL were observed. These findings suggest that in the absence of terminal B cell differentiation in vivo, certain patients with cAgamma possess precursor cells that can respond to antigen in vitro with the synthesis of specific humoral products, including IgM antibody.
The pathways and kinetics of interchain disulfide bond formation have been determined in vitro for purified myeloma proteins representing the three major subclasses of mouse immunoglobulin G(IgG) using the reoxidation system described previously (Petersen, J.G.L. & Dorrington, K.J. (1974) J. Biol. Chem. 249, 5633-5641). Mixtures of oxidized and reduced glutathione were added to act as a disulfide interchange catalyst. The pathways of covalent assembly observed in vitro were qualitatively and quantitatively similar to those followed by the various subclasses in vivo. HH and HHL were the principle covalent intermediates seen with IgG1 (MOPC 31C) and IgG2a (MOPC 173 and clone 19). With IgG2b( MPC 11C), HL, HH and HHL were all prominant intermediates. The time courses of reoxidation were simulated using a theoretical model based on second-order reaction kinetics (Percy, J.R., Percy, M.E. & Dorrington, K.J. (1974) J. Biol. Chem. 250, 2398-2400). Two distinct phases were apparent in the reoxidation sequence. The first, which lasted for the initial 5-15 min, did not confirm to the theoretical model. The second phase could be accounted for by the model and represented the remainder of the covalent assembly process. The physico-chemical basis for this biphasic phenomenon was explored. Sedimentation velocity studies showed that noncovalent association was incomplete at the beginning of the reoxidation step for all proteins except IgG2b (MOPC 11C). No dissociation was apparent in the reduced and alkylated proteins at pH 5 in the absence of prior exposure to acid conditions. Thus, exposure to acid appears to affect the affinity between the subunits in the native proteins. Transfer of the proteins from pH 5 to pH 8.2 (the pH at which reoxidation proceeds) is accompanied by the generation of an absorption difference spectrum over an 8-10 min period. These data suggest that a pH-dependent conformational relaxation process may influence the early stages of reoxidation.
A mathematical model, based on second-order reaction kinetics, has been used to describe the covalent assembly of immunoglobulin G(IgG) in vitro from its heavy (H) and light (L) chains (Percy, M.E., Baumal, R., Dorrington, K.J. & Percy, J. (1976) Can. J. Biochem. 54, 675-687). In the present paper, the same model has now been applied to the steady-state assembly of IgG in vivo. This mathematical approach permits a quantitative comparison of the pathways of covalent assembly used by given immunoglobulins in vivo and in vitro. The assumptions in the model are: the species L, H, HL, HH, HHL and LHHL belong to a common pool; incompleted IgG intermediates may freely assemble to form HL, HH, HHL and LHHL; the reaction rate for covalent linkage between any two reacting species is proportional to the products of the number densities of the reactants and to a parameter P which takes the value PHH if the reaction joins two H chains, and PHL if it joins an H and L chain. In vivo values of PHH/PHL were determined for the 18 mouse myeloma tumours and cell lines studied by Baumal et al. (Baumal, R., Potter, M. & Scharff, M. (1971) J. Exp. Med. 134, 1316-1334). From these analyses, we have arrived at the following conclusions: (1) the three major IgG subclasses have distinctive values of PHH/PHL (mean value 53 for IgG1, 12 for IgG2a and 2.8 for IgG2b); (2) for IgGs of the same subclass, the values of PHH/PHL are similar; (3) the mean in vivo values of PHH/PHL are very close to those determined from in vitro assembly experiments. Finally, the individual values of PHH/PHL have been used to simulate pulse-chase experiments in the various tumours and cell lines. Considering the sources and magnitude of experimental error, the theoretical pathways of assembly agree with those determined qualitatively from the pulse-chase experiments.
A simple theoretical model for the formation of interchain disulfide bonds (covalent assembly) in immunoglobulins has been developed. This model successfully simulates the experimentally determined sequence of disulfide bond formation in vitro for human immunoglobulin G1 and G4 (Petersen, J. G. L., and Dorrington, K. J. (1974) J. Biol. Chem. 249, 5633-5641). The model appears to be generally applicable suggesting that the various pathways of covalent assembly observed in vivo and in vitro reflect structural differences between the immunoglobulin classes and subclasses. The rate of assembly, however, is dependent upon environmental factors.
We have devised a novel technique which uses a simple theoretical model to simulate the reduction of immunoglobulin M (IgM) molecules. By fitting the results of the simulated depolymerization to experimental data, we have obtained statistical evidence which suggests that two major isomeric forms of covalently-bonded IgM monomer are liberated when human 19S IgM is reduced with dithiothreitol. The two heavy chains of isomer 1 are linked by two dilsulfide bridges, one in the segment designated the "hinge" region (position 337), the other penultimate to the COOH terminus of the chains (position 575). The half-cystines at position 414, which are free in isomer 1, form an inter-heavy chain bridge in isomer 2. Theoretically, the relative proportions of the two forms of monomer liberated in the reduction depend upon the dithiothreitol concentration, with isomer 2 predominating at higher dithiothreitol concentrations. Although in this paper we have assumed the conventional structure of the IgM molecule, the liberation of isomers depends only upon a symmetrical arrangement of the three types of inter-heavy chain bonds in the cyclic 19S pentamer.
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The partial sequence of the light chain of the myeloma-like immunoglobulin Sac shows a large deletion in its variable region. The sequence provides evidence that the corresponding gene was formed by the repair of DNA broken at nonhomologous positions. Data from other immunoglobulin (heavy) chains containing large deletions are compatible with their genes also being the result of DNA breakage and nonhomologous repair. Single homologous reciprocal exchanges in DNA networks at immunoglobulin loci could be the cause of the nonhomologous breaks. The relevance of these events to the generation of normal antibody variability remains to be determined.
The incidence of elevated serum creatine kinase (CK) and pyruvate kinase (PK) activities was compared in 20 definite carriers of Duchenne muscular dystrophy (DMD), 47 possible carriers, and 42 female controls. When adult age was not regarded as a variable, 70% of the definite carriers had elevated PK, 55% had elevated CK, and 75% had elevated PK or elevated CK or both, 38% of the possible carriers had elevated PK, 19% had elevated CK, and 40% had elevated PK or elevated CK or both. The detection efficiency of the CK test was influenced by the age of the subjects: the upper normal limit of serum CK in the adult controls was at the minimum between 21 and 35 years of age, and CK activity in some carriers declined from elevated to normal levels with increasing age. With these considerations, 70% of definite carriers had elevated CK and 80% had elevated PK and/or CK; 34% of the possible carriers had elevated CK and 43% had elevated PK and/or CK. On the basis of the PK and CK measurements, only 16 of 24 possible carrier mothers were likely to be DMD carriers, implying that the other 8 were non-carrier mothers of new mutant sons.
In both normal chicks and chicks with hereditary muscular dystrophy the BB (brain) and MB (hybrid) isozymes were the predominant forms of creatine kinase (CK) activity in embryonic skeletal muscle. As myogenesis progressed, activity due to the MM (muscle) isozyme progressively increased, and by 1 week ex ovo, the MM isozyme accounted for approximately 97% of total muscle activity in both genotypes. During this time, the proportion of the MM isozyme was slightly but significantly lower in dystrophic muscles. After hatching the proportion of the MB isozyme and its total activity decreased in normal muscle, but increased in dystrophic pectoral muscle, and by 5 months ex ovo, the MB isozyme accounted for 10% of total CK activity. Prior to hatching there was no consistent difference in total CK activity between normal and dystrophic tissues, but by 1 week after hatching and thereafter, total CK activity was significantly lower in dystrophic pectoral muscle.
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Despite increasing emphasis on advance directives, there has been little methodologic work to assess preferences about the "do not resuscitate" (DNR) order. This developmental work assessed, in a non-patient group, the performance of a probability-trade-off task designed to assess DNR attitudes, in terms of framing effects and stability of preferences. 105 female nursing students each completed one of two versions of the task. In version I (n = 58), the trade-off moved to increasingly negative descriptions of the outcomes of resuscitation (decreasing chance of survival and increasing risk of brain death), whereas in version II (n = 47), the trade-off moved to increasingly positive descriptions. One week later, repeat assessments were obtained for versions I (n = 35) and II (n = 28). The DNR preference scores were lower and more stable when the task moved to increasingly positive descriptions; perhaps this version of the task tends to weaken risk aversion. These results imply that care should be used in applying a probability trade-off task to the assessment of DNR preferences, since artefactual effects could be induced.