[Unusual polymorphic immunocytoma].
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Aged↗
Biomedical subjects
J Gergely
Publications and source records attributed to J Gergely.
pH-dependent structural transition in rabbit skeletal troponin C.
Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.
Animals↗
Alternative pathway of complement activation by stimulated T lymphocytes. I. Binding of C3 fragments.
Human blood lymphocytes cultured for 3 days with concanavalin A (Con A), phytohemagglutinin or pokeweed mitogen, in mixed lymphocyte culture with added interleukin 2 and stimulated by a lymphoblastoid cell line were found to activate and bind C3 molecules when exposed to human serum. The split products of C3 were detected in the supernatants and on the surface of the activated cells. The surface-attached C3 fragment on the Con A blast was identified as C3b by immune adherence i.e. binding of CR1 carrying human erythrocytes. In the Con A-stimulated population the majority of cells that activated and bound C3 were CD3 and Fc gamma receptor (CD16)-positive but complement receptor-negative blasts. In this cell subset both CD4 and CD8-positive cells were detected but their frequency suggested that a proportion of them carried both markers.
Antigens, Differentiation, T-Lymphocyte↗
Alternative pathway of complement activation by stimulated T lymphocytes. II. Elevation of cytotoxic potential against complement receptor-carrying cell lines.
Exposure of lectin-stimulated (concanavalin A, phytohemagglutinin and pokeweed mitogen) blood lymphocytes to human serum or to purified C3 increased their cytotoxic capacity towards complement receptor positive targets such as Raji and Daudi cells. The lysis of complement receptor-negative lymphoblastoid cell lines was not influenced. The lytic capacity of lymphocytes exposed to 12-O-tetradecanoylphorbol 13-acetate was not elevated by human serum. Lectin-stimulated lymphocytes were previously shown to activate and bind C3. The results using lymphocytes activated in different ways and targets with or without complement receptor expression suggest that the C3b deposited on lymphocytes binds to the complement receptor on the targets. This contact elevates the avidity between the two cells as indicated also by the increased frequency of the lymphocyte-target conjugates. On the basis of immune adherence the C3 fragment bound on the lymphocytes was identified as C3b. The increase of the conjugate formation and cytotoxicity was abrogated when the target cells, Raji, were pre-exposed to purified C3d which occupy the CR2 receptor. The majority of lymphocytes responsible for the cytotoxicity were CD8+.
Antigens, Differentiation, T-Lymphocyte↗
Involvement of complement in B-cell, T-cell and monocyte/macrophage activation.
In the early 70's it had been shown, that for the immune response against T-dependent antigens C3 was necessary, while T-independent antigens, although activating the alternative pathway of complement, triggered antibody formation also in C-deficient mice. During recent years functional and biochemical knowledge about complement binding structures on B-cells and monocytes/macrophages continuously increased and, also, on T-cells C3 binding entities have been detected. In the case of B-cells and, at least in special experimental conditions, in the case of T-cells C3 can exert a proliferative response as long as the cells are prestimulated (excited) by anti-Ig or IL-2, respectively. Monocytes can bind C3b- or iC3b-carrying particles, but only when progressed to macrophages can they phagocytose such particles. Thus the concept evolves that B-cells, T-cells and monocytes can acquire competence for a C3-driven response when excited properly. The involvement of molecules such as CR1, CR2, factor H, IL-2-receptor and others with a basic structure of repeating units of 61 amino acids in the triggering processes is a surprising finding and certainly suggests their functional importance. In the case of T-independent antigens the structures triggering the alternative pathway of complement are the structures triggering monocytes directly. Whether these two functions have a causal relationship has to be shown.
Animals↗
Effector or target cell selection mediated by C3 bridges.
Potential effector cells (including stimulated lymphocytes and cultured monocytes) and potential target cells of NK and AK type cytotoxic reactions (including several lymphoblastoid cell lines) cleave the third complement component (C3). As a result of expression of C3bA sites such cells are able to bind covalently the activated C3b through its metastable binding site and thereby become "armed" by the C3b. This permits C3b-bridge formation between these cells and CR1-bearing cells. The "effector selection" (i.e. when C3b is bound covalently to potential target cells) or "target selection" (when C3b is covalently bound to C3bA sites on potential effector cells) mediated by C3b bridges results in enhanced killing capacity. Macrophages activate and bind C3b as well; but the covalent binding of C3b by these cells inhibits Fc receptor mediated ADCC type killing.
Animals↗
Changes in intracellular ionized Ca concentration associated with muscle fiber type transformation.
Since increased muscle activity, which results in fast-slow fiber transformation, is associated with increases in sarcoplasmic Ca2+ concentration ([Ca2+]i), it seemed of interest to study the level of [Ca2+] after cessation of stimulation in fibers of the extensor digitorum longus muscle chronically stimulated (8 Hz). [Ca2+]i was measured in individual fibers with a Ca2+-sensitive electrode after subtracting the membrane potential, measured simultaneously from the potential of the Ca2+ electrode. During the first 14 days of stimulation, [Ca2+]i increased from approximately 0.1 to 0.5 microM and declined in approximately 3 wk to a value slightly higher than the initial one. The rise and decline of [Ca2+]i was preceded by a transient increase in total calcium. If stimulation was terminated after 7-8 wk when an essentially complete fast-to-slow transformation had taken place, a subsequent rest period led to a reverse slow-to-fast transformation, which was also preceded by a transient increase of [Ca2+]i reaching a peak at day 5 of rest. Unstimulated fast and slow fibers and fully transformed fibers do not differ in their [Ca2+] levels; thus it appears that the transformation process itself is accompanied, particularly in its earlier stages, by elevated [Ca2+]i levels. Elucidation of the relation between changes in Ca2+ and changes in gene expression will require further work.
Adenosine Triphosphatases↗
Complement-dependent triggering of B-cells, T-cells and macrophages.
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B-Lymphocytes↗
An IgM-producing immunocytoma induces large numbers of splenic T lymphocytes with Fc mu receptors.
The expression of Fc mu receptors was investigated using spleen cells from (LOU/M/Wsl X CFY) F1 rats bearing the IR202 tumour, an IgM-producing immunocytoma. It was found that the progressively growing solid tumour was accompanied by an extraordinary expansion of host splenocytes with Fc mu receptors. These cells were nylon-wool non-adherent, Thy-1-positive and sIg-negative: features they have in common with T lymphocytes. In addition, the expansion of Fc mu receptor-bearing T cells in IR202 immunocytoma is correlated to the high level of serum IgM. These observations provide further insight into the mechanism of isotype-specific T-lymphocyte Fc receptor expression, and identify a potential model with which to analyse the immunoregulatory role of Fc mu receptor-bearing T cells.
Animals↗
Calcium binding to the low affinity sites in troponin C induces conformational changes in the high affinity domain. A possible route of information transfer in activation of muscle contraction.
Residues 89-100 of troponin C (C89-100) and 96-116 of troponin I (I96-116) interact with each other in the troponin complex (Dalgarno, D.C., Grand, R.J.A., Levine, B.A. Moir, A., J.G., Scott, G.M.M., and Perry, S.V. (1982) FEBS Lett. 150, 54-58) and are necessary for the Ca2+ sensitivity of actomyosin ATPase (Syska, H., Wilkinson, J.M., Grand, R.J.A., and Perry, S.V. (1976) Biochem. J. 153, 375-387 and Grabarek, Z., Drabikowski, W., Leavis, P.C., Rosenfeld, S.S., and Gergely, J. (1981) J. Biol. Chem. 256, 13121-13127). We have studied Ca2+-induced changes in the region C89-100 by monitoring the fluorescence of troponin C (TnC) labeled at Cys-98 with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Equilibrium titration of the labeled TnC with Ca2+ indicates that the probe is sensitive to binding to both classes of sites in free TnC as well as in its complex with TnI. When Mg2 X TnC is mixed with Ca2+ in a stopped flow apparatus, there is a rapid fluorescence increase related to Ca2+ binding to the unoccupied sites I and II followed by a slower increase (k = 9.9 s-1) that represents Mg2+-Ca2+ exchange at sites III and IV. In the TnC X TnI complex, the fast phase is much larger and the Mg2+-Ca2+ exchange at sites III and IV results in a small decrease rather than an increase in the fluorescence of the probe. The possibility is discussed that the fast change in the environment of Cys-98 upon Ca2+ binding to sites I and II may be instrumental in triggering activation of the thin filament by facilitating a contact between C89-100 and I96-116.
Animals↗
Modulation of the interaction between the two halves of troponin C by the other troponin subunits.
The interactions between troponin subunits have been studied by intrinsic fluorescence and electron spin resonance (ESR) spectroscopy. The tryptophan fluorescence of troponin T (TnT) and troponin I (TnI) when complexed with troponin C (TnC) undergoes a Ca2+-dependent transition. The midpoints of such spectral changes occur at pCa approximately equal to 6, suggesting that the conformational change of TnT and TnI is induced by Ca2+ binding to the low-affinity sites of TnC. When TnC is labelled at Cys-98 with a maleimide spin probe (MSL), the spin signal is sensitive to Ca2+ binding to both the high and the low-affinity sites of TnC in the presence of either or both of the other two troponin subunits. Since Cys-98 is located in the vicinity of one of the high-affinity sites, these results are indicative of a long-range interaction between the two halves of the TnC molecule. Our earlier kinetic studies [Wang, C.-L. A., Leavis, P. C. & Gergely, J. (1983) J. Biol. Chem. 258, 9175-9177] have shown such interactions in TnC alone. Since the ESR spectral change associated with metal binding to the low-affinity sites is only observed when MSL-TnC is complexed with TnT and/or TnI, this long-range interaction within TnC appears to be mediated through the other troponin subunits.
Animals↗
Binding characteristics and isotype specificity of Fc receptors on K cells.
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Antibody-Dependent Cell Cytotoxicity↗
Contribution of activated C3 to lymphocyte-mediated cytolysis.
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Cell Line↗
Isotype distribution and fine specificity of the antibody response of inbred mouse strains to four compounds belonging to a new group of synthetic branched polypeptides.
A new group of synthetic branched polypeptides was developed to initiate a systematic study of the relationships between the chemical structure (charge, size, primary structure, configuration and conformation), the carrier potential and the antigenic properties of these biodegradable and biocompatible macromolecules. This model system has two main advantages over the previously used ones: (i) the side chains grafted to the poly(L-lysine) backbone are composed of about three DL-Ala and a single chain-terminating amino acid with different absolute configuration and/or identity, and (ii) the conformation of these polypeptides is characterized in solution. The size, charge and inside area of the four molecules selected for this study were identical; however, the identity, the absolute configuration of the chain-terminating amino acids (D-Leu, Leu, Phe or D-Phe) and, in consequence, the conformation of the macromolecules were different. The qualitative and quantitative features of the antibody response induced by the four polypeptides were characterized in inbred mouse strains by IgM and IgG type antibody levels, as well as by isotype distribution and fine specificity of antibodies produced during the primary and memory response. The intensity of the memory response and the characteristics of subclass distribution were dependent on the conformation of the branched polypeptides. These molecules carry at least two types of antigenic determinants. One is ordered to the tetrapeptide side chain, the expression of which proved to be inversely correlated with the backbone-originated helix content of the molecules. The other antigenic determinant corresponds to the common inside area of the polypeptides which is less conformation-dependent and therefore common to all four polypeptides.
Animals↗
CH2 and CH3 domain deleted IgG1 paraproteins inhibit differently Fc receptor mediated binding and cytolysis.
Domain deleted paraproteins are suitable tools to study the interaction between IgG domains and Fc receptor (FcR) binding sites. The effect of the C gamma 2 or C gamma 3 domain deleted paraproteins was compared on antibody dependent cellular cytotoxicity (ADCC) and on FcR mediated rosette formation. The C gamma 2 domain deleted paraprotein (TIM) had no significant effect on lymphocyte or monocyte mediated ADCC, while the C gamma 3 domain deleted paraprotein (SIZ) inhibited both types of cytotoxicity. FcR dependent rosette formation was also inhibited by SIZ but TIM was ineffective. The data further confirm our previous results suggesting a significant role of C gamma 2 domain in the transfer of killing signal in ADCC and that of C gamma 3 domain in the high affinity binding to lymphocyte FcR.
Antibody-Dependent Cell Cytotoxicity↗
The immunomodulatory effect of human IgG Fc fragments on the oxazolone-specific immune response of high and low responder mice.
Intravenous injection of human IgG Fc fragments in mice resulted in the stimulation or inhibition of an oxazolone-specific antibody response depending on the schedule of Fc fragment injection. High and low responder mice for oxazolone were injected with Fc fragments according to two protocols: either on the day of oxazolone priming, or together with the oxazolone boost, and the isotype composition of oxazolone-specific antibodies was analysed by solid phase radioimmunoassay. We found the primary and secondary anti-oxazolone IgM levels increased in all instances, irrespective of the schedule of Fc fragment treatment. In contrast, the oxazolone-specific IgG production was increased only if Fc fragments were injected at the time of antigen priming. Injection of Fc fragments together with a secondary injection of oxazolone resulted in the inhibition of oxazolone-specific IgG production. Both stimulation and inhibition of oxazolone-specific antibodies were more pronounced in the low responder C57BL/6 mice strain.
Animals↗
The influence of new thymopoietin derivatives on the immune response of inbred mice.
The immunomodulatory activities of new synthetic thymopoietin derivates TP4 (Arg-Lys-Asp-Val) and TP3 (Arg-Lys-Asp) have been compared to those of TP5 (Arg-Lys-Asp-Val-Tyr) which exhibits most of the biological activity of the native hormone and probably represents the active site. Both TP4 and TP3 are shown to exert similar immunomodulatory activities to TP5 affecting both humoral and cellular responses. Primary and secondary antibody responses of high responder mice were enhanced whilst the intensity of DTH reactivity was decreased. The effect on humoral immunity was particularly apparent following administration of TP4 or TP3 to mice undergoing primary antibody responses following immunization with sub-optimal doses of antigen or suppression by CY treatment. Administration of peptide(s) elicited DTH responses in mice previously shown to exhibit genetically determined unresponsiveness: in these animals antibody responses were not modulated. The data may be interpreted that the tetra- and tri-peptide representing the N-terminal sequence of TP5 possess immunomodulatory activity which is in many aspects similar to that of TP5. The experimental systems and protocols employed are shown to be appropriate for investigating the effect(s) of potential immunomodulators on humoral and cellular immunity.
Adjuvants, Immunologic↗
Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic resonance and sedimentation studies.
The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results.
Actins↗