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J Gergely

Publications and source records attributed to J Gergely.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of the Ca(2+)-triggered conformational transition in troponin C.

Troponin C is the Ca(2+)-binding subunit of troponin in vertebrate striated muscle. Binding of Ca2+ to troponin C is thought to induce a conformational change that triggers subsequent events in the initiation of muscle contraction. A molecular modeling study has proposed that, when Ca2+ binds to the N-terminal triggering sites, helices B and C separate from the helices D and A, thereby exposing a crucial interaction site for troponin I, the inhibitory subunit of troponin [Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644]. In the present study the question of whether this separation actually occurs is addressed directly. A mutant rabbit skeletal troponin C containing a pair of cysteines at position 12 in helix A and position 49 in the polypeptide segment linking helices B and C was created by site-directed mutagenesis. Pyrene excimer fluorescence and resonance energy transfer studies on the labeled mutant troponin C reveal a Ca(2+)-induced increase in distance between the two cysteines. Under certain assumptions, the distance increase could be estimated from the extent of energy transfer to be approximately 13 A, in good agreement with the distance increase predicted by molecular modeling. Our results provide further experimental support for the model proposed by Herzberg et al. (above).

Animals↗
PubMed1992

A trypsin-like serine protease activity on activated human B cells and various B cell lines.

We have studied the trypsin-like serine protease activity of human tonsillar B lymphocytes. The lysate of the low-density, in vivo activated B cells as well as the lysate of cells stimulated with anti-human IgM F(ab')2 show elevated trypsin-like serine protease activity compared to the resting subset as monitored by the cleavage of Tos-Gly-Pro-Arg-pNA. The cleavage is sensitive to N-tosyl-L-lysyl-chloromethyl ketone and benzamidine but not to iodoacetamide. Experiments with intact cells give similar results. The finding that the intact cells hydrolyze the substrate, while their supernatant does not, suggests that the protease activity is cell membrane associated. It is possible that C3 is a substrate of the enzyme since the activated B cells cleave C3, whereas the resting B cells do not, and also C3 inhibits the enzyme-substrate reaction. In addition to the ex vivo B cells, we studied the serine protease activity of certain well-characterized B cell lines. The results show a correlation between the phenotype and the enzyme expression of the cell lines. BL41, an Epstein-Barr virus (EBV)-negative Burkitt lymphoma line, with a resting phenotype, has low activity, while its EBV genome-carrying convertants E95-A-BL41, E95-C-BL41, EHR-A-BL41 and BL41/95 that have the phenotype of activated B cells, have high proteolytic activity. The lymphoblastoid cell line WW-1-LCL which has the phenotype of an immunoblast, has the highest serine protease activity. On the basis of the above data, we suggest that a rather tight correlation exists between the degree of activation and the appearance of serine protease(s) on the surface of human B cells.

B-Lymphocytes↗
PubMed1992

Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc gamma receptor.

In the present study 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific antibodies were compared for induction of antibody dependent lysis of NIP-derivatised red blood cells effected by pre-stimulated U937 or HL-60 cells and by K cells. The chimaeric antibodies have heavy chains corresponding to human IgG subclasses 1-4, and include site-directed mutants of IgG3 as well as the aglycosylated form of IgG3; a mouse IgG2b antibody and a site-directed mutant IgG2b were also examined. rIFN stimulated U937 or HL-60 cells express increased levels of Fc gamma R1 compared to unstimulated cells; PMA stimulated HL-60 and U937 cells express an increased level of Fc gamma R11 compared to unstimulated cells; K cells express Fc gamma R111. Using these effector cell populations and the target cells mentioned above, we have compared anti-NIP antibodies with different heavy chain constant domains for their ability to induce ADCC through human Fc gamma R1, Fc gamma R11 and Fc gamma R111. The results suggest that all three human Fc gamma receptors appear to recognise a binding site on IgG within the lower hinge (residues 234-237) and trigger ADCC via this site, but that each receptor sees this common site in a different way. The possibility that other amino acid residues also participate in the binding/triggering site(s) cannot be excluded.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1992

Down-regulation of murine B lymphocyte growth: arrest of B cells in G1 underlies immunosuppression induced by an IgM antibody.

Previous studies have demonstrated that culture supernatants of LPS-stimulated murine splenic B lymphocytes (BLPSSN) are able to inhibit the growth of freshly isolated B cells via an IgM antibody. In this work we investigated the progress of LPS-activated B lymphocytes through the cell cycle in the presence of this antibody. We found that the regulatory IgM did not affect the entry of LPS-stimulated B lymphocytes into G0*, as assessed by the increased expression of I-A antigens. Events that characterize the early G1 phase (G1A), such as cell enlargement and increased RNA synthesis, also occurred in the presence of the antibody. In contrast, events which mark the G1B phase, such as further cell enlargement and late RNA synthesis were inhibited. Moreover, a significant portion of the cells failed to incorporate [3H]thymidine and did not progress through S, G2, or M, as revealed by their DNA content. Therefore, our work points toward a well-defined stage of the early G1 phase at which the antibody inhibits the progression of B lymphocyte activation. This result shows a new insight into the mechanism of antibody-mediated down-regulation of polyclonal B cell responses.

Animals↗
PubMed1992

How immunology was won in Hungary.

To mark the occasion of the 8th International Congress of Immunology, this special issue surveys the latest findings from Hungarian immunology. Here, János Gergely, president of the Congress, traces the origins and development of immunology in Hungary and describes the joys and sorrows of being an immunologist in Hungary today.

Allergy and Immunology↗
PubMed1992

Autocrine regulation of murine B lymphocyte growth by an IgM antibody.

Culture supernatants of LPS-stimulated murine B lymphocytes are able to inhibit the growth of freshly isolated splenic B cells via an IgM antibody. The binding specificity of this IgM is not yet defined, but appears to be a B lymphocyte surface structure distinct from membrane immunoglobulin, MHC class II antigen, transferrin and Fc gamma receptors, and B220. The regulatory autoantibody allows the normal progression of early, but not late steps in the cycle of polyclonally-stimulated B lymphocytes and does not affect the increased antigen-presenting capacity of activated B cells. Therefore, this autoregulatory cycle is apparently ubiquitous and may be a major component of B lymphocyte homeostasis under physiological, as well as pathological conditions. Moreover, these findings bring into focus a possible regulating role of B lymphocytes in the humoral immune response.

Animals↗
PubMed1992

Fc gamma R-dependent regulation of the biosynthesis of complement C3 by murine macrophages: the modulatory effect of IL-6.

The effect of murine IgG isotypes on the gene expression and secretion of the third component of complement (C3) has been studied using the monocytoid cell line P388D1 and oil-elicited mouse peritoneal macrophages. It is demonstrated that the binding of IgG2a and IgG2b but not IgG1 and IgG3 augments the biosynthesis of C3 both in the presence and in the absence of the phorbol ester, phorbol myristate acetate in the case of both cell types. The multifunctional cytokine interleukin-6 (IL-6) alone reveals no effect on the gene expression of C3, but increases the effectiveness of mouse IgG2a and IgG2b. Confirming the role of Fc gamma RII, a strong up-regulation of C3 gene expression and C3 secretion was found when macrophages were cultured with the F(ab')2 fragment of the Fc gamma RII-specific monoclonal antibody 2.4G2.

Animals↗
PubMed1992

Regulation of antibody production mediated by Fc gamma receptors, IgG binding factors, and IgG Fc-binding autoantibodies.

Fc receptors (FcRs) are immunoglobulin-binding structures that enable antibodies to perform a variety of functions by forming connections between specific recognition and effector cells. Besides eliciting cytotoxicity, inducing secretion of mediators and endocytosis of opsonized particles, FcRs are involved in the regulation of antibody production, both as integral membrane proteins and as soluble molecules released from the cell surface. Most FcRs belong to the same family of proteins as their ligands (immunoglobulin superfamily). This review contains recent data obtained by use of monoclonal antibodies and cloning studies on FcRs and FcR-like molecules. The importance of fine specificity of receptor binding site(s)--that of the conformation of FcRs and their ligands in triggering signaling mechanisms--is analyzed. The regulatory function of membrane-bound and -released FcRs; the correlation between cell cycle, FcR expression, and release; as well as the possible mechanisms of these phenomena are discussed.

Animals↗
PubMed1991

Stabilization by a disulfide bond of the N-terminal domain of a mutant troponin C (TnC48/82).

The regulatory activity of troponin C is reversibly inhibited by a disulfide bridge between cysteine residues introduced by site-directed mutagenesis in positions 48 and 82 (TnC48/82) in the N-terminal domain of rabbit skeletal troponin C (sTnC; Grabarek, Z., Tan, R.-Y., Tao, T., and Gergely, J. (1990) Nature 345, 132-135). In the present work we have investigated the effects of the disulfide on structural properties of TnC48/82 monitored by CD spectroscopy and limited trypsinolysis. The CD spectra of the mutant protein in the oxidized form (oxTnC48/82) with and without Ca2+ are similar to the corresponding ones of the reduced and carboxamidomethylated form (CAMTnC48/82), indicating that the disulfide has essentially no effect on the overall secondary structure. The N-terminal domain of oxTnC48/82 is resistant to thermal unfolding, but that of CAMTnC48/82 is only slightly more stable than the corresponding domain of sTnC. In the presence of Ca2+ oxTnC48/82 is more resistant to trypsinolysis than sTnC whereas the rate of tryptic digestion of CAMTnC48/82 is the same as that of sTnC, indicating that peptide bonds adjacent to lysine residues at position 84 and 88, the sites of tryptic attack, are protected by the disulfide. The disulfide cross-linked N-terminal peptide of TnC48/82 does not bind TnI, unlike its reduced or carboxamidomethylated forms. Our data indicate that the disulfide between Cys48 and Cys82 stabilizes the structure of the N-terminal domain of TnC and blocks its ability to interact with TnI. The effects of the disulfide appear to be restricted to the N-terminal domain of TnC.

Animals↗
PubMed1991

Cross-linking of residue 57 in the regulatory domain of a mutant rabbit skeletal muscle troponin C to the inhibitory region of troponin I.

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca(2+)-dependent regulation of vertebrate striated muscle contraction. In the present study, we investigated the sites of interaction between the N-terminal regulatory domain of TnC and the inhibitory region (residues 96-116) of TnI, using a mutant rabbit skeletal TnC (designated as TnC57) that contains a single Cys at residue 57 in the C-helix. TnC57 was modified with the photoreactive cross-linker 4-maleimidobenzophenone (BP-Mal), and, after formation of a binary complex with TnI, cross-linking between the proteins was induced by photolysis. The resulting product was cleaved with CNBr and several proteases, and peptides containing cross-links were purified and subjected to amino acid sequencing. The results show that Cys-57 of TnC57 is cross-linked to the segment of TnI spanning residues 113-121. Previously, we showed that Cys-98 of TnC can be cross-linked via BP-Mal to TnI residues 103-110 (Leszyk, J., Collins, J.H., Leavis, P.C., and Tao, T. (1987) Biochemistry 26, 7042-7047). Taken together, these results demonstrate that both the C- and the N-terminal domains of TnC interact with the inhibitory region of TnI and are consistent with the hypothesis that, in a complex with TnI, TnC adopts a more compact conformation than in the crystal structure.

Amino Acid Sequence↗
PubMed1991

Modulation of type II Fc gamma receptor expression on activated human B lymphocytes.

We have monitored Fc gamma RII expression during the activation of human B lymphocytes by simultaneous analysis of monoclonal antibody (mAb) binding and EA rosetting. The expression of Fc gamma RII showed a biphasic time course. Initially, a transient increase of Fc gamma RII with no ligand-binding capacity was observed with mAb staining as early as 10 min after stimulation by the F(ab')2 fragment of anti-human IgM or phorbol 12-myristate 13-acetate and then after 3 to 24 h a decrease in the number of Fc gamma RII+ cells was seen. Trypsin-like serine protease activity also appeared in the lysate of activated B cells at this time. On the 2nd day of activation a significant enhancement of Fc gamma RII expression was observed, mainly on enlarged blast cells as monitored by both mAb and by ligand binding (EA rosette). At the same time, soluble fragments of Fc gamma RII with the ability to bind human Fc were detected in the supernatant of activated B cells, probably as a result of proteolytic cleavage. These findings suggest that activated B cells are identical with the population of mononuclear cells which shed Fc gamma R when incubated at 37 degrees C. The ability of activated but not resting B cells to release Fc gamma RII correlates with the expression of early activation markers and with the appearance of a trypsin-like serine protease activity of the same cells; thus, the release of Fc gamma RII occurs in the early G1 phase of cell cycle as a result of proteolysis. Later the release of Fc gamma RII is accompanied by the enhancement of Fc gamma RII expression before the cells reach the S phase. The fragments of cleaved Fc gamma RII had an apparent molecular mass of 33 and 14-18 kDa under nonreducing conditions, and upon reduction fragments of smaller size were observed.

Antigens, CD↗
PubMed1991

The role of C3 in the immune response.

The complement system, particularly the third component, plays an important modulatory role in the inductive phase of the immune response. As discussed here by Anna Erdei and colleagues, the picture that is emerging is that immobilized C3 split products facilitate the cooperation between immunocompetent cells and are co-stimulatory molecules in T- and B-cell activation, probably as a result of their ability to promote cell-cell adhesion. In contrast, soluble C3 products inhibit lymphocyte proliferation.

Antibody Formation↗
PubMed1991

An IgM antibody is a potent immunosuppressive agent that inhibits B cell proliferation.

The results of this study demonstrate that the culture supernatant of lipopolysaccharide (LPS)-stimulated murine spleen cells is able to inhibit the growth of freshly isolated B lymphocytes. The inhibition is specific for B cells because the suppression of the LPS, Fc-fragment of human IgG, dextran sulfate, and anti-mu induced proliferation of B, but not the Concanavalin A response of T lymphocytes could be shown. The cells producing the inhibitor do not adhere to plastic, and are Thy-1 negative but surface Ig positive, i.e. they are B lymphocytes. The regulatory substance is heat resistant, sensitive to trypsin treatment, and has a high molecular weight of approximately 1000 kDa. Moreover, it is specifically adsorbed on, and can be eluted from, anti-mu and anti-Ig immunoaffinity columns. Thus, it seems to be an IgM antibody. Non-specific effects were excluded by the ineffectiveness of poly- and monoclonal IgM proteins. IgM-IgG complexes were also excluded. Thus, these results suggest the existence of a novel IgM antibody mediated control mechanism regulating B cell growth during polyclonal activation.

Animals↗
PubMed1991

Direct correlation of parvalbumin levels with myosin isoforms and succinate dehydrogenase activity on frozen sections of rodent muscle.

Parvalbumin (PV) is a soluble Ca++ binding protein which is particularly concentrated in fast muscles of rodents. We have developed a new protocol to fix frozen sections of muscle by formaldehyde vapor, which enabled us to immunochemically stain serial frozen sections for PV. Fiber types were defined on the basis of myosin ATPase stability, and of isomyosins identified by a variety of antibodies because ATPase stability alone yielded ambiguous results in the mouse. Slow Type I fibers in mouse and rat were devoid of PV and had intermediate to high SDH levels. Fast fiber subtypes IIA, IIB, and IIX-like were defined in the mouse on the basis of the similarity of their myosin heavy chain immunoreactivity to these types in the rat. The soleus muscle was usually PV negative, but a small population of strongly PV-positive IIX-like fibers was present in the mouse. In mouse fast muscle, small diameter IIA fibers were PV negative with high SDH activity. In both mouse and rat, PV reactivities of IIB and IIX fibers were higher than those of IIA and I, whereas SDH levels of IIA, IIX, and I fibers were higher than those of IIB. Thus, PV content correlated with the type of myosin ATPase but not with SDH levels. The method described for immunocytochemistry of PV may be applicable to other highly soluble proteins.

Animals↗
PubMed1990

Inhibition of mutant troponin C activity by an intra-domain disulphide bond.

Triggering of contraction in striated muscles involves a conformational transition in the N-terminal domain of troponin C, the calcium-binding component of thin filaments. We have designed a mutant troponin C in which the key conformational transition and the calcium-regulatory activity are reversibly blocked by the formation of a disulphide bridge. Our results may be applicable to other proteins of the same family of calcium-binding proteins.

Animals↗
PubMed1990

Ca2(+)-dependent interactions between the C-helix of troponin-C and troponin-I. Photocross-linking and fluorescence studies using a recombinant troponin-C.

We have used in vitro mutagenesis to synthesize in Escherichia coli a recombinant rabbit skeletal troponin-C (designated as TnC57) in which Cys-98 was replaced with leucine, and Ala-57 in the C-helix of the N-terminal domain was replaced with cysteine. TnC57 labeled with the bifunctional photocross-linker benzophenone-4-maleimide could be photocross-linked with troponin-I in both the binary complex with troponin-I and in the ternary complex with troponin-I and troponin-T. The fluorescence lifetime of TnC57 labeled with the probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine decreased from 13.2 +/- 0.1 to 11.8 +/- 0.1 ns when Ca2+ bound to the low affinity triggering sites. Complexation with either troponin-I or both troponin-I and troponin-T resulted in significant increases in this lifetime both in the absence and the presence of Ca2+. In either the binary or the ternary complex, this lifetime increased from 15.5 to 18.0 ns upon Ca2+ binding to the low affinity sites. Complementary acrylamide-quenching studies yielded results that are consistent with the fluorescence lifetime results. Our results show that the C-helix of troponin-C interacts with troponin-I, in confirmation of recent zero-length cross-linking results (Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J.H. (1990) Biochemistry 29, 299-304). Moreover, they are in support of a model (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) in which the binding of Ca2+ to the triggering sites in the N-terminal domain of troponin-C results in the movement of the B- and C-helices away from the central helix, thereby exposing a putative hydrophobic binding site for troponin-I.

Animals↗