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J Gergely

Publications and source records attributed to J Gergely.

At least 55 records · Page 3Linked to original sources
PubMed1994

Structure of the troponin complex. Implications of photocross-linking of troponin I to troponin C thiol mutants.

Ca2+ regulation of vertebrate-striated muscle contraction is initiated by conformational changes in the Ca(2+)-binding protein troponin C (TnC) and subsequent changes in the interaction of TnC with the inhibitory protein TnI. We have constructed mutants of rabbit skeletal muscle TnC in which natural Cys-98 was replaced by Leu, and a single Cys residue was introduced at position 12 (TnC12) or 89 (TnC89). Cys residues of mutant TnCs were derivatized with 4-maleimidobenzophenone and photocross-linked to TnI in binary TnC.TnI complexes. After digestion with CNBr or proteases, cross-linked peptides were purified and sequenced. TnC12 cross-linked at or near TnI Met-134 in a region known to be sensitive not only to occupancy of the regulatory Ca(2+)-binding sites of TnC but also to the contractile state of the thin filament. TnC89 cross-linked to TnI(108-113) in the inhibitory region. Taken together with earlier findings, these results indicate that in the TnC.TnI complex, both domains of TnC, as well as the linker region between them, make contact with the inhibitory region of TnI. Our data also indicate that the N- and C-terminal domains of TnC interact with opposite ends of the TnI inhibitory region.

Amino Acid Sequence↗
PubMed1994

IgG isotype distribution of local and systemic immune responses induced by influenza virus infection.

The IgG isotype profile of the influenza virus-specific immune response was studied by quantitation of serum antibody (Ab) levels in correlation with the enumeration of antibody-secreting cells (ASC) detected in the lung, spleen, mediastinal lymph nodes (MLN), Peyer's patches and bone marrow (BM). Distinct isotypic patterns for serum Ab and Ab produced by cells present at or close to the site of infection were found after primary or repeated infections. An elevated number of IgM ASC was found after primary challenge in the spleen, lung and MLN. In contrast, the site of IgA and IgG production is restricted to the lung and lymph nodes draining the site of infection. In these organs IgA, IgG2a and IgG1 ASC are found as a result of primary virus infection while viral challenge induces mostly activation of IgA-producing cells and secretion of IgA to the lung lavage. In contrast, the majority (80-90%) of Ab detected in the serum belong to the IgG2a subclass and their serum level is maintained at a high level during the whole period of the response. The relative level of virus-specific serum IgG2a in correlation with the production of IgG2a Ab found predominantly in MLN and lung is highly dependent on the viral dose used for priming or challenge. As IgG2a ASC can be detected at relatively low numbers in the spleen and BM these results suggest that the production of the dominant IgG2a isotype of serum Ab occurs close to the viral challenge site. These data, however, point to distinct isotypic regulation in systemic versus local virus-specific Ab responses.

Animals↗
PubMed1994

T cell recognition of the posttranslationally cleaved intersubunit region of influenza virus hemagglutinin.

The influenza virus hemagglutinin is synthesized as a single polypeptide chain, but upon maturation it will posttranslationally be modified by a host cell related trypsin-like enzyme. The enzymatic cleavage attacks the so-called intersubunit region of the molecule giving rise to covalently linked HA1 and HA2 subunits. An I-Ed-restricted T cell epitope was identified in the highly conserved intact intersubunit region of the influenza virus hemagglutinin. T cell recognition of a 25-mer synthetic peptide comprising the intact intersubunit region does not require further processing and the elimination of the intervening Arg residue coupling the fusion peptide to the C-terminal segment of HA1 does not abolish the T cell activating capacity. The fine specificity pattern of a T cell hybridoma similar to that of the polyclonal T cell response demonstrates that a single T cell receptor is able to recognize peptides of different sizes representing not only the uncleaved but also the cleaved form of this hemagglutinin region. Based on specificity studies the epitope was localized to the C-terminal 11 amino acids of the HA1 subunit. The cross-reactivity of peptide-primed T cells with influenza virus infected antigen-presenting cells shows that fragments comprising the identified epitope of the intersubunit region can be generated as a result of natural processing of the hemagglutinin molecule. As antigen-presenting cells are lacking the enzyme which is responsible for the posttranslational modification of newly synthesized hemagglutinin molecules, the role of immature viral proteins in immune recognition is discussed.

Amino Acid Sequence↗
PubMed1994

A natural IgM antibody does inhibit polyclonal and antigen-specific IgM but not IgG B-cell responses.

Since a B-cell growth-inhibitory natural IgM antibody was identified in the culture supernatants of LPS-stimulated murine splenic B lymphocytes [11], attempts have been made to define other possible functional role(s) of this antibody. Here we show that this regulatory IgM is able to inhibit not only the proliferation of splenic B cells, but also their IgM secretion during LPS-induced polyclonal, as well as antigen (FITC-KLH)-specific antibody responses. In contrast, IgG1 production of hapten (FITC)-specific B cells neither during restimulation with LPS nor in the presence of carrier-specific T lymphocytes in vitro was affected by regulatory IgM. Therefore, whereas newly emerging naive B cells are highly susceptible, IgG-secreting B cells appear to be completely resistant to inactivation by the regulatory IgM autoantibody.

Animals↗
PubMed1994

The intersubunit region of the influenza virus haemagglutinin is recognized by antibodies during infection.

The influenza virus haemagglutinin has an important role in the infectious cycle of the virus and carries multiple B and T cell epitopes. It is synthesized as a single polypeptide chain but viral infectivity depends on its post-translational enzymatic cleavage. The cleavage site of a trypsin-like enzyme responsible for this modification is found in the most conserved intersubunit region of the molecule. In this study the role of this region in antibody recognition was investigated. Synthetic peptides comprising the intact and cleaved forms of the intersubunit segment were used to examine the specificity of virus- or peptide-induced antibodies. The immune response elicited by viral infection resulted in the appearance of antibodies capable of neutralizing the virus without interfering with its binding to the receptor. A monoclonal antibody (MoAb) of such functional properties was shown to recognize the intact intersubunit region both in the uncleaved haemagglutinin molecule and in a 25-mer synthetic peptide comprising the intact intersubunit region. Specificity and functional studies revealed the conformation-dependent recognition of the C-terminal segment of the haemagglutinin 1 subunit by this MoAb. The binding of the antibody was shown to inhibit the trypsin-mediated cleavage of the haemagglutinin molecule and the membrane fusion event. The enzymatic cleavage of the haemagglutinin was demonstrated to abolish antibody recognition of the infective virus suggesting an escape mechanism mediated by the functional destruction of this highly conserved region. The synthetic peptide corresponding to the intact intersubunit region is characterized by an ordered structure and is able to elicit an antibody response in BALB/c mice while its subfragments are nonimmunogenic. Furthermore, this peptide elicited a protective immune response demonstrated by in vivo experiments.

Amino Acid Sequence↗
PubMed1994

The influence of branched polypeptide carriers on the immunogenicity of predicted epitopes of HSV-1 glycoprotein D.

To investigate the role of synthetic polypeptide carriers in inducing an epitope-specific immune response relevant for vaccine design, peptides comprising two distinct regions of herpes simplex virus type I glycoprotein D (1-23 and 273-284) have been conjugated to the branched polypeptides with polylysine backbone, poly[L-Lys-(DL-Alam)] (AK), or poly[L-Lys-(Leui-DL-Alam)] (LAK) and to keyhole limpet haemocyanin (KLH). The magnitude, fine specificity and isotype distribution of the conjugate-, peptide-and carrier-specific antibody responses were characterized in immunized BALB/c and CBA mice. Conjugates containing the polypeptide carrier AK were the most effective in inducing HSV gD-peptide-specific antibody responses while KLH peptide conjugates resulted in conjugate-specific antibody responses without measurable peptide specificity. The efficacy of AK-peptide conjugates was verified by the dominant appearance of peptide-specific antibodies belonging to functionally efficient IgG isotopes, accompanied by low levels of carrier specific antibody responses. Preimmunization of BALB/or CBA mice with AK conjugates comprising the 1-23 or 276-284 HSV peptides resulted in prolonged survival of animals infected with a lethal dose of infectious HSV-1. The potency of these conjugates in eliciting a protective immune response shows a close correlation with the relative levels of conjugate-induced virus-specific antibodies and the neutralizing activity of sera as measured in preimmunized survivors.

Amino Acid Sequence↗
PubMed1993

The molecular switch in troponin C.

Conformational changes in troponin C (TnC) associated with Ca(2+)-induced triggering of muscle contraction are discussed in light of the model proposed by Herzberg, Moult and James (J. Biol. Chem. 261, 2638, 1986) and of our recent work on mutants of troponin C. The model involves a Ca(2+)-induced angular movement of one pair of alpha-helical segments relative to another pair of helices in the N-terminal domain. A disulfide bridge introduced into the N-terminal domain reversibly blocks the key conformational transition and the Ca(2+)-regulatory activity. Binding of troponin I (TnI) to the disulfide form of TnC is weakened owing to the blocking of its interaction with the N-terminal domain; however incorporation of the mutant into TnC-extracted myofibrils is not abolished. Introduction of a Cys residue in the C-terminal domain of TnC leads to disulfide formation between it and the indigenous Cys-98, with accompanying inhibition of regulatory activity attributable to interference with binding to TnI and, consequently, incorporation into the thin filaments. Evidence for movement of helical segments upon Ca(2+)-binding to TnC was obtained by measurements of excimer fluorescence and of resonance energy transfer with probes attached to Cys residues introduced by site-directed mutagenesis at suitable locations. Introduction of a disulfide bridge into calmodulin, another member of the super-family of Ca(2+)-binding proteins to which TnC belongs, abolishes its interaction with target enzymes. This suggests that the type of conformational change involving angular movement of helical segments that takes place in TnC is also involved in signal transmission in other Ca(2+)-dependent regulatory proteins.

Animals↗
PubMed1993

Phenylarsine oxide (PAO) blocks antigen receptor-induced calcium response and tyrosine phosphorylation of a distinct group of proteins.

Antigen receptor (AgR) crosslinking by antigens or AgR-specific antibodies induces a cascade of enzymatic events in lymphocytes which involves activation of several non-receptor tyrosine- and serine/threonine kinases, phosphatases, phospholipases, etc. Here we show data demonstrating that a thiol group-reactive protein tyrosine phosphatase (PTP) inhibitor, phenylarsine oxide (PAO), uncouples a crucial part of the signaling events induced by anti-IgM or anti-Leu-4 (CD3) in human tonsil B lymphocytes, BL41 and Daudi B cell lines and Jurkat T lymphoma cells. PAO treatment (10 microM) resulting in distinct modification of AgR-induced tyrosine phosphorylation pattern inhibited the AgR-mediated calcium response (Ca++ release and influx) of all of these cells completely. Since this treatment did not alter the cell viability and the binding capacity of the AgR crosslinking antibodies, alteration of the tyrosine phosphorylation pattern and blockage of the calcium response indicate prompt inactivation of essential signal transduction element(s).

Arsenicals↗
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↗