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Biomedical subjects

J Gergely

Publications and source records attributed to J Gergely.

At least 127 records · Page 7Linked to original sources
PubMed1986

Saturation transfer electron paramagnetic resonance study of the mobility of myosin heads in myofibrils under conditions of partial dissociation.

The rotational motion of rigidly spin-labeled myosin heads of glycerinated myofibrils as reflected in saturation-transfer EPR spectra behaves to a first approximation as though the heads consist of two populations with different rotational motions. An immobilized fraction has a correlation time (tau 2) of approximately 0.5 ms, comparable to that of spin-labeled subfragment-1 (S1) bound to thin filaments, while a mobile fraction has a tau 2 of 10 microseconds, comparable to that of the heads of purified myosin filaments. The effects of nonhydrolyzable ATP analogues, potassium pyrophosphate (PPi), or adenylyl imidodiphosphate, Ca2+, temperature, or ionic strength on the spectra can be analyzed in terms of the fraction of myosin heads immobilized by attachment to thin filaments, without requiring changes in the motion of either attached or detached heads.

Adenylyl Imidodiphosphate↗
PubMed1986

Velocity of shortening and myosin isozymes in two types of rabbit fast-twitch muscle fibers.

The fast-twitch tibialis anterior muscle of the rabbit was stimulated (10 Hz, 8 h/day for 7 wk) to cause complete transformation of the fibers from type IIb to type IIa. The velocity of unloaded shortening of permeabilized single fiber segments dissected from control and chronically stimulated tibialis anterior muscles was measured by the slack test at 20 degrees C. The myosin isozymes in these segments were separated on pyrophosphate-containing polyacrylamide gels. Peptide mapping of the myosin chain was performed on the myosin bands cut from the gels. The velocity of unloaded shortening of the IIb fibers was significantly higher (2.50 +/- 0.09 fiber length/s; n = 6) than that of the IIa fibers (1.33 +/- 0.08 fiber lengths/s; n = 6). The two groups of fibers differed with respect to their alkali light chain complement, as assessed by nondenaturing gel analyses, and with respect to their myosin heavy chain complement, as demonstrated by peptide mapping. Thus two groups of fast-twitch muscle fibers that contain distinguishable myosin isozyme contents differ in their velocities of unloaded shortening by a factor of two.

Animals↗
PubMed1986

[Comparative studies of Fc receptors for IgG on resting and activated mouse T lymphocytes using different methods].

Fc-receptors for IgG (Fc gamma R) on resting (i.e. freshly prepared) and mitogen (Con A) or alloantigen-activated mouse spleen T cells were compared using binding of different markers such as 125J-labelled immune complexes, 125J-labelled anti Fc gamma R monoclonal antibody, FITC-labelled aggr. IgG and sheep erythrocytes covered with specific antibody (EA rosetting). C3b receptors were detected by rosetting with sheep erythrocytes covered with antibody and complement (EAC rosetting). The electrophoretic mobility of the cells without or after binding of aggr. IgG was also tested. A number of differences between resting and activated T cells were found: After activation of T cells by mitogen or alloantigen, a proportion of Fc gamma R-positive cells increased two to four times. Fc gamma R number per Fc gamma R-positive cell seemed to be higher on activated then on resting cells. Fc gamma R-positive resting cells did not shed their Fc gamma R upon incubation at 4 degrees C followed by incubation at 37 degrees C, but Fc gamma R-positive activated cells shed a remarkable proportion of their Fc gamma R on the same conditions. Binding of aggr. IgG caused a decrease of electrophoretic mobility of activated but not resting cells. Fc gamma R-positive resting cells were also C3b receptor-positive, whereas Fc gamma R-positive activated cells had no detectable C3b receptors.

Animals↗
PubMed1985

Solution conformation of the C-terminal domain of skeletal troponin C. Cation, trifluoperazine and troponin I binding effects.

Proton magnetic resonance spectroscopy has been used to study the cation (Mg2+, Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.

Animals↗
PubMed1985

Troponin-C-mediated calcium-sensitive changes in the conformation of troponin I detected by pyrene excimer fluorescence.

Troponin I (TnI) from rabbit white skeletal muscle was labeled at cysteines 48 and 64 with the fluorescent reagent N-(1-pyrene)maleimide. The fluorescence spectra of pyrene-labeled TnI (pyr-TnI) exhibit peaks characteristic of pyrene in its monomeric form and an additional peak resulting from formation of excited dimers (excimers), indicating that the labeled cysteines are close together. Formation of a pyr-TnI-TnC complex in the absence of Ca2+ has little effect on the spectrum, but when Ca2+ is bound to the low-affinity sites of TnC there is a substantial decrease in excimer and a corresponding increase in monomer fluorescence. The involvement of the low-affinity sites in the Ca2+-induced effect is consistent with the fact that Mg2+ has no effect on pyrene fluorescence. On rapid mixing of the pyr-TnI-TnC complex with Ca2+ in a stopped-flow apparatus, most of the excimer decrease is complete within the instrumental dead time, indicating a rate constant k greater than 350 s-1, which is comparable to that of the conformational change in TnC resulting from Ca2+ binding to the low-affinity sites. Rapid mixing of the Mg2-TnC-pyr-TnI complex with Ca2+ yields similar results, suggesting that the type of metal ion present at the high-affinity sites has little, if any, effect on the probe. It has been suggested previously that Cys 48 and 64 are located in a TnT-binding region of TnI (Chong P.C.S. and Hodges, R.S. (1982) J. Biol. Chem. 255, 3757). Our results suggest that a Ca2+-induced structural change in the TnI-binding region of TnC could be transmitted to TnT by affecting the TnT-binding region of TnI as part of the chain of events in the regulation of muscle contraction.

Animals↗
PubMed1985

Mapping the functional topography of Fc gamma with monoclonal antibodies: localization of epitopes interacting with the binding sites of Fc receptor on human K cells.

A panel of monoclonal antibodies (mAb) specific for the C gamma 2, C gamma 3 or inter C gamma 2/C gamma 3 domain epitopes was tested for inhibition of antibody-dependent cellular cytotoxicity (ADCC) specific for anti-D IgG-coated erythrocytes. Significant inhibition of ADCC was demonstrable for some antibodies having specificity for C gamma 2 or C gamma 3 domain epitopes, while others gave no inhibition. Fab fragments of a representative C gamma 2-specific antibody (A55) and C gamma 3-specific antibody (x3a8) retained their inhibitory capacity in lymphocyte-mediated ADCC, but only A55 Fab inhibited monocyte-mediated lysis. Furthermore, the Fab portion of A55 completely abolished the complement-dependent enhancement of ADCC mediated by concanavalin A-stimulated cells, while x3a8 Fab had no effect in this system. On the other hand, x3a8 Fab inhibited the binding of anti-D IgG-sensitized erythrocytes to lymphocytes while A55 Fab did not influence this latter interaction. The results suggest that C gamma 2 domain-FcR interaction is essential for the triggering of lytic process both in lymphocyte and in monocyte-mediated ADCC, while C gamma 3 domain has no role in the latter but is responsible for the appropriate contact between effector lymphocytes and target cells. A site in the region of Lys274 appears to be critical for triggering of both lymphocyte and monocyte-mediated ADCC.

Antibodies, Monoclonal↗
PubMed1985

The influence of Igh-1 genes on the class and subclass distribution of oxazolone-specific antibodies.

Previous studies have demonstrated that the level of the oxazolone-specific antibody response induced by contact sensitization is under the control of H-2 and Igh-1-linked genes. The aim of the present study was to clarify the role of H-2 and Igh-1 genes in the regulation of antibody affinity and isotype composition of oxazolone-specific antibodies. Analysis of the antibody response to oxazolone has revealed different ratios of IgG2a and IgG2b antibodies in mice carrying the Igh-1b allele and in strains carrying alleles a, c, and e. The characteristic ratio of IgG2a and IgG2b isotypes persisted during the whole period of the primary and secondary antibody response of CBA and CBA-Igb Igh-C congenic mice. The Igh-1-linked genes influenced the isotype distribution and not the affinity of oxazolone-specific antibodies induced by contact sensitization.

Animals↗
PubMed1985

Functional cooperation of C3b-acceptors, Fc gamma-receptors and cell-surface proteases on macrophages.

Macrophages are FcR-positive cells, synthetize complement components and express proteolytic enzymes on their surface. In this paper a functional cooperation of C3b acceptor (C3bA) sites, which bind covalently nascent C3b molecules via their metastable binding site, IgG FcRs and cell surface proteases are described and the possible importance of this cooperation in regulation of immune response is discussed. It was found that isolated monocytes did not express C3bA in contrast to cultured macrophages which showed immune adherence positivity. Stimulation of macrophages resulted in enhanced expression of C3bA. C3 synthetized by macrophages was shown to be cleaved by cellular proteases which resulted in the binding of nascent C3b to C3bA. C3bA-nascent C3b interaction inhibited FcR-dependent effector functions, such as immune complex phagocytosis and antibody-dependent cellular cytotoxicity.

Animals↗
PubMed1985

Complement-dependent cellular cytotoxicity: lymphoblastoid lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte-mediated lysis in the presence of fresh human serum.

Lymphocyte-mediated lysis of cells of the Raji, Daudi, Jijoye, and Bjab lines was elevated when fresh human serum was added to the assay. A higher proportion of effector-target conjugates was observed in the presence of human serum. In similar experiments lysis of 1301, Rael, and P3HR-1 cells was unaltered. All cell lines activated the alternative pathway of complement but they varied in the expression of receptors for complement component 3 (C3) and in the ability to fix the C3 cleavage products on their membrane. The enhancement of lysis in the presence of human serum occurred only with those cells that bound C3. This characteristic was correlated to the expression of C3 receptors. Analysis of the nature of the deposited C3 was performed with Raji cells. Raji cells exposed to human serum bound C3b as indicated by the immunoadherence test. The C3b was further processed to C3bi, because the immunoadherence declined with time and conjugate formation increased with Daudi cells, which carry the C3 receptors CR2 and CR3. This suggests that in the lytic assay lymphocytes with C3bi receptors are recruited in the presence of human serum. We assume that the bridge of C3 molecules between targets and effectors increases the avidity of their interaction.

Burkitt Lymphoma↗
PubMed1985

A possible self-regulating mechanism mediated by C3b-acceptor-bound C3b generated by stimulated macrophages.

Macrophages have been shown to produce C3 and to bear Fc receptors (FcR), and besides the various C3 receptors, they possess C3b acceptors (C3bA) as well as surface proteases capable of cleaving C3. Using the immune adherence method, we demonstrated that the amount of covalently fixed (i.e., C3bA-bound) C3b is markedly increased upon cell stimulation by phorbol myristate acetate or aggregated IgG, even in the absence of C3. The enhancement of nascent C3b (C3bx) binding to C3bA on these cells could be reversed by inhibiting the process at different stages, using either cycloheximide, phenyl-methyl-sulphonyl-fluoride, salycil hydroxamic acid, or methylamine. On the basis of our present results and earlier results, we propose a self-regulatory mechanism by which activated, C3-producing macrophages cleave C3 by their surface proteases. C3bx generated in this way fixes covalently to C3bA of the producer cells, resulting in the inhibition of FcR on these cells.

Animals↗
PubMed1985

Non-covalently bound C3 enhances lysis of rabbit erythrocytes through the alternative pathway.

Rabbit red blood cells (RaRBC, 3 x 10(7)/ml PBS) were incubated with different amounts of purified human C3 at 37 degrees for 30 min and washed twice in PBS. Different amounts of normal human serum containing 2 mM Mg2+ and 5 mM EGTA were added to the C3-treated and control RaRBC. The extent of lysis was measured after a further incubation at 37 degrees for 40 min. Enhanced lysis was observed with C3-treated RaRBC as compared to control cells. The enhancing effect was dependent on the dose of C3 used for the treatment of RaRBC. Investigation the kinetics of lysis, the lag phase was observed to be significantly shorter with the C3-treated than with the control RaRBC. No enhancement was found when RaRBC were pretreated with preformed C3b fragment. KSCN-treated C3 (C3b-like C3), however, had a lysis-enhancing effect. These results suggest that noncovalently bound C3 molecules may have a role in the initiation and/or maintenance of the alternative pathway activation on activator cells.

Animals↗
PubMed1984

Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin.

The stepwise addition of Tb3+ to calmodulin yields a large tyrosine-sensitized Tb3+ luminescence enhancement as the third and fourth ions bind to the protein [Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7-12]. Since the only tyrosine residues in calmodulin are located within binding sites III and IV, these results suggest that Tb3+ binds first to sites I and II. Recent NMR studies have provided evidence that Ca2+, on the other hand, binds preferentially to sites III and IV. Kinetic studies using a stopped-flow apparatus also show that the preferential binding of Ca2+ and lanthanide ions is different. Upon rapid mixing of 2Ca-calmodulin with two Tb3+ ions, there was a small and rapid tyrosine fluorescence change, but no Tb3+ luminescence was observed, indicating that Tb3+ binds to sites I and II but not sites III and IV. When two Tb3+ ions are mixed with 2Dy-calmodulin, Tb3+ luminescence rises rapidly as Tb3+ binds to the empty sites III and IV, followed by a more gradual decrease (k = 0.4 s-1 as the ions redistribute themselves over the four sites. These results indicate that (i) both Tb3+ and Dy3+ prefer binding to sites I and II of calmodulin and (ii) the binding of Tb3+ to calmodulin is not impeded by the presence of two Ca2+ ions initially bound to the protein. Thus, the Ca2+ and lanthanide ions must exhibit opposite preferences for the four sites of calmodulin: sites III and IV are the high-affinity sites for Ca2+, whereas Tb3+ and Dy3+ prefer sites I and II.

Animals↗
PubMed1984

The nature of the trifluoperazine binding sites on calmodulin and troponin-C.

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.

Animals↗
PubMed1984

Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1.

The kinetics of tryptic breakdown of the heavy chain of chymotryptic myosin subfragment 1 (S1) according to the following scheme (where the numbers represent approximate masses in kDa) are altered at 21 degrees C by divalent (Formula: see text) cations (Me2+) and by ATP, ADP, adenosine 5'-[beta, gamma-imino]triphosphate or PPi, with or without Me2+. ATP or its analogs slow step 2 and accelerate steps 3 and 4, while Me2+ accelerates step 2. ATP and its analogs decrease the amount of a transient 27-kDa peptide [Hozumi, T. & Muhlrad, A. (1981) Biochemistry 20, 2945-2950]. We have found direct evidence for the suggestion in this reference that the 27-kDa peptide is not an obligatory precursor of the 25-kDa fragment and that ATP or ADP suppresses the formation of the larger N-terminal fragment rather than accelerates its breakdown. Cross-linking of sulfhydryl groups located in the 20-kDa fragment leads to trapping of MgADP in the N-terminal 25-kDa peptide [Wells, J.A. & Yount, R.G. (1980) Biochemistry 19, 1711-1717]; this process affects the tryptic fragmentation of S1 similarly to, but less effectively than, nucleotides. Acts-S1 formation prevents the effect of ATP on fragmentation. At 37 degrees C S1 loses ATPase activity; tryptic digestion proceeds more rapidly and the 50-kDa and 25-kDa fragments are degraded to small peptides. Nucleotides protect against the effects of higher temperature by producing conformational changes not only in the 27-kDa N-terminal portion (containing the putative nucleotide binding site) of the heavy chain of S1 but also in the 50-kDa peptide.

Actomyosin↗