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PubMed1994

Influence of solid-phase antigen in competition enzyme-linked immunosorbent assays (ELISAs) on calculated antigen-antibody dissociation constants.

The competition ELISA method described in 1985 by Friguet and colleagues has frequently been used to determine dissociation constants (KD) of antigen-antibody reactions. Subsequently Stevens suggested a correction for the bivalency of IgG. In either case, the KD is assumed to vary only with the composition of competing fluid-phase antigen and consequently should not be affected by the solid-phase antigen. However, during the course of experiments defining epitopes to the V3 loop of human immunodeficiency virus type 1, both the composition and density of solid-phase antigen were capable of significantly influencing the calculated values. With one solid-phase antigen, the calculated KD was a function of antigen density. With a second solid-phase antigen, the calculated KD did not vary with the density. With the latter antigen or with low densities of the former, KDs calculated using the Stevens correction for bivalency were close to the estimate obtained by a radiolabeled peptide precipitation assay. Accordingly, since the density and composition of solid-phase antigen may alter the KD calculated from competition ELISAs, such estimates should be confirmed by a more readily interpretable immunological method.

Amino Acid Sequence↗
PubMed1992

A kinetic model of the agglutination process.

We propose a kinetic model of the aggregation process in a system consisting of two different types of particles. Aggregating particles (cells) are polyvalent and bear on the surface a huge number of binding sites for the other type of particles, ligands. The ligand is bivalent and has two identical active sites for binding to cells. The cross-linking of the cells by the ligands causes the aggregation phenomenon called agglutination. We obtained the analytical solution of this model task describing the time dependence of the aggregate mean size versus the composition of the system. The comparison of the analytical solution with the experimental data for the agglutination of bacterial cells by bivalent antibodies shows that the main factors affecting agglutination were correctly taken into account.

Bacterial Adhesion↗
PubMed1975

Induction of translocations by cyclophosphamide in different germ cell stages of male mice: cytological characterization and transmission.

Cytological and fertility tests were performed in F1 male mice derived from different germ-cell stages of male parents treated with cyclophosphamide (350 mg/kg body weight). The objectives of the present experiment were: (I) to determine the sensitivity of the male germ-cell stages to the induction of translocations by the compound, and (2) to characterize translocation configurations in F1 and F2 males, in order to obtain information about the pattern of chromosome breakage induced and its transmission to subsequent generations. Of 508 F1 males studied, 39 were partially sterile. The group of males conceived 8-21 days after treatment contained by far the highest proportion of partially sterile animals (30%). It was also the only group in which totally sterile animals (11%) were found. Of 25 semisterile males from this group, 24 gave evidence of translocations when spermatocytes were scored at diakinesis. The translocation frequencies in F1 derived from treated spermatozoa and spermatocytes were 14 and 1%, respectively. No translocations were detected cytologically in 6 semisterile males derived from treated spermatogonial stages. These results indicate that spermatid stages are especially sensitive to the mutagenic action of cyclophosphamide. In 21 of the 31 semisterile translocation males (68%), the majority of the spermatocytes contained 18 bivalents plus a ring-of-four configuration, indicating that both breakpoints were relatively centrally located; and in several of these males, the frequency of cells with rings was close to 100%. In another 9 F1 males (29%) the predominant multivalent configuration was a chain-of-four, indicating one of the breakpoints to be relatively more terminally located; and in one male (3%), the majority of cells had two unequal bivalents, indicating both breakpoints to be fairly close to the ends of the chromosomes involved. Determination of centromere positions by the use of C-banding showed that chain-of-four configurations in any one male were predominantly of a given type..

Animals↗
PubMed1984

The synaptonemal complexes of Caenorhabditis elegans: pachytene karyotype analysis of the rad-4 radiation-sensitive mutant.

In the rad-4 mutant of C. elegans there is a specific increase in the number of 'Disjunction Regulator Regions' (DDR) present on the synaptonemal complexes (SC) in pachytene nuclei. These DRRs either promote disjunction or inhibit nondisjunction of the X-chromosome as evidenced by the 10-fold decrease in the rate of X-chromosome nondisjunction as compared to the wild-type. The structure of the tripartite SC is normal, thus, the decrease in the rate of X-chromosome nondisjunction in the rad-4 mutant is not related to the structure of the SC but may be related to the number of DRRs. Other changes are also associated with the sensitivity to irradiation, i.e. the pachytene nuclear morphology is altered such that nuclei and nucleoli are 50% the size of wild-type. In addition, the autosomal: X-chromosome size ratio is reduced in the rad-4 mutant. That there are six SCs confirms n = 6 in this mutant and of these six SCs three can be identified: (1) the XX bivalent, SC No. 1, is the shortest and pairs synchronously with the autosomes; (2) the longest bivalent, SC No. 6, carries the nucleolar organizer region at one extreme end; and (3) SC No. 5 has two DRRs located approximately one micron apart from each other.

Animals↗
PubMed1981

Cortisol protection of the granulocyte response to isoproterenol during an in vitro influenza virus incubation.

Isolated human polymorphonuclear leukocytes (PMNs) release beta glucuronidase (BG) lysosomal enzyme during incubation with complement-activated zymosan particles. In asthma, isoproterenol (ISO) and histamine (HIS) inhibition of PMN lysosomal enzyme release is impaired while the prostaglandin E1 (PGE1) response is normal. During a respiratory infection provoking asthma, the PMN response to ISO is further impaired. The PMN response to ISO, HIS, and PGE1 is similarly impaired following an in vitro incubation with a live influenza bivalent (A + B) vaccine. In the following study, granulocytes were isolated from normal (n = 10) and asthma patients (n = 29). The isolated cells were incubated with the bivalent influenza vaccine (1 PMN:1 egg infective dose50) for 30 min. Following this in vitro virus incubation, granulocytes from normal and asthma patients had an imparied response to ISO, HIS, and PGE1. Granulocytes from asthma patients having a history of wheezing with respiratory illnesses did not have a greater impairment in the agonist response following virus incubation than normals or allergic asthma patients. However, in asthma patients (n = 6) receiving prednisone (10 to 20 mg/day), the in vitro vaccine incubation did not impair the PMN ISO response. A similar protective effect was found if granulocytes were incubated with hydrocortisone (1,4 X 10(-4) M) prior to exposure to the influenza vaccine.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1981

Kinetochore structure and its role in chromosome orientation during the first meiotic division in male D. melanogaster.

An electron microscopic investigation of kinetochore structure during the first meiotic division in male Drosophila melanogaster is presented. The data suggest that the structure that is responsible for initial microtubule attachment and chromosome orientation is a single, bilaminar hemisphere on each half-bivalent. Following the initial attachment this structure undergoes morphogenesis to a double-disc structure that reflects the underlying duality of sister chromatids in the half-bivalent. Thus these data support Darlington's idea that sister chromatids disjoin to the same spindle pole because they share a single kinetochore. Additionally, these data suggest that the meiotic mutations ord and mel-S332 sometimes cause premature "doubling" of the kinetochore region though, as discussed, possibly for a trivial reason.

Anaphase↗
PubMed1989

Interaction of meningococcal group B monoclonal antibody and its Fab fragment with alpha 2-8-linked sialic acid polymers: requirement of a long oligosaccharide segment for binding.

Mouse monoclonal IgG2a antibody (735D4) and other antibodies to the capsular polysaccharide of group B meningococci have been shown to require an unusually long segment of the alpha 2-8-linked N-acetylneuraminic acid polymer for binding. This property may be due to a conformational nature of the polysaccharide epitope recognized, or alternatively due to the requirement of bivalent binding of the antibody to the polysaccharide. In order to study the binding requirements, Fab fragments were prepared from the monoclonal antibody and their binding to alpha 2-8-linked sialic acid polymers of different lengths was studied. Both the intact antibody and its Fab fragment bound to sialic acid poly- and oligomers to similar extents, the critical chain length being about 10 sialyl units for both molecules. This excluded bivalency as the explanation for the requirement of a long oligosaccharide segment for binding. Although the binding was enhanced with increasing chain length, the first 10 monosaccharides were calculated to contribute to more than 90% of the total binding energy. This is in agreement with an oligosaccharide segment with defined conformational epitope binding to the antibody combining site. The antibody preparations also bound polysialic acid containing glycopeptides isolated from developing human and rat brain, suggesting, in quantitative binding assay, an average chain length of 10 or more sialic acid residues. The interaction of the antibody with both the bacterial and the tissue derived polysialic acids suggests that the conformational epitope critical for the interaction is formed by both classes of compounds.

Animals↗
PubMed1993

Monoclonal antibodies to HIV-1 p24 core protein include pairs which exhibit synergistic binding.

Gamma and kappa chain cDNAs from four mouse monoclonal antibodies (mAbs) which bind three different sites on the core antigen (p24) of HIV-1 have been cloned and their V-region sequences determined. These mAbs are part of a larger group of seven anti-p24 mAbs analyzed in simultaneous competition assays with HIV-1 lysate as antigen and in protein blotting experiments using 10 carboxy-terminal truncations of a p24 fusion protein. One mAb, BB128, recognizes the p24 loop sequence EAAEWDRVHP and enhances the binding of two other mAbs (BI1777 and BI1279) when tested pairwise in simultaneous competition assays. The two monoclonals enhanced by BB128 recognize different antigenic sites on p24, with BI1777 binding to carboxy-terminal sequences and BI1279 to amino-terminal residues. In the pairwise assays, mAb BI1279 also acts as enhancing antibody for BI1777, as does mAb BB328, which recognizes residues in the central region of p24. Since aggregated p24 monomers form the HIV-1 capsid, p24 is a multivalent antigen in HIV-1 lysate. It seems likely, therefore, that synergistic binding of mAb pairs to p24 is effected by bivalent binding of the enhancing mAb stabilizing a conformation favorable for bivalent binding of the enhanced mAb.

Amino Acid Sequence↗
PubMed1994

Human/mouse chimeric monoclonal antibodies with human IgG1, IgG2, IgG3 and IgG4 constant domains: electron microscopic and hydrodynamic characterization.

The unique structure of the human IgG3 constant region with its greatly extended hinge can clearly be seen in electron micrographs, which compare a series of recombinant proteins with the same murine anti-dansyl variable domain but constant domains from human IgG1, IgG2, IgG3 and IgG4. The hinge region of IgG3 was found to be very long, with some measurements extending to 100 A. It exhibited considerable flexibility allowing the Fc to be displaced far toward either side. Upon addition of bivalent hapten, all of the monoclonal antibodies formed complexes. IgG1, IgG3 and IgG4 formed circular dimers, composed of two antibodies forming a ring-shaped complex, presumably through the binding of two bivalent haptens. IgG2, on the other hand, showed a distribution of complexes which was noticeably different from the other subclasses. Some circular dimers, some linear dimers and a large amount of monomer were seen. This was interpreted in terms of an energy barrier to ring closure arising from the orientation of the Fab arms of IgG2 probably leading to linear dimers as the predominate complex seen with the analytical ultracentrifuge. A substantial number of these dimers probably dissociated upon dilution for examination in the electron microscope. The distribution of the angles between the Fab arms of the monoclonal antibodies forming the circular dimers has been measured for the different subclasses. Most were open at wide angles (> 100 degrees) but some formed very shallow angles, with the Fab arms being nearly parallel to each other. The free energy for this transition was calculated from the ratio of open/closed angles, and it was found to be proportional to the length of the upper hinge of the monoclonal antibody, in agreement with previous nanosecond depolarization results (Dangl et al., Eur. molec. Biol. Org. J. 7, 1989-1994, 1988).

Animals↗
PubMed1983

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast.

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).

Kinetics↗
PubMed1986

The influence of theophylline treatment on PGE2 production, PGE2 sensitivity and the distribution pattern of lymphocyte classes relative to the immune status.

In mice, theophylline reduced immune response mechanisms in case of primed cells but stimulated the reaction if priming and treatment were performed concomitantly. The reason for these bivalent reactions is unclear. We studied the effect of theophylline on prostaglandin E2 (PGE2) production by resident macrophages (M phi), on PGE2 sensitivity of thymocytes and spleen cells and on the distribution pattern of lymphocyte classes in thymus and spleen dependent on whether animals were immunized with sheep red blood cells (SRBC) or not. We found a theophylline induced decrease of PGE2 production of M phi in normal but an increase in immunized animals which was less pronounced if given concomitantly with a booster injection. The PGE2 resistance of thymocytes and spleen cells defined by the concentration of PGE2 needed for a 50% inhibition of PHA-P induced transformation was considerably increased by theophylline in normal animals. If given concomitantly with primary sensitization, theophylline equally increased PGE2 resistance but diminished it if applied together with a booster injection. This increase or decrease of PGE2 resistance ran parallel with a decrease or increase of the relative number of Lyt 2+ cells (suppressor T cells = Ts) in the thymus and in the spleen of theophylline treated animals. These results demonstrated that the bivalent effect of theophylline on immune response dependent on the immune status could predominantly be explained by its modulating effect on PGE2 sensitivity of thymocytes and spleen cells together with a corresponding shift in the number of Ts-cells, whereas the influence on PGE2 production by M phi seemed less important.

Animals↗
PubMed1993

A comparative study of the immune responses of sheep against foot-and-mouth disease virus types Asia-1 and O PEG-concentrated aluminium hydroxide gel and oil-adjuvanted vaccines.

Foot-and-mouth disease (FMD) vaccines prepared against types Asia-1 and O PEG-concentrated and adjuvanted with aluminium hydroxide gel were compared with oil-adjuvanted types Asia-1 and O vaccines in sheep. The study was conducted by inoculating 0.5 ml of monovalent vaccine under laboratory conditions and 1 ml dose of bivalent vaccine in field conditions. The antibody responses were monitored by serum neutralization and ELISA tests. The results indicate that PEG-concentrated gel vaccine was of comparable immunogenicity to oil-adjuvanted vaccine over a study period of 8 weeks. No significant difference was observed in the antibody response with monovalent or bivalent vaccine. However, to substantiate the present findings and reach a final conclusion, further studies with both vaccines may have to be carried out over a longer period of time.

Adjuvants, Immunologic↗
PubMed1992

Biosynthesis of marsupial milk oligosaccharides. II: Characterization of a beta 6-N-acetylglucosaminyltransferase in lactating mammary glands of the tammar wallaby, Macropus eugenii.

Tammar wallaby (Macropus eugenii) mammary glands contain a UDP-GlcNAc:Gal beta 1----3Gal beta 1----4Glc beta 1----6-N-acetylglucosaminyltransferase (GlcNAcT) whose activity has been characterized with respect to the effect of pH, apparent Km for acceptor, effects of bivalent metal ions, acceptor specificity and identity of products. The enzyme did not show an absolute requirement for any bivalent metal ion but its activity was increased markedly by Mg2+, Ca2+ and Ba2+ and, to a lesser extent, by Mn2+. When Gal beta 1----3Gal beta 1----4Glc was used as acceptor, the product was Gal beta 1----3[GlcNAc beta 1----6]Gal beta 1----4Glc. With Gal beta 1----3Gal beta 1----3Gal beta 1----4Glc as acceptor, the product was shown, by 1H-NMR spectroscopy and exo-beta-galactosidase digestion, to be a novel pentasaccharide with the structure Gal beta 1----3[GlcNAc beta 1----6]Gal beta 1----3Gal beta 1----4Glc, suggesting that the enzyme recognises the non-reducing end of the acceptor substrate, rather than the reducing end.

Animals↗
PubMed1996

ATP-diphosphohydrolase activity in rat renal microvillar membranes and vascular tissue.

Ecto-nucleotidases may have a role in the regulation of purinoceptor-mediated responses. ATP-diphosphohydrolase or apyrase has been described as an ecto-nucleotidase, which is characterized by a low specificity for its substrates and bivalent cations. The aim of this work was to demonstrate the presence of apyrase as an ecto-enzyme in the rat kidney. ATPase-ADPase activities of the renal microvillar membrane preparation, which correspond to "right side out' membranes, were characterized. The detection of ATP-diphosphohydrolase in the renal vasculature was done through perfusion of isolated rat kidney. ATPase-ADPase activities of the microvillar membrane preparation and apyrase share similar kinetic properties. These include: low substrate and bivalent metal specificities and insensitivity towards inhibitors like: oligomycin, ouabain, verapamil, levamisole and Ap5A. The M(r) or native ATPase and ADPase activities was determined by the 60Co irradiation-inactivation technique being around 65 kDa for both hydrolytic activities. Immunowestern blot analysis also supports the presence of apyrase in microvilli. Perfusion of isolated rat kidney with ATP and ADP, in the presence or absence of different inhibitors or apyrase antibodies indicated the existence of this enzyme in the vascular endothelium. The identification of ATP-diphosphohydrolase as an ecto-enzyme both in microvilli and vasculature support the proposal that the enzyme may have an important role in the extracellular metabolism of nucleotides.

Adenosine Triphosphatases↗
PubMed2006

Bis-imidazoles as molecular probes for peripheral sites of the zinc endopeptidase of botulinum neurotoxin serotype A.

Botulinum neurotoxin serotype A (BoNTA) is highly toxic, and its antidote is currently unavailable. The essential light-chain subunit of BoNTA is a zinc endopeptidase that can be used as a target for developing antidotes. However, the development of high-affinity, small-molecule inhibitors of the endopeptidase is as challenging as the development of small-molecule inhibitors of protein-protein complexation. This is because the polypeptide substrate wraps around the circumference of the endopeptidase upon binding, thereby constituting an unusually large substrate-enzyme interface of 4840 angstroms2. To overcome the large-interface problem, we propose using the zinc-coordination and bivalence approaches to design inhibitors of BoNTA. Here we report the development of alkylene-linked bis-imidazoles that inhibit the endopeptidase in a two-site binding mode. The bis-imidazole tethered with 13 methylene groups, the most potent of the alkylene-linked dimers, showed 61% inhibition of the zinc endopeptidase of BoNTA at a concentration of 100 microM. The results demonstrate the presence of a peripheral binding site for an imidazolium group at the rim of the BoNTA active-site cleft. This peripheral site enables the use of the bivalence approach to improve our previously reported small-molecule inhibitors that were developed according to the zinc-coordination approach.

Binding Sites↗
PubMed2004

The effect of phospholipids on the formation of immune complexes between autoantibodies and beta2-glycoprotein I or prothrombin.

In the last decennium, it became clear that antiphospholipid antibodies found in patients with antiphospholipid syndrome (APS) are in fact antibodies against lipid-bound plasma proteins. The most frequently occurring antigens are beta2-glycoprotein I and prothrombin, although several other lipid-bound plasma proteins have been reported as antigen for antiphospholipid antibodies. Both proteins bind to anionic phospholipids, mainly phosphatidylserine, which becomes exposed at the surface of activated platelets, apoptotic cells, or cell-derived microparticles. The binding of beta2-glycoprotein I and prothrombin to these cell surfaces or to artificial lipid vesicles with comparable amounts of anionic phospholipids is rather weak. Antiphospholipid antibodies from patients are predominantly of low affinity regarding their interaction with beta2-glycoprotein I or prothrombin in solution. In the presence of a suitable phospholipid surface, however, this interaction is strongly enhanced. There is now strong evidence that formation of bivalent, trimolecular immune complexes at the lipid membrane essentially contributes to the binding of these intrinsically low affinity patient antibodies. Depending on the affinity, the epitope specificity, and the polyclonality of a particular IgG preparation, multimeric structures of lipid-bound immune complexes may form a lattice with multiple interactions on the lipid (cell) surface. It is hypothesized that the functional activity, that is, the ability of antibodies to interfere with lipid-dependent reactions, not only depends on their affinity for the antigen, but also on their ability to form multiple interconnected bivalent trimolecular complexes at the lipid (or cell) surface. It is further proposed that the rate of desorption of immune complexes may present a better indicator for the functional properties of the antibodies than the amount of adsorbed immune complexes.

Animals↗
PubMed2005

Dynamics of task sets: evidence from dense-array event-related potentials.

Prior research suggests that task sets facilitate coherent, goal-directed behavior by providing an internal, contextual frame that biases selection toward context-relevant stimulus attributes and responses. Questions about how task sets are engaged, maintained, and shifted have recently become a major focus of research on executive control processes. We employed dense-array (128-channel) event-related potential (ERP) methodology to examine the dynamics of brain systems engaged during the preparation and implementation of task switching. The EEG was recorded while participants performed letter and digit judgments to pseudorandomly-ordered, univalent (#3, A%) and bivalent (G5) stimulus trials, with the appropriate task cued by a colored rectangle presented 450 ms before target onset. Results revealed spatial and temporal variations in brain activity that could be related to preparatory processes common to both switch and repeat trials, switch-specific control processes engaged to reconfigure and maintain task set under conflict, and visual priming benefits of task repetition. Despite extensive practice and improvement, both behavioral and ERP results indicated that subjects maintained high levels of executive control processing with extended task engagement. The patterns of ERP activity obtained in the present study fit well with functional neuroanatomical models of self-regulation of action. The frontopolar and right-lateralized frontal switch effects obtained in the present study are consistent with the role of these regions in adapting to changing contextual contingencies. In contrast, the centroparietal P3b and N384 effects related to the contextual ambiguity of bivalent trials are consistent with the context monitoring and updating functions associated with the posterior cingulate learning circuit.

Adolescent↗
PubMed2004

Interaction between alginates and manganese cations: identification of preferred cation binding sites.

Algal and bacterial alginates have been studied by means of 13C NMR spectroscopy in presence of paramagnetic manganese ions in order to reveal the nature of their interaction with bivalent cations. It is found that the mannuronate blocks bind manganese cations externally near their carboxylate groups, while guluronate blocks show the capability to integrate Mn2+ into pocket-like structures formed by adjacent guluronate residues. In alternating mannuronate-guluronate blocks, manganese ions preferentially locate in a concave structure formed by guluronate-mannuronate pairs. Partial acetylation of the alginate generally reduces its capability to interact with bivalent cations, however, the selectivity of the binding geometry is conserved. The results may serve as a hint for the better understanding of the alginate gelation in presence of calcium ions.

Alginates↗