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D S Linthicum

Publications and source records attributed to D S Linthicum.

At least 19 recordsLinked to original sources
PubMed1992

Molecular mimicry of hepatitis B surface antigen by an anti-idiotype-derived synthetic peptide.

Monoclonal antibody 2F10 is an "internal-image" anti-idiotype (anti-id) antibody capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg). By mRNA sequencing and computer-assisted molecular modeling of monoclonal antibody 2F10, we identified a 15-amino acid region of the heavy-chain hypervariable region that has partial residue homology with sequences of the "a" determinant epitopes of HBsAg. We have established that a linear 15-mer peptide from a contiguous region on the anti-id antibody can (i) generate anti-HBsAg-specific antibodies when injected into mice, (ii) prime murine lymph node cells for in vitro HBsAg-specific T-cell proliferative responses, and (iii) stimulate in vitro human CD4+ T cells that were primed in vivo to HBsAg by natural infection with hepatitis B virus or vaccination with a commercially available HBsAg vaccine. Significantly, this peptide could also stimulate CD4+ T cells of human hepatitis B virus carriers. We conclude that a 15-mer peptide derived from the anti-id sequence can duplicate the B- and T-cell stimulatory activity of the intact anti-id antibody and the antigen that is mimicked, HBsAg.

Amino Acid Sequence↗
PubMed1992

Kinetics of haloperidol binding to monoclonal antibodies as measured by direct fluorescence quenching.

Monoclonal antibodies which bind small neurogenic ligands may mimic certain aspects of the stereospecific binding present in the natural biological receptor, and may prove useful in designing engineered protein receptors, provided their interactions are correctly understood. We report here the kinetic and equilibrium characteristics of ligand-antibody complexes of haloperidol, a dopaminergic antagonist, with three of its monoclonal antibodies as studied by fluorescence quenching techniques. These antibodies possessed moderate to high affinity constants, ranging from 10(5) to 10(9) M-1, and caused fluorescence quenching of a fluorescein-labeled haloperidol as well as quenching (40-60%) of the internal tryptophan fluorescence. The dissociation rates of the ligand from the complexes were measured at different conditions of temperature, pH and ionic strength. The results provide important information regarding the kinetic and thermodynamic parameters of the binding pockets of these antibodies.

Antibodies, Monoclonal↗
PubMed1992

Spectrofluorimetric study of the intermolecular complexation of monoclonal antibodies with the high potency sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid.

Molecular complexation between a set of five monoclonal antibodies (MAbs) and a N,N',N"-trisubstituted guanidinium sweetener (TGS) was studied by monitoring the intrinsic fluorescence of the MAbs. Changes in the emission spectral properties of the MAbs were found to be related to the location of tryptophan residues in the antibody complementarity determining regions (CDRs). Two of the MAbs, NC10.10 and NC10.8, showed fluorescence quenching and hypsochromic (blue) shifts in the emission maxima upon complexation with the TGS ligand. Experiments with three other MAbs, NC10.1, NC6.8 and NC2.3, revealed only monotonic fluorescence quenching. The association constants obtained by spectroscopic techniques for the different MAb-TGS complexes were found to be comparable with those determined using a conventional RIA. The thermodynamic parameters of the MAb-TGS complexation were also examined. The intermolecular complexation was found to be exothermic for four of the five MAbs in this study. However, MAb NC2.3 was found to be an exception, in that it was associated with a small positive enthalpic change. This type of spectrofluorimetric analysis can aid in the identification of interactive residues and molecular dynamics involved in TGS recognition by this set of MAb. Such information may prove useful in understanding the molecular recognition motifs responsible for the intense taste properties of high potency guanidine sweeteners.

Acetates↗
PubMed1992

Kinetic and energetic parameters of imipramine binding to monoclonal antibodies as measured by fluorescence spectroscopy.

Monoclonal antibodies which bind small drugs are useful for the study of the interactive forces involved in antibody-ligand complexation. Detailed understanding of these supramolecular forces requires a careful examination of structural and thermodynamic parameters of the interacting molecules. Fluorescence spectroscopy techniques are very useful in this regard. We report here, the kinetic and energetic parameters of four monoclonal antibodies made against the tricyclic antidepressant imipramine. These monoclonal antibodies were found to possess high to very high binding affinity constants, ranging from 10(7) to 10(10) M-1, and caused fluorescence quenching or enhancement of a fluorescein labelled imipramine. The dissociation rates of the fluorescent ligand from the complexes were measured at different temperatures in order to provide some insight regarding the kinetic and energetic (thermodynamic) parameters of the antibody-ligand binding interactions.

Animals↗
PubMed1991

Analysis of the binding site architecture of monoclonal antibodies to morphine by using competitive ligand binding and molecular modeling.

The structural features of mAb directed against the opiate morphine were analyzed by using competitive ligand analog-binding studies, examination of the V region amino acid sequence, and computer-aided molecular modeling of the fragment V region. The antibody response in BALB/c mice to morphine is relatively restricted, in that all of the mAb examined in this study contained the same lambda L chain and very similar H chain V regions. A three-dimensional model of the antimorphine-binding site was constructed by using computational and graphic display techniques. Each of the six complementary-determining regions was constructed by using fragment replacement methods employing canonical loop conformations of known "parent" structures. Experimental competitive ligand-binding data and theoretical modeling suggest that a charged glutamate residue at position H:50 and aromatic side chains of residues H:33W, H:47W, H:58F, H:95W, H:101iY, and L:91W are key features in ionic and hydrophobic interactions with the ligand. This study represents the first use of theoretical and experimental modeling techniques to describe the Ag-binding site of a mouse fragment V region containing a lambda L chain.

Amino Acid Sequence↗
PubMed1991

Binding of the neuroleptic drug haloperidol to a monoclonal antibody: refinement of the binding site molecular model using canonical structures.

The ligand binding site of a monoclonal antibody (185), which binds the neuroleptic drug haloperidol, has been modelled using canonical structures and energy minimization techniques. This refined modelling protocol has allowed us to predict the variable region loop conformations. Three key residues, H:50(W), H:100a(D) and L:96(Y) appear to create the basis of the electrostatic, pi-pi stacking interactions and hydrogen bonding required for the high affinity binding site characteristics present in this antibody. The use of computer-aided graphics techniques and appropriate three-dimensional modelling permits inspection of the predicted molecular recognition features of the ligand binding site.

Animals↗
PubMed1991

Inhibition of acetylcholinesterase by caffeine, anabasine, methyl pyrrolidine and their derivatives.

The inhibition of acetylcholinesterase (AChE) by caffeine, anabasine, methylpyrrolidine and several derivatives was examined. Most of the compounds had moderate inhibitory activity with I50 values in the range of 87-480 microM. The inhibition of AChE by these compounds has not been previously reported. A structural feature common to these compounds is the N-methyl determinant of the pyrrolidine ring which may be important in binding to the AChE.

Acetylcholinesterase↗
PubMed1991

Substrate inactivation of lung thromboxane synthase preferentially decreases thromboxane A2 production.

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The production of both products was inhibited by 1-benzylimidazole and furegrelate. Multiple additions of PGH2 dramatically reduced the ability of the enzyme to synthesize TXA2, but did not effect the synthesis of HHT. In addition, 1-benzylimidazole did not protect thromboxane synthase from inactivation with multiple additions of PGH2. When the enzyme was incubated with PGH2 in the presence of 1-benzylimidazole, the synthesis of TXA2 was inhibited. When the inhibitor was removed the enzyme had still been inactivated by PGH2 in the presence of 1-benzylimidazole. Thus the substrate inactivation of the enzyme does not require the production of TXA2. Our data suggests that the synthesis of TXA2 and HHT can be differentially inactivated and may occur at different sites on the enzyme.

Animals↗
PubMed1991

Monoclonal antibodies to sweet taste proteins. I. Analysis of antigenic epitopes on thaumatin by competitive inhibition assays.

Using a competitive inhibition binding immunoassay, we have examined some of the antigenic epitopes present on thaumatin, an intense sweet tasting protein from the African fruit katemfe. We have developed a library of monoclonal antibodies which react with different surface antigenic epitopes on thaumatin. Some of these monoclonal antibodies also cross-react with monellin, another unrelated sweet tasting protein. The competitive binding immunoassay examines the immunoreactivity of both solid-phase and liquid-phase monoclonal antibodies. At least six major antigenic epitopes on thaumatin were identified by our library of monoclonal antibodies. This type of competitive binding analysis may prove useful in the discovery of "sweet taste determinants" on plant proteins and in the development of tandem immunoassays for quantitation of sweet tasting proteins in plant extracts.

Antibodies, Monoclonal↗
PubMed1990

Measurement of PGH synthase activity using a pan-specific monoclonal antibody.

A pan-specific monoclonal antibody that recognizes a variety of prostaglandin moieties and does not recognize arachidonic acid or the hydroxy-eicosatetraenoic acids was used to assess the general ability of a tissue to produce prostaglandins. Although this assay does not give a quantitative measure of PGH synthase activity, it does provide a sensitive and convenient means of screening a large number of samples for prostaglandin production.

Animals↗
PubMed1989

Production and characterization of monoclonal idiotypes and anti-idiotypes for small ligands.

The procedures described in this chapter have enabled us to identify and characterize monoclonal antibodies and their respective anti-idiotypes. We have developed several different types of immunoassays which afford greater flexibility to the investigator, depending on the type of antibodies desired and the availability of labeled antigens. Use of the intrasplenic injection technique for the final booster immunization prior to the fusion protocol has enabled us to achieve more consistent results than the usual intravenous or intraperitoneal injection routes. Isoelectric focusing of tissue culture supernatant from monoclonal antibody-secreting clones can easily identify possible duplicate clones, and thereby reduces the amount of labor required for extensive characterization of a large number of clones. We have found that these techniques have enabled us to identify "sister clones" or redundancies in our collection of antiligand and anti-idiotype antibodies rapidly and accurately. These various techniques have allowed us to save much time, labor, and money in the search for specific antibodies with desired characteristics.

Animals↗
PubMed1989

Methysergide, a serotonin antagonist, does not inhibit the expression of autoimmune encephalomyelitis in the rabbit.

In view of recent interest in the potential role of vasoactive amines in the expression of experimental autoimmune encephalomyelitis (EAE) and neuritis (EAN), we set out to determine the effect of slow-release methysergide, a serotonin antagonist, on the effector phase of EAE/EAN in rabbits immunized with homologous spinal cord in Freund's adjuvant. On day 6 post-immunization (p.i.), slow-release pellets of methysergide maleate were implanted subcutaneously in graded doses 0-400 mg. At the highest dose, blood concentrations of methysergide were approximately 90 ng/ml on day 8 p.i. falling to 20 ng/ml by day 16 p.i. However, even at the highest dose of methysergide, rabbits developed typical clinical and histological signs of EAE/EAN. It is concluded that serotonergic mechanisms do not play a critical role in the effector phase of EAE/EAN in the rabbit.

Animals↗
PubMed1988

An immunohistological study of autoimmune encephalomyelitis and neuritis in the rabbit. Observations in the dorsal root ganglion using the freeze-dried paraffin-embedded tissue technique.

Previous studies of experimental autoimmune encephalomyelitis have shown that, in the central nervous system, the emigration of T-lymphocytes precedes that of mononuclear phagocytes during inflammatory lesion formation. In the present report, the formation of analogous lesions of autoimmune neuritis (EAN) was investigated in the dorsal root ganglia of rabbits immunized with homologous spinal cord in Freund's adjuvant. The relative time course of emigration of T-lymphocytes and mononuclear phagocytes into the ganglia was examined using monoclonal antibody labeling of both types of cells in serial sections of freeze-dried paraffin-embedded tissue. Results indicate that, unlike in the central nervous system, in the rabbit dorsal root ganglion T-lymphocytes and mononuclear phagocytes appear to emigrate simultaneously, as revealed by their concomitant presence in the earliest detectable lesions of EAN. It was also found that the cortical region of the rabbit dorsal root ganglion was a preferential site of EAN lesion formation, and that such lesions correlated well with the onset of clinical signs of paralysis. These results are discussed within the context of known "blood-tissue barriers" and the possible local modulation of inflammatory cell entry into regions of the nervous system.

Animals↗
PubMed1988

Analysis of idiotypic and anti-idiotypic antibodies as models of receptor and ligand.

Antibodies to small bioactive ligands and peptides may mimic the binding characteristics of the natural receptor; in turn, the anti-idiotypic antibodies generated against the binding sites of such anti-ligand antibodies may mimic some aspects of small bioactive ligands and peptides. Among the several levels of investigation of such antibody-receptor networks are (a) the quantitative structure-activity relationships of ligand binding to antibody as compared with natural receptor; (b) the molecular modeling of antibody-receptor binding sites and the genomic basis for such structures; and (c) the characteristics of the molecular mimicry exhibited by "mimetopes" on anti-idiotypic antibodies. To illustrate the analysis encountered at each of these levels, we discuss here antibody and anti-idiotypic systems that are directed to small neuroactive ligands and their receptors.

Amino Acid Sequence↗
PubMed1987

Simultaneous visualization of vascular permeability change and leukocyte egress in the central nervous system during autoimmune encephalomyelitis.

An unresolved issue in the study of demyelinating disease is whether blood-brain barrier damage is dependent upon the migration of inflammatory cells into the central nervous system (CNS). In a study of experimental autoimmune encephalomyelitis (EAE) in rabbits, a freeze-dried, paraffin-embedded tissue technique was exploited to enable (1) the immobilization of intravenously injected sodium fluorescein tracer, as an index of vascular permeability; and (2) an effective labeling by monoclonal antibodies of both T-lymphocytes and mononuclear phagocytes in "unfixed" neural tissue. Using these newly combined methods, evidence was found that increased vascular permeability in the CNS during EAE occurs concomitantly with, and not prior to, infiltration by mononuclear phagocytes.

Animals↗
PubMed1987

A freeze-dried paraffin-embedded tissue technique for immunohistochemical studies requiring unfixed tissue from the nervous system.

Immunohistochemical studies using monoclonal antibodies against epitopes in neural tissues frequently require tissue which has not been fixed in formalin. In the past, such studies have been performed on frozen (cryostat) sections despite the general loss of morphological preservation compared with paraffin sections. In the present report, a detailed, versatile method for obtaining improved morphological preservation of "unfixed" neural tissue is described which utilizes freeze-dried paraffin-embedded sections (a modified Altmann-Gersh technique). The advantages of using mercury flotation of freeze-dried sections are particularly stressed.

Animals↗