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D S Linthicum

Publications and source records attributed to D S Linthicum.

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

Histamine-induced microvascular leakage in pial venules: differences between the SJL/J and BALB/c inbred strains of mice.

The actions of histamine on pial venule leaky site formation were measured intravitally in two inbred strains of mice (BALB/c and SJL/J). Pial venules were visualized using a cranial window microscopy technique, and microvascular leaky site formation was assessed visually using a fluorescein-dextran indicator. SJL/J mice were found to be sensitive to histamine-induced leakage, whereas the BALB/c strain was refractory. Exposure to pertussis toxin enhanced the sensitivity to histamine in the SJL/J strain, but little effect was observed for BALB/c mice. However, the employment of a polymerase chain reaction (PCR) technique for the detection of mRNA for histamine H1 receptor identified receptor-specific message in isolated cerebrovascular endothelium from both strains of mice. The lack of pial responsiveness in the BALB/c mice remains unexplained. Mast cells in the dura mater were found to be more numerous in SJL/J mice than in BALB/c mice. This observation supports previous observations of strain-specific differences in CNS inflammation. The results support the concept that genetically controlled differences in vascular sensitivity and localization of CNS-associated mast cells may play important roles in the generation of vasogenic edema and inflammation in CNS trauma and disease.

Animals↗
PubMed1993

PROGEN: an automated modelling algorithm for the generation of complete protein structures from the alpha-carbon atomic coordinates.

A modelling algorithm (PROGEN) for the generation of complete protein atomic coordinates from only the alpha-carbon coordinates is described. PROGEN utilizes an optimal geometry parameter (OGP) database for the positioning of atoms for each amino acid of the polypeptide model. The OGP database was established by examining the statistical correlations between 23 different intra-peptide and inter-peptide geometric parameters relative to the alpha-carbon distances for each amino acid in a library of 19 known proteins from the Brookhaven Protein Database (BPDB). The OGP files for specific amino acids and peptides were used to generate the atomic positions, with respect to alpha-carbons, for main-chain and side-chain atoms in the modelled structure. Refinement of the initial model was accomplished using energy minimization (EM) and molecular dynamics techniques. PROGEN was tested using 60 known proteins in the BPDB, representing a wide spectrum of primary and secondary structures. Comparison between PROGEN models and BPDB crystal reference structures gave r.m.s.d. values for peptide main-chain atoms between 0.29 and 0.76 A, with a grand average of 0.53 A for all 60 models. The r.m.s.d. for all non-hydrogen atoms ranged between 1.44 and 1.93 A for the 60 polypeptide models. PROGEN was also able to make the correct assignment of cis- or trans-proline configurations in the protein structures examined. PROGEN offers a fully automatic building and refinement procedure and requires no special or specific structural considerations for the protein to be modelled.

Algorithms↗
PubMed1993

Cloned mouse cerebrovascular endothelial cells that maintain their differentiation markers for factor VIII, low density lipoprotein, and angiotensin-converting enzyme.

This communication describes a relatively novel cell culture technique for the isolation of cerebrovascular endothelial cells from three strains of inbred mice. Cerebrovascular endothelial cells were identified by their morphology, the presence of Factor VIII-related antigen and angiotensin-converting enzyme, and the uptake of acetylated low-density lipoprotein. Cloned cerebrovascular endothelial cells were found to maintain their differentiated state and diploid genotype through 15 serial passages. The morphology and growth characteristics of these cells were found to be altered when cultured on different extracellular matrices. The isolation and cloning methods described are simple and highly reproducible.

Animals↗
PubMed1993

Variable region sequence and characterization of monoclonal antibodies to a N,N',N"-trisubstituted guanidine high potency sweetener.

A library of monoclonal antibodies (mAb) was made against a trisubstituted guanidinium sweetener (N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidine acetic acid) that is 200,000 times sweeter than sucrose on a molar basis. The mAb were characterized in terms of their ligand affinities, H- and L-chain isotypes and V-region amino acid sequences. Nine of these mAb were found to have dissociation constants in the nanomolar range. The H-chain V-regions were cloned, sequenced and found to be derived from five different families (Q52, X24, J558, 7183 and 36-60). L-chain V-regions were found to be derived from three kappa families (V kappa-4/5, V kappa-19/28 and V kappa-1) and one lambda family (V lambda-1). Amino acid homologies with these family sequences ranged from 51-91% for heavy chains and 69-97% for light chains. Sequence comparisons with Ig structures solved by X-ray diffraction were made in order to identify canonical structures. Identification and localization of combining region tryptophans (L:96W and H:33W) for two mAb (NC10.8 and NC6.8) supported previous ligand-induced tryptophan fluorescence quenching observations.

Acetates↗
PubMed1993

Enhancement of histamine-induced vascular leakage by pertussis toxin in SJL/J mice but not BALB/c mice.

Pertussis toxin (PTX) from Bordetella pertussis is known to enhance inflammatory responses which involve histamine and serotonin, including cell-mediated delayed-type hypersensitivity reactions. In this study we examined the effects of PTX on histamine-modulated microvascular responses. The actions of histamine on arteriole diameter and post-capillary leaky site formation in the cremaster muscle were measured intra-vitally in two inbred strains of mice (viz. BALB/c and SLJ). In SJL mice the rate and extent of histamine-induced leaky site formation were greatly enhanced (from 8.3 to 21.0 leaky sites per 0.1 cm2) by pre-exposure to PTX. In sharp contrast, PTX did not alter histamine-induced leaky site formation in BALB/c mice. Histamine-mediated dilation in arterioles in both strains of mice were not enhanced by PTX. PTX may enhance the development of inflammatory responses by enhancing histamine-induced leaky site formation of the microvasculature.

Animals↗
PubMed1993

Monoclonal antibodies to sweet taste proteins: II. Development of two different immunoassays for thaumatin and monellin.

Two different types of immunoassays for the detection and quantitation of thaumatin and monellin, plant proteins with intense sweet taste properties, have been developed. A tandem enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of thaumatin, in which one monoclonal antibody (mAb) raised against thaumatin (TM-1-D) was used as the solid-phase "capture" antibody, while the second mAb antibody (TM-1-C) was labeled with biotin. A standard curve for the assay was obtained and had a correlation coefficient of 0.987; the detection limit of the immunoassay was 5 ng/ml. Using a single anti-monellin mAb, a competitive enzyme immunoassay (EIA) was developed for monellin. The competitive binding of biotinylated monellin and known monellin standards with mAb 4.2E was examined; this assay had a minimum detection limit of 30 micrograms/ml.

Antibodies, Monoclonal↗
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↗