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Biomedical subjects

Mark S Johnson

Publications and source records attributed to Mark S Johnson.

69 records · Page 4Linked to original sources
PubMed2002

Discrimination between agonists and antagonists by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-selective glutamate receptor. A mutation analysis of the ligand-binding domain of GluR-D subunit.

The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor indicate that the distal anionic group of agonist molecules are stabilized by interactions with an N-terminal region of an alpha-helix (helix F) in the lobe 2 ("domain 2," Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181) of the two-lobed ligand-binding domain. We used site-directed mutagenesis to further analyze the role of this region in the recognition of both agonists and antagonists by the AMPA receptor. Wild-type and mutated versions of the ligand-binding domain of GluR-D were expressed in insect cells as secreted soluble polypeptides and subjected to binding assays using [(3)H]AMPA, an agonist, and [(3)H]Ro 48-8587 (9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]triazolo[1,5-c] quinazoline-2,5-dione), a high affinity AMPA receptor antagonist, as radioligands. Single alanine substitutions at residues Leu-672 and Thr-677 severely affected the affinities for all agonists, as seen in ligand competition assays, whereas similar mutations at residues Asp-673, Ser-674, Gly-675, Ser-676, and Lys-678 selectively affected the binding affinities of one or two of the agonists. In striking contrast, the binding affinities of [(3)H]Ro 48-8587 and of another competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione, were not affected by any of these alanine mutations, suggesting the absence of critical side-chain interactions. Together with ligand docking experiments, our results indicate a selective engagement of the side chains of the helix F region in agonist binding, and suggest that conformational changes involving this region may play a critical role in receptor activation.

Baculoviridae↗
PubMed2002

Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin.

Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin-biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys(111)-->isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin-biotin technological applications.

Amino Acid Sequence↗
PubMed2002

The major conformational IgE-binding epitopes of hevein (Hev b6.02) are identified by a novel chimera-based allergen epitope mapping strategy.

A novel approach to localize and reconstruct conformational IgE-binding epitope regions of hevein (Hev b6.02), a major natural rubber latex allergen, is described. An antimicrobial protein (AMP) from the amaranth Amaranthus caudatus was used as an immunologically non-IgE-binding adaptor molecule to which terminal or central parts of hevein were fused. Hevein and AMP share a structurally identical core region but have different N-terminal and C-terminal regions. Only 1 of 16 hevein-allergic patients showed weak IgE binding to purified native or recombinant AMP. Chimeric AMP with the hevein N terminus was recognized by IgE from 14 (88%) patients, and chimeric AMP with the hevein C terminus was recognized by IgE from 6 (38%) patients. In contrast, chimeric AMP containing the hevein core region was recognized by IgE from only two patients. When both the N-terminal and C-terminal regions of hevein were fused with the AMP core, IgE from all 16 patients bound to the chimera. This chimera was also able to significantly inhibit (>70%) IgE binding to the native hevein. On the contrary, linear synthetic peptides corresponding to hevein regions in the AMP chimeras showed no significant IgE binding capacity in either enzyme-linked immunosorbent assay or inhibition enzyme-linked immunosorbent assay. These results suggest that the IgE binding ability of hevein is essentially determined by its N-terminal and C-terminal regions and that major IgE-binding epitopes of hevein are conformational. The chimera-based epitope mapping strategy described here provides a valuable tool for defining structural epitopes and creating specific reagents for allergen immunotherapy.

Adenosine Monophosphate↗
PubMed2002

Functional attributes of the phosphate group binding cup of pyridoxal phosphate-dependent enzymes.

Twenty-four structures of pyridoxal-5'-phosphate (PLP)-dependent enzymes that represent five different folds are shown to share a common recognition pattern for the phosphate group of their PLP-ligands. All atoms that interact with the phosphate group of PLP in these proteins are organized within a two-layer structure so that the first interacting layer contains from five to seven atoms and parallel with this is a second layer containing from three to seven interacting atoms. In order to identify features of the phosphate-binding site common to PLP-dependent enzymes, a simple procedure is described that assigns relative positions to all interacting atoms unambiguously, such that the networks of interactions for different proteins can be compared. On the basis of these diagrams for 24 enzyme-cofactor complexes, a detailed comparison of the two-layer structures of PLP-dependent enzymes, with both similar and different folds, was made. A majority of the structurally defined PLP-dependent proteins use the same atom types in analogous "key" positions to bind their PLP-ligands. In some instances, proteins use water molecules when a key position is unoccupied. A similar two-layer recognition pattern extends to protein recognition of at least one other, non-PLP ligand, glucosamine 6-phosphate. We refer to this three-dimensional recognition pattern as the phosphate-binding cup. In general, the phosphate-binding cup provides a very stable anchoring point for PLP. When numerous water molecules occur within the cup, however, then the phosphate group of PLP participates directly in the enzymatic reactions with inorganic phosphate replacing the water molecules of the cup. With glucosamine-6-phosphate synthase, the water molecules of the phosphate-binding cup facilitate the entry of substrate and the exit of product.

Binding Sites↗
PubMed2002

Antiferritin VL homodimer binds human spleen ferritin with high specificity.

The antiferritin variable light domain (VL) dimer binds human spleen ferritin ( approximately 85% L subunits) but with approximately 50-fold lower affinity, K(a)=4 x 10(7) x M(-1), than the parent F11 antibody (K(a)=2.1 x 10(9) x M(-1)). The VL dimer does not recognize either rL (100% L subunits) or rH (100% H subunits) human ferritin, whereas the parent antibody recognizes rL-ferritin. To help explain the differences in ferritin binding affinities and specificities, the crystal structure of the VL domain (2.8A resolution) was determined by molecular replacement and models of the antiferritin VL-VH dimer were made on the basis of antilysozyme antibody D1.3. The domain interface is smaller in the VL dimer but a larger number of interdomain hydrogen bonds may prevent rearrangement on antigen binding. The antigen binding surface of the VL dimer is flatter, lacking a negatively charged pocket found in the VL-VH models, contributed by the CDR3 loop of the VH domain. Loop CDR2 (VL dimer) is located away from the antigen binding site, while the corresponding loop of the VH domain would be located within the antigen binding site. Together these differences lead to 50-fold lower binding affinity in the VL dimer and to more restricted specificity than is seen for the parent antibody.

Amino Acid Sequence↗
PubMed2002

Determinants of antagonist binding at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit, GluR-D. Role of the conserved arginine 507 and glutamate 727 residues.

Previous structural and mutagenesis studies indicate that the invariant alpha-amino and alpha-carboxyl groups of glutamate receptor agonists are engaged in polar interactions with oppositely charged, conserved arginine and glutamate residues in the ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. To examine the role of these residues (R507 and E727 in the GluR-D subunit) in the discrimination between agonists and antagonists, we analyzed the ligand-binding properties of homomeric GluR-D and its soluble ligand-binding domain with mutations at these positions. Filter-binding assays using [3H]AMPA, an agonist, and [3H]Ro 48-8587, a high-affinity antagonist, as radioligands revealed that even a conservative mutation at R507 (R507K) resulted in the complete loss of both agonist and antagonist binding. In contrast, a negative charge at position 727 was necessary for agonist binding, whereas the isosteric mutation, E727Q, abolished all agonist binding but retained high-affinity binding for [3H]Ro 48-8587, displaceable by 7,8-dinitroquinoxaline-2,3-dione. Competition binding studies with antagonists representing different structural classes in combination with ligand docking experiments suggest that the role of E727 is antagonist-specific, ranging from no interaction to weak electrostatic interactions involving indirect and direct hydrogen bonding with the antagonist molecule. These results underline the importance of ion pair interaction with E727 for agonist activity and suggest that an interaction with R507, but not with E727, is essential for antagonist binding.

Animals↗
PubMed2002

A dissimilarity matrix between protein atom classes based on Gaussian mixtures.

MOTIVATION: Previously, Rantanen et al. (2001; J. Mol. Biol., 313, 197-214) constructed a protein atom-ligand fragment interaction library embodying experimentally solved, high-resolution three-dimensional (3D) structural data from the Protein Data Bank (PDB). The spatial locations of protein atoms that surround ligand fragments were modeled with Gaussian mixture models, the parameters of which were estimated with the expectation-maximization (EM) algorithm. In the validation analysis of this library, there was strong indication that the protein atom classification, 24 classes, was too large and that a reduction in the classes would lead to improved predictions. RESULTS: Here, a dissimilarity (distance) matrix that is suitable for comparison and fusion of 24 pre-defined protein atom classes has been derived. Jeffreys' distances between Gaussian mixture models are used as a basis to estimate dissimilarities between protein atom classes. The dissimilarity data are analyzed both with a hierarchical clustering method and independently by using multidimensional scaling analysis. The results provide additional insight into the relationships between different protein atom classes, giving us guidance on, for example, how to readjust protein atom classification and, thus, they will help us to improve protein--ligand interaction predictions. CONTACT: vira@utu.fi

Cluster Analysis↗
PubMed2002

Influence of dietary 2,4,6-trinitrotoluene exposure in the northern bobwhite (Colinus virginianus).

The risk to wildlife from exposure to the explosive, 2,4,6-trinitrotoluene (TNT) has been a concern at numerous military installations where it has been found in the soil. To date, no published data are available describing effects of TNT exposure in an avian species. Subchronic dietary exposure to TNT was therefore evaluated in a species of management concern at military installations, the northern bobwhite (Colinus virginianus). Adult male and female quail (n = 5/sex/dose) were given commercial feed containing 3,000, 1,500, 750, and 100 mg/kg TNT for 90 d following the determination of an acute lethal dose and a 14-d range finding study. Dietary TNT intake caused a dose-dependent decrease in total red blood cell counts, packed cell volume, total plasma protein, blood prolymphocytes, and blood lymphocytes. An increased trend in late apoptotic/necrotic blood leukocytic cells was also observed in TNT-exposed birds, as was hemosiderosis in the liver. With the exception of hemosiderosis, these trends were statistically significant yet of questionable biological significance. Since treatment-related responses in this preliminary study were variable, a conservative interpretation is suggested. However, since these treatments had concentrations that were a log-fold or more than doses in similar studies using mammals, these data suggest that northern bobwhite are less sensitive to oral exposures of TNT than mammals.

Animal Feed↗
PubMedDate not supplied

McQSAR: a multiconformational quantitative structure-activity relationship engine driven by genetic algorithms.

The generation of quantitative structure-activity relationships (QSARs) under the supervision of a genetic algorithm (GA) is a QSAR modeling approach used for more than a decade. In this paper we present McQSAR, an extension to the traditional GA approach to derive QSARs. McQSAR is able to use descriptors for multiple representations per compound, such as different conformers, tautomers, or protonation forms. Test runs show that the algorithm converges to a set of representations that describe the binding mode of the set of input molecules to a reasonable resolution provided that suitable descriptors-based on the three-dimensional structure-are used. Furthermore, the frequency of chance correlation was measured during multiple runs on a real-life data set using simulated linear relationship functions. The observed frequency of chance correlation, on average 0.3 +/- 0.5%, was found independent of the size of the calibration set and the number of terms in the underlying relationship function.

Algorithms↗
PubMedDate not supplied

Responses of oral 2,4,6-trinitrotoluene (TNT) exposure to the common pigeon (Columba livia): a phylogenic and methodological comparison.

Considerable concentrations of the explosive, 2,4,6-trinitrotoluene (TNT) have been found in the soil at many installations where explosives have been used, manufactured, assembled, or destroyed. To evaluate risk to avian receptors, measures of exposure are compared with a threshold level of sublethal toxicity. To date, a single feeding study has evaluated the responses of oral TNT exposure to birds with equivocal results regarding sublethal effects. The present study followed a controlled dosing regime comprising four dose groups and a control (200, 120, 70, 20, and 0 mg TNT/kg body weight [bw]-day) in the common pigeon (Columba livia) for 60 days. Overt signs of toxicity occurred with both sexes between 2 and 3 weeks of exposure. Signs included weight loss, neuromuscular effects (e.g., ataxia, tremors, etc.), and scant red feces (chromaturia). Emetic events following dosing were common and proportional to dose; however, attempts to quantify vomitus compound concentration suggests that birds were marginally successful at removing TNT following administration. Eight of 12 and 2 of 12 males and females died or were moribund in the 200 and 120 mg/kg-day groups, respectively. Changes in hematological parameters, liver, kidney, and ovary weights were related to treatment. Dose-related changes in plasma albumin and sodium concentrations were also observed. These results suggest that subchronic exposure to TNT can adversely affect the central nervous system and hematological parameters in birds. Chemical analysis of blood detected concentrations of the two primary reduction metabolites, but not parent compound, suggesting that toxicity may be due to the bioaccumulation of a toxic intermediate.

Administration, Oral↗
PubMedDate not supplied

Influence of oral 2,4-dinitrotoluene exposure to the northern bobwhite (Colinus virginianus).

Military activities associated with training, munitions manufacturing, and demilitarization has resulted in soil residues of munition compounds and their breakdown products. Two isomers of dinitrotoluene (2,4- and 2,6-) are often found in soil associated with those activities at considerable concentrations. Consequently, issues regarding the effects of exposure to birds that visit these habitats require evaluation. To provide data useful to a risk assessment approach, we followed a controlled dosing regime (gavage) using 2,4-dinitrotoluene (2,4-DNT) in the Northern Bobwhite (Colinus virginianus) for 60 days following a 14-day range-finding study and the determination of a LD50 using the up/down method. The LD50 was determined to be 55 mg/kg using corn oil as a vehicle. Individuals dosed exceeding this level were moribund or died within 60 h of exposure. Morbidity and death occurred during the 14-day range-finding study at dosing regimens of 35 and 55, but not at 15, 5, and 0.5 mg/kg-day. Compound-related morbidity/mortality occurred in the 60-day study during the first week of exposure at 25 and 15, but not at 5, 1, and 0 mg/kg-day. Overt signs of toxicity occurred with both sexes at the onset of exposure. Signs included weight loss, diarrhea, and lethargy. Dose-related changes in egg production, ovary, kidney, and brain mass, and body weight, but not feed consumption, were found. Changes in kidney mass and histological observations suggest accumulation of nitrogenous waste may be the cause of morbidity. These data suggest that oral 2,4-DNT exposures are more acutely toxic and has a different etiology than 2,4,6-trinitrotoluene in birds.

Administration, Oral↗