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Biomedical subjects

Mark S Johnson

Publications and source records attributed to Mark S Johnson.

At least 19 recordsLinked to original sources
PubMed2005

Food avoidance behavior to dietary octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) exposure in the northern bobwhite (Colinusvirginianus).

High-melting explosive (HMX; octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) is a widely utilized explosive component of munitions used by the military. Consequently, production and use through testing and training at military installations has resulted in deposition of HMX in soil. Since these areas are often used by birds, the oral toxicity of HMX exposure to northern bobwhite (Colinus virginianus) was evaluated. Attempts to determine the acute lethal dose were unsuccessful. Initially, 8 birds (1 male/1 female per dose group) were orally dosed at levels ranging from 125 to 2125 mg HMX/kg body weight. A single death at the midrange resulted in subsequent trials of oral doses up to 10,760 mg/kg body weight. Only a single death occurred at 7173 mg/kg. A subsequent 28-d feeding study was then conducted to evaluate the potential for toxicity resulting from repetitive oral exposures. Northern bobwhite were exposed to concentrations of HMX in feed of either 10000, 1000, 100, or 0 mg/kg. These exposures resulted in a clear concentration-related reduction in feed consumption and body mass. Reductions in egg production in females were correlated with changes in body mass and feed consumption. Other physiological indicators were consistent with a considerable reduction in feed intake. These results suggest that HMX concentration is responsible for intense feed aversion behavior and thus not likely a factor that would appreciably contribute to risk for wild birds at military ranges.

Administration, Oral↗
PubMed2005

Divergent pharmacological activity of novel marine-derived excitatory amino acids on glutamate receptors.

Kainate receptors show a particular affinity for a variety of natural source compounds, including dysiherbaine (DH), a potent agonist derived from the marine sponge Dysidea herbacea. In this study, we characterized the pharmacological activity and structural basis for subunit selectivity of neodysiherbaine (neoDH) and MSVIII-19, which are natural and synthetic analogs of DH, respectively. NeoDH and MSVIII-19 differ from DH in the composition of two functional groups that confer specificity and selectivity for ionotropic glutamate receptors. In radioligand binding assays, neoDH displayed a 15- to 25-fold lower affinity relative to that of DH for glutamate receptor (GluR)5 and GluR6 kainate receptor subunits but a 7-fold higher affinity for kainate (KA)2 subunits, whereas MSVIII-19 displaced [(3)H]kainate only from GluR5 subunits but not GluR6 or KA2 subunits. NeoDH was an agonist for kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in patch-clamp recordings; in contrast, MSVIII-19 acted as a potent antagonist for homomeric GluR5 receptor currents with weaker activity on other kainate and AMPA receptors. Neither neoDH nor MSVIII-19 activated group I metabotropic GluRs. Homology modeling suggests that two critical amino acids confer the high degree of selectivity between the dysiherbaine analogs and the GluR5 and KA2 subunits. In summary, these data describe the pharmacological activity of two new compounds, one of which is a selective GluR5 receptor antagonist that will be of use for understanding native receptor function and designing more selective ligands for kainate receptors.

Alanine↗
PubMed2005

Rapid turnover of c-FLIPshort is determined by its unique C-terminal tail.

The caspase-8 inhibitor c-FLIP exists as two splice variants, c-FLIP(L) and c-FLIP(S), with distinct roles in death receptor signaling. The mechanisms determining their turnover have not been established. We found that in differentiating K562 erythroleukemia cells both c-FLIP isoforms were inducibly degraded by the proteasome, but c-FLIP(S) was more prone to ubiquitylation and had a considerably shorter half-life. Analysis of the c-FLIP(S)-specific ubiquitylation revealed two lysines, 192 and 195, C-terminal to the death effector domains, as principal ubiquitin acceptors in c-FLIP(S) but not in c-FLIP(L). Furthermore the c-FLIP(S)-specific tail of 19 amino acids, adjacent to the two target lysines, was demonstrated to be the key element determining the isoform-specific instability of c-FLIP(S). Molecular modeling in combination with site-directed mutagenesis demonstrated that the C-terminal tail is required for correct positioning and subsequent ubiquitylation of the target lysines. Because the antiapoptotic operation of c-FLIP(S) was not affected by the tail deletion, the antiapoptotic activity and ubiquitin-mediated degradation of c-FLIP(S) are functionally and structurally independent processes. The presence of a small destabilizing sequence in c-FLIP(S) constitutes an important determinant of c-FLIP(S)/c-FLIP(L) ratios by allowing differential degradation of c-FLIP isoforms. The conformation-based predisposition of c-FLIP(S) to ubiquitin-mediated degradation introduces a novel concept to the regulation of the death-inducing signaling complex.

Amino Acid Sequence↗
PubMed2005

Novel CalphaNN structural motif for protein recognition of phosphate ions.

Phosphate is one of the most frequently exploited chemical moieties in nature, present in a wide range of naturally occurring and critically important small molecules. Several phosphate group recognition motifs have been found for a few narrow groups of proteins, but for many protein families and folds the mode of phosphate recognition remains unclear. Here, we have analyzed the structures of all fold-representative protein-ligand complexes listed in the FSSP database, regardless of whether the bound ligand included a phosphate group. Based on a phosphate-binding motif that we identified in pyridoxal phosphate binding proteins, we have identified a new anion-binding structural motif, CalphaNN, common to 104 fold-representative protein structures that belong to 62 different folds, of which 86% of the fold-representative structures (51 folds) bind phosphate or lone sulfate ions. This motif leads to a precise mode for phosphate group recognition forming a structure where atoms of the phosphate group occupy the most favorable stabilizing positions. The anion-binding CalphaNN motif is based only on main-chain atoms from three adjacent residues, has a conservative betaalphaalpha or betaalphabeta geometry, and recognizes the free phosphate (sulfate) ion as well as one or more phosphate groups in nucleotides and in a variety of cofactors. Moreover, the CalphaNN motif is positioned in functionally important regions of protein structures and often residues of the motif directly participate in the function of the protein.

Amino Acid Motifs↗
PubMed2005

Model structures of alpha-2 adrenoceptors in complex with automatically docked antagonist ligands raise the possibility of interactions dissimilar from agonist ligands.

Antagonist binding to alpha-2 adrenoceptors (alpha2-ARs) is not well understood. Structural models were constructed for the three human alpha2-AR subtypes based on the bovine rhodopsin X-ray structure. Twelve antagonist ligands (including covalently binding phenoxybenzamine) were automatically docked to the models. A hallmark of agonist binding is the electrostatic interaction between a positive charge on the agonist and the negatively charged side chain of D3.32. For antagonist binding, ion-pair formation would require deviations of the models from the rhodopsin structural template, e.g., a rotation of TM3 to relocate D3.32 more centrally within the binding cavity, and/or creation of new space near TM2/TM7 such that antagonists would be shifted away from TM5. Thus, except for the quinazolines, antagonist ligands automatically docked to the model structures did not form ion-pairs with D3.32. This binding mode represents a valid alternative, whereby the positive charge on the antagonists is stabilized by cation-pi interactions with aromatic residues (e.g., F6.51) and antagonists interact with D3.32 via carboxylate-aromatic interactions. This binding mode is in good agreement with maps derived from a molecular interaction library that predicts favorable atomic contacts; similar interaction environments are seen for unrelated proteins in complex with ligands sharing similarities with the alpha2-AR antagonists.

Adrenergic alpha-2 Receptor Agonists↗
PubMed2005

Integrin evolution: insights from ascidian and teleost fish genomes.

Integrins are a family of alphabeta heterodimeric receptors essential to cell adhesion in all metazoans. In humans, the family consists of 18 alpha and 8 beta subunits that combine to form 24 dimers. Here, we present phylogenetic reconstructions for the alpha and beta integrin subunits based on sequences from 24 invertebrate and vertebrate species, including the fully sequenced genomes of the ascidian Ciona intestinalis (a urochordate) and the pufferfish Takifugu rubripes (a teleost). Both genomes contain integrin alpha subunits that have the inserted alphaI domain. As for the one alphaI domain containing integrin alpha subunit discovered earlier from the ascidian Halocynthia roretzi, the Ciona alphaI domains are missing the distinctive characteristics of mammalian collagen receptors and segregate from all vertebrate alphaI domain integrins in a phylogenetic tree, forming a new subgroup of alpha subunits with alphaI domains. Each of the pufferfish alphaI domain sequences does have characteristics of the collagen receptor alphaI domains, but no leukocyte-specific alphaI domains were found in pufferfish. Comparative protein modeling suggests that several of these fish alphaI domains are structurally compatible with binding to a GFOGER sequence in a collagen triple helix.

Amino Acid Motifs↗
PubMed2005

Conserved structural, pharmacological and functional properties among the three human and five zebrafish alpha 2-adrenoceptors.

1. Zebrafish has five distinct alpha(2)-adrenoceptors. Two of these, alpha(2Da) and alpha(2Db), represent a duplicated, fourth alpha(2)-adrenoceptor subtype, while the others are orthologue of the human alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2. The zebrafish alpha(2)-adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist-stimulated [(35)S]GTPgammaS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3. The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G-protein coupling of the zebrafish and human alpha(2)-adrenoceptors, separated by approximately 350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish alpha(2)-adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40-fold affinity differences. 4. The alpha(2A) orthologues and the zebrafish alpha(2D) duplicates clustered as close pairs, but the relationships between the orthologues of alpha(2B) and alpha(2C) were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the alpha(2)-adrenoceptors, we suggest that the second extracellular loop--where significant sequence variability is located --might contribute significantly to the observed affinity differences.

Animals↗
PubMed2005

Genetic analysis of the HAMP domain of the Aer aerotaxis sensor localizes flavin adenine dinucleotide-binding determinants to the AS-2 helix.

HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur. The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure. The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level. Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain. In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding. AS-2 in Aer is not amphipathic and is predicted to be buried. Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding. The V264M residue replacement in this region resulted in an inverted response in which E. coli cells expressing the mutant Aer protein were repelled by oxygen. Bioinformatics analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis. Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites. We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain.

Adenylyl Cyclases↗
PubMed2005

Collaboration Between Mental Health Professionals and Family Physicians: A Survey of New Jersey Family Physicians.

Background: Mental health problems are frequent in primary care, and there are many barriers to their detection and treatment. Clinical research protocols that include close collaboration between mental health professionals and primary care physicians have been found to be beneficial. This study explores the opinions of community family physicians regarding mental health professionals working directly in the primary care office.Method: Members of the New Jersey Academy of Family Physicians (N = 709) were sent a 25-item questionnaire about collaboration with mental health professionals. Three mailings were sent, with a 62% response rate. The surveys were mailed between May and July 1999.Results: Of family physicians included in the analysis, 13.5% reported having an in-office mental health professional. Of those who did not, 60.2% responded that they would consider having one. Compared with physicians who would not consider having an in-office mental health professional, physicians with a mental health professional and those without an in-office mental health professional but who would consider one were statistically more likely (p < .01) to respond that an in-office mental health professional would result in increased use of mental health services, improved acceptance of referrals to mental health professionals, and improved detection and treatment of mental health problems.Conclusion: Although few family physicians have an in-office mental health professional, many more would consider this arrangement and recognize the potential benefits of such collaboration.

Journal Article↗
PubMed2005

Mental health, family function and obesity in African-American women.

CONTEXT: African-American women are disproportionately affected by obesity and its related diseases. How psychological and psychosocial factors that affect this population differ across weight categories remains poorly understood. PURPOSE: To determine whether poor mental health and family functioning are associated with obesity in African-American women. METHODS: African-American women patients aged 21-65 years were interviewed at three primary care centers. Four well-established assessment tools were used to measure general mental and physical health status, family functioning, depressive symptoms and anxiety levels. Demographics, health behaviors and family and personal histories of overweight were assessed. RESULTS: Among 113 patients, after controlling for age and parity, obese women had significantly higher anxiety levels, poorer perception of their physical health, more often were overweight as a child, had overweight parents or siblings and experienced more psychosocial problems in their family growing up, compared to overweight and normal weight women. CONCLUSIONS: The observed findings of poor mental health, perception of physical health and family function in obese African-American women support a need for clinical attention and further study.

Adult↗
PubMed2004

Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli.

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.

Amino Acid Sequence↗
PubMed2004

Construction of hevein (Hev b 6.02) with reduced allergenicity for immunotherapy of latex allergy by comutation of six amino acid residues on the conformational IgE epitopes.

Recently we have established that IgE Abs bind to conformational epitopes in the N- and C-terminal regions of the major natural rubber latex allergen, hevein (Hev b 6.02). To identify the critical amino acid residues that interact with IgE, the hevein sequence was scanned by using site-specific mutations. Twenty-nine hevein mutants were designed and produced by a baculovirus expression system in insect cells and tested by IgE inhibition-ELISA using sera from 26 latex allergic patients. Six potential IgE-interacting residues of hevein (Arg(5), Lys(10), Glu(29), Tyr(30), His(35), and Gln(38)) were identified and characterized further in detail. Based on these six residues, two triple mutants (Hdelta3A, Hdelta3B) and hevein mutant where all six residues were mutated (Hdelta6), were designed, modeled, and produced. Structural and functional properties of these combinatory mutants were compared experimentally and in silico with those of recombinant hevein. The IgE-binding affinity of the mutants decreased by three to five orders of magnitude as compared with that of recombinant hevein. Skin prick test reactivity of the triple mutant HDelta3A was drastically reduced and that of the six-residue mutant Hdelta6 was completely abolished in all patients examined in this study. The approach presented in this paper offers tools for identification and modification of amino acid residues on conformational epitopes of allergens that interact with IgE. Hevein with a highly reduced ability to bind IgE should provide a valuable candidate molecule for immunotherapy of latex allergy and is anticipated to have a low risk of systemic side effects.

Adolescent↗
PubMed2004

BODIL: a molecular modeling environment for structure-function analysis and drug design.

BODIL is a molecular modeling environment geared to help the user to quickly identify key features of proteins critical to molecular recognition, especially (1) in drug discovery applications, and (2) to understand the structural basis for function. The program incorporates state-of-the-art graphics, sequence and structural alignment methods, among other capabilities needed in modern structure-function-drug target research. BODIL has a flexible design that allows on-the-fly incorporation of new modules, has intelligent memory management, and fast multi-view graphics. A beta version of BODIL and an accompanying tutorial are available at http://www.abo.fi/fak/mnf/bkf/research/johnson/bodil.html.

Algorithms↗
PubMed2004

The collagen receptor subfamily of the integrins.

The four collagen receptor integrins, alpha1beta1, alpha2beta1, alpha10beta1 and alpha11beta1, form a structurally and functionally distinct subgroup when compared to other members of the integrin family. In this review, we discuss the structures of these receptors and their differences in collagen binding and signalling function.

Animals↗
PubMed2004

Lead accumulation in woodchucks (Marmota monax) at small arms and skeet ranges.

Increasing concern regarding the stewardship of US Army lands requires a proactive program to evaluate sites of potential risk. Small arms and upland skeet ranges are a potentially significant source of lead exposure for burrowing mammals. Woodchucks (Marmota monax) were evaluated for lead exposure in a previously used upland skeet range and a small arms range, respective to animals collected at two nearby reference locations. Soil lead concentrations collected at burrow entrances on the firing ranges were compared with blood, bone, kidney, liver, and fecal concentrations of woodchucks collected from the reference areas. No statistical differences were found in the lead concentrations in tissue between woodchucks in reference and firing ranges; concentrations of lead in liver and kidney were below detection limits. Levels in bone, blood, and feces suggest the bioavailability of lead at these various sites, although other factors (e.g., differences in foraging areas, age structure, habitat preferences, and environmental conditions) were also likely to influence exposure. Blood levels were below that which suggests toxicity. Further analysis of other ranges with higher lead concentrations and of small mammal species with smaller home ranges is recommended to further elucidate trends that could be extrapolated to other sites.

Analysis of Variance↗