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Biomedical subjects

A Gilchrist

Publications and source records attributed to A Gilchrist.

At least 19 recordsLinked to original sources
PubMed2004

Measuring entangled qutrits and their use for quantum bit commitment.

We produce and holographically measure entangled qudits encoded in transverse spatial modes of single photons. With the novel use of a quantum state tomography method that only requires two-state superpositions, we achieve the most complete characterization of entangled qutrits to date. Ideally, entangled qutrits provide better security than qubits in quantum bit commitment: we model the sensitivity of this to mixture and show experimentally and theoretically that qutrits with even a small amount of decoherence cannot offer increased security over qubits.

Journal Article↗
PubMed2001

G alpha minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation.

The C termini of G protein alpha subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a G alpha(i) peptide to bind the A(1) adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998 ) J. Biol. Chem. 273, 14912--14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of G alpha(i) could specifically inhibit downstream responses to agonist stimulation of the muscarinic M(2) receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610--6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of G alpha subunits. Minigene vectors expressing G alpha C-terminal peptides (G alpha(i), G alpha(q), G alpha(12), and G alpha(13)) or the control minigene vector, which expresses the G alpha(i) peptide in random order (G(iR)), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal G alpha minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.

Cell Line↗
PubMed2001

Automated detection of the G20210A prothrombin mutation using the LCx microparticle enzyme immunoassay.

BACKGROUND: The prothrombin mutation, a G/A transition at position 20210 in the 3' untranslated region of the prothrombin gene, is associated with an increased risk of deep venous thrombosis and obstetrical complications. Several methods have been developed to detect the mutation; however, given the increased demand for this test in risk factor assessment, the development of simple and efficient screening methods has become necessary. METHODS: We have used a rapid, sensitive, and precise method developed by Abbott Laboratories to detect the prothrombin mutation. The method employs a polymerase chain reaction (PCR) amplification and the Abbot LCx microparticle enzyme immunoassay (MEIA) for detection. This method is able to detect and identify both homozygous and heterozygous genotypes. RESULTS: Two hundred ninety-six patients with a history of deep venous thrombosis, pulmonary embolism, preeclampsia, or cardiovascular disease and 163 control patients were included in this study. The prevalence of the mutation was 5.74% in the high-risk group and 3.06% in the control group. There was complete agreement between the results from the MEIA detection with those obtained using other detection methodologies, namely standard PCR and restriction fragment length polymorphism (RFLP) analysis. CONCLUSIONS: The MEIA detection method of the prothrombin mutation represents a simple, fast, and reliable alternative to standard methods of detection and is well suited for use in routine clinical laboratories. The results of our study confirm others' studies showing a greater incidence of G20210A prothrombin gene mutation in patients with an increased risk of venous thrombosis and pulmonary embolism as well as patients with cardiovascular disease and pregnant women with preeclampsia. It reinforces the necessity of including the screening for prothrombin mutation in populations at risk.

3' Untranslated Regions↗
PubMed2001

Hodgkin's lymphoma of T-cell type: clonal association with a CD30+ cutaneous lymphoma.

The derivation of Reed-Sternberg cells in Hodgkin's lymphoma has been a subject of great interest. In most cases, Reed-Sternberg cells seem to be derived from germinal center B cells. In few sporadic cases, a T-cell origin has been shown. This article supports the concept of a T-cell derivation for rare cases of Hodgkin's lymphoma and provides evidence of a novel mechanism of pathogenesis from chronic inflammation in the skin.

Base Sequence↗
PubMed2001

Development and current functioning in adolescents with Asperger syndrome: a comparative study.

Adolescents with Asperger syndrome (AS: without delay in speech development, diagnosed according to ICD-10 clinical criteria) were compared with a group with high-functioning autism (HFA: all with delayed speech development), and a group with conduct disorder (CD). Family and genetic studies suggest that Asperger syndrome and autism form part of the same spectrum, whereas the social impairments in conduct disorder are assumed to have different origins. The aims were to explore the relationships between early speech development and other aspects of functioning in autistic disorders, and to compare autistic and nonautistic social impairments. Early and current behaviour and IQ profiles were investigated. The CD group were clearly different from both the AS and HFA groups. The AS group tended to have less severe early behavioural abnormalities than the HFA group, and were unlikely to have speech abnormalities, but other communicative, social, and restricted/ stereotyped behavioural difficulties were largely of a similar pattern to the abnormalities in the HFA group. Eighty per cent of the AS group met criteria for autism on the diagnostic algorithm associated with the Autism Diagnostic Interview-Revised. By adolescence, the AS group were reported to be as abnormal as the HFA group but in structured 1:1 interaction their conversation was better. IQ profile in the AS group showed relative strength on verbal measures, unlike the HFA group, but relatively good performance on the Block Design subtest of the WISC/WAIS was a feature of both the AS and HFA groups. The results indicate closely similar behavioural manifestations may arise by adolescence despite differences in speech development. Follow-up studies and further family investigations will be required to clarify the origins of these and other patterns of autistic development.

Adolescent↗
PubMed2000

Structural requirements for the stabilization of metarhodopsin II by the C terminus of the alpha subunit of transducin.

The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction and activation of its cognate G protein, transducin (G(t)). A C-terminal 11-amino acid peptide from transducin, G(talpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G(t). Using a combinatorial library we identified analogs of G(talpha)-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). We have made peptides with key substitutions either on the background of the native G(talpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine which amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(talpha)-(340-350) peptides for Meta II, whereas a decrease was observed following C-terminal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between the G(talpha) C terminus and light-activated rhodopsin.

Amino Acid Sequence↗
PubMed2000

Endothelial cell-surface gp60 activates vesicle formation and trafficking via G(i)-coupled Src kinase signaling pathway.

We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein G(i), and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial (125)I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11-amino acid G(alphai) carboxyl-terminal peptide inhibited endothelial (125)I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of G(alphai) with the caveolin-1, whereas dn-Src inhibited G(alphai) binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream G(i)-coupled Src kinase signaling pathway.

Animals↗
PubMed2000

Social and psychiatric functioning in adolescents with Asperger syndrome compared with conduct disorder.

Lack of standardized phenotypic definition has made outcome studies of Asperger syndrome (AS) difficult to interpret. This paper reports psychosocial functioning in 20 male adolescents with AS, defined according to current ICD-10 criteria, and a comparison group of 20 male adolescents with severe conduct disorder. Subjects were gathered from clinical referral. Evaluation used standardized interviewer rated assessments of social functioning and psychiatric morbidity. The AS group showed severe impairments in practical social functioning despite good cognitive ability and lack of significant early language delay. High levels of anxiety and obsessional disorders were found in AS; depression, suicidal ideation, tempers, and defiance in both groups. Results are compared with those from other studies. Relevance to clinical ascertainment and treatment is discussed.

Adaptation, Psychological↗
PubMed1999

Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway.

We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.

Amino Acid Sequence↗
PubMed1999

A dominant-negative strategy for studying roles of G proteins in vivo.

G proteins play a critical role in transducing a large variety of signals into intracellular responses. Increasingly, there is evidence that G proteins may play other roles as well. Dominant-negative constructs of the alpha subunit of G proteins would be useful in studying the roles of G proteins in a variety of processes, but the currently available dominant-negative constructs, which target Mg2+-binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein alpha subunits in both mediating receptor-G protein interaction and in receptor selectivity. Thus we have made minigene plasmid constructs that encode oligonucleotide sequences corresponding to the carboxyl-terminal undecapeptide of Galphai, Galphaq, or Galphas. To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M2 muscarinic receptor activated K+ channels in HEK 293 cells. The minigenes were transiently transfected along with G protein-regulated inwardly rectifying K+ channels (GIRK) into HEK 293 cells that stably express the M2 muscarinic receptor. The presence of the Galphai carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M2 muscarinic receptor. The Galphai minigene construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Galpha carboxyl peptide in random order, pcDNA-GalphaiR) had no effect on agonist-mediated M2 muscarinic receptor GIRK response. The inhibitory effects of the Galphai minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Galphaq or Galphas had no effect on M2 muscarinic receptor stimulation of the K+ channel.

Cell Line↗
PubMed1999

An anchoring theory of lightness perception.

A review of the field of lightness perception from Helmholtz to the present shows the most adequate theories of lightness perception to be the intrinsic image models. Nevertheless, these models fail on 2 important counts: They contain no anchoring rule, and they fail to account for the pattern of errors in surface lightness. Recent work on both the anchoring problem and the problem of errors has produced a new model of lightness perception, one that is qualitatively different from the intrinsic image models. The new model, which is based on a combination of local and global anchoring of lightness values, appears to provide an unprecedented account of a wide range of empirical results, both classical and recent, especially the pattern of errors. It provides a unified account of both illumination-dependent failures of constancy and background-dependent failures of constancy, resolving a number of long-standing puzzles.

Gestalt Theory↗
PubMed1999

PDZK1, a novel PDZ domain-containing protein up-regulated in carcinomas and mapped to chromosome 1q21, interacts with cMOAT (MRP2), the multidrug resistance-associated protein.

We recently reported the isolation and partial characterization of two novel proteins, MAP17 and PDZK1. Using in situ hybridization, we demonstrated that MAP17 and PDZK1 mRNAs are markedly up-regulated in human carcinomas. PDZK1, originally isolated as a protein interacting with MAP17, contains four PDZ protein-interaction domains and could potentially interact with as many as four target proteins. In this paper, we confirm the overexpression of PDZK1 in human carcinomas using a specific antibody and demonstrate the localization of the PDZK1 gene to human chromosome 1q21, a region frequently altered in neoplastic conditions. Using the yeast two-hybrid system, we have also determined that PDZK1 interacts with the carboxy-terminal portion of cMOAT (MRP2), the canalicular multispecific organic anion transporter associated with multidrug resistance. This is of particular interest because proteins containing PDZ domains are involved in the clustering and signaling pathways of membrane-associated proteins, including ion channels. Therefore, the protein cluster formed by the association of cMOAT, PDZK1, and MAP17 could play an important role in the cellular mechanisms associated with multidrug resistance, and PDZK1 may represent a new target in cancer cells resistant to chemotherapeutic agents.

ATP-Binding Cassette Transporters↗
PubMed1998

Antagonists of the receptor-G protein interface block Gi-coupled signal transduction.

The carboxyl terminus of heterotrimeric G protein alpha subunits plays an important role in receptor interaction. We demonstrate that peptides corresponding to the last 11 residues of Galphai1/2 or Galphao1 impair agonist binding to A1 adenosine receptors, whereas Galphas or Galphat peptides have no effect. Previously, by using a combinatorial library we identified a series of Galphat peptide analogs that bind rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). Native Galphai1/2 peptide as well as several analogs were tested for their ability to modulate agonist binding or antagonist-agonist competition using cells overexpressing human A1 adenosine receptors. Three peptide analogs decreased the Ki, suggesting that they disrupt the high affinity receptor-G protein interaction and stabilize an intermediate affinity state. To study the ability of the peptides to compete with endogenous Galphai proteins and block signal transduction in a native setting, we measured activation of G protein-coupled K+ channels through A1 adenosine or gamma-aminobutyric acid, type B, receptors in hippocampal CA1 pyramidal neurons. Native Galphai1/2, peptide, and certain analog peptides inhibited receptor-mediated K+ channel gating, dependent on which receptor was activated. This differential perturbation of receptor-G protein interaction suggests that receptors that act on the same G protein can be selectively disrupted.

Adenosine↗
PubMed1998

p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-alpha or FMLP stimulation.

Mitogen-activated protein (MAP) kinase-mediated signal-transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the roles of MAP kinases in neutrophils are not well understood yet. Protein phosphorylation analysis of cellular MAP kinases indicates that exposure of human neutrophils to chemotactic factor FMLP as well as granulocyte-macrophage CSF, PMA, or ionomycin rapidly induced the activation of p38 and p44/42 MAP kinases, but stimulation with inflammatory cytokine TNF-alpha triggered the activation of p38 MAP kinase only. To study the cellular functions of these MAP kinases, the inhibitor SB20358, which specifically inhibited enzymatic activity of cellular p38 MAP kinase, and the inhibitor PD98059, which specifically blocked the induced protein phosphorylation and activation of p44/42 MAP kinase in intact neutrophils, were utilized. Inhibition of the cellular p38 MAP kinase activation almost completely abolished the TNF-alpha-stimulated IL-8 production and superoxide generation of human neutrophils. In addition, the FMLP-induced neutrophil chemotaxis as well as superoxide generation were suppressed markedly by inhibiting the activation of cellular p38 MAP kinase, but not p44/42 MAP kinase. Moreover, RIA indicates that the activation of cellular p38 MAP kinase was required for the neutrophil IL-8 production stimulated by granulocyte-macrophage CSF or LPS as well as TNF-alpha, but not for that induced by PMA or ionomycin. These results demonstrate that the activation of cellular p38 MAP kinase is indispensable for the TNF-alpha- or FMLP-mediated cellular functions in human neutrophils, and suggest that p38 MAP kinase may play a different role in response to distinct stimulation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

High expression and activation of MAP kinase-activated protein kinase 2 in cardiac muscle cells.

Recently, three mammalian mitogen-activated protein (MAP) kinases, ERK, SAPK/JNK, and p38/HOG-1 have been identified, each with apparently unique signal transduction pathways. The p38 MAP kinase mediates an intracellular stress-activated signaling pathway by regulating down-stream molecules, such as MAP kinase-activated protein (MAPKAP) kinase 2. To study the tissue specificity of MAPKAP kinase 2, mRNA blots containing multiple human tissues were hybridized with a specific oligonucleotide probe corresponding to human MAPKAP kinase 2. The Northern blot analysis revealed that two mRNA species of MAPKAP kinase 2, with sizes of 4.8 and 3.3 kb, were expressed in high levels in both human heart and skeletal muscle tissues. To better understand how MAPKAP kinase 2 is regulated in myocardium, cultured rat cardiac myoblast (H9c2) cells were stimulated with heat shock, H2O2-induced oxidative stress, or phorbol ester (PMA). Enzymatic activity of cellular MAPKAP kinase 2 in the cell lysates was evaluated using an in vitro kinase assay. Exposure of H9c2 cells to heat shock or oxidative stress induced a transient increase of cellular MAPKAP kinase 2 activity, which reached its peak level within 5 min. In contrast, stimulation of H9c2 cells with PMA, a potential myocardial hypertrophic factor, induced a sustained increase of cellular MAPKAP kinase 2 activity that was detectable for over 1 h. In addition, in vitro protein phosphorylation analysis with recombinant MAPKAP kinase 2 showed that small heat shock protein (hsp25) served as a major substrate molecule for the kinase in H9c2 cells and the protein phosphorylation of cellular hsp25 was stimulated by H2O2-induced oxidative stress or PMA treatment in intact H9c2 cells. Moreover, exposure of H9c2 cells to H2O2-induced oxidative stress or PMA rapidly activated cellular p38 MAP kinase as detected by the induced protein phosphorylation of the kinase. Taken together, these results strongly suggest that MAPKAP kinase 2 may be involved in stress-activated signal transduction in myocardium.

Animals↗
PubMed1996

Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation.

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen-activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase-activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP-stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

Amino Acid Sequence↗