Search PubMed⌕ Search

Biomedical subjects

G A Thompson

Publications and source records attributed to G A Thompson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Purification of glycosylphosphatidylinositol-anchored proteins by modified triton X-114 partitioning and preparative gel electrophoresis.

Triton X-114 phase partitioning has frequently been used to obtain preparations enriched in glycosylphosphatidylinositol (GPI)-anchored proteins and other hydrophobic proteins from crude cellular homogenates. We have developed a new modification of this phase-partitioning technique which allows two distinct GPI-anchored proteins of Tetrahymena mimbres to be separated from other hydrophobic as well as hydrophilic proteins and recovered in approximately 90% yield. The unique feature of the new method is a 24-h incubation of the first Triton X-114 extract at -20 degrees C. This improves the partitioning of GPI-anchored proteins into the detergent phase while promoting the aggregation of other hydrophobic proteins. Individual GPI-anchored proteins in the detergent phase are then purified to near homogeneity by one-step preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. By substituting Triton X-114/water mixtures for the conventional SDS buffer used to collect proteins eluting from the preparative gel, the purified GPI-anchored protein can be rapidly concentrated from relatively large volumes of eluate by phase partitioning at 32 degrees C. The method is also effective in separating mammalian GPI-anchored alkaline phosphatase from other proteins. It is likely to be of general utility in characterizing the GPI anchor structures associated with nonabundant and abundant GPI-anchored proteins coexisting within the same cell type.

Cells, Cultured↗
PubMed1995

Novel aspects of the regulation of a cDNA (Arf1) from Chlamydomonas with high sequence identity to animal ADP-ribosylation factor 1.

ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identified in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [32P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.

ADP-Ribosylation Factor 1↗
PubMed1995

The actions of a range of excitatory amino acids at (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-depolarizing receptors on neonatal rat motoneurones.

Depolarizations induced by a range of amino acids including some sulphur-containing excitatory transmitter candidates were evoked from motoneurones in the neonatal rat spinal cord under conditions that precluded activation of known ionotropic glutamate receptors. The responses could be partially and differentially depressed by continuous application of several metabotropic glutamate receptor (mGluR) antagonists or by receptor desensitization with the mGluR agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD]. In most cases [the exceptions being (1S,3R)-ACPD and to a lesser extent, quisqualate], the major component of these depolarizations was resistant to antagonism by phenylglycine-derived mGluR antagonists or desensitization of (1S,3R)-ACPD-sensitive receptors. Of the excitatory responses observed with the tested agonists, those evoked by L-glutamate itself were generally the least affected by blockade of known glutamate receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1995

Differential actions of 3-(4-chlorophenyl) glutamic acid stereoisomers and L-trans-pyrrolidine-2,4-dicarboxylic acid upon L-homocysteic acid- and L-glutamic acid-induced responses from rat spinal motoneurones.

The four recently synthesized stereoisomers of 3-(4-chlorophenyl) glutamic acid (chlorpheg) were individually examined for their abilities to potentiate depolarizations of neonatal rat motoneurones evoked by L-homocysteic acid (L-HCA, 10 microM). This property had previously been observed using the racemate and is believed to be mediated by uptake inhibition. Both the (2S,3S)- and (2S,3R)- isomers were selective potentiators of L-HCA- (vs L-Glu) induced depolarizations although the (2S,3S)- isomer was more effective. The (2R,3S)- isomer had a slight but significant depressant action which could be attributed to N-methyl-D-aspartate (NMDA) receptor antagonism. Comparison of the potentiating properties of (2S,3S)- and (2S,3R)-chlorpheg with those of L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC, a L-Glu uptake inhibitor) upon L-HCA- and L-Glu-evoked responses revealed that both chlorpheg isomers (500 microM each) selectively potentiated responses evoked by L-HCA (10 microM) but had no significant effect upon those evoked by L-Glu (50 microM). On the other hand, use of tPDC at the same concentration significantly enhanced the depolarizations evoked by both amino acids, although its action on L-Glu-evoked responses was greater. It is concluded that (i) the (2S,3S)- isomer and to a lesser extent, the (2S,3R)- isomer of chlorpheg are responsible for the potentiating actions seen with the chlorpheg racemate used in previous studies and (ii) (2R,3S)-chlorpheg is a weak NMDA antagonist. The apparently selective action of (2S,3S)- and (2S,3R)-chlorpheg upon L-HCA-relative to L-Glu-induced depolarizations supports the existence of multiple excitatory amino acid uptake sites, some of which may yet be unidentified.

Animals↗
PubMed1994

A precise, rapid and sensitive in vitro assay to measure the adhesion of ovarian tumour cells to peritoneal mesothelial cells.

Ovarian cancer is the second most common gynaecological cancer in the UK, causing 2000 deaths per year. It spreads by shedding cells which attach to the mesothelial lining of the peritoneal cavity. In order to quantitatively study this interaction, a model system was developed in which mesothelial cells were cultured as monolayers in multiwell plates, and ovarian tumour cells were added that had been pre-labelled with a fluorescent dye (calcein). Synchronous interaction between the two populations was achieved by brief centrifugation at low g and the degree of attachment was measured on an automated fluorimeter after washing away the unbound cells. Using this procedure it was possible to measure tumour cell adhesion in 96 wells in 3-4 h. The reproducibility of the method was high even after short incubation times and the background absorbance was so low that the adhesion of less than a 1000 cells could be easily detected. The method works equally well for all ovarian tumour cell lines so far studied, and in preliminary experiments, it was shown that it can be used to screen for the effects of various blocking agents.

Cell Adhesion↗
PubMed1994

Characteristics of the gene that encodes acetyl-CoA carboxylase in the diatom Cyclotella cryptica.

Efforts are currently under way in several laboratories to develop renewable fuels from biological sources. Our group conducts research involving the production of lipid-derived "biodiesel" fuel from microscopic algae. Lipid accumulation in algae typically occurs during periods of environmental stress, including growth under nutrient-deficient conditions. Biochemical studies have suggested that acetyl-CoA carboxylase (ACCase), a biotin-containing enzyme that catalyzes an early step in fatty acid biosynthesis, may be involved in the control of this lipid accumulation process. Therefore, it may be possible to enhance lipid production rates by increasing the activity of this enzyme via genetic engineering. As a first step toward this objective, we have cloned the gene that encodes ACCase from the eukaryotic alga Cyclotella cryptica. This is the first time that this gene has been isolated from a photosynthetic organism. The amino acid sequence of ACCase deduced from this gene exhibits a high degree of similarity to the sequences of animal and yeast ACCases in the biotin carboxylase and carboxyltransferase domains, but less similarity exists in the biotin carboxyl carrier protein domain. Comparison of the genomic nucleotide sequence to the sequences of cDNA clones has revealed the presence of two introns in the gene. We are currently constructing expression vectors containing this gene and developing algal transformation protocols to enable overexpression of ACCase in C. cryptica and other algal species.

Acetyl-CoA Carboxylase↗
PubMed1994

Organization and characterization of Cucurbita phloem lectin genes.

The phloem of pumpkin and squash contains a dimeric chitin-binding lectin called PP2 (phloem protein 2). We have isolated three genomic clones from pumpkin (Cucurbita maxima Duch.) that encoded PP2. One clone, lambda gPC13-1, contained two PP2 genes that were 99.8% identical over a region of 3055 nucleotides. This conserved region included 1922 bp of 5' non-coding sequence, 844 bp of protein coding sequence (including two introns), and 289 bp of 3' non-coding sequence. To examine the conservation of the phloem lectin within the genus Cucurbita, we analyzed nine different species for PP2, its mRNA, and the genes that encode PP2. DNA blot analysis indicated that each species contained genes that encoded PP2, however, there was considerable restriction fragment length polymorphism (RFLP) among the species. PP2 gene copy number reconstructions indicated that PP2 is encoded by a small gene family (two to eight genes). Although a high level of PP2 DNA polymorphism existed among species, a single mRNA (ca. 1 kb) was detected in each species. PP2, affinity-purified from the vascular exudate of each species, reacted with PP2-specific antibodies; five species contained a single PP2 polypeptide while four species contained two PP2 polypeptides.

Amino Acid Sequence↗
PubMed1994

Effect of disinfection of custom tray materials on adhesive properties of several impression material systems.

The effects of impression tray disinfection procedures on the bond strength of impression-material adhesives to two types of resin trays were evaluated with a tensile test. Autopolymerizing acrylic resin and a visible light-curing resin were formed into one-half inch cubes. A screw eye was attached to each cube before polymerization. Perforated trays were fabricated with stops to maintain an even one-eighth inch of impression material over the resin block. Hooks on the opposite side permitted attachment of the metal plate to a mechanical testing machine. Before adhesive was applied, one third of the resin specimens were immersed in a 1:213 iodophor solution; one third in a 10% sodium hypochlorite solution, and one third were kept in the "as fabricated" condition. Polysulfide, polyether, and polyvinyl siloxane impression material-adhesive systems were evaluated. The resin-impression material-metal plate couples were attached to a mechanical testing machine and tensile forces were applied at a separation rate of 5 inches per minute. Mean values for adhesive strength ranged from 3.49 kg/cm2 for the autopolymerizing acrylic resin/iodophor/polyether combination to 10.55 kg/cm2 for the autopolymerizing acrylic resin/untreated/polyvinyl siloxane combination. Differences were detected among materials and disinfecting procedure. Clinically, disinfection of resin trays may adversely affect retention of the impression material to the tray.

Acrylic Resins↗
PubMed1993

Intracellular translocation of a 28 kDa GTP-binding protein during osmotic shock-induced cell volume regulation in Dunaliella salina.

The primary aim of this study was to determine if small GTP-binding proteins play a role in the conspicuous and much-examined volume control process in Dunaliella salina. We confirmed the previous identification by Rodriguez et al. (Rodriguez Rosales, M.P., Herrin, D.L. and Thompson, G.A., Jr. (1992) Plant Physiol. 98, 446-451) of small GTP-binding proteins in the green alga Dunaliella salina and revealed the presence of at least five such proteins, having molecular masses of approx. 21, 28, 28.5, 29 and 30 kDa. These proteins were concentrated largely in the endoplasmic reticulum (ER) and in an intermediate density organelle fraction (GA) containing mainly Golgi vesicles, mitochondria and flagella. The chloroplast fraction and plasma membrane contained the 28-kDa GTP-binding protein exclusively, while the cytosol contained both the 28-kDa component and small amounts of a 21-kDa GTP-binding protein. Immunodetection analysis showed that the D. salina 28-kDa protein cross-reacted strongly with a polyclonal antibody raised against a Volvox carteri yptV1 type GTP-binding protein. This antibody was utilized for quantitative GTP-binding protein measurements as described below. Certain anti-GTP-binding protein antibodies derived from non-plant sources, namely, monoclonal antibodies raised against yeast and mouse ypt1 GTP-binding proteins, cross-reacted not only with the D. salina 28-kDa protein but also the 29-kDa component. The 30-kDa GTP-binding protein of D. salina did not bind the antibodies mentioned above but did cross-react with an anti-yeast ypt1 polyclonal antibody. None of the D. salina GTP-binding proteins reacted positively with polyclonal antibodies raised against SEC4, rab1 or rab6 proteins. When D. salina cells were subjected to hypoosmotic swelling by abruptly reducing the NaCl concentration of their medium from 1.7 M to 0.85 M, the increase in cell surface area was accompanied by a substantial translocation of the 28-kDa GTP-binding protein from the ER and GA fractions to the plasma membrane, chloroplast and cytosolic fractions, as determined by quantitative [32P]GTP binding and [125I]antibody binding on nitrocellulose blots. This translocation increased the content of the 28-kDa component in the plasma membrane, chloroplast and cytosol by 3-4-fold. No net movement of the 30-kDa GTP-binding protein from either the ER or GA fractions was observed following hypoosmotic shock.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Size↗
PubMed1993

Insulin- and contraction-stimulated translocation of GTP-binding proteins and GLUT4 protein in skeletal muscle.

Low molecular weight GTP-binding proteins and GLUT4 protein were isolated in purified plasma membrane and low density microsome fractions from rat skeletal muscle. GTP-binding proteins were detected via the ability of these proteins to bind [32P]GTP subsequent to Western blotting. GLUT4 protein was detected via the anti-GLUT4 antibody F349 subsequent to Western blotting. The possible involvement of GTP-binding proteins in the regulation of GLUT4 protein movement was investigated by examining the subcellular distribution of GTP-binding proteins and GLUT4 protein under basal conditions and following stimulation by insulin or muscle contraction. Insulin stimulation caused a 111 +/- 34.8% increase in the plasma membrane content of GTP-binding proteins which was paralleled by a 74 +/- 19.1% increase in the plasma membrane content of GLUT4 protein. The insulin-stimulated increase in plasma membrane GTP-binding proteins and GLUT4 protein occurred coincident with 27 +/- 4.6 and 33 +/- 7.4% decreases, respectively, in the low density microsome content of these proteins. In addition, muscle contraction significantly increased the plasma membrane content of GTP-binding proteins (63 +/- 18.1%) and GLUT4 protein (67 +/- 22.2%). However, with muscle contraction the concentrations of GTP-binding proteins and GLUT4 protein were not altered in low density microsome fractions. The similar patterns with which the GTP-binding proteins and GLUT4 protein responded to stimulation by insulin and muscle contraction suggests a possible, but yet unidentified functional relationship between these proteins.

Animals↗
PubMed1993

Identification of an ARF type low molecular mass GTP-binding protein in pea (Pisum sativum).

The ARF class of low molecular mass GTP-binding proteins, originally detected as cholera toxin-activated ADP-ribosylation factors (ARF) in mammalian cells, is now known to participate in intracellular membrane vesicle trafficking. We identified a GTP-binding protein in pea plumules which resembles ARF in several respects. Like mammalian ARF, the pea protein had an apparent molecular mass of 21 kDa and was distributed mainly (approximately 97%) in the cytosol, with 1% and 1.5% occurring in the Golgi and microsomal fractions, respectively. In comparison, small GTP-binding proteins in the 26-30 kDa range were enriched in membranous fractions. The 21 kDa protein crossreacted strongly with an ARF class I antibody prepared against a mammalian ARF, but did not crossreact with ARF 5 (of class II) antibodies. An anti-Volvox yptl antibody did not show immunoreactivity to the 21 kDa pea protein, although it crossreacted strongly with a 28 kDa pea plumule protein. Our results strongly suggest that the 21 kDa pea protein is an analog of the ARF protein localized in the cytosol of mammalian cells. As far as we are aware, this is the first observation of an ARF protein in plants.

ADP-Ribosylation Factors↗
PubMed1993

Pharmacokinetics of intravenous and epidural ropivacaine in the rhesus monkey.

Ropivacaine is a new long-acting amide local anesthetic which is possibly less cardiotoxic than bupivacaine. The absorption and disposition of ropivacaine were characterized in six rhesus monkeys in an open two-way crossover study following intravenous and epidural administration. For these studies, animals were anesthetized for placement of intravenous and intraarterial catheters. For the epidural studies, a PE-10 catheter was also inserted 3 cm into the lumbar epidural space. After recovery from anesthesia, animals received ropivacaine 1 mg kg-1 intravenously over 1 min or 10 mg of ropivacaine epidurally (two 1 ml doses of 0.5%, 5 min apart), and arterial blood samples were obtained over 5 h. Serum ropivacaine concentrations were determined by gas chromatography with NP detection. Concentration-time data following i.v. and epidural administration were fitted simultaneously. Initial parameter estimates were obtained by analyzing each route separately. Input rates and their corresponding extent of absorption were estimated using deconvolution. Mean (+/- SD) disposition parameters included: Vss = 1.11 +/- 0.198 l kg-1; CL = 0.711 +/- 0.158 l h-1 kg-1; t1/2,z = 2.07 +/- 0.438 h. Mean (+/- SD) absorption parameters included: F1 = 0.506 +/- 0.221; t1/2,kal = 0.060 +/- 0.078 h; F2 = 0.444 +/- 0.182; t1/2,ka2 = 6.45 +/- 11.09 h. Ropivacaine's biphasic absorption and bioavailability are similar to those of other amide local anesthetics. The biphasic absorption may be related to partitioning into fat or regional changes in blood flow induced by the drug.

Amides↗
PubMed1993

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei.

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

Cell Nucleus↗
PubMed1993

Lipid modification during epidermal cell differentiation.

As keratinocytes differentiate into corneocytes of the stratum corneum or epidermal permeability barrier, their lipids are modified so as to fulfill totally different functions. Recent experimentation has clarified the molecular mechanisms by which lipids of membrane origin are targeted to specialized lamellar bodies, where metabolic retailoring makes them suitable for use in the water-impermeable intercellular lamellae. In this latter structure the modified lipids are bound covalently to specialized proteins in a way that encourages the formation of lipid bilayers alternating with lipid monolayers. Only now are potential clues to the molecular regulation of this dramatic lipid transformation becoming apparent.

Animals↗