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Biomedical subjects

Charles M Thompson

Publications and source records attributed to Charles M Thompson.

17 recordsLinked to original sources
PubMed2006

Inhibitors of the alpha-ketoglutarate dehydrogenase complex alter [1-13C]glucose and [U-13C]glutamate metabolism in cerebellar granule neurons.

Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), an important component of the tricarboxylic acid (TCA) cycle, occurs in several neurological diseases. The effect of specific KGDHC inhibitors [phosphonoethyl ester of succinyl phosphonate (PESP) and the carboxy ethyl ester of succinyl phosphonate (CESP)] on [1-13C]glucose and [U-13C]glutamate metabolism in intact cerebellar granule neurons was investigated. Both inhibitors decreased formation of [4-13C]glutamate from [1-13C]glucose, a reduction in label in glutamate derived from [1-13C]glucose/[U-13C]glutamate through a second turn of the TCA cycle and a decline in the amounts of gamma-aminobutyric acid (GABA), aspartate, and alanine. PESP decreased formation of [U-13C]aspartate and total glutathione, whereas CESP decreased concentrations of valine and leucine. The findings are consistent with decreased KGDHC activity; increased alpha-ketoglutarate formation; increased transamination of alpha-ketoglutarate with valine, leucine, and GABA; and new equilibrium position of the aspartate aminotransferase reaction. Overall, the findings also suggest that some carbon derived from alpha-ketoglutarate may bypass the block in the TCA cycle at KGDHC by means of the GABA shunt and/or conversion of valine to succinate. The results suggest the potential of succinyl phosphonate esters for modeling the biochemical and pathophysiological consequences of reduced KGDHC activity in brain diseases.

Animals↗
PubMed2005

Analysis and sequencing of the active-site peptide from native and organophosphate-inactivated acetylcholinesterase by electrospray ionization, quadrupole/time-of-flight (QTOF) mass spectrometry.

A method to identify and sequence recombinant mouse acetylcholinesterase (rMoAChE) including the native and organophosphate-modified active-site peptides was developed using capillary liquid chromatography with electrospray ionization, quadrupole/time-of-flight mass spectrometry. Addition of 2-propanol to the reversed-phase gradient system and a decreased gradient slope improved the peptide resolution and the signal of the active-site peptide. The highest protein coverage and active-site peptide signal were achieved when the rMoAChE:chymotrypsin ratio of 5:1 was used with digestion at 37 degrees C. rMoAChE and the active-site peptide were identified and sequenced from chymotryptic digests of native, methyl paraoxon-, and ethyl paraoxon-inactivated rMoAChE showing unequivocally that the exact modification site was the active-site serine.

Acetylcholinesterase↗
PubMed2005

Differential localization of acetylcholinesterase in neuronal and non-neuronal cells.

Acetylcholinesterase (AChE) expression is regulated in cell types at the transcriptional and translational levels. In this study, we characterized and compared AChE catalytic activity, mRNA, protein expression, and protein localization in a variety of neuronal (SH-SY5Y neuroblastoma and primary cerebellar granule neurons (CGN)) and non-neuronal (LLC-MK2, HeLa, THP-1, and primary astrocytes) cell types. All cell lines expressed AChE catalytic activity; however the levels of AChE-specific activity were higher in neuronal cells than in the non-neuronal cell types. CGN expressed significantly more AChE activity than SH-SY5Y cells. All cell lines analyzed expressed AChE protein at equivalent levels, as well as mRNA splice variants. Localization of AChE was characterized by immunofluorescence and confocal microscopy. SH-SY5Y, CGN, and nerve-growth factor-differentiated PC-12 cells exhibited a pattern of AChE localization characterized as diffuse in the cytoplasm and punctate staining along neurites and on the plasma membrane. The localization in HeLa, LLC-MK2, fibroblasts, and undifferentiated PC-12 cells was significantly different than in neuronal cells-AChE was intensely localized in the perinuclear region, without staining near or on the plasma membrane. Based on the evidence presented here, we hypothesize that the presence of AChE protein doesn't correlate with catalytic activity, and the diffuse cytoplasmic and plasma membrane localization of AChE is a property of neuronal cell types.

Acetylcholinesterase↗
PubMed2005

Characteristic mass spectral fragments of the organophosphorus agent FP-biotin and FP-biotinylated peptides from trypsin and bovine albumin (Tyr410).

A mass spectrometry-based method was developed for selective detection of FP-biotinylated peptides in complex mixtures. Mixtures of peptides, at the low-picomole level, were analyzed by liquid chromatography and positive ion, nanospray, triple quadrupole, linear ion trap mass spectrometry. Peptides were fragmented by collision-activated dissociation in the mass spectrometer. The free FP-biotin and peptides containing FP-biotinylated serine or FP-biotinylated tyrosine yielded characteristic fragment ions at 227, 312, and 329 m/z. FP-biotinylated serine yielded an additional characteristic fragment ion at 591 m/z. Chromatographic peaks containing FP-biotinylated peptides were indicated by these diagnostic ions. Data illustrating the selectivity of the approach are presented for tryptic digests of FP-biotinylated trypsin and FP-biotinylated serum albumin. A 16-residue peptide from bovine trypsin was biotinylated on the active site serine. A 3-residue peptide from bovine albumin, YTR, was biotinylated on Tyr410. This latter result confirms that the organophosphorus binding site of albumin is a tyrosine. This method can be used to search for new biomarkers of organophosphorus agent exposure.

Animals↗
PubMed2005

Phosphonate analogues of alpha-ketoglutarate inhibit the activity of the alpha-ketoglutarate dehydrogenase complex isolated from brain and in cultured cells.

The alpha-ketoglutarate dehydrogenase complex (KGDHC), a control point of the tricarboxylic acid cycle, is partially inactivated in brain in many neurodegenerative diseases. Potent and specific KGDHC inhibitors are needed to probe how the reduced KGDHC activity alters brain function. Previous studies showed that succinyl phosphonate (SP) effectively inhibits muscle and Escherichia coli KGDHC [Biryukov, A. I., Bunik, V. I., Zhukov, Yu. N., Khurs, E. N., and Khomutov, R. M. (1996) FEBS Lett. 382, 167-170]. To identify the phosphonates with the highest affinity toward brain KGDHC and with the greatest effect in living cells, we investigated the ability of SP and several of its ethyl esters to inhibit brain KGDHC, other alpha-keto acid-dependent enzymes, and KGDHC in intact cells. At a concentration of 0.01 mM, SP and its phosphonoethyl (PESP) and carboxyethyl (CESP) esters completely inhibited isolated brain KGDHC even in the presence of a 200-fold higher concentration of its substrate [alpha-ketoglutarate (KG)], while the diethyl (DESP) and triethyl (TESP) esters were ineffective. In cultured human fibroblasts, 0.01 mM SP, PESP, or CESP produced 70% inhibition of KGDHC. DESP and TESP were also inhibitory in the cell system, but only after preincubation, suggesting the release of their charged groups by cellular esterases. Thus, SP and its monoethyl esters target cellular KGDHC directly, while the di- and triethyl esters are activated in intact cells. When tested on other enzymes that bind KG or related alpha-keto acids, SP had minimal effects and its two esters (CESP and TESP) were ineffective even at a concentration (0.1 mM) 1 order of magnitude higher than that which inhibited cellular KGDHC activity. The high specificity in targeting KGDHC, penetration into cells, and minimal transformation by cellular enzymes indicate that SP and its esters should be useful in studying the effects of reduced KGDHC activity on neuronal and brain function.

Alanine Transaminase↗
PubMed2005

Binding properties of dipropyltryptamine at the human 5-HT1a receptor.

Dipropyltryptamine (DPT) is a synthetic indolealkylamine first characterized in the 1960s. Largely forgotten since the discovery of multiple serotonin receptor subtypes, some of the properties of DPT at the cloned human 5-HT1a receptor are described here. When [3H]8-OH-DPAT is bound to the receptor, DPT inhibits the interaction with an IC50 of 0.1 micromol/l. This interaction is shown to be competitive when double-reciprocal plots of the DPT/agonist interaction are analyzed. DPT's effects in the signal transduction system are complex. While DPT alone (0.1-1,000 micromol/l) activates Gi when both cAMP and gamma-S-GTP incorporation are measured, in the presence of 5-HT (0.1-10 micromol/l), DPT blocks the agonist effect. In combination, the findings suggest that DPT is a moderate affinity partial agonist at the human 5-HT1a receptor. These results provide evidence that DPT has potential as a versatile experimental tool at 5-HT1a receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2005

Reaction kinetics of biotinylated organophosphorus toxicant, FP-biotin, with human acetylcholinesterase and human butyrylcholinesterase.

A biotinylated organophosphate could be useful for identifying proteins that react with organophosphorus toxicants (OP). FP-biotin, 10-(fluoroethoxyphosphinyl)-N-(biotinamidopentyl)decanamide, was synthesized and found to be stable in methanol and chloroform but less stable in water. Because acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) are known to be sensitive targets of OP, their reactivity with FP-biotin was tested. The rate constant for reaction with human AChE was 1.8 x 10(7) M(-1) min(-1), and for human BChE, it was 1.6 x 10(8) M(-1) min(-1). A phosphorus stereoisomer, constituting about 50% of the FP-biotin preparation, appeared to be the reactive species. The binding affinity was estimated to be >85 nM for AChE and >5.8 nM for BChE. It was concluded that FP-biotin is a potent OP, well-suited for searching for new biomarkers of OP exposure.

Acetylcholinesterase↗
PubMed2005

Inhibitor of the glutamate vesicular transporter (VGLUT).

The vesicular glutamate transporter (VGLUT) is responsible for the uptake of the excitatory amino acid, L-glutamate, into synaptic vesicles. VGLUT activity is coupled to an electrochemical gradient driven by a vacuolar ATPase and stimulated by low Cl-. VGLUT has relatively low affinity (K(m) = 1-3 mM) for glutamate and is pharmacologically and structurally distinct from the Na+-dependent, excitatory amino acid transporters (EAATs) found on the plasma membrane. Because glutamatergic neurotransmission begins with vesicular release, compounds that block the uptake of glutamate into the vesicle may reduce excitotoxic events. Several classes of competitive VGLUT inhibitors have emerged including amino acids and amino acid analogs, fatty acids, azo dyes, quinolines and alkaloids. The potency with which these agents inhibit VGLUT varies from millimolar (amino acids) to nanomolar (azo dyes) concentrations. These inhibitors represent highly diverse structures and have collectively begun to reveal key pharmacophore elements that may elucidate the key interactions important to binding VGLUT. Using known inhibitor structures and preliminary molecular modeling, a VGLUT pharmacophore is presented that will aid in the design of new, highly potent and selective agents.

Amino Acid Transport Systems, Acidic↗
PubMed2004

Albumin, a new biomarker of organophosphorus toxicant exposure, identified by mass spectrometry.

The classical laboratory tests for exposure to organophosphorus toxicants (OP) are inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in blood. In a search for new biomarkers of OP exposure, we treated mice with a biotinylated organophosphorus agent, FP-biotin. The biotinylated proteins in muscle were purified by binding to avidin-Sepharose, separated by gel electrophoresis, digested with trypsin, and identified from their fragmentation patterns on a quadrupole time-of-flight mass spectrometer. Albumin and ES1 carboxylesterase (EC 3.1.1.1) were found to be major targets of FP-biotin. These FP-biotinylated proteins were also identified in mouse plasma by comparing band patterns on nondenaturing gels stained for albumin and carboxylesterase activity, with band patterns on blots hybridized with Streptavidin Alexa-680. Two additional FP-biotin targets, AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8), were identified in mouse plasma by finding that enzyme activity was inhibited 50-80%. Mouse plasma contained eight additional FP-biotinylated bands whose identity has not yet been determined. In vitro experiments with human plasma showed that chlorpyrifos oxon, echothiophate, malaoxon, paraoxon, methyl paraoxon, diazoxon, diisopropylfluorophosphate, and dichlorvos competed with FP-biotin for binding to human albumin. Though experiments with purified albumin have previously shown that albumin covalently binds OP, this is the first report of OP binding to albumin in a living animal. Carboxylesterase is not a biomarker in man because humans have no carboxylesterase in blood. It is concluded that OP bound to albumin could serve as a new biomarker of OP exposure in man.

Acetylcholinesterase↗
PubMed2004

Stereoselective addition of dimethyl thiophosphite to imines.

Dimethyl thiophosphite (DMTP) was synthesized from dimethyl phosphite, and the diastereoselective addition of DMTP to benzaldimines bearing chiral auxiliary groups was examined. Yields of the product alpha-aminophosphonothionates ranged from 17% to 75% after chromatography. The addition of DMTP to the benzaldimine derived from (S)-phenylglycinol afforded the highest diastereoselectivity (83:17), whereas addition of DMTP to the benzaldimine derived from threonine methyl ester and alanine methyl ester were far less diastereoselective, affording 38:62 and 61:39 ratios, respectively. Addition of DMTP to the benzaldimine derived from (R)-alpha-methylbenzylamine (78:22) and (S)-serine methyl ester (73:27) were intermediate in selectivity. DMTP addition to the imines formed between serine methyl ester and acetaldehyde and isobutyraldehyde gave 55:45 and 70:30 ratios, respectively, with the diastereoselectivity corresponding roughly to the size of the alpha-alkyl group. The stereochemistry of the newly formed alpha-stereocenters resulting from the addition of DMTP to (S)- and (R)-phenylglycinol benzaldimines was confirmed by conversion of the product alpha-aminophosphonothionates to the known enantiomers of phosphonophenylglycine.

Catalysis↗
PubMed2004

The synaptic vesicle proteome: a comparative study in membrane protein identification.

A proteomic analysis of the synaptic vesicle was undertaken to obtain a better understanding of vesicle regulation. Synaptic vesicles primarily consist of integral membrane proteins that are not well resolved on traditional isoelectric focusing/two-dimensional gel electrophoresis (IEF/2-DE) gels and are resistant to in-gel digestion with trypsin thereby reducing the number of peptides available for mass spectrometric analysis. To address these limitations, two complementary 2-DE methods were investigated in the proteome analysis: (a) IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) for resolution of soluble proteins and, (b) Benzyl hexadecyl ammonium chloride/SDS-PAGE (16-BAC/SDS-PAGE) for resolution of integral membrane proteins. The IEF/SDS-PAGE method provided superior resolution of soluble proteins, but could only resolve membrane proteins with a single transmembrane domain. The 16-BAC/SDS-PAGE method improved separation, resolution and identification of integral membrane proteins with up to 12 transmembrane domains. Trypsin digestion of the integral membrane proteins was poor and fewer peptides were identified from these proteins. Analysis of both the peptide mass fingerprint and the tandem mass spectra using electrospray ionization quadrupole-time of flight mass spectrometry led to the positive identification of integral membrane proteins. Using both 2-DE separation methods, a total of 36 proteins were identified including seven integral membrane proteins, 17 vesicle regulatory proteins and four proteins whose function in vesicles is not yet known.

Cell Membrane↗
PubMed2003

Differentiation between acetylcholinesterase and the organophosphate-inhibited form using antibodies and the correlation of antibody recognition with reactivation mechanism and rate.

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.

Acetylcholinesterase↗
PubMed2003

Stereoselective inactivation of Torpedo californica acetylcholinesterase by isomalathion: inhibitory reactions with (1R)- and (1S)-isomers proceed by different mechanisms.

The present study was undertaken to test the hypothesis that acetylcholinesterase (AChE) inhibition by isomalathion stereoisomers proceeds with different primary leaving groups for (1R)- and (1S)-isomers. Consistent with results obtained with enzyme from other species, AChE from Torpedo californica (TcAChE) was stereoselectively inhibited by isomalathion isomers with the (1R,3R)-isomer exhibiting greater potency than (1S,3S)-isomalathion. TcAChE modified by (1R)-isomers readily reactivated in the presence of 2-pralidoxime methiodide (2-PAM), whereas enzyme inhibited by (1S)-isomalathions was intractable toward reactivation. Computer-based molecular modeling showed that the ligand positioned as the primary leaving group was diethyl thiosuccinyl for (1R)-isomers and thiomethyl for (1S)-isomalathions. Mass spectral analysis revealed that inhibition of TcAChE by (1R)-isomers resulted in an O,S-dimethyl phosphate adduct, as expected from expulsion of the diethyl thiosuccinyl ligand. In contrast, inactivation of the enzyme by (1S)-isomalathions yielded an O-methyl phosphate adduct, consistent with initial loss of thiomethyl followed by displacement of the diethyl thiosuccinyl group. The findings demonstrate that the inhibitory reactions of TcAChE with (1R)- and (1S)-isomalathions proceed by different mechanisms involving distinct primary leaving groups.

Acetylcholinesterase↗
PubMed2002

Synthesis and in vitro pharmacology of substituted quinoline-2,4-dicarboxylic acids as inhibitors of vesicular glutamate transport.

The vesicular glutamate transport (VGLUT) system selectively mediates the uptake of L-glutamate into synaptic vesicles. Uptake is linked to an H+-ATPase that provides coupling among ATP hydrolysis, an electrochemical proton gradient, and glutamate transport. Substituted quinoline-2,4-dicarboxylic acids (QDCs), prepared by condensation of dimethyl ketoglutaconate (DKG) with substituted anilines and subsequent hydrolysis, were investigated as potential VGLUT inhibitors in synaptic vesicles. A brief panel of substituted QDCs was previously reported (Carrigan et al. Bioorg. Med. Chem. Lett. 1999, 9, 2607-2612)(1) and showed that certain substituents led to more potent competitive inhibitors of VGLUT. Using these compounds as leads, an expanded series of QDC analogues were prepared either by condensation of DKG with novel anilines or via aryl-coupling (Suzuki or Heck) to dimethyl 6-bromoquinolinedicarboxylate. From the panel of almost 50 substituted QDCs tested as inhibitors of the VGLUT system, the 6-PhCH=CH-QDC (K(i) = 167 microM), 6-PhCH2CH2-QDC (K(i) = 143 microM), 6-(4'-phenylstyryl)-QDC (K(i) = 64 microM), and 6-biphenyl-4-yl-QDC (K(i) = 41 microM) were found to be the most potent blockers. A preliminary assessment of the key elements needed for binding to the VGLUT protein based on the structure-activity relationships for the panel of substituted QDCs is discussed herein. The substituted QDCs represent the first synthetically derived VGLUT inhibitors and are promising templates for the development of selective transporter inhibitors.

Animals↗
PubMed2002

Examination of cross-antigenicity of acetylcholinesterase and butyrylcholinesterase using anti-acetylcholinesterase antibodies.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) share a high degree of homology and overlap in several biochemical properties. This study aimed to compare and contrast the antigenic reactivity of AChE and BuChE with several polyclonal antibodies. We have performed a detailed analysis of AChE and BuChE enzymatic activities with different substrates and different inhibitors. Immunoassays conducted with polyclonal amino-terminus-specific anti-AChE antibodies were selective for mouse and electric eel AChE (EEAChE). Polyclonal carboxy-terminus-specific anti-AChE antibodies reacted with EEAChE and human BuChE, indicating an unexpected cross-reactivity. Polyclonal antisera raised against the whole AChE protein cross-reacted with horse BuChE, but not human BuChE. These data demonstrate that AChE and BuChE are immunologically similar.

Acetylcholinesterase↗
PubMed2002

Two studies examining the negative effect of self-efficacy on performance.

Although hundreds of studies have found a positive relationship between self-efficacy and performance, several studies have found a negative relationship when the analysis is done across time (repeated measures) rather than across individuals. W. T. Powers (1991) predicted this negative relationship based on perceptual control theory. Here, 2 studies are presented to (a) confirm the causal role of self-efficacy and (b) substantiate the explanation. In Study 1, self-efficacy was manipulated for 43 of 87 undergraduates on an analytic game. The manipulation was negatively related to performance on the next trial. In Study 2, 104 undergraduates played the analytic game and reported self-efficacy between each game and confidence in the degree to which they had assessed previous feedback. As expected, self-efficacy led to overconfidence and hence increased the likelihood of committing logic errors during the game.

Humans↗