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W L Roberts

Publications and source records attributed to W L Roberts.

At least 55 records · Page 3Linked to original sources
PubMed1994

Solid-phase extraction combined with radioimmunoassay for measurement of zalcitabine (2',3'-dideoxycytidine) in plasma and serum.

Of the antiviral agents that are currently in clinical use in the US for therapy for human immunodeficiency virus infections, zalcitabine (ddC) is the most potent and is effective at the lowest plasma concentrations. The two reported procedures for measuring these low concentrations involve a chromatographic technique coupled with mass spectrometry. We have developed a procedure combining solid-phase extraction with a strong cation-exchange resin and commercially available RIA reagents for the quantification of ddC in plasma or serum. The method demonstrates good linearity, specificity, and precision, with overall CVs of < 10% from 2-20 micrograms/L and 17% at 0.8 microgram/L (the lower limit of quantitation). No significant cross-reactivity with nucleoside analogs other than ddC analogs was noted. The major advantages of this assay are its efficiency and relative simplicity, which should facilitate its performance in many laboratories.

Antiviral Agents↗
PubMed1993

Antimony quantification in Leishmania by electrothermal atomic absorption spectroscopy.

Tri- and pentavalent antimony were quantified in Leishmania mexicana pifanoi amastigotes and promastigotes by atomic absorption spectroscopy with electrothermal atomization. Leishmania grown in axenic culture were treated with either potassium antimony tartrate [Sb(III)] or sodium stibogluconate [Sb(V)]. The parasites were collected, digested with nitric acid, and subjected to atomic absorption spectroscopy. The method was linear from 0 to 7 ng of antimony. The interassay coefficients of variation were 9.6 and 5.7% (N = 5) for 0.52 and 3.7-ng samples of leishmanial antimony, respectively. The limit of detection was 95 pg of antimony. The assay was used to characterize Sb(III) and Sb(V) influx and efflux kinetics. Influx rates were determined at antimony concentrations that produced a 50% inhibition of growth (IC50). The influx rates of Sb(V) into amastigotes and promastigotes were 4.8 and 12 pg/million cells/h, respectively, at 200 micrograms antimony/ml. The influx rate of Sb(III) into amastigotes was 41 pg/million cells/h at 20 micrograms antimony/ml. Influx of Sb(III) into promastigotes at 1 microgram antimony/ml was rapid and reached a plateau of 175 pg/million cells in 2 h. Efflux of Sb(III) and Sb(V) from amastigotes and promastigotes exhibited biphasic kinetics. The initial (alpha) half-life of Sb(V) efflux was less than 4 min and that of Sb(III) was 1-2 h. The apparent terminal (beta) half-lives ranged from 7 to 14 h.

Animals↗
PubMed1993

Diagnosis and misdiagnosis of poisoning with the cyanide precursor acetonitrile: nail polish remover or nail glue remover?

Accurate diagnosis of acetonitrile ingestion is critical to management. Often this involves differentiating nail polish remover (acetone) from nail glue remover (acetonitrile). Initial symptoms of acetonitrile ingestion are indistinguishable from those of acetone and common alcohols. However, acetonitrile is metabolized to cyanide, producing severe delayed toxicity. Acetonitrile produced increased serum osmolality and osmolal gap, but these findings are non-specific and normal values cannot rule out potentially fatal exposure. Acetone, but not acetonitrile, was detectable in urine or serum with Acetest tablets; both were unreactive with a ketone dipstick. Acetone and acetonitrile could be detected with routine gas chromatography methods for alcohols. Both substances had identical retention times on the widely used stationary phase, 5% Carbowax 20M on graphitized carbon, and with GasChrom 254. Three other systems afforded unique retention times, but acetonitrile was easily mistaken for ethanol in two. Physicians and laboratories must take care to avoid misdiagnosis of acetonitrile ingestion as exposure to acetone, ethanol or another alcohol.

Acetone↗
PubMed1993

Antileishmanial activity of sodium stibogluconate fractions.

Sodium stibogluconate, a pentavalent antimony derivative produced by the reaction of stibonic and gluconic acids, is the drug of choice for the treatment of leishmaniasis. It has been reported to be a complex mixture rather than a single compound. We separated sodium stibogluconate into 12 fractions by anion-exchange chromatography. One fraction accounted for virtually all the leishmanicidal activity of the fractionated material against Leishmania panamensis promastigotes, with a 50% inhibitory concentration (IC50) of 12 micrograms of Sb per ml; that of unfractionated sodium stibogluconate was 154 micrograms of Sb per ml. Further analysis of this active fraction revealed that a major component was m-chlorocresol, which had been included in the sodium stibogluconate formulation as a preservative. The IC50 of pure m-chlorocresol was 1.6 micrograms/ml, a concentration equivalent to that present in unfractionated sodium stibogluconate at a concentration of 160 micrograms of Sb per ml. After ether extraction to remove m-chlorocresol, the IC50 of sodium stibogluconate was > 4,000 micrograms of Sb per ml. In contrast, when L. panamensis amastigotes were grown in macrophages, the IC50 of ether-extracted sodium stibogluconate was 10.3 micrograms of Sb per ml. The 12 fractions of ether-extracted sodium stibogluconate obtained by anion-exchange chromatography had IC50s of 10.1 to 15.4 micrograms of Sb per ml. We conclude that preservative-free sodium stibogluconate has little activity against L. panamensis promastigotes but is highly active against L. panamensis amastigotes in macrophages. This activity is associated with multiple chemical species.

Animals↗
PubMed1993

Interference in immunoassay measurements of total and free phenytoin in uremic patients: a reappraisal.

Accumulation of phenytoin metabolites in uremia has been shown to interfere with some immunoassay methods for phenytoin measurement. We evaluated the effects of uremia (serum creatinine > 13 mg/L) on free and total phenytoin concentrations measured by the Abbott TDx fluorescence polarization immunoassay, and the DuPont aca and Syva EMIT homogeneous enzyme-multiplied immunoassay methods, using HPLC as the comparison method. In uremic patients, the TDx assay showed both fixed and proportional bias in comparison with the HPLC for both total phenytoin concentration (TDx = 1.24 x HPLC + 1.9 mg/L) and free phenytoin concentration (TDx = 1.52 x HPLC + 0.24 mg/L). The total bias was as great as 100% for both total and free phenytoin. Cross-reactive substances other than 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH) and HPPH-glucuronide appeared to be responsible. In contrast, there was minimal interference with aca and EMIT assays.

Adult↗
PubMed1992

Measurement of endothelial permeability to 125I-low density lipoproteins in rabbit arteries by use of en face preparations.

A procedure of en face quantitative autoradiography of the endothelium (Hautchen preparations) was developed to examine regional variations in 125I-low density lipoprotein (125I-LDL) permeability in the arterial wall in vivo. Endothelial preparations from fixed arterial tissue and calibration standards consisting of known concentrations of 125I-albumin were dipped in nuclear emulsion, exposed for 1-3 months, developed, and stained with hematoxylin. Digital image analysis was used to analyze dark-field images of autoradiographs. Background grain densities on cold endothelial preparations were 30-100% higher than on glass, but the variability in grain densities on the two different surfaces was similar. Regression slopes of grain density versus concentration for calibration standards were the same for sections placed on cold tissue or glass. For 1-5-microns-thick calibration standards of the same concentration, the grain density was proportional to the total amount of radioactivity per unit area. The results indicated that errors arising from nonuniformities in preparation thickness were minimal, and permeabilities and intimal concentrations could be determined. Rabbits were killed 10 minutes after injection of 125I-LDL, and endothelial preparations were made. For regions of uniformly low grain density in the rabbit aorta, the 125I-LDL permeability was 1.9 +/- 0.8 x 10(-8) cm/sec, and the effective diffusion coefficient was 5.4 +/- 3.1 x 10(-10) cm2/sec. Errors in the estimated permeability arising from nonuniformities in tissue thickness were the same as the reported experimental variability. Analysis of elevated regions of permeability suggested that 125I-LDL was binding to the extracellular matrix. Approximately 25% of the sites of elevated grain density were associated with mitotic endothelial cells, and such regions had higher permeabilities than sites associated with nonmitotic cells. Around intercostal arteries, sites of highest permeability were distal and lateral to the vessels and occurred where lesions first develop in hypercholesterolemic rabbits.

Animals↗
PubMed1991

Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages.

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.

Acetylcholinesterase↗
PubMed1990

Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage.

Human decay-accelerating factor (DAF) proteins expressed on E and nucleated cells differ in their susceptibility to phosphatidylinositol (PI)-specific phospholipase C (PLC) cleavage/release. To investigate the mechanism of this difference, the glycoinositol-phospholipid anchoring moieties of E DAF, and of HeLa cell, polymorphonuclear cell, and lymphocyte DAF were structurally compared. Labeling of PI-PLC-resistant E DAF with 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)-diazirine ([125I]TID) and TLC analysis of nitrous acid deamination anchor fragments showed a predominant phospholipid species with less polar migration than the 125I-TID-labeled PI. Gas chromatographic analyses of methanolyzed E protein revealed 2.20 +/- 0.16 mol of fatty acids [16:0, 18:0, 18:1, 20:4, 22:4, and 22:5 (0.76, 0.36, 0.25, 0.15, 0.40, 0.28 mol, respectively)] and 0.86 +/- 0.05 mol of inositol per mol of N-terminal Asp. Gas chromatography-mass spectroscopy demonstrated principally myo-inositol but also variable amounts of the chiro-isomer. Nondenaturing polyacrylamide gel electrophoresis of 14C-radiomethylated E protein revealed that pretreatment with hydroxylamine, a reagent which removes ester-linked lipids, rendered it PI-PLC susceptible. In contrast, parallel analyses of 35S-cys-labeled PI-PLC-sensitive HeLa DAF protein revealed only minor amounts of the hydroxylamine-sensitive phospholipid species. Similar results were obtained with 125I-surface-labeled DAF from polymorphonuclear cells, as well as from unstimulated peripheral blood and anti-CD3-activated lymphocytes. These findings demonstrate that, rather than PI, the E DAF anchor contains an inositol alkylacylglycerol-phospholipid which is heterogeneous with respect to acyl groups and inositol isomers, that an ester-linked substitution in this inositolphospholipid underlies the resistance of E DAF protein to PI-PLC cleavage/release, and that this structural modification is cell-specific.

CD55 Antigens↗
PubMed1989

Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholipase C and serum phospholipase D.

Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.

Acetylcholinesterase↗
PubMed1989

Well-circumscribed, radiopaque, and radiolucent lesion of the anterior maxilla.

In the case reported here, compound composite odontoma was found in a 63-year-old man. Considering the growth characteristics of odontoma, it is likely that this lesion was of at least 45-50 years' duration. The typically asymptomatic nature of the odontoma and its location, apical to the canine and lateral incisor roots, explain why it was not noticed by the patient nor detected by a dentist for many years. Because definitive diagnosis of odontoma is possible only after histopathological examination of the lesion, surgical excision is advised.

Humans↗
PubMed1988

Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C.

The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) contains a novel inositol phospholipid which in this and the accompanying paper (Roberts, W.L., Santikarn, S., Reinhold, V.N., and Rosenberry, T.L. (1988) J. Biol. Chem 263, 18776-18784) is shown to be a plasmanylinositol that is palmitoylated on the inositol ring. The inositol phospholipid was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I] iodophenyl)diazirine and characterized by various chemical and enzymatic cleavage procedures whose products were analyzed by thin layer chromatography and autoradiography or gas chromatography. Acidic methanolysis of human erythrocyte acetylcholinesterase (Ehu AChE) revealed 18:0 and 18:1 alkylglycerols (0.55 and 0.20 mol/mol AChE, respectively). Acetolysis was shown by TLC to release alkylacylglycerol acetates from Ehu AChE. Analysis by gas chromatography revealed that 83% of the alkylacylglycerol acetates contained an 18:0 or 18:1 1-alkyl group and a 22:4 (n - 6), 22:5 (n - 3), or 22:6 (n - 3) 2-acyl group. The inositol phospholipid is linked to the anchor by a glucosamine in glycosidic linkage, and deamination with nitrous acid cleaved the glycosidic linkage and released the phospholipid. The deamination and acetolysis products from Ehu AChE were purified by high performance liquid chromatography, and fatty acid analysis following acidic methanolysis of the purified products revealed that 2 fatty acid residues were associated with the deamination product and only one with the alkylacylglycerol acetolysis product. The other fatty acid residue was primarily palmitate and was indicated to be in ester linkage to an inositol hydroxyl(s). This linkage was shown to be responsible for the resistance of the inositol phospholipid to cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase. Deacylation of the inositol phospholipid deamination product by treatment with base removed this palmitoyl group and facilitated release of alkyl- and alkylacylglycerol species by phosphatidylinositol-specific phospholipase C with concomitant formation of inositol 1-phosphate. In contrast, digestion of Ehu AChE with a recently reported anchor-specific phospholipase D resulted in release of plasmanic acids from the intact palmitoylated plasmanylinositol.

Acetylcholinesterase↗
PubMed1988

Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry.

The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) is composed of a glycan linked through a glucosamine residue to an inositol phospholipid that is resistant to the action of phosphatidylinositol-specific phospholipase C. Deamination cleavage of the glucosamine with nitrous acid released the inositol phospholipid which was purified by high performance liquid chromatography. Analysis by fast atom bombardment mass spectrometry with negative ion monitoring and by the complementary technique of collision-induced dissociation revealed molecular and daughter ions that indicated a plasmanylinositol with a palmitoyl group on an inositol hydroxyl. The intact membrane anchor was released from reductively methylated human erythrocyte acetylcholinesterase by proteolysis with papain or Pronase, deacylated by base hydrolysis, and purified by high performance liquid chromatography. Positive and negative ion fast atom bombardment mass spectrometry of the major products isolated by high performance liquid chromatography indicated the following structure for the complete glycoinositol phospholipid anchor. (formula; see text) Methylation of free amino groups by reduction with deuterium instead of hydrogen permitted determination of the number of free amino groups in individual fragment ions as further confirmation of structural assignments. The structure of the glycan portion of the human erythrocyte acetylcholinesterase membrane anchor appears to be similar to that described for Trypanosome brucei variant surface glycoprotein MITat 1.4 (variant 117) (Ferguson, M.A.J., Homans, S.W., Dwek, R.A., and Rademacher, T.W. (1988) Science 239, 753-759) except for the absence of a galactose antenna and the presence of a phosphorylethanolamine on the hexose adjacent to glucosamine.

Acetylcholinesterase↗
PubMed1988

Characterization of [3H]palmitate- and [3H]ethanolamine-labelled proteins in the multicellular parasitic trematode Schistosoma mansoni.

Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.

Amino Acids↗
PubMed1988

Alkylacylglycerol molecular species in the glycosylinositol phospholipid membrane anchor of bovine erythrocyte acetylcholinesterase.

Bovine erythrocyte acetylcholinesterase, a glycosylinositol phospholipid anchored membrane enzyme, was digested with phosphatidylinositol-specific phospholipase C and the released glycerol-containing moieties were identified and quantitated. About 96% of the total was alkylacylglycerol, of which sn-1-stearyl-2-stearoylglycerol, sn-1-stearyl-2-oleoylglycerol and sn-1-oleyl-2-stearoylglycerol accounted for 69%, 13% and 10%, respectively. These alkylacylglycerols are in marked contrast to the exclusively diacylglycerol species present in phosphatidylinositol from bovine erythrocyte membranes. This difference suggests that assembly of the membrane anchor of Ebo AChE involves a selected cellular pool of diradylglycerols.

Acetylcholinesterase↗
PubMed1988

Back pain: treatment and prevention in a community hospital.

Because back pain is a widespread and costly condition that tends to recur, treatment must focus on both the amelioration of acute symptoms and prevention over the long term. This paper reports a longitudinal evaluation of a program from a community hospital that emphasizes both these aspects. One hundred twenty patients routinely admitted to this program were randomly assigned to treatment and control groups. These groups were assessed for differences in demonstrated physical strength, mobility, body mechanics, and self-care knowledge, and in levels of self-reported exercise, anxiety, and pain. There were significant immediate gains on physical measures of fitness and in observed body mechanics; patients also reported significant gains in physical capabilities at home and in leisure activities. Self-care knowledge also improved. When assessed one year later, original gains in physical strength and mobility were being maintained, and self-reported physical capabilities also remained high. Although demonstrated knowledge of correct body mechanics declined over this period, it was still significantly greater than before the program. In light of these results, we believe that outpatient programs like the one reported here merit careful consideration in an era of concern about rising costs for primary health care.

Adolescent↗
PubMed1987

Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues.

Acetylcholinesterase extracted with Triton X-100 from bovine brain caudate nuclei was purified by affinity chromatography to apparent homogeneity. The purified enzyme was labeled with [3H]diisopropyl fluorophosphate at the active sites and with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a compound which has been shown to be selective for the hydrophobic membrane-binding domains of several other proteins. The subunit structure was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide reduction. After reduction, a single 3H-labeled band at 70 kDa was stained by silver, but most of the 125I label corresponded to a 20-kDa species. Prior to reduction, five 3H-labeled and silver-stained bands were apparent at 70, 140, 160, 260, and greater than 360 kDa. These species were presumed to represent monomer and disulfide-linked oligomers of 70-kDa catalytic subunits. 125I label was selectively associated with the 160-, 260-, greater than 360-, and a 90-kDa species. Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalytic subunit monomers and the second of which contains two disulfide linkages through an intervening 125I-labeled 20-kDa noncatalytic subunit. This 20-kDa subunit is proposed to contain the membrane attachment site. The brain enzyme did not contain components characteristic of the glycolipid anchors of erythrocyte acetylcholinesterases. However, part of the 125I label was associated with fatty acids, indicating that at least a portion of the brain enzyme membrane anchor is composed of nonamino acid components.

Acetylcholinesterase↗
PubMed1987

An interim prosthesis for the glossectomy patient.

The use of a simple patient-held prosthesis for improving speech in a patient with mandible and tongue resections has been described. As subjectively perceived and objectively verified, speech intelligibility improved in a test patient. There were no apparent risks or lengthy therapy. The ease of fabrication and low cost indicate that this prosthesis may be of value in aiding the speech of glossectomy patients.

Aged↗