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Biomedical subjects

J E Evans

Publications and source records attributed to J E Evans.

16 recordsLinked to original sources
PubMed1992

A human lysosomal alpha(1----6)-mannosidase active on the branched trimannosyl core of complex glycans.

Normal human fibroblasts and fibroblasts from a patient with alpha-mannosidosis were grown in the presence or absence of 100 microM swainsonine for 7 days. Accumulated oligosaccharides were isolated and analysed by high performance liquid chromatography (HPLC) and methylation analysis. Man alpha 1----3Man beta 1----4GlcNAc and Man alpha 1----2Man alpha 1----3-Man beta 1----4GlcNAc (where Man is D-mannose and GlcNAc is N-acetyl-D-glucosamine) comprised greater than 80% of the total oligosaccharides in untreated mannosidosis cells. However, Man alpha 1----6[Man alpha 1----3]Man beta 1----4GlcNAc was the major Man3GlcNAc isomer present after 7 days of swainsonine treatment. No mannose-containing oligosaccharides were detected in control fibroblasts in the absence of swainsonine but, in its presence, oligosaccharides containing 2-9 mannose residues accumulated. Man alpha 1----6[Man alpha 1----3]-Man alpha 1----6[Man alpha 1----3]Man beta 1----4GlcNAc and Man alpha 1----6-[Man alpha 1----3]Man beta 1----4GlcNAc were the major components (67%). Surprisingly, Man alpha 1----3Man beta 1----4GlcNAc was only observed in swainsonine-treated control cells during the recovery period after removal of swainsonine. These studies suggest the presence of a second lysosomal alpha-mannosidase activity which is unaffected in genetic alpha-mannosidosis, but is inhibited by swainsonine. This enzyme would cleave the alpha(1----6)-linked mannose residue from branched Man3GlcNAc to form Man alpha 1----3Man beta 1----4GlcNAc. To confirm this hypothesis, fractions from alpha-mannosidosis and control fibroblasts that bound to concanavalin A (ConA)-Sepharose and were eluted with 0.5 M alpha-methyl mannoside were incubated at pH 4.0 with Man alpha 1----6[Man alpha 1----3]Man beta 1----4-GlcNAc. As anticipated, Man alpha 1----3Man beta 1----4GlcNAc was the sole product using enzyme from mannosidosis fibroblasts, while the major product from control fibroblasts was Man alpha 1----6Man beta 1----4GlcNAc. This confirmed the presence of a swainsonine-inhibitable alpha(1----6)-mannosidase activity unaffected by the disease. The differing substrate specificities of the alpha(1----6)-mannosidase and the major lysosomal alpha-mannosidase indicate that the alpha(1----6)-mannosidase plays an important role in the generation of the oligosaccharides accumulated in alpha-mannosidosis patients.

Carbohydrate Sequence↗
PubMed1992

Bioavailability studies of drugs with nonlinear pharmacokinetics: I. Tracer dose AUC varies directly with serum concentration.

The authors show that for a drug cleared by one enzyme the area under the serum concentration-time curve from time 0 to infinity (AUC0-INF) of a test dose can be expressed as AUC0-INF = TD x F x (Km + C)/Vmax where TD is test dose size, F is fraction absorbed, C is drug serum concentration at the time of the study, and Km and Vmax are the Michaelis constant and maximum velocity of the enzyme. This equation predicts the AUC0-INF produced by a given tracer dose of drug will vary directly with C in drugs with nonlinear pharmacokinetic properties (i.e., drugs whose value for C approaches or exceeds Km) if C is held constant by administration of tracer and maintenance doses of drug. The AUC0-INF produced by intravenous tracer doses of 150 mg of 13C15N2-sodium phenytoin was determined in 15 subjects at 30 different values of C. AUC0-INF showed a high degree of direct linear correlation with C (AUC0-INF (ug x hr/mL) = 35.4 + 8.1 x C (ug/mL), r = 0.885, P < .0001). Consequences of this observation for relative bioavailability studies of drugs with nonlinear pharmacokinetic properties are discussed.

Adolescent↗
PubMed1990

Cysteine-108 is an acylation site in myelin proteolipid protein.

Myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids. A large proportion of these acyl moieties are bound in thioester linkages, as demonstrated by alkylation of newly formed SH groups upon deacylation. To identify the Cys residue(s) involved in the thioester linkage(s), reduced and carboxyamidomethylated proteolipid protein was labeled with [14C]iodoacetamide upon deacylation with neutral hydroxylamine. The labeled protein was digested with trypsin or pepsin, and peptides analyzed by RP-HPLC. Identification of the isolated radioactive peptides by amino acid analysis, peptide sequencing and/or fast-atom bombardment-mass spectrometry revealed that Cys108 in the bovine PLP sequence is an acylated site. The sequence surrounding the palmitoylation site in the myelin PLP is strikingly similar to that found in rhodopsin. Furthermore, as in rhodopsin and other members of the G protein-coupled receptor family, this Cys residue is located within a hydrophilic, basic, and possibly cytoplasmic, domain.

Acylation↗
PubMed1979

Subunit localizations of zinc(II) in DNA-dependent RNA polymerase from Escherichia coli B.

RNA Polymerase holoenzyme and core enzyme from Escherichia coli B have been shown to contain two zinc ions. Flameless atomic absorption spectroscopy of the isolated core subunits indicated that one zinc ion is localized on the beta subunit and the other is bound on the beta' subunit. Atomic fluorescence spectroscopy showed that prolonged dialysis of the metalloenzyme against 0.01 M o-phenanthroline resulted in the removal of both zinc(II) ions with accompanying loss of enzymatic activity. The activity of the apoenzyme was observed to be completely restored by readdition of zinc(II) and partially restored by cobalt(II).

Apoenzymes↗
PubMed1979

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography.

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in combination with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10(3) concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.

Chromatography, High Pressure Liquid↗
PubMed1978

Rapid and sensitive determination of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography.

The separation and quantitative analysis of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography (HPLC) are described. The substituted unsaturated disaccharides which result from digestion of chondroitin sulfates with chondroitinase are quickly separated on polar absorbents such as silica gel. The UV absorption properties of these unsaturated disaccharides permit UV measurement with detection limits of approximately 100 ng. Their separation by HPLC facilitates the use of enzymatic methods for the determination of chondroitin sulfates A, B and C. The potential of this method in clinical application is demonstrated by quantitative assays of glycosaminoglycans from a normal urine and urine from a patient with Hunter syndrome. The results are consistent with amount of isomeric chondroitin sulfates found in comparable urines by others.

Autoanalysis↗
PubMed1978

Occurrence of novel branched-chain fatty acids in Refsum's disease.

Two novel branched-chain fatty acids, which appear to be unsaturated analogs of phytanic acid, have been observed in sera and urine of patients with Refsum's disease. They occur in both phospholipids and neutral lipids, and have been isolated and characterized.

Eicosanoic Acids↗
PubMed1977

Ewing's tumour: uncommon presentation of an uncommon tumour.

A case of Ewing's sarcoma presenting with high cervical cord compression is described and disseminated disease was present at the time of diagnosis. A combination of radiotherapy and chemotherapy did not result in a cure. The current treatment of Ewing's sarcoma is briefly reviewed.

Adolescent↗
PubMed1976

High-performance liquid chromatography of phosphatidylcholine and sphingomyelin with detection in the region of 200 nm.

A sensitive method for the separation of phosphatidylcholine and sphingomyelin by high-performance liquid chromatographic analysis is described. The elution of the phospholipids from a microparticulate (10 mum) silica-gel chromatographic column was monitored with an ultraviolet spectromonitor at 203 nm. Acetonitrile/methanol/water (65:21:14, by vol.) was used as the solvent. It was shown by using synthetic phosphatidylcholines of knowm fatty acid composition and of varying degree of unsaturation that the absorption at 203 nm was primarily due to the isolated double bonds and the response measured varied with the degree of unsaturation. Approx. 1 nmol of phosphatidylcholine, containing at least one double bond per molecule, can be detected. The amounts of phosphatidylcholine and sphingomyelin could be determined by high-performance liquid chromatography and ultraviolet absorption if the apparent extinction coefficient of the material analyzed was established. Alternatively, peaks were collected and the phospholipids were determined by the analysis of phosphorus. The analysis of phosphatidylcholine and sphingomyelin present in the lipid extracts from animal tissues, blood and amniotic fluids were made without interference from other phospholipids or ultraviolet-absorbing material. The method described here is complementary to the high-performance liquid chromatographic method described previously for the analysis of ethanolamine-containing phosphoglycerides and serine-containing phosphoglycerides [Jungalwala, Turel, Evans and McCluer (1975) Biochem. J. 145, 517-526].

Amniotic Fluid↗
PubMed1976

The affinity labeling of amino acids in or about the active center of DNA-dependent DNA polymerase I.

The use of an affinity label and an inhibitor that shows relative specificity for one amino acid has led to the identification of two amino acid residues in or near the active center of DNA-dependent DNA polymerase I. [35S]-beta-D-Ribosyl-6-methylthiopurine periodate oxidation product ([35S]MMPR-OP) and [14C]phenylglyoxal ([14C]PG) were used to elucidate the presence of a single lysine and arginine in or about the active center of the enzyme.

Amino Acids↗
PubMed1976

Quantitative analysis of brain galactosylceramides by high performance liquid chromatography of their perbenzoyl derivatives.

A high performance liquid chromatographic (HPLC) method for analysis of galactosylceramides as their benzyol derivates has been devised. Samples containing 10-150 nmoles of monohexoslceramides are benzoylated by heating for 60 min at 60 degree C in 0.5 ml of 10% (v/v) Benzoyl chloride in pyridine. The products are purified by solvent distribution and analyzed by SPLC. The benzoylated cerebrosides with nonhydroxy fatty acids are separated from those with hydroxy fatty acids on a Zipax column with 7% ethyl acetate in hexane as a solvent and UV absorption at 280 nm is recorded. This isocratic procedure can be applied directly to chloroform-methanol procedure can be applied directly to chloroform-methanol extracts of adult brain with a relative standard deviation of 3.0% for cerebrosides with nonhydroxy fatty acids and 4.0% for cerebrosides with hydroxy fatty acids. Sulfatides do not interfere in the assay and can be converted to cerebrosides after desulfation by mild acid methanolysis. Benzoylated glucosyl- and galactosylceramides can be separated on a MicroPak NH2 column with 1.5% 2-propanol in cyclopentane as the chromatographic solvent.

Animals↗
PubMed1975

Sensitive analysis of ethanolamine- and serine-containing phosphoglycerides by high-performance liquid chromatography.

A highly sensitive method for the separation and quantitative measurement of phospholipids containing primary amino groups, such as phosphatidylethanolamine, phosphatidylserine and lysophosphatidylethanolamine, is described. The method involves a simple and quantitative derivative formation of the phospholipids containing amino groups to their u.v.-absorbing biphenylcarbonyl derivatives. These have molar extinction coefficients of about 23,000 at 268nm. The phospholipid derivatives are then separated and non-destructively determined by high-performance liquid chromatography. The amino phospholipids containing vinyl ether bonds (plasmalogens) can be determined separately from the diacyl- and alkylacyl-amino phospholipids. The lower limit of detection by high-performance liquid-chromatographic analysis of the phospholipid derivatives is about 10-13pmol or 0.3-0.4ng of phospholipid P. The quantitative range of derivative formation and analysis by high-performance liquid chromatography of the phospholipids containing amino groups was shown to be 10-500nmol. The method was shown to be applicable to the analysis of phospholipids containing amino groups in tissue samples.

Biphenyl Compounds↗