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T G Warner

Publications and source records attributed to T G Warner.

At least 37 records · Page 2Linked to original sources
PubMed1986

Diagnosis and characterization of GM 2 gangliosidosis type II (Sandhoff disease) by analysis of the accumulating N-acetyl-glucosaminyl oligosaccharides with high performance liquid chromatography.

The N-acetyl-glucosaminyl oligosaccharides excreted in urine and accumulating in tissues of Sandhoff disease patients have been analyzed and characterized using a combination of high performance liquid chromatography and 500 MHz proton magnetic resonance spectroscopy. Delineation between infantile and juvenile onset forms of the disease was possible, as the latter forms had 6- to 13-fold lower levels of urinary oligosaccharides. Patients from a geographically isolated population deme in the La Rioja region of Argentina had urinary oligosaccharides similar to unrelated non-Argentinean patients with identical clinical phenotype. Together, these results indicate that the urinary oligosaccharides serve as useful indicators of the mutation differences or clinical heterogeneity within this disease only in cases of markedly differing clinical presentation. Analysis of the accumulating metabolites in liver, kidney, pancreas, lung and spleen, showed a similar oligosaccharide pattern which differed dramatically from brain. These results suggest the possibility of tissue specific regulation of oligosaccharide biosynthesis since there are notable differences between neural and visceral tissues.

Acetylglucosamine↗
PubMed1985

Characterization and analysis of branched-chain N-acetylglucosaminyl oligosaccharides accumulating in Sandhoff disease tissue. Evidence that biantennary bisected oligosaccharide side chains of glycoproteins are abundant substrates for lysosomes.

Branched chain N-acetylglucosaminyl oligosaccharides accumulating in visceral and neural tissues of two patients with Sandhoff disease were isolated and quantified using high performance liquid chromatography. Detailed structural analysis of the three most abundant fractions, oligosaccharides 4, 5, and 6, was carried out using 360 MHz proton magnetic resonance spectroscopy. The biantennary bisected heptasaccharide, oligosaccharide 6, was ubiquitously distributed and a major component of the stored oligosaccharides in all tissues analyzed including, liver, spleen, kidney, lung, pancreas, and brain. This analysis indicates that glycoproteins containing biantennary bisected oligosaccharide side chains are abundant substrates for lysosomes in human tissues. Moreover, oligosaccharide 6 was the predominant storage product in brain comprising 70% of the total accumulating water-soluble glycoconjugates. Oligosaccharide 5, a triantennary heptasaccharide, had a similar distribution in visceral tissues and it was the major storage product in pancreas but was at very low levels in brain. These results suggest that the biosynthetic enzymes, GlcNAc transferase III (Narasimham, S. (1982) J. Biol. Chem. 257, 10235-10242) and IV (Gleeson, P.A., and Schachter, H. (1983) J. Biol. Chem. 258, 6162-6173), which are responsible for synthesis of these structures, have a generalized distribution with varying levels of expression in human viscera, moreover, transferase IV may have limited expression in neural tissue. The proposed structures for the branched-chain compounds are as follows. (formula; see text)

Acetylglucosamine↗
PubMed1984

Alpha-mannosidosis: analysis of urinary oligosaccharides with high performance liquid chromatography and diagnosis of a case with unusually mild presentation.

Mannose containing oligosaccharides (OS) excreted in the urine of patients with alpha-mannosidosis have been analyzed with high performance liquid chromatography (HPLC). The HPLC method provides a highly sensitive assay for detection of the urinary oligosaccharides and was employed for diagnosis of a fifteen-year-old female with an unusually mild presentation of the disease. Dysostosis multiplex and coarse facies were absent; mental impairment was particularly mild. The elution profile of the urinary OS from this patient and two, more severely affected, patients with mannosidosis were nearly identical, containing nine major OS fractions. The concentrations of the OS were eight fold lower in our patient but, when calculated relative to creatinine, the levels of the urinary OS of all patients were similar.

Adolescent↗
PubMed1984

Stable isotope dilution analysis of galactitol in amniotic fluid: an accurate approach to the prenatal diagnosis of galactosemia.

A stable isotope dilution assay for galactitol in amniotic fluid has been developed using selected ion monitoring chemical ionization gas chromatography-mass spectrometry of the hexaacetate derivative. [1,1-2H2]Galactitol was synthesized for use as the internal standard. Galactitol is a component of normal amniotic fluid with a mean concentration of 0.70 +/- 0.18 mumol/liter (n = 5). The amniotic fluid of a fetus with galactosemia had a concentration of 7.96 mumol/liter. Mannitol, sorbitol, and inositol were also found to be normal constituents of amniotic fluid. This stable isotope dilution assay is a rapid accurate method for measurement of galactitol in amniotic fluid for prenatal diagnosis of galactosemia.

Amniotic Fluid↗
PubMed1983

Diagnosis of GM1 gangliosidosis based on detection of urinary oligosaccharides with high performance liquid chromatography.

An improved, rapid, and sensitive method for the biochemical diagnosis of GM1 gangliosidosis based on the detection and quantification of urinary galactosyl-oligosaccharides with high performance liquid chromatography was developed. The oligosaccharides, in 50-100 microliters of urine, were converted to radioactively labeled oligosaccharide-alditols with NaB3H4 and fractionated on commercial silica-amine bonded, high performance liquid chromatography columns. Delineation between infantile, juvenile, and adult onset subtypes of GM1 gangliosidosis was possible by analysis of the levels of the excreted oligosaccharides and their characteristic elution profile. Infantile and juvenile patients contain identical numbers of oligosaccharide fractions (13 resolved components) but can be distinguished by 3-10-fold lower levels of oligosaccharides in juvenile patients and, in some cases by a disproportionately lower concentration of high molecular weight compounds. Adult onset patients were distinguished by substantially lower concentrations of urinary oligosaccharides, 130-180-fold below those in infantile patients, and the apparent absence of high molecular weight oligosaccharides.

Adult↗
PubMed1983

Prenatal diagnosis of GM1 gangliosidosis by detection of galactosyl-oligosaccharides in amniotic fluid with high-performance liquid chromatography.

Prenatal diagnosis of infantile GM1 gangliosidosis was accomplished by analyzing the galactosyl-oligosaccharides accumulating in amniotic fluid with high-performance liquid chromatography (HPLC) at 14 weeks gestation. The pattern of amniotic oligosaccharides was nearly identical with that in neonatal urine in GM1 gangliosidosis but the concentrations were about one-fiftieth of that in urine, necessitating a highly sensitive assay.

Amniotic Fluid↗
PubMed1982

Structure analysis of the major oligosaccharides accumulating in canine GM1 gangliosidosis liver.

A homologous series of structurally related, high molecular weight oligosaccharides have been isolated and purified from the livers of a mixed breed of Beagle dogs affected with GM1 gangliosidosis. Five individual oligosaccharide fractions were purified by charcoal chromatography, preparative silicic acid thin layer chromatography, and gel filtration chromatography. Molecular size determinations revealed that these oligosaccharides contained 6, 9, 11, and 13 sugar residues, respectively. Detailed structure analysis was carried out on the most abundant fractions, oligosaccharides 1,2 and 3 (OS 1,2 and OS 3) using permethylation analysis and 360-MHz proton magnetic resonance spectroscopy coupled with sequential exoglycosidase degradation. OS 1,2 was a mixture of two linear isomeric hexasaccharides and OS 3 was a nonasaccharide containing a bianntenary branched mannosyl core. The proposed structures are: (formula see text) These compounds are nearly identical with the oligosaccharides stored in human GM1 gangliosidosis liver but they differ from the human compounds uniquely since they contain 2 GlcNAc residues at the reducing terminus instead of 1, suggesting that there may be significant differences in glycoprotein metabolism or structure between mammalian species.

Animals↗
PubMed1981

Purification of the lysosomal acid lipase from human liver and its role in lysosomal lipid hydrolysis.

The lysosomal acid lipase has been purified 2,500-fold to near homogeneity from human liver. The enzyme was converted to a soluble form by extraction of frozen tissue with Triton X-100. The enzyme, which required Triton X-100 in buffers at all purification steps for optimal yields, was stabilized by the inclusion of 33% ethylene glycol during purification. Lectin chromatography on concanavalin A-Sepharose followed by chromatography on carboxymethyl-cellulose and Sephadex G-150 provided the highly purified enzyme in 17% yield. Sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the minimum molecular weight was about 29,000 +/- 1,000. Minor protein contaminants at Mr = 58,500, 14,700 and 13,900 were present in the final preparation. A single protein band, with enzyme activity, was observed in nondenaturing acrylamide gels containing Triton X-100. Gel filtration on Sephadex G-150 in the presence of Triton X-100 gave an apparent molecular weight of about 125,000 +/- 13,000. Trioleoylglycerol, cholesterol oleate, and 1,2- and 1,3-dioleoylglycerols were substrates for the purified enzyme giving apparent Vmax values of 5,400, 1,400, 19,400, and 22,100 nmol min-1 mg of protein-1, respectively, and Km values of 0.8, 0.8, 0.9, and 1.2 mM, respectively. The recoveries of both trioleoylglycerol and cholesterol oleate hydrolytic activities were nearly identical at each purification step, suggesting that the acid lipase as single enzyme is responsible for lysosomal hydrolysis of the neutral lipids. Monooleoylglycerols were not substrates for the enzyme.

Drug Stability↗
PubMed1980

Cystic calculi.

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Animals↗
PubMed1980

Sialidosis: delineation of subtypes by neuraminidase assay.

A sensitive assay for acid neuraminidase using 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid is described. In skin fibroblasts, patients with sialidosis Types 1 and 2 have severe deficiencies of neuraminidase activity compared with controls. Patients with Type 1 sialidosis have activities which are 10 times higher than those with Type 2 sialidosis, in keeping with their milder clinical involvement. Two Italian patients with Type I sialidosis had a Km which was one-sixth normal; the other patients had a Km in the normal range.

Cells, Cultured↗
PubMed1980

Separation and characterization of the acid lipase and neutral esterases from human liver.

Electrophoresis of human liver homogenates followed by reaction with 4-methylumbelliferyl palmitate reveals the presence of two major electrophoretic forms with esterase (lipase) activity toward this substrate. The two enzymes were isolated and partially purified based on their solubility differences and their relative affinities for the lectin column concanavalin A-Sepharose 4B. Lipase A was particulate with an acidic pH optimum (5.2) and could be solubilized with the non-ionic surfactant Triton X-100. Lipase B was soluble and had a more neutral pH optimum (6.3--6.6). Both forms bound to immobilized concanavalin A and could be specifically eluted. Buffers containing alpha-methylmannoside eluted lipase B, and buffers with alpha-methylmannoside and Triton X-100 eluted lipase A, giving a 22- and 257-fold purification, respectively, over whole-tissue homogenates. Cholesterol oleate, trioleoylglycerol, and 4-methylumbelliferyl palmitate were substrates for solubilized lipase A. Lipase B hydrolyzed 4-methylum-belliferyl palmitate but not trioleoylglycerol or cholesterol oleate. Lipase B was more thermolabile than lipase A, and it was selectively inhibited by diethyl-p-nitrophenyl phosphate at low concentrations. We conclude that lipase A and B are distinctly different enzymes and that they are probably not related polymorphic forms of one another.

Chromatography, Affinity↗
PubMed1979

Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis.

A procedure for the synthesis of the fluorogenic substrate analogue 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid for the human acid neuraminidase has been developed. The substrate was employed for the characterization of the enzyme in sonicates of cultured human skin fibroblasts and for enzymatic detection of the neuraminidase deficiency in the neurological storage disorder, sialidosis. Synthesis was accomplished by reacting 2-deoxy-2-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in acetonitrile at room temperature. The coupled product was purified on silicic acid chromatography, followed by base-catalyzed removal of the O-acetyl and methoxy blocking groups, and with additional purification of the hydrolyzed product on silicic acid. The overall yield, based on N-acetylneuraminic acid, was 37%. Under linear assay conditions, at pH 4.3, the apparent maximal velocities (nmol (mg of protein)-1 h-1) for normal fibroblasts were 58--115, 0.2--1.8 for sialidosis fibroblasts, and 28--38 for obligate heterozygotes. The apparent Km for normals was 0.13 mM.

Adult↗
PubMed1979

An improved method for the isolation and assay of the acid lipase from human liver.

An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.

Carbon Radioisotopes↗
PubMed1977

Partial characterization of the bile salt-dependent triacylglycerol lipase from the leopard shark pancreas.

Leopard shark triacylglycerol lipase has been characterized as a crude pancreatic preparation. The enzyme demonstrated an absolute requirement for trihydroxy bile salts for activity with natural bile salts of the shark giving a 4-fold greater stimulation of activity than pure sodium taurocholate. Bile salts also protected the enzyme from apparent inactivation by p-chloromercuribenzoate and trypsin treatment. The shark lipase demonstrated a temperature optimum of 36 degrees C and was rapidly inactivated at 50 degrees C even in the presence of bile salts. Divalent metal ions were required for activity with Ca2+ providing the greatest stimulation. At 22 degrees C, pH 8.5 and in the presence of natural bile salts, the apparent V was about 0.6 mumol fatty acid released/min per mg protein. The shark enzyme hydrolyzed over 90% of the fatty acids from trioleovylglycerol and methyl esters of pancreatic lipase-resistant fatty acids were hydrolyzed at the same rate as typical fatty acid methyl esters. Hydrolysis of triacylglycerol proceeded about ten-times faster than wax ester hydrolysis. The kinetic properties of the leopard shark enzyme were compared to other bile salt-dependent lipolytic enzymes. Pancreatic lipase activity was not detected.

Animals↗
PubMed1977

An improved method for the preparation of unsaturated phosphatidylcholines: acylation of sn-glycero-3-phosphorylcholine in the presence of sodium methylsulfinylmethide.

An improved method for the partial chemical synthesis of unsaturated and radioactively labeled phosphatidylcholines is described. This procedure offers advantages over conventional acylation methods in that it can be carried out on a millimole or micromole scale under mild conditions and it does not require a large excess of the fatty acid acylating reagent. In this procedure sn-glycero-3-phosphorylcholine is reacted with twice the theoretical amount of fatty acid imidazolide and sodium methylsulfinylmethide in dimethylsulfoxide for several minutes at 17 degrees C. Phosphatidylcholine, which was purified by gradient-elution chromatography on silicic acid, was isolated in 60% yield and was estimated to be about 99% pure. The preparations of 1,2-dioleoyl-, 1,2-dilinoleoyl-, and 1,2-dilinolenoyl-sn-glycero-3-phosphorylcholine are described. The reaction was also carried out on a small scale for the preparation of high specific activity 1,2-di[ 1'(-14)C]oleoyl-sn-glycero-3-phosphorylcholine in 38% yield with a specific activity of about 9.7 muCi/mumol.

Acylation↗
PubMed1975

Action of the highly purified, membrane-bound enzyme phosphatidylserine decarboxylase Escherichia coli toward phosphatidylserine in mixed micelles and erythrocyte ghosts in the presence of surfactant.

Phosphatidylserine decarboxylase, Escheichia coli, was purified to near-homogeneity by the procedure of Dowhan, W., Wickner, W. T., and Kennedy, E. P. ((1974) J. Biol. Chem. 249, 3079-3084) and assayed by following the production of CO2 using gas chromatography. The purified enzyme has an absolute requirement for the surfactant Triton X-100. The function of Triton in the assay is evaluated and a kinetic scheme describing the action of this membrane-bound enzyme in the micellar system provided by the surfactant is presented. According to this scheme, the enzyme first binds to a mixed micelle, composed of phosphatidylserine and Triton, where the dissociation constant is KSA. The enzyme, now part of the mixed micelle, then binds the substrate phosphatidylserine in its active site and this binding is related to the Michaelis constant, KMB. KSA, expressed as the sum of the molar concentrations of Triton and phosphatidylserine, is about 0.04 M. KMB, expressed as the mole fraction of phosphatidylserine in the mixed micelles, is about 0.03. Phosphatidylserine decarboxylase activity toward phosphatidylserine in human erythrocyte ghosts was also determined. The amount of phsophatidylserine converted to phosphatidylethanolamine and CO2 was found to be related to the amount of phosphatidylserine solubilized from the membrane by Triton X-100. In the absence of Triton, no significant activity of the enzyme toward the ghosts was detected even after subjecting the ghosts to lyophilization, homogenization, or sonication.

Binding Sites↗