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Biomedical subjects

G H Thomas

Publications and source records attributed to G H Thomas.

At least 91 records · Page 5Linked to original sources
PubMed1985

Accumulation of N-acetylneuraminic acid (sialic acid) in human fibroblasts cultured in the presence of N-acetylmannosamine.

Human skin fibroblasts incubated for 72 h in medium containing 10 mM N-acetyl-D-mannosamine accumulate material that yields a chromophore in the presence of thiobarbituric acid. This material was tentatively identified as free (unbound) sialic acid due to its reactivity with thiobarbituric acid prior to acid hydrolysis, its solubility in 10% trichloroacetic acid, its chromatographic properties on an anion-exchange column and its enzymatic susceptibility to acylneuraminate pyruvate-lyase. Mass spectrometry analysis established that the accumulated material was, in fact, N-acetylneuraminic acid. Loading studies demonstrated a linear relationship between the amount of N-acetylmannosamine in the medium and the level of sialic acid accumulating within the cells. Cells grown in the absence of N-acetylmannosamine contained an average of 5 nmol free sialic acid/mg protein, while cells cultured for 72 h in 20 mM amounts of this material contained an average of 156.3 nmol free sialic acid/mg protein. When the cells were removed from the N-acetylmannosamine-enriched medium and incubated in regular medium, more than 80% of the accumulated, intracellular sialic acid disappeared within the first 96 h. It was concluded from these data that normal fibroblasts cultured in medium enriched with N-acetylmannosamine store large amounts of N-acetylneuraminic acid and can thus serve as an excellent model for the study of both normal and abnormal sialic acid metabolism, transport, storage and/or metabolic (feedback) regulation in human tissue.

Biological Transport, Active↗
PubMed1985

Identification and partial purification of fibroblast growth factor from the brains of developing rats and leucodystrophic mutant mice.

High titres of fibroblast growth factor activity (assessed by mitogenicity for Balb/c 3T3 fibroblast cells in vitro) have been extracted from the brains of foetal and neonatal rats long before myelinogenesis commences, from the brains of hypomyelinated, leucodystrophic, murine mutants and from normal adult rats. Partial purification, by ion-exchange chromatography and gel filtration, indicates that the brain fibroblast growth factor activity from these sources is associated with very similar basic protein fractions. These results, together with the observation that none of the samples of partially purified basic fibroblast growth factor elicits the experimental allergic encephalomyelitis response attributed to myelin basic protein in the rat, suggest that basic fibroblast growth factor is not a degradation product of myelin basic protein.

Aging↗
PubMed1985

Overproduction of N-acetylneuraminic acid (sialic acid) by sialuria fibroblasts.

Fibroblasts from the original sialuria patient are shown to contain high levels of free sialic acid, i.e., an average of 87 versus a normal average of 2 nmol/mg of protein. Gas liquid chromatography-mass spectrometry analysis confirmed that the accumulated material is N-acetylneuraminic acid. Addition of D(+) glucosamine (0-5 mM) to the media of these cells increased the intracellular free sialic acid concentrations from 74 to 137 nmol/mg protein. In contrast, normal cells treated in an identical manner maintained their normal free sialic acid level of less than 4 nmol/mg protein. Addition of 20 mM N-acetylmannosamine in place of glucosamine resulted in a marked increase in free sialic acid in both the patient and the control, i.e., increases of 157 (from 95 to 252) and 120 (from 3 to 123) nmol/mg protein, respectively. Finally, while normal cells in the presence of glucosamine accumulated high levels of uridine diphosphate N-acetylhexosamine, the patient's cells accumulated much lower amounts of this compound. It is concluded that the elevated sialic acid level in sialuria is due, at least in part, to overproduction of free N-acetylneuraminic acid. Indirect evidence is presented that this may result from either hyperactivity or increased levels of the enzyme (uridine diphosphate N-acetylglucosamine 2-epimerase) that has been shown in other tissues to convert uridine diphosphate N-acetylglucosamine to N-acetylmannosamine.

Cells, Cultured↗
PubMed1984

Treatment of the cbl B form of methylmalonic acidaemia with adenosylcobalamin.

A 30-month-old girl was found to have the cbl B mutant form of methylmalonic aciduria by complementation analysis of fibroblasts. She was unresponsive to hydroxycobalamin and was treated with intramuscular adenosylcobalamin (AdoCbl), the deficient coenzyme, at a dose of 1 mg/24h during a period of clinical stability. Serum cobalamin increased from 770 to 54 200 pg/ml. Mean urinary methylmalonic acid excretion was 409 mg/24 h prior to therapy. There was a transient fall in methylmalonic acid excretion during the first 5 days of therapy (range 167-245 mg/24h) followed by a rise in excretion toward pretreatment levels (range 317-485 mg/24h) during the second week of AdoCbl treatment. There was no change in plasma ammonia, glycine or serum bicarbonate level. We interpret the failure of this child to have a sustained and clinically significant response to AdoCbl as indicating that AdoCbl did not reach or enter the mitochondria intact, or in some other way was unavailable as a coenzyme for the methylmalonyl CoA mutase apoenzyme.

Amino Acid Metabolism, Inborn Errors↗
PubMed1984

Arginine-responsive asymptomatic hyperammonemia in the premature infant.

We found that more than 50% of premature infants have elevated plasma ammonium levels during the first 2 months of life. Ammonium levels were twice normal and were unaccompanied by clinical symptoms of vomiting or lethargy. Ten of these infants were given supplements of arginine (1 to 2 mmol/kg/day PO) for 1 to 2 weeks preceded and followed by control periods. In each infant, plasma ammonium levels fell significantly within 2 days of start of arginine supplementation, and increased once arginine was discontinued. We studied 59 additional premature infants, of whom 26 had normal ammonium levels and 33 were hyperammonemic. Plasma arginine and ornithine levels were significantly lower in the hyperammonemic group, but there was no difference in urinary excretion of arginine or ornithine between groups. Half of the hyperammonemic infants received arginine supplementation between 2 and 8 weeks of age. Plasma ammonium levels in the arginine group was 33 + 1 mumol/L., compared to 45 + 2 mumol/L in the untreated group. Follow-up at 18 months of age showed similar IQ scores in all groups, suggesting that significant neurologic deficits do not result from this transient metabolic defect. The mechanism of the hyperammonemia is unclear.

Ammonia↗
PubMed1984

Early manifestations of multiple sulfatase deficiency.

We describe two boys, presenting by 1 year of age, with developmental delay from birth, mildly coarse facial features, and hepatomegaly. These clinical features were most suggestive of a mucopolysaccharidosis, particularly MPS II. Biochemical studies, including sulfate incorporation in fibroblasts and lysosomal enzyme analyses in fibroblasts, leukocytes, and serum, showed abnormalities in both sulfatide and mucopolysaccharide metabolism and led to the diagnosis of multiple sulfatase deficiency. With time, both patients developed an ichthyotic rash and profound intellectual deterioration. We conclude that findings in the first year of life in some patients with MSD may closely resemble those in patients with a MPS disorder rather than the late infantile form of metachromatic leukodystrophy, as is classically described. Thus, MSD should be considered in the young patient suspected of having a MPS disorder.

Cerebroside-Sulfatase↗
PubMed1983

Deposition of scar tissue in the central nervous system.

Standard parasagittal lesions were placed stereotactically in the cerebral hemispheres of neonatal and adult rats in order to compare scarring in the immature and mature animal. Lesions were examined by light and electron-microscopy and immunofluorescence to study the astrocyte reaction, collagen deposition, and the formation of the basement membrane of the glia limitans. Normal mature scarring characterized by the deposition of collagen, astrocyte end-feet alignment over a glia limitans, and the permanent presence of mesodermal cells (fibroblasts and macrophages) in the core of the lesion, does not occur in wounds before 8-10 days post-partum (dpp). Instead there is no deposition of collagen, and only a transitory astrocyte response occurs with the formation of an interrupted glia limitans. These latter features disappear with time so that the wound is ultimately obliterated by the growth of axons and dendrites through the lesion. Mature scarring is attained over 8-12 dpp when increasing amounts of collagen are deposited and a continuous permanent glia limitans is formed. The acquisition of the mature response to injury from 8-12 dpp may be correlated with the presence of increasing titres of a fibroblast growth factor (FGF), derived from autolytic digestion of injured brain tissue. We have investigated FGF activity using a 3 T 3 fibroblast tissue culture assay to detect mitogenic activity in brain extracts from rats lesioned at different ages and from leukodystrophic mice which have no myelin. Our results show that high titres of FGF are present in the developing brain long before myelination commences, and that normal levels of FGF are found in the brains of leukodystrophic mice which have no myelin. Scarring in brain lesions in these mutants is quite normal.

Animals↗
PubMed1983

Aspartylglucosaminuria in the United States.

Aspartylglucosaminuria (AGU) was diagnosed in two unrelated males with progressive mental retardation, coarse facies and skeletal abnormalities. Until now, this disorder has been described in predominantly Finnish populations with only one previous case reported in the U.S. We conclude that AGU may be more common in non-Finnish populations than the number of reported cases would indicate and should be included in the differential diagnosis in patients with suspected lysosomal storage disorders regardless of their geographical or ethnic backgrounds.

Adolescent↗
PubMed1983

Alterations in cultured fibroblasts of sibs with an infantile form of a free (unbound) sialic acid storage disorder.

Cultured fibroblasts from two sibs with generalized hypertonia, hepatosplenomegaly, and psychomotor retardation within the first year of life were found to have unusual morphologic features. When examined by phase microscopy, the unstained and unfixed cells contained a large number of vacuolated structures whose gross appearance resembled that of a honeycomb in the cell cytoplasm. Electron microscopy studies, following fixation, showed the "honeycombing" to be the result of numerous, closely packed, cytoplasmic, membrane-bound vacuoles. In some of these structures the remains of fibrilogranular material could be detected. Biochemical analysis of crude sonicates of these cells revealed increased levels (4--7 x N) of an acid soluble component that reacted with thiobarbituric acid. Analysis of trimethylsilyl derivatives of this material by gas liquid chromatography and mass spectrometry showed it to be indistinguishable from sialic acid (N-acetylneuraminic acid). Quantitation of this material from the cells of one of the sibs after isolation on a Dowex column yielded 39.8 nmoles of free (unbound) sialic acid per mg protein whereas normal fibroblasts had 1--2 nmoles per mg. Bound sialic acid levels were at the upper limits of normal (24.8 versus 11--23 nmoles per mg protein). The concentration of cytidine monophosphate-sialic acid was normal. After incubation of the patient's fibroblasts with [3H]-N-acetylmannosamine for 72 h, there was a 7-fold increase (compared to normal fibroblasts) in the amount of radioactivity in free sialic acid present in the acid soluble fraction. The amount of labeled, bound sialic acid in the acid-insoluble pool, however, was the same in both patient and control fibroblasts.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1983

Impaired cerebroside sulfate hydrolysis in fibroblasts of sibs with "pseudo" arylsulfatase A deficiency without metachromatic leukodystrophy.

Low arylsulfatase A levels are reported in two siblings, one with a neurologic disability not typical for metachromatic leukodystrophy, the other a healthy 18-year-old female with a normal developmental history. In both individuals, arylsulfatase A levels in white blood cells were 7-8% of control values. Cultured fibroblasts gave low values (8-10% of normal) for both cerebroside sulfatase and arylsulfatase A activities. Other family members had enzyme levels consistent with heterozygote or normal status. Cerebroside sulfate loading tests of cultured fibroblasts in 199-CO2 media were normal for all family members who were tested. In MEM-HEPES media, however, cells from the two arylsulfatase A deficient siblings showed attenuated sulfolipid catabolism. Additional clinical and laboratory studies on these individuals failed to demonstrate any features suggestive of metachromatic leukodystrophy, i.e., normal nerve conduction velocities, normal sural nerve biopsy results, and normal urinary sulfatide excretion. It is concluded that the neurologic abnormalities in the one sibling are not the result of the low enzyme activity and that both individuals represent examples of pseudo arylsulfatase A deficiency (arylsulfatase A deficiency without metachromatic leukodystrophy). These results thus call into question the ability of the high-sensitivity cerebroside sulfate loading test as carried out in MEM-HEPES media to differentiate pathologically significant defects i.e., metachromatic leukodystrophy from benign "pseudo-deficiencies."

Adolescent↗
PubMed1982

Early events in the response of fast skeletal muscle to chronic low-frequency stimulation. Polyamine biosynthesis and protein phosphorylation.

The concentrations of putrescine, spermidine and spermine and the activities of ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAM-D) were investigated in fast muscle subjected to chronic low-frequency electrical stimulation. Both ODC and SAM-D activities increased markedly between 18 and 48 h of stimulation. Changes in enzyme activities were followed by phasic elevations in the concentrations of putrescine, spermidine and spermine. Peak levels were reached first by putrescine at 3-4 days, followed by spermidine at about 9 days and then by spermine at about 11 days. A possible relationship was sought between these events and changes produced in vitro in the phosphorylation pattern of cytoplasmic proteins and the total activity of cyclic AMP-dependent protein kinase. However, during the early stages of stimulation, no prominent changes were seen either in the phosphorylation pattern or in the activity of cyclic AMP-dependent protein kinase. These characteristics changed significantly at a later stage (by 12 days of stimulation) and became indistinguishable from those of slow muscle by 3 to 4 weeks of stimulation.

Adenosylmethionine Decarboxylase↗
PubMed1982

Restriction enzyme cleavage of ultraviolet-damaged DNA.

SV40 and pBR322 DNAs damaged by ultraviolet light were cleaved abnormally by several restriction enzymes because of damage to pyrimidines in the recognition sequences. The use of a tandemly duplicated plasmid provided a particularly sensitive target molecule for detecting pyrimidine dimers and other possible photoproducts. The relative efficiency with which cleavage was blocked (HindIII greater than TaqI greater than EcoRI greater than BamI greater than SalI much greater than Hha I, Hae III) corresponds approximately to the relative frequency of pyrimidine dimer formation in the recognition sequences, but at a slightly higher frequency in potential sites for the non-cyclobutane T-C product. The pyrimidine dimers appear to have a range of influence that extends 1 to 3 basepairs along the DNA molecule. These effects provide clues to the way DNA damage from mutagens and carcinogens can interfere with specific enzyme-DNA interactions.

Base Sequence↗
PubMed1982

Infantile form of sialic acid storage disorder: clinical, ultrastructural, and biochemical studies in two siblings.

We describe two sibs with coarse facies, hepatosplenomegaly, prominent psychomotor retardation and unexpectedly fair complexion. Ultrastructural studies of conjunctival, skin, bone marrow and liver biopsies from these individuals showed generalized lysosomal storage of polysaccharide-like material, i.e., membrane bound inclusions containing sparse, fibrillo-granular material. Biochemical analyses of urine and cultured fibroblasts from these patients revealed increased levels of free (unbound) sialic acid. The ultrastructural and biochemical findings in these sibs are similar to those previously found in Salla disease, however, the clinical course is much more severe. It is concluded that these children represent a new pathogenetic entity whose basic defect is still to be defined.

Bone Marrow↗
PubMed1982

Nonuniform deficiency of hexosaminidase A in tissues and fluids of two unrelated individuals.

Serum samples from two unrelated, clinically normal individuals lacked detectable hexosaminidase A by heat inactivation and electrophoretic analysis. In contrast, 15 and 17% of the hexosaminidase in their leukocytes and 23 and 26% of the hexosaminidase of their cultured fibroblasts had the heat stability and electrophoretic properties of the A form of this enzyme. An in vitro measurement of fibroblasts GM2 ganglioside-beta-galactosaminidase was in the range expected for Tay-Sachs disease (TSD) heterozygotes (2.5 and 3.1 versus a normal mean of 3.7). In contrast, fibroblasts from a patient with TSD, analyzed in an identical fashion, contained no detectable activity. Ten days after addition of labeled GM2 ganglioside to the medium of the cultured fibroblasts, 43 and 59% of the radioactivity taken up by the cells of these patients remained as unhydrolyzed ganglioside as compared with 94% in TSD fibroblasts and 42% in control cells. An analysis of sphingolipid composition by high performance liquid chromatography although the endogenous level of GM2 was elevated in TSD fibroblasts (0.39 nmoles/mg protein) there was no increase in the cells of these patients (0 and 0.12 versus control of 0.17 nmoles/mg protein). Finally, the synthesis of hexosaminidase was examined by an electrophoretic analysis of immunoprecipitates of the enzyme precursors that had been radiolabeled by culturing fibroblasts in medium containing [3H]-leucine. These studies revealed a normal pattern of biosynthesis, processing and secretion of the alpha and beta chains. The ratio of the alpha chain to the beta chain, however, was in the range expected for TSD heterozygotes.

Adult↗
PubMed1982

Two clonal cell populations (mosaicism) in a 46,XY male with mucolipidosis II (I-cell disease)--an autosomal recessive disorder.

Cultured fibroblasts from a 46,XY male with an atypical form of mucolipidosis II (I-cell disease) had two distinct phenotypes. One population of these fibroblasts had the morphological and biochemical features characteristic of I-cell disease, while the remaining cells were indistinguishable from normal fibroblasts. Direct evidence that the patient was a mosaic, having two cell populations, was provided by the establishment of pure, stable clones of both wild type and I-cell fibroblasts from each of two biopsies obtained several months apart. Additionally, it was shown that the I-cell fibroblasts lacked UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosaminylphosphotransferase while the morphologically normal cells contained levels of this enzyme just below or at the lower end of the normal range.

Cells, Cultured↗