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G H Thomas

Publications and source records attributed to G H Thomas.

At least 73 records · Page 4Linked to original sources
PubMed1989

Correction of sphingomyelinase deficiency in Niemann-Pick type C fibroblasts by removal of lipoprotein fraction from culture media.

The average sphingomyelinase activity of fibroblasts obtained from 12 Niemann-Pick type C patients was 37.9% of that of normal fibroblasts (27.2 versus 72 nmol (mg protein)-1 h-1) when the cells were cultured in minimum essential medium containing 13% fetal bovine serum. Following replacement of the above medium with medium in which the lipoprotein fraction had been removed from the fetal bovine serum, the sphingomyelinase activity rose over a 7-day period from about 1/3 of normal to normal or above. Upon reintroduction of medium containing 10% fetal bovine serum which had not been extracted, the sphingomyelinase activity of the Niemann-Pick type C cells again fell within 48 h to 30% of the normal controls. In contrast, cell lines from patients with either Niemann-Pick Type A or B were not influenced by the presence or the absence of lipoprotein, i.e. lacked sphingomyelinase activity under all culture conditions examined. Histochemical staining with filipin showed an inverse relationship between the sphingomyelinase activity and intracellular, free, unesterified, cholesterol level. Moreover, immunochemical staining with an antibody against a lysosomal membrane protein provided direct evidence that the accumulation of unesterified cholesterol in cells cultured in regular (non-extracted) medium occurred within lysosomes and/or related organelles.

Cells, Cultured↗
PubMed1989

Evidence for non-lysosomal storage of N-acetylneuraminic acid (sialic acid) in sialuria fibroblasts.

The results of the investigations reported here indicate that patients affected with the infantile sialic acid storage disorder (ISSD) and the original French sialuria patient suffer from distinct and fundamentally different disorders. While phase microscopy and immunochemical studies demonstrated abnormal storage within intracellular inclusions in ISSD cells, no morphological evidence of storage within any subcellular organelles was found in the sialuria cells. Moreover, comparative subcellular fractionation studies on gradients of colloidal silica showed the excess sialic acid in ISSD cells to be located within the light (buoyant) lysosomal fraction, while the excessive, free sialic acid in the sialuria cells was found in the cytoplasmic fraction with no increased storage within the lysosomal fractions. It is concluded that the sialic acid abnormalities in ISSD and the French type of sialuria are the result of very different biochemical and genetically unrelated abnormalities.

Fibroblasts↗
PubMed1989

A deletion map of the WAGR region on chromosome 11.

The WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) region has been assigned to chromosome 11p13 on the basis of overlapping constitutional deletions found in affected individuals. We have utilized 31 DNA probes which map to the WAGR deletion region, together with six reference loci and 13 WAGR-related deletions, to subdivide this area into 16 intervals. Specific intervals have been correlated with phenotypic features, leading to the identification of individual subregions for the aniridia and Wilms tumor loci. Delineation, by specific probes, of multiple intervals above and below the critical region and of five intervals within the overlap area provides a framework map for molecular characterization of WAGR gene loci and of deletion boundary regions.

Abnormalities, Multiple↗
PubMed1989

Cancer in relatives of leukemic patients with chromosomal rearrangements at rare (heritable) fragile-site locations in their malignant cells.

The cancer occurrence in relatives (N = 407) of 40 case probands (who had leukemia and rearrangements at the same chromosomal location as at least one of 23 recognized rare [heritable] autosomal fragile sites [Sutherland and Mattei 1987]) was compared both to cancer occurrence in relatives (N = 390) of 40 control probands (who had leukemia or other hematologic illness but no recognized chromosomal rearrangements) and to cancer incidence in the general population of the United States. Fragile-site carrier status was not determined in case or control probands. No significant excess of cancer in case relatives, compared with either control relatives or to general (SEER) population expectancies, was found. Furthermore, there was neither evidence of cancer at younger ages, when cases were compared with control relatives, nor an excess of cancer at multiple sites. Male relatives of cases did, however, show a small excess of cancer, especially in older age groups. There was a slight, but not statistically significant, excess of lung cancer in case relatives, with this deviation occurring almost exclusively in relatives of probands having rearrangements at 11q23 and having lymphoid leukemia. It is possible that heritable tendency to chromosomal rearrangement--and thus to cancer--is expressed in such a small proportion of family members that cancer excess in these families could not be detected with the numbers of relatives analyzed in this study, although there was no significant evidence for a hereditary predisposition to cancer in the families of probands with leukemia and with chromosomal rearrangements at the same apparent chromosomal location as rare fragile sites.

Adolescent↗
PubMed1988

Two anonymous DNA segments distinguish the Wilms' tumor and aniridia loci.

The association of Wilms' tumor with aniridia (the WAGR complex) in children with 11p13 chromosomal abnormalities has been established, but the paucity of molecular probes in 11p13 has hampered identification of the responsible genes. Two new anonymous DNA segments have been identified that map to the WAGR region of 11p13. Both DNA probes identify a cytologically undetectable deletion associated with a balanced chromosome translocation inherited by a patient with familial aniridia, but not Wilms' tumor. The same two DNA segments are also included in the distal p13-p14.1 deletion of another patient, who has aniridia, Wilms' tumor, and hypogonadism, but they are not included in the p12-p13 deletion of a third patient, who does not have aniridia but has had a Wilms' tumor. The discovery of this aniridia deletion and these two DNA segments that physically separate the Wilms' tumor and aniridia loci should facilitate identification of the genes in the WAGR locus, beginning with the aniridia gene.

Animals↗
PubMed1988

Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter.

Genomic footprinting on the Drosophila hsp26 promoter in isolated nuclei has shown that a TATA box binding factor is present before and after induction by heat shock, while three of the seven heat shock consensus sequences 5' of the gene are occupied (presumably by heat shock factor, HSF) specifically on heat shock. The sites of HSF interaction are separated by greater than 200 bp of which approximately 150 bp are bound to the surface of a nucleosome. The juxtaposition of these various macromolecules on the DNA suggests a basis for the major DNase I hypersensitive site 5' of hsp26 and a novel tertiary structure for the promoter complex.

Animals↗
PubMed1988

Rapid diagnosis of sensitivity to ultraviolet light in fibroblasts from dermatologic disorders, with particular reference to xeroderma pigmentosum.

A rapid and simple method for determining the sensitivity of human fibroblasts to ultraviolet light is described. As an alternative to the colony formation assay, this method can be used for the rapid diagnosis of ultraviolet light sensitivity in fibroblasts from photosensitive disorders. The method is based on growth of small numbers of cells in 1-cm wells of culture trays for 4 or more days after irradiation and determination of cell survival by the incorporation of [3H]hypoxanthine. D37 values (the dose at which 37% of the control level of incorporation remains) obtained from this procedure showed the same relative sensitivity of normal and xeroderma pigmentosum fibroblasts as was obtained by colony formation. Untransformed and SV40-transformed fibroblasts, which have different growth rates and different responses to high cell densities, gave different D37 values by this assay in culture trays as compared with colony formation. Comparison of relative sensitivities to irradiation should therefore be made only between cell types with similar growth characteristics. The similar sensitivity of normal and xeroderma pigmentosum cells to mitomycin C was also determined by this culture tray method. By increasing cell density at the beginning of the experiments, a greater capacity of group C compared with group D fibroblasts for recovery from potentially lethal damage was also detected.

Cells, Cultured↗
PubMed1987

Purification and characterization of sialic acid containing materials accumulated in cultured skin fibroblasts from a patient with type II sialidosis.

Sialyl conjugates which accumulate in sialidase deficient fibroblasts were made radioactive in the sialic acid moiety by culturing the cells in the presence of [3H]N-acetylmannosamine. The accumulated radioactive compounds were fractionated by anion exchange chromatography and gel filtration. At least nine chromatographically distinct sialyl conjugates were obtained. Neutral and amino sugar analysis of these compounds indicated that all were sialyloligosaccharides of the type which could be derived from glycoproteins containing complex, N-asparagine-linked carbohydrate groups.

Cells, Cultured↗
PubMed1986

Characterization of a supercoil-dependent S1 sensitive site 5' to the Drosophila melanogaster hsp 26 gene.

We have analyzed the prominent supercoil-dependent S1 nuclease cleavage site 5' to hsp 26 in the plasmid 88B13, which contains 11.7 kilobases from the Drosophila locus 67B1. The double stranded cleavage product is generated by initial nicking on the purine strand, six preferred sites occurring between positions -96 and -90 (relative to the start of transcription) with weaker ones extending to position -84, followed by cleavage on the pyrimidine strand at positions -86 and -84. A derivative of 88B13, 88B13-X, was generated by insertion of an Xho I linker at position -84; this does not affect the positions or strand specificity of the S1 cleavage in that region. A small deletion, delta 41.1, removes the homopurine/homopyrimidine stretch from positions -86 to -132 and is no longer sensitive to cleavage by S1 nuclease 5' to hsp 26. Mung bean and P1 nucleases recognize the same site 5' to hsp 26 and give the same general pattern of cleavage. All three nucleases show an initial cleavage of 88B13 DNA at this site at pH 5.5 but not at pH 6.5, indicating that the DNA structure there may be pH dependent in vitro.

Animals↗
PubMed1986

Chromosome studies in 10 patients with the Rett syndrome.

Chromosomes of white blood cells from 10 girls with the Rett syndrome, 9 of their mothers and 17 unrelated controls (5 girls and 12 women) were examined for the presence of the fragile site on Xp22 under different culture conditions. Six of the 10 Rett patients were fra(X)(p22) positive while 4 failed to express this fragile site. In the 6 patients expressing the fragile site it was present in 1 to 3% of all cells. Similarly, 5 of the mothers and 6 of the control women showed this fragile site, with a frequency of 1 to 9%. Concordance analysis showed that in the 9 mother-daughter pairs examined, 5 had concordant results while 4 of the pairs were discordant. These findings are very similar to those found in non-Rett individuals by ourselves and other investigators. These data agree with published findings that fra(X)(p22) is a common fragile site in normal individuals. Therefore, we conclude that the incidence of the Xp22 fragile site in the Rett syndrome does not differ from that found in various non-Rett individuals, including normal persons. This indicates to us that fra(X)(p22) cannot serve as a chromosomal marker for the Rett syndrome.

Adult↗
PubMed1986

Unusual supernumerary chromosomes: types encountered in a referred population, and high incidence of associated maternal chromosome abnormalities.

In a 6-year period 128 patients with supernumerary autosomes were identified in our laboratory. The majority had "primary" trisomy, but 19 (15%) had extra, unusual chromosomes, not just a normal chromosome present in an extra copy. Of these, 18 were complex and did not resemble any one part of the standard chromosome complement. There was a preponderance of females among the 19 cases. Chromosome analysis of the parents in the 14 most recent cases revealed maternal chromosome abnormalities in 11 (79%). Of these 11, eight mothers had balanced reciprocal translocations; nondisjunction led to the smaller of their translocation chromosomes being passed on as the supernumerary chromosome in their offspring. Thus, nondisjunction of maternal translocations accounts for a major proportion of the unusual supernumerary chromosomes found by our laboratory. Advanced maternal age was noted in this group of mothers. Three mothers had supernumerary chromosomes themselves. We conclude that unusual supernumerary chromosomes (1) are not rare among patients referred for chromosome studies; (2) are generally not simple products of breakage; (3) are very frequently the result of malsegregation of a balanced maternal reciprocal translocation; and (4) are very difficult to characterize unless a balanced parental translocation is identified. Parental karyotypes should be obtained whenever a patient has an extra, unusual chromosome.

Adolescent↗
PubMed1986

Familial isolated aniridia associated with a translocation involving chromosomes 11 and 22 [t(11;22)(p13;q12.2)].

Isolated aniridia segregated as an autosomal dominant trait in a family with 11 affected members spanning five generations. Four of the eight individuals studied had aniridia associated with glaucoma and cataracts. Cytogenetic studies revealed an apparently balanced reciprocal translocation between chromosomes 11 and 22 [t(11;22)(p13;q12.2)], while four unaffected relatives had normal karyotypes. There is no evidence of Wilms tumor or genitourinary abnormalities in any members of the family. Restriction enzyme analysis of the human catalase gene revealed no abnormalities in the individuals with the translocation. A summary of phenotypic abnormalities in 61 cases associated with aniridia is presented, as well as a comparison of breakpoints in 44 cases of 11p deletion. These data indicate that single breaks at 11p13 are associated with isolated aniridia, while deletion of 11p13 results in aniridia combined with Wilms tumor, genitourinary abnormalities, and/or mental retardation.

Abnormalities, Multiple↗
PubMed1986

Accumulation of [3H]sialyl-conjugates in sialidosis (sialidase-deficient) fibroblasts cultured in the presence of [3H]-N-acetylmannosamine.

Skin fibroblasts from normal individuals and a patient with the infantile form of sialidosis were cultured for up to 72 h in medium containing [3H]-N-acetylmannosamine. The sialidosis fibroblasts consistently accumulated more labeled compound(s) than the control cells, i.e. 37-88% more cpm per mg protein. Precipitation of sonicates of these cells with 10% trichloracetic acid, TCA, demonstrated that the excess radioactivity in the sialidosis fibroblasts was in one or more TCA soluble compounds. There was no detectable difference in the amount of label in the TCA insoluble material. The TCA soluble, labeled, material from the sialidosis and the control fibroblasts was separated, isolated and purified on AG1-X8, QAE Sephadex A-25 and Bio-Gel P-4 chromatography columns. Analysis of the isolated material showed the excess radioactivity in the sialidosis fibroblasts to be due to increased levels of [3H]sialic acid covalently bound to a variety of anionic sialyl conjugates. These compounds have been separated and partially purified. Finally, acid hydrolysis and chromatographic analysis of the TCA insoluble fractions showed that greater than 80% of the label in this material was also due to [3H]sialic acid. There was no detectable difference between the control and the sialidosis patient in the amount of label in this fraction.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1986

Disturbances in DNA precursor metabolism associated with exposure to an inhibitor of poly(ADP-ribose) synthetase.

3-Aminobenzamide (3AB) is widely used as an inhibitor of poly(ADP-ribose) synthetase to study the effect of protein ribosylation on various cellular processes, but the specificity of its inhibition has not been demonstrated. We found that 3AB has a wide range of effects on DNA precursor metabolism, as determined by high-performance liquid chromatographic separation of deoxynucleosides derived from enzymatic digestion of cellular DNA. 3AB (10-20 mM) significantly reduced cell growth in human lymphoblastoid cells. Furthermore, the incorporation of [3H]deoxycytidine into DNA was significantly enhanced relative to incorporation of [3H]deoxythymidine, [3H]deoxyguanosine, and [3H]deoxyadenosine. Incorporation of fragments of [3H]glucose into the pyrimidine fraction of DNA was significantly inhibited relative to incorporation into the purine fraction. At only 1 mM, 3AB had a major inhibitory effect on the incorporation of the methyl group from [3H]methionine into deoxyguanosine, deoxyadenosine, and deoxycytidine, with 50% inhibition into deoxyguanosine and deoxyadenosine and 90% inhibition into deoxycytidine. The specificity of 3AB inhibition to poly(ADP-ribose) synthetase is therefore doubtful in view of this variety of metabolic effects, involving pyrimidine synthesis and de novo synthesis via the one-carbon pool.

Benzamides↗
PubMed1986

Anorexia and altered serotonin metabolism in a patient with argininosuccinic aciduria.

We studied serotonin metabolism in a metabolically stable 7-year-old girl with argininosuccinic aciduria who had severe anorexia. The CSF concentration of 5-hydroxyindoleacetic acid (HIAA), the metabolite of serotonin, was markedly elevated at 79 ng/ml (normal 33 +/- 11 ng/ml). Altered serotonin metabolism was also reflected in the sleep EEG, which showed decreased REM sleep. Reducing her intake of tryptophan, the precursor of serotonin, from 35 mg/kg/day to 7 mg/kg/day resulted in a decrease in CSF concentration of HIAA to 20 ng/ml and the onset of spontaneous eating for the first time in 4 1/2 years. REM sleep increased from 3% to 9.5% of total sleep time. Two days after increasing tryptophan intake to 25 mg/kg/day, spontaneous feeding stopped, associated with a rise in CSF HIAA to 45 ng/ml. Caloric/carbohydrate intake was found to affect CSF HIAA levels and food intake in an additive manner with tryptophan intake. These observations suggest that altered serotonin metabolism affected feeding behavior in this child, and that this effect could be modified by changing tryptophan or carbohydrate intake.

Amino Acid Metabolism, Inborn Errors↗
PubMed1986

Behavioral and neurotransmitter changes in the urease-infused rat: a model of congenital hyperammonemia.

Rats implanted with subcutaneous or intraperitoneal osmotic minipumps infusing 0.8-1.25 IU urease/kg/h develop sustained hyperammonemia (range 137-497 microM, controls 88 +/- 51 microM +/- SD) for 5-7 days. Glutamine levels are also significantly elevated in plasma (677 +/- 166 versus 428 +/- 122 microM) and cerebral cortex (13.2 +/- 9.8 versus 4.7 +/- 2.8 nmol/mg tissue). Neurobehavioral abnormalities include decreased food intake and increased stereotypic activity. Increased serotonin turnover was suggested by elevated levels of tryptophan and 5-hydroxyindoleacetic acid in cerebral cortex, brain stem, and cerebellum of urease-infused compared to sham-operated animals. There were no changes in norepinephrine or gamma aminobutyric acid, and there was no correlation between the degree of hyperammonemia or glutaminemia and brain levels of tryptophan or biogenic amines. Animals receiving a tryptophan-deficient diet had significantly lower levels of tryptophan and 5-hydroxyindoleacetic acid in brain regions compared to animals receiving a normal tryptophan intake, under both control and hyperammonemic conditions. Despite the prevention of increased serotonin flux in hyperammonemic animals receiving a tryptophan-deficient diet, food intake and weight declined and there was increased stereotypic behavior.

Ammonia↗