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Biomedical subjects

A L Beaudet

Publications and source records attributed to A L Beaudet.

At least 199 records · Page 11Linked to original sources
PubMed1983

Sequence for human argininosuccinate synthetase cDNA.

The nucleotide sequence for human argininosuccinate synthetase cDNA was determined by analysis of six clones isolated from a single experiment. The sequence covered 1623 nucleotides including 76 bases of poly(A) and contained a 1236 nucleotide open reading frame encoding a protein of 46,434 daltons. In one cDNA isolate, a cloning artifact or perhaps RNA polymerase error involving addition of an A in a region of six A's within the coding sequence was documented. Single base variations in the 3' untranslated region were examined in detail since detection of DNA polymorphisms in the cDNAs could imply over-expression of both alleles at the active locus in canavanine-resistant cells, i.e. a trans-acting mechanism for enzyme overproduction. However, the sequence from five cDNAs suggested some single base artifacts, and DNA polymorphism remains uncertain. The occurrence of three tandem arginine codons in the 5' untranslated region of the cDNA suggested the possibility of an interaction of arginyl-tRNA with mRNA to regulate RNA processing or half-life as a mechanism for arginine-mediated repression.

Amino Acid Sequence↗
PubMed1983

Abnormal mRNA for argininosuccinate synthetase in citrullinaemia.

Citrullinaemia is a human inborn error of metabolism resulting from the deficiency of argininosuccinate synthetase. In a previous study of cultured skin fibroblasts from citrullinaemia patients, we showed that the presumed defects in DNA were not detectable by Southern blotting analysis, and that only 2 of 11 cell lines contained detectable enzyme antigen. All citrullinaemia cell lines contained hybridizable mRNA but slight size heterogeneity was noted. Here we report the extension of the analysis of the RNA using S1 nuclease mapping techniques. Among six cell lines examined, five showed an abnormality of mRNA detectable by S1 nuclease analysis. The data indicate that a minimum of three out of five non-consanguineous patients represent compound heterozygotes. The S1 nuclease detectable defects may represent deletions or rearrangements in the genomic DNA, or more probably represent examples of abnormal RNA splicing. The approach used here is useful for molecular analysis of genetic defects, for prenatal diagnosis, and for study of genetic variation.

Amino Acid Metabolism, Inborn Errors↗
PubMed1983

Lactic acidosis, neurological deterioration and compromised cellular pyruvate oxidation due to a defect in the reoxidation of cytoplasmically generated NADH.

Two patients, one dying at 25 days and one at 20 months had 'chronic' lactic acidaemia with a high lactate to pyruvate ratio. Both showed EEG abnormalities and seizure activity and both died of respiratory failure. Investigation of cultured skin fibroblasts from these patients revealed normal pyruvate dehydrogenase and pyruvate carboxylase activities but the cells showed a decreased ability to oxidase pyruvate which was returned to normal on the addition of methylene blue. Subsequent investigations revealed that the mitochondria from the patients' cells could oxidase pyruvate normally but that the cells had an abnormal NAD to NADH ratio under standard conditions of incubation. It was concluded that both children had a redox disequilibrium in the cytoplasmic compartment due to a problem in transporting reducing equivalents from the cytoplasmic to the mitochondrial compartments.

Acidosis↗
PubMed1983

Repair response of human fibroblasts to bleomycin damage.

The ability of human fibroblasts to repair the specific types of DNA damage caused by bleomycin (BLM) was examined in whole-cell experiments. The method utilized for analysis was alkaline sucrose-gradient centrifugation of DNA. The results of these studies show that a repair pathway exists for the damage produced in DNA by bleomycin. DNA from BLM-treated cells shows a decrease in molecular weight, caused by chemical or enzymatic incision at sites of drug action. If the drug is removed, the DNA rapidly returns to high molecular weight, demonstrating reformation of damaged DNA. This repair response to BLM-damage was also confirmed in fibroblasts isolated from patients with putative DNA-repair defects. We observed that the response (to BLM) of cells from patients with Fanconi anemia was altered in that the fall in molecular weight of DNA from treated cells was not as great as that observed in other cell strains after drug treatment.

Bleomycin↗
PubMed1983

Stable low molecular weight DNA in xeroderma pigmentosum cells.

Xeroderma pigmentosum (XP) cells from several complementation groups contained more low molecular weight DNA upon alkaline sucrose gradient centrifugation than did other human cells examined. Under conditions in which only 5% of the DNA in normal cells sedimented at 16 S or less, 20% of the DNA in XP cells from complementation group A sedimented at 16 S or less. Because cells were layered directly onto the gradients for lysing of cells and denaturing of the DNA, it appears that this low molecular weight material is due to naturally existing gaps or alkali-sensitive sites, or both, in the cellular DNA. The increase in low molecular weight DNA seen in XP complementation group A cells also is seen in complementation groups C, D, and E. When prelabeled cells were incubated for increasing times after removal of the radioactive label, the amount of low molecular weight material remained constant over a 3-hr period. The introduction of the DNA-damaging agent, bleomycin, to prelabeled XP cells produced a surprising effect. The normal response of human cells to bleomycin is an increase in low molecular weight DNA, dependent on the dose of the drug and time of treatment. In XP cells the reverse was observed. That is, the low molecular weight DNA observed in untreated XP cells disappeared upon addition of the drug. The process responsible for the unusual response of XP cells to bleomycin is unknown, but these results are compatible with an inducible repair process.

Bleomycin↗
PubMed1982

Dispersion of argininosuccinate-synthetase-like human genes to multiple autosomes and the X chromosome.

DNA sequences closely homologous to argininosuccinate synthetase are present at ten or more distinct locations in the human genome, including sites on chromosomes 6, 9 and X. Argininosuccinate synthetase thus represents one of the most widely dispersed multigene families described to date, the first instance of a multigene family associated with an enzyme of intermediary metabolism and, perhaps most striking, the first instance of a multigene family with members on both autosomes and sex chromosomes.

Argininosuccinate Synthase↗
PubMed1982

Glycogen storage disease: long-term follow-up of nocturnal intragastric feeding.

Nocturnal intragastric feeding of patients with certain hepatic forms of glycogen storage disease has been advocated as an effective treatment, resulting in improved blood chemical values and linear growth. We are reporting the long-term follow-up of five patients with glycogen storage diseases; three with type Ia, one with type Ib, and one with type III disease. All had improvement in one or more of the following: linear growth, serum glutamic oxaloacetate transaminase, total lipids, cholesterol, phospholipids, or triglycerides. None had significant improvement in venous CO2, serum lactate or urate. One of the patients in this study died after 1.1 years of treatment, and another patient developed hepatocellular carcinoma after 4.4 years of treatment. Nocturnal intragastric feeding, in conjunction with appropriate daytime feeding, is helpful in the management of patients with glycogen storage disease but response to treatment is variable, and it remains to be determined whether the ultimate prognosis of the diseases can be improved.

Adolescent↗
PubMed1982

Molecular analysis of argininosuccinate synthetase deficiency in human fibroblasts.

We have analyzed cultured skin fibroblasts derived from patients with argininosuccinate synthetase deficiency for alterations in gene structure, mRNA content, and protein structure. Genomic DNA was digested with the endonucleases EcoRI or HindIII, and the fragments were analyzed by Southern blotting and hybridization with a cDNA probe for argininosuccinate synthetase. The blot pattern is complex because there are at least 10 copies of argininosuccinate synthetase-like genes scattered over multiple human chromosomes. All nine patients studied showed patterns of DNA fragments that were indistinguishable from the normal control cell lines, and despite the possibility that the complexity could mask some changes, major deletions of the active gene(s) were not present. Blot hybridization of RNA indicated the presence of hybridizable mRNA of approximately normal size in seven of seven individuals examined with a suggestion of some heterogeneity. Analysis of enzyme antigen by protein transfer from NaDodSO4 containing polyacrylamide gels revealed considerable heterogeneity. This analysis revealed no cross-reacting material (CRM) in nine cell lines, CRM of normal molecular weight in one cell line, and CRM of reduced molecular weight in one cell line. These findings suggest that the genes for argininosuccinate synthetase in most citrullinemia patients are transcribed and produce stable mRNA. These mRNA either are not translated, or the translation product (enzyme) is rapidly degraded or is immunologically nonreactive. Defective gene expression in this disorder appears to involve abnormal mRNA, which may be altered by point mutations, frame shift mutations, deletions, insertions or particularly by abnormal RNA processing.

Argininosuccinate Synthase↗
PubMed1981

Cloning of cDNA for argininosuccinate synthetase mRNA and study of enzyme overproduction in a human cell line.

Previous studies of the human cell line RPMI-2650 (wild type) and its canavanine-resistant variants have demonstrated differences in argininosuccinate synthetase activity as follows: canavanine-resistant much greater than wild type grown in citrulline greater than wild type grown in arginine (Su, T.-S., Beaudet, A. L., and O'Brien, W. E. (1981) Biochemistry 20, 2956-2960). A recombinant plasmid containing a 1.55-kilobase insert complementary to the mRNA for human argininosuccinate synthetase was isolated by the combined use of differential colony hybridization and immunoprecipitation of the products of plasmid-selected mRNA translation. Both blot and dot hybridization analysis of polyadenylated RNA indicated a major mRNA species of 1.67 kilobase in all cells, and the levels of mRNA correlated well with the levels of enzyme activity: canavanine-resistant, 180; wild type grown in citrulline, 7; and wild type grown in arginine, 1. One major mRNA species of 1.67 kilobase and one minor species of 2.68 kilobase were observed in wild type and canavanine-resistant cell lines. Reassociation kinetics of pAS1 with genomic DNA from human liver, canavanine-resistant cells, and wild type cells were not significantly different. Blot hybridization of genomic DNA revealed no detectable differences between wild type cells, canavanine-resistant cells, and human leukocytes. The data demonstrated that there were multiple copies, perhaps 10 or more, of argininosuccinate synthetase-like sequences in human DNA and that the canavanine-resistant phenotype was not due to gene amplification.

Argininosuccinate Synthase↗
PubMed1981

Increased translatable messenger ribonucleic acid for argininosuccinate synthetase in canavanine-resistant human cells.

The level of argininosuccinate synthetase activity in the human tissue culture cell line RPMI 2650 was 6-fold higher when citrulline was substituted for arginine in the culture medium. Canavanine-resistant (Canr) variants were isolated and had enzyme activity up to 25 nmol min-1 (mg of protein)-1 or 180-fold higher than that of the wild-type cells grown in arginine. The differences in enzyme activity were paralleled by differences in the amount of enzyme determined immunologically. The micrograms of enzyme per milligrams of protein, determined by complement fixation, were 0.03 for wild-type cells grown in arginine, 0.29 for wild-type cells grown in citrulline, and 6.73 for a Canr variant. In vivo labeling studies suggested increased synthesis of argininosuccinate synthetase in Canr cells, and in vitro translation of poly(adenylic acid) [poly(A)] messenger ribonucleic acid (mRNA) from wild-type and Canr cells confirmed a quantitatively compatible increase in translatable poly(A) mRNA for the enzyme in Canr cells. No precursor for the enzyme was recognized by using in vitro translation, and the poly(A) mRNA for the enzyme had a sedimentation value of 16 S by sucrose-gradient analysis. The levels of argininosuccinate synthetase activity in the Canr cells were similar to those found in normal liver.

Animals↗
PubMed1981

Repair of Bleomycin-damaged DNA by human fibroblasts.

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.

Bleomycin↗
PubMed1981

Apolipoprotein C-III-1 activates lysosomal sphingomyelinase in vitro.

Apolipoprotein (apo)C-III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphingomyelinase from human fibroblasts. At the sphingomyelin concentrations tested, maximal stimulation was obtained with 5 microM apoC-III-1 or apoC mixture. Apolipoproteins A-I, A-II, B, and C-I conferred little or no stimulation. Sphingomyelinase was stimulated 20-fold by lysophosphatidylcholine with an optimum concentration of 70 microM using 0.3 mM substrate. Sphingomyelinase activity was inhibited by concentrations of apoC-III-1 and lysophosphatidylcholine three- to fivefold above stimulatory levels. Triton X-100 activated sphingomyelinase 300-fold with a pH optimum of 5.0, while the pH optimum with the biological activators was 4.0. These results raise the possibility of an in vivo activity for the biological activators. The proteins that enter lysosomes as part of a lipoprotein complex may activate lysosomal enzymes that degrade the lipid components.

Apolipoprotein C-III↗
PubMed1980

Acidic phospholipases in cultured human fibroblasts: deficiency of phospholipase C in Niemann-Pick disease.

Radioactive phosphatidyl choline substrates specifically labeled in the one position or two position fatty acid were used to establish conditions for the detection of acidic phospholipase A1, A2 and C activities in extracts of cultured human fibroblasts. Maximal activity was detected at a pH of 3.0, 4.0 and 5.0 respectively, suggesting that the enzymes are of lysosomal origin. None of the activities were stimulated or inhibited markedly by Ca2+ or EDTA. The A1 and A2 activities, but not the C activity, were inactivated by the sulfhydryl reactive Ellman reagent. All three enzyme activities were in the normal range for cultured fibroblasts which were deficient in acid lipase, indicating that these activities are not attributable to the acid lipase gene product. Phospholipase A activity was deficient in fibroblast extracts from patients with Niemann-Pick disease, types A, B and C. These data suggest either identity or a genetic relationship between sphingomyelinase and phospholipase C. The activities examined were within the normal range in fibroblasts from patients with neuronal ceroid lipofuscinosis, sea blue histiocyte disease and selected uncharacterized degenerative diseases.

Calcium↗
PubMed1980

Neutropenia and impaired neutrophil migration in type IB glycogen storage disease.

The association of neutropenia with type IB glycogen storage disease was observed in siblings. Biochemical studies of liver demonstrated a defect in glucose-6-phosphate transport. Neutrophil mobilization in vivo was impaired but the bone marrow was normal histologically. In vitro studies of neutrophils indicated a defect in random and directed cell migration. The studies suggest that a role for glucose-6-phosphate transport in neutrophils should be considered.

Agranulocytosis↗