Molecular analysis of a woman with late onset cystic fibrosis.
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Chromosome Deletion↗
Biomedical subjects
R Rozen
Publications and source records attributed to R Rozen.
Cystic fibrosis mutations in North American populations of French ancestry: analysis of Quebec French-Canadian and Louisiana Acadian families.
A 3-bp deletion (delta F508) in the cystic fibrosis (CF) gene is the mutation on the majority of CF chromosomes. We studied 112 CF families from North American populations of French ancestry: French-Canadian families referred from hospitals in three cities in Quebec and from the Saguenay-Lac St. Jean region of northeastern Quebec and Acadian families living in Louisiana. delta F508 was present on 71%, 55%, and 70% of the CF chromosomes from the major-urban Quebec, Saguenay-Lac St. Jean, and Louisiana Acadian families, respectively. A weighted estimate of the proportion of delta F508 in the French-Canadian patient population of Quebec was 70%. We found that 95% of the CF chromosomes with delta F508 had D7S23 haplotype B, the most frequent haplotype on CF chromosomes. In the Saguenay-Lac St. Jean families, 86% of the CF chromosomes without delta F508 had the B haplotype, compared with 31% for the major-urban Quebec and Louisiana Acadian families. The incidence of CF in the Saguenay-Lac St. Jean population was 1/895 live-born infants.
Blotting, Southern↗
Regulation of ornithine aminotransferase in retinoblastomas.
Ornithine-delta-aminotransferase (OAT) is a nuclear-encoded, mitochondrial enzyme that converts ornithine to glutamate semialdehyde. Although OAT is expressed in most tissues of the rat, liver, kidney, and retina have the highest levels of OAT activity. Studies of OAT regulation in liver and kidney have indicated transcriptional and translational controls for the enzyme in a tissue-specific manner. Little is known about OAT modulation in retinal tissue, although chorio-retinal degeneration is the predominant feature in a hereditary disorder of OAT deficiency, gyrate atrophy. To characterize OAT regulation in retinal lines, we studied its synthesis in two retinoblastoma strains, Y79 and RB355. Baseline OAT mRNA levels were similar in the two cell lines, yet Y79 expressed 3-fold more immunoreactive OAT protein and enzyme activity than RB355; this finding suggested the presence of a post-transcriptional mechanism for the regulation of steady-state OAT levels. Treatment of the two strains with estradiol or thyroid hormone for 24 h resulted in approximately 5-fold increases in OAT protein and activity. Since similar increases in OAT mRNA levels were observed in both strains after Northern blotting, it is likely that these hormones exert their effects at the transcriptional level. Finally, primer extension analysis revealed two OAT mRNA species in both strains, due to the presence of an additional exon (exon 2) in one of the transcripts. The absence of this exon in other tissues reflects the unique mechanisms which govern OAT in retinoblastomas.
Blotting, Northern↗
Effects of a low dose of naltrexone on glucose-induced allesthesia and hunger in humans.
In order to study the effects of a low dose of the opioid antagonist naltrexone on ingestive behavior for sucrose in humans, preference for sucrose solutions and feelings of hunger were scored on visual analogical scale by 14 healthy subjects with or without naltrexone. Effects of intragastric glucose load or water, and naltrexone (25 mg) or placebo were tested. At this low dose, naltrexone alone had a slight effect on allesthesia, and it produced a strong potentiation of glucose-induced allesthesia.
Adult↗
[Anorexia nervosa: absence of sensitivity to nutritional protein markers. Study of 23 patients and comparison to a paired group with colonic Crohn's disease].
One of the most important therapeutic goal in anorexia nervosa is to define with the patient a body weight to obtain. To do so, objective criteria are needed. For this purpose, we studied in a longitudinal way, 9 nutritional parameters in 23 patients with anorexia nervosa, as compared with 23 age - and sex - matched patients with Crohn's disease: body weight, tricep's skinfolds, mid-arm muscle circumferences; serum albumin, prealbumin, transferrin, hemoglobin, cholesterol; urinary creatinine and calcium, magnesium, zinc and copper serum levels and urinary outputs. Despite losses of body weight, of lean body mass and of fatty mass higher in patients with anorexia nervosa than in those with Crohn's disease, the former had higher nutritional protein's serum levels than the latter. These nutritional protein markers were not in anorexia nervosa different from normal values. In anorexia patients, zinc and copper serum levels and urinary outputs were very low. This study suggests that nutritional protein markers of hepatic synthesis are not sensitive markers for malnutrition evaluation and can not be used to follow renutrition efficacy.
Adult↗
Novel PKU mutation on haplotype 2 in French-Canadians.
We analyzed DNA from nine French-Canadian probands from eastern Quebec province; all had hyperphenylalaninemia (phenylketonuria [PKU] or non-PKU forms) caused by mutations at the phenylalanine hydroxylase locus. Analysis of RFLP haplotypes and mutations revealed a novel mutation, an A-to-G transition (met----val) in codon 1 (the translation-initiation codon). It occurred on 5 of the 18 mutant chromosomes and was associated each time with haplotype 2. A proband homozygous for this mutation had the PKU phenotype. In other probands, the codon 1 mutation was inherited once with the splice junction mutation in exon 12 (on haplotype 3), conferring PKU, and was inherited twice with a mutation on haplotype 1, conferring PKU in one proband and non-PKU hyperphenylalaninemia in the other. The other five probands carried mutations, conferring PKU, on the following haplotype combinations: 1/3 (twice), 1/9, 3/4, and 1/1. The mutations on haplotypes 1, 4, and 9 are not yet characterized. This preliminary study reveals a novel PKU mutation and considerable genetic heterogeneity at the phenylalanine hydroxylase locus in French-Canadians.
Exons↗
Energy expenditure economy induced by decrease in lean body mass in anorexia nervosa.
In anorexia nervosa, the low energy input associated with the classic overactivity during the malnourished state needs a sparing of energy expended at rest and also during physical activity. Therefore, we measured energy expenditure, both at rest and during moderate bicycling exercise (30W, 6 min), in 11 adult anorectic patients (weight 35.15 +/- 4.30 kg, mean +/- s.d.) at the beginning of their treatment and again after a mean weight gain of about 8.4 kg. During the malnourished state, the resting energy expenditure (REE) was lower than that predicted according to Harris and Benedict (P less than 0.001). Although it was significantly increased after weight gain (P less than 0.05), the REE per kg of lean body mass remained unchanged after repletion. The total oxygen consumption related to exercise remained unchanged after refeeding (2114 +/- 487 ml/15 min, basal vs 2168 +/- 394 ml/15 min, repletion) (n.s.). Thus in anorexia nervosa, weight loss and malnutrition did not induce economy either in energy expended at rest per unit of lean body mass nor in the energy expended in moderate cycling activity.
Adolescent↗
Chromosomal localization of the gene for the human trifunctional enzyme, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.
A trifunctional protein in man, 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of tetrahydrofolate derivatives; these derivatives supply one-carbon units for intermediary metabolism. Somatic cell hybridization and in situ hybridization were used to localize the functional gene coding for this protein--to human chromosome 14q24, near the c-fos and TGF-beta 3 loci. A second hybridizing sequence, possibly a pseudogene, was identified near the centromere of the X chromosome, at Xp11.
Aminohydrolases↗
Primary structure of a human trifunctional enzyme. Isolation of a cDNA encoding methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.
A DNA clone complementary to the messenger RNA encoding the human trifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyl-tetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase has been isolated from a lambda gt10 library. In vitro transcription-translation of the 3.1-kilobase cDNA clone yields a protein of 101 kDa, which is of identical size and exhibits the same immunoreactivity as the enzyme purified from human liver. A coding region of 2805 base pairs in the cDNA encodes a protein of 935 amino acids. The initiator methionine is absent from the purified enzyme and the amino-terminal 30 amino acids derived by automated sequence analysis are identical (arginine at position 18 was not identified) with that deduced from the nucleotide sequence. The amino acid sequence of the human enzyme shows extensive homology with that of the yeast enzyme, although the amino-terminal bifunctional dehydrogenase-cyclohydrolase domain is less homologous than the carboxyl-terminal synthetase domain. A region was identified which probably serves as a link between these two major domains of the human enzyme. The synthetase domain contains two regions that are homologous to consensus sequences for an ATP-binding site.
Amino Acid Sequence↗
Renal tubular cells are potential targets for epidermal growth factor.
Epidermal growth factor (EGF) is a potent polypeptide mitogen with various receptor-mediated growth effects on cells from the skin, breast, and gastrointestinal tract. Recent studies indicate that EGF is produced in the kidney and is excreted in the urine, but the biological significance of renal EGF is uncertain. We demonstrate in vitro mitogenicity of EGF for LLC-PK1 cells, a tubular epithelial cell line derived from pig kidney cortex. Furthermore, when subconfluent monolayers of LLC-PK1 cells are exposed to EGF for 24 h, sodium-dependent phosphate transport is stimulated (209-410% of control). These cells possess EGF-specific high-affinity binding sites at their surface (Kd 300-700 pM) but cannot synthesize the growth factor. EGF binding sites are not a peculiarity of the LLC-PK1 cell line, since similar sites are present on MDCK cells (derived from dog kidney distal tubule or collecting duct), primary cultures of mouse proximal tubular cells, and freshly prepared membrane fractions from mouse kidney. Cortical basolateral membranes are highly enriched in EGF binding sites, whereas EGF binding by brush-border membrane fractions is minimal and is compatible with contamination.
Animals↗
Permanent administration of d-fenfluramine in rats: paradoxical effects.
In order to test the hypothesis of Levitzky that d-fenfluramine (d-F) acts by modifying the ponderal set-point, we compared the effects of a permanent infusion of d-F on food intake and body weight (BW). The effect on the weight persisted as long as the infusion; the clear-cut anorectic effect lasted only a few days. This paradox is compatible with the set-point hypothesis. In rats rendered overweight by insulin treatment, the d-F-induced decrease in BW was approximately four times smaller than in controls. In rats rendered overweight by a cafeteria diet, the decrease in BW was twice as large in permanently cafeteria fed rats as in cafeteria, then, ad lib fed rats. In rats rendered underweight by a restricted chow diet and then returned to an ad lib feeding, the final BW depended only on the doses of d-F (0.6 or 12 mg/kg BW/day), whatever the weight at the beginning of infusion. Thus, the underweight paradigm fits well with the set-point hypothesis; the overweight paradigm fits only partially.
Animals↗
The effects of a constant T3 level and thermoneutrality in diet-induced hyperphagia.
Rats were thyroidectomized, then fitted with a miniosmotic pump infusing T3, thereby assuring a constant circulating level of T3. After a ten-day recovery period, they were submitted either to a chow or to a cafeteria diet. Body weight, food intake, and energy expenditure were recorded during a thirty day period. Thyroidectomized T3 supplemented rats did not exhibit hyperphagia when fed a cafeteria diet. Despite this puzzling normophagia, they still chose nutrients in a distribution similar to that of other cafeteria-fed rats and, though maintaining the same weight as chow-fed rats, increased their proportion of fatty weight compared to these rats. The relationship between energy expenditure, T3 concentration, and cafeteria diet are discussed.
Adipose Tissue↗
Prenatal diagnosis of ornithine transcarbamylase deficiency with use of DNA polymorphisms.
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Amino Acid Metabolism, Inborn Errors↗
Clinical application of DNA analysis in a family with OTC deficiency.
We describe the clinical application of DNA restriction fragment analysis to the genetic evaluation of a family with a child deficient in ornithine transcarbamylase (OTC). The results of protein loading studies and the interpretation of the DNA haplotype profiles for the human OTC gene are reported. DNA restriction fragment analysis may be a reliable technique for the prenatal diagnosis of OTC deficiency and identification of obligate carriers of this gene.
Amino Acid Metabolism, Inborn Errors↗
DNA analysis for ornithine transcarbamylase deficiency.
We have utilized the Southern blotting technique to analyse genomic DNA from males with ornithine transcarbamylase (OTC) deficiency and their families. Using a nearly full-length human cDNA probe, we have identified 3 patients with deletions at this locus and have characterized 4 different restriction fragment length polymorphisms that can be used as linkage markers for the OTC mutation. These polymorphisms occur at sufficiently high frequencies so as to enable us to distinguish the two X-chromosomes in approximately 80% of OTC carriers. As a direct consequence of these findings, prenatal diagnosis and carrier assessment can be offered to a large fraction of families at risk for OTC deficiency.
Chromosome Deletion↗
Effects of a 'physiological' dose of triiodothyronine on obese subjects during a protein-sparing diet.
Twenty obese euthyroidian women followed an exclusively proteic diet for 18 days (74 g/day). Half received placebo, and half received 10 micrograms L-triiodothyronine. In the control group, as expected, mean weight loss was 7 percent of initial body weight; serum T3 and TSH decreased; rT3 increased; basal oxygen consumption diminished by 11 percent; nitrogen balance reached equilibrium at day 11. As compared to this group, the T3-treated group lost significantly more weight; serum T3 and TSH increased; rT3 decreased; oxygen consumption remained stable and nitrogen balance did not deteriorate. Thus, the physiological decrease in thyroid hormones provoked by a restricted diet is linked to energy expenditure but not to nitrogen balance equilibrium.
Adult↗
A gamma-aminobutyric acid-specific transport mechanism in mammalian kidney.
We describe high-affinity, sodium-dependent transport of gamma-aminobutyric acid in slices exposing basal lateral membranes and brush-border membrane vesicles prepared from rat renal cortex. In the presence of aminooxyacetic acid, to block gamma-aminobutyric acid oxidation, uptake into the intracellular space of slices was saturable (apparent Kt, 26 +/- 4 microM, mean and S.E.) and concentrative (steady-state distribution ratio at 50 microM gamma-aminobutyric acid, 47.7 +/- 2.4, mean and S.E.). Brush-border membrane vesicles accumulated gamma-aminobutyric acid in the presence of an inward-directed sodium chloride gradient, (apparent Kt, 30-36 microM) with the peak of 'overshoot' at 10 min. Uptake by vesicles responded to manipulation of the transmembrane potential gradient with valinomycin or impermeant anion. beta-Alanine inhibited gamma-aminobutyric acid transport by slices and brush-border membrane vesicles; inhibitors of neuronal-type gamma-aminobutyric acid transport (e.g., nipecotic and diaminobutyric acids) did not. An 'ABC test' indicated that gamma-aminobutyric acid and beta-alanine do not share a single carrier in either the brush-border or basal-lateral membrane of renal cortex. Influx of gamma-aminobutyric acid into brush-border membrane vesicles, at transequilibrium NaCl, was stimulated by trans-gamma-aminobutyric acid but not by trans-taurine. Ion gradient-driven gamma-aminobutyric acid co-transport was unaffected in freeze-thawed brush-border membrane vesicles; this treatment abolished beta-alanine and taurine co-transport. We conclude that rat kidney membranes (brush-border and basal-lateral) possess a gamma-aminobutyric acid-preferring, high-affinity transport mechanism.
Animals↗
In vitro synthesis of the trifunctional protein, methylenetetrahydrofolate dehydrogenase--methenyltetrahydrofolate cyclohydrolase--formyltetrahydrofolate synthetase, in normal and transformed cells.
The trifunctional eucaryotic protein, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of folate derivatives which are required for thymidine and purine synthesis. The protein was synthesized in vitro using a reticulocyte lysate cell-free translation system. The immunoprecipitated translation product programmed by normal rat liver and rat brain mRNA was identical in size to that programmed by mRNA from transformed cells (HeLa, Morris hepatamas 5123D and 3924A). The protein synthesized by normal rat liver slices or Reuber H35 hepatoma cells in culture was similar in size to the translation product, suggesting that there is no significant posttranslational modification in vivo that alters the molecular weight. Differences in the level of synthesis were observed among the tissues examined. Since no consistent pattern emerged, however, it appears that transformation per se does not alter the structure or expression of this polypeptide. Nonetheless, a two- to three-fold increase in synthesis by Morris hepatoma 5123D may be an important biological observation with respect to studying the regulation of this protein that is critical for nucleotide synthesis.
Aminohydrolases↗