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L J Elsas

Publications and source records attributed to L J Elsas.

At least 91 records · Page 5Linked to original sources
PubMed1988

Influx and efflux of 3-O-methyl-D-glucose by cultured human fibroblasts.

We measured the initial rates of 3-O-methyl-D-glucose (OMG) fluxes by cultured human fibroblasts. D-Glucose (300 mM) and cytochalasin B (5 microM) inhibited approximately 80% of OMG (1 mM) influx. OMG rapidly entered human fibroblasts, and influx was linear up to 20 s. OMG influx and efflux were about equal. Cytochalasin B inhibited OMG (1 mM) influx and efflux within 20 s of exposure. Cytochalasin B half maximally inhibited OMG influx and efflux at 0.51 and 0.75 microM, respectively. In zero trans conditions, the kinetics of OMG influx and efflux were similar. However, when OMG was present on the trans side of the membrane, OMG influx but not OMG efflux was stimulated. Trans stimulation of OMG influx increased the maximal velocity of this transport process, without affecting its Km. These results suggest that 1) OMG influx and efflux occur through the same transporter, and 2) the glucose transporter of cultured human fibroblasts presents a functional asymmetry when substrate is present on the trans side of the membrane.

3-O-Methylglucose↗
PubMed1987

Congenital hypothyroidism: increased risk of neonatal morbidity results in delayed treatment.

In a population-based screen of 617,913 infants, primary congenital hypothyroidism (CH) was confirmed in 100 children. 32 of the 100 infants with CH had an additional defect or complication. In the group with CH the rates of congenital heart disease, non-cardiac malformations, respiratory distress syndrome, and death were higher than in the general population of the same age. Black infants were less likely than whites to have CH, but were at twice the risk of additional impairment. Infants with CH who had an additional complication were screened (12.7 vs 4.8 days) and treated (32.4 vs 19.7 days) significantly later than those infants with isolated CH. Congenital malformations and neonatal complications should not be reasons for deferring screening for CH.

Congenital Abnormalities↗
PubMed1987

Ascorbate regulation of collagen biosynthesis in Ehlers-Danlos syndrome, type VI.

We studied two unrelated individuals with Ehlers-Danlos syndrome type VI, which is characterized by congenital hypotonia, lax joints, severe kyphoscoliosis, friable skin, and hemorrhagic hypotrophic scars. The diagnosis was confirmed by decreased hydroxylysine residues in dermal collagen and decreased collagen lysyl hydroxylase activities in their cultured skin fibroblasts. Despite the diminished hydroxylysine residues in dermal collagen from the probands, we found no differences in hydroxylysyl residues of collagen synthesized by fibroblasts in culture. When patient 1 was given oral sodium ascorbate (5 g/d) for 3 weeks, ascorbate concentrations increased two-fold in plasma and 300-fold in urine. Urinary excretion of hydroxylysine and hydroxyproline increased during ascorbate administration. After a 1-year interval, bleeding time, wound healing, and muscle strength improved. Ascorbate supplementation (50 micrograms/mL) to confluent fibroblasts cultured from the two patients and controls increased hydroxyprolyl and hydroxylysyl residues of fibroblasts four to seven and three to four-fold respectively. Total protein associated with the cell layer increased 14% to 32% without concomitant change in cellular DNA. Total soluble collagenous material recovered from culture media increased 61% to 103% with ascorbate supplementation. These studies demonstrate that ascorbate improves the clinical status of patients with impaired collagen lysyl hydroxylase activity by enhancing lysyl and prolyl hydroxylation and total collagen production.

Amino Acids↗
PubMed1987

Impaired insulin binding and excess glucose transport in fibroblasts from a patient with leprechaunism.

The insulin receptor, though understood in fine molecular detail, remains problematic with regards to its mechanism(s) of cellular signalling. To better understand the normal mechanisms of plasma membrane signalling by insulin, we studied the family of a patient with insulin resistance, hypoglycemia and leprechaunism. Skin fibroblasts were cultured from the proband and his parents. High-affinity insulin binding to the proband's cells was absent. Both parents' fibroblasts showed high-affinity insulin binding intermediate between the controls and the proband. Thus impaired binding conformed to an autosomal recessive pattern of inheritance. The transport of neutral amino acids by the proband's cells was comparable to controls and was insulin-sensitive. By contrast, hexose transport by the proband's fibroblasts was very high and insulin-insensitive. This increased uptake was due to an increased number of glucose transporters on the plasma membrane and to a posttranslational mechanism. Similar levels of glucose transporter mRNA were observed in control and mutant cells. Thus, this inborn error of the insulin receptor offers unique insight into a regulatory signal by its alpha subunit which, when altered, results in constitutively increased glucose transport.

Abnormalities, Multiple↗
PubMed1987

Structural analysis of normal and mutant insulin receptors in fibroblasts cultured from families with leprechaunism.

Leprechaunism is an inherited disorder characterized by insulin resistance and intrauterine growth restriction. In this study we analyze insulin binding and subunit structure of the insulin receptor in dermal fibroblasts cultured from three unrelated families whose probands (Ark-1, Atl, and Minn) were affected by leprechaunism. Cells cultured from all three probands had markedly reduced insulin binding at equilibrium. Fibroblasts cultured from the parents of Ark-1 and Atl had partial and differing degrees of impairment in insulin binding. The structure of the alpha subunit of insulin receptors was analyzed by cross-linking 125I-insulin to plasma membranes. A major band of 350 kilodaltons (kD) (corresponding to the heterotetrameric insulin receptor alpha 2 beta 2) was observed in control and leprechaun fibroblasts. The relative amount of radioactivity cross-linked to plasma membranes reflected the genetic variations seen in insulin binding to intact cells. In reducing gels, 125I-insulin was cross-linked equally to a 250-kD (alpha-alpha dimer) and a 125-kD (alpha monomer) protein in cells from controls, the parents of Ark-1 and Atl, and probands Atl and Minn. By contrast, cells from the Ark-1 proband had diminished cross-linking of alpha-alpha dimers. The ratio of dimer to monomer in cells from controls was 0.93 +/- 0.06, and that in cells from Ark-1 was 0.31 +/- 0.19 (P less than .01). Beta-subunit structure and function was analyzed by studying insulin-enhanced autophosphorylation. Although maximal stimulation of beta-subunit phosphorylation was reduced to 30% in proband Ark-1 fibroblasts, this reduction was quantitatively related to reduced insulin binding. These results indicate that mutations causing severe insulin resistance and defective insulin binding are transmitted with autosomal recessive patterns of inheritance and that heterogeneity exists for these mutations. The mutation in pedigree Ark-1 most likely produces conformational changes in alpha-subunit interaction.

Cells, Cultured↗
PubMed1986

Localisation of the gene for Emery-Dreifuss muscular dystrophy to the distal long arm of the X chromosome.

The linkage relationships of the gene for Emery-Dreifuss muscular dystrophy have been analysed in a large American kindred using DNA probes from different regions of the X chromosome. Close linkage was found with the locus for factor VIII, with no recombinants in 12 opportunities (maximum lod score 4.3), and with locus DXS15 (two recombinants in 17 opportunities, maximum lod score 2.9 at 0 = 10 cM). No linkage was found with probes pERT87 and 754, which are closely linked to Duchenne and Becker muscular dystrophies at Xp21. These results confirm a separate localisation on the distal part of the long arm at q27-28 for Emery-Dreifuss muscular dystrophy and should provide the basis for prenatal diagnosis and improved carrier detection in this disorder if the linkage is confirmed to be close.

Chromosome Mapping↗
PubMed1985

A [3H]lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase.

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.

Cells, Cultured↗
PubMed1985

Absence of branched chain acyl-transferase as a cause of maple syrup urine disease.

Decreased function of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex results in branched chain ketoacidemia or maple syrup urine disease. Activity of this multienzyme complex varies from 0 to approximately 15% of wild type branched chain alpha-ketoacid dehydrogenase complex activity within the population of homozygous affected individuals. We used the technique of Western Blotting with antibodies against purified bovine liver branched chain alpha-ketoacid dehydrogenase complex to screen mitochondrial proteins from cultured human fibroblasts for immunocrossreactive proteins. This method probes the physical structure of the proteins forming this multienzyme complex. One patient with branched chain ketoacidemia lacked an immunoreactive transacylase protein. This protein catalyzes the transfer of the branched chain acyl group from the decarboxylase to reduced coenzyme A. Kinetic analysis of the enzyme activity in cell lysates from this patient confirmed that the complex would not utilize coenzyme A. Thus, we have defined a structural basis for an impaired multienzyme complex of mitochondria in man.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1985

Thiamine response in maple syrup urine disease.

We measured the biochemical response for four patients with maple syrup disease to pharmacologic doses of thiamine, and correlated their response to their branched chain alpha-ketoacid dehydrogenase activity. We observed a linear correlation between the concentrations of each plasma branched-chain amino acid and its corresponding ketoacid analogue. In addition, the renal tubular reabsorption of branched-chain amino and ketoacids was nearly complete within these physiologic concentrations. Three children responded to thiamine therapy with a reduction in concentration of plasma and urinary branched-chain amino and ketoacids. Each responder had at least 5% activity for branched chain alpha-ketoacid dehydrogenase in their mononuclear blood cells and in whole cell fibroblasts from cultured skin when compared to the activity in normal control cells. We propose that each child with maple syrup urine disease be assessed for their response to thiamine by quantifying the concentration of branched-chain amino acids in plasma before and after vitamin supplementation.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1985

Leprechaunism: an inherited defect in a high-affinity insulin receptor.

We examined in vivo oral glucose tolerance tests and in vitro insulin binding, cellular response, and insulin-receptor structure of fibroblasts cultured from the skin of a patient with leprechaun syndrome and her parents. In response to oral glucose, the proband exhibited marked hyperinsulinism (maximum plasma insulin = 4,120 microU/ml), the father had mild hyperinsulinism (maximum plasma insulin = 240 microU/ml), and the mother was normal. [125I]insulin binding to monolayers of intact fibroblasts demonstrated complex kinetics that were interpreted using a two-receptor model. Normal high-affinity binding had an apparent KA of 1.6 X 10(10)/molar with 1,100 sites/cell. The proposed low-affinity state receptor had an apparent KA of 6.8 X 10(7)/molar with approximately 30,000 sites/cell. Insulin binding to the proband's cells had no high-affinity binding but had normal low-affinity binding. Cells from the mother had 60%, and cells from the father, 2%, of control insulin binding to the high-affinity receptor, but normal, low-affinity site binding. Two different, insulin-stimulable responses were evaluated under experimental conditions identical with those used for insulin binding. Insulin stimulation of 2-methylaminoisobutyric acid uptake occurred with half-maximal responses between 25 and 50 ng/ml insulin. This response was similar in cells from controls and the patient. By contrast, the uptake and phosphorylation of 2-deoxy-D-glucose was stimulated at half-maximal insulin concentrations between 1 and 10 ng/ml in control cells but was not significantly increased in the proband's cells until 1,000 ng/ml concentrations of insulin were attained. In affinity crosslinking experiments, [125I]insulin was covalently bound to insulin receptors of fibroblast membranes using disuccinimidylsuberate. [125I]insulin specifically bound to 125,000 dalton monomeric subunits and 250,000 dalton dimers. In control cells, the ratio of monomer to dimer was approximately one, but significantly fewer dimers were crosslinked in insulin receptors from the patient's cells. We conclude that in this family two different recessive mutations impair high-affinity insulin-receptor binding and that the proband with leprechaunism is a compound heterozygote for these mutations. The two mutations produced structural changes in the receptor that altered subunit interactions and loss of high-affinity binding and cellular responsivity.

Binding Sites↗
PubMed1984

Insulin-specific receptor-mediated slowing of beat rate in embryonic heart cells.

Spheroidal aggregates of embryonic heart cells showed their spontaneous beat rate when exposed to insulin. The concentration that produced a half-maximal response (1.7 nM) corresponded to the dissociation constant of binding to a specific high-affinity insulin receptor. The pace-maker phase of action potentials recorded during insulin perfusion was preceded by a prolonged or flattened after hyperpolarization, and its slope was less steep than controls. The action potential duration was also prolonged. These results indicate that physiological concentrations of insulin can regulate the embryonic heart rate.

Action Potentials↗
PubMed1984

Structural analysis and subunit interaction of insulin receptor from membranes of cultured embryonic chick heart cells.

In previous studies of cultured embryonic chick heart, insulin hyperpolarized cells and slowed their beat rate through occupancy of a high affinity receptor. In the present studies, we chemically characterize the native structure and subunit interactions of this insulin receptor. A stokes radius of 87 A and an apparent molecular mass of 350,000 daltons were found for membrane proteins specifically cross-linked to [125I] insulin by disuccinimidyl suberate. A primary subunit of 125,000 daltons in dithiothreitol or 115,000 daltons in its absence (alpha-subunit) was heavily cross-linked. A smaller subunit had a size of 90,000 daltons (beta-subunit). This beta-subunit was not readily labeled by [125I]insulin cross-linking, but insulin enhanced 32P incorporation from [gamma-32P]ATP, enabling its visualization. Subunit interaction could be studied, because alkaline conditions produced dissociation of the native 350,000-dalton receptor. This spontaneous dissociation was not a result of proteolysis and was prevented by acid conditions, oxidants, or N-ethylmaleimide. Using alkaline conditions followed by chemical reduction in two-way gels, we directly visualized the native complex of 350,000 daltons dissociating into combinations of subunits of apparent sizes of 290,000 (alpha 2 beta), 220,000 (alpha alpha), and 195,000 (alpha beta) daltons. Dithiothreitol produced combinations of subunits, which differed from alkaline dissociation. In alkaline conditions, the 290,000 (alpha 2 beta) and 220,000 (alpha alpha) dalton combinations predominated, whereas dithiothreitol produced 190,000 (alpha beta)-dalton proteins, which suggested that alpha-S-S-alpha disulfide bonds existed and were susceptible to chemical reductants, while alpha-S-S-beta disulfide bonds were more sensitive to alkaline lysis. We conclude from these observations that the native insulin receptor of embryonic chick heart cell exists on the sarcolemmal membrane as a relatively homogeneous tetramer of nonhomologous subunits in an alpha 2 beta 2 configuration. The alpha-subunits are the primary sites for insulin binding, and a beta-subunit is autophosphorylated. alpha-S-S-alpha and alpha-S-S-beta bonding exist, and these disulfide bonds have different sensitivities to chemical reducing agents and alkaline lysis.

Adenosine Triphosphate↗
PubMed1984

Genotyping and prenatal assessment of collagen lysyl hydroxylase deficiency in a family with Ehlers-Danlos syndrome type VI.

Collagen lysyl and prolyl hydroxylase activities were measured in cultured fibroblasts from a child with clinical features of Ehlers-Danlos syndrome. Lysyl-to-prolyl hydroxylase activity ratios in cells from the proband, mother, father, and control were .24, .86, .52, and 1.00, respectively, providing a biochemical diagnosis of Ehlers-Danlos syndrome type VI and indicating an autosomal recessive mode of inheritance in this family. Prenatal assessment of lysyl hydroxylase deficiency was requested and accomplished for the first time during a subsequent pregnancy in the family. A series of control cultures established lysyl hydroxylase activity to be similar in cultured amniotic fluid cells (AF and F cells) and in cultured dermal fibroblasts. Cultured F and AF cells from the monitored pregnancy had enzyme activity similar to controls, indicating that the fetus should not be affected by lysyl hydroxylase deficiency. This finding was confirmed by demonstration of normal lysyl hydroxylase activity in fibroblasts cultured from the newborn baby. These studies show that cells cultured from second trimester amniotic fluid have collagen lysyl hydroxylase activity similar to that in dermal fibroblasts, making prenatal diagnosis of lysyl hydroxylase deficiency possible.

Adult↗