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J H Priest

Publications and source records attributed to J H Priest.

At least 37 records · Page 2Linked to original sources
PubMed1988

Molecular evidence for true isochromosome 21q.

In one family a duplicated 21q was shown to be a true isochromosome, which segregates from mosaic mother to non-mosaic child with full Down syndrome phenotype. Densitometric analysis of Southern blots, using probe pPW228C for the distal long arm of chromosome 21, indicated that the 21q duplication contains two copies of the allele detected by the probe. Maternal mosaic karyotype of 45,XX,-21/46,XX/46, XX,-21,+21i(21q) also suggested transverse mitotic centromere division as the origin of the 21q isochromosomes. Morphologic analysis of chromosome heteromorphisms strengthened this interpretation because the free 21 missing in the cell line with 45 chromosomes was also missing in cells with the isochromosome. In a second family the cytogenetic data also suggested transmission of an i(21q) from mosaic mother to non-mosaic Down syndrome child but molecular evidence did not prove identity of alleles in the duplicated chromosome 21.

Chromosome Aberrations↗
PubMed1987

H-Y typing by ELISA in a 46,X,dic(Y)(q11.2101) male: effects of a nonmosaic Yp duplication.

A new ELISA was used to measure H-Y antigen in cultured fibroblasts from a male with 46,X,dic(Y)(q11.2101) involving duplication of Yp. Aliquots of monoclonal H-Y antibody were absorbed with 1, 2, 4, or 8 X 10(6) cells from the 46,X,dic(Y) male, or with corresponding numbers of cells from a normal XY male and a normal XX female, and then were tested for residual activity against a soluble antigen source. Portions of the antibody absorbed with cells of the 46,X,dic(Y) male were found to be less reactive than portions absorbed with cells of the normal XY male, for all numbers of cells and both dilutions of plated antigen. The results, quantified in an electronic optical density reader, imply presence of excessive H-Y in cells of the 46,X,dic(Y) male, and suggest presence of a genetic determinant of H-Y on Yp or proximal Yq near the centromere.

Antibodies, Monoclonal↗
PubMed1987

Synthesis and properties of vinyl monomer/enzyme conjugates. Conjugation of L-asparaginase with N-succinimidyl acrylate.

Monomer conjugation of an enzyme followed by copolymerization with free monomer is a useful method of enzyme immobilization. L-asparaginase was conjugated with N-succinimidyl acrylate. Analysis of the conjugated enzyme via isoelectric focusing showed that a molar ratio of 9.5 free monomers per enzyme was needed during the conjunction for each vinyl group bound. Only 3% of the enzyme activity was lost per vinyl group added, and conjugation of an average of four monomers per enzyme thermally destabilized the enzyme only at temperatures above 50 degrees C. Activity of the enzyme at physiological temperatures was relatively unaffected.

Acrylates↗
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

Phase-separation immunoassays.

Solid-phase-based immunoassays have traditionally been plagued by nonspecific binding to the solid phase and by slow reaction kinetics relative to reactants that are free to diffuse in solution. We have developed two novel immunoassays in which the solid phase is generated in situ after the specific binding reaction has occurred, thereby enhancing reaction kinetics and minimizing the opportunities for non-specific binding. In the first system, the capture antibody is conjugated to an organic monomer, polymerization of which to form insoluble polymer particles is initiated by a reaction involving free radicals. The amount of signal-labeled antibody incorporated into the resulting particles is directly proportional to the concentration of antigen. The principle is illustrated for the simultaneous assay of IgG and IgM in a single sample. In the second system, capture antibody is conjugated to a polymer, the solubility of which is a function of temperature. Specific binding is conducted below the critical solution temperature of the polymer, which is then separated from solution by increasing the temperature above the critical temperature. The incorporation of signal-labeled antibody into the precipitated polymer is directly proportional to the concentration of antigen. This principle is illustrated for the assay of hepatitis B surface antigen and Chlamydia trachomatis.

Acrylic Resins↗
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

Growth factor and cytogenetic abnormalities in cultured nevi and malignant melanomas.

It has been proposed that benign nevi that fail to differentiate normally may undergo stepwise growth and morphologic changes resulting in progression toward dysplastic nevi, which in some cases progress into malignant melanoma. In this study, we sought to determine the relationship between production of endogenous growth factors and the appearance of chromosomal abnormalities in cultured nevi and melanomas. Newly established cultures from 8 nevi with benign histology and 6 malignant melanomas, and 2 malignant melanoma cell lines were studied. Assays for mitogenic growth factors were based on stimulation of [3H]thymidine incorporation into DNA in Hs0294 malignant melanoma cells, produced by serum-free conditioned medium from nevus or melanoma cultures. Karyotypes were examined in cultures of an equivalent passage. Three of the 8 nevus cultures were mitogen-negative and displayed normal karyotypes; one nevus culture was mitogen-positive and had a normal karyotype, although the biopsied tissue demonstrated histologic evidence of benign melanocytic proliferation; one was mitogen-negative initially, but had an extra chromosome 8 in 2 of 50 cells; 3 were mitogen-positive and chromosomally abnormal. Each of the cultures in this latter group exhibited reciprocal translocation (rcpt) as the only identifiable abnormality [rcpt(6;15), rcpt(10;15), rcpt(15;20)], or a constitutional rcpt(4;5). Thus, there was direct correlation between growth factor production and chromosome abnormality in 6 of 8 benign nevus cultures. In the newly established melanoma cultures there was also concordance between growth factor and chromosomal status; conditioned media from 4 of 6 were mitogen-positive by at least one assay, and all 4 of the mitogen-positive cultures had chromosomally abnormal cell populations. Of the 2 melanoma cultures negative for growth factors, one was also negative for chromosome abnormality; the other had chromosomal change consisting of increased polyploidy. Both melanoma cell lines had abnormal karyotypes and were mitogen-positive. Though numerous chromosome changes were noted in the karyotypically abnormal melanoma cells, 6 of the 8 cultures exhibited abnormalities in chromosomes 1, 6, and/or 7. These data suggest that steps in the progression from benign nevi toward dysplastic nevi or malignant melanoma include: proliferation resulting from altered production of endogenous mitogenic growth factors; and development of specific chromosomal abnormalities.

Cells, Cultured↗
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

Analysis of crossing-over in a family with translocation 9;10 involving a chromosome 9 with a pericentric inversion.

The presence of two markers on chromosome 9, both a balanced reciprocal translocation and an inversion, allows morphologic demonstration of recombination between the normal and rearranged homologues. In the family under discussion 50% of the progeny studied (two of four) received a translocated 9 without the inversion from a parent with a translocated and inverted 9, indicating crossing-over between members of the chromosome 9 pair. Thus the morphology of the chromosomes allows a recombinant event which is normally invisible to be seen cytologically. Theoretically after crossing-over the balanced reciprocal translocation heterozygote results from adjacent-1 segregation and unbalanced derivative chromosome combinations from alternate segregation. Therefore it cannot be assumed that the balanced progeny necessarily result from alternate segregation and the unbalanced from adjacent-1. The prenatal diagnostic studies presented in this report also show that chromosome analysis of other family members is required when the recombination between homologues produces differences in chromosome morphology between parent and fetus.

Adult↗
PubMed1985

Oxygen dependence of oestrogen production by human placental microsomes and cultured choriocarcinoma cells.

The oxygen dependence of oestrogen (oestrone and 17 beta-oestradiol) formation from androstenedione and testosterone was studied in term human placental microsomes and in cultured human choriocarcinoma cells (BeWo line). Incubations were performed under various steady-state oxygen concentrations and the production of oestrone and 17 beta-oestradiol quantitated by specific radioimmunoassays. The aromatization of C19-steroids by both placental microsomes and choriocarcinoma cells was shown to be oxygen dependent over a wide range of O2 concentrations. The results indicate that placental oxygenation may be a critical factor in determining oestrogen production in vivo. Therefore, impaired oestrogen biosynthesis due to hypoxia could be an important factor in a variety of physiological and pathological conditions.

Androstenedione↗
PubMed1985

Evaluation of rebreathing in various modifications of the Mapleson-D system.

Several modifications of the Bain system for nitrous oxide analgesia or general anesthesia were tested for rebreathing and retention of CO2. The classic Bain circuit causes the most rebreathing as measured by the fractional concentration of CO2 in a sample of inspired gas (FICO2), but stimulation of the respiratory system seems to allow near normal end tidal CO2 and arterial CO2 tensions. The most predictable systems that have the least amount of rebreathing are those that have the least dead space.

Anesthesia, Dental↗
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

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

Metabolism of [4-14C]androstenedione by cells cultured from human amniotic fluid.

The conversion of [14C]androstenedione by human fibroblast (F) and amniotic fluid (AF) cells obtained by amniocentesis was investigated using human dermal fibroblasts (DF) as controls. Cell suspensions were incubated with [14C]androstenedione in the presence of a NADPH generating system. Steroid reaction products were separated from unreacted substrate by chromatography on micro-columns of magnesium oxide, partially resolved by partition chromatography on celite and further characterized by thin-layer chromatography and recrystallization to constant specific activity. In the case of F cells the pattern of metabolism was qualitatively similar to that of DF cells; the predominant metabolite was testosterone and several uncharacterized metabolites were detectable. However testosterone was the only metabolite isolated from incubations with AF cells. The results demonstrate distinct differences in the capacity of AF and F type cells to metabolize [14C]androstenedione and support the view that F cells resemble typical fibroblasts from dermis or other connective tissues.

Amniotic Fluid↗
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↗
PubMed1984

Reversal of DNA methylation with 5-azacytidine alters chromosome replication patterns in human lymphocyte and fibroblast cultures.

Prior studies demonstrated that developmental or induced methylation of DNA can inactivate associated gene loci. Such DNA methylation can be reversed and specific genes reactivated by treatment with 5-azacytidine (5- azaC ). The present cytogenetic studies using replication banding methods show that 5- azaC treatment also results in an increase or decrease in replication staining at one or more band locations in human lymphocyte and fibroblast chromosomes. New replication band locations are not formed. These changes in replication staining, which reflect changes in timing of replication, are different between these two tissues. However, in both tissues, the delayed onset of replication in the heterocyclic, inactive X is shortened by 5- azaC . A correlation is thus suggested between the induced temporal change to earlier DNA replication, and induced hypomethylation and gene activation. The temporal effect on chromosome replication in 5- azaC -treated cells depends on the portion of the S-period studied. Toward the beginning of S, early-replication patterns are increased in both lymphocytes and fibroblasts. Toward the end of S, late-replication patterns are increased only in lymphocytes, suggesting a differential effect of 5- azaC in: (1) early-vs. late-S, and (2) lymphocytes vs. fibroblasts. Generally, 5- azaC has its greatest effect on the inactive chromosome regions that are typically late-replicating prior to 5- azaC treatment. These observed changes in replication band staining suggest that DNA methylation may modify regional groups of genes in concert.

Azacitidine↗