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Biomedical subjects

R J Desnick

Publications and source records attributed to R J Desnick.

At least 253 records · Page 14Linked to original sources
PubMed1984

First-trimester prenatal diagnosis of Tay-Sachs disease.

The prenatal diagnosis of Tay-Sachs disease was made in two at-risk fetuses by the analysis of chorionic villi obtained at 9 and 11 menstrual weeks, respectively. The diagnoses were based on the absence of beta-hexosaminidase A activity as determined by: (1) specific enzyme assays, (2) anion-exchange chromatography, and (3) cellulose acetate gel electrophoresis. The enzymatic diagnoses were confirmed on fetal tissue as well as by ultrastructural demonstration of the first-trimester fetal neuropathology. Optimal assay conditions for beta-hexosaminidase A in chorionic villi were established for the prenatal diagnosis of Tay-Sachs disease. Importantly, it was noted that a small amount of decidua or maternal blood could lead to misdiagnosis. Thus, extreme care must be taken in the preparation of chorionic villi for Tay-Sachs as well as other prenatal metabolic diagnoses.

Brain↗
PubMed1984

Assignment of the structural gene encoding human aspartylglucosaminidase to the long arm of chromosome 4 (4q21----4qter).

The structural gene for the human lysosomal enzyme aspartylglucosaminidase (AGA) has been assigned to chromosome 4 using somatic cell hybridization techniques. The human monomeric enzyme was detected in Chinese hamster-human cell hybrids by a thermal denaturation assay that selectively inactivated the Chinese hamster isozyme, while the thermostable human enzyme retained activity. Twenty informative hybrid clones, derived from seven independent fusions, were analyzed for the presence of human AGA activity and their human chromosomal constitutions. Without exception, the presence of human AGA in these hybrids was correlated with the presence of human chromosome 4. All other human chromosomes were excluded by discordant segregation of the human enzyme and other chromosomes. Two hybrid clones, with interspecific Chinese hamster-human chromosome translocations involving the long arm of human chromosome 4, permitted the assignment of human AGA to the region 4q21----4qter.

Amidohydrolases↗
PubMed1984

Human alpha-L-iduronidase. II. Comparative biochemical and immunologic properties of the purified low and high uptake forms.

The physicokinetic and immunologic properties of the purified low and high uptake forms of the human lysosomal hydrolase, alpha-L-iduronidase, have been determined and compared. The apparent Km and Vmax values for the low and high uptake forms were similar toward two artificial substrates, 4-methylumbelliferyl-alpha-L-iduronide (0.07 and 0.06 mmol/l; 16.15 and 14.85 mumol/min/mg, respectively), and phenyl-alpha-L-iduronide (1.42 and 1.66 mmol/l; 0.83 and 1.05 mumol/min/mg, respectively), and one natural substrate, anhydro-[3H]-mannitol-iduronide (0.86 and 1.04 mmol/l; 2.50 and 2.79 mumol/min/mg, respectively). The pH optima for both purified forms also were similar for each of the three substrates ( approximately 3.50, approximately 3.50, and approximately 4.50, respectively). Heparin markedly inhibited the 4-methylumbelliferyl-alpha-L-iduronide activities of both the low and high uptake forms, while dermatan sulfate and heparan sulfate were more inhibitory toward the low uptake activity. EDTA was a potent inhibitor of both enzyme forms; the divalent cations, Mg2+ and Ca2+, could recover up to 30% of the enzymatic activities after EDTA treatment. p-Chloromercuribenzoate and maleate also were inhibitory, whereas dithiothreitol and 2-mercaptoethanol were stimulatory. Both enzyme forms had similar thermostabilities ; the half-lives at 45, 52, and 60 degrees C were about 38, 24 and 12 min, respectively. The low and high uptake forms were immunologically cross-reactive as demonstrated by Ouchterlony double immunodiffusion and immunotitration studies using anti-human low uptake antibodies.

Endocytosis↗
PubMed1983

Purification and properties of human hepatic aspartylglucosaminidase.

Aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which hydrolyzes the N-acetylglucosamine-asparagine linkages in glycoproteins, was purified from human liver to homogeneity. The purification procedure included chromatography on DEAE-cellulose and concanavalin A-Sepharose, gel filtration on Sephadex G-200, and high performance liquid chromatography. The purified enzyme had a final specific activity of 1,200,000 units/mg of protein, a pH optimum of 6.1, a pI of 5.7, a Vmax of 1,240,000 units/mg, and a Km of 1.25 mM toward a natural substrate, aspartylglucosamine. The purified enzyme was remarkably thermostable, retaining 90% of initial activity after 1 h at 60 degrees C. The molecular weight of the native enzyme was estimated to be 80,000 by gel filtration and 84,000 by analytical polyacrylamide gel electrophoresis. Under denaturing conditions, the molecular weight was 76,000, indicating that the native enzyme was a monomer. Amino acid composition revealed only 2 methionine residues/enzyme molecule.

Adult↗
PubMed1983

Determination of delta-aminolevulinate dehydratase activity by a specific fluorometric coupled-enzyme assay.

A coupled-enzyme assay for the specific and sensitive determination of delta-aminolevulinate dehydratase activity has been developed. The assay specifically measured picomole quantities of the product, porphobilinogen, by its enzymatic conversion to uroporphyrinogen I and the fluorometric detection of oxidized uroporphyrin I. The coupled-enzyme assay was linear with time and protein concentration and required less than 3 h for 20 individual determinations. Under the standard assay conditions, 10 to 100 pmol of uroporphyrin I was reliably measured, representing 0.085 to 0.850 nmol/h of delta-aminolevulinate dehydratase activity per assay. In addition, the fluorometric assay was more sensitive than either the standard or the semimicro colorimetric methods. The specificity, rapidity, and sensitivity of this new fluorometric method facilitates the reliable determination of low levels of aminolevulinate dehydratase activity in small amounts of crude tissue homogenates or in cultured cells.

Animals↗
PubMed1983

Microautoradiographic study on the tissue localization of liposome-entrapped or unentrapped 3H-labeled beta-galactosidase injected into rats.

The localization of intravenously injected liposome-entrapped or unentrapped 3H- beta-ga lactosidase in various tissues of rats was investigated by microautoradiography. The microautoradiographic silver grains, indicating the uptake of both forms of the enzyme were observed in all of the rat tissues studied, such as the liver, kidneys, spleen, lungs, heart, muscle and brain. The liver was found to be the most active in taking up both forms of the enzyme and probably Kupffer cells were primarily involved in the uptake of the enzyme. Also in the brain, the uptake of both forms of the enzyme was observed, though far less extensively, through the blood-brain barrier. The uptake of the enzyme in the brain was somewhat greater in the gray matter than in the white matter.

Animals↗
PubMed1983

The pathology of the feline model of mucopolysaccharidosis I.

Five cats with feline alpha-L-iduronidase-deficient mucopolysaccharidosis were studied. Membrane-bound cytoplasmic inclusions were present in central nervous system neurons, hepatocytes, chondrocytes, vascular and splenic smooth muscle cells, bone marrow leukocytes, and fibroblasts of the skin, eye, and cardiac valves. The lesions in these cats closely resemble those described in human patients with mucopolysaccharidosis I H (Hurler syndrome).

Animals↗
PubMed1982

Purification and properties of feline and human arylsulfatase B isozymes. Evidence for feline homodimeric and human monomeric structures.

Normal feline and human arylsulfatase B isozymes were purified to homogeniety from liver. The specific activities of the feline and human enzymes toward p-nitrocatechol sulfate were 1,100,000 and 800,000 units/mg of protein, and toward UDP-N-acetylgalactosamine-4-sulfate were 5,500 and 4,000 units/mg of protein, respectively. Although both enzymes had the same pH optimum (5.7), the feline enzyme was more electronegative than the human enzyme when electrophoresed on polyacrylamide gels. Compared to the human isozyme, feline arylsulfatase B had a lower Km toward p-nitrocatechol sulfate (1.2 versus 3.6 mM), was more thermostable at 60 degrees C (68 versus 30 min), and had a slightly lower pI (7.8 versus 8.0). The native molecular weight of the feline enzyme was estimated to be about twice that the human isozyme by gel filtration, analytical polyacrylamide gel electrophoresis, and sucrose density-gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single protein bands of Mr = 41,000 and 38,000 for the feline and human isozymes, respectively. Alkylation and cross-linking experiments were consistent with the feline enzyme being a homodimer and the human enzyme a monomer. Amino acid compositional analyses revealed few significant differences between the two isozymes.

Amino Acids↗
PubMed1982

Gaucher type I (Ashkenazi) disease: a new method for heterozygote detection using a novel fluorescent natural substrate.

A new acid beta-glucosidase assay for the detection of heterozygotes for Gaucher Type I disease has been developed using isolated lymphocytes as enzyme source and a novel fluorescent natural substrate, NBD-glucosyl ceramide. The procedure for optimal heterozygote discrimination was established by systematic evaluation of the effect of various solubilization techniques, detergent concentrations, pH, enzyme sources and artificial vs. natural substrates. A pilot screening study indicated the feasible application of this assay for heterozygote detection in the high-risk Ashkenazi Jewish population.

Clinical Enzyme Tests↗
PubMed1982

Assignment of human alpha 1-antitrypsin to chromosome 14 by somatic cell hybrid analysis.

Human alpha 1-antitrypsin ( alpha-1-AT;Pi) production was analyzed in 11 primary mouse hepatoma-human lymphoid cell hybrids and in 14 secondary rat hepatoma-human fetal liver fibroblast hybrids. The presence of human alpha-1-AT was determined by Laurell immunoelectrophoresis of concentrated and isotopically labeled supernatant medium. Human alpha-1-AT production segregated in the mouse-human hybrids concordantly with human purine nucleoside phosphorylase and with chromosome 14. All rat-human hybrids that were alpha-1-AT positive were also positive for human purine nucleoside phosphorylase and chromosome 14. Our study demonstrated the usefulness of rodent hepatoma cell hybrids for mapping human liver-specific genes because differentiated functions are expressed despite the fact that the human parental cells did not express these functions. Our study also showed that human alpha-1-AT gene product can be processed for secretion in the rodent hepatoma cellular environment. The mouse-human hybrids showed that no other human chromosome carries genes necessary for processing or secretion of human alpha-1-AT in the hybrid cell milieu.

Animals↗
PubMed1982

Assignment of the gene for cytosolic alanine aminotransferase (AAT1) to human chromosome 8.

The segregation of human cytosolic alanine aminotransferase (AAT1) and the individual human chromosomes has been studied in 27 secondary and tertiary rat hepatoma-human (liver) fibroblast hybrids. The staining solution used to visualize AAT activity on starch gels was specific for AAT since it was visualized only when all components of the stain were present. The locus for human AAT1 has been assigned to chromosome 8.

Alanine Transaminase↗
PubMed1982

Regional assignment of the structural gene for human acid beta-glucosidase to q42 leads to qter on chromosome 1.

The structural gene for human acid beta-glucosidase (GBA) has been regionally assigned to a narrow region on chromosome 1 using somatic cell hybridization, specific immunoprecipitation, and assay with the natural substrate. A human fibroblast line, 46,XX,del(1)(pter leads to q42:), was fused with mouse RAG fibroblasts and the heterokaryons were subcloned. All hybrid subclones containing a normal chromosome 1 were positive for GBA. In contrast, subclones with a single del(1) were negative for GBA by both immunoprecipitation and natural substrate assays. These results were consistent with the previous assignment of GBA to the region 1p11 leads to qter and further localized the gene to the narrow region 1q42 leads to qter.

Chromosome Mapping↗
PubMed1982

Fluorometric coupled-enzyme assay for delta-aminolevulinate synthase.

The rapid and specific determination of picomole quantities of gamma-aminolevulinate has been accomplished by its enzymatic conversion to uroporphyrinogen I and subsequent fluorometric detection of the oxidized uroporphyrin I. The coupled-enzyme assay was linear with time and protein concentration and required less than 3 h for 25 individual determinations. Under the standard assay conditions, 5-100 pmol of uroporphyrin I was reliably quantitated; these values corresponded to a range of gamma-aminolevulinate synthase activities from 0.2 to 15 nmol/h/ml enzyme. The sensitivity of this method was comparable to the more time-consuming radiochemical determinations of gamma-aminolevulinate synthase activity. The gamma-aminolevulinate synthase activity of liver homogenates from uninduced rats was 8.6 U/g liver (37 degrees C).

5-Aminolevulinate Synthetase↗
PubMed1982

Porphobilinogen deaminase: methods and principles of the enzymatic assay.

Porphobilinogen (PBG) deaminase catalyzes the formation of the linear tetrapyrrole, hydroxymethylbilane, from four molecules of PBG. The tetrapyrrole is either converted to uroporphyrinogen III by uroporphyrinogen cosynthetase or nonenzymatically cyclized to uroporphyrinogen I. Methods for the biosynthesis of gram quantities of PBG and for the determination of PBG-deaminase specific activity in various tissues were developed. Using this assay, the PBG-deaminase activities in human erythrocytes, brain, liver and cultured fibroblasts and lymphoblasts as well as various rat tissues were determined as reference values. In addition, the conditions for the use of this assay in the enzymatic diagnosis of heterozygotes for acute intermittent porphyria were established.

Ammonia-Lyases↗
PubMed1982

Enhancement of residual arylsulfatase B activity in feline mucopolysaccharidosis VI by thiol-induced subunit association.

The molecular pathology of the deficient arylsulfatase B activity in feline mucopolysaccharidosis (MPS) VI was investigated. Compared with the highly purified normal feline hepatic enzyme, the purified MPS VI residual activity had a 100-fold higher Michaelis constant (K(m)), an altered electrophoretic mobility, half the apparent native molecular weight, and markedly decreased thermo-, cryo-, and pH stabilities. Molecular weight and alkylation studies were consistent with the normal enzyme being a homodimer and the residual MPS VI enzyme a monomer. When incubated with various sulfhydryl reagents, the residual specific activity was enhanced several-fold, whereas the activity of the purified normal enzyme was un-affected or slightly inhibited. In the presence of dithiothreitol (DTT) and cysteamine, a lysosomotropic aminothiol, the residual activity had an electrophoretic mobility and native molecular weight similar to those of the normal feline enzyme. These findings suggested that the monomeric residual enzyme was dimerized in the presence of thiol-reducing agents. To determine if thiol-induced subunit association could therapeutically increase the residual activity and degrade the accumulated dermatan sulfate, in vitro and in vivo experiments were undertaken. When 2 mM DTT or cysteamine was incubated with heparinized whole blood from an MPS VI cat, the leukocyte residual arylsulfatase B activity increased 11- and 20-fold, respectively, and the accumulated dermatan sulfate was degraded in the presence of both thiol reagents. Intravenous administration of DTT (50 mg/kg) effected an immediate, but transient, increase in leukocyte residual activity; however, the substrate levels were not significantly decreased. In contrast, intravenous administration of cysteamine (15 mg/kg) increased leukocyte residual activity more than sixfold 30 min postinfusion; concomitantly, the leukocyte substrate was decreased to 35% of the initial level immediately after infusion and to about 45% of preinfusion values during the 120-min period studied. These results suggest that the defective residual activity in feline MPS VI can be therapeutically manipulated by thiol-induced subunit association. Furthermore, this animal analog provides a prototype for the investigation of human inborn errors of metabolism resulting from enzymatic defects that might be amenable to enzyme manipulation therapy.

Animals↗