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).

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.

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.

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.

Rapid loss of delta-aminolevulinic acid dehydratase activity in primary cultures of adult rat hepatocytes: a new model of zinc deficiency.

Nonproliferating cultures of adult rat hepatocytes were found to lose 60-70% of cell-associated zinc during their first 24 h of incubation in standard, serum-free medium. The loss of zinc was accompanied by a profound loss (greater than 95%) in the activity of the zinc metalloenzyme, delta-aminolevulinic acid dehydratase, as well as a loss (greater than 85%) in the cellular content of immunoreactive delta-aminolevulinic acid dehydratase protein. Restoration of cellular zinc content by the addition of zinc to the culture medium partially prevented the losses of both delta-aminolevulinic acid dehydratase activity and immunoreactive protein.

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.

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.

Oculomotor abnormalities in chronic GM2 gangliosidosis.

The clinical and electro-oculographic eye abnormalities in chronic GM2 gangliosidosis have been described. The most prominent oculomotor disturbances in our patients involved the pursuit system. This was evident during performance of eye tracking and vestibulo-ocular reflex suppression.

Ocular pathology of Fabry's disease in a hemizygous male following renal transplantation.

The ocular pathology of a hemizygous male with Fabry's disease after renal transplantation is reported. The ocular pathology in this patient was essentially identical to that which has previously been reported for both hemizygotes and heterozygotes afflicted with Fabry's disease. Glycolipid deposits and/or osmophilic inclusion bodies were found universally throughout the ocular vasculature.

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.

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.

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.

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.

Ultrastructure of the eye in fetal type II glycogenosis (Pompe's disease).

Type II glycogenosis is an autosomal recessive storage disease characterized by absence of the enzyme acid alpha-1,4-glucosidase. The eye of a 16 week fetus, aborted after diagnosis by amniocentesis, was studied by light and electron microscopy. Extensive deposits of lysosomal and cytoplasmic glycogen were present in virtually all ocular tissues examined, with the notable exception of pigment epithelia (iris and retina).

Gaucher disease: a membranous enzymopathy.

Inhibitors and activators of acid beta-glucosidase activity were used as probes to characterize the components of the active site of the membrane bound enzyme, acid beta-glucosidase. Three components of the active site were defined: (1) a catalytic site, (2) an aglycon binding site, and (3) a hydrophobic binding site (Fig. 5A).

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.