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R J Desnick

Publications and source records attributed to R J Desnick.

At least 217 records · Page 12Linked to original sources
PubMed1987

Gaucher disease: genetic heterogeneity within and among the subtypes detected by immunoblotting.

The genetic heterogeneity of Gaucher disease subtypes and variants was investigated by immunoblotting of fibroblast extracts. For these studies polyclonal and monoclonal antibodies were raised to acid beta-glucosidase preparations containing a single N-terminal amino acid sequence that was colinear with that encoded by the beta-Glc cDNAs. Three forms (Mr approximately equal to 67,000, 64,000-61,000, and 58,000) of cross-reacting immunologic material (CRIM) were observed in control individuals. Decreased amounts of the same CRIM forms were detected in most type 1 Gaucher disease patients, but single CRIM forms of variable molecular weight were observed in several non-Jewish type 1 variants. One or two CRIM forms of variable molecular weight were found in neuronopathic (type 2 and type 3) patients. The amount of CRIM was severely decreased in the majority of the type 2 and type 3 patients; one American black type 2 patient was CRIM negative. With this one exception, one CRIM form was detected in the cell-free culture media from all normal or Gaucher disease fibroblasts that had an Mr approximately 2,000 greater than the highest respective intracellular molecular-weight form. All intra- or extracellular CRIM forms were reduced to a single form after deglycosylation with N-Glycanase. In addition, the radioactivity from [3H]Br-conduritol B epoxide, a specific covalent inhibitor of beta-Glc, localized to the CRIM forms of beta-Glc on immunoblots. These results indicate that all subtypes and variants of Gaucher disease result from mutations that alter the stability and/or processing of beta-Glc. Furthermore, the heterogeneity of the CRIM patterns within and among the variants of Gaucher disease cause the diagnostic usefulness of immunoblotting to be restricted to those families in which the phenotype has been well established.

Antibodies, Monoclonal↗
PubMed1986

Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes.

Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.

Gaucher Disease↗
PubMed1986

Acceptability of chorionic villi sampling for prenatal diagnosis.

The factors that influence women in choosing between first-trimester chorionic villi sampling and second-trimester amniocentesis for prenatal diagnosis were investigated. Five hundred twenty women of advanced maternal age who had previously undergone prenatal diagnosis by amniocentesis and were delivered of a normal infant were requested to complete a questionnaire concerning their attitudes toward amniocentesis and chorionic villi sampling. The majority of respondents indicated that the time at which chorionic villi sampling is performed (76%), the rapid availability of diagnostic results (72%), and the type of abortion procedure available (68%) would make them choose this method. In contrast, the factors that influenced women to choose amniocentesis included the known low risk of spontaneous abortion (76%) and confidence in the skill of the obstetrician who would perform the procedure (56%). When all factors were considered together, 68% of the respondents chose amniocentesis based on the known low risk of spontaneous abortion, whereas for those who chose chorionic villi sampling (32%), the major criterion was the fact that the procedure is performed in the first trimester. However, 87% of women who preferred amniocentesis indicated that if the risk of spontaneous abortion associated with chorionic villi sampling. These results indicate that for many women of advanced maternal age, the acceptability and the use of chorionic villi sampling will be dependent on the demonstration that the risk of fetal loss is low, approaching that of amniocentesis.

Abortion, Spontaneous↗
PubMed1986

Molecular cloning of a cDNA for human delta-aminolevulinate dehydratase.

A cDNA encoding human delta-aminolevulinic acid dehydratase (ALA-D; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, was isolated from a human liver cDNA expression library. Of the original 17 clones selected with anti-ALA-D antibody, only four expressed anti-ALA-D epitopes as assessed by rescreening with antibody preabsorbed with purified antigen. Subsequent screening of the antibody-positive clones with mixed oligodeoxynucleotide (oligo) probes, synthesized to correspond to human N-terminal and bovine active-site peptide sequences, identified three clones which hybridized only with the oligo probes for the bovine amino acid (aa) sequences. Restriction endonucleases analysis revealed that these three clones contained the same 800-bp cDNA insert. This insert was recloned into bacteriophage M13mp18 and mp19 and sequenced by primer extension. The aa sequence predicted from the partial nucleotide sequence was found to be essentially colinear with the sequences of four bovine ALA-D peptides, totaling 35 non-overlapping aa residues.

Amino Acid Sequence↗
PubMed1986

Echocardiographic abnormalities and disease severity in Fabry's disease.

Fabry's disease is an X-linked recessive genetic deficiency of the enzyme alpha-galactosidase A, which leads to the pathologic deposition of neutral glycosphingolipids in lysosomes of the vascular endothelium of the heart, brain and kidney. The disease is progressive in hemizygous male patients, with increasing involvement of the major organs leading to death. Because cardiac involvement is a constant feature, echocardiograms were performed on 35 patients with Fabry's disease, 23 hemizygotes (aged 28.6 +/- 14 years) and 12 heterozygotes (aged 31.6 +/- 6 years), to determine whether cardiac involvement could be detected noninvasively. The results demonstrated that hemizygous male patients had a greater aortic root diameter, thicker interventricular septum and greater ventricular mass than did heterozygous female patients. Left ventricular mass per square meter of body surface area correlated well with clinical disease severity (r = 0.68, p less than 0.05), suggesting progressive glycosphingolipid deposition. Older heterozygotes (greater than 25 years old) had more severe evidence of cardiac disease than did younger male patients. Although mitral valve prolapse was identified in 12 (54%) of 23 male hemizygotes and in 7 (58%) of 12 female heterozygotes its presence did not correlate with clinical disease severity or other echocardiographic variables. Therefore, echocardiographic evidence of Fabry's disease appears to correlate with age-related disease severity and may be a useful noninvasive marker to follow disease progression and possible regression when appropriate therapy becomes available.

Adult↗
PubMed1986

Human alpha-galactosidase A: nucleotide sequence of a cDNA clone encoding the mature enzyme.

The complete nucleotide sequence has been determined for a lambda gt11 cDNA clone (lambda AG18) containing the full-length coding region for the mature lysosomal form of human alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The lambda AG18 insert contained a 1226-base-pair sequence with an open reading frame encoding 398 amino acids of the mature polypeptide (predicted Mr = 45,356) and the last 5 amino acids of the propeptide sequence. The poly(A) signals AATACA and ATTAAA occurred 28 and 11 nucleotides prior to the TAA stop codon, respectively. There was no 3' untranslated region as the poly(A) sequence immediately followed the TAA termination codon; a second independently cloned cDNA confirmed this finding. The predicted amino acid sequence was colinear with 86 nonoverlapping residues (22% of the mature subunit) determined by microsequencing amino-terminal, tryptic, and cyanogen bromide peptides of the purified mature enzyme. Four potential N-glycosylation sites were identified, all of which occurred at predicted beta turns in hydrophilic regions of secondary structure. RNA transfer hybridization analysis of HeLa poly(A)+ RNA demonstrated a single 1.45-kilobase band whose signal was decreased by prior immunoabsorption of polysomes with monospecific alpha-Gal A antibodies. Searches of nucleic acid and protein data bases did not reveal significant homology even with the limited sequences available for mammalian lysosomal enzymes.

Amino Acid Sequence↗
PubMed1986

Human delta-aminolevulinate dehydratase: nucleotide sequence of a full-length cDNA clone.

Two cDNAs encoding human delta-aminolevulinate dehydratase (ALA-D; porphobilinogen synthase; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, were identified, recloned into bacteriophage M13, and sequenced by primer extension. The first clone with an 827-base-pair (bp) pEX-ALA-D cDNA insert, shown to contain DNA sequences that were colinear with four bovine ALA-D peptide sequences, was used to screen a pKT218 human liver library. A second clone containing a 1200-bp insert was identified that contained an open reading frame of 990 bp as well as 5' (66 bp)- and 3' (94 bp)-untranslated regions, the latter terminating in poly(dA). The predicted N-terminal amino acid sequence was colinear with the first 13 residues of microsequenced ALA-D purified from human erythrocytes. The ATG initiation codon was preceded by ACGCC, a functional initiation sequence, while an upstream (position -32), in-phase AACTG ATG sequence was entirely nonhomologous with the initiation consensus sequence and, therefore, presumed to be nonfunctional. The unusual polyadenylylation signal, AGTAAA, has been reported only in the human HRAS1 gene. The nucleotide sequences of the two cDNA clones differed at position 730 or 733 and encoded two differently charged amino acids. This nucleotide difference may be the basis for the polymorphic charge isozymes of human ALA-D. The sequence encoding this zinc metalloenzyme contained a cysteine- and histidine-rich binding site for zinc and an unusual region of charge complementarity surrounding the active lysine residue in the catalytic site.

Amino Acid Sequence↗
PubMed1986

Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site.

Human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic beta-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3H-labeled conduritol B epoxide (3H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V8 protease digestion of the 3H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (Mr, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid beta-glucosidase.

Amino Acid Sequence↗
PubMed1986

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.

1-Deoxynojirimycin↗
PubMed1986

Gaucher's disease type 1: assessment of bone involvement by CT and scintigraphy.

The effectiveness of CT and technetium-99m sulfur colloid (99mTc SC) bone-marrow scans in determining the extent and severity of skeletal involvement in 23 patients with type 1 Gaucher's disease was compared with the effectiveness of conventional radiographic techniques and technetium-99m methylene diphosphonate (99mTc MDP) bone scintigrams. Density measurements obtained by CT proved sensitive in differentiating normal marrow (-50 to -120 H). Scintigrams with the sulfur colloid nuclide demonstrated three distinct patterns of uptake: peripheral expansion of normal marrow (profile B), greater marrow expansion with patchy areas lacking uptake (profile C), and greater loss of uptake with retention of the nuclide in other reticuloendothelial organs and circulation (profile D). CT scans provided greater sensitivity in resolving the extent of marrow involvement in affected areas, while the 99mTc SC scintigrams were more effective in overall assessment of the severity of bone-marrow involvement. Both conventional radiographic techniques and 99mTc MDP bone scans were useful primarily as screening procedures or for evaluating specific involved areas. 99mTc MDP scans were useful in evaluating regional defects (i.e., ischemic necrosis) in certain cases, but no consistent patterns were observed. CT and 99mTc SC scans are useful for determining the extent and severity of Gaucher's disease involvement of bone marrow.

Bone Diseases↗
PubMed1986

Arylsulfatase B activity in cultured retinal pigment epithelium: regional studies in feline mucopolysaccharidosis VI.

Feline mucopolysaccharidosis VI (MPS VI) is a recessively inherited lysosomal storage disease resulting from a deficiency of arylsulfatase B (ASB). Previous histopathologic findings have indicated that the disease is expressed morphologically in non-pigmented retinal pigment epithelial cells (RPE) in the posterior pole and superior equatorial regions by the accumulation of vacuolated inclusions and eventual cellular hypertrophy, while pigmented regions in the periphery are minimally affected. To determine if the regional and age-dependent variations in disease severity result from differences in residual enzyme activity, primary cultures of feline MPS VI-affected RPE were initiated from defined regions of the eye and maintained in vitro for 14 days. Cultures initiated from nonpigmented areas of affected adult eyes (posterior pole, superior equatorial) were more diseased than those from pigmented (inferior-equatorial, peripheral) areas. In the nonpigmented cultures, the disease was expressed by the accumulation of single membrane-bound inclusions and cellular hypertrophy. These inclusions were indistinguishable in their morphologic appearance and distribution from those found in situ. In contrast, the cultures initiated from pigmented areas remained normal or minimally affected. The same spatial disease distribution was present in young affected eyes, but the expression of the disease was much less severe. It is apparent that temporal, spatial, and pigmentation factors were correlated with disease expression in vitro as well as in situ. Arylsulfatase B activity was measured biochemically, and found to be deficient in all regions of young and adult eyes. It was notable that there was no correlation between the level of residual enzyme activity, and the pigmentation or spatial position from which the cells were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Genetic heterogeneity in acute intermittent porphyria: characterisation and frequency of porphobilinogen deaminase mutations in Finland.

The occurrence of different porphobilinogen deaminase mutant types in 68 patients with acute intermittent porphyria from 33 unrelated families in Finland was studied with biochemical and immunological techniques. In this fairly homogenous population four different porphobilinogen deaminase mutant types were identified and their frequencies determined. Most (about 80%) of the mutations were cross reacting immunological material (CRIM) negative, including a large kindred with normal erythrocyte porphobilinogen deaminase activities. The remainder of the families had CRIM positive mutations, including an unusual type (type 2) that had an immunoreactive, non-catalytic porphobilinogen deaminase level considerably greater than the maximal theoretical ratio of CRIM to activity of 2.0 for a single mutant allele. Correlations of the amount of residual porphobilinogen deaminase activity and the occurrence of acute clinical manifestations in each mutant type suggested that CRIM positive type 2 patients may have fewer acute symptoms.

Ammonia-Lyases↗
PubMed1985

Genetic heterogeneity in Gaucher disease: physicokinetic and immunologic studies of the residual enzyme in cultured fibroblasts from non-neuronopathic and neuronopathic patients.

To elucidate the genetic heterogeneity in the three major phenotypic subtypes of Gaucher disease, the residual acid beta-glucosidase in fibroblasts from patients with all three subtypes from different ethnic and demographic groups was investigated by comparative kinetic, thermostability, and immunotitration studies. The kinetic studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to the enzyme modifiers, taurocholate (or phosphatidylserine), and glucosyl sphingosine (or N-hexyl glucosyl sphingosine); Group A residual enzymes responded normally to these modifiers. All neuronopathic patients (types 2 and 3) and most non-Jewish, non-neuronopathic patients (type 1) had group A residual activities and thus could not be distinguished by their kinetic properties. Group B residual enzymes had markedly abnormal responses to these modifiers. All Ashkenazi and only two non-Jewish type 1 patients had group B residual activities. Group C residual activity had an intermediate response to all modifiers and represented a single Afrikaner type 1 patient. Pedigree studies indicated that this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. Thermostability studies showed additional heterogeneity of the residual activities within the three kinetic groups. Group A (type 2) and group B (type 1) enzymes had similarly decreased thermostabilities. In contrast, group A (type 1) residual activities were heterogeneous; three classes of thermostabilities were found among these enzymes: normal, decreased, and increased. Immunotitration of equal amounts of the normal or Gaucher disease beta-glucosidase activities with monospecific IgG indicated that the enzyme proteins from most Gaucher disease patients were antigenically altered and/or that large amounts of catalytically abnormal or inactive antigen were present. A decreased amount of antigenically and catalytically normal enzyme was present in a group A, type 1 African black patient, suggesting decreased stability or synthesis of his mutant acid beta-glucosidase. These kinetic, immunologic, and thermostability studies indicated that 1) type 1 Gaucher disease is biochemically heterogeneous and results from at least four distinct allelic acid beta-glucosidase mutations that alter enzyme structure and/or function, 2) neuronopathic and non-Jewish non-neuronopathic phenotypes cannot be distinguished reliably by kinetic analyses alone, and 3) the Ashkenazi type 1 Gaucher disease results from a unique mutation that alters a specific active site domain of acid beta-glucosidase.

Cells, Cultured↗