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Biomedical subjects

R L Stevens

Publications and source records attributed to R L Stevens.

At least 163 records · Page 9Linked to original sources
PubMed1979

Enzyme replacement therapy by fibroblast transplantation: long-term biochemical study in three cases of Hunter's syndrome.

We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunter's syndrome, over periods ranging from 2.5 to 3.75 yr. The level of Hunter corrective factor excreted by all three patients increased after transplantation, as did the activity of alpha-L-idurono-2-sulfate sulfatase in serum, when measured directly with a radioactive disulfated disaccharide substrate. Sulfatase activity was also raised in leukocyte homogenates from the two patients that we were able to assess. These increases in enzyme activity were accompanied by corresponding increases in catabolism of heparan and dermatan sulfates, as shown by (a) a decrease in sulfate:uronic ratios of urinary oligosaccharides, (b) an increase in iduronic acid monosaccharide, and (c) a normalization of Bio-Gel P-2 gel filtration profiles. Both the increase in enzyme activity and increased catabolism were maintained during the period of study and were not affected by either a gradual decrease or total withdrawal of immunosuppressive therapy.

Child↗
PubMed1979

Metachromatic leukodystrophy without arylsulfatase A deficiency.

Two siblings of consanguinous parents were noted to have a neurologic syndrome marked by developmental delay, regression of psychomotor performance, marked spasticity and progressive central nervous system degeneration. Markedly delayed nerve conduction times and a sural nerve biopsy which demonstrated changes typical of metachromatic leukodystrophy (MLD) were evident. Impairment of sulfated glycolipid metabolism was documented by analysis of glycospingolipid in urinary sediment. In spite of these findings, activities of arylsulfatase A and cerebroside sulfatidase in white blood cells and cultured skin fibroblasts were near normal. However, when intact growing fibroblasts were loaded with 35SO4-sulfatide a clear defect in sulfatide cleavage, comparable to that seen in MLD patients, was observed. Thus, these patients represent a new form of sulfatide storage disease -- MLD characterized by intact enzyme activity in cell homogenates but defective sulfolipid metabolism in vivo and in intact fibroblasts.

Adult↗
PubMed1979

Arysulfatase A modulation with pH in multiple sulfatase deficiency disorder fibroblasts.

It has been observed that multiple sulfatase deficiency disorder (MSDD) fibroblasts contained from profoundly deficient to near normal amounts of arylsulfatase (ARS) A depending on the medium in which they were cultured. Our present findings show that the major factor determining the enzyme level is the pH of the medium during growth. In media which became acidic or was maintained at low pH (less than 7), the cells expressed the enzymopathy, while in high pH media (7.4), the cells produced enzyme. The high and low enzyme states were reversible. The ARS A deficiency in MSDD must, therefore, be a secondary manifestation of a mutation in another system.

Cerebroside-Sulfatase↗
PubMed1978

Sequence comparison of human plasma alpha1-acid glycoprotein and the immunoglobulins.

The amino acid sequence of human plasma alpha1-acid glycoprotein, upon comparison with the sequences of other blood proteins, was shown to possess significant similarity with the immunoglobulins. Employing direct and corrected sequence identity, the average mutation value and two different computer comparisons for the evaluation of sequence similarity, the following two regions of this alpha-globulin, which account for approximately half of the total amino acid sequence of the protein, were found to possess sequence similarity with the immunoglobulins. a) The region from residues 77 through 125 proved to be related to the variable region of several human H and L chains, and b) the region from residues 136 through 166 was found to be related not only to the constant region of a human and a mouse L chain but also to the third and fourth constant region of a rabbit and a human H chain, respectively. These results suggest that alpha1-acid glycoprotein is probably related to the immunoglobulins and further suggest that it possibly diverged from the immunoglobulin evolutionary tree prior to the formation of the primitive L chain.

Amino Acid Sequence↗
PubMed1978

Iduronate sulfatase analysis of hair roots for identification of Hunter syndrome heterozygotes.

Iduronate sulfatase, the enzyme deficient in Hunter syndrome, can be readily measured in individual hair roots. Samples from Hunter syndrome hemizygotes had activities at or near the limits of detection. Samples from two mothers of Hunter syndrome patients, one an obligate heterozygote, had lower average iduronate sulfatase activity than the normal mean, and a significant number of hair roots had activity in the pathognomic range. A third mother showed a normal distribution of enzyme activity, and no hair roots were in the range of those from an affected individual. These results are similar to studies on the distribution of other X-linked enzymes in individual hair root samples from heterozygotes. This suggests that hair root iduronate sulfatase assessment is useful in the detection of Hunter syndrome carrier status, but further refinement of the test system is necessary.

Female↗
PubMed1978

Presence of arylsulfatase A (ARS A) in multiple sulfatase deficiency disorder fibroblasts.

Multiple deficiency disorder fibroblasts cultured in MEM-CO2 showed deficiencies of arylsulfatase A(ARS A) comparable to the deficiency in metachromatic leukodystrophy fibroblasts. However, the MSDD fibroblasts cultured in MEM-HEPES contained near normal levels of ARS A. Moreover, the enzyme from the latter fibroblasts was indistinguishable from ARS A of control fibroblasts on DEAE-cellulose chromatography, ratio of activity with several substrates, thermal inactivation, sensitivity to inhibitors, and precipitation by antiserum to human ARS A. These data support the conclusion that the ARS A genome is intact in MSDD fibroblasts and, by extension, in MSDD patients. Other sulfatases were present at levels ranging from mildly deficient to near normal but never as low as seen in the corresponding specific sulfatase deficient disorders.

Cell Line↗
PubMed1977

A low molecular weight glycosaminoglycan from the human aorta.

A low mol. wt, dialyzable glycosaminoglycan was isolated from human aorta and was found to be homogeneous on 2 dimensional electrophoresis. As judged by its electrophoretic mobilities and its hydrolysis by chondroitin sulfatase ABC, it was concluded that this hitherto unknown glycosaminoglycan is an oversulfated chondroitin sulfate.

Aorta↗
PubMed1977

Arylsulfatase of human tissue. Studies on a form of arylsulfatase B found predominantly in brain.

The distribution of soluble arylsulfatase (aryl-sulfate sulfohydrolases, EC 3.1.6.1) in human tissues was investigated by DEAE-cellulose chromatography, All tissues examined contained arylsulfatase A and arylsulfatase B. In addition, brain singularly contained significant quantities (15-25% of total arylsulfatase) of a minor anionic arylsulfatase from designated arylsulfatase Bm, whereas only trace amounts of arylsulfatase Bm were found in liver, kidney, testis and placenta. Arylsulfatase B and arylsulfatase Bm had equal activity toward methyl-umbelliferyl sulfate, nitrocatechol sulfate and a physiological substrate UDP-N-acetylgalactosamine 4-sulfate, but both forms were inactive toward the arylsulfatase A substrates cerebroside sulfate and ascorbic acid 2-sulfate. Purified preparations of placental arylsulfatase B, brain arylsulfatase Bm, and urinary arylsulfatase A did not hydrolyze estrone sulfate, dehydroepiandrosterone sulfate or pregnenolone sulfate. The physico-chemical properties of arylsulfatase Band arylsulfatase Bm differed with respect to thermal lability, DEAE-cellulose chromatography, polyacrylamide gel electrophoresis and isoelectric focussing. In the latter technique, utilizing thin polyacrylamide slab gels, the isoelectric point for placental arylsulfatase B was 8.2, while brain arylsulfatase Bm resolved into 3 activity bands with pI values 6.8, 7.0 and 7.2. Although the physico-chemical properties differed, arylsulfatase B and arylsulfatase Bm appear to be functionally equivalent as well as generically related.

Brain↗
PubMed1977

Steroid sulfatase deficiency.

Placental steroid sulfatase deficiency is a genetic disorder only recently reported in the medical literature. Most documented cases of placental sulfatase deficiency have been marked by delay in onset of labor, lack of cervical dilatation, and relative refractoriness of oxytocic agents and amniotomy. We have studied the placenta, cultured fibroblasts, and amniotic fluid cells from an affected patient. The activities of estrone sulfatase, pregnenolone sulfatase, dehydroepiandrosterone sulfatase, and arylsulfatase C in the placenta from the patient were severely deficient. Arylsulfatases A and B were present at levels within the normal range for this tissue. Fibroblast dehydroepiandrosterone sulfatase activity was virtually absent in the patient's cells and present at normal levels in individuals with a variety of lysosomal disorders. It would thus appear that the mutation responsible for steroid sulfatase deficiency is genetically and biochemically distinct from those involved in the lysosomal sulfatase deficiency states. The cell culture studies further suggest that the defect is a generalized one which should be detectable in midtrimester of pregnancy and may have phenotypic consequences in later postnatal life.

Estriol↗
PubMed1976

Microheterogeneity of arylsulfatase A from human tissues.

Human arylsulfatase A (cerebroside-3-sulfate 3-sulfohydrolase, EC 3.1.6.8) exhibited microheterogeneity on isoelectric focusing in polyacrylamide gels. Pure urinary enzyme gave 3 bands of activity with pI values of 4.7, 4.8 and 4.9, whereas purified liver enzyme yielded six equally spaced bands from pI 4.4 to 4.9. Detection of enzyme in the gel was made by either methylumbelliferyl sulfate or nitrocatechol sulfate. Crude enzyme preparations from human liver, kindey, placenta, brain and testis showed the six-banded pattern with varying amounts of activity in the different bands. The banding pattern of cultured human fibroblast extracts was distinctive: in addition to activity in the area of Bands 1-6 a sharp band at pI 5.1 was observed with both enzyme stains. This latter band was also present in metachromatic leukodystrophy fibroblast extracts. However, in this case the band did not appear when the specific aryl-sulfatase A stain was used. Enzyme Bands 1, 2 and 3 from urine were isolated by extraction of the gel. The three bands refocused in their initial positions; showed nearly identical enzymatic activities toward methylumbelliferyl sulfate, mitrocatechol sulfate, cerebroside sulfate and ascorbic acid 2-sulfate; and demonstrated equivalent immunological competence by antibody titration. The banding pattern of urinary arylsulfatase A was unchanged with neuraminidase treatment, whereas Bands 4-6 of the liver enzyme were converted to Bands 1-3 by this treatment. It appears that Bands 4-6 are due to sialylation of aryl-sulfatase A but that Bands 1-3 are probably due to some other type of post-ribosomal protein modification.

Brain↗
PubMed1976

Two alpha-glucosidases in cultured amniotic fluid cells and their differentiation in the prenatal diagnosis of Pompe's disease.

A sensitive fluorometric assay utilizing 4-methylumbelliferyl-alpha-D-glucopyranoside has been developed for the determination of alpha-glucosidase. The enhanced sensitivity was achieved by increasing the solubility of the substrate with a water miscible organic solvent. With this system, cultured amniotic fluid cells were found to have two major forms of alpha-glucosidase with somewhat overlapping acidic pH optima; one with pH optimum at 4.5 is deficient in Pompe's disease (type II glycogenosis), while one with pH optimum at 6.0 is not affected in this disease. Specificity for the pH 4 form of alpha-glucosidase was achieved by exploiting the greater thermal lability of the pH 6 enzyme. The pH 6 form of the enzyme was also detectable in freshly prepared extracts of cultured fibroblasts. The procedure is direct and simple and has been applied to the prenatal diagnosis in two pregnancies at risk for Pompe's disease.

Amniotic Fluid↗
PubMed1976

Ascorbic acid-2-sulfate sulfhohydrolase activity of human arylsulfatase A.

Pure human arylsulfatase A (EC 3.1.6.1) was found to hydrolyze ascorbic acid 2-sulfate to ascorbic acid and inorganic sulfate at rates from 200 to 2000 mumol/mg per h depending on the method of assay. This rate was lower than that observed with the synthetic substrate 4-nitrocatechol sulfate, but higher than that seen with the physiological substrate cerebroside sulfate. Extracts of cultured fibroblasts from normal subjects were also shown to hydrolyze ascorbic acid 2-sulfate; extracts of fibroblasts from patients with metachromatic leukodystrophy, known to be deficient in arylsulfatase A, did not. Similarly, hydrolysis of ascorbic acid 2-sulfate was not observed when a partially purified preparation of human arylsulfatase B was tested under a variety of conditions. Thus, in the human, arylsulfatase A appears to be the major, if not the only, ascorbic acid-2-sulfate sulfohydrolase.

Ascorbic Acid↗
PubMed1976

The glycosaminoglycans of the human artery and their changes in atherosclerosis.

The changes in levels of glycosaminoglycans (GAGs) of the intima and media of the human artery in atherosclerosis were determined by a recently introduced two-dimensional electrophoresis technique that permits direct measurments of each of these macromolecules. To identify the arterial GAGs, they were fractionated by chromatography on a DEAE-Sephadex A-25 column, and the resulting three fractions (hyaluronic acid [HA], heparan sulfate [HS], and the partially separated chondroitin sulfates B [CSB] and C [CSC]) were analyzed for their electrophoretic mobilities by this electrophoretic method, for their digestability by highly specific hydrolases (leech hyaluronidase, heparinase, and chondroitinases ABC and AC) and for their iduronic acid content. From these studies we concluded that normal and atherosclerotic human aortas contain CSB, CSC, HA, and HS. Further, we demonstrated that CSB is a hybrid consisting of approximately 40% CSA and 60% CSB and that CSC appears to be a polymer consisting essentially of glucuronic acid and N-acetylgalactosamine-6-sulfate. Classical CSA as well as chondroitin (CH) were not present in detectable amounts. In the relatively normal intima, the mean concentrations of the GAGs were found to be 4.7, 20.9, 1.3, and 5.1 mg/g of dry, defatted, decalcified tissue for CSB, CSC, HA, and HS, respectively. With the progression of atherosclerosis, there was a pronounced decrease in the total GAG content (from 32 to 18 mg) associated with a decrease in the CSC and HS levels but without a change in the HA concentrations. Of particular interest, however, was the increase in the CSB level. In the media whose total GAG content averaged approximately 20 mg, no significant changes in these GAG levels were noted with the progression of the disease except for that of CSC. These findings may be important in explaining the increased lipoprotein and collagen deposition in the diseased aorta.

Aorta↗
PubMed1975

Purification and properties of arylsulfatase. A from human urine.

Arylsulfatase A (cerebroside sulfate sulfohydrolase) was purified 3500-fold at a 7% yield from human urine. A crude urinary protein concentrate was prepared by treating pooled urine with ammonium sulfate and subsequently drying the precipitate with acetone. The powder thus obtained was extracted with buffer and was subjected to chromatographic and electrophoretic procedures as follows: (a) ammonium sulfate reverse gradient solubilization chromatography; (b) DEAE-cellulose chromatography; (c) Sephadex G-200 gel filtration; (d) preparative polyacrylamide gel electrophoresis; (e) SP-Sephadex chromatography; and (f) antialbumin-Sepharose chromatography. The enzyme was judged to be essentially homogeneous by: (a) a single band on polyacrylamide gel electrophoresis at two pH values; (b) formation of a single precipitin line on immunodiffusion against its antiserum: (c) complete freedom from albumin, the major contaminating protein; and (d) a single band on sodium dodecyl sulfate gel electrophoresis.

Catechols↗