Alpha-L-iduronidase activity in cultured skin fibroblasts and amniotic fluid cells

Using phenyl-α-l-iduronide as substrate, we have examined the level of α-l-iduronidase activity in homogenates of fibroblasts derived from normal individuals, from patients affected with α-l-iduronidase deficiency disorders (Hurler syndrome, Scheie syndrome, and a disease of intermediate severity presumed to be a Hurler/ Scheie compound) and from parents of such patients. Extracts derived from the affected individuals had no detectable α-l-iduronidase activity, whereas those derived from heterozygotes varied between 20% and 95% of the normal mean. Overlap between normal and heterozygous levels was reduced if the α-l-iduronidase activity was expressed on the basis of the β-galactosidase activity in the same homogenate. Cultured amniotic fluid cells from normal pregnancies had less than half as much α-l-iduronidase activity as fibroblasts from normal adults; this might cause problems in distinguishing a heterozygous fetus from an affected one by the enzyme assay alone.     

Polyethylene glycol-conjugated adenosine phosphorylase: development of alternative enzyme therapy for adenosine deaminase deficiency

Purine nucleoside phosphorylase had previously been engineered to accept 6-amino substituted purine nucleosides by two active site substitutions, Asn243Asp; Lys244Gln. In the present study, recombinant adenosine phosphorylase (AP) has been conjugated to branched polyethylene glycol (PEG) polymers of approximately 42.5 kDa. Matrix-assisted laser desorption/ionization analysis and SDS acrylamide electrophoresis analysis indicated a subunit composition of greater than 205 kDa consistent with the conjugation of as many as four PEG molecules per AP subunit. The PEG-conjugated enzyme retained greater than 90% of the native catalytic activity. Administration of the enzyme to mice demonstrated the PEG-AP to have a 67-fold increased plasma half-life compared to the native enzyme, 65.1+/-2.9 h versus 57.8+/-1.1 min, respectively. PEG-AP was principally confined to the plasma with minimal activity detected in tissues and of these spleen had the greatest activity and essentially no activity was found in urine. PEG-AP has retained activity with inosine and its injection into PNP-deficient mice resulted in a 2.7-fold increase in urine urate. AP was also shown to protect human CEM cells in culture from the toxic effects of 2'-deoxyadenosine. These studies provide evidence for consideration of PEG-AP as an alternative enzyme therapy for the inherited deficiency of adenosine deaminase.     

Expression of different isoenzymes of adenylate deaminase in cultured human muscle cells. Relation to myoadenylate deaminase deficiency.

Adenylate deaminase activity was determined in cultured muscle cells of different maturation grades and muscle biopsies from normal subjects and four patients with a primary myoadenylate deaminase (MAD) deficiency. Adenylate deaminase activity was much lower in cultured human muscle cells than in normal muscle. The activity increased with maturation. The ratio of activities measured at 5 and 2 mM AMP decreased in the order: immature muscle cells greater than more mature muscle cells greater than muscle. Adenylate deaminase activity was detectable in muscle cell cultures of MAD-deficient patients. However, both at 2 and 5 mM AMP this activity was significantly lower than in cultured cells with the same high maturation grade obtained from control subjects, whereas the ratio between the activities at 5 and 2 mM AMP was higher. The observations indicate that transition from a fetal to an adult muscle isoenzyme of adenylate deaminase takes place in human cultured muscle cells during maturation. In cultures obtained from MAD-deficient patients this transition does not occur and only the fetal isoenzyme is present.     

Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells

1. 1. The specific activity of the lysosomal hydrolases in cultured skin fibroblasts varies according to the phase of growth in culture.

2. 2. Diagnosis of heterozygous genotypes for lysosomal enzyme deficiency diseases is unreliable with cultured fibroblasts, at least partly because of the growth curve-associated variations in specific activity.

3. 3. Fluctuations in specific activity during the beginning of the growth curve in vitro can be avoided by initiating cultures with cells which are in the early log phase of growth.

4. 4. Primary amniotic fluid cell cultures show no relationship between length of time in culture and lysosomal enzyme specific activity.

5. 5. Secondary amniotic fluid cell cultures exhibit growth curve-related variations in lysosomal enzyme specific activity as they assume fibroblast-like growth kinetics.

6. 6. Prenatal and postnatal diagnosis on cultured amniotic fluid cells and fibroblasts requires the use of appropriate controls which are matched for stage of growth and length of time after the last trypsinization.

     

Improved technique for the expression of fragile-X in cultured amniotic fluid cells

Summary An improved technique for inducing fra(X) expression in cultured cells was obtained by using diazepam for mitotic arrest and 5-fluorodeoxyuridine (FUdR) for the induction of fra(X) expression. The method was developed using cultured fibroblast and urinary cells from fra(X) patients. Prenatal studies were performed on cultured amniotic fluid cells in five pregnancies at risk for fra(X). In two cases the cultured cells showed a 46,XY, fra(X) karyotype. One of the pregnancies was terminated and the diagnosis was confirmed by chromosome studies on several fetal tissues including chorionic villi and by histopathologic changes in the lymphatic vessels of the fetal testes. The fra(X) was also demonstrated in chorionic villi in a case in which amniotic fluid cells were not studied. Chorionic villi were isolated after a spontaneous abortion, the cultured cells had a 45,X karyotype and in addition 5% of the cells were fra(X) positive.     

Characterization of hCG regulation in cultured human amniotic fluid cells: II. Mechanisms for stimulation

We showed previously that sodium butyrate stimulated human chorionic gonadotropin (hCG) measured by radioimmunoassay of medium from human second trimester amniotic fluid cell cultures, termed AF cells. We now find that stimulation of hCG in the presence of sodium butyrate takes as long as 20 h. When AF cells are preincubated with sodium butyrate, hCG levels increase in direct relation to length of the preincubation period. These findings suggest that elevation of hCG is not due merely to a release of hormone from the cells. Addition of cycloheximide or Actinomycin D inhibited protein synthesis and RNA synthesis, respectively, and prevented the stimulation of hCG by sodium butyrate. These results lend support for a mechanism of regulation involving protein and RNA synthesis, the increase in hCG levels being due to new synthesis of the hormone. Other agents reported to influence hCG production by different types of cell cultures include dibutyryl cyclic AMP, epidermal growth factor (EGF), methotrexate, and hydroxyurea. Dibutyryl cyclic AMP and EGF have no effect on hCG production in our AF cells: methotrexate causes a minimal increase, hydroxyurea causes a further increase, but sodium butyrate has the strongest stimulatory effect. We conclude that amniotic fluid cells in culture are susceptible to environmental agents capable of modulating synthesis of hCG by mechanisms involving synthesis of RNA and protein.     

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

Summary l-Carnitine transport was studied in cultured muscle cells and skin fibroblasts of patients with primary systemic carnitine deficiency and control subjects. In both cell culture types, two systems for carnitine transport were identified. The kinetic parameters for carnitine transport were remarkably similar in cultured muscle cells and skin fibroblasts. Normal rates and kinetic properties of carnitine transport were observed for both cell lines from patients with systemic carnitine deficiency. These studies do not rule out a defect in carnitine transport in vivo. This study was supported by research grants AM27451 and NS06277 from the National Institutes of Health and by a Research Center Grant from the Muscular Dystrophy Association.     

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a K_m value for dCMP of 0.91mm. dCTP activation required the presence of Mg²⁺ or Mn²⁺ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg²⁺ or Mn²⁺ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).     

Characterization of hCG regulation in cultured human amniotic fluid cells

Summary We showed previously that sodium butyrate stimulated human chorionic gonadotropin (hCG) measured by radioimmunoassay of medium from human second trimester amniotic fluid cell cultures, termed AF cells. We now find that stimulation of hCG in the presence of sodium butyrate takes as long as 20 h. When AF cells are preincubated with sodium butyrate, hCG levels increase in direct relation to length of the preincubation period. These findings suggest that elevation of hCG is not due merely to a release of hormone from the cells. Addition of cycloheximide or Actinomycin D inhibited protein synthesis and RNA synthesis, respectively, and prevented the stimulation of hCG by sodium butyrate. These results lend support for a mechanism of regulation involving protein and RNA synthesis, the increase in hCG levels being due to new synthesis of the hormone. Other agents reported to influence hCG production by different types of cell cultures include dibutyryl cyclic AMP, epidermal growth factor (EGF), methotrexate, and hydroxyurea. Dibutyryl cyclic AMP and EGF have no effect on hCG production in our AF cells: methotrexate causes a minimal increase, hydroxyurea causes a further increase, but sodium butyrate has the strongest stimulatory effect. We conclude that amniotic fluid cells in culture are susceptible to environmental agents capable of modulating synthesis of hCG by mechanisms involving synthesis of RNA and protein. Research supported by Grant HD 11379 from the National Institutes of Health.     

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

The level of adenosine deaminase (ADA; EC 3.5.4.4) was estimated at different passages in six confluent fibroblast cultures established from forearm skin biopsies of healthy adult normal volunteers. After determination of the zinc concentration in standard growth medium, ADA activity was estimated at different passages of subculture in media with different zinc concentrations. The results indicated that the specific activity of ADA in control confluent skin fibroblast cultures (passage 2) cultivated in standard growth medium containing 15.4 microM zinc (similar to that present in normal human plasma) was equal to 226.6+/-19.64 micromol min(-1) mg(-1) protein. The results showed that there were no significant changes in ADA specific activity in any of the control cultures as the zinc concentration of the medium was increased. To characterize the passage of subculture at which fibroblasts enter the ageing phase, three marker enzymes were assayed namely, phosphofructokinase, lactate dehydrogenase and glycogen phosphorylase. The result showed that the cells enter the ageing phase at passage 20 and beyond. Further investigation showed that ADA activity of serially subcultured confluent cultures cultivated in standard growth medium significantly dropped at passages 20, 25 and 30. ADA activity however was not significantly altered in cells at passage 2, 10 and 15 cultivated in standard growth medium and in the presence of higher zinc levels (23.1, 34.6, 53.8 and 73.1 microM). Furthermore there was significant lowering of ADA activities in cells at passages 20, 25 and 30 when cells were cultured in the presence of 15.4, 23.1 and 34.6 microM zinc. Such lowered activities of ADA were restored to normal when the cells were cultured in the presence of higher zinc concentration equal to 53.8 and 73.1 microM. From the results we concluded that it is possible to restore ADA activity in aged skin fibroblasts to normal levels by raising the zinc concentration in the culture medium to four or five times the control normal plasma zinc level.     

Genetic expression in partial adenosine deaminase deficiency. mRNA levels and protein turnover for the enzyme variants in human B-lymphoblast cell lines

A severe genetic deficiency of adenosine deaminase is causally associated with an autosomal recessive form of severe combined immunodeficiency disease, while subjects with absent erythrocyte but partial lymphocyte enzyme activity remain immunocompetent. The genetic expression of adenosine deaminase in B-lymphoblast cell lines derived from four unrelated subjects with the "partial" enzyme deficiency was examined. Enzymatic activity among these cell lines ranged from 5 to 50% of normal with the level of immunoreactive adenosine deaminase protein either proportional to enzyme activity or elevated in two of the cases. Northern blot analysis using a cDNA probe showed that adenosine deaminase mRNA in each of these cell lines was of normal expected size (1.6-1.8 kilobases) and was present in normal to above normal amounts. Rates of enzyme synthesis varied from 165 to 15% of normal. Adenosine deaminase protein degradation rates in these cell lines were 1.5 to almost 3 times faster than normal, consistent with the observed absence of the enzyme in erythrocytes. From these analyses apparent abnormalities in mRNA regulation, translation, and protein degradation can be identified among the partially adenosine deaminase-deficient cell lines studied. Ultimately, it will be essential to determine the nature of the protein mutation and the gene defect to define the structural alterations and functional abnormalities of enzyme variants isolated from subjects with partial adenosine deaminase deficiency.     

Epidermolysis bullosa simplex: Expression of gelatinase activity in cultured human skin fibroblasts

We have measured the baseline level of gelatinase in fibroblast-conditioned medium from 41 Scandinavian individuals. They comprised 12 healthy persons, 11 individuals with the skin disorder dominant epidermolysis bullosa simplex (DEBS), 16 patients with other types of epidermolysis bullosa (EB) and 2 siblings with prolidase deficiency. These results divide the cell strains into those with low and those with high activity levels. Although this dual biochemical trait occurred in all the groups of individuals, the high-activity trait was more frequent among the DEBS patients. The localized DEBS forms showed an elevated activity level, in contrast to the previously reported generalized DEBS Köbner forms. Although a high level was found in some individuals with other EB forms, the high incidence in four families with localized DEBS Weber-Cockayne (eight of eight) and a single family with generalized DEBS—mottled pigmentation (two of two) may result from a close linkage between an EB gene and a gene responsible for the biochemical trait. In addition, in the single complete family tested, the level of gelatinase activity in cultured fibroblasts seemed to be regulated by codominant alleles or genes. A raised baseline level of gelatinase activity in cultured skin fibroblasts may be the result of either an altered expression of gelatinase or an allelic variant of this enzyme with increased specific activity. Further studies of gelatinase in cultured fibroblasts may provide insight into the regulatory mechanism and genetics behind this activity and allow formal linkage studies versus DEBS.This work was supported in part by the Norwegian Research Council for Science and the Humanities (NAVF) and the Concerted Action on Hereditary Connective Tissue Diseases of the European Community (1990–1992, project leader, M. Matton).Part of this work was performed at the Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo.