Abnormal copper metabolism in menkes cultured fibroblasts

Menkes fibroblast cultures were established and copper metabolism was investigated. Menkes fibroblasts contained four to six times higher concentrations of copper than control cells. In Menkes cells more than 90% of the intracellular copper was present in cytosol (105,000 x g supernatant fraction); in control fibroblasts the corresponding value was about 67%. During cultivation of fibroblasts in medium supplemented with 100 ng/ml of copper (270 ng CuCl₂·2H₂O /ml), the amount of copper increased continuously in Menkes cells, at least up to 4 days, while in control cells in reached a maximum after 24 h, followed by a gradual decrease. When the medium was replaced with one without copper chloride, copper concentrations in Menkes cells returned to the original level in three days, whereas those in control cells returned to the normal level in one day. Using Sephadex G-75 column chromatography of cytosol, two copper-containing peaks were observed (peaks 1 and 3, corresponding to the peaks from rat liver cytosol). Approximately 75% of the copper in the cytosol from Menkes cells was eluted in peak 3. The corresponding copper peak was very small in control cells. Copper peaks 1 and 3 from both cells increased after treating cells with copper chloride and the increase was inhibited by cycloheximide, an inhibitor of protein biosynthesis.Metallothionein purified from human kidneys was eluted as a single copper-protein from a Sephadex G-75 column in the same fractions as peak 3. SDS polyacrylamide-slab gel electrophoresis of the purified metallo-thionein and the material eluted in peak 3 from Menkes fibroblasts showed single peaks for copper at identical migration distances.     

Cholesterol ester and triglyceride metabolism in intact fibroblasts from patients with Wolman's disease and cholesterol ester storage disease

Cholesterol ester and triglyceride metabolism was examined in intact fibroblast monolayers from normal individuals and patients with Wolman's disease and cholesterol ester storage disease. Cholesterol esters were introduced into cells by incubation in medium containing [3H]cholesteryl linoleate (CL) bound to human low density lipoprotein. Triglycerides were introduced by incubation with glycerol tri[1-14C]oleate (triolein) bound to human very low-density lipoprotein. Both types of mutant cell lines accumulated the unhydrolyzed substrates to a greater extent than did normal cells with the greatest accumulation observed in Wolman's disease cells. Wolman's disease cells hydrolyzed CL at 10-22% and triolein at 11-19% the rate of normal cells; cholesterol ester storage disease cells hydrolyzed these substrates at 28-49 and 30-47% the normal rate, respectively. In contrast, assays of acid lipase activity in cell lysates revealed less than 1% of control activity in both disorders. The data suggest that the mutant acid lipase present in Wolman's disease and cholesterol ester storage disease is more active in the intact cell than assays of cell lysates would indicate. In addition, the differences observed between the two disorders provide a biochemical explanation for the different phenotypes associated with the two disorders.     

Trace metal metabolism in cultured skin fibroblasts of the mottled mouse: response to metallothionein inducers

Menkes' kinky hair syndrome is a lethal X-linked disorder marked by tissue-specific increases in copper content. An animal model of kinky hair syndrome is provided by mice mutant at the X-linked mottled locus. The basic defect is unknown. In order to discriminate among potential etiologies, we asked whether the expression of the mottled mutation causes abnormalities in the metabolism of trace metals other than copper in hemizygous mottled (blotchy) cultured skin fibroblasts, and whether we can differentiate mutant and normal cells according to their response to metal inducers of metallothionein. Blotchy fibroblasts accumulated up to 12 times more 64Cu than control (littermate) cells, over time and over a range of 64Cu concentrations. A saturable high affinity component to 64Cu accumulation over a fixed time interval was revealed in these studies. While 64Cu uptake kinetics were indistinguishable in mutant and control cells, the patterns of 64Cu exit differed. In both cell types, the rate of release of a rapidly exchangeable fraction of newly acquired 64Cu was similar. However, in mutant cells, a larger fraction of recently accumulated 64Cu is retained. In contrast to the results for 64Cu, accumulation and exit of 65Zn and 109Cd were not distinguishable in mutants and controls. With exposure to either a strong (cadmium) or weaker (zinc) inducer of metallothionein, 64Cu accumulation was increased in normal cells, while there was no change from the already elevated level of 64Cu accumulation in blotchy cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schilder's disease: non-invasive diagnosis and successful treatment with human immunoglobulins

Schilder's disease (SD) is a rare variant of multiple sclerosis with a predilection to children. It is characterized by focal neurological abnormalities, which are atypical for MS, in conjunction with tumor-like white matter lesions on MRI. We report the case of an 11-year-old girl that demonstrates two important features of the disease: a) the clinical presentation and subsequent course in conjunction with the serial neuroradiological findings stress the feasibility of a non-invasive diagnosis of SD; and b) we report a significant clinical response to treatment with intravenous human Immunoglobulins.     

Maple syrup urine disease: analysis of branched chain ketoacid decarboxylation in cultured fibroblasts.

Kinetic data are presented for the decarboxylation of branched chain alpha-ketoacids (BCKA) by intact human fibroblasts. Cultured cells of normal individuals and nine patients with different clinical pictures of maple syrup urine disease (MSUD) are studied with both alpha-ketoisocaproic acid (2-oxo-4-methylpentanoic acid (KIC)) and alpha-ketoisovaleric acid (2-oxo-3-methylbutanoic acid (KIVA)) as substrates. One normal cell strain and one patient cell strain is analyzed with alpha-keto-beta-methyl-n-valeric acid (2-oxo-3-methylpentanoic acid (MEVA)) as a substrate. A biphasic degradation kinetic for each BCKA is obtained for normal control subjects. The component with higher substrate affinity is affected in MSUD: for KIC the normally hyperbolic substrate curve is changed to sigmoid shape, for KIVA and MEVA as substrates this component is not detectable at all. Considering qualitative aspects of the BCKA decarboxylation kinetics intact fibroblasts yield the same results as our recent studies with the decarboxylase moieties of partially purified kidney BCKA dehydrogenase of normal individuals and one patient with classic MSUD (27). The decarboxylation velocities for normal and patient fibroblasts with one exception differ widely at low but not at high substrate concentrations of BCKA. To get meaningful data on the residual substrate degradation activities with intact fibroblasts of different phenotypes of MSUD physiologically low substrate concentrations are required in the assay.     

Elevated cystine levels in cultured skin fibroblasts from patients with I-cell disease

Cultured skin fibroblasts from patients with I-cell disease (mucolipidosis II) exhibit multiple deficiency of acid hydrolase activities associated with a defect in the mechanism of packaging of these enzymes into lysosomes. The authors have examined such cells to ascertain whether the impairment of lysosomal function is of so broad a nature as to result in the storage of the amino acid cystine in a manner similar to that seen in cells derived from patients with cystinosis, an unrelated lysosomal storage disease of unknown etiology. Of 10 I-cell lines examined by automated amino acid analysis, seven were found to possess abnormally high levels of total free cyst(e)ine (i.e., greater than 1 nmole 1/2 Cys/mg protein). The mean half-cystine content of those I-cell lines subjected to multiple analysis ranged from 3-10 nmole/mg protein. levels which are comparable to those seen in homozygous cystinotic cells. The cystine content of several of these lines appeared to increase with subculture. Cultured fibroblasts from two patients with the biochemically similar, but clinically less severe, mucolipidosis III (pseudo-Hurler polydystrophy) exhibited normal to marginally elevated levels of cystine, whereas cells from individuals with three different mucopolysaccharide storage disorders contained normal levels of the amino acid. It was concluded that cystine, and not cysteine, was the predominant form of this amino acid in these cells, because previous reaction of I-cell extracts with N-ethylmaleimide did not alter the observed cystine levels. The further identification of excess cystine in these cells was corroborated by analytical results obtained with a highly specific cystine-binding protein method as well as by high-voltage electrophoresis of extracts from cells pulsed with 35S-cystine. Comparative analysis of intracellular amino acids in normal and I-cell fibroblasts indicated that the elevation of csystine seen in the latter was unique to this amino acid and did not reflect a generalized increase in the total free amino acid content of these mutant cells.     

Niemann-Pick-like liver disease and reduced cholesterol esterification in fibroblasts of two male infants

Niemann-Pick disease type C (NPC) presents in about half of the cases in the newborn period with jaundice, hepato-splenomegaly, and a clinical pattern similar to neonatal hepatitis. The definitive diagnosis can in most instances be made by the appropriate biochemical testing of lipoprotein stimulated cholesteryl ester synthesis and cholesterol accumulation in cultured patient fibroblasts. We report two infants who by liver biopsy had classical findings of NPC and a cholesteryl ester synthesis level about 50% of the normal lower limit. On the other hand neither of these patients' fibroblasts showed any evidence of low density lipoprotein-induced cholesterol accumulation, precluding the possibility of a definitive diagnosis. These cases demonstrate the importance of the appropriate biochemical testing before final counseling is carried out. The possibility of our patients representing allelic or nonallelic variants of NPC are discussed.     

Inherited lactic acidosis: correction of the defect in cultured fibroblasts

We report a case of familial lactic acidosis, lethal in the newborn period. Studies in intact fibroblasts identified a defect in the oxidative pathway of pyruvate metabolism. Although assay of pyruvate dehydrogenase on cell sonicates was not appreciably reduced, flux through the enzyme and other mitochondrial multienzyme dehydrogenases was severely impaired in intact cells. Deficient lactate conversion to carbon dioxide could be repaired by the addition to the incubation medium of electron acceptors such as methylene blue (25 micrograms/ml) or dichlorophenolindophenol (25 micrograms/ml).     

Sphingomyelinases in human tissues. III. Expression of Niemann-Pick disease in cultured skin fibroblasts.

Sphingomyelinase was effectively extracted cultured fibroblasts homogenized in 1% glycine. The average specific activity was 24.6 nmol substrate hydrolyzed per hr per mg protein. Cultured cells from two cases of Niemann-Pick disease type A and one case of type B had markedly reduced enzyme activity, whereas, in type E cells, total activity was twice normal. Sphingomyelinase was resolved by isoelectric focusing into three peaks of activity (I-II), where I and II were the major forms. Species I had a pI of 4.6, II a pI of 4.8, and III a pI of 5.0. All three peaks, although markedly reduced, were present in cell extracts from both types A and B. All species of enzyme in B cells were found at higher levels than in A cells. Cultured cells from a case of type E contained sphingomyelinase I at a level several fold higher than normal, species II was absent, whereas III was found at near normal levels.     

Inducibility of metallothionein biosynthesis in cultured normal and Menkes kinky hair disease fibroblasts: effects of copper and cadmium.

Metallothionein biosynthesis is not induced by extracellular copper in Menkes Kinky hair disease (MKHD) or in normal cultured fibroblasts under the conditions of these experiments. In the presence of copper, MKHD fibroblasts also incorporated less cysteine than did normal fibroblasts. Extracellular cadmium greatly enhanced the uptake of cysteine in both normal and MKHD cultures. By the technique of polyacrylamide gel electrophoresis, it was demonstrated that metallothionein is induced by cadmium in normal and MKHD-cultured fibroblasts.