Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Danlos syndrome

Cultured fibroblasts of 13 patients with the Menkes syndrome and two with a new subtype (type IX) of the Ehlers-Danlos syndrome (E-D IX patients) showed many very similar abnormalities in their copper and collagen metabolism. Both cell types had markedly increased copper concentrations and 64Cu incorporation, and this cation accumulated in metallothionein or a metallothionein-like protein, as previously established for Menkes cells. Histochemical staining indicated that copper was distributed diffusely throughout the cytoplasm in both cell types, this location being consistent with the accumulation in metallothionein. Both fibroblast types also had markedly low lysyl oxidase activity and distinctly increased extractability of newly synthesized collagen, whereas no abnormalities were present in cell viability, duplication rate, prolyl 4-hydroxylase activity, or collagen synthesis rate. A high negative correlation (P less than 0.001) was found in the pooled group of Menkes and E-D IX cells between cellular copper concentration (r = 0.804) or 64Cu incorporation (r = 0.863) and the logarithm of lysyl oxidase activity. There was also a high positive correlation (P less than 0.001) between cellular copper concentration and incorporation (r = 0.869). One of the two E-D IX patients was also shown to have similar changes in lysyl oxidase activity and collagen extractability in the skin biopsy specimen, suggesting that the abnormalities observed in cultured cells are similar to those present in vivo. The only distinct abnormality found in the cells of the parents of the E-D IX patients was an increased 64Cu incorporation in those of the mother, this finding being consistent with X-linked inheritance of the disorder.     

Type IX Ehlers-Danlos syndrome and Menkes syndrome: the decrease in lysyl oxidase activity is associated with a corresponding deficiency in the enzyme protein.

Type IX of the Ehlers-Danlos syndrome (E-D IX) and the Menkes syndrome are X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a severe reduction in the activity of lysyl oxidase, the extracellular copper enzyme that initiates crosslinking of collagens and elastin. No increase in this deficient enzyme activity was obtained when culture media from fibroblasts of patients with E-D IX or the Menkes syndrome were incubated with copper under various conditions in vitro. A distinct, although small, increase in lysyl oxidase activity was obtained, however, when copper-supplemented media were used during culturing of the fibroblasts, although even under these conditions, the enzyme activity in the media from the affected cells remained markedly below that of the controls. Immunoprecipitation, dot-blotting, and immunoperoxidase staining experiments with antisera to human lysyl oxidase indicated that fibroblasts from patients with E-D IX or the Menkes syndrome do not secrete into their medium, or contain inside the cell, any significant amounts of a copper-deficient, catalytically inactive lysyl oxidase protein. These findings appear to be consistent with the hypothesis that synthesis of the lysyl oxidase protein itself is impaired. The possibility is not excluded, however, that a copper-deficient enzyme protein may be synthesized in normal amounts but become degraded very rapidly inside the cell. The failure to obtain any large increase in the deficient lysyl oxidase activity upon various forms of copper administration suggests that it may not be possible to obtain any significant improvement in the connective tissue manifestations of these disorders by copper therapy.     

Pyruvate dehydrogenase activity is not deficient in the brain of three autopsied cases with Leigh disease (subacute necrotizing encephalomyelopathy,SNE)

Summary In autopsied brain tissue from three cases with Leigh disease (subacute necrotizing encephalomyelitis, SNE) and controls, the activity of pyruvate dehydrogenase complex (PDHC) was determined under different conditions. It was found to be at the control level or increased, but not deficient. The activities of succinate dehydrogenase, fumarase, succinate cytochrome c reductase, cytochrome c oxidase, and glutamate dehydrogenase were measured as additional mitochondrial markers and showed no essential differences between SNE and control tissue. The metabolic defect in SNE remains unknown. According to the literature, the defect may be localized to the mitochondrial systems. However, the reported results indicate that it cannot be ascribed to PDHC function. Extensive biochemical studies are necessary for understanding of the pathogenesis in the fatal genetic metabolic disease.     

A novel syndrome affecting multiple mitochondrial functions, located by microcell-mediated transfer to chromosome 2p14-2p13

We have studied cultured skin fibroblasts from three siblings and one unrelated individual, all of whom had fatal mitochondrial disease manifesting soon after birth. After incubation with 1 mM glucose, these four cell strains exhibited lactate/pyruvate ratios that were six times greater than those of controls. On further analysis, enzymatic activities of the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, NADH cytochrome c reductase, succinate dehydrogenase, and succinate cytochrome c reductase were severely deficient. In two of the siblings the enzymatic activity of cytochrome oxidase was mildly decreased (by approximately 50%). Metabolite analysis performed on urine samples taken from these patients revealed high levels of glycine, leucine, valine, and isoleucine, indicating abnormalities of both the glycine-cleavage system and branched-chain alpha-ketoacid dehydrogenase. In contrast, the activities of fibroblast pyruvate carboxylase, mitochondrial aconitase, and citrate synthase were normal. Immunoblot analysis of selected complex III subunits (core 1, cyt c(1), and iron-sulfur protein) and of the pyruvate dehydrogenase complex subunits revealed no visible changes in the levels of all examined proteins, decreasing the possibility that an import and/or assembly factor is involved. To elucidate the underlying molecular defect, analysis of microcell-mediated chromosome-fusion was performed between the present study's fibroblasts (recipients) and a panel of A9 mouse:human hybrids (donors) developed by Cuthbert et al. (1995). Complementation was observed between the recipient cells from both families and the mouse:human hybrid clone carrying human chromosome 2. These results indicate that the underlying defect in our patients is under the control of a nuclear gene, the locus of which is on chromosome 2. A 5-cM interval has been identified as potentially containing the critical region for the unknown gene. This interval maps to region 2p14-2p13.     

Synthesis of a metallothionein-like protein in cultured human skin fibroblasts: Relation to abnormal copper distribution in Menkes' disease

A metallothionein-like protein (MTP) is synthesized in normal diploid human skin fibroblasts cultured in Zn- or Cu-supplemented medium. Synthesis of MTP is not detected in cells cultured without metal supplementation of complete tissue-culture medium. Cultured fibroblasts from patients with Menkes' disease accumulate excess Cu which chromatographs both with highmolecular-weight protein(s) and with a Cu-MTP. Under normal culture conditions, the Menkes' MTP incorporates [³⁵S]-cystine, but not appreciable amounts of ⁶⁵Zn. However, Menkes fibroblasts retain the ability to incorporate ⁶⁵Zn into MTP in response to Zn supplementation of the medium. The results do not support the idea that Menkes' disease results from a failure of Cu to bind to MTP, but rather that an elevated intracellular Cu concentration in Menkes' disease fibroblasts leads to association of excess Cu with high-molecular-weight protein, stimulating synthesis of a Cu-binding MTP.     

Freezing injury in the yeast respiratory system

The effect of freeze-thawing on the yeast respiratory system was studied at rapid rates of cooling. Freezing of whole cells with liquid nitrogen induced decrease of respiratory activity to under 20% of that of original cells. Mitochondria harvested from freeze-thawed cells have markedly decreased succinate oxidizing activity. Activity of succinate cytochrome c reductase was reduced significantly after freeze-thawing of whole cells while activities of succinate dehydrogenase and cytochrome c oxidase were reduced slightly. By spectrophotometric analysis it was found that about one-half the amount of cytochrome c + c1 was eluted from mitochondria to cytosol after freeze-thawing of cells. The activities of succinate oxidation in mitochondria from freeze-thawed cells were restored to normal levels by the addition of cytochrome c. Freeze-thawing of isolated mitochondria did not induce deactivation of succinate oxidizing activities and succinate cytochrome c reductase, and no elution of cytochrome c was observed. It was concluded that the decreased respiratory activities of yeast cells by freezing of cells with liquid nitrogen can be attributed primarily to the elution of cytochrome c from mitochondria.     

Distribution of the ATPase inhibitor proteins of mitochondria in mammalian tissues including fibroblasts from a patient with Luft's disease.

The mitochondrial ATPase inhibitor proteins--the Pullman-Monroy inhibitor (PMI) and the Ca(2+)-binding protein (CaBI)--have a wide distribution, both being present in mitochondria of bovine heart and kidney, rat liver and brain, two mitochondrial populations of rabbit skeletal muscle, and mitochondria from human fibroblasts and the human breast cancer cell line T-47-D. The ratio of CaBI to PMI was highest in heart and skeletal muscle mitochondria. The subsarcolemmal fraction of skeletal muscle had 2.6-times as much CaBI as did the intermyofibrillar. The ratio of CaBI to PMI in the mitochondria of the other normal tissues and fibroblasts was close to 1. In contrast, mitochondria from T-47D cells had 1.5-times as much PMI as CaBI whilst mitochondria from fibroblasts from a patient with Luft's disease showed a virtual lack of PMI. The specific ATPase, ATP-synthetase and succinate dehydrogenase activities of the Luft's mitochondria were, however, in the normal range. The specific ATP synthetase activity of the T-47D cells was significantly higher than normal. We conclude that tissues like heart and skeletal muscle which experience wide fluctuations in intracellular Ca2+ have a greater need for CaBI. Why lack of PMI could lead to 'loose' coupling of oxidative phosphorylation in skeletal muscle of Luft's patients, but not in fibroblasts is discussed.     

Studies on sphingomyelinase and beta-glucosidase activities in Niemann-Pick disease variants. Phosphodiesterase activities measured with natural and artificial substrates

Cultured fibroblasts were studied from 12 cases of Niemann-Pick disease group C. In 11, sphingomyelinase and glucocerebrosidase (and beta-glucosidase) activities were reduced to around 50% of those of controls. On isoelectric focusing, all 12 strains lacked sphingomyelinase activity in the major cathodic region (pI 8.0). The defect was also demonstrated with the artificial phosphodiester substrates bis(4-methylumbelliferyl) phosphate and 4-methylumbelliferyl pyrophosphate diester. In control fibroblasts and those heterozygous for types A or B or group C Niemann-Pick disease, the major sphingomyelinase peak electrofocused at pI 8.0. No direct interaction could be demonstrated by mixing experiments between group C Niemann-Pick extracts and those of type A disease or Gaucher disease. Profiles for beta-glucosidase activity appeared normal in Niemann-Pick group C fibroblasts. No reduction of sphingomyelinase or glucocerebrosidase activities was found in Niemann-Pick group C liver, nor any attenuation of cathodic sphingomyelinase activity in the affected tissue. Results suggest that sphingomyelinase expression differs in fibroblasts and liver. Enzyme defects associated with Niemann-Pick disease group C were only observed in cultured cells.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Deficiency of complex III of the mitochondrial respiratory chain in a patient with facioscapulohumeral disease.

Facioscapulohumeral disease (FSHD), an inherited neuromuscular disorder, is characterized by progressive wasting of specific muscle groups, particularly the proximal musculature of the upper limbs; the primary defect in this disorder is unknown. We studied a patient with FSHD to determine whether the mitochondrial respiratory chain was functionally abnormal. Muscle biopsy revealed fiber atrophy with patchy staining for oxidative enzymes. Electron microscopy of a liver section showed many enlarged mitochondria with paracrystalline inclusions. Decreased oxidation of the respiratory substrates-alanine and succinate-in skin fibroblasts suggested a deficiency of complex III of the electron-transport chain; cytochrome c oxidase activity (complex IV) was in the normal range. Biochemical analysis of liver supported the fibroblast data, since succinate oxidase activity (electron-transport activity through complexes II-IV) was reduced, whereas complex IV activity was normal. Furthermore, analysis of the cytochrome spectrum in liver revealed typical peaks for cytochromes cc1 and aa3, whereas cytochrome b (a component of complex III) was undetectable. Southern blot analysis of fibroblast mtDNA revealed no major deletions or rearrangements. Our study provides the first documentation of a specific enzyme-complex deficiency associated with FSHD.     

Biochemical effects of mild iron deficiency and cold acclimatization on rat skeletal muscle

Most of the previous studies on the effects of iron deficiency on skeletal muscle respiratory capacity and work performance have been investigated in severe or moderate iron-deficiency anemia. We report here that even in mild iron deficiency where the hemoglobin concentration was 10 g/dl and the iron stores in livers and spleen were not completely depleted, a marked reduction in succinate dehydrogenase was observed in skeletal muscles but not in heart. Similarly, cytochrome oxidase activities were reduced. Although no significant change in glycerophosphate dehydrogenase was detected in the iron-deficient rats, exposure to cold in this group greatly reduced this enzyme activity. As cold acclimatization accelerates marrow erythropoiesis (20) which in turn, demands more iron, it seems that in the iron-insufficient state, this iron demand for marrow activity may persist at the expense of the tissue iron pool, resulting in a marked reduction in glycerophosphate dehydrogenase activities. Since succinate dehydrogenase plays a significant role in the impairment of mitochondrial function and early fatigue of iron-deficient muscle (11), the present study shows that even in mild iron deficiency, some loss of muscle functions could result as succinate dehydrogenase activities were greatly reduced.     

A biochemically distinct form of cytochrome oxidase (COX) deficiency in the Saguenay-Lac-Saint-Jean region of Quebec.

We report the results of biochemical and molecular investigations on a group of patients from the Saguenay-Lac-Saint-Jean region of Quebec who have an unusual form of cytochrome oxidase deficiency and Leigh disease. This group can be distinguished from the classical presentation of cytochrome oxidase deficiency with Leigh disease, by the severity of the biochemical defect in different tissues. The activity in skin fibroblasts, amniocytes, and skeletal muscle of cytochrome oxidase is 50% of normal, while in kidney and heart it is close to normal values. Brain and liver, on the other hand, have very low activities. The defect in activity appears to result from a failure of assembly of the cytochrome oxidase complex in liver, but levels of mRNA for both mitochondrially encoded and nuclear-encoded subunits in liver and skin fibroblasts were found to be the same as those in controls. The cDNA sequence of the liver-specific cytochrome oxidase subunits VIa and VIIa were determined in samples from patient liver and skin fibroblasts and showed normal coding sequence.     

Toxic effects of ozone on murine L929 fibroblasts. Enzyme inactivation and glutathione depletion.

Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enzymes. Most sensitive to ozone exposure were glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. The activities of another cytosolic enzyme, lactate dehydrogenase, the mitochondrial enzymes glutamate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and the activity of the lysosomal enzymes acid phosphatase and beta-glucuronidase were, initially, not or only slightly affected. The localization of the lysosomal enzymes did not change during ozone exposure. After prolonged exposure complete deterioration of the cells was observed and all enzyme activities declined. The activity of the enzymes was also monitored during ozone exposure of a sonicated cell suspension and it was shown that all these enzymes are in fact susceptible to ozone. These observations clearly demonstrate that, besides the structure and amino acid composition of an enzyme, the localization in the cell plays an important role in its susceptibility to ozone. The intracellular levels of reduced and oxidized glutathione were affected as well. The ATP content, however, proved to be insensitive to ozone exposure.     

Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient.     

Respiratory systems and cytochromes in Campylobacter fetus subsp. intestinalis.

Cell suspensions of Campylobacter fetus subsp. intestinalis grown microaerophilically in complex media consumed oxygen in the presence of formate, succinate, and DL-lactate, and membranes had the corresponding dehydrogenase activities. The cells and membranes also had ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity which was cyanide sensitive. The fumarate reductase activity in the membranes was inhibited by p-chloromercuriphenylsulfonate, and this enzyme was probably responsible for the succinate dehydrogenase activity. Cytochrome c was predominant in the membranes, and a major proportion of this pigment exhibited a carbon monoxide-binding spectrum. Approximately 60% of the total membrane cytochrome c, measured with dithionite as the reductant, was also reduced by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. A similar proportion of the membrane cytochrome c was reduced by succinate under anaerobic conditions, whereas formate reduced more than 90% of the total cytochrome under these conditions. 2-Heptyl-4-hydroxyquinoline-N-oxide inhibited reduction of cytochrome c with succinate, and the reduced spectrum of cytochrome b became evident. The inhibitor delayed reduction of cytochrome c with formate, but the final level of reduction was unaffected. We conclude that the respiratory chain includes low- and high-potential forms of cytochromes c and b; the carbon monoxide-binding form of cytochrome c might function as a terminal oxidase.     

Selection of succinic dehydrogenase mutants of Neurospora crassa.

A method is described which permits the selection of mutants of Neurospora crassa that are deficient in succinic dehydrogenase activity. The method relies on the observation that succinic dehydrogenase-deficient strains fail to reduce the dye nitrotetrazolium blue when overlaid with the dye in the presence of succinate and phenazine methosulfate. Wild-type colonies reduced the dye and turned blue, whereas mutant colonies remained colorless. In this communication we present studies of a mutant, SDH-1, isolated by this method. The mutant had 18% of the succinic dehydrogenase activity of the parent strain used in the mutation experiments as determined from the ratio of Vmax activities obtained from Lineweaver-Burk plots. The SDH-1 mutant segregated in a Mendelian manner when back-crossed to its parent strain. Succinate oxidase activity in SDH-1 was low and was markedly inhibited by adenosine 5'-diphosphate. The succinate oxidase activity of the parent strain was high and was not affected by the presence of adenosine 5'-diphosphate.     

Mucopolysaccharidosis 3 A (Sanfilippo A disease): deficiency of a heparin sulfamidase in skin fibroblasts and leucocytes

Cultured skin fibroblasts and peripheral leucocytes from patients with Sanfilippo A disease are strikingly deficient in sulfamidase activity (sulfamatase, EC 3.1.6.?), as measured with heparin - N³⁵SO₄. A partial sulfamidase deficiency was found in the cells of the heterozygote carriers. Since Sanfilippo A fibroblasts have normal sulfate ester hydrolase activities towards oligosaccharides prepared from ³⁵SO₄-labelled heparan sulfate by nitrous acid treatment, the basic defect in Sanfilippo A disease is considered to be the inactivity of a heparin (heparan sulfate) sulfamidase.     

Function and Regulation of the Mammalian Copper-transporting ATPases: Insights from Biochemical and Cell Biological Approaches

Copper is an essential trace element that plays a very important role in cell physiology. In humans, disruption of normal copper homeostasis leads to severe disorders, such as Menkes disease and Wilson's disease. Recent genetic, cell biological, and biochemical studies have begun to dissect the molecular mechanisms involved in transmembrane transport and intracellular distribution of copper in mammalian cells. In this review, we summarize the advances that have been made in understanding of structure, function, and regulation of the key human copper transporters, the Menkes disease and Wilson's disease proteins.     

Loss of electron-dense lamellar material from Fabry's disease fibroblasts after enzyme replacement

Cultured fibroblasts from a patient with Fabry's disease were treated with alpha-galactosidase A. The cells internalized the enzyme via a receptor-mediated transport system, resulting in the uptake of enzyme to 50% of the activity of normal cells. Following uptake of the enzyme and incubation for 9 days, a loss of electron-dense lamellar material within membrane-bound residual bodies was detected by electron microscopy. Morphometric analysis of electron micrographs showed that the percentage volume of cytoplasm occupied by electron-dense lamellar material in Fabry's disease fibroblasts decreased to near normal after treatment with enzyme. These results indicate that the ultrastructural abnormalities of Fabry's disease cells can be corrected by enzyme replacement, at least in cultured fibroblasts.     

Application of electron spin resonance for evaluation of the level of free radicals in the myometrium in full-term pregnancy with normal labour and uterine inertia

In order to identify and quantify free radicals in the tissues of patients with normal physiological and pathological states of births, we developed a method to evaluate the amount of free radicals in myometrium of subplacental area and from body of uterus, using electron spin resonance spectroscopy. Analysis of the concentration of free radicals in the myometrium in full-term pregnancy with normal labour and during uterine inertia was studied. The activities of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase in samples of these tissues were tested too. Low free radical concentrations in these tissues were associated with disturbances in contractile activity of myometrium along with reduction of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase activity. There proved to be an association between the level of free radicals in the tissues and alteration in the physiological processes.