Uptake and metabolism of free cyanocobalamin by cultured human fibroblasts from controls and a patient with transcobalamin II deficiency

We have investigated the uptake and metabolism of free cyanocobalamin (CN-Cbl; vitamin B₁₂) by intact cultured human skin fibroblasts. Monolayers of control fibroblasts take up free CN-[⁵⁷Co]Cbl via a saturable, calcium-independent process that is inhibited by sulfhydryl reagents, inhibitors of protein synthesis, and inhibitors of electron transport, but not by inhibitors of glycolysis. CN-Cbl taken up in this manner is converted to active cobalamin (Cbl) coenzymes (adenosylcobalamin and methylcobalamin) and becomes associated with intracellular Cbl-dependent apoenzymes (methylmalonyl CoA mutase and homocysteine:methyltetrahydrofolate methyltransferase). Since fibroblasts from controls were also found to synthesize transcobalamin II (TC II), a plasma protein shown previously to facilitate the cellular uptake of Cbl, it seemed possible that the observed uptake of free CN-Cbl was TC II-mediated. This thesis was rejected by demonstrating that cells from a patient with complete TC II deficiency took up free CN-Cbl as well as control cells did. Finally, we propose a mechanism by which an uptake process for free Cbl might serve a function in intracellular metabolism of Cbl.     

Abnormal collagen metabolism in cultured skin fibroblasts from patients with Duchenne muscular dystrophy.

Total collagen synthesis is decreased by about 29% (P less than 0.01) in skin fibroblasts established in vitro from male patients with Duchenne muscular dystrophy (DMD) as compared with that in normal male skin fibroblasts in vitro. The reduction in collagen synthesis is associated with an approximately 2-fold increase in collagen degradation in DMD fibroblasts. Correlated to these alterations in the metabolism of collagen, DMD fibroblasts express a significantly higher hydroxyproline/proline ratio (DMD: 1.36-1.45; P less than 0.01) than do normal fibroblasts (controls: 0.86-0.89). The increased hydroxylation of proline residues of collagen (composed of type I and type III) could be the cause for the enhanced degradation of collagen in DMD fibroblasts.     

Identification of GM2-gangliosidosis B1 variant carriers

Summary GM2-gangliosidosis B1 variant, considered a rare disorder with a wide geographical and ethnic distribution, appears to be exceptionally frequent in Portugal. In order to establish a carrier detection method for this disease we have determined the ratio of enzymatic activities against 4MUGS and 4MUG in urine from B1 variant obligate carriers and controls, using the total extract and the Hex A immunobound to a monoclonal antibody. The Hex A immunoassay was applied to the identification of carriers in B1 variant families and the results obtained were compared with those from DNA analysis. The reliability and feasibility of the Hex A immunoassay make it a suitable method for B1 variant carrier screening, which is particularly important for the prevention of this severe neurological disease in the population at risk.     

Glucose metabolism in fibroblasts from patients with erythrocyte hexokinase deficiency

Four different hexokinase (HK) isoenzymes are distributed in different proportions in human tissues. Fibroblasts contain HK type I as the predominant glucose phosphorylating activity, the same isoenzyme that predominates in red blood cells (RBC). We have established cell lines from two patients homozygous for RBC HK deficiency but carrying different mutations. In one case (HK-Melzo) the residual RBC enzyme shows a marked heat instability but possesses normal kinetic and regulatory properties; in the other (HK-Napoli), the enzyme is characterized by an increased Ki for glucose-1,6-diphosphate. These properties are also retained by the fibroblasts' hexokinase. Glucose utilization by cultured fibroblasts from these patients was markedly reduced in the cell lines where HK deficiency was more pronounced. However, cells with only 30% HK activity retained their full ability to utilize glucose in the hexose monophosphate pathway. This was shown to be true not only under basal conditions but also in the presence of oxidative agents such as methylene blue. Significant reduction of the ATP level was also found in HK-Melzo fibroblasts. Thus, HK deficiency is associated with reduced glucose utilization and normal hexose monophosphate shunt rates. Results previously obtained on RBC support similar conclusions.     

Phosphofructokinase activity in fibroblasts from patients with Alzheimer's disease and age- and sex-matched controls

The activity of the enzyme phosphofructokinase (PFK) was comparable in cultured skin fibroblasts from eight patients with Alzheimer's disease and eight age- and sex-matched controls. Mean activities were similar in the two groups whether measured under nonallosteric conditions at pH 8.0 or under allosteric conditions at pH 7.0, in the presence of 0.1 or 1 mM ATP. Activities of PFK in Alzheimer's disease and control cells also showed a similar temperature dependence and similar isozyme patterns on column chromatography. These results argue against the existence of significant structural variations of PFK in Alzheimer's disease.     

Dolichol metabolism in cultured skin fibroblasts from patients with “neuronal” ceroid lipofuscinosis (Batten's disease)

Dolichol metabolism was investigated in skin fibroblast cultures from normal individuals and patients with Batten's disease. Incorporation of [³H]mevalonolactone and [¹⁴C]acetate into the dolichol fraction of total lipid extracts was similar in cells from normal individuals and patients with Batten's disease. [¹⁴C]Acetate incorporation into dolichol in non-saponifiable lipid extracts was compared with incorporation into dolichol in total lipid extracts, and no difference in the proportion of dolichol esterified to fatty acids was found in Batten's cells as compared to normal cells. The rate of loss of radioactivity from the dolichol pool after prelabelling with [¹⁴C]acetate was also similar in cells from Batten's and normal individuals. Thus, in the fibroblast system used, no evidence was found to support the hypothesis that Batten's disease is due to a defect in dolichol metabolism.     

Abnormal copper metabolism in cultured fibroblasts from patients with Wilson's disease

Skin fibroblasts derived from patients with Wilson's disease have, under certain conditions, elevated concentrations of copper. However, the levels of intracellular copper varied from one experiment to another and the reason for this inconsistency has not yet been determined. 64Cu retention after 24 hours and its release in "chase" experiments was not abnormal, thus distinguishing these fibroblasts from Menkes' syndrome fibroblasts. The data provides evidence that the mutant gene responsible for Wilson's disease is expressed in fibroblasts under certain conditions.     

Abnormal copper metabolism in cultured fibroblasts from patients with Wilson's disease

Skin fibroblasts derived from patients with Wilson's disease have, under certain conditions, elevated concentrations of copper. However, the levels of intracellular copper varied from one experiment to another and the reason for this inconsistency has not yet been determined.⁶⁴Cu retention after 24 hours and its release in ‘chase’ experiments was not abnormal, thus distinguishing these fibroblasts from Menkes' syndrome fibroblasts. The data provides evidence that the mutant gene responsible for Wilson's disease is expressed in fibroblasts under certain conditions. This study was supported by a grant from the Australian National Health and Medical Research Council.     

Peripheral and autonomic nervous system involvement in chronic GM2-gangliosidosis

GM2-gangliosidosis (McKusick 268800 and 272800) is a rare hereditary, progressive disorder of ganglioside metabolism caused by deficiency of lysosomal -hexosaminidase (EC 3.2.1.52) activity. It is characterized by severe central nervous system involvement. Involvement of the peripheral and autonomic nervous system has been suspected but rarely documented in published case reports in the chronic form of the disease. Four patients, aged 24–29 years, with chronic GM2-gangliosidosis were examined prospectively for evidence of peripheral and autonomic nervous system dysfunction. All had nerve conduction studies, sympathetic skin responses and cardiac monitoring during the head tilt-table test. Three patients had objective evidence of autonomic dysfunction with abnormal sympathetic nervous skin responses and axonal sensorimotor polyneuropathy. None of the patients had evidence of significant cardiovascular autonomic dysfunction on the head tilt-table test. The peripheral and autonomic nervous system may be involved in patients with chronic GM2-gangliosidosis. In some cases, this may be clinically significant. On the other hand, cardiovascular autonomic instability is apparently not a significant problem in young adult patients with the disease.     

Oestrogen formation in genital and non-genital skin fibroblasts cultured from patients with hypospadias

OBJECTIVE Hypospadias is the most common birth defect in males. in most cases the aetiology is unknown. Since penile development is androgen dependent and oestrogen can modify androgen action, we compared the formation of oestrogen In penile tissue from patients with hypospadias to those with normal penile development. DESIGN AND PATIENTS Oestrogen formation was assessed in fibroblast monolayers grown from biopsies of genital and non-genital skin from 11 males with normal genital development (controls) and 18 males with severe hypospadias utilizing the incorporation of tritium into H₂O resulting from the aromatization of 1β-³H-androstenedione. RESULTS in paired fibroblast strains from genital and non-genital skin of nine males with hypospadias, oestrogen formation was significantly (P < 0·025) lower In non-genital skin. Rates of oestrogen formation were also higher in a subset of foreskins from subjects with hypospadias than In normal controls and the remaining hypospadias subjects. In addition, oestrogen formation in this subset of fibroblast strains from patients with hypospadias was markedly enhanced by incubation of Intact monolayers with either cholera toxin or forskolin, agents known to stimulate CAMP formation. Oestrogen formation in the remaining cell strains (controls and hypospadias) was also enhanced in most instances by cholera toxin and forskolin, although to a much lower degree. Thus, we identified in the hypospadias group a subgroup of fibroblast strains in which unstimulated and stimulated oestrogen formation was markedly higher than in other strains examined. CONCLUSIONS Since oestrogen can modify certain androgen effects within cells and since formation of the male genitalia during embryogenesis is mediated by androgens, elevated oestrogen formation in male genital tissue might be a causative factor of hypospadias in some Instances.     

Increased aromatase activity in pubic skin fibroblasts from patients with isolated gynecomastia

Aromatase activity (AR) was studied in pubic skin fibroblasts from eight patients with isolated gynecomastia (PSFG) and five normal subjects (PSFC). Cell monolayers were incubated in the presence of [3H]androstenedione (2 nM) for 4 or 24 h. Culture medium was extracted after addition of [14C] carriers to monitor recovery. Metabolites were separated by two successive chromatographic steps. Estrone (E1) and estradiol (E2) were characterized by crystallization, the other metabolites: 16-hydroxyestrone (16 alpha-OHE1) estriol (E3), and epiestriol (epiE3) by their chromatographic migration. AR was expressed either as femtomoles of E2 per microgram DNA (ARE2) or as total aromatized metabolites (ART = E1 + E2 + 16 alpha-OHE1 + E3 + epiE3/microgram DNA). After 4 h of incubation, no ARE2 could be measured in PSFC; it was low but significant in PSFG (0.03 +/- 0.02 (SEM) fmol/microgram DNA, P less than 0.01). The difference in ART was even more striking: 0.28 +/- 0.1 fmol/microgram DNA in PSFC, 3.15 +/- 2.88 in PSFG (P less than 0.05). 16 alpha-OHE1 represented in this latter group 62.5% of total aromatized metabolites vs. 39% in PSFC. After 24 h, ART was 4.17 +/- 3.70 and 1.02 +/- 0.42 fmol/microgram DNA in PSFG and PSFC, respectively (P less than 0.05); E3 + epiE3 represented 50% of the metabolites in both groups. In conclusion, AR is increased in PSFG relative to PSFC and an important oxidative metabolism of estrogens exists in both types of cells. This increased peripheral AR could result in increased formation of estrogens at the target cell site and represent an element of androgen-estrogen imbalance which would favor the development of gynecomastia.     

Impaired collagen gel contraction with cultured skin fibroblasts from patients with systemic sclerosis

We investigated the capacity of skin fibroblasts, derived from 9 patients with systemic sclerosis (SSc), to contract collagen lattices in a three-dimensional culture system. In comparison with control fibroblasts (N = 8), more than 30% of SSc fibroblasts exhibited markedly impaired ability to contract collagen lattices. The expression of alpha2beta1 integrins and integrin-mediated signals were not significantly different between normal and SSc fibroblasts. Although the underlying mechanisms remain to be determined, our present data provide evidence that certain aspects of interaction with collagen are impaired in SSc fibroblasts.     

Defective organization of actin in cultured skin fibroblasts from patients with inherited adenocarcinoma.

In the cytoplasm of well-spread cultured normal fibroblasts, actin is organized into a network of cables that run the length of the cell just inside the adherent cell membrane. A diffuse matrix replaces the cables in fibroblasts that have become tumorigenic as a result of oncogenic transformation. We have found a similar disruption in actin organization in cultured skin fibroblasts (passage 6-10) obtained by biopsy from patients with the inherited colonic cancer, adenomatosis of the colon and rectum (ACR). Because ACR is inherited as an autosomal dominant trait, about half the children of ACR patients will develop colon cancer, but they typically remain asymptomatic until at least the second decade of life. Actin distribution within cultured cells from children of ACR patients was identical either to that seen in cultured cells from normal persons or to that seen in cultured cells from ACR patients. The two different patterns were independent of age, sex, drug treatment, or infections of the donors. Apparently, this class of colonic carcinoma is accompanied by a systemic aberration in the organization of fibroblast cytoplasm, and this aberration can be detected by immunofluorescent localization of actin within cultured skin fibroblasts, prior to manifestation of any colonic symptoms.     

Deficiency of gamma-ray excision repair in skin fibroblasts from patients with Fanconi''s anemia.

The capacity of preparations of skin fibroblasts from normal individuals and patients with Fanconi's anemia to excise gamma-ray products of the 5,6-dihydroxydihydrothymine type from exogenous DNA was investigated. The excision capacity of whole-cell homogenates of fibroblasts from two of four patients with Fanconi's anemia was substantially below normal. This repair deficiency was further pronounced in nuclear preparations from cells of the same two patients.     

Copper deficiency in the mitochondria of cultured skin fibroblasts from patients with menkes syndrome

Summary The mitochondrial copper concentrations and cytochrome C oxidase activity of the fibroblasts from the patients with Menkes syndrome were investigated. Both the mitochondrial copper concentrations and cytochrome C oxidase activity of fibroblasts from patients with Menkes syndrome were lower than those of the control fibroblasts. These data indicate that the mitochondria of fibroblasts from patients with Menkes syndrome are in a state of copper deficiency. The activity decline of cytochrome C oxidase, a mitochondrial cuproenzyme, seems to be caused by copper deficiency in the mitochondria.     

Alterations in cholesterol metabolism in cultured fibroblasts from patients with Niemann-Pick disease type C

Summary Cholesterol synthesis, esterification and efflux were investigated in cultured fibroblasts from patients with Niemann-Pick disease type C. Sterol synthesis from sodium acetate was markedly increased in the two Niemann-Pick disease type C strains as compared to controls, either in the presence or absence of exogenous cholesterol supply by low-density lipoproteins. By contrast, cholesterol esterification was about 2–3-fold reduced when measured by oleic acid incorporation into cholesteryl esters and 10–15-fold reduced when measured with labelled free cholesterol as precursor, although acylcoenzyme-A: cholesterol acyltransferase activity was normal when studiedin vitro on cell homogenates. Chase experiments with¹⁴C-cholesterol demonstrated that the rate of cholesterol efflux was decreased by about 3–4-fold in fibroblasts from patients with Niemann-Pick disease type C. These results provide further evidence for alterations of sterol metabolism in Niemann-Pick disease type C and support the hypothesis of a trapping of exogenous cholesterol, which cannot enter the regulatory intracellular pools.