Extracts Of Skin Fibroblasts Research Articles

Liquid chromatography/electrospray ionisation–tandem mass spectrometry quantification of GM2 gangliosides in human peripheral cells and plasma

GM2 gangliosidosis is a group of inherited neurodegenerative disorders resulting primarily from the excessive accumulation of GM2 gangliosides (GM2) in neuronal cells. As biomarkers for categorising patients and monitoring the effectiveness of developing therapies are lacking for this group of disorders, we sought to develop methodology to quantify GM2 levels in more readily attainable patient samples such as plasma, leukocytes, and cultured skin fibroblasts. Following organic extraction, gangliosides were partitioned into the aqueous phase and isolated using C18 solid-phase extraction columns. Relative quantification of three species of GM2 was achieved using LC/ESI–MS/MS with d35GM1 18:1/18:0 as an internal standard. The assay was linear over the biological range, and all GM2 gangliosidosis patients were demarcated from controls by elevated GM2 in cultured skin fibroblast extracts. However, in leukocytes only some molecular species could be used for differentiation and in plasma only one was informative. A reduction in GM2 was easily detected in patient skin fibroblasts after a short treatment with media from normal cells enriched in secreted β-hexosaminidase. This method may show promise for measuring the effectiveness of experimental therapies for GM2 gangliosidosis by allowing quantification of a reduction in the primary storage burden.

Age-associated modifications of Base Excision Repair activities in human skin fibroblast extracts

Base Excision Repair (BER) is the predominant repair pathway responsible for removal of so-called small DNA lesions such as abasic sites (AP site), uracil (U), 8-oxo-7,8-dihydroguanine (8oxoG), thymine glycol (Tg). In this study, we investigated effect of aging on excision efficacy of several endogenous base lesions and AP sites using an in vitro multiplexed fluorescent approach on support (parallelized oligonucleotide cleavage assay). Human fibroblasts nuclear extracts from 29 donors of different ages were characterized in their ability to simultaneously excise the different lesions. Clearly, three different groups of lesions emerged according to the efficiency of their cleavage: one exhibited very high cleavage efficiency (AP sites and U paired with G), one showed intermediate cleavage efficiency (U paired with A and Tg). The third group included 8oxoG, A paired with 8oxoG, T at CpG site and hypoxanthine (Hx) and displayed poor repair. Aging was significantly associated with modification of excision efficiency for AP sites, uracil, Tg and 8oxoG. Repair rate decreased for the first three lesions and the most drastic effects were observed for repair of U:A. Surprisingly, excision of 8oxoG increased with aging suggesting a completely different regulation or adaptation for the initiation step of this related specific repair pathway.

Poorly specific binding of thyroglobulin to orbital fibroblasts from patients with Graves’ ophthalmopathy

It has been proposed that thyroglobulin (Tg) may be involved in the pathogenesis or the progression of Graves' ophthalmopathy (GO). According to this hypothesis, following its release from the thyroid, Tg would reach orbital tissues, thereby eliciting an autoimmune aggression. In support of this, we recently found that intact Tg is present in orbital tissues of patients with GO, where it is complexed with glycosaminoglycans. In this study, we searched for additional Tg binding sites in orbital tissues, using primary cultures of orbital and skin fibroblasts from 7 GO patients who had undergone orbital decompression. Biotin-labeled Tg bound to both skin and orbital fibroblasts in a saturable manner, with constants of dissociation of approximately 75 nmol/l for skin fibroblasts and approximately 40 nmol/I for orbital fibroblasts. In an attempt to identify Tg binding sites, fibroblast extracts were blotted onto membranes that were incubated with biotin-labeled Tg, which bound especially to a protein migrating at approximately 300 kDa, present in both orbital and skin fibroblast extracts. Because no appreciable inhibition of binding of biotin-labeled Tg was produced by unlabeled Tg, we concluded that binding was poorly specific and it is unlikely to be involved in the pathogenesis of GO.

Disordered methionine/homocysteine metabolism in premature vascular disease. Its occurrence, cofactor therapy, and enzymology.

Mild homocysteinemia occurs surprisingly often in patients with premature vascular disease. We studied the possible enzymatic sources of this mild hyperhomocysteinemia and the control of homocysteine levels in plasma by treatment of patients with the cofactors and cosubstrates of homocysteine catabolism. We assessed homocysteine metabolism in 131 patients who had premature disease in their coronary, peripheral, or cerebrovascular circulation by using a standard oral methionine-load test. Impaired homocysteine metabolism occurred in 28 patients. We assayed levels of the primary enzymes of homocysteine catabolism in cultured skin fibroblast extracts from 15 of these 28 patients. The patients' cystathionine beta-synthase levels (3.68 +/- 2.52 nmol/h per milligram of cell protein, mean +/- SD) were markedly depressed compared with those from 31 healthy adult control subjects (7.61 +/- 4.49, P < .001). The patients' levels of 5-methyltetrahydrofolate: homocysteine methyltransferase were normal. While betaine: homocysteine methyltransferase was not expressed in skin fibroblasts, 24-hour urinary betaine and N,N-dimethylglycine measurements were consistent with normal or enhanced remethylation of homocysteine by betaine: homocysteine methyltransferase in the 13 patients tested. When treated daily with choline and betaine, pyridoxine, or folic acid, there was a normalization of the postmethionine plasma homocysteine level in 16 of 19 patients. Our results indicate that mild homocysteinemia in premature vascular disease may be caused by either a folate deficiency or deficiencies in cystathionine beta-synthase activity. It does not necessarily involve deficiencies of either 5-methyltetrahydrofolate:homocysteine methyltransferase or betaine:homocysteine methyltransferase. Effective treatment regimens are also defined.

Properties of Acid Phosphatase in Human Stratum corneum

Sheets of stratum corneum were prepared by a trypsinization procedure from human skin samples, homogenized with a freeze press and then fractionated into a soluble fraction and a sediment by centrifugation at 50,000 g. Acid phosphatase (AcP) activity was found in both fractions but the bulk of the activity was detected in the supernatant. Highest activities were observed after treatment with Triton X-100. The bulk of the AcP activity remained bound to the pellet, if suspension and fractionation of the homogenized stratum corneum were performed in acetate buffer in the range between pH 4.0-5.0, probably due to ionic or hydrophobic interactions. AcP activity was totally lost if homogenates or fractions were stored frozen at -20 degrees C in buffers with pH values lower than 4.0. Triton X-100 extracts from whole skin, epidermis, stratum corneum, cultured skin fibroblasts and leukocytes were compared by isoelectric focusing. Extracts from whole skin, epidermis and stratum corneum yielded almost identical patterns with one main AcP activity band at pI of 5.65, whereas a second pronounced band from whole skin behaved similarly to one band from cultured skin fibroblasts and leukocytes (pI 6.1). The prominent band from extracts of stratum corneum and epidermis was not observed in extracts of skin fibroblasts and leukocytes. Hence, we conclude that stratum corneum and epidermis contain a tissue-specific AcP.

Sphingolipid activator protein deficiency in a 16-week-old atypical Gaucher disease patient and his fetal sibling: Biochemical signs of combined sphingolipidoses

We describe a patient who presented shortly after birth with hyperkinetic behaviour, myoclonia, respiratory insufficiency and hepatosplenomegaly. Gaucher-like storage cells were found in bone marrow. A liver biopsy showed massive lysosomal storage morphologically different to that in known lipid storage disorders. Biochemically, the patient had partial deficiencies of beta-galactocerebrosidase, beta-glucocerebrosidase and ceramidase in skin fibroblast extracts, but the sphingomyelinase activity was normal. Glucosyl ceramide and ceramide were elevated in liver tissue. Loading of cultured fibroblasts with radioactive sphingolipid precursors indicated a profound defect in ceramide catabolism. Immunological studies in fibroblasts showed a total absence of cross-reacting material to sphingolipid activator protein 2 (SAP-2). The patient died at 16 weeks of age. The fetus from his mother's next pregnancy was similarly affected. The possibility that the disorder results from a primary defect at the level of SAP-2 is discussed. We have named this unique disorder SAP deficiency.

Synthesis of pyrene derivatives of cerebroside sulfate and their use for determining arylsulfatase A activity

Two fluorescent derivatives of cerebroside sulfate (‘sulfatide’) have been synthesized and used as substrates for determining arylsulfatase A activity. These were 12-(1-pyrene)dodecanoyl cerebroside sulfate (P12-sulfatide) and 12(1-pyrenesulfonylamido)dodecanoyl cerebroside sulfate (PSA12-sulfatide). When incubated at pH 5.0 in the presence of 5 mM MnCl 2 and 5.5 mM of taurodeoxycholate, either substrate was hydrolyzed by arylsulfatase A of human leukocytes. The rate of hydrolysis was proportional to the incubation time and concentration of enzyme; Michaelis-Menten type kinetics were observed with increasing concentrations of substrate. For determining the rate of hydrolysis, each of the two products (i.e., P12- and PSA12-cerebrosides) were separated from the bulk of respective unreacted sulfatide on small columns of DEAE-Sephadex A-25 and their fluorescence intensities read at 343–378 and 350–380 nm for the excitation and emission wavelengths for P12- and PSA12-cerebrosides, respectively. When extracts of skin fibroblasts derived from normal individuals and patients with Maroteaux-Lamy (lacking arylsulfatase B) or metachromatic leukodystrophy (lacking arylsulfatase A) were used as source of enzyme, P12-sulfatide was hydrolyzed by the former two but not by the latter cell extract. Several derivatives of cerebroside sulfate were also synthesized and found to inhibit the hydrolysis of pyrenesulfatide by leukocyte arylsulfatase A. The results demonstrate that these two pyrene containing sulfatides can be effectively used as specific substrates for the determination of arylsulfatase A activity in extract of cells and most probably also of tissues.

Detection of cystic fibrosis protein by isoelectric focusing and silver staining in serum, dried whole blood, and cultured fibroblasts

AbstractExperimental conditions were developed for visualizing cystic fibrosis protein (CFP) in cultured fibroblasts from patients with cystic fibrosis (CF) while improving the visualization of CFP in serum. Serum, whole dried blood and cultured skin fibroblast extracts derived from CF homozygotes, heterozygotes, and normal individuals were submitted to isoelectric focusing (IEF) in polyacrylamide gels using a pH gradient of 7.1 to 9.3 and stained for protein with silver. The results showed an improvement in the separation of bands in the cathodal region over that obtained with a pH gradient of 3.5 to 10 and a better visualization of CFP bands than provided with Coomassie blue. Serum IgG could be removed from the cathodal region by including Protein A on the gel during IEF, thus enabling better visualization of CFP bands. With cultured fibroblast extracts, CFP was only observed when gels were stained with silver. This study presents improved sensitivity of a potential diagnostic tool in CF and its use with cultured skin fibroblasts.

Chromatofocusing of skin fibroblast sphingomyelinase: Alterations in Niemann-Pick disease type C shared by G M1-gangliosidosis

Sphingomyelinase activity of cultivated skin fibroblast extracts from normal individuals was resolved by Chromatofocusing in the pH range 8-5 into three major components with pI's of 7.3, 6.3 and 5.9, respectively. Chromatofocusing proved a more efficient and reproducible separation technique than preparative flat-bed isoelectric focusing and it gave a constant profile even when detergent concentration varied. In skin fibroblasts from five patients with Niemann-Pick disease type C, a varying degree of reduction in the proportion of the 7.3 peak was observed. In a patient with clinical features of Niemann-Pick disease type C, the finding of such a profile would thus be a good argument for the diagnosis, but it is not pathognomonic as we found similar changes in two cases with G M1-gangliosidosis, while some cases of Niemann-Pick disease type C have borderline normal profiles. These results challenge the concept of a specific sphingomyelinase isoenzyme deficiency as the basic defect in Niemann-Pick disease type C.

Diagnosis of Tay-Sachs disease using [ 3H] N-acetylneuraminic acid labelled G M2 ganglioside as substrate

G M2 ganglioside labelled with tritium in the N-acetylneuraminic acid moiety was prepared and used to measure β-hexosaminidase A activity in cultured human skin fibroblast extracts. The latter convert this substrate to the correspondingly labelled G M3 ganglioside which can easily be separated from the substrate by thin-layer chromatography. No cleavage of the N-acetylneuraminic acid group was observed under our conditions. Two methods are described for the determination of G M2-β-hexosaminidase A activity in fibroblasts. The application of these methods to the diagnosis of Tay-Sachs disease is discussed.

24] Assay of enzymes of lipid metabolism with colored and fluorescent derivatives of natural lipids

Publisher Summary This chapter discusses assay of enzymes of lipid metabolism with colored and fluorescent derivatives of natural lipids. Fatty acid containing the colored or fluorescent probe was condensed with cholesterol, or glycerol, or deacylated sphingolipid, or glycerophospholipid. The colored or fluorescent lipid product was then isolated from the reaction mixture and purified by column or by thin-layer chromatography. The yellow substrates are dispersed and incubated with lipase. The reaction is terminated with alkaline ethylene glycol, and the unreacted neutral substrate is extracted into benzene. Acid is then added, the fatty acid produced by enzymatic hydrolysis is extracted into benzene, and its absorbance is determined spectrophotometrically. The activity of the lysosomal sphingomyelinase was determined at pH 5.0 using a solubilized preparation of enzyme from a lysosome-enriched preparation of rat brain, as well as extracts of skin fibroblasts of normal humans and Niemann-Pick patients and of amniotic cells. Fluorescent sphingomyelin and nonfluorescent sphingomyelin are mixed in the molar ratio used in the assay procedure, and the concentration is estimated by determining the phosphorus content.

Increased phosphoribosylpyrophosphate synthetase activity in fibroblasts of hypoxanthine-guanine phosphoribosyl transferase deficient patients

An increased activity of phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate is demonstrated in extracts of skin fibroblast cultures derived from a patient with Lesch-Nyhan syndrome. This eccessive response of the phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate results in increased levels of phosphoribosylpyrophosphate and thus contributes to purine overproduction characteristic of this disorder. The level of enzyme response in skin fibroblast extracts from the carrier mother was between activity of the patient and normals, further suggesting the x-linkage of human phosphoribosylpyrophosphate synthetase.

Heterozygote expression in propionyl coenzyme A carboxylase deficiency. Differences between major complementation groups.

We measured propionyl coenzyme A carboxylase (PCC) activity in extracts of skin fibroblasts and peripheral blood leukocytes from controls and obligate heterozygotes for PCC deficiency. 6 heterozygotes were from the pcc A complementation group; 12 were from the other major complementation group, designated pcc C. Mean PCC activity in fibroblast extracts from pcc A heterozygotes was 52% of that in controls, whereas mean PCC activity in pcc C heterozygotes was indistinguishable from that of controls. Similar results were obtained with extracts of peripheral blood leukocytes. In none of eight families (three pcc A and five pcc C) in which PCC activity was studied in both parents of an affected child were significant intrafamilial differences observed. The activities of two other mitochondrial enzymes (beta-methyl-crotonyl CoA carboxylase and glutamate dehydrogenase) were comparable in controls and both groups of heterozygotes. Whereas the data from pcc A heterozygotes are consistent with expected gene dosage effects, those from pcc C heterozygotes are not. Inasmuch as mammalian PCC is a large molecular weight tetramer, each protomer of which is probably composed of two nonidentical subunits, the latter results are most consistent with unbalanced rates of synthesis and(or) degradation of the two subunits in normal cells with compensatory balancing in pcc C heterozygotes.

A spectrophotometric method for determination of sphingomyelinase

A colored derivative of sphingomyelin was synthesized and used as substrate for several sphingomyelinases. The compound is N-ω-trinitrophenyl aminolaurylsphingosylphosphorylcholine. The rate of hydrolysis of this substrate was compared to that of bovine brain sphingomyelin, labelled with tritium in the choline moiety. The following enzyme preparations were used: homogenate-less debris of brain, assayed at pH 5.0 or 7.4; a solubilized preparation derived from rat brain lysosomes, assayed at pH 5.0 and a purified enzyme of Staphylococcus aureus. With all preparations, the rates of hydrolysis of the yellow derivative were very similar to those of the brain sphingomyelin. Extracts of skin fibroblasts of normal and Niemann-Pick patients as well as amniotic cells were also used. Again, the rates of hydrolysis of the yellow derivative practically equalled those using brain sphingomyelin.

561 ISOELECTRIC FOCUSING OF α-L-FUCOSIDASE FROM CYSTIC FIBROSIS AND CONTROL SKIN FIBROBLASTS

α-L-Fucosidase activity is elevated in cystic fibrosis (CF) skin fibroblasts while nine other acid hydrolases including neuraminidase have activities similar to those in the control fibroblasts (Biochem. Biophys. Res. Commun. 79,869,1977). Extracts of skin fibroblasts from CF individuals and age, sex, and race matched controls were analyzed by isoelectric focusing on thin layer polyacrylamide gels to determine if the elevated activity of α-L-fucosidase in the CF cells resulted from a difference in a specific isoenzyme. Fibroblasts were seeded at a density of 3.0 × 106 cells/150 cm2 and harvested after 7 days with trypsin. Cell pellets were suspended in Triton X-100, broken in a Dounce homogenizer and centrifuged at 10,400g for 20 min. The supernatant solutions were electrofocused for 2 h in an LKB Multiphor apparatus both before and after treatment with Vibrio cholerae neuraminidase. Two separate ampholine gradients, pH 3.5-10 and pH 4-7, were used. The gels were cut in 2 mm slices and α-L-fucosidase activity was assayed using 4-methylumbelliferyl-α-L-fucopyranoside as substrate in citrate buffer, pH 5.8. The CF and control fibroblasts had similar isoenzyme patterns under all of the conditions described with the exception that several CF preparations showed a predominance of the isoenzymes with higher isoelectric points. USPHS GM07025 and The National Foundation-March of Dimes.