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Cultured Fibroblasts (cultured + fibroblast)
Selected AbstractsIsolated sulfite oxidase deficiency: mutation analysis and DNA-based prenatal diagnosisPRENATAL DIAGNOSIS, Issue 5 2002J. L. Johnson Abstract Isolated sulfite oxidase deficiency is an autosomal recessive, neurological disorder resulting from a defect in SUOX, the gene encoding the enzyme that catalyzes the terminal reaction in the sulfur amino acid degradation pathway. In its classical, severe form, sulfite oxidase deficiency leads to intractable seizures, severe and progressive brain pathology and death at an early age. We report here on clinical features and mutational analysis of the genetic defect in a newborn with sulfite oxidase deficiency. Cultured fibroblasts from this patient exhibited no detectable sulfite oxidase activity, and a unique four base pair deletion was present in the cDNA isolated from the same source. Identification of the same genetic defect in a heterozygous state in each of the parents and the monitoring of subsequent pregnancies in this family by DNA-based prenatal diagnosis are also described. The deletion mutation was identified in a homozygous state in uncultured chorionic villus tissue from the second pregnancy that was subsequently terminated. In the third pregnancy, the presence of sulfite oxidase activity and identification of the mutation in a heterozygous state suggested that the fetus was not affected. This pregnancy resulted in the birth of a normal child. Copyright © 2002 John Wiley & Sons, Ltd. [source] Pyruvate dehydrogenase deficiency presenting as dystonia in childhoodDEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 10 2004R A Head MA Two individuals with pyruvate dehydrogenase (PDH) deficiency due to missense mutations in the gene for the E1, subunit (PDHA1) presented during childhood with dystonia. The first patient, a male, presented at age 4 years with dystonia affecting the lower limbs, which responded to treatment with combined carbidopa and levodopa. The second patient, a female, was first investigated at age 6 years because of a dystonic gait disorder. In both patients, the main clue to the biochemical diagnosis was a raised concentration of lactate in the cerebrospinal fluid. PDH activity was significantly reduced in cultured fibroblasts in both cases. Dystonia is a previously unrecognized major manifestation of PDH deficiency and is of particular interest as the mutations in the PDHA1 gene in these patients have both been identified previously in individuals with typical presentations of the condition. [source] Sterol-induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double-bond position in the sterol tetracyclic ring structureFEBS JOURNAL, Issue 21 2000Petra Leppimäki We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7-dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 µm cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol·mg,1 protein (from 90 nmol·mg,1 to 190 nmol·mg,1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol-enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7-dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane-bound CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme, was increased by 47 ± 4% in cholesterol-enriched cells whereas its activity was unchanged in lathosterol-enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down-regulated acetate-incorporation into cholesterol, and upregulated sterol esterification in the sterol-enriched fibroblasts. Using 31P-NMR to measure the lamellar-to-hexagonal (L,,HII) phase transition in multilamellar lipid dispersions, lathosterol-containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine and either cholesterol or lathosterol (70 : 30 mol/mol), differential scanning calorimetry also revealed that the L,,HII -transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the L,,HII -transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells. [source] Sjögren-Larsson syndrome: Diversity of mutations and polymorphisms in the fatty aldehyde dehydrogenase gene (ALDH3A2),HUMAN MUTATION, Issue 1 2005William B. Rizzo Abstract Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by ichthyosis, mental retardation, and spastic diplegia or tetraplegia. The disease is caused by mutations in the ALDH3A2 gene (also known as FALDH and ALDH10) on chromosome 17p11.2 that encodes fatty aldehyde dehydrogenase (FALDH), an enzyme that catalyzes the oxidation of long-chain aldehydes derived from lipid metabolism. In SLS patients, 72 mutations have been identified, with a distribution that is scattered throughout the ALDH3A2 gene. Most mutations are private but several common mutations have been detected, which probably reflect founder effects or recurrent mutational events. Missense mutations comprise the most abundant class (38%) and expression studies indicate that most of these result in a profound reduction in enzyme activity. Deletions account for about 25% of the mutations and range from single nucleotides to entire exons. Twelve splice-site mutations have been demonstrated to cause aberrant splicing in cultured fibroblasts. To date, more than a dozen intragenic ALDH3A2 polymorphisms consisting of SNPs and one microsatellite marker have been characterized, although none of them alter the FALDH protein sequence. The striking mutational diversity in SLS offers a challenge for DNA-based diagnosis, but promises to provide a wealth of information about enzyme structure,function correlations. Hum Mutat 26(1), 1,10, 2005. © 2005 Wiley-Liss, Inc. [source] A biodegradable copolymer for the slow release of growth hormone expedites scarring in diabetic ratsJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2007Francisco García-Esteo Abstract In many diseases wound healing is impaired. This study was designed to establish whether the healing process in diabetes could be improved using a site-specific polymer delivery system containing hGH. The system was first optimized in in vitro experiments performed on cultured fibroblasts taken from healthy and diabetic rats and then tested in an incisional wound model created in the diabetic Wistar rat. In the in vitro experiments using cultured fibroblasts, cell viability, growth, and proliferation were determined, along with polymer degradation, hormone release rates and the expression of TGF,1 in the culture medium. For the in vivo experiments, polymer discs with/without GH were inserted through 3 cm incisions made on the backs of the animals. Wound specimens were obtained 7 and 30 days after surgery to evaluate inflammatory/apoptotic cells, metalloprotease expression and neoangiogenesis using microscopy and immunohistochemical techniques. The local administration of GH using a polymer delivery system did not affect the normal wound healing process. Conversely, when used in diabetic animals, epidermal and dermal repair was expedited. Our findings indicate that GH induces cell proliferation, enhances CD4+ infiltration; increases extracellular matrix protein deposition; stimulates angiogenesis; and diminishes apoptosis at the diabetic wound site. These effects give rise to a comparable wound healing process to that observed in healthy animals. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006 [source] Cerebrotendinous xanthomatosis: molecular characterization of two Scandinavian sistersJOURNAL OF INTERNAL MEDICINE, Issue 3 2002E. Rystedt Abstract.,Rystedt E, Olin M, Seyama Y, Buchmann M, Berstad A, Eggertsen G, Björkhem I (Karolinska Institutet, Stockholm, Sweden; OchanomizuUniversity, Tokyo, Japan; Medisinsk avdeling, Haukeland sykehus, Bergen). Cerebrotendinous xanthomatosis: molecular characterization of two Scandinavian sisters (Case report). Journal of Internal Medicine 2002; 252: 259,264. Cerebrotendinous xanthomatosis (CTX) is a hereditary disorder, which is inherited as an autosomally recessive disease, causing production of cholesterol and cholestanol xanthomas and mental retardation. The disease is caused by mutations in the gene for sterol 27-hydroxylase (CYP27A1). The only CTX patients diagnosed in Scandinavia are two Norwegian sisters from a consanguineous marriage. Here we have characterized the mutation and its functional consequences for the enzyme. Analysis of genomic DNA from cultured fibroblasts identified a base exchange C > T in position 1441, causing arginine at amino acid position 441 to be replaced by tryptophan. The same mutation was introduced by mutagenesis in the complimentary DNA (cDNA) for CYP27, ligated into the expression vector pcDNA4/HisMax and transfected into HEK293 cells. The mutated enzyme had less than 5% of the enzyme activity compared with the native enzyme. No abnormal catalytic products could be identified in the cell culture medium. Probably the mutation affects the haem binding within the holoenzyme. The mutation has also previously been reported in a Japanese family. This is the second example of a CTX-causing mutation that has been recognized in more than one population. [source] Prostaglandin E2 secretion from gingival fibroblasts treated with interleukin-1,: effects of lipid extracts from Porphyromonas gingivalis or calculusJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2001Frank C. Nichols Complex lipids of Porphyromonas gingivalis have been identified in lipid extracts from calculus-contaminated root surfaces and in diseased gingival tissues. However, little is known about the biological effects of these complex lipids on host cells. The purpose of this study was to evaluate the effects of P. gingivalis or calculus lipids on prostaglandin secretion from gingival fibroblasts. Lipids were extracted from paired subgingival plaque and teeth samples, and calculus-contaminated root surfaces before and after scaling and root planing, in order to determine the relevant levels of lipid extracts for the treatment of gingival fibroblasts in culture. Primary cultures of gingival fibroblasts were exposed to lipid extracts from either P. gingivalis or calculus/teeth for a period of 7 days. Control and lipid-treated cultures were exposed to human recombinant interleukin-1, for 48 h and prostaglandin secretion from interleukin-1,-treated fibroblasts was compared with control and lipid-treated fibroblasts without interleukin-1, treatment. These experiments demonstrated that P. gingivalis lipids or calculus-tooth lipids potentiate interleukin-1,-mediated prostaglandin secretory responses from gingival fibroblasts. Additionally, P. gingivalis or calculus-tooth lipid extracts were readily taken up by gingival fibroblasts as measured by bacterial fatty acid recovery in lipid extracts of cultured fibroblasts. These results indicate that bacterial lipid penetration into gingival tissues in combination with a chronic inflammatory response may substantially potentiate prostaglandin secretion from gingival fibroblasts, thereby promoting tissue destructive processes associated with adult periodontitis. [source] Novel Aspects of Intrinsic and Extrinsic Aging of Human Skin: Beneficial Effects of Soy Extract,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2005Kirstin M. Südel ABSTRACT Biochemical and structural changes of dermal connective tissue substantially contribute to the phenotype of aging skin. To study connective tissue metabolism with respect to ultraviolet (UV) exposure, we performed an in vitro (human dermal fibroblasts) and an in vivo complementary DNA array study in combination with protein analysis in young and old volunteers. Several genes of the collagen metabolism such as Collagen I, III and VI as well as heat shock protein 47 and matrix metalloproteinase-1 are expressed differentially, indicating UV-mediated effects on collagen expression, processing and degradation. In particular, Collagen I is time and age dependently reduced after a single UV exposure in human skin in vivo. Moreover, older subjects display a lower baseline level and a shorter UV-mediated increase in hyaluronan (HA) levels. To counteract these age-dependent changes, cultured fibroblasts were treated with a specific soy extract. This treatment resulted in increased collagen and HA synthesis. In a placebo-controlled in vivo study, topical application of an isoflavone-containing emulsion significantly enhanced the number of dermal papillae per area after 2 weeks. Because the flattening of the dermal-epidermal junction is the most reproducible structural change in aged skin, this soy extract appears to rejuvenate the structure of mature skin. [source] Different amounts of isoflavones in various commercially available soy extracts in the light of gene expression-targeted isoflavone therapyPHYTOTHERAPY RESEARCH, Issue S1 2010Ewa Piotrowska Abstract Isoflavones are plant-derived, biologically active compounds that are commonly used as natural drugs or diet supplements in the treatment of menopausal symptoms and as antioxidants. Recently, it was proposed that genistein (4,,5,7-trihydroxyisoflavone) may be used in the treatment of patients suffering from Sanfilippo disease (mucopolysaccharidosis type III), a severe genetic disorder for which no therapy is available. A pilot clinical study with this novel therapy, called ,gene expression-targeted isoflavone therapy' (GET IT), indicated that a standardized, genistin-rich soy isoflavone extract is effective in the treatment of such patients. Since various isoflavone-containing products are commercially available, the content of the main isoflavones were measured in such products. Extremely different amounts of isoflavones were determined in various products, from 0.13 to 39 mg per tablet. Only some of these products were found to be effective in inhibition of the synthesis of glycosaminoglycans (compounds whose degradation is severely impaired in mucopolysaccharidoses, including Sanfilippo disease) in cultured fibroblasts. Since in GET IT the dose of genistein is calculated per patient's body weight, the amount of this isoflavone in a tablet is crucial for this therapy. Therefore, the results presented in this report indicate that a careful choice of a proper isoflavone extract is necessary for GET IT. Copyright © 2009 John Wiley & Sons, Ltd. [source] Methylmalonic acidaemia leads to increased production of reactive oxygen species and induction of apoptosis through the mitochondrial/caspase pathway,THE JOURNAL OF PATHOLOGY, Issue 4 2007E Richard Abstract Methylmalonic acidaemia (MMA) is a heterogeneous group of rare genetic metabolic disorders caused by defects related to intracellular cobalamin (vitamin B12) metabolism. Increasing evidence has emerged suggesting that free radical generation is involved in the pathophysiology of neurodegenerative diseases, including some inborn errors of metabolism. We have previously identified in MMA patients several differentially expressed proteins involved in oxidative stress [mitochondrial superoxide dismutase (MnSOD) and mitochondrial glycerophosphate dehydrogenase (mGPDH)] and apoptosis by a proteomic approach. We have now extensively evaluated various parameters related to oxidative stress and apoptosis in cultured fibroblasts from a spectrum of patients with methylmalonic acidaemia. Fibroblasts from several MMA patients showed a significant increase in intracellular reactive oxygen species (ROS) content and in MnSOD expression level with respect to controls, suggesting a cellular response to intrinsic ROS stress. Moreover, we have demonstrated, using siRNA, that mGPDH is an important ROS generator in MMA patients. Cells from patients with MMA had a higher rate of apoptosis than those of controls and there was evidence that this process primarily involves the mitochondrial/caspase-dependent pathway. ROS level,phenotype correlation revealed that patients with severe neonatal cblB disorder had elevated intracellular ROS content. These findings support the possible role of oxidative stress in the pathophysiology of methylmalonic acidaemia. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] SPARC, an upstream regulator of connective tissue growth factor in response to transforming growth factor , stimulationARTHRITIS & RHEUMATISM, Issue 12 2006X. D. Zhou Objective To differentiate the effects of inhibition of specific small interfering RNA (siRNA) of SPARC (secreted protein, acidic and rich in cysteine) and siRNA of connective tissue growth factor (CTGF) in cultured human fibroblasts, and to identify potential interrelationships between SPARC and CTGF. Methods Fibroblasts from skin biopsy specimens of 2 normal individuals were transfected with siRNA of SPARC and siRNA of CTGF. The fibroblasts were stimulated with or without transforming growth factor ,1 (TGF,1) and examined by real-time quantitative reverse transcription,polymerase chain reaction to determine the transcription levels of several extracellular matrix genes. Results After exogenous TGF,1 stimulation, both SPARC siRNA and CTGF siRNA showed a protective role against overexpression of collagen genes. Following TGF,1 stimulation, SPARC siRNA,transfected fibroblasts showed a greater reduction in expression of the collagen genes compared with CTGF siRNA,transfected fibroblasts, as well as a significantly decreased expression of CTGF (P < 0.05). Using linear structure equations to quantitatively model a genetic network based on expression levels of each gene, a positive regulatory role of SPARC on CTGF, COL1A2, COL3A1, COL11A1, and TIMP3 was observed. However, the regulatory role of CTGF on SPARC appeared to be negative and very small, while the positive regulatory effects of CTGF on COL1A2, COL3A1, COL11A1, and TIMP3 were less than those of SPARC. Conclusion The results of this quantitative comparison support the hypothesis that in these cultured fibroblasts, the regulatory effects of SPARC on some major extracellular matrix structural components are greater than those of CTGF. In addition, SPARC appears to regulate CTGF in a predominantly positive manner, while CTGF may act as a negative feedback control on SPARC following TGF, stimulation. [source] Western blotting analysis of the ,-hexosaminidase ,- and ,-subunits in cultured fibroblasts from cases of various forms of GM2 gangliosidosisACTA NEUROLOGICA SCANDINAVICA, Issue 6 2002K. Utsumi Objectives, The GM2 gangliosidoses are a group of genetic disorders caused by the accumulation of ganglioside GM2 in neuronal cells. We examined the , - and , -subunits of , -hexosaminidases by a non-radioisotopes detecting system to evaluate whether it was a useful method for understanding of the pathophysiologies of GM2 gangliosidoses. Materials and methods, We investigated the , - and , -subunits of , -hexosaminidases in cultured fibroblasts from cases of various forms of GM2 gangliosidosis by means of Western blotting and a chemiluminescence detection system. Results, In a patient with infantile Tay-Sachs disease [HEXA genotype, Int5-SA(g,1,t)/Int5-SA(g,1,t)], the mature , -subunit was undetectable. In a patient with infantile Sandhoff disease (HEXB genotype, C534Y/C534Y), the mature , -subunit was deficient. However, a small amount of the mature , -subunit was detected in a patient with adult Sandhoff disease (HEXB genotype, R505Q(+I207V)/R505Q(+I207V)), which may have resulted in the residual enzyme activity and mild clinical course. Normal amounts of , - and , -subunits were detected in a patient with GM2 activator deficiency. Conclusion, This method is easy and sensitive for detecting target proteins, and is useful for clarification of the pathophysiologies of GM2 gangliosidoses. [source] | |||||||||||||