ABSTRACT[pSJMethionine Labeling, Immunoprecipitation, and Polyacrylamide Gel Electrophoresis. Labeling, immunoprecipitation, and PAGE were done as described (26). Rabbit antibodies against chicken type II collagen were generously provided by B. Vertel (Syracuse University) (27).Immunofluorescence. Normal and MC29-infected chondrocytes or fibroblasts were plated on coverslips, and immunofluorescence staining was performed as described (28). Ascorbic acid was added to a final concentration of 50 ,ug/ml 24 hr before staining. Rabbit antibodies against the core protein of chick cartilage-specific proteoglycans were a gift from B. Vertel (27).RNA Extraction and Dot Blot Hybridization. Total cellular RNA was extracted by the guanidine HCl procedure (29). To assess the integrity of RNA, 5 ,g of total RNA from each sample was denatured, fractionated on formaldehyde/agarose gels, and stained with ethidium bromide. Samples containing 5, 2, and 1 ,ug of total cell RNA each with, respectively, 0,3,and 4 ,ug
The Sanfilippo type A syndrome, one of the most frequent forms of mucopolysaccharidosis III, is characterized by severe mental retardation, progressive neurological degeneration, and mild somatic changes. It is due to a deficiency of heparan-N-sulfatase (sulfamidase) activity and consequent excretion of heparan sulfate in the urine. The disease is transmitted through an autosomal recessive mechanism, and more than 60 gene mutations have been identified. Up to now, only 10 cases of attenuated form of Sanfilippo type A syndrome have been described, and the specific mutation has been identified only in two of them. We report here on a female patient, 20 years old, with Sanfilippo type A syndrome presenting with a mild clinical phenotype characterized essentially by a moderate nonevolving mental retardation. The genetic analysis demonstrated that the patient is homozygous for mutation R206P; presence of polymorphism R456H was also found. This study places R206P as a mild mutation underlying Sanfilippo type A disease.
MPS VI (mucopolysaccharidosis type VI) is a lysosomal storage disease in which deficient activity of the enzyme N-acetylgalactosamine 4-sulfatase [ASB (arylsulfatase B)] impairs the stepwise degradation of the GAG (glycosaminoglycan) dermatan sulfate. Clinical studies of ERT (enzyme replacement therapy) by using rhASB (recombinant human ASB) have been reported with promising results. The release of GAG into the urine is currently used as a biomarker of disease, reflecting in some cases disease severity and in all cases therapeutic responsiveness. Using RNA studies in four Italian patients undergoing ERT, we observed that TNFalpha (tumour necrosis factor alpha) might be a biomarker for MPS VI responsive to therapy. In addition to its role as a potential biomarker, TNFalpha expression could provide insights into the possible pathophysiological mechanisms underlying the mucopolysaccharidoses.
Growth of quail chondrocytes in the presence of retinoic acid (RA) results in the suppression of the differentiated phenotype. RA-treated chondrocytes recover their differentiated phenotype if they are cultured for an additional 15 days in the absence of RA. A few days after removal from RA, treated chondrocytes acquire the polygonal morphology characteristic of chondrocytes growing as attached cells; they also gradually resume collagen II expression and synthesize cultures. The levels of collagen X mRNA decrease during the second week of culture in the absence of RA. Finally, at the end of 15 days, the absolute levels of collagen II and collagen X mRNAs are very similar in control and recovering chondrocytes.
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