Feline mucopolysaccharidosis type VI: correction of glycosaminoglycan storage in myoblasts by retrovirus-mediated transfer of the feline N-acetylgalactosamine 4-sulfatase gene

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal storage disorder characterised by the deficiency of N-acetylgalactosamine 4-sulfatase (4S). MPS VI has also been described in the cat. As an initial step toward muscle-mediated gene therapy in the MPS VI cat, we have made two retroviral constructs (pLf4S and pLf4SSN) that transduce the feline 4S gene. Both constructs were designed to express the feline 4S sequence from the viral long terminal repeat promoter. In addition pLf4SSN expressed the neomycin resistance gene from the SV40 early promoter. Amphotrophic virus was produced for each construct and used to transduce feline MPS VI myoblasts. Lf4S- and Lf4SSN-transduced MPS VI feline myoblasts demonstrated correction of glycosaminoglycan storage and contained 55-fold and 3.5-fold elevated levels of 4S activity when compared with normal feline myoblasts respectively. Recombinant feline 4S (rf4S) secreted by Lf4S-transduced MPS VI myoblasts was shown to be endocytosed by MPS VI feline cells via the mannose-6-phosphate receptor system, leading to metabolic correction. The results from this study demonstrate that muscle-mediated gene replacement therapy may be a viable method for achieving circulating levels of recombinant f4S (rf4S) in the MPS VI cat.     

Structure of a human lysosomal sulfatase

BACKGROUND:. Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase. RESULTS:. The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme. CONCLUSIONS:. The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.     

Analysis of prostate tissue DNA for the presence of human papillomavirus by polymerase chain reaction, cloning, and automated sequencing

BACKGROUND AND OBJECTIVE: As photorefractive keratectomy (PRK) becomes more widely used, the incidence of repeated PRK increases. The present study was conducted to evaluate results of repeated PRK in view of the meager data on this topic. PATIENTS AND METHODS: In this retrospective study, the authors reviewed the records of 1028 eyes that had undergone PRK, and analyzed the results of 66 eyes that required a second PRK for undercorrection according to baseline refraction. RESULTS: A second PRK was performed in 6.3%, 13.7%, and 10.1% of low, moderate, and high myopes, respectively. The mean refraction 1 year after repeated PRK was similar in both myopic groups: less than -1.00 D. Of the low myopes, 87.50% had residual refraction within 1 D after 1 year. Of the moderate myopes, 88.23% had residual refraction within 1 D after 1 year. All of the low myopes achieved uncorrected visual acuity (VA) of 20/25 or better 1 year after repeated PRK, compared with 58.82% of the moderate myopes. Loss of best-corrected VA never exceeded two lines. CONCLUSION: The overall results of PRK appear to be satisfactory.     

Expression of p53 protein in normal, dysplastic, and malignant gastric mucosa: an immunohistochemical study

Mutations in the p53 nuclear oncogene are the most frequent genetic abnormalities encountered in human malignancies. Using the polyclonal antibody CM-1, we have examined the expression of the p53 oncoprotein immunohistochemically in archival material of normal, dysplastic, and malignant gastric mucosa. Abnormal expression of this protein was not observed in biopsies of normal gastric tissue (n = 30) but was detected in 22 of the 36 gastric cancers analysed (61 per cent). Nuclear staining was diffuse in 15 of the positive cancer cases, the remaining seven showing a more varied heterogeneous staining pattern. Abnormal p53 protein was not detected in mild (n = 14) or moderate (n = 16) gastric dysplasia but was present in 3 out of 15 severe dysplasia cases. The results suggest that expression of the p53 oncoprotein is a common finding in gastric cancer and occurs as a late event in the malignant transformation process.     

Molecular weight distribution in alkyd resins. Part II. Castor oil and hydrogenated castor oil alkyds

Alkyds prepared from castor and hydrogenated castor oils have been prepared direct from the oil and by first subjecting the oil to a glycerolysis reaction. The molecular weight distributions of the alkyds have been measured in solvent systems designed to separate predominantly on polarity and molecular weight. The properties of the alkyds in stoving enamels have been evaluated. The results are discussed in relation to existing theories relating processing conditions to molecular weight distribution in alkyd resins. Previous suggestions regarding the reactivity of the hydroxyl groups in the oil molecules are not consistent with the results obtained in this study.     

Chloramphenicol acetylation in Streptomyces

Twenty-one strains of actinomycetes were screened for the presence of chloramphenicol acetyltransferase, the enzyme responsible for chloramphenicol resistance in many species of bacteria. Only five strains, belonging to three species, yielded mycelial lysates which catalysed the formation of chloramphenicol acetates in the presence of acetyl-coenzyme A: Streptomyces coelicolor Müller, S. acrimycini, and S. griseus. A mutant of S. acrimycini selected for an increase in resistance to chloramphenicol had a higher specific activity for chloramphenicol acetyltransferase than that found in the parental strain; the enzyme was not inducible in the mutant, the parental strain, or any other strain tested. Chloramphenicol was not acetylated by lysates of a strain of S. venezuelae, the organism known to produce it.     

Comparing the temporal stability of self-report and interview assessed personality disorder.

Findings from several large-scale, longitudinal studies over the last decade have challenged the long-held assumption that personality disorders (PDs) are stable and enduring. However, the findings, including those from the Collaborative Longitudinal Personality Disorders Study (CLPS; Gunderson et al., 2000), rely primarily on results from semistructured interviews. As a result, less is known about the stability of PD scores from self-report questionnaires, which differ from interviews in important ways (e.g., source of the ratings, item development, and instrument length) that might increase temporal stability. The current study directly compared the stability of the Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM–IV) PD constructs assessed via the Schedule for Nonadaptive and Adaptive Personality (SNAP–2; Clark, Simms, Wu, & Casillas, in press) with those from the Diagnostic Interview for DSM–IV Personality Disorders (Zanarini, Frankenburg, Sickel, & Yong, 1996) over 2 years in a sample of 529 CLPS participants. Specifically, we compared dimensional and categorical representations from both measures in terms of rank-order and mean-level stability. Results indicated that the dimensional scores from the self-report questionnaire had significantly greater rank-order (mean r = .69 vs. .59) and mean-level (mean d = 0.21 vs. 0.30) stability. In contrast, categorical diagnoses from the two measures evinced comparable rank-order (mean κ = .38 vs. .37) and mean-level stability (median prevalence rate decrease of 3.5% vs. 5.6%). These findings suggest the stability of PD constructs depends at least partially on the method of assessment and are discussed in the context of previous research and future conceptualizations of personality pathology. (PsycINFO Database Record (c) 2011 APA, all rights reserved)