Search PubMed⌕ Search

Biomedical subjects

J J Hopwood

Publications and source records attributed to J J Hopwood.

At least 19 recordsLinked to original sources
PubMed2005

Over-expression of human lysosomal alpha-mannosidase in mouse embryonic stem cells.

Alpha-mannosidosis is a lysosomal storage disorder characterised by the lysosomal accumulation of mannose-containing oligosaccharides and a range of pathological consequences, caused by a deficiency of the lysosomal enzyme alpha-mannosidase. One of the major features of alpha-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment. Implantation of stem cells into the central nervous system has been proposed as a potential therapy for these disorders. We report the construction and characterisation of mouse embryonic stem cell lines for the sustained over-expression of recombinant human lysosomal alpha-mannosidase (rhalphaM). Two vectors (involving recombinant human alpha-mannosidase expression driven by either the chicken beta-actin promoter/CMV enhancer or by the elongation factor 1-alpha promoter) were constructed and used to transfect mouse D3 embryonic stem cells. Selected clonal cell lines were isolated and tested to evaluate their expression of recombinant human alpha-mannosidase. Stem cell clones transfected with the chicken beta-actin promoter/CMV enhancer maintained rhalphaM expression levels throughout differentiation. This expression was not markedly elevated above background. In contrast, the vector incorporating the elongation factor 1-alpha promoter facilitated substantial over-expression of alpha-mannosidase when analysed out to 21 days of differentiation in stably transfected cell lines. The highest expressing cell line was found to qualitatively retain a similar differentiation potential to untransfected cells, and to secrete alpha-mannosidase that could mediate a reduction in the level of oligosaccharides stored by human alpha-mannosidosis skin fibroblasts. These results suggest potential for the use of this cell line for investigation of a stem cell therapy approach to treat alpha-mannosidosis.

Animals↗
PubMed2005

Survival and engraftment of mouse embryonic stem cell-derived implants in the guinea pig brain.

alpha-Mannosidosis is a lysosomal storage disease resulting from a deficiency of the enzyme alpha-D-mannosidase. A major feature of alpha-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment available. We have a guinea pig model of alpha-mannosidosis that models the human condition. This study investigates the feasibility of implanting differentiated mouse embryonic stem cells in the neonatal guinea pig brain in order to provide a source of alpha-mannosidase to the affected central nervous system. Cells implanted at a low dose (1.5 x 10(3)cells per hemisphere) at 1 week of age were found to survive in very low numbers in some immunosuppressed animals out to 8 weeks. Four weeks post-implantation, cells implanted in high numbers (10(5) cells per hemisphere) formed teratomas in the majority of the animals implanted. Although implanted cells were found to migrate extensively within the brain and differentiate into mature cells of neural (and other) lineages, the safety issue related to uncontrolled cell proliferation precluded the use of this cell type for longer-term implantation studies. We conclude that the pluripotent cell type used in this study is unsuitable for achieving safe engraftment in the guinea pig brain.

Animals↗
PubMed2005

Pharmacokinetic profile of recombinant human N-acetylgalactosamine 4-sulphatase enzyme replacement therapy in patients with mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): a phase I/II study.

AIM: Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) is a lysosomal storage disease caused by a deficiency of the enzyme-N-acetylgalactosamine 4-sulphatase (ASB). Enzyme replacement therapy with recombinant human ASB (rhASB) has been studied in a randomized, double-blind, two-dose (0.2 and 1.0 mg/kg/week) phase I/II study (n = 7) followed by an open-label single dose (1.0 mg/kg/week) extension study. We report the pharmacokinetic profile of rhASB and the impact of antibody development. METHODS: Pharmacokinetic analysis was performed at weeks 1, 2, 12, 24, 83, 84 and 96. Infusions were administered over 4 hours using a ramp-up protocol. Plasma ASB and rhASB antibody concentrations and urine glycosaminoglycan (GAG) concentrations were determined. RESULTS: The area under the plasma concentration-time curve (AUC(0-t)) for the high-dose group increased from week 1 to week 2, but remained unchanged at weeks 12 and 24. A large difference in mean AUC(0-t) was observed between the low- and high-dose groups. Pharmacokinetic results at weeks 83, 84 and 96 were similar to those at week 24. Six patients developed antibodies to rhASB. One patient developed high antibody levels in combination with a high ASB concentration, while a second patient also developed high antibody levels with undetectable ASB concentrations. Antibodies from the second patient blocked detection of ASB. By week 72, antibody levels had decreased in all patients. The high-dose rhASB produced a more rapid and greater percentage reduction in urinary GAG concentrations than the lower dose (70% versus 55% at 24 weeks). Antibody levels did not appear to influence urinary GAG concentrations. CONCLUSION: Pharmacokinetic parameters appear to be independent of the duration of treatment and are not linear between the 0.2 and 1.0 mg/kg/week doses. Antibodies to rhASB develop in most patients, but their concentration decreases over time. Antibody formation may influence pharmacokinetic parameters during the early phases of treatment, although it appears to have limited impact on biochemical efficacy.

Adolescent↗
PubMed2004

Identification and molecular characterization of alpha-L-iduronidase mutations present in mucopolysaccharidosis type I patients undergoing enzyme replacement therapy.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase (IDUA). Mutations in the gene are responsible for the enzyme deficiency, which leads to the intralysosomal storage of the partially degraded glycosaminoglycans dermatan sulfate and heparan sulfate. Molecular characterization of MPS I patients has resulted in the identification of over 70 distinct mutations in the IDUA gene. The high degree of molecular heterogeneity reflects the wide clinical variability observed in MPS I patients. Six novel mutations, c.1087C>T (p.R363C), c.1804T>A (p.F602I), c.793G>C, c.712T>A (p.L238Q), c.1727+2T>A, and c.1269C>G (p.S423R), in a total of 14 different mutations, and 13 different polymorphic changes, including the novel c.246C>G (p.H82Q), were identified in a cohort of 10 MPS I patients enrolled in a clinical trial of enzyme-replacement therapy. Five novel amino acid substitutions and c.236C>T (p.A79V) were engineered into the wild-type IDUA cDNA and expressed. A p.G265R read-through mutation, arising from the c.793G>C splice mutation, was also expressed. Each mutation reduced IDUA protein and activity levels to varying degrees with the processing of many of the mutant forms also affected by IDUA. The varied properties of the expressed mutant forms of IDUA reflect the broad range of biochemical and clinical phenotypes of the 10 patients in this study. IDUA kinetic data derived from each patient's cultured fibroblasts, in combination with genotype data, was used to predict disease severity. Finally, residual IDUA protein concentration in cultured fibroblasts showed a weak correlation to the degree of immune response to enzyme-replacement therapy in each patient.

Amino Acid Substitution↗
PubMed2004

Purification and characterization of recombinant murine sulfamidase.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in the lysosomal enzyme sulfamidase, which is required for the degradation of heparan sulfate. The disease is characterized by neurological dysfunction but relatively mild somatic manifestations. A naturally occurring mouse model to MPS IIIA exhibits a similar disease progression to that observed in patients. Disease in the mice results from a base substitution at codon 31 in the sulfamidase gene, altering an aspartic acid to an asparagine (D31N). This aspartic 31 is involved in binding of the divalent metal ion needed for catalytic function, and as such reduces the specific activity of the enzyme to about 3% of that of wild-type. The mutant protein has decreased stability and shows increased degradation over a 24 h chase period when compared to wild-type mouse sulfamidase. Mouse sulfamidase that was purified using a two-step ion exchange procedure was shown to have similar kinetic properties to that of purified human sulfamidase. Recombinant murine sulfamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.

Animals↗
PubMed2003

Mass spectrometry in the study of lysosomal storage disorders.

Lysosomal storage disorders represent a group of over 45 distinct genetic diseases, each one resulting from a deficiency of a particular lysosomal protein or, in a few cases, from non-lysosomal proteins that are involved in lysosomal biogenesis. A common biochemical feature of this group of disorders is the accumulation within lysosomes of undegraded or partially degraded substrates that are normally degraded within, and transported out of the lysosome. The particular substrates stored and the site(s) of storage vary with disease type and enzyme/protein deficiency. The nature of the substrate can be used to group the disorders into broad categories including the mucopolysaccharidoses, lipidoses, glycogenoses and oligosaccharidoses. These categories show many clinical similarities within groups as well as significant similarities between groups. For most lysosomal storage disorders the relationship between the stored substrates (type, amount and location) and the disease pathology is not well understood. The use of mass spectrometry and in particular tandem mass spectrometry provides a powerful tool for the investigation of stored substrates in this group of disorders. In this review we will describe the use of mass spectrometry for the analysis of stored substrates. We will discuss progress in the field, limitations of current methods, and summarise issues relating to the diagnosis and treatment of some of the more prevalent lysosomal storage disorders.

Glycogen Storage Disease↗
PubMed2001

Conditional tissue-specific expression of the acid alpha-glucosidase (GAA) gene in the GAA knockout mice: implications for therapy.

Both enzyme replacement and gene therapy of lysosomal storage disorders rely on the receptor-mediated uptake of lysosomal enzymes secreted by cells, and for each lysosomal disorder it is necessary to select the correct cell type for recombinant enzyme production or for targeting gene therapy. For example, for the therapy of Pompe disease, a severe metabolic myopathy and cardiomyopathy caused by deficiency of acid alpha-glucosidase (GAA), skeletal muscle seems an obvious choice as a depot organ for local therapy and for the delivery of the recombinant enzyme into the systemic circulation. Using knockout mice with this disease and transgenes containing cDNA for the human enzyme under muscle or liver specific promoters controlled by tetracycline, we have demonstrated that the liver provided enzyme far more efficiently. The achievement of therapeutic levels with skeletal muscle transduction required the entire muscle mass to produce high levels of enzyme of which little found its way to the plasma, whereas liver, comprising <5% of body weight, secreted 100-fold more enzyme, all of which was in the active 110 kDa precursor form. Furthermore, using tetracycline regulation, we somatically induced human GAA in the knockout mice, and demonstrated that the skeletal and cardiac muscle pathology was completely reversible if the treatment was begun early.

Animals↗
PubMed2001

Gentamicin-mediated suppression of Hurler syndrome stop mutations restores a low level of alpha-L-iduronidase activity and reduces lysosomal glycosaminoglycan accumulation.

Hurler syndrome is the most severe form of a lysosomal storage disease caused by loss of the enzyme alpha-L-iduronidase (encoded by the IDUA gene), which participates in the degradation of glycosaminoglycans (GAGs) within the lysosome. In some populations, premature stop mutations represent roughly two-thirds of the mutations that cause Hurler syndrome. In this study we investigated whether the aminoglycoside gentamicin can suppress stop mutations within the IDUA gene. We found that a Hurler syndrome fibroblast cell line heterozygous for the IDUA stop mutations Q70X and W402X showed a significant increase in alpha-L-iduronidase activity when cultured in the presence of gentamicin, resulting in the restoration of 2.8% of normal alpha-L-iduronidase activity. Determination of alpha-L-iduronidase protein levels by an immunoquantification assay indicated that gentamicin treatment produced a similar increase in alpha-L-iduronidase protein in Hurler cells. Both the alpha-L-iduronidase activity and protein level resulting from this treatment have previously been correlated with mild Hurler phenotypes. Although Hurler fibroblasts contain a much higher level of GAGs than normal, we found that gentamicin treatment reduced GAG accumulation in Hurler cells to a normal level. We also found that a reduced GAG level could be sustained for at least 2 days after gentamicin treatment was discontinued. The reduction in the GAG level was also reflected in a marked reduction in lysosomal vacuolation. Taken together, these results suggest that the suppression of premature stop mutations may provide an effective treatment for Hurler syndrome patients with premature stop mutations in the IDUA gene.

Anti-Bacterial Agents↗
PubMed2001

Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: Diagnostic, clinical, and biological implications.

Mucopolysaccharidosis (MPS) types IIIA, B, C, and D are a group of autosomal recessive lysosomal storage diseases caused by mutations in one of four genes which encode enzyme activities required for the lysosomal degradation of heparan sulfate. The progressive lysosomal storage of heparan sulfate eventually results in the clinical onset of disease, which is predominantly characterized by severe central nervous system degeneration. MPS-IIIA and MPS-IIIB involve deficiencies of heparan sulfate sulfamidase (SGSH) and alpha-N-acetylglucosaminidase (NAGLU), respectively. Both the SGSH and NAGLU genes have been cloned and characterized, thereby permitting mutation analysis of MPS-IIIA and MPS-IIIB patients. A total of 62 mutations have now been defined for MPS-IIIA consisting of 46 missense/nonsense mutations, 15 small insertions/deletions, and one splice site mutation. A total of 86 mutations have been identified in the NAGLU gene of MPS-IIIB patients; 58 missense/nonsense mutations, 27 insertions/deletions, and one splice site mutation. Most of the identified mutations in the SGSH and NAGLU genes are associated with severe clinical phenotypes. Many of the missense, nonsense, and insertion/deletion mutations have been expressed in mammalian cell lines to permit the characterization of their effects on SGSH and NAGLU activity and intracellular processing and trafficking. For MPS-IIIA and MPS-IIIB many of the reported mutations are unique making screening the general population difficult. However, molecular characterization of MPS-IIIA patients has revealed a high incidence of particular mutations of different geographical origins, which will be beneficial for the molecular diagnosis of MPS-IIIA.

Acetylglucosaminidase↗
PubMed2001

Mucopolysaccharidosis type VI: Structural and clinical implications of mutations in N-acetylgalactosamine-4-sulfatase.

Mucopolysaccharidosis type VI (MPS-VI) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-4-sulfatase (4S; or ARSB). Mutations in the 4S gene are responsible for 4S deficiency, which leads to the intralysosomal storage of partially degraded glycosaminoglycans, dermatan sulfate, and chondroitin 4-sulfate. To date, a total of 45 clinically relevant mutations have been identified in the human 4S gene. Missense mutations are the largest group, with 31 identified mutations. Nonsense mutations and small insertions or deletions comprise the remainder, with seven mutations each. Six polymorphisms have also been reported: two amino acid substitutions and four silent transitions. Mapping of the missense mutations onto the 4S structure shows that they are distributed throughout the three subunits of the mature 4S polypeptide. Mutations have been identified in active site residues, in residues adjacent to the active site, in potential substrate binding residues, in residues exposed on the surface, and in residues buried within the protein core. Missense mutations have also been identified in disulfide crosslinks. Molecular modeling of MPS-VI mutations onto the 4S structure suggests that the majority cause 4S deficiency via destabilization and the consequent reduction of 4S protein concentration. The vast majority of MPS-VI mutant alleles are either unique to a patient or are present in a small number of patients. So far, no common mutations have been described. Therefore, screening of the general population for MPS-VI alleles will be difficult.

Animals↗
PubMed2001

Purification and characterization of recombinant human lysosomal alpha-mannosidase.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is required in the degradation of the asparagine-linked carbohydrates of glycoproteins. Deficiency of this enzyme leads to the lysosomal storage disorder alpha-mannosidosis. As an initial step toward enzyme replacement therapy for alpha-mannosidosis, the human lysosomal alpha-mannosidase cDNA was cloned into the pcDNA 3.1 vector and expressed in Chinese hamster ovary cells. Dimethyl sulfoxide (DMSO) added to the cell culture media to induce growth arrest led to a 4-fold increase in the enzyme production, with an average yield of 3.2 mg L(-1) day(-1). alpha-Mannosidase was secreted as an active homodimer of a 130-kDa precursor that was proteolyzed into two polypeptides of 55 and 72 kDa during the subsequent purification of the enzyme. N-terminal sequence analysis of the purified enzyme revealed that the proteolysis occurred close to a cleavage site previously identified in the intracellular form of lysosomal alpha-mannosidase. Generation of monoclonal antibodies against the recombinant enzyme made it possible to develop a single-step immunoaffinity purification procedure for alpha-mannosidase. The immunoaffinity-purified enzyme which mainly consisted of the 130-kDa precursor, displayed specific activity and kinetics similar to those of the processed form. Recombinant alpha-mannosidase was taken up by cultured alpha-mannosidosis fibroblasts and was trafficked to the lysosomes via the mannose 6-phosphate pathway where it reduced the amounts of stored mannose-containing oligosaccharides.

Animals↗
PubMed2001

Prediction of Sanfilippo phenotype severity from immunoquantification of heparan-N-sulfamidase in cultured fibroblasts from mucopolysaccharidosis type IIIA patients.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of heparan-N-sulfamidase (NS; EC 3.10.1.1), resulting in defective degradation and subsequent storage of heparan sulfate and leading to a clinical phenotype known as Sanfilippo syndrome. A sensitive and specific monoclonal/polyclonal-based immunoquantification assay has enabled the determination of NS protein, down to approximately 3 pg NS protein, in cultured fibroblasts from control and MPS-IIIA patients. Cultured skin fibroblasts from 15 normal controls contained 11.9 to 105 ng of NS protein/mg extracted cell protein, whereas NS protein ranged from "none detected" to 11 ng/mg in fibroblasts from 35 MPS-IIIA patients. A relationship between genotype/phenotype and amount of NS protein present in these MPS-IIIA fibroblasts was established. Immunoquantification, in combination with a specific and highly sensitive tetrasaccharide-based assay of NS activity, enabled the determination of residual specific NS activity in these fibroblasts. Specific NS activity ranged from 28 to 1289 nmol/min/mg NS protein for MPS-IIIA patients, compared to 870 nmol/min/mg of recombinant human NS. It is proposed that this immunoquantification method, in conjunction with the specific NS activity assay, may be used to predict clinical severity in MPS-IIIA patients, allowing for the selection of individuals best suited for gene- and enzyme-replacement therapy when these methods become available. Also proposed is that an enzyme-replacement therapy achieving a correction of approximately 10% of normal NS activity is required to avoid the onset of a Sanfilippo clinical phenotype.

Adolescent↗
PubMed2001

Expression and characterization of human recombinant and alpha-N-acetylglucosaminidase.

Mucopolysaccharidosis type IIIB (MPS-IIIB, Sanfilippo type B Syndrome) is a heterosomal, recessive lysosomal storage disorder resulting from a deficiency of [alpha]-N-acetylglucosaminidase (NAGLU). To characterize this enzyme further and evaluate its potential for enzyme replacement studies we expressed the NAGLU-encoding cDNA in Chinese hamster ovary cells (CHO-K1 cells) and purified the recombinant enzyme from the medium of stably transfected cells by a two-step affinity chromatography. Two isoforms of recombinant NAGLU with apparent molecular weights of 89 and 79 kDa were purified and shown to differ in their glycosylation pattern. The catalytic parameters of both forms of the recombinant enzyme were indistinguishable from each other and similar to those of NAGLU purified from various tissues. However, compared to other recombinant lysosomal enzymes expressed from CHO-K1 cells, the mannose-6-phosphate receptor mediated uptake of the secreted form of recombinant NAGLU into cultured skin fibroblasts was considerably reduced. A small amount of phosphorylated NAGLU present in purified enzyme preparations was shown to be endocytosed by MPS-IIIB fibroblasts via the mannose-6-phosphate receptor-mediated pathway and transported to the lysosomes, where they corrected the storage phenotype. Direct metabolic labeling experiments with Na(2) (32)PO(4) confirmed that the specific phosphorylation of recombinant NAGLU secreted from transfected CHO cells is significantly lower when compared with a control lysosomal enzyme. These results suggest that the use of secreted NAGLU in future enzyme and gene replacement therapy protocols will be severely limited due to its small degree of mannose-6-phosphorylation.

Acetylglucosaminidase↗
PubMed2001

A novel missense mutation in lysosomal sulfamidase is the basis of MPS III A in a spontaneous mouse mutant.

Sanfilippo syndrome type III A (Mucopolysaccharidosis (MPS) III A) is a rare, autosomal recessive, lysosomal storage disease, characterized by the accumulation of heparan sulfate and the loss of function of lysosomal heparan N-sulfatase activity. The disease leads to devastating mental and physical consequences and a mouse model that can be used to explore gene therapy and enzyme or cell replacement therapies is needed. We have previously identified a mouse with low sulfamidase activity and symptoms and pathologies typical of MPS III A (Bhaumik, M., Muller, V. J., Rozaklis, T., Johnson, L., Dobrenis, K., Bhattacharyya, R., Wurzelmann, S., Finamore, P., Hopwood, J. J., Walkley, S. U., and Stanley, P. [1999] A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome). Glycobiology 9, 1389--1396). We now show that the sulfamidase gene of the MPS III A mouse carries a novel mutation (G91A) that gives an amino acid change (D31N) likely to interfere with the coordination of a divalent metal ion in the active site of this sulfatase. This spontaneous mouse mutant is an excellent model for MPS III A in humans as this disease often arises due to a missense mutation in lysosomal sulfamidase.

Amino Acid Sequence↗
PubMed2001

Glycosidase active site mutations in human alpha-L-iduronidase.

Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.

Amino Acid Sequence↗
PubMed2001

Management of Chlamydia trachomatis in a women's hospital: a review of current practice.

OBJECTIVE: To establish a measure of testing for Chlamydia trachomatis within the Liverpool Women's Hospital with a view to optimising both testing and management of infection. DESIGN: Prospective observational study to review the outcome of Chlamydia testing and subsequent management of patients between September 1997 - September 1998. RESULTS: It was observed that opportunities for detecting infection were missed and testing was undertaken predominantly for diagnostic purposes. RECOMMENDATION: Consideration be given to a centralised system for overview of positive results linking with audit/education to reduce sequelae of Chlamydia within gynaecology.

Adult↗