22 results on '"Mattaliano, Robert"'
Search Results
2. Pompe disease in a Brazilian series: clinical and molecular analyses with identification of nine new mutations
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Oba-Shinjo, Sueli M., da Silva, Roseli, Andrade, Fernanda G., Palmer, Rachel E., Pomponio, Robert J., Ciociola, Kristina M., S. Carvalho, Mary, Gutierrez, Paulo S., Porta, Gilda, Marrone, Carlo D., Munoz, Verônica, Grzesiuk, Anderson K., Llerena, Jr., Juan C., Berditchevsky, Célia R., Sobreira, Claudia, Horovitz, Dafne, Hatem, Thamine P., Frota, Elizabeth R. C., Pecchini, Rogerio, Kouyoumdjian, João Aris, Werneck, Lineu, Amado, Veronica M., Camelo, Jr., José S., Mattaliano, Robert J., and Marie, Suely K. N.
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- 2009
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3. Conjugation of Mannose 6-Phosphate-containing Oligosaccharides to Acid α-Glucosidase Improves the Clearance of Glycogen in Pompe Mice
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Zhu, Yunxiang, Li, Xuemei, Kyazike, Josephine, Zhou, Qun, Thurberg, Beth L., Raben, Nina, Mattaliano, Robert J., and Cheng, Seng H.
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- 2004
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4. Identification and quantitation of vesivirus 2117 particles in bioreactor fluids from infected Chinese hamster ovary cell cultures.
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Qiu, Yongchang, Jones, Nathan, Busch, Michelle, Pan, Peng, Keegan, Jesse, Zhou, Weichang, Plavsic, Mark, Hayes, Michael, McPherson, John M., Edmunds, Tim, Zhang, Kate, and Mattaliano, Robert J.
- Abstract
The prevention of adventitious agent contamination is a top priority throughout the entire biopharmaceutical production process. For example, although viral contamination of cell banks or cell cultures is rare, it can result in serious consequences (e.g., shutdown and decontamination of manufacturing facilities). To ensure virus free production, numerous in vivo and in vitro adventitious agent assays and biophysical characterizations such as electron microscopy are conducted on cell banks, raw materials, process materials, and drug substances throughout the manufacturing process. Molecular assays such as PCR and other nucleotide-based techniques are also routinely used for screening and identification of any viral agents. However, modern techniques in protein identification of complex protein mixtures have not yet been effectively integrated throughout the industry into current viral testing strategies. Here, we report the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures by global protein sequencing using mass spectrometry in combination with multi-dimensional liquid-chromatography. Following mass spectrometric data acquisition and rigorous data analysis, six virus specific peptides were identified. These peptides were fragments of two structural proteins, capsid protein pre-cursor (four unique peptides) and small structural protein (two unique peptides), from the same species: Vesivirus 2117. Using stable heavy isotope-labeled peptides as internal standards, we also determined the absolute concentration of Vesivirus particles in the bioreactor fluid and the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. The positive identification of Vesivirus 2117 was subsequently confirmed by RT-PCR. Biotechnol. Bioeng. 2013; 110: 1342-1353. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
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- 2013
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5. New developments in the treatment of Pompe disease
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Mattaliano, Robert J.
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- 2010
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6. Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity.
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Wallner, Barbara P., Mattaliano, Robert J., Hession, Catherine, Cate, Richard L., Tizard, Richard, Sinclair, Lesley K., Foeller, Carl, Chow, E. Pingchang, Browning, Jeffrey L., Ramachandran, K. L., and Pepinsky, R. Blake
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- 1986
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7. The state of the N-terminus of recombinant proteins: Determination of N-terminal methionine (formylated, acetylated, or free)
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Rose, Keith, Savoy, Luc-Alain, Simona, Marco G., Offord, Robin E., Wingfield, Paul T., Mattaliano, Robert J., and Thatcher, David R.
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- 1987
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8. Sequencing of peptides and proteins from the carboxy terminus
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Boyd, Victoria L., Bozzini, MeriLisa, Zon, Gerald, Noble, Richard L., and Mattaliano, Robert J.
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- 1992
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9. Disulfide scrambling of interleukin-2: HPLC resolution of the three possible isomers
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Browning, Jeffrey L., Mattaliano, Robert J., Chow, E.Pingchang, Liang, Shu-Mei, Allet, Bernard, Rosa, Joseph, and Smart, John E.
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- 1986
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10. Inhibition of glycogen biosynthesis via mTORC1 suppression as an adjunct therapy for Pompe disease
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Ashe, Karen M., Taylor, Kristin M., Chu, Qiuming, Meyers, Elizabeth, Ellis, Allen, Jingozyan, Varvara, Klinger, Katherine, Finn, Patrick F., Cooper, Christopher G.F., Chuang, Wei-Lien, Marshall, John, McPherson, John M., Mattaliano, Robert J., Cheng, Seng H., Scheule, Ronald K., and Moreland, Rodney J.
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GLYCOGEN storage disease type II , *ETIOLOGY of diseases , *GLUCOSIDASES , *GLYCOGEN , *BIOSYNTHESIS , *RAPAMYCIN , *IMMUNOSUPPRESSIVE agents , *THERAPEUTICS - Abstract
Abstract: Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1–2years of age to a more slowly progressive course that causes significant morbidity and early mortality in children and adults. Recombinant human GAA (rhGAA) improves clinical outcomes with variable results. Adjunct therapy that increases the effectiveness of rhGAA may benefit some Pompe patients. Co-administration of the mTORC1 inhibitor rapamycin with rhGAA in a GAA knockout mouse reduced muscle glycogen content more than rhGAA or rapamycin alone. These results suggest mTORC1 inhibition may benefit GSDs that involve glycogen accumulation in muscle. [Copyright &y& Elsevier]
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- 2010
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11. Glycoengineered Acid α-Glucosidase With Improved Efficacy at Correcting the Metabolic Aberrations and Motor Function Deficits in a Mouse Model of Pompe Disease.
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Yunxiang Zhu, Ji-Lei Jiang, Gumlaw, Nathan K., Jinhua Zhang, Bercury, Scott D., Ziegler, Robin J., Lee, Karen, Kudo, Mariko, Canfield, William M., Edmunds, Timothy, Jiang, Canwen, Mattaliano, Robert J., and Cheng, Seng H.
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GLUCOSIDASES , *GLYCOGEN storage disease type II , *MOTOR ability , *OLIGOSACCHARIDES , *OXIMES , *MUSCLE cells , *LYSOSOMES , *LABORATORY mice , *PHYSIOLOGY - Abstract
Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid α-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.Molecular Therapy (2009) 17 6, 954–963 doi:10.1038/mt.2009.37 [ABSTRACT FROM AUTHOR]
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- 2009
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12. Autophagy and Mistargeting of Therapeutic Enzyme in Skeletal Muscle in Pompe Disease.
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Fukuda, Tokiko, Ahearn, Meghan, Roberts, Ashley, Mattaliano, Robert J., Zaal, Kristien, Ralston, Evelyn, Plotz, Paul H., and Raben, Nina
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GLYCOGEN storage disease type II , *ENZYMES , *GLUCOSIDASES , *LYSOSOMES , *ENDOCYTOSIS , *LIPOFUSCINS - Abstract
Enzyme replacement therapy (ERT) became a reality for patients with Pompe disease, a fatal cardiomyopathy and skeletal muscle myopathy caused by a deficiency of glycogen-degrading lysosomal enzyme acid α-glucosidase (GAA). The therapy, which relies on receptor-mediated endocytosis of recombinant human GAA (rhGAA), appears to be effective in cardiac muscle, but less so in skeletal muscle. We have previously shown a profound disturbance of the lysosomal degradative pathway (autophagy) in therapy-resistant muscle of GAA knockout mice (KO). Our findings here demonstrate a progressive age-dependent autophagic buildup in addition to enlargement of glycogen-filled lysosomes in multiple muscle groups in the KO. Trafficking and processing of the therapeutic enzyme along the endocytic pathway appear to be affected by the autophagy. Confocal microscopy of live single muscle fibers exposed to fluorescently labeled rhGAA indicates that a significant portion of the endocytosed enzyme in the KO was trapped as a partially processed form in the autophagic areas instead of reaching its target—the lysosomes. A fluid-phase endocytic marker was similarly mistargeted and accumulated in vesicular structures within the autophagic areas. These findings may explain why ERT often falls short of reversing the disease process and point toward new avenues for the development of pharmacological intervention.Molecular Therapy (2006) 14, 831–839; doi: 10.1016/j.ymthe.2006.08.009 [ABSTRACT FROM AUTHOR]
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- 2006
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13. Replacing acid α-glucosidase in Pompe disease: recombinant and transgenic enzymes are equipotent, but neither completely clears glycogen from type II muscle fibers
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Raben, Nina, Fukuda, Tokiko, Gilbert, Abigail L., de Jong, Deborah, Thurberg, Beth L., Mattaliano, Robert J., Meikle, Peter, Hopwood, John J., Nagashima, Kunio, Nagaraju, Kanneboyina, and Plotz, Paul H.
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GLUCANS , *INBORN errors of metabolism , *CARDIOMYOPATHIES , *GLYCOGEN - Abstract
Abstract: Pompe disease (type II glycogen storage disease) is an autosomal recessive disorder caused by a deficiency of lysosomal acid α-glucosidase (GAA) leading to the accumulation of glycogen in the lysosomes primarily in cardiac and skeletal muscle. The recombinant human GAA (rhGAA) is currently in clinical trials for enzyme replacement therapy of Pompe disease. Both clinical data and the results of preclinical studies in our knockout model of this disease show that rhGAA is much more effective in resolving the cardiomyopathy than the skeletal muscle myopathy. By contrast, another form of human GAA—transgenic enzyme constitutively produced in liver and secreted into the bloodstream of knockout mice (Gaa−/−)—completely prevented both cardiac and skeletal muscle glycogen accumulation. In the experiments reported here, the transgenic enzyme was much less efficient when delivered to skeletal muscle after significant amounts of glycogen had already accumulated. Furthermore, the transgenic enzyme and the rhGAA have similar therapeutic effects, and both efficiently clear glycogen from cardiac muscle and type I muscle fibers, but not type II fibers. Low abundance of proteins involved in endocytosis and trafficking of lysosomal enzymes combined with increased autophagy in type II fibers may explain the resistance to therapy. [Copyright &y& Elsevier]
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- 2005
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14. Conjugation of Mannose 6-Phosphate-containing Oligosaccharides to Acid α-Glucosidase Improves the Clearance of Glycogen in Pompe Mice.
- Author
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Yunxiang Zhu, Xuemei Li, Kyazike, Josephine, Qun Zhou, Thurberg, Beth L., Raben, Nina, Mattaliano, Robert J., and Cheng, Seng H.
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ENZYMES , *THERAPEUTICS , *GLYCOGEN , *GLUCANS , *PROTEINS , *ABSORPTION (Physiology) , *MUSCLE cells - Abstract
Clinical studies of enzyme replacement therapy for Pompe disease have indicated that relatively high doses of recombinant human acid α-glucosidase (rhGAA) may be required to reduce the abnormal glycogen storage in cardiac and skeletal muscles. This may be because of inefficient cation-independent mannose 6-phosphate receptor (CI-MPR)-mediated endocytosis of the enzyme by the affected target cells. To address this possibility, we examined whether the addition of a high affinity ligand to rhGAA would improve its delivery to these cells. Chemical conjugation of high mannose oligosaccharides harboring mono- and bisphosphorylated mannose 6-phosphates onto rhGAA (neo-rhGAA) significantly improved its uptake characteristics by muscle cells in vitro. Infusion of neo-rhGAA into Pompe mice also resulted in greater delivery of the enzyme to muscle tissues when compared with the unmodified enzyme. Importantly, this increase in enzyme levels was associated with significantly improved clearance of glycogen (∼5-fold) from the affected tissues. These results suggest that CI-MPR-mediated endocytosis of rhGAA is an important pathway by which the enzyme is delivered to the affected lysosomes of Pompe muscle cells. Hence, the generation of rhGAA containing high affinity ligands for the CI-MPR represents a strategy by which the potency of rhGAA and therefore the clinical efficacy of enzyme replacement therapy for Pompe disease may be improved. [ABSTRACT FROM AUTHOR]
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- 2004
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15. Comparison of Two In Vitro Methods for the Measurement of Recombinant Human TSH Bioactivity
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Sendak, Rebecca A., Wang, Fei, Geagan, Laura B., Armstrong, Lori A., Thyne, Charles D., Cole, Edward S., and Mattaliano, Robert J.
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THYROTROPIN , *GLYCOPROTEIN hormones - Abstract
Thyroid stimulating hormone (TSH), a pituitary glycoprotein hormone, is a potent inducer of intracellular cAMP production. Two methods for measuring TSH bioactivity were evaluated and compared. One assay is based on using a radioimmunoassay (RIA) to measure the recombinant human TSH-induced increase in cAMP using a bovine thyroid membrane isolate. The other is based on a Chinese hamster ovary (CHO) cell line that has been transfected with the TSH receptor and a cAMP-responsive luciferase reporter. The within-assay coefficient of variation for the membrane-based assay was determined to be approximately 35% compared with approximately 25% for the cell-based assay. Twenty-one preparations of recombinant human TSH (rhTSH) were tested using both methods. No significant difference was detected between the data sets and no assay bias was present. Both assay systems provide a suitable means for measuring the activity of rhTSH. The advantage of the membrane-based assay is the relatively small quantity of TSH needed for analysis. However, the average time required to analyse a sample using the membrane-based method was more than twice as long as that needed to test a sample in the cell-based assay. Other advantages of the cell-based method include the use of a 96-well format, which facilitates the analysis of several concentrations of rhTSH within one assay plate, and the use of a non-radioactive endpoint. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved. [Copyright &y& Elsevier]
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- 2002
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16. Inhibiting glycogen biosynthesis by mTORC1 suppression as an adjunct therapy for Pompe disease
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Moreland, Rodney, Taylor, Kristin, Ashe, Karen, Meyers, Elizabeth, Ellis, Allen, Jingozyan, Varvara, Finn, Patrick, Cheng, Seng, Klinger, Katherine, Marshall, John, McPherson, John, Mattaliano, Robert, and Scheule, Ronald
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- 2011
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17. Detection of adventitious agents using next-generation sequencing.
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Richards B, Cao S, Plavsic M, Pomponio R, Davies C, Mattaliano R, Madden S, Klinger K, and Palermo A
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- Animals, Biopharmaceutics standards, Bioreactors, CHO Cells, Cell Culture Techniques, Cricetulus, DNA, Bacterial genetics, DNA, Viral genetics, Humans, Limit of Detection, RNA, Viral genetics, Reference Standards, Virology standards, Bacteria genetics, Bacteriological Techniques standards, Biological Products analysis, Biopharmaceutics methods, Drug Contamination prevention & control, High-Throughput Nucleotide Sequencing standards, Virology methods, Viruses genetics
- Abstract
Next-generation sequencing has been evaluated at Genzyme as a means of identifying bioreactor contaminants due to its capability for detection of known and novel microbial species. In this approach, data obtained from next-generation sequencing is used to interrogate databases containing genomic sequences and identities of potential adventitious agents. We describe here the use of this approach to help identify the causative agent of a bioreactor contamination. We also present the results of spiking experiments to establish the limits of detection for DNA viruses, RNA viruses, and bacteria, in a background of Chinese hamster ovary cells, a cell line used for production of many human therapeutics. Using Illumina sequencing-based detection, all of the viruses included in this study were detected at less than 1 copy per cell, and bacteria were detected at 0.001 copy per cell. Thus, next-generation sequencing-based detection of adventitious agents is a valuable approach that can fill a critical unmet need in the detection of known and novel microorganisms in biopharmaceutical manufacturing., Lay Abstract: Because biological products are manufactured in cells, the living environment must be kept sterile. Any introduction of microorganisms into the culture vessel may affect the growth and other biological properties of the cells or contaminate the product. It is therefore important to monitor the culture for such contaminants, but many methods can only detect a specific microorganism. In this study, we show that next-generation sequencing-based detection is a sensitive and complementary approach that can potentially detect a wide range of organisms., (© PDA, Inc. 2014.)
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- 2014
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18. Glycoengineered acid alpha-glucosidase with improved efficacy at correcting the metabolic aberrations and motor function deficits in a mouse model of Pompe disease.
- Author
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Zhu Y, Jiang JL, Gumlaw NK, Zhang J, Bercury SD, Ziegler RJ, Lee K, Kudo M, Canfield WM, Edmunds T, Jiang C, Mattaliano RJ, and Cheng SH
- Subjects
- Animals, Disease Models, Animal, Glycogen metabolism, Glycogen Storage Disease Type II metabolism, Humans, Mice, Mice, Inbred C57BL, Muscle, Skeletal drug effects, Muscle, Skeletal metabolism, Muscle, Skeletal pathology, Protein Binding, Receptor, IGF Type 2 metabolism, alpha-Glucosidases chemistry, alpha-Glucosidases genetics, alpha-Glucosidases pharmacology, Glycogen Storage Disease Type II drug therapy, Mannosephosphates chemistry, Oligosaccharides chemistry, Protein Engineering methods, alpha-Glucosidases metabolism, alpha-Glucosidases therapeutic use
- Abstract
Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.
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- 2009
- Full Text
- View/download PDF
19. Dysfunction of endocytic and autophagic pathways in a lysosomal storage disease.
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Fukuda T, Ewan L, Bauer M, Mattaliano RJ, Zaal K, Ralston E, Plotz PH, and Raben N
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- Age Factors, Animals, Blotting, Western methods, Cells, Cultured, Disease Models, Animal, Fluorescent Antibody Technique methods, Glucan 1,4-alpha-Glucosidase deficiency, Green Fluorescent Proteins metabolism, In Vitro Techniques, Lysosomal Storage Diseases genetics, Lysosomal Storage Diseases metabolism, Lysosomal-Associated Membrane Protein 1 metabolism, Mice, Mice, Knockout, Microscopy, Electron, Transmission methods, Muscle Fibers, Skeletal metabolism, Muscle Fibers, Skeletal ultrastructure, Myoblasts metabolism, Myoblasts ultrastructure, Time Factors, Transfection methods, Transport Vesicles metabolism, Transport Vesicles ultrastructure, Tubulin metabolism, Autophagy physiology, Endocytosis physiology, Lysosomal Storage Diseases physiopathology
- Abstract
Objective: To understand the mechanisms of skeletal muscle destruction and resistance to enzyme replacement therapy in Pompe disease, a deficiency of lysosomal acid alpha-glucosidase (GAA), in which glycogen accumulates in lysosomes primarily in cardiac and skeletal muscles., Methods: We have analyzed compartments of the lysosomal degradative pathway in GAA-deficient myoblasts and single type I and type II muscle fibers isolated from wild-type, untreated, and enzyme replacement therapy-treated GAA knock-out mice., Results: Studies in myoblasts from GAA knock-out mice showed a dramatic expansion of vesicles of the endocytic/autophagic pathways, decreased vesicular movement in overcrowded cells, and an acidification defect in a subset of late endosomes/lysosomes. Analysis by confocal microscopy of isolated muscle fibers demonstrated that the consequences of the lysosomal glycogen accumulation are strikingly different in type I and II muscle fibers. Only type II fibers, which are the most resistant to therapy, contain large regions of autophagic buildup that span the entire length of the fibers., Interpretation: The vastly increased autophagic buildup may be responsible for skeletal muscle damage and prevent efficient trafficking of replacement enzyme to lysosomes.
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- 2006
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20. Carbohydrate-remodelled acid alpha-glucosidase with higher affinity for the cation-independent mannose 6-phosphate receptor demonstrates improved delivery to muscles of Pompe mice.
- Author
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Zhu Y, Li X, McVie-Wylie A, Jiang C, Thurberg BL, Raben N, Mattaliano RJ, and Cheng SH
- Subjects
- Aging, Animals, Glucan 1,4-alpha-Glucosidase therapeutic use, Glycogen metabolism, Glycogen Storage Disease Type II metabolism, Mice, Molecular Structure, Muscle, Skeletal enzymology, Myocardium enzymology, Myocardium metabolism, Oligosaccharides, Protein Binding, Receptor, IGF Type 2 chemistry, Recombinant Proteins, alpha-Glucosidases, Glucan 1,4-alpha-Glucosidase chemistry, Glucan 1,4-alpha-Glucosidase metabolism, Glycogen Storage Disease Type II drug therapy, Muscle, Skeletal metabolism, Receptor, IGF Type 2 metabolism
- Abstract
To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid alpha-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.
- Published
- 2005
- Full Text
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21. The effect of posttranslational modifications on the in vitro activity of recombinant human thyroid-stimulating hormone.
- Author
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Sendak RA, Ganesa C, Lee KL, Harrahy JJ, Théberge R, Morgan CJ, Cole ES, Kohn LD, and Mattaliano RJ
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- Chromatography, Liquid, Circular Dichroism, Fluorescence, Humans, Mass Spectrometry, Oligosaccharides analysis, Oxidation-Reduction, Peptide Mapping, Recombinant Proteins chemistry, Recombinant Proteins drug effects, Recombinant Proteins metabolism, Thyrotropin chemistry, Thyrotropin drug effects, tert-Butylhydroperoxide pharmacology, Protein Processing, Post-Translational, Thyrotropin metabolism
- Abstract
Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of beta-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitro activity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. beta-Subunit C-terminal truncation up to residue 113 did not effect the in vitro activity of the hormone. The relationship between the heterogeneity of oligosaccharide structures on rhTSH and specific activity of the glycoprotein hormone was also examined. Oligosaccharide profiles were generated for preparations of rhTSH containing similar sialic acid levels. A weak correlation was observed between relative levels of monosialylated biantennary, bisialylated biantennary, and trisialylated triantennary oligosaccharide species and in vitro activity of the recombinant hormone (p < 0.05). To examine the effect of chemically induced methionine oxidation on the activity of rhTSH, the hormone was treated with tert-butyl hydroperoxide and then characterized. Using peptide mapping and mass spectrometry, the degree of oxidation of the five methionine residues within rhTSH was measured. Met-71 in the alpha-subunit was the most susceptible to oxidation whereas Met-9 in the beta-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the beta-subunit, and Met-29 and Met-47 in the alpha-subunit were less than half of that observed for Met-71. The in vitro activity of rhTSH initially declined with increasing oxidation; however, the loss in activity plateaued at approximately 50% of the control sample activity. In summary, despite the possible effects that posttranslational modifications may have on the bioactivity of a protein, a limited degree of variation in bioactivity was observed for the rhTSH preparations described in this study.
- Published
- 2003
- Full Text
- View/download PDF
22. Comparison of two in vitro methods for the measurement of recombinant human TSH bioactivity.
- Author
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Sendak RA, Wang F, Geagan LB, Armstrong LA, Thyne CD, Cole ES, and Mattaliano RJ
- Subjects
- Animals, CHO Cells, Cattle, Cricetinae, Genes, Reporter, Humans, In Vitro Techniques, Luciferases genetics, Radioimmunoassay methods, Receptors, Thyrotropin genetics, Recombinant Proteins analysis, Recombinant Proteins pharmacology, Thyroid Gland drug effects, Transfection, Biological Assay methods, Thyrotropin analysis, Thyrotropin pharmacology
- Abstract
Thyroid stimulating hormone (TSH), a pituitary glycoprotein hormone, is a potent inducer of intracellular cAMP production. Two methods for measuring TSH bioactivity were evaluated and compared. One assay is based on using a radioimmunoassay (RIA) to measure the recombinant human TSH-induced increase in cAMP using a bovine thyroid membrane isolate. The other is based on a Chinese hamster ovary (CHO) cell line that has been transfected with the TSH receptor and a cAMP-responsive luciferase reporter. The within-assay coefficient of variation for the membrane-based assay was determined to be approximately 35% compared with approximately 25% for the cell-based assay. Twenty-one preparations of recombinant human TSH (rhTSH) were tested using both methods. No significant difference was detected between the data sets and no assay bias was present. Both assay systems provide a suitable means for measuring the activity of rhTSH. The advantage of the membrane-based assay is the relatively small quantity of TSH needed for analysis. However, the average time required to analyse a sample using the membrane-based method was more than twice as long as that needed to test a sample in the cell-based assay. Other advantages of the cell-based method include the use of a 96-well format, which facilitates the analysis of several concentrations of rhTSH within one assay plate, and the use of a non-radioactive endpoint., (Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.)
- Published
- 2002
- Full Text
- View/download PDF
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