10 research outputs found
Simultaneous measurements of knee motion using an optical tracking system and radiostereometric analysis (RSA)
A Rapid and Sensitive Method for Measuring NAcetylglucosaminidase Activity in Cultured Cells
A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly
desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with
Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available
techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide
a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell
disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone
procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure
for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-
Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell
disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the
NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a
rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG
activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master
regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity
and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput
screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of
candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination
therapies