Uridine diphosphate glucose

Preparation Method

The reaction mixture for 20S proteasome inhibitory assay contained 0.1 M Tris-acetate, pH 7.0, 20S proteasome, MG-132 and 25 μM substrate dissolved in DMSO in a final volume of 1 ml.

Reaction Conditions

After incubation at 37 ℃ for 15 min, the reaction was stopped by the addition of 0.1 ml of 10% SDS and 0.9 ml of 0.1 M Tris-acetate, pH 9.0.

Applications

After measurement of fluorescence of reaction products, the IC₅₀ of MG-132 against m-calpain and 20S proteasome can be determined. MG-132 inhibits 20S proteasome with IC₅₀ of 100 nM.

Cell lines

KIM-2

Preparation Method

MG-132 were diluted in Me₂SO. Cells were treated with protease inhibitors or dilutant alone. Supernatant and monolayer cells were harvested by centrifugation and fixed in 70% ethanol in PBS for staining with acridine orange. Equal volumes of cells and acridine orange (5 mg/ml in PBS) were mixed on a microscope slide and examined by fluorescence microscopy.

Reaction Conditions

0, 1.5, 5 μM, 24 h

Applications

MG-132 treatment induces apoptosis in a cell cycle dependent manner.

Animal models

C57BL/10ScSn DMD mdx mice

Preparation Method

Localized administration was performed by injection of MG-132 into the gastrocnemius muscles of mdx mice. To visualize the injected muscle, MG-132 (final concentration of 20 μmol/L) was pre-mixed with 1% India ink in phosphate-buffered saline (PBS) for a total volume of 100 μl. Mice were sacrificed 24 hours after injection, and skeletal muscles were quickly isolated for further analysis. To systemically administer MG-132, Alzet Minipumps was subcutaneously implanted in the anterior back region of mdx mice. Experiments were conducted on 6-month-old mdx mice. For 8 days, administration of either different concentrations of MG-132 (delivered at rate of either 1 μg, or 5 μg or 10 μg/kg/24 hours) or the inhibitor-diluent (PBS only) was enforced, as a negative control. Skeletal muscle tissues were collected from untreated (PBS only) and MG-132-treated mdx mice for further analysis.

Dosage form

1 μg, 5 μg, 10 μg/kg, injection into the gastrocnemius muscles or subcutaneously implanted Alzet Minipumps

Applications

MG-132, as a proteasomal inhibitor, effectively rescues the expression levels and plasma membrane localization of dystrophin, β-dystroglycan, α-dystroglycan, and α-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, MG-132 reduces muscle membrane damage, as revealed by vital staining of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice.

References:

[1]. Tsubuki, S et al. Differential inhibition of calpain and proteasome activities by peptidyl aldehydes of di-leucine and tri-leucine. Journal of biochemistry vol. 119,3 (1996): 572-6.

[2]. MacLaren, A P et al. p53-dependent apoptosis induced by proteasome inhibition in mammary epithelial cells. Cell death and differentiation vol. 8,3 (2001): 210-8.

[3]. Bonuccelli, Gloria et al. Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins. The American journal of pathology vol. 163,4 (2003): 1663-75.