The first goal of this study was to understand the role of u-calpain in skeletal muscle
protein degradation in cultured muscle cells. Several strategies were developed to down-regulate
endogenous u-calpain activity and m-calpain activity in rat myotubes. These
included over-expression of antisense u-calpain (AnsL), dominant negative u-calpain
(DN-u-CL), antisense 30K subunit (AnsS) and fused antisense u-calpain/30K (AnsLS,
i.e., 80K/30K). The ability to regulate calpain activity was confirmed by fodrin
degradation (an index of calpain activity). Our data supported the contention that u-calpain
contributes significantly to total protein degradation in myotubes. Specifically,
over-expressing DN-u-calpain reduced total protein degradation by 7.9% (P<0.01) at 24
hr time point and by 10.6% (P<0.01) at a 48 hr time point. Similarly, over-expression of
antisense u-CL and the 30K subunit reduced total protein degradation significantly at the
24 hr time point (P<0.05). However, over-expression of the fused antisense (80K/30K)
did not affect (P>0.05) the total protein degradation. In addition to this we determined
that desmin was a calpain substrate and that calpain could not degrade tropomyosin.
The second goal of this study was to evaluate the relationships among u- and m-calpain
and the 30KD subunit. The rationale for this study was that our earlier work indicated
coordinated regulation of the calpain subunits. Our data demonstrated for the first time
that the transcription and translation of u-calpain and 30K, and m-calpain and 30K are
coordinately regulated, respectively. However, the expression of u-calpain did not affect
the expression of m-calpain
The third goal of this study was to develop a skeletal muscle-specific inducible
expression system that may be used in transgenic animal research. A skeletal muscle a-actin
promoter was used to replace the cytomegalovirus immediate-early promoter
(pCMV) in the ecdysone inducible mammalian expression system. LacZ was used as a
reporter gene. A beta-galactosidase staining assay and high-sensitivity B-gal activity
assay indicated that the skeletal muscle-specific expression system functioned in
myotubes. After 48 hr of administration of ponasterone A (inducer), the treated cells had
15-fold higher B-gal activity than the control cells. / Graduation date: 2002
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/32264 |
| Date | 15 March 2001 |
| Creators | Xiao, Ying-Yi |
| Contributors | Forsberg, Neil E. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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