Age‐Related changes and tissue distribution of parvalbumin in normal and dystrophic mice of strain 129 ReJ

Abstract

In murine muscular dystrophy, hindlimb muscle contains a functionally defective thiol protease inhibitor (TPI) which has been implicated in the onset and progression of the disease in mice. More recently, this protease inhibitor has been identified as parvalbumin, a calcium binding protein. In this study, a polyclonal antibody against mouse muscle parvalbumin was used to study the concentration and distribution of this protein in normal and dystrophic male mice at various ages. Immunodetection assays were used to screen extracts of hindlimb, forelimb, brain, heart, lung, liver, and kidney in 60-day-old normal and dystrophic male mice for parvalbumin content. Parvalbumin was detected in relatively high amounts in both hindlimb and forelimb muscle extracts, while much lower concentrations were detected in brains of normal and dystrophic animals. No parvalbumin was detected in the lung, liver, heart, or kidney extracts using the immunoassay. With aging, the parvalbumin concentration in hindlimb muscle of normal mice remained fairly constant for 90 days, whereupon the level increased at 120 days. In contrast, the parvalbumin concentration in hindlimb muscle of dystrophic mice decreased steadily with age to about 22%% of normal animals at 120 days. The parvalbumin content was also reduced in dystrophic brain.

Characterization of parvalbumin cDNA clones and gene expression in normal and dystrophic mice of strain 129 ReJ

“…The increase in calpain activity in skeletal muscle of dystrophic animals may be due, in part, to enhanced influx of c a 2 + into muscle, resulting in activation of calpain and elevation in the rate of protein degradation (Leberer et al 1988;Fong et al 1990). Reduced levels of PV have been found in skeletal muscle of aging mice with hereditary muscular dystrophy (Jasch and Moase 1985;Klug et al 1985;Greaves et al 1991). Moreover, there appears to be a correlation among high intracellular c a 2 + levels, increased calpain activity, and low PV content in skeletal muscle of dystrophic mice compared with normal littermate control animals.…”

“…The anti-PV antibody was supplied by D.S. Greaves of this laboratory; it has been characterized previously (Greaves et al 1991). The biotinylated goat anti-rabbit antibody, streptavidin-alkaline phosphatase, NBT, BCIP, restriction endonucleases, T, DNA ligase, guanidine isothiocyanate, and phenol were purchased from GIBCOJBRL (Burlington, Ont.).…”

“…The cDNA library was plated on host Escherichia coli Y1090 and approximately 4 X 10' plaques were screened with a 1:100 dilution of PV antiserum prepared in this laboratory (Greaves et al 1991) according to an established procedure (Young and Davis 1983). Phage DNA was isolated from 13 putative positive tertiary clones by the plate lysate method (Sambrook et al 1989).…”

“…In a previous study in this laboratory, polyclonal antibodies for mouse PV were isolated and used to study the concentration and tissue distribution of the protein in normal and dystrophic mice of strain 129 ReJ of various ages (Greaves et al 1991). In this paper we describe a study which characterizes two PVAs and the expression of the Pva gene in normal and dystrophic mice of strain 129 ReJ.…”

Immunodetection of thiol proteinase levels in various populations of Artemia cysts and during development

“…One month later a second set of injections at the same dose was performed. Blood was sampled from the ear vein weekly and the serum prepared from these samples was tested for the presence of antibody using a modified Ouchterlony system (Ouchterlony 1949;Greaves et al 1991). While the preimmune serum showed no antibody reaction with purified proteinase, serum from immunized rabbits 75 days after the initial injection gave a strong precipitin line on agar plates.…”