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ABSTRACT: Alteration of skeletal muscle protein breakdown is a hallmark of a set of pathologies, including sepsis, with negative consequences for recovery. The aim of the present study was to search for muscle markers associated with protein loss, which could help in predicting and understanding pathological wasting. With the use of differential display reverse transcription-PCR, we screened differentially expressed genes in muscle from septic rats in a long-lasting catabolic state. One clone was isolated, confirmed as being overexpressed in septic skeletal muscle and identified as encoding the lysosomal cysteine endopeptidase cathepsin L. Northern- and Western-blot analysis of cathepsin L in gastrocnemius or tibialis anterior muscles of septic rats confirmed an elevation (up to 3-fold) of both mRNA and protein levels as early as 2 days post-infection, and a further increase 6 days post-infection (up to 13-fold). At the same time, the increase in mRNAs encoding other lysosomal endopeptidases or components of the ubiquitin-proteasome pathway did not exceed 4-fold. Cathepsin L mRNA was also increased in tibialis anterior muscle of rats treated with the glucocorticoid analogue, dexamethasone, or rats bearing the Yoshida Sarcoma. The increase in cathepsin L mRNA was reduced by 40% when the tumour-bearing animals were treated with pentoxifylline, an inhibitor of tumour necrosis factor-alpha production. In conclusion, these results demonstrate a positive and direct correlation between cathepsin L mRNA and protein level and the intensity of proteolysis, and identify cathepsin L as an appropriate early marker of muscle wasting. Cathepsin L presumably participates in the pathological response leading to muscle loss, with glucocorticoids and tumour necrosis factor-alpha potentially being involved in the up-regulation of cathepsin L.
Biochemical Journal 12/2001; 360(Pt 1):143-50. · 4.90 Impact Factor
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ABSTRACT: Nine days of hindlimb suspension resulted in atrophy (55%) and loss of protein (53%) in rat soleus muscle due to a marked elevation in protein breakdown (66%, P < 0.005). To define which proteolytic system(s) contributed to this increase, soleus muscles from unweighted rats were incubated in the presence of proteolytic inhibitors. An increase in lysosomal and Ca 2+-activated proteolysis (254%, P < 0.05) occurred in the atrophying incubated muscles. In agreement with the measurements in vitro, cathepsin B, cathepsins B + L and m-calpain enzyme activities increased by 111%, 92% and 180% (P < 0.005) respectively in the atrophying muscles. Enhanced mRNA levels for these proteinases (P < 0.05 to P < 0.001) paralleled the increased enzyme activities, suggesting a transcriptional regulation of these enzymes. However, the lysosomal and Ca 2+-dependent proteolytic pathways accounted for a minor part of total proteolysis in both control (9%) and unweighted rats (18%). Furthermore the inhibition of these pathways failed to suppress increased protein breakdown in unweighted muscle. Thus a non-lysosomal Ca 2+-independent proteolytic process essentially accounted for the increased proteolysis and subsequent muscle wasting. Increased mRNA levels for ubiquitin, the 14 kDa ubiquitin-conjugating enzyme E2 (involved in the ubiquitylation of protein substrates) and the C2 and C9 subunits of the 20 S proteasome (i.e. the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates) were observed in the atrophying muscles (P < 0.02 to P < 0.001). Analysis of C9 mRNA in polyribosomes showed equal distribution into both translationally active and inactive mRNA pools, in either unweighted or control rats. These results suggest that increased ATP-ubiquitin-dependent proteolysis is most probably responsible for muscle wasting in the unweighted soleus muscle.
Biochemical Journal 05/1996; 316 ( Pt 1):65-72. · 4.90 Impact Factor
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ABSTRACT: A rapid purification procedure is described for cathepsin B from bovine liver. After preparation of crude lysosomal extracts, the method only involves DEAE Zeta-Prep-Disk chromatography, gel filtration, and fast protein liquid chromatography on Mono-S column. Two active peaks (P1 and P2) of cathepsin B were distinguished. Both presented uncleaved (relative mass (Mr) 30,000) and cleaved (Mr 25,000 + Mr 5000) chains, but different isoforms as revealed by isoelectrofocusing. These two different populations of cathepsin B isoforms nevertheless exhibited similar enzymatic properties. Km and kcat were 114 microM and 52 s-1, and 125 microM and 75 s-1, for hydrolysis of Z-Arg-Arg-NMec by P1 and P2, respectively. Both were rapidly inhibited by low concentrations of E-64 or leupeptin, but were unaffected by cathepsin-L-specific inhibitor Z-Phe-Phe-CHN2.
Biochemistry and Cell Biology 05/1990; 68(4):822-6. · 2.67 Impact Factor
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ABSTRACT: We postulated that Fibroblast Growth Factor (FGF) involved in fetal or regenerative morphogenesis of skeletal muscle originated from this tissue. Using a bovine retina cDNA probe encoding acidic FGF, we showed that growing muscles from bovine fetuses express this mRNA, but that this expression is reduced in neonate muscles. Cultures of proliferating satellite cells isolated from adult rat muscles expressed aFGF mRNA strongly but bFGF mRNA weakly; these mRNAs disappeared in cells differentiated into myotubes. 10(-7)M 12-O-tetradecanoyl phorbol -13-acetate (TPA) increased aFGF mRNA expression in both proliferating and differentiated satellite cells. Contrastingly, proliferating L6 myogenic cells only expressed aFGF mRNA significantly under TPA treatment. Therefore, the satellite cells did seem to be a possible source for FGF, especially aFGF, which might regulate the myogenic process.
Biochemical and Biophysical Research Communications 03/1990; 166(3):1205-12. · 2.48 Impact Factor
