Mu-calpain is involved in the regulation of TNF-alpha-induced matrix metalloproteinase-3 release in a rheumatoid synovial cell line

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.     

Enhanced alpha-amylase production in recombinant Bacillus brevis by fed-batch culture with amino acid control

Fed-batch culture with controlled L-amino acid composition was performed to improve production of a recombinant gene product in Bacillus brevis. The maximum recombinant protein (alpha-amylase) level and specific activity increased from 5.14 kU/mL and 0.77 kU/mg dry cell in conventional fed-batch culture to 12.01 kU/mL and 2.64 kU/mg dry cell, respectively, when L-amino acid concentration was controlled at 5 mM using an asparagine (Asn)- and isoleucine (Ile)-enriched nitrogen source. The L-amino acid concentration in the culture was monitored by an automatic biotech analyzer and controlled at 2-20 mM using a mixture of polypeptone and yeast extract. Although L-amino acid concentrations were controlled at low levels, the alpha-amylase activity increased only 1.3 times compared to an uncontrolled batch culture; accumulation of ammonium ion was not reduced. When L-amino acid was controlled at the high level, more cell mass and less recombinant gene product were produced than in those with low control level. To overcome ammonium ion inhibition, the specific amino acids Asn and Ile were substituted to improve the production of gene product. Addition of these amino acids to a flask culture led to an improvement in the enzyme production level and specific activity to 2.9 and 5.1 times, respectively, as high as that without them. Both the control of amino acids at low concentrations and the enrichment of Asn and Ile were effective for the improvement of recombinant protein production from recombinant B. brevis cells. (c) 1996 John Wiley & Sons, Inc.