Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-twohybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcomaderived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/ CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGFenhancing effect on cell adhesion to FN was abolished by a blocking antibody against a5b1 integrin (a5b1), but not by one against anti-avb3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that a5b1 is involved in this adhesion.
Natto is a traditional Japanese fermented food made by fermenting boiled soy beans with Bacillus natto. Its contents of inhibitors against the angiotensin converting enzyme (ACE, EC3.4.15.1) were investigated. Relatively strong inhibitory activity (IC50: 0.4 mg/ml, 11.8 inhibition units/g natto) was detected in natto extracts and the inhibitory activity observed in the viscous fraction was more potent than in the bean extract. Two groups of inhibitors in the viscous material, high and low molecular weight inhibitors, were resolved by dialysis test. The inhibitor of high molecular weight was a protein with low IC50 value (0.12 mg/ml). The two types of low molecular weight inhibitors were detected in ethanol extracts (IC50: 0.53 mg/ml and 0.95 mg/ml) and they were found to be stable over a wide range of pH and temperature up to 100 degrees C. They were different in the mode of ACE inhibition. One is competitive, and the other noncompetitive against the hydrolysis of Bz-Gly-His-Leu by ACE.
Brevibacillus expression system is an effective bacterial expression system for secretory proteins. The host bacterium, Brevibacillus choshinensis, a gram-positive bacterium, has strong capacity to secrete a large amount of proteins (approximately 30 g/L), which mostly consist of cell wall protein. A host-vector system that utilizes such high expression capacity has been constructed for the production of secretory proteins and tested for various heterologous proteins, including cytokines, enzymes, antigens, and adjuvants.
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