Intracellular levels of calmodulin are increased in transformed cells

By using Hoechst 33342, rabbit anti calmodulin antibody ,FITC-1abeled goat anti rabbit IgG and SR101(sulfo rhodamine 101) simultaneously to stain individual normal and transformed cells, t-he micrbspectrophotometric analysis demonstrated that 3 markers' which represented the nucleus, calrnodulin and total protein respectively, could be recognized in individualj cells without interference. The phase of the cell cycle was determined by DNA content (Hoechst 33342). We found that in transformed cells (NIH3T3) tsRSV-LA90,cultured at 33 C and transformed C3H10T1/ 2 cells), the ratio of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV - LA90 cells, cultured at 39C and C3H10T1/2 cells) in every cell cycle phase. This ratio increased obviously only from G1 to S phase in either normal or transformed cells. The results showed that calmodulin really increased during the transformation, and its increase was specific. In the meantime when cells proceeded from G1 to S, the intracellular calmodulin content also increased specifically.

Osteoinductivity assay of the variability of repeated extractions of bone morphogenetic proteins from bovine bone at different times

Objective: To observe the activity of repeated extracts of bone matrix and the production of purified bone morphogenetic proteins (BMPs). Methods: BMPs were extracted 1- 4 times from fresh bovine cortical bone by the modified Urist’s method, with each collected precipitate separated and lyophilized as partially purified BMPs. Another fresh bovine bone was extracted three times and the precipitates were mixed and lyophilized. Meanwhile,the alkaline phosphatase (ALP) activity was measured by an in vitro assay employing cultured C2C12 mouse myoblast cells through the osteoinductivity of bovine BMPs extracted four times at days 1, 4, 7, and 14, and the correlation between BMPs quantities and costing during extraction processes was analyzed.Results: The BMPs purified and the cost showed a positive correlation (r=(0.969)). To separate and lyophilize each collected precipitate as partially purified BMPs raised the cost, and mixed precipitates also cost much. ALP activities of the 1st and mixed extractions of BMPs were shown to be highly osteoinductive and keep a significantly high level (P<(0.05)-(0.01)) 4 days after culturing, compared with the 2nd, 3rd and 4th extractions, especially the control group. However, the more times the extraction was done, the less activity of BMPs was shown and more costing was. The x-ray and histological analysis also showed that the 1st extraction of BMPs induced more ossicles and new bone formation.Conclusions: The results indicated that BMPs enhanced the abilities of osteoinductivity in C2C12 culture in vitro. The first extraction of BMPs from bone is fitfull, the second extraction should be enough, while, the 3rd and 4th extractions are unnecessary for they cost more and waste more time, say nothing of mixed extractions.