Purification and characterization of phosphoenolpyruvate phosphomutase from Pseudomonas gladioli B-1.

Phosphoenolpyruvate phosphomutase (PEPPM) catalyzes C-P bond formation by intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate (PnPy). We purified PEPPM from a gram-negative bacterium, Pseudomonas gladioli B-1 isolated as a C-P compound producer. The equilibrium of this reaction favors the formation of the phosphate ester by cleaving the C-P bond of PnPy, but the C-P bond-forming reaction is physiologically significant. The C-P bond-forming activity of PEPPM was confirmed with a purified protein. The molecular mass of the native enzyme was estimated to be 263 and 220 kDa by gel filtration and polyacrylamide gel electrophoresis, respectively. A subunit molecular mass of 61 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the native protein was a tetramer. The optimum pH and temperature were 7.5 to 8.0 and 40 degrees C, respectively. The Km value for PnPy was 19 +/- 3.5 microM, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 microM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the Km values were 3.5 +/- 1.4 microM for Mg2+, 16 +/- 5 nM for Mn2+, 3.0 +/- 1.5 microM for Zn2+, and 1.2 +/- 0.2 microM for Co2+.

Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor.

Cartilage calcification at specific sites is a key event that leads to skeletal development and growth. To obtain insights into the control of cartilage calcification, we examined whether cells distributed in permanent cartilage regions might have the ability to express the calcification-related phenotype in a permissive environment. Chondrocytes were isolated from the permanent and growth plate cartilages of 4-week-old rabbit ribs. They were seeded as a pelleted mass in a centrifuge tube and cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum. These cells proliferated for several generations, and then synthesized large amounts of proteoglycans, yielding a cartilage-like tissue in 16 days. Cultures from the permanent and growth plate cartilages showed similar time courses for increases in DNA synthesis and proteoglycan production that reached similar maximal levels. Thereafter, they initiated the syntheses of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor and induced matrix calcification without additional phosphate. The increases in alkaline phosphatase, 1,25-dihydroxycholecalciferol receptor, and calcium contents in cultures from the permanent cartilage were consistently delayed for 4-7 days relative to the growth plate-derived cells, but caught up by Day 28. The maximal levels of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor in the cultures from the permanent cartilage were 40- to 100-fold higher than that of the in vivo permanent cartilage. These results provide evidence that permanent cartilage cells in postnatal young rabbit ribs have the capacity to express alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor and induce calcification in a permissive environment, although they never express these calcification-related phenotypes in vivo.

Development of alumina ceramic bipolar hip prosthesis and clinical application.

The concept of the bipolar type prosthesis is that the articular motion is observed primarily between the HDP bearing insert and the inner head, thereby avoiding the greater frictional shear force between the prosthetic head and acetabulum found with the traditional one piece endoprosthesis. A newly developed ceramic bipolar hip prosthesis utilizes an outer head composed of bio-inert fine alumina ceramic rather than a metal head. The mechanical strength of this prototype bipolar hip prosthesis was tested with static load and impact force, and the dynamic fatigue strength was also tested with repeated loads. Results were judged to show that the device has strength adequate for clinical use. The hip prostheses has been clinically applied in replacement of the femoral head of a neck fracture, femoral head necrosis and osteoarthritic hip. Finally, using a mathematical model, the so-called self-centering effect due to center offset between outer and inner heads is discussed.

Original surface replacement total hip prosthesis with alumina ceramic prosthetic head without cement.

We have developed a surface replacement total hip prosthesis of H.D.P. socket to ceramic head type having a polycrystal alumina ceramic prosthetic head and without bone cement for fixation. The strength test was done on this hip prosthesis, and comparative studies were made between the metal prosthetic head and the ceramic prosthetic head, using calculation models prepared in consideration of friction and the prosthetic head's inside diameter. Besides, the cadaver femur was replaced by the prosthetic head, and the impact test and the fatigue test by means of repeated loading were done. As we could consequently confirm that it could be fully used for clinical practice, we have manufactured various instruments to be used for surgery, and we are now actually practicing it.