We developed a novel technique to improve tendon-bone attachment by hybridizing calcium phosphate (CaP) with tendons using an alternate soaking process. We characterized the deposited CaP on or in tendons and determined the healing process of anterior cruciate ligament (ACL) grafts by implanting CaP-hybridized free tendons in bone tunnels intra-articularly. Tendons to be implanted were alternately soaked 10 times in a Ca-containing solution and a PO(4)-containing solution for 30 s each. Treated tendons had ash contents threefold that of untreated tendons. Low-crystallinity apatite was found on or in treated tendons. In animal experiments, the CaP-hybridized tendon exhibited osteoclasts at the tendon-bone interface at 5 days after operation. At 2 weeks after operation, there were more osteoclasts and osteoblasts around the tendon than at 5 days after operation. Directly bonded areas were partially found between the implanted tendon and newly formed bone. The formation of a cartilage layer was partially apparent at 3 weeks after operation. The newly formed bone was observed almost around the tendon. We conclude that CaP-hybridized tendons clearly enhance the healing process of ACL grafts at the tendon-bone interface and regenerate a direct insertion-like formation of tendons similar to a normal healthy ACL insertion within 3 weeks after operation.

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Inhibitory action of a variety of quinoid compounds on neuronal nitric oxide synthase (nNOS) activity was examined with a 20000g rat cerebellar supernatant preparation and purified nNOS. The inhibition of citrulline formation from l-arginine by quinones, which exhibit one-electron reduction potentials (E17) ranging between -240 and -100 mV, increased at a more positive one-electron reduction potential, suggesting that quinone appears to act as an electron acceptor for nNOS. Among the quinones tested, 9,10-phenanthraquinone (PQ), corresponding to an E17 value of -124 mV, exhibited the most potent inhibiton of citrulline formation (IC50 value = 10 microM). A kinetic study revealed that PQ is a competitive inhibitor with respect to NADPH, with a Ki value of 0.38 +/- 0.12 microM, and a noncompetitive inhibitor with respect to l-arginine, with a Ki value of 9.63 +/- 0.20 microM. Partial purification of the enzymes which are responsible for reducing PQ in 20000g supernatant of rat cerebellum by anion-exchange column chromatography indicated that one catalyst for PQ reduction was nNOS. Reductase activity of PQ by purified nNOS required CaCl2/calmodulin and was markedly suppressed by the flavoprotein inhibitor diphenyleneiodonium but not by l-nitroarginine which is a specific inhibitor for NO formation. nNOS effectively reduced the quinones as well as PQ causing a marked decrease in the production of NO from l-arginine, while 1, 4-benzoquinone, 9,10-anthraquinone, mitomycin C, and lapachol, which show negligible inhibitory action on nNOS activity, were poor substrates for the enzyme on reduction. These results indicate that PQ and other quinones used in the present study interact with the NADPH-cytochrome P450 reductase domain on nNOS and thus probably inhibit NO formation by shunting electrons away from the normal catalytic pathway. Therefore, our study suggests that quinones could possibly affect NO-dependent physiological and/or pathophysiological actions in vivo.

Expression of interferon-tau (IFNT), necessary for pregnancy establishment in ruminant ungulates, is regulated in a temporal and spatial manner. However, molecular mechanisms by which IFNT gene transcription is regulated in this manner have not been firmly established. In this study, DNA microarray/RT-PCR analysis between bovine trophoblast CT-1 and Mardin-Darby bovine kidney (MDBK) cells was initially performed, finding that transcription factors GATA2, GATA3, and GATA6 mRNAs were specific to CT-1 cells. These mRNAs were also found in Days 17, 20, and 22 (Day 0 = day of estrus) bovine conceptuses. In examining other bovine cell lines, ovary cumulus granulosa (oCG) and ear fibroblast (EF) cells, GATA2 and GATA3, but not GATA6, were found specific to the bovine trophoblast cells. In transient transfection analyses using the upstream region (-631 to +59 bp) of bovine IFNT gene (bIFNT, IFN-tau-c1), over-expression of GATA2/GATA3 did not affect the transcription of bIFNT-reporter construct in human choriocarcinoma JEG3 cells. Transfection of GATA2, GATA3, ETS2, and/or CDX2, however, was effective in the up-regulation of the bIFNT construct transfected into bovine oCG and EF cells. One Point mutation studies revealed that among six potential GATA binding sites located on the upstream region of the bIFNT gene, the one next to ETS2 site exhibited reduced luciferase activity. In CT-1 cells, endogenous bIFNT gene transcription was up-regulated by over-expression of GATA2 or GATA3, but down-regulated by siRNA specific to GATA2 mRNA. These data suggest that GATA2/3 is involved in trophoblast-specific regulation of bIFNT gene transcription.

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