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To investigate the mechanism of bone formation by EP4 activation, an osmotic pump was implanted subcutaneously into the backs of rats and an EP4 receptor agonist was administered at a dose of 100 ng/kg/min for up to 28 days. The histology of the femur (including bone marrow) and the serum and/or urinary bone metabolism parameters were examined on Day 0, 1, 3, 5, 7, 14, and Day 28. In EP4 receptor agonist treated-rats, increase of osteoclasts in the metaphysis was observed on Day 1 and the number of osteoblast showed an increase from Day 3. In addition, cancellous bone and endosteal bone formation were observed in the metaphysis and diaphysis from Day 5 and this peaked on Day 28. Serum alkaline phosphatase activity showed a transient decrease on Day 1, but thereafter showed an increase. The Gla-type osteocalcin level showed an increase from Day 1. Moreover, Gla/Glu osteocalcin ratio showed an increase on Day 5. The urinary excretion of deoxypyridinoline increased on Day 3, showed a transient decrease on Day 5, and increased once again from Day 7. These results indicate that EP4 receptor agonist-induced bone formation is related to an increase of osteoclasts at the initial stage and a subsequent increase of osteoblasts.

Bone remodeling, comprising resorption of existing bone and de novo bone formation, is required for the maintenance of a constant bone mass. Prostaglandin (PG)E 2 promotes both bone resorption and bone formation. By infusing PGE 2 to mice lacking each of four PGE receptor (EP) subtypes, we have identified EP4 as the receptor that mediates bone formation in response to this agent. Consistently, bone formation was induced in wild-type mice by infusion of an EP4-selective agonist and not agonists specific for other EP subtypes. In culture of bone marrow cells from wild-type mice, PGE 2 induced expression of core-binding factor α1 (Runx2/Cbfa1) and enhanced formation of mineralized nodules, both of which were absent in the culture of cells from EP4-deficient mice. Furthermore, administration of the EP4 agonist restored bone mass and strength normally lost in rats subjected to ovariectomy or immobilization. Histomorphometric analysis revealed that the EP4 agonist induced significant increases in the volume of cancellous bone, osteoid formation, and the number of osteoblasts in the affected bone of immobilized rats, indicating that activation of EP4 induces de novo bone formation. In addition, osteoclasts were found on the increased bone surface at a density comparable to that found in the bone of control animals. These results suggest that activation of EP4 induces bone remodeling in vivo and that EP4-selective drugs may be beneficial in humans with osteoporosis.

The constituent amino acids of plusbacins A1-A4 were determined to be two moles of L-trans-3-hydroxyproline and one mole each of D-threo-beta-hydroxyaspartic acid, L-threo-beta-hydroxyaspartic acid, D-allo-threonine, D-serine, D-alanine and L-arginine. In plusbacins B1-B4, one mole of L-trans-3-hydroxyproline is replaced by L-proline. The fatty acid residue of A1 and B1 was determined to be 3-hydroxy-tetradecanoic acid, for A2 and B2 to be 3-hydroxy-isopentadecanoic acid, for A3 and B3 to be 3-hydroxy-isohexadecanoic acid, and for A4 and B4 to be 3-hydroxy-hexadecanoic acid. A lactone linkage was suggested to reside between L-threo-beta-hydroxyaspartic acid and 3-hydroxy-fatty acid residues by degradation experiments. The amino acid sequences of plusbacins A2 and B2 were confirmed by Edman degradation of their deacylated products, and supported by mass spectrometric studies. From the above, structures of all components of plusbacins were concluded.

New antibiotics, plusbacins A1-A4 and B1-B4, were isolated from the culture broth of a strain of Pseudomonas sp. These antibiotics were isolated as a complex by column chromatographies on Diaion HP-20 and silica gel, and then separated by HPLC. They are amphoteric in nature. The hydrochlorides are obtained as colorless powders, soluble in methanol and alkaline water. From their physico-chemical properties, these antibiotics are presumed to be acyloctapeptides containing a lactone linkage, and their differences occur in amino acid and fatty acid residues. The antibiotics are active against Gram-positive bacteria in vitro and in vivo.

New antibiotics, resorcinomycins A and B, were isolated from the culture broth of a streptomycete strain identified as Streptoverticillium roseoverticillatum. The antibiotics are water-soluble amphoteric substances, positive to SAKAGUCHI'S reagent. The molecular formulas C14H20N4O5 and C13H18N4O5 for A and B were indicated by elemental analysis and secondary ion MS. The structures of these antibiotics were determined by 1H and 13C NMR spectrometry and some chemical evidences to be N-[(S)-alpha-guanidino-3,5-dihydroxy-4-isopropylphenylacetyl]glyci ne and N-[(S)-alpha-guanidino-3,5-dihydroxy-4-ethylphenylacetyl]-glycine, respectively.

Production of superoxide anion (O-2) induced by tetradecanoyl phorbol acetate (TPA) in intact guinea pig polymorphonuclear leukocytes (PMNL) was markedly inhibited by a seleno-organic compound, 2-phenyl-1, 2-benzisoselenazol-3 (2H)-one (Ebselen), with glutathione peroxidase-like activity. The compound almost completely inhibited O-2 production by a particulate fraction prepared from TPA-treated PMNL at a concentration as low as 250 nM.