Previously, we identified two pro‐phenol oxidase‐activating factors, named PPAF‐I and PPAF‐II, directly involved in the activation of the purified pro‐phenol oxidase (pro‐PO) from the hemolymph of the coleopteran, Holotrichia diomphalia larvae [Lee, S. Y., Kwon, T. H., Hyun, J. H., Choi, J. S., Kawabata, S. I., Iwanga, S, & Lee, B. L. (1998) Eur. J. Biochem. 254, 90−97]. Here, we report molecular cloning of cDNA for PPAF‐I. Based on the sequence of the cloned cDNA, the PPAF‐I gene appears to encode a member of serine protease zymogen consisting of 365 amino acid residues with a molecular mass of 40 193 Da. The 109 amino acid residues preceding the amino‐terminus Ile residue of the mature protein seem to constitute a prepro‐sequence. The mature protein is a serine protease composed of 256 amino acids with a calculated molecular mass of 28 009 Da. The overall structure is highly similar to that of Drosophila easter serine protease (42.9 % identity), an essential serine protease zymogen for pattern formation in normal embryonic development. The locations of disulfide linkages in the pro‐segment of PPAF‐I were similar to those of Tachypleus proclotting enzyme and the mammalian neutrophil‐derived defensin. Furthermore, [3H]diisopropylphosphate (iPr2P)‐labeled PPAF‐I was specifically produced from the crude preparation of PPAF‐I zymogen by incubation with lipopolysaccharide or 1,3‐β‐glucan, whereas [3H]iPr2P‐labeled PPAF‐I was not produced under the same conditions in the absence of these microbial polysaccharides. These results indicate that the pro‐PO‐activation system in H. diomphalia larvae may proceed with the activation of PPAF‐I zymogen by microbial polysaccharides.

We previously reported that a synthetic anti-bacterial peptide, KLKLLLLLKLK-NH 2 (L5), showed significant chemotherapeutic activity in methicillin-resistant Staphylococcus aureus-infected mice, and its ability to activate human neutrophils was related to its chemotherapeutic activity. In this study, we found that activation of neutrophils by L5 was inhibited by pertussis toxin, suggesting that GTP-binding protein (G-protein) participates in this process. We isolated an L5-binding protein, which turned out to be human calreticulin, with a molecular mass of 60 kDa from neutrophil membranes. From experiments using an anti-calreticulin antibody, we proposed that calreticulin is partly localized on the surface of neutrophils, and L5-bound calreticulin transmits a signal into cells via G-protein to activate neutrophils to generate superoxide anion.

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A cDNA for mouse prolyl endopeptidase (PEP) was cloned and its nucleotide sequence determined. The overall amino acid sequence identity between mouse and other mammalian PEPs was about 96%. A specific inhibitor of PEP, N-benzyloxycarbonyl-thioprolyl-thioprolinal- dimethylacetal (ZTTA), inhibited DNA synthesis by Swiss 3T3 cells. Mouse PEP was shown to be localized partly in restricted nuclear regions. These results suggest that PEP participates in mammalian DNA synthesis.

A prolyl endopeptidase that hydrolyses Suc-Gly-Pro-MCA (Suc, succinyl; MCA, methyl-coumaryl-7-amide) was purified to near homogeneity from NIH-Sape-4 cells derived from the flesh fly (Sarcophaga peregrina). The molecular mass of the purified enzyme was 84 kDa, and its activity was inhibited almost completely by 1 mM diisopropyl fluorophosphate (DFP). Immunoblotting and DFP-labeling experiments revealed that the leg imaginal discs of Sarcophaga contained this enzyme as a major serine proteinase. This prolyl endopeptidase is suggested to be involved in the differentiation of imaginal discs, because 2 mM DFP and 0.1 mM N-benzyloxycarbonyl-thioprolyl-thioprolynal-dimethylaceta l (ZTTA), a specific inhibitor for prolyl endopeptidase, inhibited differentiation of the imaginal discs from the eversion to the elongation stage.

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We have been developing a passive motion exercise device for ankle dorsiflexion/plantarflexion that can be applied to patients with complicated ankle joint deformity. In conventional ankle motion exercise devices, a footplate remains parallel to the axis of ankle dorsiflexion/plantarflexion. This may cause pain by forcing the ankle position to fit in the device and may also cause uneven contact of the sole to the footplate during the exercise. Therefore, we proposed to make a device which enables passive dorsiflexion/plantarflexion movements of ankle with adjustable inversion/eversion. In this study, the effect of passive joint of inversion/eversion is examined using pressure distribution on the footplate.

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