Masahiro Iwaki

Characterization of the proteases in the crude mite extract.

Characterization of the proteases was performed in the crude mite extract fractionated by Sephacryl S-200 gel filtration. Three peaks of protease activities were detected in the fractions. From the results of substrate specificity and susceptibility to the inhibitors, PK.1 protease (about 60 kD) is suggested to be a trypsin-like protease of mites. From the results of susceptibility to various agents, PK.2 (about 30 kD) and PK.3 (about 20 kD) proteases may be cysteine proteases, e.g., papain and cathepsin B. PK.3 protease existed in the precipitate of 60% ammonium sulfate fractionation. The data in the present study suggest the possibility that Dermatophagoides farinae I allergen might be a cysteine protease probably derived from the gastrointestinal tract of the house dust mite.

Isolation of Cysteine Protease in the Crude Mite Extract, Dermatophagoides farinae

In order to study the relationship between cysteine protease and Der f I, which is one of the major allergens in the mite, Dermatophagoides farinae, isolation of cysteine protease was attempted using various column chromatographies. Both the potent cysteine protease activity and the allergenic activity were detected in the same fractions by anion exchange chromatography on a DEAE-Sephacel, gel chromatographies and chelating Sepharose 6B chromatography. In the double immunodiffusion test, the finally isolated fraction and rabbit anti-Der f I sera reacted to give a single precipitation line which fused completely with the precipitation line formed by Der f I and anti-Der f I sera. Sequence analysis for the first 10 N-terminal amino acids from cysteine protease and Der f I were identical. These results strongly suggest that cysteine protease of mites may be Der f I allergen and that measuring cysteine protease activity may possibly become a beneficial method for detecting Der f I allergens.

Increased monoamine turnover in the subfornical organ area following body fluid depletion

To clarify whether monoaminergic inputs to the subfornical organ (SFO) area participate in fluid regulatory systems, we examined the effects of body fluid depletion on monoamine turnover in the region of the SFO using microdialysis techniques in rats. An iso-osmotic reduction of fluid volume following subcutaneous treatment with polyethylene glycol (PEG) significantly increased dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the SFO area. 5-hydroxytryptamine (5-HT) in the SFO area could be detected after the PEG treatment, while 5-HT was undetectable before the treatment. The data imply that both dopaminergic and serotonergic systems in the SFO area may be involved in controlling body fluid balance.

The role of plasma proteases in inflammation. The study by using FUT-175, a selective serine-protease inhibitor.

Out of numerous factors relating to the pathogenesis and development of inflammation, the roles of plasma serine-proteases including, kallikrein, Clr, Cls, trypsin and plasmin are not thoroughly elucidated. To investigate the role of an enzyme, it is effective to use the selective inhibitor on it.In the present studies, authors used two pharmacological tools, i.e., FUT-175 (nafamostat mesilate), a selective inhibitor on serine-proteases and indomethacin, a selective inhibitor on cyclooxygenase and investigated the participation of serine-proteases in inflammation.At first, the pharmacological profile of FUT-175 was investigated in comparison with indomethacin. It was made clear that FUT-175 had selective inhibitory activities only on serine-proteases and no indomethacin-like activities.In the second, FUT-175 had significant suppressive effects on the representative models of inflammation including carrageenin-induced pleurisy and foot edema and adjuvant-arthritis in rats.These results suggest that plasma serine-proteases, especially complement system and kallikrein-kinin system play an important part in the processes of either acute or chronic inflammatory reactions.