Seventy-four peptide amides of 7-amino-4-methylcoumarine (Mec) of the type Boc-Xaa-Yaa-Arg-NH-Mec were newly synthesized and tested to find specific substrates for blood-clotting proteases and trypsin. The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively. Among these peptides, the followings were found to be most sensitive substrates for individual enzymes: Boc-Asp(OBz1)-Pro-Arg-NH-Mec (k,,, = 160 s-', K, = 11 pM, k,,,/K, = 15000000 M-' s-') for human a-thrombin, Z-< Glu-Gly-Arg-NH-Mec (k,,, = 19 s-', K, = 59 pM, k,,,/K, = 320000 M-' s-') for bovine factor Xa, BocGln-Gly-Arg-NH-Mec (k,,, = 5.8 s-', K, = 140 pM, k,,,/K, = 42000) for bovine factor XIIa, Boc-Asp(OBz1)-Ala-Arg-NH-Mec (k,,, = 9.2 s-', K,,, = 120 pM, k,,,/K, = 77000 M-' s -l ) for bovine activated protein C, and Boc-Gly-Phe-Arg-NH-Mec (k,,, = 29 s-', K, = 230 pM, kcat/Km = 130000 M-' s-') for bovine plasma kallikrein. Moreover, Boc-Glu(OBz1)-Ala-Arg-NH-Mec (k,,, = 46 s-', K, = 370 pM, kcat/Km = 120000 M-' s-'> was newly found as a good substrate for human factor XIa. Bovine trypsin effectively hydrolyzed peptide-NH-Mec substrates containing Ala and Pro at the P2 site. The most reactive substrate was Boc-Gln-Ala-Arg-NH-Mec (k,,, = 120 sK1, K, = 6.0 pM, k,,,/K, = 20000000 M-' s-').Peptide amides of 7-amino-4-methylcoumarine (Mec) originally developed for the sensitive assay for a-chymotrypsin [l] [7], and horseshoe crab clotting factors [12]. Most of these substrates were designed based on the information from the COOH-terminal sequences of the 'activation peptides', which are liberated during the conversions of plasma serine protease zymogens to their active enzymes. The utility of these fluorogenic peptide substrates has been established for the assay of trace amounts of enzymes because of their high sensitivities. Moreover, the specific substrates for a-thrombin, factor Xa, plasma kallikrein and urokinase so far developed by our group [7] are now commercially available.

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SummarySeasonal changes in the percentage of macropterous individuals in natural population of Microvelia douglasi in permanent habitats were investigated in Shimane, western part of Japan. Macropterous forms of 5th instar nymphs occurred most frequently from late June to early July. However, the seasonal appearance of macropterous adults differed between habitats and years. There were significant effects of density, temperature, photoperiod and food availability on the percentage of macropters under laboratory conditions. The percentage of macropterous forms generally increased with density, but the mode of the response was modified by temperature, photoperiod and food availability. When insects were reared under long day photperiod (16L8D) with abundant food (0.5–1.0 g/container/day), the percentage of macroptery varied most among the different density treatments at the intermediate temperature (24°C) and the difference was reduced at higher temperature (28° and 32°C). Effects of temperature were small under the short day photoperiod (12L12D). Under 12L12D photoperiod sensitivity of density responses were higher at 32°C and lower at 24°C than those under 15L8D photoperiod. When the insects were supplied with food every other day, the density response became obscure. Ecological significance of environmental responses in the wing morph determination was discussed in relation to the heterogeneous environment of water surface.

Phenolic melanin precursors can be utilized for the development of anti-melanoma agents. The sulphur homologue of tyrosine, 4-S-cysteinylphenol (CP) and its decarboxylation product, 4-S-cysteaminylphenol (CAP) were shown to be substrates of melanoma tyrosinase, forming melanin-like pigment. Both, but in particular the 4-S-CAP, exhibited a significant in vivo depigmenting effect. Here, we report on the in vivo anti-melanoma effect of 4-S-CP, and 4-S-CAP and its N-acetyl derivative. In a previous in vitro study, it was shown that 4-S-CP and 4-S-CAP required a catalytic amount of dopa for optimal mammalian tyrosinase activity. To enhance the potential anti-melanoma effect of these two compounds. L-dopa and a decarboxylase inhibitor (carbidopa) were given concomitantly. We found that 4-S-CAP showed a significant growth inhibition of B16 melanoma inoculated s.c. into C57BL/6J mice. The anti-melanoma effect was increased significantly by combination of L-dopa and carbidopa. In addition, we tested the in vivo anti-melanoma effect of an N-acetyl derivative of 4-S-CAP (N-Ac-4-S-CAP). We found that N-Ac-4-S-CAP was the tyrosinase substrate and potent inhibitor of melanoma growth. N-acetyl 4-S-CAP showed a marked increase in water solubility. We suggest that N-Ac-4-S-CAP may prove to be a valuable model for the development of anti-melanoma agent using a metabolic pathway of melanin synthesis.

A unique biological property of the melanocyte, melanin synthesis may permit a rational approach to design agents for better management of malignant melanoma. This in vivo and in vitro study examined the selective melanocytotoxicity and antimelanoma effects of phenolic compounds, cysteinylphenol (CP), cysteaminylphenol (CAP), and related compounds, and found (1) that both 4-S-CP and 4-S-CAP are melanin precursors, (2) that 4-S-CAP possesses a marked depigmenting potency with selective destruction of melanocytes in black follicles, and (3) a significant inhibition in the protein synthesis and tumor growth of B16 melanoma. Importantly, a whole body autoradiography indicated that these phenolic melanin precursors are selectively incorporated into melanoma tissues after i.p. administration.