A novel method to observe the autolytic activation of a mammalian cytoplasmic calcium protease, mu-calpain, was developed using a set of antipeptidic antibodies capable of distinguishing between the pre- and post-autolysis forms of the enzyme. Antibodies raised against synthetic peptides designed to match the N-terminal sequences of the pre- and post-autolysis forms of the mu-calpain large subunit reacted specifically with the corresponding form of calpain and not with the other. The antibodies were specific and sensitive enough to detect the antigens in crude cell lysates. The relevance of the immunochemical detection of calpain activation was confirmed by the observation that proteolysis of a substrate protein by purified mu-calpain paralleled autolysis at various pCa as probed by these antibodies and that autolysis preceded substrate proteolysis. We also observed calcium-dependent autolysis of calpain accompanying subsequent proteolysis of substrate in intact cells using the antibodies. The method will provide a novel approach to assess the physiological targets of the enzyme by determining the local intracellular sites of calpain activation.

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34+ cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-γ production. However, NK cell cytotoxicity and IFN-γ production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-γ Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-γ production plays an important role in Fas-mediated cytotoxicity.

Achacin is an antibacterial glycoprotein puri¢ed from the mucus of the giant snail, Achatina fulica Fe ¤russac, as a humoral defense factor. We showed that achacin has L-amino acid oxidase activity and can generate cytotoxic H 2 O 2 ; however, the concentration of H 2 O 2 was not su⁄cient to kill bacteria. The antibacterial activity of achacin was inhibited by various H 2 O 2 scavengers. Immunochemical analysis revealed that achacin was preferentially bound to growth-phase bacteria, accounting for the important role in growth-phase-dependent antibacterial activity of achacin. Achacin may act as an important defense molecule against invading bacteria.

cDNAs encoding aquaporins PIP1;1, PIP2;1, and TIP1;1 were isolated from Mimosa pudica (Mp) cDNA library. MpPIP1;1 exhibited no water channel activity; however, it facilitated the water channel activity of MpPIP2;1 in a phosphorylation-dependent manner. Mutagenesis analysis revealed that Ser-131 of MpPIP1;1 was phosphorylated by PKA and that cooperative regulation of the water channel activity of MpPIP2;1 was regulated by phosphorylation of Ser-131 of MpPIP1;1. Immunoprecipitation analysis revealed that MpPIP1;1 binds directly to MpPIP2;1 in a phosphorylation-independent manner, suggesting that phosphorylation of Ser-131 of MpPIP1;1 is involved in regulation of the structure of the channel complex with MpMIP2;1 and thereby affects water channel activity.

The changes in viscoelasticity of natural actomyosin and myosin during thermal gelation were investigated by dynamic viscoelasticity measurements. Thermal gelation of natural actomyosin could be divided into four characteristic temperature ranges. The storage modulus increased considerably in the 32-43"C range, decreased sharply in the 43-52°C range, and then increased again in the .52-80°C range. For the thermal gelation of myosin, the storage modulus increased in two steps at two temperature ranges, i.e., 30-41"C and 51-80°C. An increase in the loss modulus was observed at an early stage of each of the two ranges.

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