While investigating the effect of marine products on cell growth, we found that visceral extracts of Chub mackerel, an ocean fish, had a powerful and dose-dependent apoptosis-inducing effect on a variety of mammalian tumor cells. This activity was strikingly dependent on infection of the C. mackerel with the larval nematode, Anisakis simplex. After purification of the protein responsible for the apoptosis-inducing activity, we cloned the corresponding gene and found it to be a flavoprotein. This protein, termed apoptosis-inducing protein (AIP), was also found to possess an endoplasmic reticulum retention signal (C-terminal KDEL sequence) and H2O2-producing activity, indicating that we had isolated a novel reticuloplasimin with potent apoptosis-inducing activity. AIP was induced in fish only after infection with larval nematode and was localized to capsules that formed around larvae to prevent their migration to host tissues. Our results suggest that AIP may function to impede nematode infection.

FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-jB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-jB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.

It was demonstrated that the IgG with anti-0:-galactosyl specificity (anti-0:-Gal IgG) occurs in normal human serum. Subsequently, we isolated the IgG with anti-,8-galactosyl specificity (anti-,6'-Gal IgG) from normal human serum by affinity chromatography using lactoseSepharose as affinity adsorbent (15). In this study, the carbohydrate specificity of anti-,6'-Gal IgG was further examined. Purified human anti-,6-Gal IgG showed distinct specificity with the structure of Gal,6'1-4Glc in oligosaccharide inhibition experiments. However, it did not react with Gal,6'1-4Glc,6'l-1'Cer nor NeuAca2-3Gal)81-4Glc,8 1-1'Cer(GM3) in thin-layer chromatography (TLC)*, suggesting that besides Galfll-4Glc, more inner structure may be important. The ELISA* for anti-,6'-Gal IgG and anti-0:-Gal IgG using synthetic lactose or melibiose-BSA* conjugates was developed, and the levels of these antibodies in normal and patient sera were measured. Their concentrations were found to vary in the range of 10-280 ,ug/ml and 10-220 ,ug/ml, respectively, in normal subjects. Distinctly higher levels over these ranges were found in two terminal patients with malignant tumor. Anti-,6'-Gal antibodies were also found in the sera from various non-primates. Purified calf anti-ii'-Gal antibodies showed the same specificity as that of humans.

The lgG class antibodies with anti-,6-galactosyl specificity were isolated from normal human serum by affinity chromatography, using lactose-Sepharose as immunoadsorbent. The sugar specificity for binding to the isolated antibodies was examined with neoglycoproteins and was found to be different from that of the known anti-galactosyl antibody (anti-ac-Gal IgG). Since expression of the terminal galactosyl groups on cell surface is closely associated with metastatic ability of tumor cell, the anti-,6'-galactosyl antibody (anti-,6'-Gal IgG) might have potential in various fields of cancer research.

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