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.

Keywords: S I protein/hnRNP/intermediate filament/microtubule/micro filament/vimentin ABSTRACT. SI proteins A-D are hnRNPproteins which were originally isolated from cell nuclei of various tissues, by selective extraction at pH 4.9 from the supernatants of nuclei mildly treated with DNase I or RNase A. In the present study, a hybridoma was isolated which produced a monoclonal antibody that reacted specifically with SI proteins C2 and D2. Whenthe antibody was used in indirect immunofluorescence staining of cultured cells, it stained, in addition to the nuclei, the cytoskeleton-like fibrous structures in the cytoplasm. Wedemonstrate that the cytoskeletal filaments are vimentin intermediate filaments. This is the first report on the hnRNP protein-association with cytoskeleton, and will help to clarify cytoplasmic mRNA localization as well as cytoplasmic distribution of hnRNPproteins.SI proteins constitute a group of nuclear proteins (8) present at sites sensitive to RNaseas well as DNase,and can be extracted at pH4.9 from the reaction supernatant of nuclei mildly treated with either enzyme (6, 9 (44.5); Dl (41.5), and D2 (39.5). They were found in all rat tissues examined, and in cells from various species including mammals, bird (chicken), fish (carp), amphibian (frog) (8 and unpublished), and echinoderm (starfish) (2). A polyclonal antibody was raised in a rabbit against rat liver SI protein B reacted with the proteins B2, Cl and Dl (7,14). Withthis antiserum, the SI proteins were detected in the extranucleolar nucleoplasm, localizing in the euchromatin bordering the heterochromatic areas (14), where most of the RNApolymerase II transcription takes place (3). In a molecular cloning from a rat liver CDNA expression library using the polyclonal antibody, a clone was isolated that encoded a new class of RNA-binding protein, Sl-1, of 852 amino acid residues (10). Although the Sl-1 protein did not coincide with the SI proteins, the Sl-1 and B2, Cl, and Dl were similar in that they possess the same or similar epitope structures as well as the RNA-bindingactivities. A hybridoma that produces a monoclonal antibody specific for the SI proteins C2 and D2 was isolated.With the mono-and polyclonal antibodies, all the SI proteins (C2 and D2, and B2, Cl and Dl) were demonstrated to occur in the nucleus as a soluble form as well as a chromatin-bound form. By sedimentation and immunoprecipitation analyses of the soluble fraction from rat liver nuclei, the antibody-reacting SI proteins were demonstrated to be in association with RNA-protein complexes of heterogeneous sizes, with S-values up to 200 S or more. Four regions of C2 and D2 were microsequenced, and each region gave the identical amino acid sequence between C2 and D2, indicating that they are homologous proteins (probably resulting from alternative splicing). Moreover, these peptide sequences were identical to those contained by the human type A/B hnRNP protein (12). Thus, the SI proteins C2 and D2 are identical or closely related to the type A/B hnRNPprotein that has tw...

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Using affinity chromatography on lactosyl-Sepharose, a beta-galactoside-binding protein of 38 kDa was detected in mouse L1210 lymphocytic leukemia cells. Immunoblotting analysis revealed that it is distinct from any known larger molecular weight galectin. The partial amino acid sequences of the 38 kDa protein indicated that it is a novel member of the galectin family. This 38 kDa lectin is expressed in lymphocytic cell lines but not macrophage-like cell lines.

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