We have previously reported that the cabbage butterf ly, Pieris rapae, contains a 98-kDa protein, named pierisin, that induces apoptosis in a variety of human cancer cell lines. In the present study, sequencing and cloning of a cDNA encoding pierisin was accomplished. PCR-direct sequencing showed that the gene encodes an 850-amino acid protein with a calculated molecular weight of 98,081. An intact clone at the amino acid level encompassing the entire coding region was obtained by recombination of two independent clones, and the molecular mass of its in vitro expressed protein was about 100 kDa on SDS͞PAGE, the same as that of purified native pierisin. The expressed protein induced apoptosis in human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, like the native protein, indicating functional activity. The deduced amino acid sequence of pierisin showed 32% homology with a 100-kDa mosquitocidal toxin from Bacillus sphaericus SSII-1. In addition, pierisin showed regional sequence similarities with ADP-ribosylating toxins, such as the A subunit of cholera toxin. A glutamic acid residue at the putative NAD-binding site, conserved in all ADPribosylating toxins, was also found in pierisin. Substitution of another amino acid for glutamic acid 165 resulted in a great decrease in cytotoxicity and induction of apoptosis. Moreover, inhibitors of ADP-ribosylating enzymes reduced pierisininduced apoptosis. These results suggest that the apoptosisinducing protein pierisin might possess ADP-ribosylation activity that leads to apoptosis of the cells.

Weyl semimetals are characterized by the presence of massless band dispersion in momentum space. When a Weyl semimetal meets magnetism, large anomalous transport properties emerge as a consequence of its topological nature. Here, using in−situ spin- and angle-resolved photoelectron spectroscopy combined with ab initio calculations, we visualize the spin-polarized Weyl cone and flat-band surface states of ferromagnetic Co2MnGa films with full remanent magnetization. We demonstrate that the anomalous Hall and Nernst conductivities systematically grow when the magnetization-induced massive Weyl cone at a Lifshitz quantum critical point approaches the Fermi energy, until a high anomalous Nernst thermopower of  ~6.2 μVK−1 is realized at room temperature. Given this topological quantum state and full remanent magnetization, Co2MnGa films are promising for realizing high efficiency heat flux and magnetic field sensing devices operable at room temperature and zero-field.

Cytotoxic activity in extracts of pupae and adults of various kinds of butterflies and moths was tested in vitro against the human gastric carcinoma cell line, TMK‐1, which was chosen as an example of human carcinoma cells. Among the species examined, cytotoxicity was limited to Pieris rapae, Pieris napi and Pieris brassicae. Activity was found down to a dilution of 1/104, while with the other butterflies and moths no activity was observed, even at 1/102. When the cytotoxicity of the three developmental stages, larvae, pupae and adults, of Pieris rapae was compared, the pupae showed the strongest activity, the IC50 against TMK‐1 cells being at the 1/106 dilution. For larvae and adults, the respective IC50 values were at the 1/105 and 5/105 dilutions. The active principle in the pupae of Pieris rapae was found to be heat‐labile and not extractable with organic solvents, but precipitated with ammonium sulfate and digested by proteases, suggesting that it is a protein. This cytotoxic factor was named pierisin.

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1-233 or 234 -850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N-and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing ''nicked'' pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N-and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity. W e recently found that a potent cytotoxic factor against human cancer cells is present in the body f luids of larvae and pupae of cabbage butterf ly, Pieris rapae (1). The factor responsible for this cytotoxicity has been identified as a 98-kDa protein, and we named it pierisin-1 (2). Subsequent studies showed that pierisin-1 exhibited potent cytotoxic effects against various types of human cancer cell lines and human umbilical vein endothelial cells, with IC 50 values ranging from 0.043 ng/ml to 150 ng/ml, and induced typical apoptosis of most cell lines up to 48 h (3). The pierisin-1 gene has been cloned from P. rapae larval mRNA. Pierisin-1 shows regional sequence similarities with ADP-ribosylating toxins such as the A-subunit of cholera toxin. Disruption of a possible NADbinding site by site-directed mutagenesis results in the loss of its cytotoxic activity (4). The mRNA of pierisin-1 is highly expressed in fifth-instar larvae, and the concentration of pierisin-1 reached highest before pupation (4). From these observations, it w...