Kazunori Nishi

Relaxin-3-Deficient Mice Showed Slight Alteration in Anxiety-Related Behavior

Relaxin-3 is a neuropeptide belonging to the relaxin/insulin superfamily. Studies using rodents have revealed that relaxin-3 is predominantly expressed in neurons in the nucleus incertus (NI) of the pons, the axons of which project to forebrain regions including the hypothalamus. There is evidence that relaxin-3 is involved in several functions, including food intake and stress responses. In the present study, we generated relaxin-3 gene knockout (KO) mice and examined them using a range of behavioral tests of sensory/motor functions and emotion-related behaviors. The results revealed that relaxin-3 KO mice exhibited normal growth and appearance, and were generally indistinguishable from wild genotype littermates. There was no difference in bodyweight among genotypes until at least 28 weeks after birth. In addition, there were no significant differences between wild-type and KO mice in locomotor activity, social interaction, hot plate test performance, fear conditioning, depression-like behavior, and Y-maze test performance. However, in the elevated plus maze test, KO mice exhibited a robust increase in the tendency to enter open arms, although they exhibited normal performance in a light/dark transition test and showed no difference from wild-type mice in the time spent in central area in the open field test. On the other hand, a significant increase in the acoustic startle response was observed in KO mice. These results indicate that relaxin-3 is slightly involved in the anxiety-related behavior.

Dnacin A1 and dnacin B1 are antitumor antibiotics that inhibit cdc25B phosphatase activity

The p80cdc25 protein is a protein phosphatase directly involved in p34cdc2 protein kinase activation by dephosphorylation. The cdc25B gene is one of three human cdc25 homologs which can complement the temperature-sensitive cdc25 mutation of Schizosaccharomyces pombe, and is expressed a high levels in human cell lines, particularly in some cancer cells. A fusion protein of glutathione-S-transferase (GST) and the catalytic domain of cdc25B protein was constructed and found to retain phosphatase activity in the manner of a p80cdc25 phosphatase by using a chromogenic substrate, p-nitrophenylphosphate. Two benzoquinoid antitumor compounds, dnacin A1 and dnacin B1, inhibited phosphatase activity in a non-competitive manner.

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.

Both the Conserved and the Unique Gene Structure of Stomach-Specific Calpains Reveal Processes of Calpain Gene Evolution

Abstract. The proteins nCL-2 and nCL-2′ are members of the Ca2+-dependent cysteine protease (calpain) superfamily, with stomach-specific expression. Like other typical calpains, nCL-2 has three distinct domains, a protease, a C2-like, and a 5EF-hand Ca2+-binding domain, as well as the N-terminal propeptide region. On the other hand, nCL-2′ lacks the C2-like and 5EF-hand domains but is otherwise identical to nCL-2, except for the three C-terminal residues. To examine the stomach-specific and presumed alternative expression mechanisms of nCL-2 and nCL-2′, we have cloned and characterized the mouse gene for nCL-2 and nCL-2′. The mouse nCL-2 gene contains at least 23 exons, spanning more than 50 kb, and possesses an exon specific for nCL-2′ in the middle. Therefore, nCL-2 and nCL-2′ are generated by alternative splicing of the same gene, Capn8. Capn8 shows the highly conserved gene organization of the other typical calpain large subunit genes, CAPN1, CAPN2, CAPN3, CAPN9, CAPN11, and Capn12, except for the unique exon between exon 9 and exon 10 of Capn8, which encodes the 3′ half of the nCL-2′ transcript. No such exon in the corresponding regions was found in CAPN1, CAPN2, CAPN3, CAPN9, or CAPN11. Gene and cDNA structures of a presumed human orthologue of mouse nCL-2, CAPN8, were determined, revealing that it overlaps human CAPN2, the gene for the m-calpain large subunit, in head-to-head orientation at 1q32-41. These features of Capn8 and CAPN8 illustrate a process of calpain gene evolution, i.e., the protease, C2-like, and 5EF-hand domains presumably functioned as independent genes, and the calpain superfamily has evolved by ordered fusions of these ancestral gene units, with subsequent amplifications.