Naomi Sano

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae.

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.

Mitochondrial DNA diversification, molecular phylogeny, and biogeography of the primitive rhacophorid genus Buergeria in East Asia.

In this study we sought to clarify the evolutionary relationships and biogeographic history of the bell-ring frog, Buergeria buergeri (family Rhacophoridae), and two congeneric species Buergeria japonica and Buergeria robusta, by analyzing three mitochondrial (mt) genes: 12S rRNA, Cytb, and ND5. Phylogenetic analyses based on gene data showed the mt clades corresponding to the Buergeria species and three major haplogroups within B. buergeri. Each haplogroup corresponded clearly to the area in which it was distributed, namely eastern Japan (excluding Hokkaido; Hg I), central Japan (Hg II), and western Japan (including the Shikoku and Kyushu regions; Hg III). The estimated divergence time suggested that the lineage splits of the Buergeria species occurred before the formation of the island of Taiwan and the Japan Archipelago (including the Ryukyu islands). The differentiation among the genealogical lineages of B. buergeri seems to have begun in the Late Miocene (approx. 7-5Mya), and the formation of their present distribution pattern might have been influenced by climatic changes and geographical events such as the formation of a wide peneplane and expansions of certain basins.

Hagfish C1q: Its unique binding property

Hagfish C1q (HaC1q) was identified and characterized as a pattern-recognition molecule (PRM) in the hagfish complement system. The serum from hagfish, Eptatretus burgeri, was applied to a GlcNAc-agarose column and eluted sequentially with GlcNAc and EDTA. Four (31, 27, 26, and 19 kDa) and one (26 kDa) proteins were detected as bound molecules in the GlcNAc- and the EDTA-eluates, respectively. Among these, the 26 kDa protein from the EDTA eluate was found to be a homologue of mammalian C1q through cDNA analysis. HaC1q had an ability to bind to various microbes in a Ca(2+)-dependent manner and its target ligands on the microbes were lipopolysaccharide, lipoteichoic acid, and peptidoglycan. The binding of HaC1q to GlcNAc-agarose was not inhibited by an excess amount of monosaccharide such as GlcNAc. While HaC1q bound to Sepharose 6B with a matrix of GlcNAc-agarose (polymer of agarobiose), it did not bind to Sepharose 4B that contained lower concentration of agarobiose than Sepharose 6B. Therefore, the target of HaC1q on GlcNAc-agarose was concluded to be agarobiose and high density of the target moiety seemed to be required for the stable binding. This finding was in accordance with the known behavior of other lectins involved in the complement system. We have concluded that HaC1q recognizes agarobiose-like structures present on the surface of microbes and acts as a pattern-recognition molecule in the process for elimination of invading microbes.

Artificial Production and Natural Breeding of the Endangered Frog Species Odorrana ishikawae, with Special Reference to Fauna Conservation in the Laboratory

Odorrana ishikawae is listed as a class IB endangered species in the IUCN Red List and is protected by law in both Okinawa and Kagoshima Prefectures, Japan. Here, in an effort to help effectively preserve the genetic diversity of this endangered species in the laboratory, we tested a farming technique involving the artificial breeding of frogs, and also promoted natural breeding in the laboratory. Field-caught male/female pairs of the Amami and Okinawa Island populations were artificially bred using an artificial insemination method in the 2004, 2006, and 2008 breeding seasons (March to April). Although fewer than 50% of the inseminated eggs achieved metamorphosis, approximately 500, 300, and 250 offspring from the three respective trials are currently being raised in the laboratory. During the 2009 and 2010 breeding seasons, second-generation offspring were produced by the natural mating activities of the first offspring derived from the two artificial matings in 2004. The findings and the methods presented here appear to be applicable to the temporary protection of genetic diversity of local populations in which the number of individuals has decreased or the environmental conditions have worsened to levels that frogs are unable to survive by themselves.

The first see-through frog created by breeding: description, inheritance patterns, and dermal chromatophore structure

We have succeeded in creating see-through frogs from natural color mutants of the Japanese brown frog Rana japonica, which usually possesses an ochre or brown back; this coloration enables the organs, blood vessels, and eggs to be observed through the skin without performing dissection. We crossed two kinds of recessive color mutant (black-eyed and gray-eyed) frogs through artificial insemination, and F2 offspring produced frogs whose skin is translucent throughout the life cycle. Three kinds of dermal chromatophores—xanthophores, iridophores, and melanophores—are observed in a layered arrangement in the skin of wild-type frogs, but few chromatophores were present in the skin of the see-through frogs. The translucent skin enables observation of organ growth and cancer formation and progression in the animal, which can be monitored over its entire life without the need for dissection. See-through frogs thus provide a useful animal model for environmental, medical, and biological research.