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Background & Aims: University students are prone to changes in their health status and lifestyle due to changes in their living environment and associated stress and anxiety. These changes may affect them in later life. This study utilized a cross-sectional study among Japanese female university students to examine dietary factors affecting their fecal microbiota. Methods: Sixty-eight healthy female university students were evaluated using an eating behavior assessment and diet history questionnaire. The 12-component Japanese diet index (JDI-12) was then calculated. A quantitative real-time PCR method was used to analyze the predominant bacterial species in the gut, and the Firmicutes/Bacteroidetes ratio (F/B ratio) at the phylum level was calculated. The partial correlation between the fecal microbiota and eating behavior abnormality score was assessed, and dietary habits associated with the F/B ratio were analyzed. Results: A significant correlation was identified between F/B ratios and the eating behavior abnormality score (r = 0.26, FDR = 0.064). Additionally, multiple regression analysis identified a negative correlation trend between the F/B ratio and JDI-12 score (β = −0.22; p = 0.091), and exploratory analysis found a negative association between the F/B ratio and consumption of beef and pork, one of the less beneficial JDI-12 components (β = −0.33, FDR = 0.120). Conclusion: In healthy female university students, there was a positive correlation between eating behavior abnormality and the F/B ratio, indicating that adherence to the Japanese diet pattern may be associated with a lower F/B ratio.

Background: Patients undergoing surgery for esophageal cancer are at risk of prolonged hospital stay for postoperative malnutrition. Postoperative early oral feeing is a part of the &ldquo, enhanced recovery after surgery protocol&rdquo, for coping with this risk. However, the usefulness of early oral intake during perioperatively is questionable. Methods: In total, 117 patients treated surgically for esophageal cancer were analyzed in the study. We assessed the oral energy sufficiency rate per nutritional requirement (oral-E/NR) at the fourth week postoperatively and classified the patients into two groups: Poor oral intake group (POI group, &lt, 25% oral-E/NR) and the control group (&ge, 25% oral-E/NR). We analyzed the relationship among postoperative oral intake and prognoses. Results: The POI group had worse postoperative nutritional status and a lower survival rate than the control group. In a multivariate analysis, &lt, 25% oral-E/NR was one of the independent factors contributing to negative outcomes postoperatively (adjusted hazard ratio: 2.70, 95% confidence interval: 1.30&ndash, 5.61). Conclusions: In patients undergoing surgery for esophageal cancer, poor postoperative oral intake negatively affected not only on their postoperative nutritional status but also their overall prognosis. It is necessary to improve the adequacy of oral intake postoperatively for patients with esophageal cancer.

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.

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.

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.

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.

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.

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of an endangered Japanese frog, Odorrana ishikawae (family Ranidae). We also sequenced partial mt genomes of three other Odorrana and six ranid species to survey the diversity of genomic organizations and elucidate the phylogenetic problems remaining in this frog family. The O. ishikawae mt genome contained the 37 mt genes and single control region (CR) typically found in vertebrate mtDNAs, but the region of Light-strand replication origin (OL) was triplicated in this species. Four protein-encoding genes (atp6, nd2, nd3, and nd5) were found to have high sequence divergence and to be usable for population genetics studies on this endangered species. Among the surveyed ranids, only two species (Rana and Lithobates) manifested the typical neobatrachian-type mt gene arrangement. In contrast, relatively large gene rearrangements were found in Amolops, Babina, and Staurois species; and translocations of single tRNA genes (trns) were observed in Glandirana and Odorrana species. Though the inter-generic and interspecific relationships of ranid taxa remain to be elucidated based on 12S and 16S rrn sequence data, some of the derived mt gene orders were found to have synapomorphic features useful for solving problematic ranid phylogenies. The tandem duplication and random loss (TDRL) model, the traditional model for mt gene rearrangement, failed to easily explain several of the mt gene rearrangements observed here. Indeed, the recent recombination-based gene rearrangement models seemed to be more suitable for this purpose. The high frequency of gene translocations involving a specific trn block (trnH-trnS1) and several single tRNA genes suggest that there may be a retrotranslocation in ranid mt genomes.

The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of a Malagasy poison frog, Mantella madagascariensis (family Mantellidae), and partial sequences of two Mantella (M. baroni and M. bernhardi) and two additional mantellid species (Boophis madagascariensis and Mantidactylus cf. ulcerosus). The M. madagascariensis genome was shown to be the largest (23 kbp) of all vertebrate mtDNAs investigated so far. Furthermore, the following unique features were revealed: (1) the positions of some genes and gene regions were rearranged compared to mitochondrial genomes typical for vertebrates and other anuran groups, (2) two distinct genes and a pseudogene corresponding to transfer RNA gene for methionine (tRNA-Met) were encoded, and (3) two control regions with very high sequence homology were present. These features were shared by the two other Mantella species but not the other mantellid species, indicating dynamic genome reorganization in a common ancestor linage before divergence of the Mantella genus. The reorganization pathway could be explained by a model of gene duplication and deletion. Duplication and deletion events also seem to have been responsible for concerted sequence evolution of the control regions in Mantella mt genomes. It is also suggested that the pseudo tRNA-Met gene sustained for a long time in Mantella mt genomes possibly functions as a punctuation marker for NADH dehydrogenase subunit (ND) 2 mRNA processing. Phylogenetic analyses employing a large sequence data set of mt genes supported the monophyly of Mantellidae and Rhacophoridae and other recent phylogenetic views for ranoid frogs. The resultant phylogenetic relationship also suggested parallel occurrence of two tRNA-Met genes, duplicated control regions, and ND5 gene translocation in independent ranoid lineages.

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin pathway.

The complete nucleotide sequence (21,359 bp) of the mitochondrial DNA of the rhacophorid frog Rhacophorus schlegelii was determined. The gene content, nucleotide composition, and codon usage of this genome corresponded to those typical of vertebrates. However, the Rh. schlegelii genome was unusually large due to the inclusion of two control regions and the accumulation of lengthy repetitive sequences in these regions. The two control regions had 97% sequence similarity over 1,510 bp, suggesting the occurrence of concerted sequence evolution. Comparison of the gene organizations among anuran species revealed that the mitochondrial gene arrangement of Rh. schlegelii diverged from that of typical vertebrates but was similar to that of Buergeria buergeri. The positions of the tRNA-Leu(CUN) and tRNA-Thr genes were exchanged between Rh. schlegelii and B. buergeri. Based on parsimonious consideration and the basal phylogenetic position of B. buergeri, these genes seemed to have been rearranged in an ancestral lineage leading to Rh. schlegelii.

The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used next-generation sequencing technology of Ion Torrent PGM™. Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the three-related species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM™ for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies.

The sedative properties of linalool were examined using the optically active linalools, (R)-(−)-, (S)-(+)- and (RS)-(±)-forms. (R)-(−)-linalool with specific rotation of [α]D= −15.1° was isolated by repeated flash column chromatography from lavender oil, while (S)-(−)-linalool with [α]D= +17.4° and (RS)-(±)-linalool with [α]D=0° and content of (R)-form 50.9% and (S)-form 49.1% were obtained from coriander oil and commercial linalool, respectively, by using the same method. The effect of (RS)-(±)-linalool in our experiments, which was quite similar to that of linalool, with much more favorable impressions accompanied by a tendency to a greater decrease of the beta wave after hearing environmental sound after, rather than before work, appeared to be identical to that observed for (R)-(−)-linalool. The feature was just the reverse in the case of (S)-(+)-linalool.

An opsonic molecule that is designated the third component of hagfish complement (HC3), and a fragment of HC3 known as HC3b have recently been identified in the hagfish, Eptatretus burgeri. These proteins were purified from plasma and generated a set of several bands and/or smears during SDS-PAGE under standard, non-reducing conditions. Two-dimensional electrophoretic analysis of the proteins under non-reducing and reducing conditions revealed the breakdown of polypeptides at the site of a thioester bond and the concomitant partial release of a split product, depending on the weak covalent or non-covalent association of polypeptide chains, in a large fraction of molecules of HC3 during SDS-PAGE. Moreover, the heterogeneity of HC3b can be ascribed to the different configurations of subunits. A similar phenomenon was not observed in the case of lamprey C3, even though breakdown of polypeptides at a thioester bond did occur in some molecules.

Normal serum from the hagfish Eptatretus burgeri strongly enhanced the phagocytic activity of homologous peritoneal macrophages against rabbit erythrocytes (RRBC). Such phagocytosis was effectively but incompletely inhibited by rabbit antibodies against hagfish C3 (HC3). However, when RRBC were pretreated with purified HC3 or normal hagfish serum in the presence of EDTA, little enhancement of ingestion was observed. The results indicate that HC3 binds to RRBC, in collaboration with other serum factor(s), and that bound HC3 functions as an opsonin. Additionally, we obtained evidence for the possible presence of another opsonin (HOP) that was resistant to EDTA in C3-depleted hagfish serum. HOP prepared from plasma had a molecular mass of approximately 1,000 kDa, consisting of various small polypeptides that were able to form aggregates via disulfide bonds and/or noncovalent interactions. The functions of HC3 and HOP might be essential to immunity in hagfish because no clear evidence for the product...

Anderson’s crocodile newt (Echinotriton andersoni) is distributed in the Central Ryukyu Islands of southern Japan, but environmental degradation and illegal collection over the last several decades have devastated the local populations. It has therefore been listed as a class B1 endangered species in the IUCN Red List, indicating that it is at high risk of extinction in the wild. The species is also protected by law in both Okinawa and Kagoshima prefectures. An artificial insemination technique using hormonal injections could not be applied to the breeding of this species in the laboratory. In this study we naturally bred the species, and tested a laboratory farming technique using several male and female E. andersoni pairs collected from Okinawa, Amami, and Tokunoshima Islands and subsequently maintained in near-biotopic breeding cages. Among 378 eggs derived from 17 females, 319 (84.4%) became normal tailbud embryos, 274 (72.5%) hatched normally, 213 (56.3%) metamorphosed normally, and 141 (37.3%) became normal two-month-old newts; in addition, 77 one- to three-year-old Tokunoshima newts and 32 Amami larvae are currently still growing normally. Over the last five breeding seasons, eggs were laid in-cage on slopes near the waterfront. Larvae were raised in nets maintained in a temperature-controlled water bath at 20 °C and fed live Tubifex. Metamorphosed newts were transferred to plastic containers containing wet sponges kept in a temperature-controlled incubator at 22.5 °C and fed a cricket diet to promote healthy growth. This is the first published report of successfully propagating an endangered species by using breeding cages in a laboratory setting for captive breeding. Our findings on the natural breeding and raising of larvae and adults are useful in breeding this endangered species and can be applied to the preservation of other similarly wild and endangered species such as E. chinhaiensis., This work was supported by Grants-in-Aid for Scientific Research (B and C) (Nos. 24310173 and 20510216) to Masayuki Sumida and a Grant-in-Aid for Young Scientists (B) (No. 23710282) to Takeshi Igawa from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

We recently reported the primary structures, antimicrobial activities and cDNA precursors of nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorranaishikawae. Their cDNA clones revealed a highly conserved approximately 60 bp region upstream of the start codon. This conserved region was used in the "shotgun" cDNA cloning method to reveal additional cDNAs encoding novel antimicrobial peptides of O.ishikawae. After sequencing 344 clones, we identified novel 13 cDNAs encoding dermal peptides in addition to the previously identified nine antimicrobial peptides. These 13 unique cDNAs encoded precursor proteins each containing a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg/Lys processing site and a dermal peptide at the C-terminus. The dermal peptides were members of the palustrin-2 (two peptides; termed palustrin-2ISc and palustrin-2ISd), nigrocin-2 (one peptide; nigrocin-2ISc), brevinin-1 (one peptide; brevinin-1ISa), odorranain-M (one peptide; odorranain-MISa) and entirely novel peptides (eight peptides; ishikawain-1-8). Although palustrin-2ISd and odorranain-MISa had few antimicrobial activities, palustrin-2ISc and nigrocin-2ISc possessed a broad-spectrum of growth inhibition against bacteria. Brevinin-1ISa had the most potent antimicrobial activities against the Gram-positive bacteria and the fungus but not the Gram-negative bacterium, Escherichiacoli. However, eight novel peptides showed no growth inhibition against these microorganisms.

Developing lampreys were fixed at frequent intervals between the gastrula stage (6 days) and the earliest ammocoete larva (31 days). Expression of lamprey engrailed (en) gene was studied by labeling with a polyclonal antiserum (αEnhb-1) raised against mouse en protein. Western blotting of proteins from developing lampreys reveals a major band (40±103Mr), which is probably lamprey en protein. Expression domains of en were demonstrated in developing lampreys by immunohistochemistry of whole mounts and histological sections. Expression of en first becomes detectable at the head protrusion stage (11/12 days) in neural tube cells at the mid/hindbrain boundary and soon thereafter in some mesodermal cells of the mandibular arch. These en-expressing cells of the mandibular arch are located in the walls of vesicles of paraxial mesoderm that originate by enterocoely on either side of the pharynx. At the tailbud stage (15 days), en expression is also detectable in mesodermal cells of the anterior lip and in some mesodermal and epidermal cells in the region of the tailbud. By the eye spot stage (18 days), detectable en expression in the mandibular arch becomes limited to cells of the velothyroideus muscles, which drive the power stroke of the recently formed velum. At later stages, while the preceding expression domains fade, en expression begins in some epidermal cells associated with the lip papillae, gill slits, and nostril. We suggest that the velothyroideus muscles of lampreys may be homologous to certain jaw muscles of teleosts–namely, the levator arcus palatini and the dilator operculi, which express en continuously while differentiating from the myogenic mesoderm into identifiable muscle types.

We determined the complete nucleotide sequences of mitochondrial (mt) genomes from two dicroglossid frogs, Hoplobatrachus tigerinus (Indian Bullfrog) and Euphlyctis hexadactylus (Indian Green frog). The genome sizes are 20462 bp in H. t igerinus and 20280 bp in E. hexadactylus. Although both genomes encode the t ypical 37 mt genes, the following unique features are observed: 1) the ND5 genes are duplicated in H. tigerinus that have completely identical sequences, whereas duplicated ND5 genes in E. hexadactylus possessed dissimilar substitutions; 2) duplicated control region (CR) in H. tigerinus has almost identical sequences whereas single control region (CR) was found in E. hexadactylus; 3) the tRNA-Leu (CUN) gene is translocated from the LTPF tRNA cluster to downstream of ND5-1 in H. tigerinus, and the tRNA-Pro gene is translocated from the LTPF tRNA cluster to downstream of CR in E. hexadactylus; 4) pseudo tRNA-Leu (CUN) and tRNA-Pro genes are observed in E. hexadactylus; and 5) two tRNA-Met genes are encoded in both species, as observed in the previously reported dicroglossid mt genomes. Almost all observed gene rearrangements in H . tigerinus and E. hexadactylus can be explained by the tandem duplication and random loss model, except translocation of tRNA-Pro in E. hexadactylus. The novel mt genomic f eatures found in this study may be useful for future phylogenetic studies in the dicroglossid taxa. However, the mt genome with interesting features found in the p resent study reveal a high level of variation of gene order and gene content, inspiring more research to understand the mechanisms behind gene and genome evolution in the dicroglossid and as well as in the amphibian taxa in future studies.

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. 7–5 Mya), 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.

An epidemiological survey on the vector mites of Tsutsugamushi disease was conducted in Taiwan and the Ryukyu Islands. On the Penghu Islands and Taitung of Taiwan, the only trombiculid mite belonging to the subgenus Leptotrombidium collected from rodents and insectivores was L. (L.) deliense, the potential vector of Rickettsia tsutsugamushi in the Asia-Pacific region south of Japan. On the other hand, on Mt. Alishan three species of the subgenus Leptotrombidium including L. (L.) kawamurai were collected from rodents, suggesting that the reservoirs of R. tsutsugamushi may be in this highland. On Okinawa Island, Japan, the trombiculid mite was not collected from rodents and insectivores in the present survey., 論文

We sequenced the whole mitochondrial (mt) genomes of three neobatrachian species: Japanese tree frog Hyla japonica, Japanese common toad Bufo japonicus, and narrow-mouthed toad Microhyla okinavensis. The gene arrangements of these genomes diverged from that of basal anurans (suborder Archaeobatrachia), but are the same as that of the members of derived frogs (i.e., superfamily Hyloidae and Ranoidae in suborder Neobatrachia), suggesting the one-time occurrence of a gene rearrangement event in an ancestral lineage of derived anurans. Furthermore, several distinct repeat motifs including putative termination-associated sequences (TASs) and conserved sequence blocks (CSBs) were observed in the control regions (CRs) of B. japonicus and H. japonica, while no repeat motifs were found in that of M. okinavensis. Phylogenetic analyses using both nucleotide and amino acid data of mt genes support monophyly of neobatrachians. The estimated divergence time based on amino acid data with multiple reference points suggests that the three living amphibian orders may have originated in the Carboniferous period, and that the divergences of anurans had occurred between the Permian and Tertiary periods. We also checked the influence of the data types and the settings of reference times on divergence time estimation. The resultant divergence times estimated from several datasets and reference time settings suggest that the substitution saturation of nucleotide data may lead to overestimated (i.e., older) branching times, especially for early divergent taxa. We also found a highly accelerated substitution rate in neobatrachian mt genes, and fast substitution possibly resulted in overestimation. To correct this erroneous estimation, it is efficient to apply several reference points among neobatrachians.

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organizations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid frogs, indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mitochondrial genes also supported the gene rearrangement pathway.

Cytological observation and artificial crossing experiments were used to examine the geographic differences in the sex-determining mechanism and mode of inheritance of the sex-linked AAT-1 gene in the bell-ring frog, Buergeria buergeri. The AAT-1 phenotypes were also examined by allozyme analysis using field-caught females and males collected from 19 populations from the Honshu, Shikoku, and Kyushu islands of Japan, in order to comprehensively elucidate the geographic variability in the expression of the sex-linked AAT-1 gene of B. buergeri. The results showed that the Aomori population of B. buergeri from the northern end of Honshu was female heterogametic in sex determination, that chromosome No. VII was a sex chromosome of the ZZ/ZW type, and that the sex-linked AAT-1 gene was expressed on both the Z and W chromosomes. This mode of AAT-1 expression in the Aomori population was different from that in the Hiroshima population from western Honshu, in which the AAT-1 gene was expressed on the Z chromosome but not on the W chromosome. The results also showed that there was no differentiation among populations in the expression of the AAT-1 genes on the Z chromosome, whereas two populations, the Hiroshima and Aomori frogs, exhibited distinct modes of expression of the AAT-1 gene on the W chromosome. These two modes of expression may be widely distributed in western and eastern Japan, and coexist in the central part of Honshu.

The third component of complement (C3) of a newt, Cynops pyrrhogaster, was purified using a fast protein liquid chromatography technique. The purified newt C3 consists of two polypeptide chains (the molecular masses of the alpha and beta-chains of C3 were 120,000 and 70,000, respectively) linked by disulfide bonds. The alpha-chain retained an internal thiolester bond that was cleaved with methylamine, and the N-terminal amino acid sequence of the alpha-chain was XVQLIDAKAGKAAKF. Digestion of newt C3 with trypsin yielded fragments that induced significant histamine release from newt peritoneal cells. These results indicate that newt C3 retains structural and functional properties shared with mammalian C3.

A protein from hagfish serum that cross-reacted with the third component of hagfish complement (C3) was purified to homogeneity and its structural properties were compared with those of C3 which has a two-subunit chain structure (115 and 72 kDa). This protein (designated C3b), when purified from plasma, consisted of three disulfide-linked polypeptide chains (77, 72 and 30 kDa). On immunoelectrophoresis, purified C3b migrated more rapidly towards the anode than the beta mobility of C3. However, immunochemical analysis revealed that C3b, after the first step in its purification, consisted of two disulfide-linked polypeptide chains (105 and 72 kDa). Treatment of C3b with methylamine, prior to spectrophotometric titration of the free sulfhydryl groups, did not significantly affect the end-point of the titration, suggesting the absence of a thioester bond in this molecule. Analysis of the amino acid sequences of the amino-termini of the subunits of C3b revealed that 77 amino acid residues at the amino-terminus of the native alpha chain were missing from both the 77-kDa and the 105-kDa polypeptides from C3b. These results indicate that the C3b in this study was analogous to mammalian C3b. Furthermore, amino acid sequencing data indicated that most of the native C3 from hagfish serum has an irregular two-subunit (alpha+gamma and beta)-linked structure, as a result of one-sided processing of putative hagfish pro-C3 at the beta-alpha processing site exclusively. Moreover, it appears that only the molecular features of degenerated hagfish C3 (C3b) are altered during its purification to generate a three-chain structure.

Serum from the arctic lamprey contains natural agglutinins which react in different degrees with the heterogenetic antigens on various vertebrates erythrocytes. These natural haemagglutinins were undetectable after heating the serum at 46°C for 30 min. However, no significant haemolytic activity was found in the sera of the arctic lamprey and brook lamprey. Although production of specific antibodies against sheep erythrocytes (SRBC) was regularly inducible in the fall (in early anadromous migrants), the anti-SRBC response rose only modestly in the spring. Arctic lampreys, which received 0.5 ml of a 20 % SRBC suspension per week for 6 weeks at 15°C, showed a rise of 26- to 212-fold in specific haemagglutination titers in the 4th week after the last injection. The induced antibodies were heat stable and displayed a high degree of specificity to SRBC antigens. This antibody interacted with suitable complement to lyse SRBC. A remarkable secondary response was produced. Similar experiments were performed on all stages of the brook lamprey's life cycle, indicating that transforming larvae have the most sensitive reactivity against SRBC.

The present studies were designed to determine whether cellular immune responses of ammocoete (larval lamprey), Lampetra reissneri , to heterogenous erythrocytes is comparable in complexity to and shares similar recognition mechanisms with those of other gnathostomes. These responses were measured by means of immunocytoadherence. The levels and kinetics of antigen-binding activity in the peripheral blood and primitive spleen (typhlosole) were established by following a single high (10 %) or low (0.001 %) SRBC challenge. Antigen-binding responses were similar in some respects to those previously reported for other poikilothermic vertebrates. Rabbit anti-arctic lamprey Ig inhibited antigen binding of immunocytes. Hapten-carrier immunization was used to explore lymphoid heterogeneity in this species. The anti-hapten (2,4,6-trinitrophenyl;TNP) response was not specifically enhanced by carrier priming, however, there were many lymphoid cells bearing anti-TNP receptors in untreated ammocoetes. Consequently, it is doubtful whether there is cell-cell collaboration in the lamprey immune system.

The rejection of skin allografts by the larval lamprey, Lampetra reissneri, was studied by light- and electron-microscopy, with particular attention to the cell types involved in the reaction. In all allografts, melanophores were destroyed within 20-60 days (the mean survival time, 36 +/- 12 days). Neither the epidermis nor the underlying collagenous lamella was invaded by host cells until the 60th day. A heavy infiltration of host leucocytes was observed in the allografts in melanophore and adipose layers and in the bundles of muscles. Throughout all stages from 10 to 60 days after the grafting, the cells of the polymorphonuclear leucocyte (PMN) series and eosinophilic granulocytes predominated, but macrophages were not observed at any stages examined. Plasma cells occurred occasionally at later stages (40-60 days) of allograft rejection, but lymphocytes were rarely found at any stages of graft rejection. These observations, combined with the recent finding of the antibody-enhanced phagocytic activity of granulocyte-series cells in the lamprey, indicate that PMNs, but not lymphocytes, function as the major effector cells in allograft rejection in this phylogenetically oldest class of contemporary vertebrates.

The serum of Arctic lampreys (Lampetra japonica) lyses rabbit erythrocytes (ER), with optimal activity at 4°C. This lytic activity is relatively heat stable, is inactivated after incubation with zymosan (5 mg/ml) for 1 hr at 15°C, and is controlled by Mg++ dependent factors present in normal lamprey serum. Hemolysis of sheep erythrocytes (ES) was obtained with lamprey hemolysin using bullfrog serum as complement (C). The optimal conditions were established for sensitizing ES with lamprey hemolysin to fix bullfrog C. Under the same conditions hemolysis was not induced with zymosan-treated bullfrog C and lamprey serum. These findings suggest that lamprey serum possesses components that function via the alternate pathway, instead of the classical pathway usually found in more advanced vertebrates.

Centrifugation on Ficoll-Paque of the blood from the Arctic lamprey, Lampetra japonica, yielded a uniform population of leucocytes containing about 90% polymorphonuclear leucocytes (PMNs). The PMNs showed a significantly enhanced phagocytic activity in vitro against sheep erythrocytes (ES), provided they were opsonized with the specific lamprey antibody (AL) against ES. This enhanced phagocytosis by PMN was more active at 4 degrees C than 15 degrees C, being dependent on Ca++; the opsonic activity of lamprey antibody was not replaced by bullfrog antibody. By the use of electron microscopy, it was found that the PMNs possess a well developed Golgi complex and rough ER, as well as highly variable cytoplasmic granules. Observations by both SEM and TEm revealed the formation of prominent pseudopodia, lamellipodia and phagosomes by the PMN during the process of engulfing erythrocytes.