Using double-immunolabeling, we attempted to localize the glucocorticoid receptor (GR) and methionine-enkephalin-octapeptide (ENK-8) in the rat spinal cord. The 2nd to 8th cervical segments were analyzed in intact and in colchicine-injected animals. GR immunoreactivity was detected in the nuclei of neurons in all segments, and was most intense in small neurons in the superficial layers (laminae 1–3) of the dorsal horn. Medium-sized neurons in laminae 4–6 and the reticular formation, as well as the large ventral horn neurons, all showed moderate immunoreactivity. Double-immunolabeling revealed that, in all regions examined except the ventral horn, some of the GR-positive neurons were also ENK-8-positive. These findings suggest that enkephalinergic neurons in the spinal cord may be regulated by glucocorticoids, each of which is presumably related to the neuronal antinociceptive mechanism in the dorsal horn.

In order to establish the physiological saline suitable for the giant marine isopod Bathynomus doederleini, we analyzed ionic compositions, pH and osmotic pressure of the hemolymph. We made three test salines on the basis of ionic compositions of the hemolymph. Saline A contains surplus Cl⁻ (89.5 mM over in comparison with the serum) and no SO₄²⁻. Saline B contains surplus Cl⁻ (71.4 mM over) and normal SO₄²⁻. Saline C contains normal Cl⁻ and surplus SO₄²⁻ (35.7 mM over). We examined the effects of the test solutions and other salines on activities of isolated hearts. The heart in the salines continued to beat regularly more than 20 hr at 20°C. Heart rate in the test solutions was significantly higher (saline A: 66.5±5.1/min, n=8, saline B: 60.4±7.0/min, n=9, saline C: 67.9±8.4/min, n=8) than that in sea water (SW: 50.2±4.8/min, n=10) and was significantly lower than that in the saline for decapod which was originally designed by Pantin (Pantin's saline : 77.6 ±8.9 /min, n=8). Among the test salines, there were no significant differences in heart rate, systolic force and membrane potential of cardiac ganglionic neuron and myocardial cell. The standard physiological saline for Bathynomus was determined as follows (mM) ; Na , 479.4; K , 15.7; Ca² , 14.6; Mg² , 60.7; Cl⁻, 627.6; SO₄²⁻, 9.1; HEPES, 5; pH 7.9.

We have isolated a cDNA clone for a zinc-requiring metallopeptidase in Xenopus oocytes from Xenopus ovary library using oligonucleotides synthesized on the basis of the partial amino acid sequence. The full-length 2,055 bp cDNA encodes a protein of 685 amino acid residues with a predicted molecular mass of 78,136 Da. The deduced amino acid sequence of this protein exhibits high similarity to that of human (74.1%), pig (75.3%) and rat (74.1%) thimet oligopeptidase (TOP) [EC 3.4.24.15]. Expression of the cDNA in bacterial cells resulted in the production of an active metalloenzyme. Thus, we concluded that the metallopeptidase purified from Xenopus oocytes is a member of the TOP family.

In Northern blot analyses, one major species of Xenopus-TOP (X-TOP) mRNA of 3.0 kb was expressed relatively strongly from early stage (III) of Xenopus oogenesis, its level decreasing in later stages (V and VI). This result suggests that the expression of X-TOP mRNA is regulated during Xenopus oogenesis.

A transient activation of dihydropteridine reductase (DHPR), which is the regenerating enzyme of tetrahydrobiopterin in the system of aromatic amino acid hydroxylases, was studied during the incubation of DHPR with Ca² -activated protease, m-calpain. The DHPR subunit (29 k) was cleaved by m-calpain just before the 35th (Ser) and 48th (Val) residue from the N-terminus, generating two new fragments of 21 k and 19 k. By determining kinetic parameters, we found that 21 k and 19 k were more active than the native enzyme and that the activation of them was more remarkable and transient against the natural substrate of quinonoid dihydrobiopterin than against a synthesized substrate. Phosphorylation of DHPR by Ca² /calmodulin-dependent protein kinase II controlled the sensitivity of the enzyme to the Ca² -activated protease.

A cDNA encoding a matrix metalloproteinase-related protein has been cloned from a porcine ovary cDNA library. This putative protein comprises 119 amino acid residues, and exhibits sequence similarity with the NH₂-terminal half of rat matrilysin with 264 amino acid residues. A porcine ovary matrilysin cDNA clone was also isolated and characterized. A structural comparison of the matrilysin-related protein with porcine matrilysin suggests that both proteins are the products of the same gene. To further clarify their relationship, PCR amplification was carried out using porcine genomic DNA with two appropriate primers. The sequence analysis of a resulting 3.5kb PCR product revealed that a processing step involving internal exons of the gene produces the matrilysin-related protein. Relative amounts of the two distinct mRNAs in the porcine ovary were estimated by the competitive PCR method. The levels of mRNA for the matrilysin-related protein were approximately 4% of the wild-type matrilysin. The results indicate that the matrilysin-related protein is specifically expressed in the porcine ovary.

In R. cyanophlyctis intratesticular efferent ductules arise from seminiferous tubules and join vasa efferentia. The latter, whose number varies from 7–9 are connected to a lateral kidney canal (LKC). From the LKC sperm traverse to cranial nephrons through Bowman's capsules. The urinogenital (Wolffian) duct transports both urine and sperm to cloaca. In R. limnocharis the sperm from seminiferous tubules traverse to the cranial nephrons through intratesticular efferent ductules followed by 6–8 branched vasa efferentia. The nephrons release sperm into the Wolffian duct. The LKC is absent. By contrast, in P. maculatus the testes are composed of seminiferous lobules that radiate from a longitudinal collecting ducts (LCD) situated along the inner aspect of the testes. The LCD is drained by 8–12 vasa efferentia that are connected to nephrons directly; intratesticular efferent ductules and LKC are absent. In M. rubra the intratesticular efferent ductules arise from the seminiferous tubules. From testis 3–4 branched vasa efferentia traverse through the mesorchium to join the ipsilateral kidney. The vasa efferentia transport sperm directly to the cranial uriniferous tubules. From there the sperm are released in the Wolffian duct. Both LCD and LKC are absent. In B. melanostictus 25–30 branched vasa efferentia arise from each testis and are connected directly to nephrons. In B. fergusonii 14–18 branched vasa efferentia connect testis and the ipsilateral kidney. The sperm from vasa efferentia directly pass to the nephrons and then into the Wolffian duct. Intratesticular efferent ductules, LCD and LKC are absent in both B. melanostictus and B. fergusonii. The Wolffian duct is remarkably glandular all along its length in P. maculatus unlike in other species studied presently in which only seminal vesicle (ampulla) is glandular. The present study shows a diversity in the organization of urinogenital system in anurans and lack of a phylogenetic trend.

We studied diets of an Asian ranid, Rana rugosa, inhabiting three different environments (reservoir, river shoreline, and paddy fields) in Kyoto, Japan. In all the three habitats, ants were the most frequently consumed prey, representing surprisingly similar proportions in both the frequency of occurrence (81.9–85.7%) and the number of total prey items (56.8–59.4%). These values are exceptionally large for Rana, and equivalent to those reported for ant specialists in other families of frogs such as dendrobatids or bufonids. However, R. rugosa consumed ants lower in proportions than those found in the environment, and could not be regarded as purely ant specialists. Instead, we conclude that this species tends to avoid ants more weakly than other species of Rana. Other than ants, larger prey were more and smaller ones less frequently taken in proportion to frog body size, indicating that the frog consumes ants because of its weak avoidance of these abundant potential prey.

Field studies on the reproductive traits of a population of a gnathiid isopod Elaphognathia cornigera (Nunomura) were conducted at a rocky intertidal shore on the Izu Peninsula, southern Japan. Fortnightly surveys for two years showed some peculiar seasonal trends in the female reproductive characteristics. While adult males and juveniles of the species occurred throughout the year, semelparous adult females occurred only from spring to autumn. Based on the occurrence patterns of the females in six different reproductive stages, there seemed to be four generations in 1994 and three in 1995. Though the reproductive investment of a female showed no significant variation through each year, there were marked seasonal variations in the female broods. Female body size and brood size became the largest in early summer, but egg size reached its maximum in autumn. Maximum food availability (high density of gobies) in spring and the unfavorable environmental conditions in winter were thought to be the major controlling factors of the size and number of female broods.

The gray red-backed vole, Clethrionomys rufocanus, from Far East Asia including Sakhalin and Hokkaido is known to harbor intraspecific morphological and cytogenetic variations. Here we analyzed geographic variation in the mitochondrial cytochrome b (Cytb) gene and Y chromosome specific Sry gene by a PCR direct sequencing technique. Determination of sequences in the Cytb gene (1140 bp) in 29 individuals provided 28 haplotypes and construction of a neighbor joining tree clearly indicated that they were grouped into four major lineages, which predominated in Primorskyi territory, Kamchatska-Magadan, Sakhalin, and Hokkaido-Kuril, with 0.02–0.04 sequence divergence (Kimura's distance, all substitutions, all codon positions). The sequences for the Sry region (336–366 bp) included a variable repetitious region of microsatellites such as TG(TC)₂. In total five sequences were recognized which can be grouped into two forms, continental and insular (Sakhalin and Hokkaido) based on the presence or absence of a segment of TG(TC)₃TG(TC)₄. It was revealed that, therefore, the population of Sakhalin possessed their own type of mitochondrial DNA but the same Sry gene as Hokkaido. A similar trend can be seen in the continental populations. Our results suggested that the population of C. rufocanus in each of the geographic domains accumulated own genetic elements in part but genetic exchanges between neighboring populations occurred during the course of evolution. It is noteworthy that the insular domains, Sakhalin and Hokkaido, have played an important role in raising the amount of genetic diversity in small rodent species.

Petauristinae (flying squirrels) consists of 44 extant species in 14 recent genera, and their phylogenetic relationships and taxonomy are unsettled questions. We analyzed partial mitochondrial cyto-chrome b gene sequences (1,068 base pairs) to investigate the phylogenetic relationships among six flying squirrel genera (Belomys, Hylopetes, Petaurista, Petinomys, and Pteromys from Asia and Glaucomys from North America). Molecular phylogenetic trees, constructed by neighbor-joining and maximum likelihood methods, strongly indicated the closer relationship between Hylopetes and Petinomys with 100% bootstrap values. Belomys early split from other flying squirrels. Petaurista was closely related to Pteromys, and Glaucomys was most closely related to the cluster consisting of Hylopetes and Petinomys. The bootstrap values supporting branching at the deeper nodes were not always so high, suggesting the early radiation in the evolution of flying squirrels.

This study obtained 18S rDNA sequence data to investigate the extent of genetic divergence in the Japanese dendrocoelid group, and applied the data to the estimation of phylogenetic relationships and taxonomy. The sequence data, consisting of 558 base positions in an aligned data set, were obtained by using the polymerase chain reaction and direct sequencing. For comparative analysis, the 18S rRNA gene sequence data on dendrocoelids from different distant geographical places (Europe, Lake Baikal in Siberia, Kamchatka) published previously were used. Such analysis showed that: (1) Bdellocephala species from Japan, Kamchatka and Lake Baikal are closely related ; (2) Japanese species classified within the genus Dendrocoelopsis do not form a single cluster of closely related organisms; (3) Dendrocoelopsis ichikawai differs minimally from Bdellocephala baicalensis (from Lake Baikal), reliably groups with all Bdellocephala, and should therefore be classed with this genus.

Taxonomic conclusions. (1) The genus Dendrocoelopsis should be revised after molecular typification of European and American representatives. (2) The current generic status of D. ichikawai based on the presence of a well-developed penis papilla should be reconsidered. (3) Sequence divergence amongst representatives of B. brunnea indicates that representatives should be closely examined for the presence of morphological characters by which they might be distinguished.

Phylogenetic relationships among the East Asian species of the genus Crocidura (Mammalia, Insectivora, Soricidae) were inferred from the partial sequences (402 base pairs) of the mitochondrial cyto-chrome b gene. Six species of Crocidura and one representative of another insectivoran genus Suncus were examined. In the neighbor joining tree, four distinct lineages were recognized: C. lasiura from northeastern China and Korea, and C. dsinezumi from the main-islands of Japan; C. attenuata from Taiwan; C. watasei from the Amami and Okinawa Groups, and S. murinus from the Miyako Group; and C. suaveolens from Tsushima and Taiwan, and C. sibirica from Central Asia in Russian territory. All these lineages excluding C. lasiura and C. dsinezumi were also recognized by the maximum parsimony analysis. Clustering of C. watasei with S. murinus suggested the non-monophyly of the genus Crocidura as of the present definition. Biogeo-graphical implications of the present results were also discussed.

Enchytraeus japonensis, a terrestrial enchytraeid, reproduces asexually by fragmentation and subsequent regeneration. Because of its extreme ability to regenerate, the species has recently been proposed as a new experimental material for regeneration study, and a method has been developed for inducing sexual reproduction in the laboratory. The inducibility of sexual reproduction allowed us to perform, for the first time, a taxonomical analysis of E. japonensis based on the morphology of the genital organs, and to compare it with the morphology of the highly similar E. bigeminus, another fragmenting enchytraeid. Comparisons between the two species were also made for general protein patterns and isozyme patterns of seven enzymes using PAGE-IEF. The morphological investigation revealed that the two species are discriminable by only one character; glandular bodies associated with the male copulatory organs that are present in E. bigeminus but absent in E. japonensis. In contrast to this morphological similarity, protein patterns were highly divergent between the two species; 29 (55%) out of 53 distinguishable bands of general proteins and 25 (86%) out of 29 isozymes of the enzymes examined (EST, MDH, PGM, GPI, LDH, ME, and HE) were species-specific. These results suggest that Enchytraeus japonensis and E. bigeminus are closely related but different species.

Mygalomorph spider's hemocyanins examined contain monomer subunits which are the component parts of hemocyanin hexamers and dimer subunits which bond two hexamers together. We determined the N-terminal amino acid sequences of twenty-two monomer subunits and ten constituent monomers of the dimer subunits prepared from hemolymph of seven mygalomorph spiders. We classified these subunits into eight groups based on the sequence comparison. From the distribution of monomer subunits in mygalomorph spiders, it is evident that duplications and losses of monomer subunits occurred frequently. This is the reason why we cannot detect an orthologous monomer subunit derived from a common ancestral sequence in the seven spiders. These monomer subunits were found in either Atypoidea or Avicularioidea. On the other hand, the dimer subunits having similar N-terminal sequences were found widely in both mygalomorph spider groups, showing that the dimer subunits are more conservative than monomer subunits for their function different from monomer subunits.

Phylogenetic relationships of the family Agamidae were inferred from 860 base positions of a mitochondrial DNA sequence of 12S and 16S rRNA genes. Results confirmed the monophyly of this family including Leiolepis and Uromastyx (Leiolepidinae), and indicated the sister relationship between Agamidae and Chamaeleonidae. Our results also indicated the presence of two major clades in Agamidae. In one of these major clades, “Leiolepidinae” was first diverged, followed by the Lophognathus and Hypsilurus in order, leaving Physignathus, Chlamydosaurus and Pogona as monophyletic. This result contradicts the currently prevailing hypothesis for the agamid phylogeny, which, on the basis of morphological data, assumes the primary dichotomy between Leiolepidinae and the remainder (Agaminae). The phylogenetic diversity of agamid lizards in the Australian region is supposed to have increased through an in situ continental radiation rather than through multiple colonizations from Southeast Asia. Distributions of some species in Asia and Melanesia are attributed to the secondary dispersals subsequent to this radiation.

In Japan, Eurema hecabe (L.) has two geographically separated groups (subtropical and temperate) differing not only in the color pattern expression of the wings associated with ovarian arrest, but also in the fringe color of the wings and host-plant utilization. In a field survey (Kato, 1999), it was found that individuals with different fringe color of the forewing (brown or yellow type) inhabit Okinawa-jima Island located in north of the subtropical region, and that fringe-color is linked with seasonal morph expression. In the present study, rearing experiments were done to investigate whether these sympatric individuals are different forms within one species or belong to different species. The results show that each population of brown- and yellow-types maintained its own seasonal morph response to photoperiod and temperature through three generations. Host-plant utilization by the larvae and the fringe-color also differed between them and was maintained. These findings strongly suggest that E. hecabe of Okinawa-jima Island is divided into two distinct populations, and that one belongs to the temperate and the other to the subtropical group.

Interspecific differences in the ability to excrete urea in four gobiid fishes, Mugilogobius abei, M. chulae, M. sp. 1 and M. sp. 2, in response to elevated external ammonia, were compared and set against the phylogenetic relationships of species, following molecular phylogenetic analyses using a portion of mitochondrially-encoded 12S ribosomal RNA and tRNA-Val genes, which were sequenced from five species of Mugilogobius. The resulting tree, based on 710 base pairs of aligned sequences, was statistically robust and indicated two major clades, (M. abei, M. sp. 1 and M. chulae) and (M. parvus and M. sp. 2), respectively. High ureagenic ability was demonstrated in M. abei and M. sp. 1, being sister species according to the phylogenetic analysis, while the remaining species having a low level of ability. The two former species are restricted to temperate and subtropical Japan, whereas the others are widely distributed throughout tropical Asia. The most parsimonious reconstruction of these features suggested that ancestral Mugilogobius, probably widely distributed in tropical Asia, may had low ureagenic ability. The high ureagenic ability of the temperate species may have evolved concurrently with the northward spread of Mugilogobius from the tropical zone.