The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium cal-cite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.

A novel one-step microplate cytotoxicity assay using the cytoplasmic fluorescent viability dye calcein AM was established for simple, rapid, sensitive, and quantitative measurements of the allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the ascidian Halocynthia roretzi. The mutual and directional ACR was distinguishable by the assay using the hemocytes from pairs of animals with different alloreactivities. The ACR assay may allow more precise genetic analysis of the gene that controls allore-activity of hemocytes, since the mutual and directional ACR may be related to levels of expression or numbers of the gene product or products on the target cells. The directional ACR will be useful in elucidating the cellular and molecular mechanisms of self-recognition in H. roretzi, since it allowed us to equate hemocytes from one animal with “effector cells” and those from the other animal of the pair with “target cells”. In addition, the quantitative ACR assay in a large number of samples is possible and it will allow production of monoclonal antibodies that may recognize receptors or ligands functioning in self-recognition processes by the H. roretzi hemocytes.

Cells derived from ovotestis tissue of pigmented Biomphalaria glabrata, Puerto Rican strain were cultured in double diluted GIT medium supplemented with modification of amino acids components of pigmented B. glabrata, ovotestis and mid-gut region and 3% inactivated fetal calf serum. As a result, two types of cells, epithelial and fibroblastic like cells increased in number during the cultivation. It seem that the medium used in this study is a suitable medium for cultivation of cells from ovotestis of pigemeted B. glabrata. These two types of cells have been maintained by successive transplantation for over 3 passages.

Recently, we have reported a novel tropomyosin (TM) -binding protein, glutamate dehydrogenase (GDH) and demonstrated by affinity column chromatography that chicken liver TM interacts with GDH in an ATP-dependent manner. To elucidate the physiological roles of the interaction between TM and GDH, we performed co-sedimentation assays of TM and GDH with F-actin, because it is known that TM exerts its physiological functions by associating with actin filaments. The results showed that TM and GDH co-pelleted with F-actin. GDH alone also co-precipitated with F-actin, but the amount of GDH sedimenting with F-actin was increased in the presence of chicken liver TM, suggesting that GDH is involved in the regulation of the actin cytoskeleton. We also prepared crude GDH from the nuclear and mitochondrial fractions obtained by subcellular fractionation of the chicken liver cells. Semi-nondenaturing 2D-PAGE revealed that partially purified GDH from the nuclear fraction was associated with TM, but not GDH from the mitochondrial fraction, suggesting preferential binding of TM to GDH. We determined the nucleotide sequence of chicken GDH cDNA and showed that the GDH transcript was widely expressed in the chicken organs. We examined the localization of TM and GDH by immunohistochemistry and revealed that they were distributed in the cytoplasm of the adult chicken liver. From these results, we propose two hypotheses on the physiological roles of the interaction between TM and GDH in nonmuscle cells.

Allatotropin is a 13-residue amidated neuropeptide isolated from pharate adult heads of the tobacco hornworm, Manduca sexta and strongly stimulates biosynthesis of juvenile hormones in adults, but not larval, lepidopteran corpora allata. From a Bombyx mori midgut cDNA library, a cDNA that encodes a 130-amino-acid polypeptide containing M. sexta allatotropin sequence was isolated. The B. mori allatotropin cDNA consists of 1196 nucleotides. The encoded allatotropin peptide is identical to that isolated from M. sexta and that predicted from Pseudaletia unipuncta, with 84% and 81% identity in the amino acid sequence of the allatotropin peptide precursor, respectively. M. sexta allatotropin is flanked by two different endoproteolytic cleavage sites within the precursor of the B. mori allatotropin peptide. Evidence from northern blotting of B. mori tissues showed that the allatotropin gene is expressed in the cells of midgut, head and integument with different transcription amount, but not in the fat body and silk gland. Midgut has also a number of allatotropin-immunoreactive cells and nerve fibers. These results will provide valuable information in understanding the AT gene of insects.

On the basis of our preliminary observation that a crude extract of the stomach of the toad Bufo japonicus exhibited a chitinase activity with its optimum pH around 3.0, we undertook molecular cloning of a cDNA encoding this putative gastric chitinase. By use of 2 degenerate oligonucleotide primers derived from the 2 conserved regions of the vertebrate chitinases, a reverse transcription-PCR product was obtained. This product was used as a probe to screen a cDNA library constructed from the toad stomach. The longest positive clone was revealed to contain an open reading frame for a putative chitinase protein of 484 amino acids, which protein exhibited sequence similarity to the known vertebrate chitinases. Our data also revealed this putative gastric chitinase to be distinct from the chitinase that we had previously isolated from the pancreas of the same species. In this putative gastric chitinase, both the N-terminal catalytic domain and the C-terminal chitin-binding domain were perfectly conserved, suggesting this protein to function as chitinase in the toad stomach.

Effects of LiCl on the specification process of pigment founder cells were examined in the sea urchin Hemicentrotus pulcherrimus. If embryos were treated with 30 mM LiCl during 4–7 or 7–10 hours postfertilization, pigment cells increased significantly. Aphidicolin treatment indicated that this increase was due to the increase in the pigment founder cells. Interestingly, if the embryos were treated sequentially with LiCl and Ca² -free seawater during 4–7 and 7–10 hr, respectively, they differentiated only about the same number of pigment cells as control embryos. Further, the increase was scarcely discerned when the embryos were treated with LiCl in the absence of Ca² during 7–10 hr. These results suggested that effect of LiCl would be ascribed to the increase in cell adhesiveness. In fact, LiCl-treated embryos were more difficult to be dissociated into single cells. Cell electrophoresis showed that the amount of the negative cell surface charges decreased considerably in LiCl-treated embryos. It was also found that the number of pigment cells seldom exceeded 100, even if embryos were exposed to a higher concentration of LiCl. This suggested that only a subpopulation of the descendants of veg2 blastomeres received the inductive signal emanated from the micromere progeny.

The aryl hydrocarbon receptor (AHR) is a member of ligand-activated transcription factors and conserved among vertebrates. To investigate the role of AHR in fish development, medaka embryos were treated with agonist (2,3,7,8-tetrachlorodibenzo-p-dioxin), antagonists (α- naphthoflavone and resveratrol), and inhibitor (piperonyl butoxide) of cytochromes (Cyts) P450 encoded by a battery of target genes. These embryos were found to have similar abnormal phenotypes. Among the most consistent phenotypes were blood clotting and malformation of bone that were associated with vascular damages. These results thus indicate that control of AHR is important for proper development of fish embryos. AHR may control levels of Cyts P450 that are responsible for synthesis and metabolism of a toxic compound that caused the abnormal phenotypes. Complementary DNA fragments encoding AHR homologs were cloned from medaka embryos. AHR-specific mRNA was ubiquitously expressed in embryos and adult tissues.

In order to check the quality of in vitro spermatogenesis of Japanese eel, in vitro11-ketotestosterone (11-KT) induced spermatogenesis was compared with in vivo spermatogenesis induced by a single injection of human chorionic gonadotropin (hCG) in detail. DNA contents of germ cells from in vitro and in vivo testicular fragments were compared using flow cytometry. Since the in vitro result of flow cytometry showed prominent 1C peak including spermatozoa and spermatids, the reduction of DNA by meiosis was assumed to progress normally, (i.e., haploid spermatozoa were produced in this in vitro system). In the testes of in vitro culture, however, spermatozoa were not released into lumen. Furthermore, the number of mitotic divisions of the in vitro experiment (6 divisions) was fewer than that of in vivo (10 divisions). In electron microscopy observations, both of in vivo and in vitro spermatozoon had a crescent-shaped nucleus with a flagellum, and a single large spherical mitochondrion. However, the elongation of the sperm head was not sufficient and the mitochondrion was not always located at the anterior end as is observed for the spermatozoa obtained from hCG injected eels. Eel spermatogenesis related substance-11 (eSRS11) is homologue of histone H1 which is up-regulated during spermatogenesis. Using this probe, in vitro spermatogenesis was also evaluated in molecular levels. In Northern blot analysis, eSRS11 mRNA was detected in both in vivo and in vitro testes. However, the expression of in vitro was much weaker than that of in vivo. These differences indicate that the stimulation of 11-KT is not sufficient, and another factors are needed to induce complete spermatogenesis in vitro.

Contents of mRNAs encoding LHβ-, FSHβ-, TSHβ- and common a-subunit precursor molecules were measured in male Japanese quail deprived of food for three days. Plasma LH, FSH, thyroxine and triiodothyronine levels were also measured in the same birds. Plasma LH levels declined during the period of food deprivation. Levels in starved birds were not different from those in control birds after one day of starvation but were significantly lower after three days. Plasma FSH levels showed a similar decline, although the changes were not significant. Plasma thyroxine levels did not decrease during starvation, whilst plasma triiodothyronine levels decreased drastically and significantly soon after the start of starvation. All the hormone subunit mRNA contents in starved birds also decreased, with differences from control birds significant 3 days after the start of starvation. Plasma FSH levels showed a strong positive correlation with pituitary FSHβ mRNA levels, while plasma LH levels had a strong positive correlation with common α mRNA levels and practically no correlation or even a negative correlation with LHβ mRNA levels. These results suggest that starvation suppresses not only gonadotropin and thyrotropin secretion but also their synthesis in the pituitary gland. Furthermore, these results showed that FSH and LH have different synthesis and secretion dynamics in the Japanese quail. Contradicting results with TSHβ mRNA and thyroid hormones lead us to assume that starvation affects thyroid hormone metabolism in peripheral tissue, presumably in the liver.

A breeding population of Rana japonica was studied at a marsh on the campus of Hiroshima University in Higashi-Hiroshima during the five years 1995-1999. The mark-recapture study showed that the size of the breeding population varied from year to year, and increased more than twofold in 1999 in comparison with the preceding years. The sex ratio of the breeding population (male/female) was from nearly 1.0 to 1.6. Frogs of both sexes were estimated to breed for the first time at the age of one or two years, and their maximum age was four years according to skeletochronology using phalanges and mark-recapture. Modes of the estimated ages were one year for males during the study years except 1997, but one or two years for females. Two thirds of breeding frogs, irrespective of their sex, were estimated to breed only once throughout their lives.

We conducted a quantitative analysis of geographic color variation in two species of dung beetles: Geotrupes auratus and G. laevistriatus. The reflectance of the dorsal surfaces was measured from 300 to 700 nm using a spectrophotometer. The reflectance curves for both beetles were bimodal; there were two distinct peaks, namely, the α peak, between 400 and 700 nm, and the β peak at around 300 nm. A stepwise discriminant analysis indicated that geographic color variation in Geotrupes beetles was primarily characterized by a shift of the α peak. Using beetles from three locations, we compared the wavelength (nm) of the α peak (λmax(α)) and its reflectance intensity (R(α)) to investigate sex and population differences. Intraspecific geographic variation in coloration was effectively detected by discriminant analysis of spectral reflectance curves. Our results showed that G. auratus and G. laevistriatus had similar coloration within each sampling location. Our study also revealed hidden sex differences in R(α); R(α) of males were significantly higher than those of females in both species. Since the dorsal surface of the beetles shows remarkable color variation, and coloration can be assessed objectively using reflectance spectra, Geotrupes beetles may be good model organisms to investigate geographic color variation.

The type species of Loxomitra Nielsen, 1964, L. kefersteinii (Claparède, 1867), has following characters: A) the buds have a pair of terminal wings at the base of the stalk, and B) the liberated buds crawl on the substratum by attaching the two terminal wings to the substratum and twisting the whole body. By contrast, some other members of the genus have following characters: A′) the buds lack the terminal wings but have a foot with foot groove, and B′) the liberated buds glide over the substratum using the foot with foot groove. I thus propose to divide Loxomitra (sensu lato) into two genera–Loxomitra (sensu stricto) characterized by A) and B), and Loxocorone gen. nov. by A′) and B′). I also describe two new Loxomitra (sensu stricto), L. mizugamaensis sp. nov., and L. tetraorganon sp. nov., and one new Loxocorone, L. allax sp. nov. from the Ryukyu Archipelago, Japan. All of the currently recognized species of the Loxomitra (sensu lato) are reviewed to specify their generic allocations in response to the above change.

Allozyme analysis for 41 populations of brown frog species, Rana dybowskii, R. huanrenensis, and R. amurensis from Korea and three reference species (Chinese R. chensinensis and Japanese R. dybowskii and R. tsushimensis), were performed to clarify taxonomic status of Korean brown frogs. The level of average genetic differentiation (Nei's D) among local populations of each species in Korea was very low (D<0.012) and Korean and Japanese R. dybowskii also showed conspecific level of differentiation (D=0.070). Whereas, much larger, discrete genetic differences were detected in the interspecific comparisons (D>0.370). In the genetic relationships among five species examined, the 24 chromosome brown frogs (R. dybowskii, R. huanrenensis, and R. chensinensis) did not form a monophyletic group. Rana dybowskii with the chromosome number of 2n=24 was grouped together with R. amurensis with the chromosome number of 2n=26. The hypothesis of reversal change from 24 to 26 in Korean R. amurensis seems to better explain the phylogenetic relationships of east Asian brown frogs than the assumption of parallel reduction in chromosome number from 2n=26 to 24 in R. dybowskii and in the common ancestor of R. huanrenensis and R. chensinensis. The genetic, morphological, and reproductive divergences between Korean R. dybowskii and R. huanrenensis were compared.