Secretory activity of the corpora allata (CA) and corpora cardiaca (CC) in the pars-intercerebralis complex has been suggested to be controlled by biogenic amines, despite a lack of quantitative information. The situation is similar to the frontal ganglion (FG), a member of the same complex. Hence, the levels of biogenic amines and related substances in these organs were measured in cricket, Gryllus bimaculatus, by high performance liquid chromatography with electrochemical detection. All organs examined contained considerable amounts of major biogenic amines, such as octopamine (OA), dopamine, and 5-hydroxytryptamine (5-HT). They also included epinephrine. The amounts of OA and 5-HT werehigher than those of other biogenic amines whose presence was detected in the examined organs. The distributional patterns of biogenic amines were different among the CA, CC and FG, e.g., the highest content of OA in the CC. The presence of precursors and metabolites indicates that biogenic amines are synthesized and metabolized in neuronal elements in the examined organs. It is probable that they are functional and also involved in the neural control of hormone secretion in CA and CC, because their turnover rates estimated from the present analysis are comparable to those in the nervous system.

A specific polyclonal antibody against Na , K -ATPase α-subunit was developed using a synthetic oligopeptide as antigen. By Western blot analysis under non-reducing conditions, this antibody recognized a protein band of approximately 150 kDa corresponding to the intact form (αβ-complex) of Na , K -ATPase in rabbit kidney. Furthermore, this antibody recognized a 150 kDa protein band corresponding to the intact form of Na , K -ATPase and some bands of about 60-65 kDa corresponding to fragments of the α-subunit in gill and kidney of masu salmon. This antibody did not recognize the α-subunit under reducing conditions. By immunohistochemical analysis, cells immunoreactive with this antibody were observed in renal tubular epithelial cells in kidney sections of rabbit, masu salmon, eel and rockfish. In addition, large spherical eosinophilic cells in gills of masu salmon, eel and rockfish were immunoreactive with the antibody. It is likely that these immunoreactive cells correspond to gill chloride cells. These data indicate that this antibody is a useful tool for studying changes in and the function of Na , K -ATPase during osmoregulatory adaptaion in a variety of fish species.

Developmental changes in the swimming behavior and underlying motoneuron activities of the larval angelfish, Pterophyllum scalare, were investigated in the course of post-embryonic development of the fish. For a few days after hatching, angelfish larvae showed spontaneous wriggling movement, which was alternating lateral bending of the tail without locomotion. On the 5th or 6th day after hatching, the larvae started swimming (locomotive activity). Spontaneous ventral root activity was recorded extracellularly from the body surface of the immobilized larvae as a monitor of the spinal motoneuron activity underlying wriggling and swimming. The number of motoneuron impulses and the duration of the burst in each cycle during the spontaneous rhythmic ventral root activity were increased as the larvae developed. In addition, the duration of the motoneuron bursts in older larvae was much more variable compared with that in early larvae, suggesting that the force of tail beat could be regulated flexibly as the larvae developed. In early larvae, two distinguishable patterns of spontaneous ventral root activity were observed: a low frequency bursting and a high frequency bursting. Such bursting patterns were consistent with the behavioral observations of free moving larvae. After the start of swimming, the frequency of the motoneuron bursting converged into a narrow range, corresponding to the stable, continuous swimming rhythm of the free moving larvae. We propose that the early stages of the angelfish larvae provide us with a useful model for investigating the developmental changes in neuronal substrates underlying swimming activity of the teleost fish.

We have isolated mutants of fission yeast defective in correct positioning of septum. In visual screening, we obtained 16 clones showing unequal septation at restrictive temperature, which were classified into three complementation groups. At restrictive temperature, all the mutants underwent nuclear division normally. In cytokinesis, however, a contractile ring was formed at the site independent of the mitotic spindle. These results suggest that positional information for cytokinesis are not accurately transmitted to the cell equator. Furthermore, all the mutants frequently displayed incorrect orientation and/or distortion of septum, which suggests that the septum positioning is closely related to correct orientation and organization of septum.

To investigate the differentiation of test cells and their roles during oogenesis and embryo-genesis in the ascidian Ciona intestinalis, we prepared two monoclonal antibodies (UA165 and UA464) that specifically recognize test cells. In the ovary, these antibodies recognized different cytoplasmic contents of test cells surrounding a vitellogenic oocyte. The recognition of the antibodies was retained after they were released from the surface of the oocyte into the perivitelline space and throughout embryogenesis. After hatching, however, these antibodies also recognized the larval tunic. These findings suggested that during oogenesis, test cells synthesize antigens recognized by the antibodies and that these antigens are stored in the cytoplasm through embryogenesis, then they are used to form the larval tunic.

The test cells, one of the two accessory cell groups of the ascidian oocyte, persist around the developing embryos and the hatched tadpole larvae, but their function during embryogenesis is not fully understood. We have produced monoclonal antibodies that specifically recognize the test cells in Ciona intestinalis (Takamura et al., Zool. Sci. 13: 241-251, 1996). In the present paper, we demonstrate the ultrastructural localization of the antigen reactive to one of the test cell-specific antibody UA165 by immunogold labeling. The mature test cells in Ciona intestinalis contain two types of cytoplasmic inclusions, i.e., irregular vacuoles with electron-dense cores and spherical vesicles with a moderately electron-dense content. UA165-immunogold particles specifically labels the content of the spherical vesicles. Concomitantly with larval tunic formation at the early tailbud stage, the spherical vesicles begin to rupture and the UA165 antigen is exocytosed into the perivitelline space surrounding the embryo. The released antigen gradually deposits on the outermost layer (outer cuticular layer) of the larval tunic. Shortly after the beginning of exocytosis, the test cells scatter in the perivitelline space, move to the embryonic surface, and adhere to the outer surface of the larval tunic. These findings indicate that during the tailbud stage the test cells secrete a component that is necessary for the completion of the larval tunic.

Regulatory mechanisms of cytokeratin expression in the stomach (the proventriculus and the gizzard) epithelia of chicken embryos were examined by epithelial-mesenchymal recombination experiments. In the proventriculus, antigens detected by monoclonal antibodies (PKK1 and AE3) against cytokeratins invariably decreased in gland cells which synthesize pepsinogen, while luminal and duct epithelial cells were positive to these antibodies. When recombined and cultured with the proventricular mesenchyme, the esophageal, proventricular and gizzard epithelia formed proventriculus-type, pepsinogen-expressing glands and the expression patterns of PKK1 and AE3 antigens were identical to that in the normal proventriculus. Under the same experimental conditions, the small-intestinal and allantoic epithelia also formed complex glands but did not express pepsinogen, and PKK1 and AE3 antigens were actively expressed throughout the epithelia. These results indicate that the proventricular mesenchyme can regulate cytokeratin expression and that the expression of cytokeratins in epithelial cells is closely related to the physiological characteristics of cells such as pepsinogen production.

In eggs of the medaka Oryzias latipes, we recently found that the changes in chorion proteins that occur upon fertilization could be induced in vitro by an egg exudate including cortical alveolar contents. In the present study, the time course of the changes in chorion proteins induced in vitro by egg exudate was examined by SDS-PAGE analysis and was found to be quite similar to that of in vivo chorion hardening at the time of fertilization. The activity of egg exudate was dose-dependent. Moreover, when the exudate was heated for 30 min at 60°C, it lost its activity. On the other hand, in heat-treated chorions, the intermediate proteins appeared following incubation with egg exudate, although chorion hardening did not occur. These results suggest the presence of a chorion protein-modifying enzyme(s) in the exudate re-leased from the cortex during egg activation in Oryzias latipes.

An efficient gene trap strategy was devised for identifying the genes that are expressed in the mouse developing nervous system. Mouse embryonic stem (ES) cell lines that carried independent integrations of a gene trap vector, pSneolNlacZA, were allowed to differentiate in a suspension culture system. To select cells containing neurons, astrocytes or neuronglia precursors, cell lines were immunohistochemically examined with antibodies against neuron-specific proteins (neurofilament protein 150 kD and microtubule associated protein 2), glial fibrillary acidic protein or nestin. Three cell clones (GT3-8, 11 and 12) were immunoreactive to either of the antibodies employed and at the same time positive for β-galactosidase activity. When chimeric embryos were generated by the use of the above 3 cell lines, some cells in their nervous system showed X-gal staining. Thus the major advantage of the present gene trap method lies in its prescreening step of manipulated ES cells prior to generation of chimeric animals. This method holds promise as a useful tool for investigating the genes involved in the development of the nervous system.

A cDNA clone encoding the membrane form of guanylyl cyclase was isolated from a Hemicentrotus pulcherrimus testis cDNA library and its nucleotide sequence was determined. The cDNA was 4123 bp long and an open reading frame predicted a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids which divides the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl-terminal, intracellular domain of 594 amino acids. Three potential N-linked glycosylation sites were present in the extracellular domain. Northern blot analysis of poly(A) RNA from testes, ovaries, eggs and embryos at various developmental stages showed that the cDNA encoding guanylyl cyclase hybridized to a mRNA of 4.4 kb from the testes.

We developed a large scale purification method of the phosphorylated (131 kDa) and dephosphory-lated (128 kDa) forms of the membrane-bound guanylyl cyclase from H. pulcherrimus spermatozoa. The purified 131 kDa and 128 kDa forms of the guanylyl cyclase contained 26.0 ± 1.3 and 4.3 ± 0.7 moles of phosphate per mol protein (mean ± S.D.; n=6), respectively. The amino-terminal amino acids of both the 131 kDa and 128 kDa forms of the guanylyl cyclase could not be detected, suggesting that they were blocked.

Molt-inhibiting hormone (MIH) activity was tested in terms of the inhibitory activity of ecdysteroid secretion by cultured Y-organs of the crayfish, Procamharus clarkii. Several neuropeptides were separated from a crude extract of sinus glands of P. clarkii by means of reverse-phase high-performance liquid chromatography (HPLC) and tested for MIH activity. An extremely high level of activity was found in one fraction in reverse-phase HPLC, suggesting that the novel neuropeptide in this fraction may be a P. clarkii MIH.

The divergency of endogenous mouse mammary tumor virus (MMTV) in European mouse strains indicates acquisition of exogenous MMTV into their genomes during evolution. In the present study, we selected two strains of Asian wild mice, Cas-Hmi (Mus musculus castaneus) and Sub-Kjr (M. m. subspecies) to investigate a possible divergency of endogenous MMTVs among Asian mouse strains. Southern blot analysis and polymerase chain reaction (PCR) demonstrated that Cas-Hmi and Sub-Kjr have the full structure of integrated endogenous MMTVs, suggesting that these mice were diverged during evolution after MMTV endemics.

The batagurid turtle, Mauremys mutica, is widely distributed in tropical to temperate East Asia. Analyses of morphometric characters and coloration revealed that the southern Ryukyu populations of this species are much diverged from the other populations, presumably as a result of their long geographical isolation. We describe those populations as a new subspecies, M. m. kami. Analysis of geographic variation also suggested that distinctly isolated populations of the central and northern Ryukyus, and Kyoto and Shiga Prefectures of central Japan have originated from animals artificially introduced from the Yaeyama Group, and Taiwan, respectively. We confirmed the absence of “larger female” sexual size dimorphism (SSD) in M. mutica unlike most other aquatic batagurids, and further demonstrated variation in SSD pattern between the subspecies: in M. m. kami, the adult male has a significantly greater carapace length than adult females, whereas the adult carapace length does not differ significantly between sexes in the nominotypical subspecies. It is hypothesized that these dimorphic patterns evolved from the widely prevailing “larger female” condition through epigamic selection involving forcible copulatory behavior.

Nucleotide sequences encoding GroEL homologs of intracellular symbionts in three closely interrelated aphids were compared with one another and that for GroEL. It was suggested that in these proteins a particular position is highly susceptible to amino acid substitution, through which the GroEL homologs of symbionts seemed to have acquired a unique function on top of the activity as molecular chaperone. This may represent a rare example of non-neutral evolution of molecule under the positive selection pressure.

Protogynous sex change in Gnorimosphaeroma naktongense, inhabiting brackish water and freshwater bodies, was examined by culturing precopulatory guarding pairs. The transitional stages of masculinizing of a female were observed clearly after releasing juveniles from a female marsupium. About forty percent of the females cultured showed direct evidence of sex change to males in the outer morphology at the end of laboratory experiments. As histological observations were not done to make clear the processes of gonad changes, the possibility of protogynous sex change was strongly suggested. Precopulatory mate guarding may be a primary selective pressure favoring protogyny in G. naktongense.