I have reported previously that axotomy of an identifiable anal motoneuron of crayfish Procambarus clarkii induces a long-lasting firing and that a prolonged depolarizing pulse to its cut end can induce a similar response. In this study, I confirmed that this stimulus is comparable to axotomy; the frequency of stimulus-induced firing increases linearly with the stimulus intensity and its firing pattern is the same as that following axotomy. Then, when the cut end was bathed for more than 1 hr in test solutions, it was examined whether the stimulus to the cut end induces or blocks the response. Na -free saline (Tris replaced Na ) or TTX (3 × 10^–7 M) reversibly blocked the response within 30 min. By contrast, Mn² saline (40 mM Mn² replaced Ca² ) or Ca² -free salines (Mg² or 1 mM EDTA replaced Ca² ) cannot block the response, but instead increased the firing frequency. These results obtained with stimulus were confirmed also by those with axotomy. I concluded that axotomy-induced firing, which occurs locally at its cut region, is primarily responsible for voltage-dependent Na conductances, but not for Ca² ones.

Innervation of the heart muscle by the cardioacceleratory neurons was morphologically and electrophysiologically examined in the isopod crustacean, Ligia exotica. Intracellular injection of neurobiotin into the first and second cardioacceleratory neurons (CA1 and CA2) revealed their peripheral axonal projections. Inside the heart, the CA1 and CA2 axons ran along the trunk of the cardiac ganglion. Finely arborized branches with many varicosities arose from the axon and projected over the heart muscle. Stimulation of either the CA1 or CA2 axon caused an overall depolarization in the muscle of a quiescent heart. The amplitude of the depolarization increased with increasing stimulus frequency. During stimulation, the membrane resistance of the heart muscle decreased. In a beating heart, the cardioacceleratory nerve stimulation caused multiple effects on the heart muscle activity and the heartbeat. The results suggest that the cardioacceleratory neurons of Ligia exotica regulate the amplitude of the heartbeat (inotropic effect) and the heart tonus (tonotropic effect) via the synaptic contacts on the heart muscle, while the heartbeat frequency (chronotropic effect) is regulated via the synapses on the cardiac ganglion neurons.

Cerebella from suckling and adult rats were examined histochemically with 19 different biotinlabeled lectins. Purkinje cells from postnatal rats had a marked ability to combine with many lectins, but minimal ability was found in adult rats except for Con-A, LEL, and MAL lectins. The cell body of Purkinje cells on postnatal day 7 was strongly labeled with 6 lectins (Con-A, LTL, MAL, SJA, UEA-I, and VVA). Only moderate staining was observed with these lectins on postnatal day 5. The dendritic tree of the cells showed a moderate labeling ability with LTL and UEA-I on postnatal days 15 and 20. The dendritic tree was strongly labeled with MAL on postnatal days 10, 15, 20 and adult. Positive reactions were observed in the cells when cerebellar sections from rats on postnatal day 7 were incubated with 3 other lectins (AAL, LEL, and SBA). The cells on postnatal day 7 were rarely labeled with BSL-II, DBA, DSL, LCA, PNA, PSA, RCA120, SSA, STL, and WGA. Purkinje cells on postnatal day 7 may be rich in N-linked oligosaccharides, with terminal sugar structures that resemble blood-group-related antigens (type H) and/or tumor-related antigens. These glycoconjugates may be present at low levels in the Purkinje cells of adult rats. Dendrites of Purkinje cells of adult rats were strongly labeled by Con-A, LEL, and MAL. The dendrites of Purkinje cells may be rich in highly branched oligosaccharides.

The relationships of salinity tolerance to immunolocalization of Na ,K -ATPase in the gill epithelium were examined during seawater and freshwater adaptation of the guppy. In fresh water, immunoreactivity for Na ,K -ATPase appeared in two types of chloride cells, which are located on the primary lamellae of the gills. Immunoreactivity was strong in the chloride cells located at the base of the secondary lamellae and weak in the chloride cells located at the interlamellar region. During seawater adaptation, the strongly-immunoreactive chloride-cells increased in number and size while the weakly-immunoreactive chloride-cells decreased in number with an increase in salinity tolerance. In the fish of the seawater-adapted strain, on the other hand, most of the chloride cells were located at the base of the secondary lamellae and showed strong immunoreactivity. During freshwater adaptation, the strongly-immunoreactive chloride-cells decreased in number and size while the weakly-immunoreactive chloride-cells increased in number with a decrease in salinity tolerance. A positive correlation was observed between the salinity tolerance and the occupying area of the strongly-immunoreactive chloride-cells while a negative correlation was observed between the salinity tolerance and the occupying area of the weakly-immunoreactive chloride-cells during the seawater and freshwater adaptation. These results directly suggested that not only the occupying area of chloride cells but also the expression of Na ,K -ATPase protein in the cells is important with respect to the osmoregulatory function in the gills and hypoosmoregulatory ability at the individual level.

The 20S proteasome purified from animal cells has various latent peptidase activities. Fatty acids such as linoleic, linolenic and oleic acids strongly activate both the chymotrypsin-type and peptidylglutamylpeptide (PGP) hydrolase-type activities, but have been reported to have little activation or inhibition of the trypsin-type activity. We show here that an increase of the fatty acid concentration produces activation of chymotrypsin-type and PGP hydrolase-type in a biphasic fashion: no effect until the threshold concentration and then a sharp activation. In contrast, the trypsin-type activity was markedly inhibited at low concentrations of fatty acid, slightly activated at higher concentrations, and inhibited again at even higher concentrations. The inhibition was removed when the concentration of fatty acid was reduced by dilution after pre-incubation with the fatty acid. As a result, the activation pattern became biphasic, which was identical to that of chymotrypsin-type and PGP hydrolase-type activities. These results suggest that in the chymotrypsin-type and PGP hydrolase-type peptidases fatty acids bind first to a class of sites without direct effect on the peptidase activity, but after saturation of this class it permits more fatty acid to bind to another class of sites involved in the activation. In the trypsin-type peptidase an additional class of fatty acid binding sites is uniquely present, which is involved in the enzyme inhibition. The dilution procedure described above removes the fatty acid molecules bound to the inhibition sites, but not the fatty acid molecules bound to the activation sites; this results in the fatty acid activation profile indistinguishable from that of the chymotrypsinand PGP hydrolase-type peptidases.

This paper aims at examining the effect of colchicine, a microtubular poison, on the process of furrow formation in whole eggs and egg fragments as well as the process of artificial induction of furrow-like dents, in eggs of the newt, Cynops pyrrhogaster. To apply colchicine locally to eggs, the eggs were slit across or along a furrow in a colchicine solution during first cleavage. When a slit was made across or in front of a growing furrow at the onset of its growth, the furrow quickly ceased growing and often regressed. Cortices containing an entire growing furrow were isolated along with a thin layer of subcortical cytoplasm immediately after the start of the first cleavage. Furrows in the cortices degenerated when the cortices were cultured in a colchicine solution, whereas they continued growing when they were cultured in Holtfreter's saline. Furrow-inducing cytoplasm was injected to a site beneath the cortex in the animal half of the egg during first cleavage. When a small slit was made close to the site of the injection in a colchicine solution, no furrow-like dent was induced. These results imply that microtubules are directly involved in the generation and growth of cleavage furrows.

Teleost fish develop seven pharyngeal arches (mandible, hyoid and five gill arches) which give rise to the jaw and gills, and skeletal cell populations which originate from the cranial neural crest. The anterior border of expression of the Deformed (Dfd) group is located in the hindbrain and pharyngeal region. To investigate pharyngeal skeletal formation in the teleost fish, we cloned the cDNA coding Hoxd-4 from a cDNA library for flounder (Paralichthys olivaceus) embryos, and analyzed gene expression pattern during embryogenesis and the effects of retinoic acid (RA) on this gene expression. Between the 21-somite and prim-5 stages, Hoxd-4 was expressed in the central nervous system from rhombomere 7 to the spinal cord, and in the pharyngeal region posterior from gill arch 2. Its expression then became restricted to cartilage precursor cells of gill arches 2-5. When embryos in the early shield stage were exposed to RA at concentrations above 10⁻⁷ M, the anterior border of Hoxd-4 expression shifted anteriorly in a dose-dependent manner, both in the central nervous system and pharyngeal region. We propose that, during gill skeleton formation, Hoxd-4 functions in the specification of regional identity between gill arches 1 and 2, and that their identity is affected by treatment with RA.

The developmental roles of factors associated with the nuclear complex of Halocynthia roretzi and Boltenia villosa oocytes were investigated by cutting mature oocytes into animal and vegetal merogons before and during GVBD. Animal and vegetal merogons were cultured in sea water until the GV cytoplasm had dispersed within the cytoplasm of control oocytes and then they were cross-fertilized and scored for their ablility to undergo normal development. Halocynthia oocyte fragments produced from the animal region of oocytes containing intact GVs exhibited a low frequency of polyspermy, a high frequency of fertilization and cleavage, and a high frequency of expressing an epidermal antigen, Epi-2. In contrast, merogons produced from the vegetal region of Halocynthia oocytes in which GVs were intact exhibited a high frequency of polyspermy, did not undergo cell division, and expressed a high frequency of Epi-2 expression. When vegetal fragments were produced after the dispersal of approximately 50-70% of the GV nucleoplasm, these merogons exhibited a low frequency of polyspermy, high frequencies of cell division (including the formation of epidermal layer), and in most cases expressed Epi-2. Vegetal Boltenia fragments produced during GVBD in some cases developed into larvae. These results suggest that the ascidian GV nucleoplasm may contain factors required for fertilization and cell division and that epidermal determinants reside in the oocyte cytoplasm.

In Dictyostelium discoideum Ax-2, the cell-cycle progression from the early aggregate to mound stage has been proposed to have some connection with prespore differentiation. Hereupon, we examined the role of cell-cycle progression during the development on cell differentiation, using two kinds of cell-cycle inhibitors. Nocodazole, an inhibitor of microtubule formation, was found to inhibit greatly cell division around the mound stage as well as during the vegetative growth phase, when applied to exponentially growing Ax-2 cells. Essentially the same inhibition was attained by treatment of starved Ax-2 cells with calyculin A, an inhibitor of serine/threonine protein phosphatases. It is noteworthy that the nocodazole-or calyculin A-treated cells exhibit abnormal morphogenesis to form a stick-like multicellular structure on non-nutrient agar, and also that prespore differentiation as exemplified by the prespore-specific Dp87 gene expression and prespore specific vacuole (PSV) formation was greatly suppressed. In contrast, the differentiation of prestalk (pstA) cells was scarcely affected by the drug treatments. Taken together these results seem to indicate that the cell-cycle progression around the mound stage is important for prespore differentiation.

The spawning and early embryogenesis of the hemichordate, Ptychodera flava, in Hawaii are described in detail and illustrated with photographs of living material. Natural spawning in the evenings of early December was induced by a shift of seawater temperature from about 22°C to about 26°C. The fertilized egg divides equally and slowly at first, reaching 8 cells at about 5 hr after insemination at room temperature (20-24°C). Divisions then appear to become slightly unequal and by 9 hr the embryo has divided into about 100 cells. The blastocoel forms during cleavage as an irregular space that, when viewed from the side, tends to appear oblate and ultimately appear crescent-shaped as the vegetal plate thickens into the blastocoel. The archenteron forms at about 18 hr as a cleft beginning at the vegetal pole and extending into the vegetal plate. As development proceeds, the embryo expands and by 24 hr forms a typical deuterostome gastrula with an outer sphere of ectoderm and a inner tube of endoderm connected at the blastopore. An out-pocketing of the gut appears at the tip of the archenteron over the next 4 hr to form the protocoel which will become the proboscis coelom. Approaching 40 hr the gut becomes asymmetric and over the next few hr contacts the ectoderm to form a mouth. Hatching occurs during this time at about 45 hr of development. Morphogenesis continues to produce an early tornaria larva by about 60 hr.

Lectin cytochemistry was carried out on thin sections of 6th-instar cricket testis using two GalNAc-specific lectins, Dolichos biflorus agglutinin (DBA) and Helix pomatia agglutinin (HPA), and the binding sites in primary spermatocytes were surveyed. Gold particles showing DBA-binding are observed specifically in dense-body clusters. These bodies are about 100-300 nm in diameter and exhibit multilaminar or multivesicular structure. HPA can bind to the dense-body clusters and another kind of larger multivesicular structures. These bodies seem to contain some heterogeneous substances, and sometimes show an autophagosome-like structure. The ultrastructures of these organelles surveyed in conventional Epon sections confirmed the structures of these multilaminar/vesicular bodies in cricket spermatocytes, which may play certain roles in intracellular circulation and degradation of glycoconjugates.

Effects of a soft diet (reduction in mastication activity: an exogenous factor) and hypothyroidism (endogenous factors) on the oxidative capacity of the masseter muscles in the young Japanese field vole Microtus montebelli, consisting only of fast-twitch oxidative (FO) fibers in the adult vole, was investigated histochemically and electron microscopically. Oxidative enzyme activity and mitochondrial development in the muscle fibers were not affected by a soft diet, while they were suppressed by doses of propylthiouracil (PTU) (hypothyroidism). Thus, it was suggested that acquisition of the sustained contraction ability in the masseter muscles of the young vole is induced by the endogenous factors rather than the exogenous factor.

Penguins are highly adapted to marine life. Their hydrodynamic efficiency depends on feathers which wear with age and need to be replaced regularly. During molting, penguins can not enter the sea to forage and are forced to fast. Therefore the duration of molting is necessarily brief. To better understand molting in penguins, we collected plasma samples from 16 (8 pairs) Humboldt penguins kept in an open display pen at Tokyo Sea Life Park from May to September, 1994 and estimated circulating concentrations of LH, testosterone, estradiol, thyroxine (T4), triiodothyronine (T3) and corticosterone. Body mass was also measured at each blood sampling. Throughout the year, reproductive activities (egg laying, incubation, hatching and rearing) and molting were observed and recorded. Humboldt penguins maintained reproductive activity from January to December except during molting. Each pair started molting between the end of July and early August; usually males started earlier. The duration of molting was 13.4 ± 0.8 days for males and 12.9 ± 0.3 days for females. Body masses were highest just before the start of molting in both sexes. Plasma concentrations of LH were high, (> 2 ng/ml) in May in both sexes, then gradually decreased, to 0.53 ± 0.38 ng/ml in males and 0.72 ± 0.11 ng/ml in females by the end of July. Testosterone and estradiol concentrations in plasma decreased and were lowest during molting. On the other hand, plasma concentrations of T4 were low until early July (ca. 20 ng/ml) and then doubled within 10 days; the high levels were maintained for one month and then decreased greatly in males and slightly in females. When the plasma concentrations of T4 started to decrease, plasma concentrations of LH increased. Changes in plasma T3 were not consistent with molting. These results indicate that the decrease of plasma levels of sex steroid hormones and the sharp increase of T4 induced molting, which lasted only for a short period.

Changes in cystine aminopeptidase (CAP) activity in serum, placenta, uterus and liver in mice under various physiological conditions were examined by enzymatic analysis using S-benzyl-L-cysteine-4-methylcoumaryl-7-amide as a substrate. Changes in serum CAP activity during pregnancy were less remarkable than those reported in humans; the activity was highest at day 13 of pregnancy and lowest at day 17. During the estrous cycle, the serum CAP level was high at diestrus, as high as that at day 13 of pregnancy. The activity in the male serum was significantly low compared to that in the diestrous serum. Although the CAP level was especially high in the maternal placenta, levels were also high in the fetal placenta, uterus and liver as compared with the serum, suggesting CAP synthesis in these tissues. The CAP activity in the uterine tissue was lower in pregnant than normal cycling mice. Furthermore, the serum CAP activity was modulated by estrogen and progesterone both in females and males, and by androgen in males. The relevance of these findings to the physiological role of CAP was discussed.

1-Methyladenine (1-MeA) has been identified as the oocyte maturation-inducing substance (MIS) in starfish, but little is known about its biosynthesis. This study showed that starfish MIS activity was present in a reactant derived from S-adenosylmethionine (SAM) by heat treatment. In vitro MIS production was markedly dependent on the temperature of the SAM solution: it increased as the temperature was raised, and reached a plateau within 5 min upon boiling, although hardly only MIS was observed upon incubation below 20°C. MIS production was also dependent on the solution pH. Analyses by high-performance liquid chromatography and thin-layer chromatography showed that the MIS was 1-MeA, though the maximum amount of 1-MeA obtained from SAM by boiling was only 0.3% of the initial SAM amount. Furthermore, use of S-[¹⁴C-methyl]SAM showed that a methyl group of 1-MeA was transferred from the SAM. Thus, it is possible that 1-MeA may be produced from SAM in vivo.

The lf (leucophore free) locus was previously reported autosomal recessive in the medaka fish (Oryzias latipes). However, extensive linkage analyses in this study using various strains revealed that the lf locus was closely sex-linked. The recombination frequency between lf and the male determining factor (y) was 2.2% (10 recombinants out of 464 progeny). Because the lf/lf homozygous fish do not have visible leucophores, they are distinguishable from wild type in early developmental stages. In the Qurt strain with heterozygous sex chromosomes (X^lf/X^lf in females and X^lf/Y in males), we can predict sex of each embryo on second day after fertilization. The strain should be a very useful material for studying sex determination or differentiation mechanisms in the medaka fish.

The effects of the consumption of the spermatophylax produced by males on female fitness were studied in the decorated cricket, Gryllodes sigillatus. An increase in the number of spermatophylaces presented to females did not increase the total number of eggs made by females, the number of eggs laid, or the hatchability of eggs laid by females, but increased the number of eggs laid in the early stage of adult life of females. The duration of the egg stage decreased with the number of spermatophylaces presented to females. The implication of the results on the sham hypothesis that the spermatophylax does not have nutritional value is discussed.

The behavior of Heterolepidoderma sp. was studied with the same approach as those already used for many species of ciliates. The ethogram we drew comprehends both helicoidal swimming (n = 20, r = 52.5 ±12.2 μm, pitch = 512 ± 101 μm, v→ = 215 ± 43 μm/sec), periodically interrupted by irregular patterns changing the direction of the swimming of random angles and creeping on the substrate. The latter behavioral state, very common for the species we studied, occurs along tracks formed by successive elements (circular, C, vs linear segments, S) joined to each other by two kinds of reactions, which change their trajectory. The surprising similarities and the unexpected differences between the behavior of this gastrotrich and those of the ciliates already studied from this point of view are discussed, on the basis of the dimensional ranges and ecological niches shared by these two, definitely unrelated groups of organisms.

For the elucidation of the phylogenetic position of insectivora in eutheria, we have sequenced the cytochrome c oxidase subunit II (COII) gene of mitochondria for three insectivoran species [musk shrew (Suncus murinus), shrew mole (Urotrichus talpoides), Japanese mole (Mogera wogura)] and analyzed these amino acid sequences with neighbor-joining (NJ) method and maximum likelihood (ML) method. NJ analysis shows polyphyly of Insectivora and Chiroptera. Assuming that each of Primates, Ferungulata, Chiroptera, Insectivora and Rodentia is a monophyletic group, ML analysis suggests that Chiroptera is a sister group of Insectivora and that Ferungulata is the closest outgroup to the (Insectivora and Chiroptera) clade.

We determined the nucleotide sequences of a part of the mitochondrial cytochrome c oxidase I gene (1,000 bp) for twelve species of Asian phytophagous ladybird beetles belonging to the genus Epilachna, and constructed molecular phylogenetic trees for ten “Henosepilachna” species, using two “Epilachna” species as outgroups. Based on the suggested phylogenetic trees, we discussed taxonomic issues and the direction of host shift in these epilachnines.

A new oribatid mite belonging to the family Oppiidae is described from the province of Biscay in the Basque Country, Northern Spain. We propose the name Corynoppia papillisetigera for it, whose shape and size of setae ta (c₂) bearing papillae is the main and distinctive feature to separate it from allied species, such as Corynoppia foliatoides ^{Subías et Rodríguez, 1986}. For the moment it has been found in a coastal meadow and eucaliptus, evergreen-oak, and pine forests.

Okinawan sea urchins, the genus Echinometra, are four independent species. But which species are the same species as E. mathaei and E. oblonga as described by Blainville 1825 is still open to question. To answer this question, a field survey of genus Echinometra was made in Mauritius (the type locality of E. mathaei) according to the characteristics used to classify Okinawan Echinometra: appearance, pore pairs, spicules in gonads and tubefeet, sperm shape, and distribution on a reef. The results of crossfertilization between Echinometra from Mauritius and Okinawa are also reported. Mauritian Echinometra are classified into three groups which resemble Okinawan Echinometra sp. B, D, and violet spine color Echinometra. The latter has almost the same characteristics as Okinawan Echinometra sp. B but with violet spines, a spine color not found in Okinawa. In cross-fertilization experiments, the sperm of Mauritian Echinometra sp. B-like and violet Echinometra fertilized Okinawan Echinometra sp. B with almost 100% success. However, fertilization was unsuccessful with other Okinawan Echinometra species. Therefore, it could be said that Okinawan Echinometra sp. B is the same as Mauritian Echinometra sp. B-like, and the counterparts of Okinawan Echinometra sp. A and C are not distributed in Mauritius. The descriptions of E. mathaei most match Mauritian Echinometra sp. B-like and the type locality of E. mathaei is Mauritius. Thus, it is probable that Mauritian Echinometra sp. B-like is E. mathaei. Therefore, Okinawan Echinometra sp. B, which most resembles Mauritian Echinometra sp. B-like, would be also E. mathaei. Echinometra sp. A and C would be considered to be new species. Echinometra sp. D is thought to be the same species as Mauritian black Echinometra. Whether Okinawan Echinometra sp. D is the same as E. oblonga remains as a problem for future research. Because the type locality of E. oblonga is not known and, it is suggested that the E. oblonga described in the Indo-West Pacific is a complex species.