Zoological Science

The response of neurons of the budgerigar (Melopsittacus undulatus) was obtained in Field L, which is a laminated auditory structure of the caudal neostriatum in the telencephalon. Warble song of this species is comprised of a number of sounds (elements). The influence of sequence and silent intervals between elements on neuronal response was investigated. First, neurons in Field L were examined to know if neuronal response to isolated elements differed from that to the same elements in warble song, a sequence of elements. Sixty-two percent of the neurons exhibited increases of spike activity in response to elements in isolation compared with that to elements in warble song. These neurons thus exhibited temporally suppressed response. We then examined neuronal activity using the stimulation with paired elements, separated by various silent intervals (delta t msec). The spike activity in response to a specified element decreased as the interval was shortened. The responses of most neurons were strongly suppressed at the delta t of 80 msec, which is often seen in element intervals of warble song. In some neurons in Field L the response was suppressed, although they did not respond to the preceding sound. We hypothesize that temporally suppressed neurons may play a role in vocal discrimination.

The present work was performed to clarify the photosensitive stage for wing-form determination and reproduction in Aquarius paludum. Only 40–60% of the adults were long winged when they were reared under 12 hr light-12 hr dark (12L-12D) during the first two instars and under 15.5L-8.5D during the rest of nymphal stage. However, all became long-winged, when they were exposed to 12L-12D until the end of the third instar. The exposure to 15.5L-8.5D till the end of the third instar induced short-winged adults of less than 15%, but long days until the fourth instar promoted about 40%. The exposure to long days either during the third, fourth or fifth instar promoted brachyptery, whereas all became long-winged when they were reared under long days either during the first or second instar. The exposure during the fifth instar induced maturing oocytes. The results indicate that the photosensitive stage for brachyptery occurs from the third to the beginning of the fifth instar, whereas that for oocyte maturation occurs during the fifth instar. Such a difference may refute the supposition of the oogenesis-flight syndrome that the two factors are tightly linked.

Formoguanamine hydrochloride (FG) is known as a potent chemical to induce blindness in chick eyes by disrupting the pigment epithelium and visual cells in the retina. In this study, we examined the effect of FG on the structure and function of the compound eyes of the butterfly, Papilio xuthus (Lepidoptera, Insecta). We administrated 2 mg FG per 1 g body weight of the pupa at about the first one third of the whole pupal period, because accomplishment of morphogenesis of the compound eye occurs in the last half of the pupal period. As a result, unusual membranous structures such as trophospongium-like structures and myeloid bodies were observed in the cytoplasm of the retinular cells besides the normal rhabdom. This result suggests that FG treatment influences on some steps in the formation of rhabdom membranes. However, the amount of chromophore, 3-hydroxyretinal and the responses to white light recorded by the ERG method from FG-treated specimens were not different from the control animals.

Ascidians are known to accumulate vanadium in their blood cells (vanadocytes) at extremely high levels which correspond to about 10⁶ to 10⁷ times the levels of vanadium ions in seawater. The route for the accumulation of vanadium ions from the outside environment into the blood system in ascidians has not yet been discovered. In the present experiments, using a combined technique of anion exchange column and atomic absorption spectrometry, we first extracted a vanadium-associated protein (VAP) from the blood cells of the ascidian Ascidia sydneiensis samea. VAP was estimated to associate with vanadium at an approximate ratio of 1 mol: 16 mols. SDS-PAGE and a polyclonal antibody against VAP (anti-VAP) revealed that VAP is composed of at least two types of peptides estimated to be 12.5 kDa and 15 kDa with a minor peptide of 16 kDa and that VAP is localized in the cytoplasm of the vanadocytes.

The polychaete Pseudopotamilla occelata is the first animal revealed to contain high levels of vanadium besides ascidians. The present experiment disclosed that P. occelata has the same antigens with those in the ascidian Ascidia sydneiensis samea, which were recognized by two types of antibodies, a polyclonal antibody against vanadium-associated proteins extracted from blood cells and a monoclonal antibody against vanadocytes in the vanadium-rich ascidian A. sydneiensis samea. There is, therefore, a possibility that similar mechanism works on the accumulation of vanadium between the Polychaeta and the Ascidiidae.

The mudskipper, Periophthalmus modestus, inhabits the intertidal zone and is active on the surface of mud flat during low tides. We investigated relationships between the behavior and environmental variables during low tides from daily and annual aspects. The observation was done in two distant sites where tidal cycles and population densities are different. The emergence of the mudskipper on the surface of the tidelands was influenced by ambient temperature; low temperature inhibited emergence and locomotor activity of the mudskipper. The mudskipper retreated into the mud as the time went on after emersion of the tideland in order to avoid increase of body temperature and desiccation of the epidermis. Therefore, the emergence of the mudskipper seems to be directly suppressed by drying up of the mud surface. There were no differences in the diel and annual changes in the activity pattern between the populations. The emergence exhibited synodic changes in both areas, and it was correlated not with lunar phase but with tidal amplitude, peaking at the time of lower ebb tides with high air temperature.

The small Japanese field mouse Apodemus argenteus has the diploid chromosome number of 46, carrying rather large centromeric C-heterochromatin in most of the 44 autosomes and a large amount of C-heterochromatin in the sex chromosomes: the largest subtelocentric X was heterochromatic in almost two-fifth (whole short arm and proximal part of the long arm) of its entire length and the medium-sized acrocentric Y was totally heterochromatic. The C-heterochromatin (C-positive) areas, other than those of the Y and smallest three pairs, had a unique property of “delayed QM-fluorescence”, which has not been reported to-date, showing dull QM-fluorescence immediately after exposure to blue light (BL), but gradually turning to bright fluorescence in a few minutes. The fluorescence intensity gradually decreased after attaining its peak, and finally became extinct. A similar pattern of fluorescence was also obtained in DA/DAPI-stained X chromosome C-heterochromatin, but not in autosomal C-heterochromatin. No such dull-to-bright transition of QM-fluorescence could be obtained by CMA₃ staining, for which the C-positive areas were apparently negative even after overexposure to BL. These facts indicate that the C-positive areas of A. argenteus showing dull-to-bright transition of QM-fluorescence contain A-T rich DNA. The delayed QM-fluorescence was found only in A. argenteus, in thirteen mammalian species so-far examined. Furthermore, this unique property of QM-fluorescence could be artificially altered to non-delayed ordinary type of fluorescence by sequentially pretreating the fixed chromosomes with hydrochloride and barium hydroxide solutions. The cytological implication of the delayed fluorescence in the C-heterochromatin of A. argenteus is briefly discussed.

To identify and characterize a chitinase related to molting in the Kuruma prawn Penaeus japonicus, we searched for chitinase-encoding cDNAs expressed in cuticular tissues. Using two degenerate oligonucleotide primers derived from the two conserved regions of the chitinase protein family, a RT(reverse transcription)-PCR product was obtained. This product was used as a probe to screen a cDNA library from a mixture of the tail fan and blade — two tissues which consist mainly of chitinous exoskeleton and underlying epidermis. A positive cDNA clone was analyzed for the sequence. This clone contains an open reading frame for a protein (named Pjchi-2) of 527 amino acids which exhibits sequence similarity to known chitinases. A typical signal sequence could not be found in the Pjchi-2 sequence. Significant accumulation of Pjchi-2 mRNA was detected in the mixture of the tail fan and blade prior to molting, whereas the transcript level was much lower during the intermolt stage. This observation suggests that Pjchi-2 plays a role in molting. The mRNA was not detected in the hepatopancreas. This expression pattern of Pjchi-2 makes a contrast to that of Pjchi-1 which encodes another chitinase family protein in P. japonicus, and is expressed in the hepatopancreas but not in the tail fan or blade.

We purified a 5.4 kDa peptide which is present in the intermediate and infectious form, but not in the reproductive form of a symbiotic bacterium Holospora obtusa of the ciliate Paramecium caudatum. Sequencing of its gene revealed that it encodes a peptide composed of 49 amino acids, and that the peptide is preceded by a putative signal peptide of 19 amino acids. We determined the transcription start point for the gene by primer extension analysis, indicating that the transcription starts with a G nucleotide located 33 nucleotides upstream from the translational initiation codon. Northern blot hybridization showed that the gene is highly expressed in the intermediate form, a transitional stage from the reproductive to infectious form of the bacterium. Immunoelectron microscopy with anti-5.4 kDa peptide antiserum revealed that the 5.4 kDa peptide is localized in the periplasm of the infectious form.

Different types of actin-related proteins which constitute an actin-superfamily together with conventional actin have recently been described (Mullins et al., 1996). Among them, Arp1 exhibits the highest homology with conventional actin. With the aim of clarifying the cellular function of Arp1 in mammalian cells, we cloned the cDNA encoding mouse α-Arp1, one of the variants of Arp1, from a mouse diaphragm cDNA library; two types of α-Arp1 cDNAs, which are probably generated by alternative RNA splicing from a single gene, were obtained and the entire sequences were determined. They differed only in the presence or absence of an insertion of 1.3 kb in the 3'-non-coding region but shared a common open reading frame. The deduced amino acid sequence was identical with that of human α-Arp1. Northern blot analysis showed that the a-Arp1 mRNA corresponding to the longer cDNA is transcribed not only in various non-muscle tissues but also in muscle tissues, while the transcript corresponding to the shorter one becomes expressed only in skeletal muscle as development progresses. It is suggested that α-Arp1 may play some role in muscle, as judged by the significant level of its expression.

β-Giucosidase ‘EC 3.2.1.21’ and endo-β-1,4-gIucanase ‘EC 3.2.1.4’ activities were measured in the wood-eating higher termite Nasutitermes takasagoensis. β-Glucosidase activity was present mainly in the salivary glands (66.7%) and midgut (22.2%), whereas endo-β-1,4-glucanase activity was detected mainly in the midgut (90.1%). Specific activity of endo-β-1,4-glucanase was also the highest in the midgut, indicating that cellulose is digested in the midgut. The major endo-β-1,4-glucanase component of N. takasagoensis was purified from whole termites by gel filtration on Sephacryl S-200 HR, Superdex-75 and hydroxyapatite column chromatography. Subsequently, the endo-β-1,4-glucanase activity from a crude midgut extract was eluted in an identical volume (Kd=0.68) to that from whole termites, suggesting the purified endo-β-1,4-glucanase is identical to that in the midgut. The molecular weight of the purified endo-β-1,4-glucanase was 47 kDa, and its specific activity was 1,200 units/mg. The optimal pH and temperature were 5.8 and 65°C, respectively. The K_m and V_max values on carboxymethyl cellulose were 8.7 mg/ml and 2,222 units/mg, respectively. The purified endo-β-1,4-glucanase hydrolyzed cellopentaose to cellotriose and cellobiose, and cellotetraose to cellobiose and a trace of cellotriose and glucose, but cellotriose and cellobiose were not hydrolyzed. The activity and stability on pH and temperature of the purified endo-β-glucanase are prominent among those from various organisms.

Membrane type matrix metalloproteinase (MT-MMP), which possesses a C-terminal transmembrane domain, is expressed on the ceil membrane (Sato et al., 1994, Nature 370: 61-65). It was suspected, therefore, that the expression of MT-MMPs might be regulated by cell-cell interactions. We examined the patterns of MT1-MMP expression in a mouse mammary gland epithelial cell line, HC11, which is capable of responding to prolactin in vitro. HC11 cells form well-differentiated monolayer of cuboidal epithelium at confluence. During the log growth phase, cells which are well dispersed and seemingly migrating actively, or located at the periphery of small colonies, reacted strongly with an anti-MT1-MMP antibody, whereas no MT1-MMP immunoreactivity was detected in the cells which established cell-cell contact with adjacent cells. At confluence, the HC11 cells lost MT1-MMP immunoreactivity completely. Northern blot analysis revealed that MT1-MMP mRNA is present at a high level in HC11 cells during the log phase of growth. Although MT1-MMP immunoreactivity disappeared by the 1st day confluence was reached, the decline of MT1-MMP mRNA levels started only a few days later. The discrepancy in the timing of decrease of MT1-MMP protein and that of the transcripts suggests the presence of translational control mechanisms for MT1-MMP expression during cell-cell interaction.

The medium in which dejellied prehatching Xenopus laevis embryos were cultured (hatching medium) can solubilize the vitelline envelope (VE) of dejellied eggs and dimethyl casein. Western blot analysis using antibodies against the hatching enzyme revealed the presence of 60 kDa and occasionally 40 kDa molecules in the hatching medium. Ion-exchange chromatography or gel-filtration followed by enzyme activity assays indicated that the fractions containing 60 kDa molecules exhibit VE-solubilizing activity but those containing 40 kDa alone do not, although both fractions exhibit the same level of proteolytic activity. However, solubilization of the VE was obtained when the 40 kDa fraction was mixed with an extremely low concentration of the 60 kDa fraction that cannot solubilize VE by itself, or when the 40 kDa fraction was applied to the VE that had been pretreated with a low concentration of the 60 kDa fraction. We propose that recognition and/or processing of the VE by 60 kDa molecules make solubilization of the VE possible by 40 kDa molecules.

Two-dimensional distributions of calcium, phosphorus, strontium, and zinc in regenerating scales of carp, Cyprinus carpio, were observed over a period of 90 days by utilizing synchrotron radiation-excited X-ray fluorescence (SRXRF) imaging. On the 15th day, these elements were highly accumulated in the part that is exposed to the ambient water, and the accumulated amount decreased linearly toward the basal edge through the focal area of the scale. As the regeneration progressed, this characteristic pattern changed such that the amount of each element in the rim part, which was exposed to the ambient water, obviously increased, while the amount in the basal edge decreased. However, the relative amounts of phosphorus, strontium, and zinc with respect to calcium in the regenerating scale remained almost constant during the regeneration. In addition, it was found that the diameter of, and the number of ridges in, the regenerating scales increased rapidly during the first 15 days, and increased at a slower pace later on.

Cells prepared from chicken skeletal muscles of early developmental stages were cultured to study their troponin T isoform expression, using antisera specific to fast- and slow-muscle-type isoforms, and compared with the cells from later stages described in the previous study (Mashima et al., 1996). We found that cultured myogenic cells from chickens and chick embryos could be classified, as in the previous study, into two types, fast type and fast/slow type in which fast- and slow-muscle-type isoforms were coexpressed. Ratios of these two types of muscle cells varied depending on their origins and developmental stages, and fast/slow type cells were in the majority at early stages. Since two distinct populations of cells committed to myogenic cell lineages were supposed to give rise to the two types of myotubes, we investigated the intrinsic stability of troponin T expression of the cultured myogenic cells using the serial subcloning method. The results of clonal analysis suggested that the expression pattern of troponin T isoform in cultured muscle cells is stable and that myogenic cell lineages play an important role in giving rise to different muscle types.

A million of insect species have been identified so far, displaying a staggering variety of adult morphologies. To elucidate mechanism how such insect morphologies are developed at a molecular level, we investigated developmental process of the two-spotted cricket, Gryllus bimaculatus, as a typical hemimetabola, and compared with that of Drosophila as an extensively studied typical holometabola. We analyzed expression patterns of homeobox genes of engrailed (en) and Distal-less (Dll) during development. In early embryos, en is expressed in the posterior compartments of body segments and developing appendages, while Dll is expressed in the distal region corresponding to the telopodite of developing appendages. Interestingly, these expression patterns are very similar to those observed in Drosophila imaginal discs. In the case of Dll, we found that its expression pattern, which is similar to each other in various appendages at early stages, changes in progress with elongation and segmentation, depending on the type of appendages. Late expression patterns of Dll are classified into three types: Dll expression in the entire region of the antenna, in a distal region of the cercus, and in distal and middle regions of the leg, maxillary and labial palpus, indicating that Dll expression patterns are closely related to segmentation patterns of the appendages. Furthermore, since Dll is intensely expressed in both sides of the femur-tibia articulation of the leg, we considered that Dll is involved in positioning of articulation during the late appendage development. Hence, our results indicated that although common molecules are involved in development of insect appendages, the variety of the morphologies depends on pattern and timing of their expressions.

The cabbage armyworm, Mamestra brassicae, enters winter and aestival diapause in the pupal stage under 10L-14D and 16L-8D, respectively, at 23°C. It develops without pupal dormancy under 14L-10D at 23°C. In 6th-instar larvae, the hemolymph ecdysteroid titers as well as the activity and sensitivity of the prothoracic glands (PGs) were nearly the same in the diapause- and nondiapause-programmed larvae. In non-diapause pupae (ND-pupae), the ecdysteroid titer peaked in the first half of the pupal stage. By contrast, in winter and aestival diapause pupae (WD- and AD-pupae), the ecdysteroid titer remained low after pupation. The ecdysteroid titer in diapausing AD-pupae was 8- to 10-times higher than that in diapausing WD-pupae. PGs from 5th-instar and 6th-instar larvae, as well as those from pupae, secreted ecdysteroids and showed a far stronger response to big prothoracicotropic hormone (PTTH) than to small PTTH in vitro. PG activity and its sensitivity to big PTTH fluctuated in a stage-dependent manner, and the pattern was nearly the same for 6th-instar larvae committed to develop into ND-, WD- or AD-pupae. When ecdysteroids for larval-pupal metamorphosis appears in the hemolymph of 6th-instar larvae, the activity and sensitivity of the PGs may be suppressed quickly. After pupation, the titer of hemolymph ecdysteroids as well as the activity and sensitivity of the PGs may be related to the strength (or duration) of pupal dormancy which differs between WD-pupae and AD-pupae.

A re-evaluation of the role of prostaglandins (PGs) in releasing egg-laying behaviour in the silkmoth, Bombyx, was performed. The results revealed that PGs do not play a crucial role as oviposition behaviour stimulants. Injection of prostaglandin E₂ (PGE₂) had no effect on egg-laying behaviour of virgin females. The oviposition behaviour of females which were injected with PG biosynthesis inhibitors such as indomethacin, quinacrine and NDGA was not interrupted or suppressed. Sterilized males of Bombyx can be induced by heat treatment (32°C) for 72 hr during the wandering stage. The rate of oviposition of female moths mated with sterilized males was suppressed at a great deal, compared to the control group, in the ensuing mating period of 24 hr. PGE₂ injection into the females mated with the sterilized males had no effect on the restoration of oviposition behaviour. There were no significant difference in the PGE₂ content in the testes of normal and sterilized males. After mating, the amount of PGE₂ increased with age in the bursa copulatrix of females which had mated with either normal or sterilized males. These results indicate that PGs do not seem to be directly involved in the egg-laying behaviour of Bombyx.

A major objective of the present study was to examine the possibility that non-granular folliculo-stellate (FS) cells in the rat anterior pituitary are involved in the myogenesis that occurs during pituitary cell culture. Enzymatically dissociated anterior pituitary cells were fractionated by use of the Percoll gradient method. The proportion of FS cells was 5.8% on average before cell fractionation. After employing the Percoll gradient procedure, FS cells were enriched to a ratio of 12.2%. Three of five cell fractions were separately cultured, and the incidence of striated muscle fibers was quantitatively investigated. There was a good correlation between the numbers of muscle fibers and the proportions of FS cells in the fractions obtained from the Percoll gradient. These results suggest that FS cells are the cells that transform into striated muscles in pituitary monolayer cultures.

To investigate the roles of the amino acids of rat prolactin (rPRL), the structure of which is presumed to consist of an antiparallel, four-α-helix bundle, mutations constructed by site-directed mutagenesis were assayed in terms of their receptor binding and Nb2 cell proliferation activities. Replacement of P64L (replacing proline at position 64 with leucine) and K67E, which are located in the long loop region between helices 1 and 2, produced drastic decreases in the binding and proliferation activities. Mutations at D91 and E118 in the second and third helices, respectively, resulted in increased Nb2 cell proliferation activity with a slight increase in receptor binding activity. Mutations at L81 in the second helix and Y145 and W148 in the second loop between helices 3 and 4 produced no marked changes. Mutation at D158N in helix 4 markedly increased receptor binding activity with a slight loss of Nb2 cell proliferation activity, although two other mutations, D158H and D158R, produced a decrease of receptor binding activity without notable changes in Nb2 cell proliferation activity. These results demonstrate that P64 and K67 are crucial for PRL function while replacement of D91, E118 and D158 possibly leads to functional enhancement.

The number of nucleolar organizer regions reflects nuclear and cellular activity, such as the proliferation and differentiation of cells. Prolonged administration of estrogen (E₂) induces the hyperplasia of prolactin (PRL) cells and the development of PRL-secreting pituitary tumors in rats. Dopamine agonists are known to reduce the effects of E₂ treatment. The purpose of this study was to investigate whether the changes of argyrophilic nucleolar organizer regions (AgNORs) in PRL cells are related to circulating levels of PRL or to the proliferative activity of PRL cells during the administration of stimulatory (E₂) or inhibitory (dopamine agonist) treatment in rats. E₂ increased the size and number of AgNORs per nuclear profile in PRL cells in rats. Bromocriptine and cabergoline, which are dopamine agonists, each reduced the number and size of AgNORs in PRL cells treated with E₂ for 10 weeks. In rats treated with E₂ alone or dopamine agonists followed by E₂, the number and size of AgNORs were correlated more closely with serum levels of PRL than with the proliferative activity of PRL cells. However, neither the number nor size of AgNORs in PRL cells was related with these parameters in different ages of the control. The number and size of AgNORs may be useful in evaluating the secretory activity of pituitary cells during the administration of stimulatory or inhibitory agents.

We studied the effects of hormones on expression of prolactin receptor (PRL-R) mRNA in pancreatic islets of adult female mice in vitro. We quantified mRNA expression in small amounts of the islet tissue by competitive PCR and one-sided competitive PCR. Fifty pancreatic islets from adult female mice were cultured in a well for 4 days with or without ovine prolactin (PRL), bovine growth hormone or estradiol-17β. PRL (1 μg/ml) significantly increased the insulin secretion and the amount of PRL-R mRNA relative to that of β-actin mRNA. Growth hormone (1 μg/ml) also increased the relative amount of PRL-R mRNA, although it did not significantly increase the insulin secretion. Neither insulin secretion nor the relative amount of PRL-R mRNA was affected by estradiol (100 ng/ml). The ratio of the short form to the long form of PRL-R mRNA was not altered by these hormones. The present observation that PRL increased PRL-R mRNA expression in pancreatic islets thus suggests the possibility that PRL up-regulates the tissue sensitivity to PRL itself during lactation.

The Japanese dormouse, Glirulus japonicus, is the only extant lineage that represents this genus and it has been classified as a single species distributed on the three main islands of Japan, namely Honshu, Shikoku and Kyushu. However, individuals collected from Fukui, Wakayama and Kochi Prefectures (southwestern part of Japan) yielded distinctly different profiles of restriction fragments of the nuclear ribosomal DNA (rDNA) spacer from those collected from Yamanashi and Nagano Prefectures (central Japan). The estimated sequence divergence between the two groups was 2.8% on average, which corresponds to a putative divergence some two million years ago. Representing mitochondrial DNA (mtDNA) sequences, 402 bases of cytochrome b gene were determined by direct sequencing and the estimated extent of the sequence divergence between the two groups was 6.5–7%. Differences between the two geographic groups were also substantial in the sequences of about 300 base-fragments from the Y-linked, sex-determining locus, Sry. To assess the phylogenetic relationships between the Japanese dormouse and members of the family Myoxidae, we compared sequences of mitochondrial 12S rRNA gene of Japanese dormice with those of the forest dormouse (Dryomys nitedula) and the common dormouse (Muscardinus avellanarius), two continental genera thought to be closely related to the genus Glirulus. The results showed that the sequences from Japanese dormice were distinct from any sequences of the two continental species and the extent of the differences were somewhat similar to that between the rat (Rattus norvegicus) and the hamster (Mesocricetus auratus).