Since the discovery of vanadium in the blood cells (coelornic cells) of an ascidian by Henze in 1911, this unusual phenomenon has attracted the interest of many investigators. We started our studies by examining the vanadium contents of several tissues from 20 ascidian species, collected not only from Japanese waters but also from the Mediterranean since about 18 years ago, using an extremely sensitive method, namely, neutron-activation analysis. We found the highest concentration of vanadium, 350 mM, in the blood cells of Ascidia gemmata which belongs to the suborder Phiebobranchia. This concentration of vanadium is 107 times higher than that in seawater. Among the approximately ten types of blood cells, the signet ring cells were revealed to be the true vanadocytes by a combination of cell fractionation and neutron-activation analysis. Of the vanadium in these vanadocytes, 97.6% was in the 3 oxidation state while the rest was in the 4 oxidation state. The contents of the vanadocytes in A gemmata had a low pH of 2.4 and these cells contained the highest levels of vanadium. These observations suggested the possibility that protons, concentrated by a H -ATPase, might be linked energetically to the accumulation of vanadium. Antibodies raised against a vacuolar-type H -ATPase were found to react with the vacuolar membranes of signet ring cells and the addition of bafilomycin A₁ a specific inhibitor of vacuolar-type H -ATPases, inhibited the uptake of protons by the vacuoles of signet ring cells, with resultant neutralization of the contents of the vacuoles. A monoclonal antibody, S4D5, prepared for the purpose of identifying signet ring cells, reacted with the signet ring cells not only of A. sydneiensis samea, which had been used as the antigen, but also with those of other species. During embryogenesis, a vanadocyte-specific antigen, recognized by this monoclonal antibody, appeared for the first time in the body wall at the same time as the significant accumulation of vanadium became apparent. Characterization of vanadium-binding proteins extracted from the blood cells of vanadium-rich ascidians is in progress and shows to help us determine the way in which ascidians selectively accumulate high levels of vanadium from seawater. The unusual phenomenon whereby some ascidians accumulate vanadium to levels more than ten million times higher than those in seawater has attracted the interest of researchers in various fields. Studies of ascidians with this unusual physiological property may help us to clarify not only how ascidians but also other organisms accumulate transition metals, as well as the physiological roles of these metals.

An attempt was made to determine electrophysiologically and immunocytochemically the neurotransmitter at neuromuscular junctions (NMJs) in cricket abdominal muscles. One mM bath-applied Lglutamate reduced the amplitudes of large and small excitatory junctional potentials (I- and s-EJPs) reversibly by about 75% of the control on the average. It also produced a slow, transient depolarization of about 10 mV. Joro spider toxin, which is an antagonist of L-glutamate, depressed the amplitudes of I- and s-EJPs almost completely and irreversibly at 3.5 × 10⁻⁶ M. By using the antibody to glutamate, glutamate-immunoreactive processes whose configuration resembled that of NMJs revealed by nickel staining were obtained. The present results strongly suggest that the neurotransmitter at cricket NMJs is L-glutamate.

Monoclonal antibodies (McAb) to actin were prepared to analyze the assembly of actin isoforms in developing muscle cells in vitro. One of the antibodies (SkA-06) was specific for α-sarcomeric actin isoforms in skeletal and cardiac muscles, while the others recognized cytoskeletal (β, γ) actin isoforms in smooth muscle and non-muscle tissues as well as the sarcomeric (α) actins. Using SkA-06 and a polyclonal antibody (PcAb) specific for cytoskeletal actins, the subcellular localization of the actin isoforms was examined by immunocytochemical methods. While in developing young myotubes, cytoskeletal and sarcomeric actins were co-localized in nascent myofibrils or stress-fiber-like structures, sarcomeric actins predominated in striated myofibrils in more developed myotubes. When FITC-labeled cytoskeletal and sarcomeric actins were introduced into young myotubes by a microinjection method, the latter became detectable in striated structures sooner than the former but they were finally incorporated into striated myofibrils. These results suggest that α-actin(s) as well as β- and γ-actins can be incorporated into myofibrils, but α-actin(s) is assembled preferentially into myofibrils in developing muscle cells.

In a colonial ascidian Clavelina miniata, physical stimulations induce strong luminescence in the tunic. We described here the tunic cell morphology and bacterial distribution in the tunic that is a luminous tissue of this species. Three types of tunic cells are morphologically discriminated as morula-like tunic cells, tunic phagocytes, and tunic granulocytes, and they correspond, respectively, to the Type I, Type II, and Type III cells described by Aoki et al. (1989). Morula-like tunic cells are similar in morphology to morula cells that are hemocytes commonly found in ascidians. Tunic phagocytes contain round granules, clear vacuoles, and occasionally phagosomes. Tunic granulocytes characterized by a number of elliptical granules and they occasionally contain phagosomes and round granules that are similar in structure to tunic phagocytes. According to the description by Aoki et al. (1989), tunic phagocytes are supposed to be luminous cells. Elongated bacteria of unique forms are found in tunic phagocytes. However, these bacteria are probably not luminous ones, since they also are distributed in tunic granulocytes and outside of the tunic cells. Because other bacteria-like inclusions are not present in tunic phagocytes, we found no structural evidence to support the bacterial origin of bioluminescence in C. miniata. The clear vacuoles of tunic phagocytes may be a possible candidate for the subcellular site producing bioluminescence.

A highly ordered nanocomposite was discovered in the transparent wing of a hawkmoth, Cephonodes hylas. The protuberances, nipple-like shaped and 250 nm in height, are regular-hexagonally arranged with the center-to-center distance of 200 nm. This morphology is almost the same as those of “corneal nipple arrays” of some insect eyes. Nanocomposites closely similar to that of the Cephonodes wing were also found in transparent wings of some other moths (Sphingidae and Sesiidae) and cicadae.

Tetrahymena thermophila has two nuclei: a micronucieus is transcriptionally silent during vegetative growth and a macronucleus is active. Extensive programmed DNA rearrangement is known to occur during the development of the somatic macronucleus from the germ-line micronucieus. We previously found a 1.4 kb micronucleus-specific sequence, C-element, which was located upstream of the micronuclear calmodulin gene and was eliminated from the macronuclear genome during macronuclear development. Here, using gel mobility shift assays, we show that C-element binding factors, CBFs, are present in the nuclear extract prepared from vegetative cells. Competition experiments demonstrate that CBFs bind to two regions within the C-element. A sequence motif common to these regions is 5′-ATAGATTT-3′.

Three-dimensional structures of the microvascular network of the fungiform papillae (FuP) of the cat tongue were observed by the corrosion cast method under a scanning electron microscope (SEM). FuP were found to be distributed sporadically among the different types of numerous filiform papillae (FiP). A single FuP consisted of the main process (MP) and the accessory process (AP) which were arranged at the anterior basal margin of the MP. FuP can be classified into four types (I∼IV) according to the shape and size of the MP and the number of AP within each FuP. FuP-I to FuP-III contained a large, medium and small MP, showing the small fishnet-ball and oval-ball shapes, and these were surrounded at the anterior basal margin by AP inclining toward the anterior part of the tongue. FuP-IV contained only the small fishnet-ball-shaped MP.

Fifteen enzymes and two blood proteins encoded by 24 presumptive loci were analyzed using starch-gel electrophoresis in 136 frogs of 16 populations of Rana ornativentris and 21 frogs of a sympatric population of Rana japonica, in order to elucidate the degree of geographic divergence of R. ornativentris in Honshu and its genetic relationships to R. japonica. The UPGMA dendrogram constructed from Nei's genetic distances showed that R. ornativentris from Honshu was divided into two distinct groups, western and eastern, and that the latter split into three subgroups, southern, central and northern. Genetic divergence was distinct between western and eastern populations of R. ornativentris at three loci, PEP-A, SOD-1 and Hb-1, with the Fst values of Wright of 0.624, 0.635 and 0.876, respectively. The average value of Fst (Fst), excluding the five invariant loci, was 0.306. Nei's genetic distances among the four western populations of R. ornativentris were 0.015∼0.061, 0.043 on average. Those among the 12 eastern populations were 0.011∼0.179, 0.063 on average, whereas those between the four western and 12 eastern populations were 0.128∼0.313, 0.225 on average. The genetic distances between the 16 populations of R. ornativentris and one population of R. japonica were 0.579∼0.956, 0.793 on average. The UPGMA dendrogram showed that R. ornativentris was distinctly separated from R. japonica.

The complete amino acid sequence of the monomer subunit of Pheretima hilgendorfi hemoglobin was determined: It consists of 140 amino acid residues, including a disulfide bond but no methionine, and has a molecular weight of 16,107 Da. Using computed analyses (amino acid maximum homology) with known sequences of monomer subunits of earthworm's hemoglobins, 115 (82%) were found to be identical with those in the corresponding positions of chain I (monomer subunit) of Pheretima sieboldi hemoglobin; 81 residues (55%), 71 residues (47%), and 66 residues (43%) were found to be in identical positions of the sequences of chain I of Lumbricus terrestris hemoglobin, chain I of Tubifex tubifex hemoglobin and chain I of Tylorrhynchus heterochaetus hemoglobin. Orthologous sequence data of monomer globins that belong to the strain A of annelid hemoglobins are discussed as useful clues for investigation of the divergence pattern of Pheretima species.

The right ovary of chick embryos is known to degenerate during embryogenesis, while the left one advanced its normal growth. The present study demonstrates germ cell death in the degenerating right ovary. In the process of germ cell death, chromatin condensation, reduction of cell size, and formation of apoptotic bodies were observed. The cell organelles were almost normal in the early stages of the process. Fragments of broken germ cells were phagocytosed by neighboring cells or macrophages. From these observations, it may be assumed that germ cell death in the right ovary of chicks is related to apoptosis. In the normal left ovary, similar cell death was only observed in the germ cells in the medulla, but not in the cortex. Therefore, germ cell death seems to be a phenomenon common to the medullae of both the right and left ovaries.

Paired pineals were observed as an anomaly in embryonic quail brains between 7 and 9 days of incubation. The size of each pineal was almost the same as that of the normal pineal and it was located slightly lateral of the midline. Histological examination of these paired pineals revealed that both had similar cytological features in comparison with the normal pineal of the same developmental stage. No abnormal features were detected in brains and eyes of the embryos with paired pineals. Since the presumptive pineal rudiments are considered to exist in the neural folds and to fuse in the midline during the formation of the neural tube, the paired pineals may be interpreted as a result of incomplete fusion of the pineal anlagen. This report describes for the first time the symmetrical occurrence of pineal glands in the developing avian brain.

Cells prepared from chicken skeletal muscles of different developmental stages were cultured to study their troponin T isoform expression, using antisera specific to the fast- and slow-muscle-type isoforms. We found that the cultured myogenic cells from chickens and chick embryos were classified into two types, fast type and fast/slow type in which fast- and slow-muscle-type isoforms were coexpressed. Cells expressing only slow-muscle-type troponin T isoforms could not be found. Most cells prepared from pectoralis major (fast muscle) and gastrocnemius (mixed muscle) of 11-day old embryos belonged to the latter, with only a small fraction belonging to the former. The percentage of fast type cells in those cells prepared from pectoralis major increased along development to over 90% by the 17th day of incubation, while, in the cells prepared from gastrocnemius, it reached a plateau of 30-40% by the 13th day of incubation. All the cells from anterior latissimus dorsi (slow muscle) belonged to the fast/slow type. Ratios of these two types of muscle cells varied depending on their origins and stages. The in vitro expression of troponin T isoforms was different from the in vivo expression, and each muscle seems to be determined differently in the composition of cell types during the developmental course.

Immunohistochemical techniques were employed to investigate the distribution of galanin-like immunoreactivity in the central nervous system (CNS) of the snail, Indoplanorbis exustus, and immunoreactivity with an antibody against choline acetyltransferase (CAT) in galanin-immunoreactive somata was also examined. Galanin-immunoreactive (Gal-IR) cells were observed in all ganglia. They were classified into large (50 μm), medium (21 μm), and small (13 μm)-sized neurons and small Gal-IR cells dominated over large and medium Gal-IR cells in number. Gal-IR cells were most abundant in the cerebral ganglion. Several immunoreactive somata were seen in the parietal, visceral, and pedal ganglia, and only a few Gal-IR cells in the pleural and buccal ganglia. Densely arranged Gal-IR fiber bundles were observed in the cerebro-pleural, parieto-pleural and bucco-cerebral connectives. Of the peripheral nerves, the pallial and pharyngeal nerves contained more numerous Gal-IR fibers than other peripheral nerves. By Western blot analysis, the galanin antibody detected approximately 6.1 kDa band, suggesting a pre-form or a long form of galanin. Some GalIR cells in the cerebral ganglion and a few in the pleural and parietal ganglia were also immunoreactive for CAT. These results indicate that a distinct neuronal system with galanin or galanin-like peptide is present in the CNS of the snail, and suggest that a part of the galanin neuronal system is cholinergic. The peptide may function as a neurotransmitter/neuromodulator and/or neurohormone in the snail.

Mammary epithelial cells isolated from pregnant mice were plated on a collagen gel matrix and cultured in a serum-free medium supplemented with combinations of insulin, dexamethasone and prolactin (PRL). After the cells formed a monolayer, the collagen gel was detached from the culture dish and allowed to float in the medium. Quantification of γ-casein mRNA by a competitive PCR method revealed that the cells on the floating gel accumulated considerably larger amounts of γ-casein mRNA than the cells on the gel remained attached to the dish. Under these floating collagen gel culture conditions, addition of both dexamethasone and PRL to the insulin-supplemented basal medium maximally stimulated the accumulation of γ-casein mRNA. These observations strongly suggest that the status of the extracellular matrix as well as hormones controls the differentiation of mouse mammary epithelial cells at the mRNA level.

Vasotocin (VT) and isotocin (IT) are teleost neurohypophysial hormones produced by neurosecretory neurons in the magnocellular part of preoptic nucleus (PM) of the hypothalamus. Several previous studies indicated that neurohypophysial hormones are involved in teleost reproductive behavior. The changes in the expression of VT and IT genes were thus studied by an in situ hybridization technique and an immunohistochemical avidin-biotin-complex method in pre-spawning chum salmon (Oncorhynchus keta). Male and female fish were caught at Atsuta, the mouth of the Ishikari Bay, and at Chitose, upstream to the Ishikari River in October, 1994. The former and the latter are referred to as bay fish and river fish, respectively. The intensity of autoradiographic hybridization signals were determined in individual neurosecretory neurons in the rostroventral, middle, and dorsocaudal parts of the PM. in the females, the levels of VT and IT mRNAs in the river fish were significantly lower than those in the bay fish in all the three loci in the PM, whereas VT and IT immunoreactivities in the river fish were higher than those in the bay fish. These results suggest that both the rates of transcription and release of VT and IT were decreased in pre-spawning female chum salmon. Contrary, in the males, the levels of IT mRNA and IT immunoreactivity in the river fish were greater than those in the bay fish particularly in the rostroventral part of the PM, whereas conspicuous changes were not seen in the levels of VT mRNA and VT immunoreactivity. The present results thus revealed sexually different expression of neurohypophysial hormone genes in the preoptic nucleus of pre-spawning chum salmon when compared between bay and river fish. The regulation of neurohypophysial hormone gene expression may be different between the male and the female during the last stages of spawning migration.

The innervation of the tilapia ovary was examined histochemically and ultrastructurally. Thick nerve bundles were localized in the area near the ovarian artery and vein in the ovarian wall on the side facing the mesentery. Groups of a few axons ramified from the thick nerves and were terminated in the proximity of clusters of steroid-producing cells which are distributed in the interstitial area among yolky oocytes. The axon terminals were in intimate relation with the steroid-producing cells. The terminals contained many clear vesicles, a few dense-cored vesicles and some mitochondria. Moreover, a few terminals were observed on the surface of steroid-producing theca cells surrounding yolky oocytes. Histochemical results using a nerve-specific stain were in agreement with the ultrastructural observations. Our observations of direct innervation of steroid-producing cells bring to light a possible new avenue for regulation of steroid production in the tilapia ovary.

The inhibitory effect of progesterone (P) injected at various times on female sexual behavior was investigated in estradiol benzoate (EB) treated ovariectomized rats. Four behavioral tests were carried out at two-week intervals. All females received 5 μg/kg b.w. EB and 0.5 mg P 44 hr after the EB. In the P-control group, an additional 5 mg P was administered at the same time as the injection of EB in four tests. Instead of P, oil was given concurrently with EB in the Oil control group. In the experimental groups, female rats were treated with 5 mg P from 1 to 40 hr before (PB group) or after (PA group) the EB-injection. A sexual behavioral test was started 4 hr after 0.5 mg P. The results show that low levels of lordosis and soliciting behavior were observed in the P group, compared to the Oil-control group. In the PB groups, lordosis quotient (LQ) was low when P was given from 1 to 24 hr before EB. Moreover, animals in which P was given 27-40 hr before EB showed lower LQ than Oil-control animals, but higher LQ than rats in the P-control group. In the PA groups, when P was administered from 1 to 24 hr after EB, low levels of lordosis response were observed, whereas animals which received P 27-40 hr after EB showed'a high score of LQ, being comparable to that in the Oil control. These results suggest that the period of 24 hr before and after EB injection is a critical period for inhibitory action of P on female sexual behavior in female rats.

In order to investigate phylogenetic relationships of the family Sciuridae living in Japan, we sequenced partial regions (379 bases) of mitochondrial 12S rRNA genes in six species of Japanese and other Asian squirrels. Phylogenetic trees constructed by sequence data indicated that two genera of flying squirrels (Petaurista and Pteromys) were clustered in a group distinct from non-flying squirrels, suggesting a possible monophyletic relationships of these flying squirrels. The evolutionary distance between the Japanese squirrel (Sciurus lis) from Honshu island and the Eurasian red squirrel (Sciurus vulgaris) from Hokkaido island was comparable to intraspecific distances of the remaining species examined.

A 224 bp fragment of the mitochondrial cytochrome b gene has been amplified from a 30-year-old mummy-like specimen of the Japanese river otter Lutra nippon by polymerase chain reaction (PCR). The amplified products were subcloned in the Smal site of pUC18 and sequenced. The sequence was different from those of the congeneric Eurasian otters Lutra lutra (Latvia) and Lutra lutra (China) in 7-9 nucleotides, all of which were located at the third position of a codon and identified as transitional differences A↔G or C↔T. The phylogenetic analysis using the 224 bp sequences of Lutra nippon, Lutra lutra (Latvia), Lutra lutra (China), Aonyx cinerea (Asian small-clawed otter), Mustela sibirica and Mustela itatsi (weasels) supports the recent morphological study that the Japanese river otter is not a subspecies of Lutra lutra, but a distinct species, Lutra nippon. We found that Lutra nippon and Lutra lutra contain the cytochrome b-like sequences, that appear to be a pseudo-form of cytochrome b gene. The sequences are characterized by the presence of deletion and termination codons, by the presence of several types of sequences with minor variations, and by the faster evolutionary rate compared with that of the mitochondrial cytochrome b gene, The genes would present in the nuclear DNA rather than in the mitochondrial DNA, as in the case of the nonfunctional cytochrome b-like sequences previously reported in a rodent.