The vomeronasal system is one of the chemosensory systems. It is believed to play an important role in the processing of pheromonal signals and the expression of reproductive function. The sensory mechanism in the vomeronasal system has not been studied as well as those in the other chemosensory systems such as the main olfactory system and the gustatory system. Recently information on the neuronal development and plasticity in the vomeronasal system has gradually increased. Neuronal development in the rat vomeronasal system continues until about the 4th week after birth. The vomeronasal system exhibits a high degree of plasticity. In this article we shall review neuronal development and plasticity in the mammalian vomeronasal system.

Angiotensin II (Ang II) exhibits a variety of physiological actions, related mainly to the regulation of blood pressure and fluid osmolarity. Recent identification of the multiple types of the Ang II receptors raises the possibility that Ang II has other unknown functions. The Ang II type 1 receptor (AT₁) mediates most of the known physiological functions of Ang II, whereas the type 2 receptor (AT₂)-mediated functions remain unclear, AT₂ is particularly interesting because it is expressed abundantly in fetal tissues and in cells undergoing apoptosis.AT₁and AT₂ exhibit unique signaling pathways among the superfamily of seven membrane-spanning receptors: i.e. the coupling of AT₁ to the Janus kinase-signal transducers and activators of transcription pathway and the coupling of AT₂ to phosphatase activation. Also, the two subtypes induce several opposite intracellular events. AT₁ mediates activation of Ca² channels and inhibition of K channels, whereas AT₂ induces inhibition of Ca² channels and activation of K channels. Therefore, it is of great importance to compare the two receptor subtypes with respect to their distribution, signaling pathways, and physiological functions.

Immunocytochemical and immunoelectron microscopic localizations of a salmonid olfactory system-specific protein (N24) were investigated in the olfactory system (the olfactory epithelium, the olfactory nerve and the olfactory bulb) of kokanee salmon (Oncorhynchus nerka) by using a specific antiserum to N24. N24 immunoreactivities were observed in the cytoplasm of ciliated and microvillous olfactory receptor cells but were not observed in the supporting and the basal cells in the olfactory epithelium. Gold particles showing immunoreactivities for N24 were scattered in the cytoplasm of the dendrites of olfactory receptor cells. Some particles were concentrated on vesicular structures, but none were observed in the membrane of olfactory receptor cells. N24 immunoreactive axons were terminated at the glomerular layer near the mitral cells in the olfactory bulb. In an olfactory rosettectomy experiment, N24 immunoreactivity in the olfactory bulb vanished fifteenth day after the excision of the olfactory rosette. These results reveal that the olfactory receptor cells produce N24 which exists in both dendrites and axons of the olfactory receptor cells, and suggest that N24 may participate in neuromodulation in the olfactory system of kokanee salmon.

Cellular differentiation and renewal in the gill chloride cells were examined in freshwater (FW) and seawater (SW)-adapted chum salmon (Oncorhynchus keta) fry and in fry during SW adaptation using 5-bromo-2′-deoxyuridine (BrdU) as a marker for newly-differentiated cells. Chloride cells and BrdU-labeled nuclei were immunocytochemically detected by using antisera specific for Na ,K -ATPase and BrdU, respectively. Although the number of chloride cells located at the base of the lamellae and in the interlamellar region (filament chloride cells) was constant in FW, SW and SW-transferred groups, chloride cells located in the lamellar epithelium (lamellar chloride cells) were fewer in SW than in FW, and decreased during SW adaptation. Newly-differentiated cells with BrdU-immunoreactive nuclei were detected mainly in the filaments, and rarely observed in the lamellae. The turnover rates of filament chloride cells for FW, SW and SW-transferred fish during the first 24 hr were 8%, 21% and 28%, respectively. These results indicate that chloride cells in the filament are replaced continuously by newly-differentiated cells in both FW and SW, and that the turnover was about 3 times greater in SW than in FW. More frequent turnover of filament chloride cells in SW suggests a specific role, presumably in salt excretion.

The oxygen equilibrium curves of human fetal and adult hemoglobins were reconstructed from the published Adair constants. The curves were then analyzed theoretically with respect to the amount of transferred oxygen, which is directly related to the saturation difference (▵S) of hemoglobin with oxygen in the artery and vein. In fetal blood, the oxygen affinity is optimized so as to provide the maximal ▵ S value in the fetus oxygen environment. In adult blood, on the other hand, the ▵ S value is far smaller than the theoretically obtained maximum, but it is most sensitive to the P₅₀ changes around its physiological value of 27 torr. The present results imply that adult blood reserves an oxygen transport capacity for increased oxygen demands under resting conditions, and that the oxygen affinity is optimized so as to make the Bohr effect most effective to gaseous exchange.

In the experiment of mouse transforming growth factor alpha (TGFα) gene expression in mammary tumors, various sizes of amplified products by reverse transcriptase-polymerase chain reaction (RT-PCR) using mouse TGFα primers were detected in addition to a predicted size in four strains of mice. During the further analysis of these RT-PCR products in mouse mammary tumors, the transcript of neurocan gene was detected in the mammary tumor from SHN mice by the cloning and nucleotide sequence analysis after RT-PCR reaction using mouse TGFα primers. The 5′-nucleotide sequence of sequential 246bp in the amplified cDNA of 527bp was completely identical to a middle part of mouse neurocan cDNA sequence, one of the chondroitin-sulfate proteoglycan expressed in the nervous tissue.

A heterotrichous ciliate Spirostomum is known to show characteristic rapid contraction of the cell body accompanied by twisting of ciliary lines which run longitudinally on the cell surface. The ciliary lines of Stentor, a closely related heterotrichous ciliate, are known to become shorter by themselves when the cell contracts, resulting in sliding between adjacent longitudinal microtubular sheets (LMSs) running just beneath the ciliary lines. In Spirostomum, in contrast, there is controversy over whether lengths of the ciliary lines alter or not. In this study, we examined changes of the ciliary line lengths by scanning electron microscopy, to measure the distance between the proximal ends of two neighboring cilia which are lining up along each ciliary line (inter-kinetosomal distances). We found that the inter-kinetosomal distance remained constant regardless of cell contraction and elongation, indicating that the cell body contraction of Spirostomum results from a cellular twisting motion with constant inter-kinetosomal distance. It can thus be concluded that the contraction of Spirostomum does not involve shortening of the inter-kinetosomal distance along ciliary lines, which is quite different from the case in Stentor.

Structural proteins in the mammalian epidermis contain citrulline residues generated by enzymatic deimination of arginine residues. We analyzed deiminated proteins solubilized from sequentially stripped layers of guinea pig epidermis. Deiminated proteins were localized in the granular and cornified layers. Those in the inner layer enriched with granular cells were resolved into numerous components by two-dimensional gel electrophoresis. An arc-shaped high-molecular-weight smear and two series of charged isomers among them coincided with filaggrin immunoreactivity. Several groups of filaggrin-negative spots appeared to be generated by further deimination and proteolysis of these filaggrins. Deiminated protein spots co-migrating with type II and type I keratins were also detected. Deiminated filaggrins and their further processed derivatives disappeared in the outer layer, while deiminated keratins persisted. These data suggested that filaggrin as well as profilaggrin were deiminated during the posttranslational processing in guinea pig skin, and that some keratins were deiminated preferentially during the cornification of epidermis, Possible biological significance of protein deimination in guinea pig skin was discussed in comparison with our recent finding on deiminated proteins in rat skin.

Holospora recta is a micronucleus-specific symbiotic bacterium of the ciliate Paramecium caudatum. This bacterium cannot grow outside the host cell. We isolated the infectious form of this bacterium from the host homogenates by 70% Percoll density gradient centrifugation. DNA in the infectious form of H. recta appeared as a large cluster in the cytoplasmic region near the large periplasm. This was not observed with the infectious form of a macronucleus-specific symbiont of P. caudatum, H. obtusa. The isolated infectious form could infect the micronucleus of P. caudatum and differentiated into the reproductive form within 18 hr after the infection at 25°C. However, not only was this bacterium unable to infect the micronucleus of P. bursaria, but it was also unable to infect the micronucleus of P. multimicronucleatum or P. novaurelia, even though the latter two species are morphologically closely related to P. caudatum. We succeeded in the cryopreservation of this bacterium, as cells stored at −85°C for 127 days maintained their infectivity and reproducibility.

Paramecium bursaria contain several hundred cells of the green algae Chlorella as endosymbionts and are designated green. Chlorella-free white cells can be obtained from natural green cells by rapid growth in constant darkness (DD). Chlorella were isolated easily from their host cells and re-infected. The infection of Chlorella was restrained by a photosynthesis inhibitor (DCMU). This result can be related with the fact that symbiotic Chlorella release their photosynthetic products. Furthermore, when green cells were cultured in DD, the number of endosymbiotic Chlorella decreased and the density of host cells increased. On the other hand, the mating reactivity rhythm of green cells disappeared in DD. The photosynthetic products of symbiotic Chlorella, maltose and oxygen, induced the rhythms of their host cells in DD, but they could not shift the phase of the rhythms. Moreover, arrhythmic mutant white cells reverted to rhythmicity after infection with Chlorella isolated from wild type green cells. Thus the photosynthetic products released by endosymbiotic Chlorella have important roles in the establishing of the endosymbiosis and the expression of circadian rhythms in P. bursaria cells.

We developed a new method for the assay of cathepsins D and E. The method was based on the different hydrolytic activities of cathepsins D and E against β-endorphin and substance P. The method was applied to the determination of the levels of cathepsins D and E in various tissues and cells of rat, monkey, and man, and was clarified to be much more specific, sensitive, and quantitative than the ordinary hemoglobin-digestion method. The levels of cathepsin D were high in adrenal and spleen, and the levels of cathepsin E were high in gastrointestinal tissues, bone marrow, and lymph node. The variations in level were much wider in the case of cathepsin E than in the case of cathepsin D. This might reflect that cathepsin D is a house-keeping lysosomal enzyme in a variety of cells and cathepsin E is involved in the physiological activities of certain types of tissues and cells.

During embryogenesis of the ascidian Halocynthia roretzi, exactly eight-hundred epidermal cells are formed in the larva, and the lineage of the cells has been almost completely described. In the present study, we examined the spatio-temporal expression patterns of eight epidermis-specific genes which we already isolated. In situ hybridization of whole-mount specimens unambiguously demonstrated that the expression patterns of the eight genes were not identical, and that they were categorizable into several types. Transcripts of seven genes were restricted to presumptive epidermal cells, although transcripts of one gene were evident in the presumptive neural cells in addition to the presumptive epidermal cells. Therefore, most of the epidermis-specific genes in ascidian embryos are expressed in lineage-associated manner. We discuss these results in relation to the question of whether (a) epidermis-specific genes are expressed exclusively in presumptive epidermal cells prior to neural induction, or (b) the genes are first expressed in both epidermal precursors and precursors of the central nervous system, then the gene expression is downregulated in the latter after the completion of neural induction. Interestingly, cells of the anterior-most region as well as the dorsal midline of the tailbud embryo did not express most of the epidermis-specific genes, suggesting regional differences in the embryonic epidermis. Some other genes might be expressed in a complementary pattern in these regions.

Seven protein phosphatases were isolated from extracts of spermatozoa and sperm tails of the sea urchin Hemicentrotus pulcherrimus by ion exchange and gel filtration chromatographies using [³²P]-histone and/or p-nitrophenyl phosphate (pNPP) as substrates and characterized for their enzymatic and molecular nature. Two of them isolated from the particulate fraction correspond to the mammalian type 1 and 2B protein phosphatases. Another two obtained from the soluble fraction were similar to the mammalian protein phosphatases, type 2A and 2C. A protein phosphatase corresponding to the mammalian type 1 enzyme (molecular mass of 43 kDa) dephosphorylated the [³²P]-autophosphorylated regulatory subunit of H. pulcherrimus sperm cAMP-dependent histone kinase.

Development and distribution of melanocytes were histologically examined with the aid of dopa reaction in the feather germs of Bh (black at hatch) quail embryos. In the feather germs of 10-day wild-type embryos, two types of melanocytes were observed; strongly dopa reaction-positive melanocytes (with black pigment) and moderately positive melanocytes (with brown pigment), which were located in the black parts and yellow parts of the feather germs, respectively. These melanocytes were arranged in a regular pattern in the barb ridges of whole feather germs. By contrast, in the heterozygotes, the distribution of the two types of melanocytes were intermingled in the correspondent to the yellow parts of the wild-type feather germs. In the parts corresponding to the black parts of the wild-type feather germs, melanocytes with black pigment were mainly seen. The distribution of melanocytes in heterozygous feather germs resulted in the black coating of the heterozygotes. The homozygous feather germs mostly contained melanocytes with moderate staining of the dopa reaction and brown pigment, leading to the brown appearance of the feather germs. Development of homozygous feather germs was delayed 1 or 2 days compared to those of the wild-type and heterozygotes, but homozygous melanocytes were also arranged at regular intervals in the barb ridges of the feather germs, which resembled the pattern seen in the wild-type and heterozygous feather germs. Therefore, the distribution of the two types of melanocytes in the feather germs might determine plumage pigmentation patterns in Bh quail embryos.

We established a monoclonal antibody, designated CiNot-1, that recognizes ascidian Ciona notochordal cells in the early tailbud embryo. It distinguished two types of cells in the notochord, according to whether or not they expressed CiNot-1 antigen. Only A-line notochord cells expressed CiNot-1 antigen in cleavage-arrested embryos. Since A- and the B-line notochord precursor cells respond differently to inductive signals, CiNot-1 distinguished two specification mechanisms in the notochord. After the middle tailbud stage, CiNot-1 recognized the nerve cord and endodermal strand in addition to the notochord. In these tissues, several cells in the distal tip of the tail did not express CiNot-1 antigen. This revealed the distinct feature of unity in the distal tip of the larval tail and suggests the specification mechanism of this region.

In order to examine whether calcitonin plays an important role in Ca homeostasis of teleosts, such as suppressing hypercalcemia at food intake, we compared the plasma Ca levels and calcitonin levels in eels fed normally with eels starved for one week (Experiment I), and in goldfish administered with a high Ca-consomme solution into the digestive tract with goldfish given physiological saline solution (Experiment II). In Experiment I, the plasma Ca levels and calcitonin levels in the fed eels were significantly higher than those in the starved eels after one week. In Experiment II, the plasma Ca levels in the high Ca-treated goldfish were significantly higher than those in the saline-treated goldfish after 1 hr and 3 hr. The number of goldfish showing over 500 pg/ml of plasma calcitonin was significantly higher in the high Ca-treated group than in the saline-treated group. From the results of both experiments, we conclude that in these two species, Ca and/or nutriment absorbed via the digestive tract may affect plasma calcitonin levels. However, more experiments are needed to directly demonstrate that calcitonin suppresses hypercalcemia at food intake.

Changes in expression of vasotocin (VT) and isotocin (IT) genes were analyzed in chum salmon during the last stages of spawning migration. Pre-spawning chum salmon were caught at following four locations in the Sanriku coast of the Pacific Ocean in Japan: 1) the off-coast area north to the Otsuchi Bay, 2) the mouth of the Otsuchi Bay, 3) inside of the Otsuchi Bay, and 4) the place 500 m upstream to the mouth of the Otsuchi River. In addition, effects of hypo-osmotic stimulation by transition from sea water (SW) to freshwater (FW) were examined in animals caught at the mouth of the Otsuchi Bay. The levels of VT and IT mRNAs in the forebrains were determined by Northern blot analysis. The plasma osmolality and the levels of Na and Cl⁻ were also analyzed. Expression patterns of VT and IT genes were different between the males and the females. In the males, VT and IT gene expression were maintained essentially at the same levels from the off-coast area to the Otsuchi River. In contrast, in the females, the level of VT-I mRNA was significantly increased in the fish caught at the mouth of the bay. After entering the bay, the level of VT-l mRNA was decreased and maintained at a low level through the final stages of spawning migration. Such sexual difference in VT and IT gene expression found in the field fish was further analyzed by a SW to FW transition experiment, in which fish were divided into two groups, those retained in SW and others replaced with FW. In the FW-replaced fish, the levels of VT and IT mRNAs were decreased in both sexes, although much more conspicuous in the females. In the SW-retained animals, changes in the levels of VT and IT mRNAs were sexually different. The levels of VT and IT mRNAs were increased in the males, whereas they were decreased in the females, when compared to the initial levels just before the experimental treatments. These results suggest that regulation of VT and IT gene expression is sexually dimorphic in pre-spawning chum salmon.

The turbellarian flatworm is a key group to understand the origin and the early evolution of triploblastic, bilaterally symmetrical animals, but phylogenetic relationships among turbellarian orders have been a subject of debates for decades, especially on the position of the acoel turbellarians. Some workers have considered the acoel representing the most primitive turbellarian order but others have regarded them as regressive. We determined almost the entire lengths of the nucleotide sequences of 18S ribosomal RNA gene (rDNA) in 17 species from 9 turbellarian orders (the Acoela, Catenulida, Macrostomida, Lecithoepitheliata, Rhabdocoela, Prolecithophora, Proseriata, Tricladida, and Polycladida). After adding the sequences of a cestode, two trematodes and some diploblastic animals obtained from databases, we reconstructed phylogenetic trees using the neighbor-joining, maximum-likelihood and maximum-parsimony methods. All trees significantly indicated that the Acoela is the earliest divergent group among the turbellarian orders. The trees also suggested that the Tricladida evolved in the separate lineage from that of a cluster of the Catenulida, Macrostomida, Lecithoepitheliata, Rhabdocoela, Polycladida, Trematoda and Cestoda after the divergence of the Acoela.

From a comparative morphological viewpoint, we examined the development of gonads of 9 species of teleost fish, genus Oryzias, in which three types of bilateral asymmetry were discriminated. The first type occurs in O. celebensis, O. javanicus, O. marmoratus, O. mekongensis, O. melastigma, and O. minutillus, in which testes are completely asymmetrical. Their gonadal rudiments are constructed only on the right side and develop into testes of a unilobate form. The second type is exhibited in O. curvinotus and O. luzonensis, where gonadal rudiments appear unilateral only on the right side. But testicular tissue occurs on the left side of the dorsal mesentery during the development, and adult testes become biiobate. The testes of O. latipes belong to the third type. They initiated from bilateral gonadal rudiments to develop into bilobate testes, These results are almost correspond with the phylogenetical classification of Oryzias fishes based on the chromosome morphology, indicating that alleles of the gene(s) controlling the symmetry of gonads in these fish originated just after the time when the phylogenetic differentiation occurred. The possible process of the development of the various types of the asymmetrical gonads was discussed.