Messenger RNA transcription levels of neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex components in the olfactory nerve system of the anadromous Pacific salmon, masu salmon Oncorhynchus masou.

Anadromous Pacific salmon (genus Oncorhynchus) are known for homing behavior to their natal rivers based on olfactory imprinted memories during seaward migration. The SNARE complex is a regulator of vesicle exocytosis from the presynaptic membrane. Our previous study suggested that its component genes (Snap25, Stx1, and Vamp2) are more highly expressed in the olfactory nervous system (ONS) during the migration stages associated with olfactory imprinting in the evolutionary species of Pacific salmon, such as chum (O. keta) and pink (O. gorbuscha) salmon. Masu salmon (O. masou) has a significantly different life history from these species, living longer in rivers and being a more primitive Pacific salmon species. In this study, the transcription of snare mRNAs in the ONS was analyzed using mainly male wild masu salmon. Five cDNAs encoding masu salmon SNAREs, which are well conserved among vertebrates, were isolated and sequenced. Each snare mRNA was highly expressed in age 1+ (yearling) parr prior to smoltification, particularly in the olfactory bulb. Their transcription status was significantly different from that of chum and pink salmon, which showed high expression in earlier under-yearling juveniles. The present results and our previous studies indicate that snare mRNAs are highly transcripted until the seaward migration, reflecting neural development and neuroplasticity of the ONS for olfactory imprinting.

Olfactory behavioural and neural responses of planktivorous lacustrine sockeye salmon (Oncorhynchus nerka) to prey odours.

Fish use a variety of sensory systems when foraging. Salmonids are generally considered visual feeders. However, some species feed on zooplanktons under dark conditions, suggesting they also detect prey using nonvisual cues. Under experimental conditions, hatchery-reared rainbow trout (Oncorhynchus mykiss) have been shown to use olfaction when searching for food pellets, but olfactory foraging has not been documented in wild salmonids. In the present study, to examine their behavioural response and neural activity in the olfactory nervous system using c-fos expression as a neural molecular marker, immature wild-caught lacustrine sockeye salmon (Oncorhynchus nerka) in a flow-through aquarium were exposed to zooplanktons (Daphnia spp.) extract including zooplanktons odorant and to dimethyl sulfide. The salmon exposed to zooplanktons odour increased their total swimming distance and time, numbers of turns and ascents, and c-fos expression in the olfactory bulb, suggesting that they can detect zooplanktons extract to locate prey in the laboratory experiments. However, no response was seen in those exposed to dimethyl sulfide. The results of this study suggest that prey odour may serve as a chemosensory cue for wild immature salmonids.

Gene expression levels of synaptic exocytosis regulator synaptophysin in the brain and the olfactory organ of anadromous salmon.

Anadromous Pacific salmon (genus Oncorhynchus) are known for their homing behavior based on olfactory imprinting, which is formed during their seaward migration. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE/Snare) complex is a minimum unit of vesicle exocytosis from the pre-synaptic membrane. Its component genes (synaptosome-associated protein 25, syntaxin 1, and vesicle-associated membrane protein 2) are more strongly expressed in the olfactory nervous system (olfactory epithelium, olfactory bulb, and telencephalon) at the migration stages related to olfactory imprinting and/or retrieval in salmon. This study focused on the mRNA synthesis of synaptophysin (Syp), one of the Snare regulatory factors. syp is strongly expressed in chum salmon (Oncorhynchus keta) olfactory nervous system during the seaward migration and temporarily increased during the homeward migration. In reference to our previous studies, these expression changes were similar to the snare genes in the chum salmon. Therefore, syp and Snare component genes were synchronously expressed reflecting the development and short-term plasticity of the olfactory nervous system that is essential for olfactory imprinting.

Gene expression of neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex components in the olfactory organ and brain during seaward and homeward migration by pink salmon (Oncorhynchus gorbuscha).

The expression of synaptic vesicle exocytosis-regulator SNARE complex component genes (snap25, stx1 and vamp2) was examined in the olfactory nervous system during seaward and homeward migration by pink salmon (Oncorhynchus gorbuscha). The expression levels of snares in the olfactory organ were higher in seaward fry than in feeding and homeward adults, reflecting the development of the olfactory nervous system. The expression of snap25a, b and stx1a was upregulated or stable in the adult olfactory bulb and telencephalon. This upregulated expression suggested alterations in olfactory neuronal plasticity that may be related to the discrimination of natal rivers. The expression of stx1b was downregulated in the adult olfactory bulb, but remained stable in the adult telencephalon. The expression of vamp2 was initially strong in seaward fry, but was downregulated in adults in both the olfactory bulb and telencephalon. Pink salmon has the lowest diversity of maturation age, the largest population, and the most evolutional position in Pacific salmon (genus Oncorhynchus). The expression of snares in the olfactory center of pink salmon reflected the timing of sexual maturation and homeward migration. The present results and our previous studies indicate that snares show distinct expression patterns between two salmon species that depend on physiological and ecological features of migration.

Molecular characterization and gene expression of syntaxin-1 and VAMP2 in the olfactory organ and brain during both seaward and homeward migrations of chum salmon, Oncorhynchus keta.

Anadromous Pacific salmon (Genus Oncorhynchus) imprint odorants from their natal streams during their seaward migration, and adult salmon use olfaction to identify their natal streams during their homeward migration. However, little is known about the molecular mechanisms of olfactory imprinting in the salmon nervous system. Our previous study suggested that the snap25s gene (encoding a soluble N-ethylmaleimide-sensitive factor attachment protein receptor [SNARE] protein) is involved in pre-synaptic functions for olfactory imprinting and/or olfactory memory retrieval in chum salmon (O. keta). In this study, the expression of other SNARE proteins was analyzed in chum salmon brains. Three cDNAs, encoding salmon SNARE proteins (STX-1a, STX-1b, and VAMP2), were isolated and sequenced, which are well-conserved among vertebrates. Quantitative PCR detected the expression of stx1s and vamp2 in all regions of the brain, and especially highly in the olfactory bulb (OB) and telencephalon. The expression levels of snares in the olfactory rosette (OR) were higher during seaward migration than in adult life stages, subsequently vamp2 in the OB and telencephalon increased during seaward migration, corresponding well with development of the olfactory nervous system. Both stx1s in the OB and stx1b in the telencephalon were elevated in the seaward period, whereas stx1a in the telencephalon increased continuously until the feeding period. Both stx1s in the telencephalon increased in the last phase of upriver migration, possibly related to the retrieval of imprinted memory. Our results indicated the involvement and distinct roles of upregulated snares in synaptic plasticity for olfactory imprinting and/or olfactory memory retrieval in Pacific salmon.

Detection and localization of the thyroid hormone receptor beta mRNA in the immature olfactory receptor neurons of chum salmon.

Thyroid hormone (TH) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in various organs and tissues of vertebrates. It is generally accepted that anadromous Pacific salmon (Oncorhynchus spp.) imprint odorants from their natal stream during their seaward migration, and they then use olfaction to discriminate their natal stream during the spawning migration. Both serum TH levels and the specific binding values of TH in the salmon olfactory epithelium were markedly increased during the seaward migration. However, thyroid hormone receptor (TR) expression in the olfactory epithelium has not been confirmed in vertebrates. We investigated gene expression of TR isoforms in chum salmon (O. keta) by both molecular biological and histochemical techniques. Expression of TRß mRNA was detected in the olfactory epithelium by reverse transcriptase polymerase chain reaction (RT-PCR). Nucleotide sequencing demonstrated the existence of a remarkable homology between the RT-PCR product and part of the ligand-binding domain of other teleost TRß isoforms. By in situ hybridization using a digoxygenin-labeled salmon olfactory TRß cRNA probe, signals for salmon olfactory TRß mRNA were observed preferentially in the perinuclear regions of immature olfactory receptor neurons (ORNs), as protein gene product 9.5 (PGP9.5)-immunopositive ORNs. Our results provide the first detection of TRß gene expression in the olfactory epithelium, and suggested the possibility that TRß may be involved in cell maturation and/or cell differentiation of the ORNs in Pacific salmon.

Involvement of 11-ketotestosterone in hooknose formation in male pink salmon (Oncorhynchus gorbuscha) jaws.

Mature male Pacific salmon (Oncorhynchus spp.) develop a hooknose, as a secondary male sexual characteristic, during the spawning period. It is likely that androgens regulate hooknose formation. However, endocrinological and histochemical details about the relationship between androgens and hooknose formation are poorly understood. In this study, we performed assays of serum androgens, detection of androgen receptor (AR) in hooknose tissues, external morphological measurement of hooknose-related lengths, and microscopic observation of hooknose tissues of pink salmon (O. gorbuscha) at different stages of sexual maturation. Expression of the arß gene was detected in hooknose tissues of males but not females. The elongation of these tissues was mediated directly via androgens. Serum 11-ketotestosterone (11-KT) concentrations indicated a significant positive correlation with both jaw lengths during sexual maturation of males. In the upper jaw, cartilage tissue developed during hooknose formation, and AR-immunoreactive chondrocytes were located in the rostal-vetral regions of hooknose cartilage in maturing male. The chondrocytes in maturing males before entering into rivers exhibited rich-cytoplasm with high cell activity than at other sexual development stages. On the other hand, in the lower jaw, the development of the spongiosa-like bone meshworks. AR-immunoreactivity was detected in a proportion of the osteocytes and osteoblast-like cells in the spongiosa-like bone meshworks. These results indicate that hooknose formation in pink salmon, which is associated with the buildup of a structure with sufficient strength that it can be used to attack other males on the spawning ground, is regulated by 11-KT.

Molecular characterization and gene expression of synaptosome-associated protein-25 (SNAP-25) in the brain during both seaward and homeward migrations of chum salmon Oncorhynchus keta.

It is generally accepted that information about some of the odorants in the natal streams of anadromous Pacific salmon (Genus Oncorhynchus) is imprinted during their seaward migration, and that anadromous Pacific salmon use olfaction to identify their natal streams during the homeward migration. However, little is known about the molecular mechanisms of the various pre-synaptic functions that are important for olfactory imprinting and memory retrieval in the salmon brain. Synaptosome-associated protein-25 (SNAP-25) mediates pre-synaptic vesicle exocytosis and regulates synaptic transmission and neuronal plasticity. Despite the importance of synaptic plasticity for memorization, the expression of SNAP-25 in the salmon brain is not well understood. In this study, snap25 expression was detected in chum salmon (O. keta) brains using molecular biological techniques. Two cDNAs encoding salmon SNAP-25 were isolated and sequenced (SNAP-25a and SNAP-25b). These cDNAs encoded proteins with 204 amino acid residues, which showed marked homology with each other (97%). The protein and nucleotide sequences demonstrated a high level of homology between salmon SNAP-25s and those of other teleost species. By quantitative PCR, the expression of snap25a and snap25b was detected in all regions of the salmon brain, especially in the telencephalon. The expression levels of snap25a in the olfactory blub were higher during seaward migration than in upriver and post-upriver migrations, reflecting synaptogenesis in the olfactory nervous system, and snap25b in the telencephalon was increased during upriver period. Our results indicated that snap25s gene is involved in synaptic plasticity for olfactory imprinting and/or olfactory memory retrieval in Pacific salmon.

Comparative anatomy of the dorsal hump in mature Pacific salmon.

Mature male Pacific salmon (Genus Oncorhynchus) demonstrate prominent morphological changes, such as the development of a dorsal hump. The degree of dorsal hump formation depends on the species in Pacific salmon. It is generally accepted that mature males of sockeye (O. nerka) and pink (O. gorbuscha) salmon develop most pronounced dorsal humps. The internal structure of the dorsal hump in pink salmon has been confirmed in detail. In this study, the dorsal hump morphologies were analyzed in four Pacific salmon species inhabiting Japan, masu (O. masou), sockeye, chum (O. keta), and pink salmon. The internal structure of the dorsal humps also depended on the species; sockeye and pink salmon showed conspicuous development of connective tissue and growth of bone tissues in the dorsal tissues. Masu and chum salmon exhibited less-pronounced increases in connective tissues and bone growth. Hyaluronic acid was clearly detected in dorsal hump connective tissue by histochemistry, except for in masu salmon. The lipid content in dorsal hump connective tissue was richer in masu and chum salmon than in sockeye and pink salmon. These results revealed that the patterns of dorsal hump formation differed among species, and especially sockeye and pink salmon develop pronounced dorsal humps through both increases in the amount of connective tissue and the growth of bone tissues. In contrast, masu and chum salmon develop their dorsal humps by the growth of bone tissues, rather than the development of connective tissue.

Sulfurovum aggregans sp. nov., a hydrogen-oxidizing, thiosulfate-reducing chemolithoautotroph within the Epsilonproteobacteria isolated from a deep-sea hydrothermal vent chimney, and an emended description of the genus Sulfurovum.

A novel mesophilic, strictly hydrogen-oxidizing, sulfur-, nitrate- and thiosulfate-reducing bacterium, designated strain Monchim33(T), was isolated from a deep-sea hydrothermal vent chimney at the Central Indian Ridge. The non-motile, rod-shaped cells were Gram-stain-negative and non-sporulating. Growth was observed between 15 and 37 °C (optimum 33 °C; 3.2 h doubling time) and between pH 5.4 and 8.6 (optimum pH 6.0). The isolate was a strictly anaerobic chemolithoautotroph capable of using molecular hydrogen as the sole energy source and carbon dioxide as the sole carbon source. The G+C content of the genomic DNA was 42.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolate belonged to the genus Sulfurovum and was closely related to Sulfurovum sp. NBC37-1 and Sulfurovum lithotrophicum 42BK(T) (95.6 and 95.4 % similarity, respectively). DNA-DNA hybridization demonstrated that the novel isolate could be differentiated genotypically from Sulfurovum sp. NBC37-1 and Sulfurovum lithotrophicum. On the basis of the molecular and physiological traits of the new isolate, the name Sulfurovum aggregans sp. nov. is proposed, with the type strain Monchim33(T) ( = JCM 19824(T) = DSM 27205(T)).

Dorsal hump morphology in pink salmon (Oncorhynchus gorbuscha).

Mature male Pacific salmon (Genus Oncorhynchus) develop a dorsal hump, as a secondary male sexual characteristic, during the spawning period. Previous gross anatomical studies have indicated that the dorsal humps of salmon are mainly composed of cartilaginous tissue (Davidson [1935] J Morphol 57:169-183.) However, the histological and biochemical characteristics of such humps are poorly understood. In this study, the detailed microstructures and components of the dorsal humps of pink salmon were analyzed using histochemical techniques and electrophoresis. In mature males, free interneural spines and neural spines were located in a line near to the median septum of the dorsal hump. No cartilaginous tissue was detected within the dorsal hump. Fibrous and mucous connective tissues were mainly found in three regions of the dorsal hump: i) the median septum, ii) the distal region, and iii) the crescent-shaped region. Both the median septum and distal region consisted of connective tissue with a high water content, which contained elastic fibers and hyaluronic acid. It was also demonstrated that the lipid content of the dorsal hump connective tissue was markedly decreased in the mature males compared with the immature and maturing males. Although, the crescent-shaped region of the hump consisted of connective tissue, it did not contain elastic fibers, hyaluronic acid, or lipids. In an ultrastructural examination, it was found that all of the connective tissues in the dorsal hump were composed of collagen fibers. Gel electrophoresis of collagen extracts from these tissues found that the collagen in the dorsal hump is composed of Type I collagen, as is the case in salmon skin. These results indicate that in male pink salmon the dorsal hump is formed as a result of an increase in the amount of connective tissue, rather than cartilage, and the growth of free interneural spines and neural spines.

Androgens and very low density lipoprotein are essential for the growth of previtellogenic oocytes from Japanese eel, Anguilla japonica, in vitro.

To investigate the regulation of lipid uptake into the eel oocyte in more detail, effects of 11-ketotestosterone (11-KT) and lipid transporters (lipoproteins) were determined in vitro. Ovarian explants from previtellogenic Japanese eels (Anguilla japonica) were incubated for 28 days with 11-KT and/or with very low density lipoproteins (Vldl), low density lipoproteins (Ldl), or high density lipoproteins (Hdl) purified from eel plasma. The androgen 11-KT induced notable increases in oocyte diameter, which were accompanied by the appearance of vacuoles rather than lipid. Ldl and Hdl increased oocyte diameters, whereas Vldl did not. However, coincubation of 11-KT and Vldl, but not of Ldl or Hdl, resulted in dramatic increases in oocyte size and lipid droplet surface area. Effects of both 11-KT (oocyte size) and Vldl (lipid droplet surface area) were dose-dependent between 1 and 100 ng/ml and between 0.5 and 5 mg/ml, respectively. Interestingly, abnormal oocyte cytology under conditions of coculture with 11-KT and Vldl could essentially be prevented if Vldl concentrations were high enough (≥ 5 mg Vldl/ml medium). Unlike 11-KT, estradiol-17beta had no effect on oocyte diameter or lipid droplet surface area. We conclude that Vldl is a key transporter of neutral lipids that accumulate into the eel oocyte during oogenesis and that Vldl-dependent lipid uptake is stimulated by the androgen 11-KT.

Expressions of the multidrug resistance-related proteins in the rat olfactory epithelium: a possible role in the phase III xenobiotic metabolizing function.

The xenobiotic metabolizing system is considered to play important roles in the olfaction by the chemical homeostasis. Several phase I and phase II xenobiotic metabolizing enzymes are expressed in the olfactory epithelium in vertebrates. Multidrug resistance-related proteins (MRPs) are the phase III xenobiotic metabolizing pumps that eliminate some conjugated ligands from cells. However, the MRP-expressions in the olfactory epithelium have not been confirmed in the mammals. We investigated gene and protein expressions of MRP type 1 (MRP1) and type 2 (MRP2) isoforms in the adult rat olfactory epithelium in order to clarify the existence of phase III xenobiotic metabolizing pumps in the olfactory organs. Expressions of MRP1 mRNA were detected in the nasal cavity by reverse transcriptase polymerase chain reaction (RT-PCR). The nucleoside sequence of the RT-PCR products were completely identical to that found in other organs of rat. On the contrary, the analysis did not detect expressions of MRP2 mRNA in the nasal cavity. By in situ hybridization using a digoxigenin-labeled MRP1 cRNA probe, signals for MRP1 mRNA were observed preferentially in the perinuclear regions of supporting cells. However, the respiratory epithelial cells did not show the signals for MRP1 mRNA. By immunohistochemistry using a specific antibody to MRP1, MRP1-immunoreactivities were seen mainly on the supporting cells. These findings suggest that MRP1 is involved in olfaction as a part of the "olfactory signal termination" by the chemical homeostasis in the "perireceptor events" of the olfactory epithelium.

Family relationships of self-care-dependent older people and institutionalized rate to nursing homes.

BACKGROUND: The government recommends home care for self-care-dependent older people in order to suppress care expenditure. Family relationships between primary caregivers and self-care-dependent older people might be one of the factors influencing the institutionalized rate. METHOD: We investigated family relationships between primary caregivers and self-care-dependent older people at home in the rural town of Oodate, Akita Prefecture, and the urban district of Katsushika, Tokyo, in 2003. One thousand and thirty-six primary caregivers completed the questionnaire and entered the present study. Two years later, we prospectively followed how the family relationship between them influenced the institutionalized rate in 2005. Finally, 556 primary caregivers completed the questionnaire in 2005. RESULTS: The institutionalized rate of subjects with poor family relationships (31%) was significantly higher than that of subjects with good family relationships (12%). CONCLUSION: Good or poor family relationships were significantly related to psychological strains and might determine the institutionalized rate in nursing homes.

Morphometry of olfactory lamellae and olfactory receptor neurons during the life history of chum salmon (Oncorhynchus keta).

It is generally accepted that anadromous Pacific salmon (genus Oncorhynchus) imprint to odorants in their natal streams during their seaward migration and use olfaction to identify these during their homeward migration. Despite the importance of the olfactory organ during olfactory imprinting, the development of this structure is not well understood in Pacific salmon. Olfactory cues from the environment are relayed to the brain by the olfactory receptor neurons (ORNs) in the olfactory organ. Thus, we analyzed morphometric changes in olfactory lamellae of the peripheral olfactory organ and in the quantity of ORNs during life history from alevin to mature in chum salmon (Oncorhynchus keta). The number of lamellae increased markedly during early development, reached 18 lamellae per unilateral peripheral olfactory organ in young salmon with a 200 mm in body size, and maintained this lamellar complement after young period. The number of ORNs per olfactory organ was about 180,000 and 14.2 million cells in fry and mature salmon, respectively. The relationship between the body size (fork length) and number of ORNs therefore revealed an allometric association. Our results represent the first quantitative analysis of the number of ORNs in Pacific salmon and suggest that the number of ORNs is synchronized with the fork length throughout its life history.

Increased immunoreactivities against endothelin-converting enzyme-1 and monocyte chemotactic protein-1 in hepatic stellate cells of rat fibrous liver induced by thioacetamide.

The progression of rat liver fibrosis induced by intraperitoneal administration of thioacetamide (TAA) was evaluated by immunocytochemistry using anti-alpha-smooth muscle actin (alpha-SMA), antiendothelin-converting enzyme (ECE)-1, and anti-monocyte chemotactic protein (MCP)-1 antibodies. The fibrous septal spaces gradually increased after administration of TAA, and pseudolobules were established in the 7-week TAA-treated groups. Immunoreactivities against alpha-SMA were not detected in hepatic stellate cells (HSCs) of the control group without TAA treatment, although they were observed in the HSCs around the fibrous septal spaces in all TAA-treated groups, indicating that activation of HSCs occurs during the establishment of pseudolobules. Immunoreactivities against ECE-1 and MCP-1 were seen in such HSCs of the TAA-treated groups, but few or no immunoreactivities were detected in the HSCs of the control group. The most significant increase in the ECE-1 immunoreactivities was detected in the 1-week TAA-treated group, whereas that in MCP-1 was observed in the 7-week TAA-treated group. The present immunocytochemistry indicated a difference in the accelerated expression period between immunoreactivities against ECE-1 and MCP-1 in the HSCs during the progression of TAA-induced liver fibrosis, suggesting that ECE-1 is involved in the early phase of liver fibrosis and that MCP-1 plays a role during the later phase.

R352Q mutation of the DHCR7 gene is common among Japanese Smith-Lemli-Opitz syndrome patients.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive malformation syndrome characterized by microcephaly, syndactyly of toes, ambiguous genitalia, and mental retardation. The underlying DHCR7 gene has been identified and a wide variety of distinct mutations were reported in USA and European SLOS patients. A significant difference has been suggested in the frequency of SLOS among different ethnic populations. Here, we report mutational analysis of seven Japanese SLOS patients. Five mutations, R352Q, R242H, G303R, X476Q, and S192F, were identified, and R352Q appeared most frequent, since nine out of the 13 mutations of Japanese origin were the same R352Q. These results suggest that R352Q is a predominant founder mutation in Japanese SLOS patients.

Strongyloides ratti: chemokinesis of glycolytic enzyme- and lectin-treated third-stage infective larvae in vitro.

The infective third-stage larvae (L3s) of Strongyloides ratti, a parasitic nematode in rodents, showed two types of chemokinesis on a gradient of sodium chloride (NaCl) in an in vitro agarose tracking assay. The types were a consistent directional avoidance behavior under unfavorable environmental conditions and a reduced avoidance behavior under favorable conditions. We examined the effects of treatments with glycolytic enzymes and lectins by analyzing the avoidance behavior. L-Fucose dehydrogenase, hyaluronidase, beta-glucosidase, alpha-mannosidase, beta-galactosidase, concanavalin A, wheat germ agglutinin and soybean agglutinin exhibited inhibitory or enhancive effects on chemokinesis. We also confirmed the sites of the amphids of L3s aside from the mouth at the anterior end by scanning electron microscopy, and that concanavalin A-binding sites existed in the vicinity of the amphids using lectin-histochemistry. The carbohydrate moieties in the amphids of S. ratti L3s may play an important role as chemosensors in perceiving environmental cues.