Language：English   Publishing type：Research paper (scientific journal)  

Beard worms from the family Siboglinidae, are peculiar animals and are known for their symbiotic relationships with sulfur bacteria. Most Siboglinids inhabit the deep-sea floor, thus making difficult to make any observations in situ. One species, Oligobrachia mashikoi, occurs in the shallow depths (24.5 m) of the Sea of Japan. Taking advantage of its shallow-water habitat, the first ecological survey of O. mashikoi was performed over a course of 7 years, which revealed that its tentacle-expanding behavior was dependent on the temperature and illuminance of the sea water. Furthermore, there were significantly more O. mashikoi with expanding tentacles during the nighttime than during the daytime, and the prevention of light eliminated these differences in the number of expending tentacles. These results confirmed that the tentacle-expanding behavior is controlled by environmental light signals. Consistent with this, we identified a gene encoding a photoreceptor molecule, neuropsin, in O. mashikoi, and the expression thereof is dependent on the time of day. We assume that the described behavioral response of O. mashikoi to light signals represent an adaptation to a shallow-water environment within the predominantly deep-sea taxon.

DOI： 10.1038/s41598-023-33309-6

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Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-hour light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight. This article is protected by copyright. All rights reserved.

DOI： 10.1111/jpi.12834

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.aquaculture.2022.738437

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Publishing type：Research paper (scientific journal)   Publisher：Zoological Society of Japan  

DOI： 10.2108/zs210107

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ecoenv.2022.113401

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Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Biological Sciences in Space  

DOI： 10.2187/bss.36.9

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Publishing type：Research paper (scientific journal)   Publisher：ST Bio-life  

We previously reported that the oral administration of melatonin from 4 to 20 months to male mice improved femoral bone strength and bone density during the aging. Additionally, melatonin receptor, MT2, was immunologically detected in both osteoblasts and osteoclasts of the mouse femoral bone. Thus, melatonin can act on both osteoblasts and osteoclasts to maintain bone strength during the aging process. Here, we analyzed plasma calcium (Ca2+), magnesium (Mg2+), and inorganic phosphorus ([PO4]3-) in 20-month-old male mice with or without administration melatonin (15-20 mg/kg/day) in drinking water. We found that plasma Ca2+ and Mg2+ levels in melatonin-treated mice increased significantly as compared with control mice. In [PO4]3-, melatonin administration tended to increase its plasma level, but did not reach statistical significance. The potential association between these divalent ions and metabolism markers of femoral bone was also examined. In the femoral diaphysis, the plasma Ca2+ and Mg2+ concentrations were positively correlated with periosteal and endosteal circumference which were significantly associated with the Strength Strain Index. Therefore, melatonin treatment enlarged femoral diaphysis and enhanced bone strength by increasing mineral depositions. In addition, the plasma melatonin levels were significantly positive correlation with total bone density and critical thickness in the femoral diaphysis. Since we had not observed the primary trabecular bone and osteoclasts in 20-month-old mice previously, it is suggested that plasma Ca2+ and Mg2+ are not elevated due to bone resorption. The increased plasma Ca2+ and Mg2+ by melatonin may originate from the intestinal absorption of these ions since melatonin binds to the vitamin D3 receptor, its activation is known to promote the intestinal absorption of Ca2+.

DOI： 10.32794/mr112500113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

<title>Abstract</title>
Potential health benefits of melatonin have been suggested. Although melatonin is present in various foods, little is known about the health effects of dietary melatonin intake. We estimated habitual dietary melatonin intake and examined its association with total and cause-specific mortality in a population-based cohort study in Japan. Study subjects included 13,355 men and 15,724 women aged ≥35 years who responded to a self-administered questionnaire in 1992. Their diets were assessed via a food frequency questionnaire at baseline. The melatonin content in various foods on the questionnaire was measured to estimate melatonin intake. Mortality was ascertained during 16 years of follow-up (1992–2008). Hazard ratios (HRs) and 95% confidence intervals (CIs) for total and cause-specific mortality were calculated according to melatonin quartiles. A total of 5,339 deaths occurred during follow-up. Melatonin intake was significantly associated with decreased risks of total mortality, cardiovascular mortality, and noncancer, noncardiovascular mortality after controlling for covariates; HRs for the highest quartile of melatonin intake versus the lowest were 0.90 (95% CI: 0.82, 0.98; P for trend = 0.05), 0.85 (95% CI: 0.72, 0.99; P for trend = 0.10), and 0.77 (95% CI: 0.67, 0.90; P for trend = 0.003), respectively. The data suggest a potential benefit of dietary melatonin with regard to mortality rates.

DOI： 10.1093/aje/kwab213

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Language：English  

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Language：English   Publishing type：Research paper (scientific journal)  

The high plasma glucose induced in glucose metabolism disorders leads to the non-enzymatic glucose-dependent modification (glycation) of type 1 collagen, which is an essential component of bone tissue. The glycation of proteins induces the formation of advanced glycation end-products, such as carboxymethyl arginine, which is preferentially generated in glycated collagen. However, the effect of advanced glycation end-product formation on the characteristics of type 1 collagen remains unclear due to the lack of suitable in vitro experimental systems analyzing type 1 collagen. Here, we show that the glycation of type 1 collagen can be analyzed in vitro using a goldfish-scale bone model. Our study using these scales provides evidence that the advanced glycation end-product formation in type 1 collagen induced by glyoxal, the carboxymethyl arginine inducer, facilitates the crosslinking of type 1 collagen, decreasing both its strength and flexibility.

DOI： 10.1111/jdi.13528

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KYUSHU UNIV, FACULTY AGRICULTURAL PUBLICATIONS  

The fish scales are well known as a major source of internal calcium storage. Therefore, we developed an original assay system using goldfish scales in which both osteoclasts (bone resorption cells) and osteoblasts (bone formation cells) coexist. In our bioassay system, we utilized the activities of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as respective indicators of each activity in osteoclasts and osteoblasts. Using this bioassay system, the influence of cadmium chloride (CdCl2) on osteoclastic and osteoblastic activities in the scales of goldfish was examined in an in vivo experiment. Goldfish were kept in tap water containing CdCl2 (10(-7) M) for 2 days. TRAP activity in the scales of goldfish decreased with 2 days of exposure to CdCl2. This inhibitory effect for osteoclasts continued even at 4 days of exposure to CdCl2. In the case of osteoblasts, CdCl2 inhibited ALP activity in the goldfish scales at 4 days after exposure, although ALP activity in the goldfish scales had not changed after 2 days of exposure to CdCl2. In in vitro cultured goldfish scales, we previously reported that ALP activity decreased after exposures of 64 and 96 hrs, although their activities did not change after 6, 18, and 36 hrs. These results were supported by our in vivo experiment. This is the first report to indicate that both osteoclasts and osteoblasts in fish were suppressed by cadmium (Cd) treatments in vivo. Considering both in vivo and in vitro experiments, we concluded that Cd inhibited both osteoclastic and osteoblastic activities in goldfish. This suggests that Cd leads to a disturbed calcium metabolism and then induces bone anomalies.

DOI： 10.15017/4486551

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Melatonin (MEL) has been reported to enhance cognitive processes, making it a potential treatment for cognitive decline. However, the role of MEL's metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in these effects are unknown. The current study directly investigated the acute effects of systemic MEL, AFMK, and AMK on novel object recognition. We also analyzed MEL, AFMK, and AMK levels in hippocampus and temporal lobe containing the perirhinal cortex following systemic MEL and AMK treatment. AMK administered post-training had a more potent effect on object memory than MEL and AFMK. AMK was also able to rescue age-associated declines in memory impairments when object memory was tested up to 4 days following training. Results from administering AMK at varying times around the training trial and the metabolism time course in brain tissue suggest that AMK's memory-enhancing effects reflect memory consolidation. Furthermore, inhibiting the MEL-to-AMK metabolic pathway disrupted object memory at 24 hours post-training, suggesting that endogenous AMK might play an important role in long-term memory formation. This is the first study to report that AMK facilitates long-term object memory performance in mice, and that MEL crosses the blood-brain barrier and is immediately converted to AMK in brain tissue. Overall, these results support AMK as a potential therapeutic agent to improve or prevent memory decline.

DOI： 10.1111/jpi.12703

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/jpi.12703

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Osteocytes, osteoblasts (bone-forming cells), and osteoclasts (bone-resorbing cells) are the primary types of cells that regulate bone metabolism in mammals. Sclerostin produced in bone cells activates osteoclasts, inhibiting bone formation; excess production of sclerostin, therefore, leads to the loss of bone mass. Fish scales have been reported to have morphological and functional similarities to mammalian bones, making them a useful experimental system for analyzing vertebrate bone metabolism in vitro. However, whether fish scales contain cells producing sclerostin and/or osteocytes has not been determined. The current study demonstrated, for the first time, that sclerostin-containing cells exist in goldfish scales. Analysis of the distribution and shape of sclerostin-expressing cells provided evidence that osteoblasts produce sclerostin in goldfish scales. Furthermore, our results found that osteocyte-like cells exist in goldfish scales, which also produce sclerostin. Finally, we demonstrated that microgravity in outer space increased the level of sclerostin in the scales of goldfish, a finding suggesting that the induction of sclerostin is the mechanism underlying the activation of osteoclasts under microgravity.

DOI： 10.2220/biomedres.41.279

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Skeletal muscles have a high metabolic capacity, which play key roles in glucose metabolism. Although periodontal disease increases the risk of metabolic syndrome, the relationship between periodontal bacterial infection and skeletal muscle metabolic dysfunction is unclear. We found that anti-Porphyromonas gingivalis (Pg) antibody titers positively correlated with intramuscular adipose tissue content (IMAC), fasting blood glucose, and HOMA-IR in metabolic syndrome patients. In C57BL/6J mice fed a high-fat diet, recipients of oral Pg (HFPg) had impaired glucose tolerance, insulin resistance, and higher IMAC compared to recipients of saline (HFco). The soleus muscle in HFPg mice exhibited fat infiltration and lower glucose uptake with higher Tnfa expression and lower insulin signaling than in HFco mice. Gene set enrichment analysis showed that TNFα signaling via NFκB gene set was enriched in the soleus muscle of HFPg mice. Moreover, TNF-α also decreased glucose uptake in C2C12 myoblast cells in vitro. Based on 16S rRNA sequencing, Pg administration altered the gut microbiome, particularly by decreasing the abundance of genus Turicibacter. Microbial network of the gut microbiome was dramatically changed by Pg administration. Our findings suggest that infection with Pg is a risk factor for metabolic syndrome and skeletal muscle metabolic dysfunction via gut microbiome alteration.

DOI： 10.1096/fj.202001158R

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Astronauts are inevitably exposed to two major risks during space flight, microgravity and radiation. Exposure to microgravity has been discovered to lead to rapid and vigorous bone loss due to elevated osteoclastic activity. In addition, long‑term exposure to low‑dose‑rate space radiation was identified to promote DNA damage accumulation that triggered chronic inflammation, resulting in an increased risk for bone marrow suppression and carcinogenesis. In our previous study, melatonin, a hormone known to regulate the sleep‑wake cycle, upregulated calcitonin expression levels and downregulated receptor activator of nuclear factor‑κB ligand expression levels, leading to improved osteoclastic activity in a fish scale model. These results indicated that melatonin may represent a potential drug or lead compound for the prevention of bone loss under microgravity conditions. However, it is unclear whether melatonin affects the biological response induced by space radiation. The aim of the present study was to evaluate the effect of melatonin on the expression levels of genes responsive to space radiation. In the present study, to support the previous data regarding de novo transcriptome analysis of goldfish scales, a detailed and improved experimental method (e.g., PCR duplicate removal followed by de novo assembly, global normalization and calculation of statistical significance) was applied for the analysis. In addition, the transcriptome data were analyzed via global normalization, functional categorization and gene network construction to determine the impact of melatonin on gene expression levels in irradiated fish scales cultured in space. The results of the present study demonstrated that melatonin treatment counteracted microgravity‑ and radiation‑induced alterations in the expression levels of genes associated with DNA replication, DNA repair, proliferation, cell death and survival. Thus, it was concluded that melatonin may promote cell survival and ensure normal cell proliferation in cells exposed to space radiation.

DOI： 10.3892/mmr.2020.11363

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Melatonin has been implicated in the regulation of bone metabolism; however, the molecular mechanisms underlying its involvement in fracture healing are still obscure. We previously developed an in vivo fracture healing model using the scale of a double-transgenic zebrafish, trap:GFP; osterix:mCherry, which labels osteoclasts and osteoblasts with GFP and mCherry, respectively. Here we show using this model that melatonin inhibits both osteoblast and osteoclast differentiation under fracture stress through the repression of Erk signaling in epidermal cells of the scale. Melatonin treatment resulted in reduced numbers of both osteoblasts and osteoclasts in the fractured scale. Immunochemistry analysis revealed that Erk signals in epidermal cells, which express melatonin receptors, were greatly enhanced in response to fracture stress, but this enhancement was blocked by melatonin treatment. Moreover, inhibition of Erk signaling phenocopied the effects of melatonin treatment in the fractured scale. Collectively, these data suggest that the activation of epidermal Erk signaling is required for both osteoblast and osteoclast differentiation in the early stage of fracture healing, and melatonin suppresses epidermal Erk signaling, leading to impaired fracture healing.

DOI： 10.1016/j.bbrc.2020.07.075

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Melatonin has recently been found to be a possible new regulator of bone metabolism. However, the influence of melatonin in natural age-related osteoporosis has not been fully elucidated yet, although there have been some reports regarding postmenopausal osteoporosis with melatonin treatments. The present study investigated the effects of long-term melatonin administration during the aging process on bone metabolism. Using quantitative computed tomography methods, we found that the total bone density of both the femur metaphysis and diaphysis decreased significantly in 20-month-old male mice. In the metaphysis, both trabecular bone mass and Polar-Strength Strain Index (SSI), which is an index of bone strength, decreased significantly. Judging from bone histomorphometry analysis, trabecular bone in 20-month-old male mice decreases significantly with age and is small and sparse, as compared to that of 4-month-old male mice. Loss of trabecular bone is one possible cause of loss of bone strength in the femoral bone. In the metaphysis, the melatonin administration group had significantly higher trabecular bone density than the non-administration group. The Polar-SSI, cortical area, and periosteal circumference in the diaphysis was also significantly higher with melatonin treatments. Since the melatonin receptor, MT2, was detected in both osteoblasts and osteoclasts of the femoral bone of male mice, we expect that melatonin acts on osteoblasts and osteoclasts to maintain the bone strength of the diaphysis and metaphysis. Thus, melatonin is a potential drug for natural age-related osteoporosis.

DOI： 10.1016/j.acthis.2020.151596

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Differentiation of osteoclasts (OCs) from hematopoietic cells requires cellular interaction with osteoblasts (OBs). Due to the difficulty of live-imaging in the bone, however, the cellular and molecular mechanisms underlying intercellular communication involved in OC differentiation are still elusive. Here, we develop a fracture healing model using the scale of trap:GFP; osterix:mCherry transgenic zebrafish to visualize the interaction between OCs and OBs. Transplantation assays followed by flow cytometric analysis reveal that most trap:GFPhigh OCs in the fractured scale are detected in the osterix:mCherry+ fraction because of uptake of OB-derived extracellular vesicles (EVs). In vivo live-imaging shows that immature OCs actively interact with osterix:mCherry+ OBs and engulf EVs prior to convergence at the fracture site. In vitro cell culture assays show that OB-derived EVs promote OC differentiation via Rankl signaling. Collectively, these data suggest that EV-mediated intercellular communication with OBs plays an important role in the differentiation of OCs in bone tissue.

DOI： 10.1038/s42003-020-0925-1

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It has been reported that spinal deformity was induced in developing fish by the addition of polycyclic aromatic hydrocarbons (PAHs). To examine the mechanism of the disruption of fish bone metabolism, the effect of benz[a]anthracene (BaA), a kind of PAH, on plasma calcium, inorganic phosphorus, osteoblasts, and osteoclasts was investigated in this study. We also measured several plasma components to analyze the toxicity of BaA on other metabolisms. BaA (1 or 10 ng/g body weight) was intraperitoneally injected (four times) into nibbler fish during breeding, for 10 days, and it was indicated, for the first time, that injecting high doses of BaA to nibbler fish induced both hypocalcemia and hypophosphatemia. Furthermore, in the scales of nibbler fish treated with high doses of BaA, both osteoclastic and osteoblastic marker messengerRNA (mRNA) expressions decreased. These results are a cause of disruption of bone metabolism and, perhaps, the induction of spinal deformities. In addition, we found that total protein, metabolic enzymes in the liver, total cholesterol, free cholesterol, and high-density lipoprotein cholesterol levels significantly decreased in BaA-injected fish. These results indicate that BaA may affect liver diseases and emphasize the importance of prevention of aquatic PAH pollution.

DOI： 10.3390/ijerph17041391

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Circadian clocks are intrinsic, time-tracking processes that confer a survival advantage on an organism. Under natural conditions, they follow approximately a 24-h day, modulated by environmental time cues, such as light, to maximize an organism's physiological efficiency. The exact timing of this rhythm is established by cell-autonomous oscillators called cellular clocks, which are controlled by transcription-translation negative feedback loops. Studies of cell-based systems and wholeanimal models have utilized a pharmacological approach in which chemical compounds are used to identify molecular mechanisms capable of establishing and maintaining cellular clocks, such as posttranslational modifications of cellular clock regulators, chromatin remodeling of cellular clock target genes' promoters, and stability control of cellular clock components. In addition, studies with chemical compounds have contributed to the characterization of light-signaling pathways and their impact on the cellular clock. Here, the use of chemical compounds to study the molecular, cellular, and behavioral aspects of the vertebrate circadian clock system is described.

DOI： 10.2174/1389450120666190926143120

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Publishing type：Research paper (scientific journal)  

© 2019 The Authors. Journal of Pineal Research Published by John Wiley & Sons Ltd Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

DOI： 10.1111/jpi.12594

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Language：English   Publishing type：Research paper (scientific journal)  

Purpose: Light-emitting diodes that emit high-intensity blue light are associated with blue-light hazard. Here, we report that blue light disturbs circadian rhythms by interfering with the clock gene in the suprachiasmatic nucleus (SCN) and that suppression of blue light at night ameliorates metabolic abnormalities by controlling circadian rhythms. Methods: C57BL/6J mice were exposed to 10-lux light for 30 minutes at Zeitgeber time 14 for light pulse with blue light or blue-light cut light to induce phase shift of circadian rhythms. Phase shift, clock gene expression in SCN, and metabolic parameters were analyzed. In the clinical study, healthy participants wore blue-light shield eyewear for 2 to 3 hours before bed. Anthropometric data analyses, laboratory tests, and sleep quality questionnaires were performed before and after the study. Results: In mice, phase shift induced with a blue-light cut light pulse was significantly shorter than that induced with a white light pulse. The phase of Per2 expression in the SCN was also delayed after a white light pulse. Moreover, blood glucose levels 48 hours after the white light pulse were higher than those after the blue-cut light pulse. Irs2 expression in the liver was decreased with white light but significantly recovered with the blue-cut light pulse. In a clinical study, after 1 month of wearing blue-light shield eyeglasses, there were improvements in fasting plasma glucose levels, insulin resistance, and sleep quality. Conclusions: Our results suggest that suppression of blue light at night effectively maintains circadian rhythms and metabolism.

DOI： 10.1167/iovs.19-27195

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© 2019, The Author(s). To elucidate the physiological role of calcitonin (CT) in stingrays (cartilaginous fish), an enzyme-linked immunosorbent assay (ELISA) system using a specific antibody against stingray CT has been developed. Synthetic stingray CT was subcutaneously injected into mice four times—once every 2 weeks—together with an adjuvant. We purified the IgG antibody fraction using the protein A affinity chromatography from collected antiserum. Evaluating the antibody titer, we found the antibody’s optimum dilution ratio to be 600 times. Competitive ELISA has been developed using the antibody diluted 600 times. Our antibody did not cross-react with teleost CTs and muscle extraction, but cross-reacted with stingray plasma and the extract of the ultimobranchial gland, the secretary organ of stingray CT. Using this ELISA, we measured the plasma CT level in stingrays and examined its correlation with several mineral concentrations. Plasma CT did not show significant correlation to calcium, magnesium, inorganic phosphorus, sodium, chlorine, or urea, although there was a correlation among the factors involved in osmoregulation, such as sodium, chlorine, and urea. On the other hand, plasma CT was significantly correlated to body weight and length. Furthermore, there was a significant correlation between plasma CT and gonad weight. Since plasma CT was correlated with the weight of liver, which is involved in the synthesis of egg yolk protein, we examined the influence of 17β-estradiol (E2) on CT secretion. After E2 injection, the plasma CT level increased significantly. This is the first study to demonstrate that E2 induced plasma CT secretion in cartilaginous fish.

DOI： 10.1007/s40071-019-00236-0

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Circadian clocks are intrinsic, time-tracking systems that bestow upon organisms a survival advantage. Under natural conditions, organisms are trained to follow a 24-h cycle under environmental time cues such as light to maximize their physiological efficiency. The exact timing of this rhythm is established via cell-autonomous oscillators called cellular clocks, which are controlled by transcription/translation-based negative feedback loops. Studies using cell-based systems and genetic techniques have identified the molecular mechanisms that establish and maintain cellular clocks. One such mechanism, known as post-translational modification, regulates several aspects of these cellular clock components, including their stability, subcellular localization, transcriptional activity, and interaction with other proteins and signaling pathways. In addition, these mechanisms contribute to the integration of external signals into the cellular clock machinery. Here, we describe the post-translational modifications of cellular clock regulators that regulate circadian clocks in vertebrates.

DOI： 10.2174/1389202919666191014094349

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Many studies have investigated the actions of melatonin on osteoblasts and osteoclasts. However, the underlying mechanisms, especially regarding osteocyte function, remain largely unknown. Therefore, this study aimed to clarify the underlying mechanisms of melatonin action on bone tissue via osteocyte function. Chick calvariae were employed as a model. In ovo injection of melatonin (5, 50 and 500 µg) dose-dependently decreased the mRNA expression levels of cathepsin K and matrix metalloproteinase 9 (MMP9) in chick calvariae without affecting the expression levels of receptor activator of NF-κB ligand or osteoprotegerin. Surprisingly enough, the expression of calcitonin mRNA in chick calvariae was significantly raised. After 3 days of in vitro treatment of melatonin (10-7 and 10-5 M) on newly hatched chick calvariae, both calcitonin mRNA expression in calvariae and the concentration of calcitonin in cultured medium were augmented in a dose-dependent manner, coincident with the decreased mRNA expression levels of cathepsin K and MMP9. Immunohistochemical analyses revealed expression of melatonin receptors and calcitonin by osteocytes buried in bone matrix. Moreover, the mRNA expression levels of melatonin receptors, calcitonin and sclerostin (a marker of osteocyte), were strongly and positively correlated. In conclusion, we demonstrated the expression of melatonin receptors and calcitonin expression in osteocytes for the first time and suggest a new mechanism underlying the suppressive effect of melatonin on osteoclasts via upregulation of calcitonin secretion by osteocytes.

DOI： 10.1530/JOE-18-0707

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DOI： 10.24659/gsr.6.1_049

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioscientifica  

Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary BALB/c mouse chondrocytes caused enhanced cell growth and increased expression of <italic>Col2a1</italic>, <italic>Aggrecan</italic> and <italic>Sox9</italic>, but inhibited <italic>Col10a1</italic> expression. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of <italic>Aanat</italic>, <italic>Mt1</italic>, <italic>Mt2</italic> and <italic>Pthrp</italic> expression, followed by <italic>Sox9</italic> and <italic>Ihh</italic>. Furthermore, expression of the clock gene <italic>Bmal1</italic> was induced, while that of <italic>Per1</italic> was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of <italic>Aanat</italic> and modified the cyclic rhythm of <italic>Bmal1</italic>, <italic>Mt1</italic> and <italic>Mt2</italic>. In contrast, <italic>Mt1</italic> and <italic>Mt2</italic> showed different rhythms from <italic>Bmal1</italic> and <italic>Aanat</italic>, indicating the existence of different regulatory genes. Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock.

DOI： 10.1530/JOE-19-0022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KYUSHU UNIV, FACULTY AGRICULTURAL PUBLICATIONS  

To analyze the influences of environmental pollutants and bioactive substances on fish bone metabolism, we have developed an original in vitro bioassay using goldfish scales that have osteoclasts, osteoblasts, and bone matrix. We used tartrate-resistant acid phosphatase (TRAP) for osteoclasts and alkaline phosphatase (ALP) for osteoblasts as respective markers. Using this assay system, we have reported the influences of heavy metals such as cadmium (Cd) and mercury (Hg) on fish bone metabolism. In the case of Cd, we found that its concentration (even at 10(-13) M) influenced osteoclastic activity in goldfish scales only at 6 hrs of incubation. Thus, in the present study, we examined the effects of polluted seawater on osteoclastic and osteoblastic activities with this scale in vitro bioassay. Polluted seawater was collected from the Suez Gulf site on the Red Sea. Polluted seawater was added into culture medium at dilution rates of 50,100, and 500 times and incubated with the goldfish scales for 6 hrs. Subsequently, the influences of polluted seawater on TRAP and ALP activities were compared with that of artificial seawater as a non-polluted seawater. As a result, TRAP activity was significantly suppressed by the polluted seawater samples diluted at least 500 times; in contrast, ALP activity did not show any change. This response was similar to the response of Cd and Hg. As heavy metal contamination of the Suez Gulf site has been reported, there is a high possibility that heavy metals are contained in the seawater. Considering our data together with environmental pollution in Egypt that has been reported until now, we should conduct a heavy metal risk assessment to protect the ecosystem in the Red Sea.

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The circadian clock generates behavioral rhythms to maximize an organism's physiological efficiency. Light induces the formation of these rhythms by synchronizing cellular clocks. In zebrafish, the circadian clock components Period2 (zPER2) and Cryptochrome1a (zCRY1a) are light-inducible, however their physiological functions are unclear. Here, we investigated the roles of zPER2 and zCRY1a in regulating locomotor activity and behavioral rhythms. zPer2/zCry1a double knockout (DKO) zebrafish displayed defects in total locomotor activity and in forming behavioral rhythms when briefly exposed to light for 3-h. Exposing DKO zebrafish to 12-h light improved behavioral rhythm formation, but not total activity. Our data suggest that the light-inducible circadian clock regulator zCRY2a supports rhythmicity in DKO animals exposed to 12-h light. Single cell imaging analysis revealed that zPER2, zCRY1a, and zCRY2a function in synchronizing cellular clocks. Furthermore, microarray analysis of DKO zebrafish showed aberrant expression of genes involved regulating cellular metabolism, including ATP production. Overall, our results suggest that zPER2, zCRY1a and zCRY2a help to synchronize cellular clocks in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish. Further, zPER2 and zCRY1a regulate total physical activity, likely via regulating cellular energy metabolism. Therefore, these circadian clock components regulate the rhythmicity and amount of locomotor behavior.

DOI： 10.1038/s41598-018-37879-8

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© 2018 Elsevier Inc. This study aimed to investigate the precise data of gene expression, functions, and chronological relationships amongst communication molecules involved in the bone remodeling process with an in vivo model using autologous transplanted scales of goldfish. Autotransplantation of methanol-fixed cell-free scales triggers scale resorption and regeneration, as well as helps elucidate the process of bone remodeling. We investigated osteoclastic markers, osteoblastic markers, and gene expressions of communicating molecules (RANKL, ephrinB2, EphB4, EphA4, Wnt10b) by qPCR, in situ hybridization for Wnt10b, and immunohistochemistry for EphrinB2 and EphA4 proteins to elucidate the bone remodeling process. Furthermore, functional inhibition experiments for the signaling of ephrinB2/Eph, ephrin/EphA4, and Wnt10b using specific antibodies, revealed that these proteins are involved in key signaling pathways promoting normal bone remodeling. Our data suggests that the remodeling process comprises of two successive phases. In the first absorption phase, differentiation of osteoclast progenitors by RANKL is followed by the bone absorption by mature, active osteoclasts, with the simultaneous induction of osteoblast progenitors by multinucleated osteoclast-derived Wnt10b, and proliferation of osteoblast precursors by ehprinB2/EphB4 signaling. Subsequently, during the second formation phase, termination of bone resorption by synergistic cooperation occurs, with downregulation of RANKL expression in activated osteoblasts and Ephrin/EphA4-mediated mutual inhibition between neighboring multinucleated osteoclasts, along with simultaneous activation of osteoblasts via forward and reverse EphrinB2/EphB4 signaling between neighboring osteoblasts. In addition, the present study shows that autologous transplantation of methanol-fixed cell-free scale is an ideal in vivo model to study bone remodeling.

DOI： 10.1016/j.cbpa.2018.06.011

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© 2018, The Author(s). The effects of α-melanocyte-stimulating hormone (α-MSH) on calcium metabolism were examined with goldfish. The scales on the left side of goldfish bodies were removed to allow the regeneration of scales under anesthesia. Thereafter, the influences of α-MSH injection (low dose: 0.1 μg/g body weight; high dose: 1 μg/g body weight) on plasma calcitonin (calcium-regulating hormone) and the calcium content of the scales were investigated. Ten days after removing the scales, we measured the plasma calcitonin and calcium content of both regenerating scales on the left side and ontogenic scales on the right side. At both doses of α-MSH injection, plasma calcitonin concentrations in the α-MSH-treated group were significantly higher than those in the control group. The mRNA expressions of α-MSH-receptors were detected in the ultimobranchial glands (secretory organ of calcitonin), indicating that α-MSH directly functions in ultimobranchial glands and promotes calcitonin secretion. Furthermore, we found that the calcium content of regenerating scales in α-MSH-treated goldfish was higher than that in control goldfish, while the calcium content of ontogenic scales on the right side was significantly decreased by α-MSH injection. There was a significant co-relationship between plasma calcitonin and the calcium content of regenerating scales. The mRNA expression of calcitonin receptors in regenerating scales was remarkably higher than that in ontogenic scales. These results imply that calcitonin functions to promote scale regeneration resulting from the inhibition of bone resorption because calcitonin suppresses osteoclastic activity. Thus, we are the first to demonstrate the interaction between α-MSH and calcitonin in teleosts.

DOI： 10.1007/s40071-018-0206-5

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Language：English   Publishing type：Research paper (scientific journal)  

J-GLOBAL

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Language：English   Publishing type：Research paper (scientific journal)  

We examined the effects of α-melanocyte-stimulating hormone (α-MSH) on bone metabolism using regenerating goldfish scales. Normally developed scales on the bodies of goldfish were removed to allow the regeneration of scales under anesthesia. Thereafter, the influence of α-MSH on the regeneration of goldfish scales was investigated in vivo. In brief, α-MSH was injected at a low dose (0.1 μg/g body weight) or a high dose (1 μg/g body weight) into goldfish every other day. Ten days after removing the scales, we collected regenerating scales and analyzed osteoblastic and osteoclastic activities as respective marker enzyme (alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase for osteoclasts) activity in the regenerating scales as well as plasma calcium levels. At both doses, osteoblastic and osteoclastic activities in the regenerating scales increased significantly. Plasma calcium concentrations in the α-MSH-treated group (high doses) were significantly higher than those in the control group. Next, in vitro experiments were performed to confirm the results of in vivo experiments. In the cultured regenerating scales, osteoblastic and osteoclastic activities significantly increased with α-MSH (10-7 and 10-6 M) treatment. In addition, real-time PCR analysis indicated that osteoclastogenesis in α-MSH-treated scales was induced by the receptor activator of the NF-κB/receptor activator of the NF-κB ligand/osteoprotegerin pathway. Furthermore, we found that α-MSH receptors (melanocortin receptors 4 and 5) were detected in the regenerating scales. Thus, in teleosts, we are the first to demonstrate that α-MSH functions in bone metabolism and promotes bone resorption via melatonin receptors 4 and/or 5.

DOI： 10.1016/j.ygcen.2018.03.020

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Language：Japanese  

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Language：Japanese   Publisher：(株)メディカルレビュー社  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KYUSHU UNIV, FACULTY AGRICULTURAL PUBLICATIONS  

We examined immunoreactive calcitonin (iCT) cells in the nervous system of the polychaete Perinereis aibuhitensis using immunohistochemical methods. We found the most iCT cells (53-70 cells) in the cerebral ganglion. We also detected iCT cells (4-6 cells) in the subpharyngeal ganglion. Furthermore, it was noted that iCT cells were present bilaterally in each segment of the ventral nerve cord. These results suggest that iCT cells play some functional roles in the nervous system. Next, the molecular weight (MW) of the iCT substance was examined using the western blotting method. Cerebral ganglia were collected from 200 individuals. These ganglia were homogenized and centrifuged. The separated supernatants were treated with 66% acetone and then dialyzed to remove low MW substances (less than 2,000) at 4 degrees C. After lyophilization, the sample was reconstituted with 1 M acetic acid and then fractionated with an ultrafiltration membrane system into MWs of 3,000 to 10,000. Thereafter, the specimen was separated by SDS-polyacrylamide gel electrophoresis and analyzed by western blotting with anti-salmon calcitonin (CT) antiserum. Our results indicated that the MW of the iCT substance was close to that of teleost fish CT (3.5 kDa). The annelid Capitella teleta has two genes encoding CT-like peptides. This suggests that the iCT substance in P. aibuhitensis includes amino acid residues similar to fish CT and belongs to the CT family.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The nucleotide sequence of a sardine preprocalcitonin precursor has been determined from their ultimobranchial glands in the present study. From our analysis of this sequence, we found that sardine procalcitonin was composed of procalcitonin amino-terminal cleavage peptide (N-proCT) (53 amino acids), CT (32 amino acids), and procalcitonin carboxyl-terminal cleavage peptide (C-proCT) (18 amino acids). As compared with C-proCT, N-proCT has been highly conserved among teleosts, reptiles, and birds, which suggests that N-proCT has some bioactivities. Therefore, both sardine N-proCT and sardine CT were synthesized, and their bioactivities for osteoblasts and osteoclasts were examined using our assay system with goldfish scales that consisted of osteoblasts and osteoclasts. As a result, sardine N-proCT (10(-7) M) activated osteoblastic marker enzyme activity, while sardine CT did not change. On the other hand, sardine CT (10(-9) to 10(-7) M) suppressed osteoclastic marker enzyme activity, although sardine N-proCT did not influence enzyme activity. Furthermore, the mRNA expressions of osteoblastic markers such as type 1 collagen and osteocalcin were also promoted by sardine N-proCT (10(-7) M) treatment; however, sardine CT did not influence their expressions. The osteoblastic effects of N-proCT lack agreement. In the present study, we can evaluate exactly the action for osteoblasts because our scale assay system is very sensitive and it is a co-culture system for osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT seem to influence osteoblasts and osteoclasts and promote bone formation by different actions in teleosts.

DOI： 10.1016/j.cbpa.2017.06.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

The influence of sodium fluoride (NaF) on calcium metabolism was examined in nibbler fish (marine teleosts). Two days after the administration of NaF (5 mu g/g of body weight) (around 10(-4) M in fish), we showed that plasma calcium levels significantly decreased in NaF-treated nibbler fish. In addition, we detected fluoride in the treated scales by use of a scanning electron microscope with an energy-dispersive X-ray microanalysis, indicating that NaF directly affects their scales. Therefore, the influence of NaF on osteoblasts and osteoclasts in the scales was examined. In the scales of NaF-injected nibbler fish, tartrate-resistant acid phosphatase (TRAP) (osteoclastic marker enzyme) decreased, although alkaline phosphatase (osteoblastic marker enzyme) was activated. To confirm the effect of NaF on osteoclasts, furthermore, the mRNA expressions of osteoclastic markers (matrix metalloproteinase-9 and TRAP) were decreased significantly 2 days after incubation. In barred knifejaws, plasma calcium levels decreased as they did in nibbler fish. Therefore, NaF functions in both osteoblasts and osteoclasts and then influences calcium metabolism in marine fish. In the marine environment, high levels of fluoride (1.2-1.5 mg F-/l) (around 10(-5)-10(-4) M) are present in seawater. It is probable that teleosts living in seawater efficiently use fluoride to regulate their blood calcium levels.

DOI： 10.1007/s12562-017-1086-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Calcitonin (CT) is a hormone that decreases serum calcium level by suppressing osteoclastic activity in the vertebrate bone. In vertebrates, the structure-function relationship of CTs has been studied extensively. We recently identified three CT superfamily peptides, Bf-CTFP1 to 3, and clarified the molecular and functional characteristics of their receptor and receptor activity-modifying protein in amphioxus, Branchiostoma floridae. However, the CT activity of Bf-CTFPs has yet to be investigated. In the present study, a functional analysis of Bf-CTFPs was performed using goldfish scales having both osteoclasts and osteoblasts. All Bf-CTFPs suppressed osteoclastic activity via a goldfish CT receptor. Although the primary amino acid sequences of the Bf-CTFPs showed low sequence similarity to vertebrate CTs, Bf-CTFP1 to 3 share three amino acids, Thr(25), Thr(27), and Pro(32)-NH2, that are required for receptor binding, with salmon CT. Moreover, homology model analysis revealed that the Bf-CTFPs form alpha-helical structures. The alpha-helical position and length of Bf-CTFP1 and 2 were conserved with those of a highly potent ligand, teleost CT. Interestingly, the composition of the alpha-helix of Bf-CTFP3 differed from those of teleost CT, despite that the action of Bf-CTFP3 on goldfish scales was the same as that of Bf-CTFP1 and 2. Collectively, the present study provides new insights into the structure-function relationship of CT and its functional evolution in chordates. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2017.01.004

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DOI： 10.1080/23312025.2017.1294550

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

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The effects of low-intensity pulsed ultrasound (LIPUS) on osteoclastogenesis were examined using fish scales that had both osteoclasts and osteoblasts. The binding of the receptor activator of NF-κB ligand (RANKL) in osteoblasts to the receptor activator of NF-κB (RANK) in osteoclasts induced osteoclastogenesis. Therefore, we focused on RANK/RANKL signaling. After 6 h of incubation following LIPUS treatment, mRNA expression of RANKL increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 h of incubation, and then the mRNA expression of functional genes was significantly up-regulated after 12 h of incubation. However, the mRNA expression of osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 h of incubation and tended to further increase after 12 h of incubation. At 24 h of incubation, osteoclastic functional genes' mRNA expression decreased to the level of the control. Furthermore, we performed an in vivo experiment with goldfish. Two weeks after daily LIPUS exposure, osteoclastic marker enzymes tended to decrease while osteoblastic marker enzymes were activated. The regeneration rate of the LIPUS-treated scales was significantly higher than that of the control scales. Thus, LIPUS moderately activates osteoclasts and induces bone formation.

DOI： 10.2220/biomedres.38.71

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Increased risk of fracture associated with type 2 diabetes has been a topic of recent concern. Fracture rislc is related to a decrease in bone strength, which can be affected by bone metabolism and the quality of the bone. To investigate the cause of the increased fracture rate in patients with diabetes through analyses of bone metabolism and bone matrix protein properties, we used goldfish scales as a bone model for hyperglycemia. Using the scales of seven alloxan-treated and seven vehicle-treated control goldfish, we assessed bone metabolism by analyzing the activity of marker enzymes and mRNA expression of marker genes, and we measured the change in molecular weight of scale matrix proteins with SDS-PAGE. After only a 2-week exposure to hyperglycemia, the molecular weight of alpha- and beta-fractions of bone matrix collagen proteins changed incrementally in the regenerating scales of hyperglycemic goldfish compared with those of euglycemic goldfish. In addition, the relative ratio of the gamma-fraction significantly increased, and a delta-fraction appeared after adding glyceraldehyde a candidate for the formation of advanced glycation end products in diabetes to isolated type 1 collagen in vitro. The enzymatic activity and mRNA expression of osteoblast and osteoclast markers were not significantly different between hyperglycemic and euglycemic goldfish scales. These results indicate that hyperglycemia is likely to affect bone quality through glycation of matrix collagen from an early stage of hyperglycemia. Therefore, non-enzymatic glycation of collagen fibers in bone matrix may lead to the deterioration of bone quality from the onset of diabetes. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpa.2016.09.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The influence of sodium fluoride (NaF) on calcium metabolism was examined in goldfish (fresh water teleost). At 2 days after administration of NaF (500 ng/g body weight; 5 pg/g body weight) (around 10(-5) to 10(-4)M in goldfish), we indicated that plasma calcium levels upregulated in both doses of NaF-treated goldfish. To examine the mechanism of hypercalcemia by NaF treatments, therefore, direct effects of NaF on osteoblasts and osteoclasts in goldfish were investigated by an original assay system using teleost scale which has osteoblasts, osteoclasts and bone matrix. Alkaline phosphatase activity in the scales increased with the treatment of NaF (10(-6) and 10(-5) M) during 6 h of incubation. Also, tartrate-resistant acid phosphatase activity increased after exposure to NaF (10(-5) M) at the 6 h of incubation. To investigate the osteoclastic activation, the mRNA expression of osteoclastogenesis related factors were examined. The receptor activator of the nuclear factor-kappa B ligand (RANKL) which is known as a factor for osteoclastogenesis, increased in the NaF-treated scales after 6 h of incubation. The ratio of RANKL/osteoprotegerin (osteoclastogenesis inhibitory factor) significantly increased after 6 h of incubation. Resulting from the increase of RANKL mRNA level, the expression of transcription-regulating factors was significantly increased. Furthermore, the expression of functional genes, cathepsin K and matrix metalloproteinase-9 mRNA, was significantly increased. In our knowledge, this is the first report concerning the effects of NaF on osteoblasts and osteoclasts in teleosts. We concluded that NaF influences calcium metabolism via osteoclastic activation in goldfish. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpc.2016.07.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biomedical Research Press  

Mechanical stress promotes osteoblast proliferation and differentiation from mesenchymal stem cells (MSCs). Although numerous growth factors and cytokines are known to regulate this process, information regarding the differentiation of mechanically stimulated osteoblasts from MSCs in in vivo microenvironment is limited. To determine the significant factors involved in this process, we performed a global analysis of differentially expressed genes, in response to tensile stress, in the mouse cranial suture wherein osteoblasts differentiate from MSCs. We found that the gene expression levels of several components involved in bone morphogenetic protein, Wnt, and epithelial growth factor signalings were elevated with tensile stress. Moreover gene expression of some extracellular matrices (ECMs), such as cysteine rich protein 61 (Cyr61)/CCN1 and galectin-9, were upregulated. These ECMs have the ability to modulate the activities of cytokines and are known as matricellular proteins. Cyr61/CCN1 expression was prominently increased in the fibroblastic cells and preosteoblasts in the suture. Thus, for the first time we demonstrated the mechanical stimulation of Cyr61/CCN1 expression in osteogenic cells in an ex vivo system. These results suggest the importance of matricellular proteins along with the cytokine-mediated signaling for the mechanical regulation of MSC proliferation and differentiation into osteoblastic cell lineage in vivo.

DOI： 10.2220/biomedres.37.117

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Other Link： http://search.jamas.or.jp/link/ui/2016362875

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

We have developed an original in vitro bioassay using teleost scale, that has osteoclasts, osteoblasts, and bone matrix as each marker: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Using this scale in vitro bioassay, we examined the effects of seawater polluted with highly concentrated polycyclic aromatic hydrocarbons (PAHs) and nitro-polycyclic aromatic hydrocarbons (NPAHs) on osteoblastic and osteoclastic activities in the present study. Polluted seawater was collected from two sites (the Alexandria site on the Mediterranean Sea and the Suez Canal site on the Red Sea). Total levels of PAHs in the seawater from the Alexandria and Suez Canal sites were 1364.59 and 992.56 ng/l, respectively. We were able to detect NPAHs in both seawater samples. Total levels of NPAHs were detected in the seawater of the Alexandria site (12.749 ng/l) and the Suez Canal site (3.914 ng/l). Each sample of polluted seawater was added to culture medium at dilution rates of 50, 100, and 500, and incubated with the goldfish scales for 6 hrs. Thereafter, ALP and TRAP activities were measured. ALP activity was significantly suppressed by both polluted seawater samples diluted at least 500 times, but TRAP activity did not change. In addition, mRNA expressions of osteoblastic markers (ALP, osteocalcin, and the receptor activator of the NF-kappa B ligand) decreased significantly, as did the ALP enzyme activity. In fact, ALP activity decreased on treatment with PAHs and NPAHs. We conclude that seawater polluted with highly concentrated PAHs and NPAHs influences bone metabolism in teleosts.

DOI： 10.2108/zs150211

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

High calcium intake may increase hip fracture (HF) incidence. This phenomenon, known as the calcium paradox, might be explained by vaccenic acid (18:1t n-7, VA), the highly specific trans fatty acid (TFA) present in dairy products. First, we ecologically investigated the relationship between 18:1 TFA intake and HF incidence using data from 12 to 13 European countries collected before 2000; then we measured the effects of VA and elaidic acid (18:1t n-9, EA) on osteoblasts from goldfish scales (tissues very similar to mammalian bone), with alkaline phosphatase as a marker; and finally we measured the effect of VA on mRNA expression in the scales for the major bone proteins type I collagen and osteocalcin. HF incidence was significantly correlated with 18:1 TFA intake in men (r=0.57) and women (r=0.65). Incubation with 1 mu mol/L VA and EA for 48 h significantly decreased alkaline phosphatase activity by 25% and 21%, respectively. Incubation of scales with 10 mu mol/L VA for 48 h significantly decreased mRNA expression for type I collagen and osteocalcin (by about 50%). In conclusion, VA may be causatively related to HF and could explain the calcium paradox. It may be prudent to reduce 18:1 TFA intake, irrespective of trans positions, to prevent HF. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.plefa.2016.04.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Using fish scales in which osteoclasts and osteoblasts coexist on the calcified bone matrix, we examined the effects of low-intensity pulsed ultrasound (LIPUS) on both osteoclasts and osteoblasts. At 3 h of incubation after LIPUS treatment, osteoclastic markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expressions decreased significantly while mRNA expressions of osteoblastic markers, osteocalcin, distal less homeobox 5, runt-related transcription factor 2a, and runt-related transcription factor 2b, increased significantly. At 6 and 18 h of incubation, however, both osteoclastic and osteoblastic marker mRNA expression did not change at least present conditions. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated with LIPUS treatment. Real-time PCR analysis indicated that the expression of apoptosis-related genes also increased significantly. To confirm the involvement of apoptosis in osteoclasts with LIPUS, osteoclasts were induced by autotransplanting scales in goldfish. Thereafter, the DNA fragmentation associated with apoptosis was detected in osteoclasts using the TUNEL (TdT-mediated dUTP nick end labeling) method. The multi-nuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was significantly elevated by treatment with LIPUS at 3 h of incubation. Thus, we are the first to demonstrate that LIPUS directly functions to osteoclasts and to conclude that LIPUS directly causes apoptosis in osteoclasts shortly after exposure. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpa.2016.01.022

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Language：English   Publisher：Biomedical Research Press  

Mechanical stress promotes osteoblast proliferation and differentiation from mesenchymal stem cells (MSCs). Although numerous growth factors and cytokines are known to regulate this process, information regarding the differentiation of mechanically stimulated osteoblasts from MSCs in in vivo microenvironment is limited. To determine the significant factors involved in this process, we performed a global analysis of differentially expressed genes, in response to tensile stress, in the mouse cranial suture wherein osteoblasts differentiate from MSCs. We found that the gene expression levels of several components involved in bone morphogenetic protein, Wnt, and epithelial growth factor signalings were elevated with tensile stress. Moreover gene expression of some extracellular matrices (ECMs), such as cysteine rich protein 61 (Cyr61)/CCN1 and galectin-9, were upregulated. These ECMs have the ability to modulate the activities of cytokines and are known as matricellular proteins. Cyr61/CCN1 expression was prominently increased in the fibroblastic cells and preosteoblasts in the suture. Thus, for the first time we demonstrated the mechanical stimulation of Cyr61/CCN1 expression in osteogenic cells in an ex vivo system. These results suggest the importance of matricellular proteins along with the cytokine-mediated signaling for the mechanical regulation of MSC proliferation and differentiation into osteoblastic cell lineage in vivo.

DOI： 10.2220/biomedres.37.117

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Other Link： http://search.jamas.or.jp/link/ui/2016362875

Language：English   Publishing type：Research paper (scientific journal)  

We investigated sleep quality and melatonin in 12 adults who wore blue-light shield or control eyewear 2 hours before sleep while using a self-luminous portable device, and assessed visual quality for the two eyewear types. Overnight melatonin secretion was significantly higher after using the blue-light shield (P < 0.05) than with the control eyewear. Sleep efficacy and sleep latency were significantly superior for wearers of the blue-light shield (P < 0.05 for both), and this group reported greater sleepiness during portable device use compared to those using the control eyewear. Participants rated the blue-light shield as providing acceptable visual quality.

DOI： 10.3109/07420528.2015.1119158

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

We previously demonstrated that monohydroxylated polycyclic aromatic hydrocarbons (OHPAHs), which are metabolites of polycyclic aromatic hydrocarbons (PAHs), act on calcified tissue and suppress osteoblastic and osteoclastic activity in the scales of teleost fish. The compounds may possibly influence other calcified tissues. Thus, the present study noted the calcified spicules in sea urchins and examined the effect of both PAHs and OHPAHs on spicule formation during the embryogenesis of sea urchins. After fertilization, benz[a]anthracene (BaA) and 4-hydroxybenz[a]anthracene (4-OHBaA) were added to seawater at concentrations of 10(-8) and 10(-7) M and kept at 18 degrees C. The influence of the compound was given at the time of the pluteus larva. At this stage, the length of the spicule was significantly suppressed by 4-OHBaA (10(-8) and 10(-7) M). BaA (10(-7) M) decreased the length of the spicule significantly, while the length did not change with BaA (10(-8) M).The expression of mRNAs (spicule matrix protein and transcription factors) in the 4-OHBaA (10(-7) M)-treated embryos was more strongly inhibited than were those in the BaA (10(-7) M)-treated embryos. This is the first study to demonstrate that OHPAHs suppress spicule formation in sea urchins. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpc.2015.02.004

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Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or "brake" of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.

DOI： 10.1371/journal.pone.0119960

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DOI： 10.3389/fnins.2015.00009

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DOI： 10.1111/jne.12165

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

To analyze the effect of polychlorinated biphenyl (PCB) 118 on fish bone metabolism, we examined osteoclastic and osteoblastic activities, as well as plasma calcium levels, in the scales of PCB (118)-injected goldfish. In addition, effect of PCB (118) on osteoclasts and osteoblasts was investigated in vitro. Immature goldfish, in which the endogenous effects of sex steroids are negligible, were used. PCB (118) was solubilized in dimethyl sulfoxide at a concentration of 10 ppm. At 1 and 2 days after PCB (118) injection (100 ng/g body weight), both osteoclastic and osteoblastic activities, and plasma calcium levels were measured. In an in vitro study, then, both osteoclastic and osteoblastic activities as well as each marker mRNA expression were examined. At 2 days, scale osteoclastic activity in PCB (118)-injected goldfish increased significantly, while osteoblastic activity did not change significantly. Corresponding to osteoclastic activity, plasma calcium levels increased significantly at 2 days after PCB (118) administration. Osteoclastic activation also occurred in the marker enzyme activities and mRNA expressions in vitro. Thus, we conclude that PCB (118) disrupts bone metabolism in goldfish both in vivo and in vitro experiments.

DOI： 10.1007/s11356-012-1347-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

To evaluate the effects of inorganic mercury (InHg) and methylmercury (MeHg) on bone metabolism in a marine teleost, the activity of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as indicators of such activity in osteoclasts and osteoblasts, respectively, were examined in scales of nibbler fish (Girella punctata). We found several lines of scales with nearly the same TRAP and ALP activity levels. Using these scales, we evaluated the influence of InHg and MeHg. TRAP activity in the scales treated with InHg (10(-5) and 10(-4) M) and MeHg (10(-6) to 10(-4) M) during 6 hrs of incubation decreased significantly. In contrast, ALP activity decreased after exposure to InHg (10(-5) and 10(-4) M) and MeHg (10(-)6 to 10(-4) M) for 18 and 36 hrs, although its activity did not change after 6 hrs of incubation. As in enzyme activity 6 hrs after incubation, mRNA expression of TRAP (osteoclastic marker) decreased significantly with InHg and MeHg treatment, while that of collagen (osteoblastic marker) did not change significantly. At 6 hrs after incubation, the mRNA expression of metallothionein, which is a metal-binding protein in osteoblasts, was significantly increased following treatment with InHg or MeHg, suggesting that it may be involved in the protection of osteoblasts against mercury exposure up to 6 hrs after incubation. To our knowledge, this is the first report of the effects of mercury on osteoclasts and osteoblasts using marine teleost scale as a model system of bone.

DOI： 10.2108/zs130265

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KYUSHU UNIV, FACULTY AGRICULTURAL PUBLICATIONS  

We recently demonstrated that prostaglandin E, (PG(2)) increases osteoclastic activity and induces bone resorption in both in vitro and in vivo experiments using goldfish. In the fish reproductive period, the plasma calcium (Ca) level in female teleosts increases remarkably to make vitellogenin, which is a major component of egg protein and a Ca binding protein. In this period, however, there is no reported relationship between PGE(2) and Ca metabolism in fish. To clarify the Ca metabolism in fish reproduction, we examined plasma PGE(2) and Ca levels and measured tartrate resistant acid phosphatase (TRAP) activities as an indicator of osteoclastic activity in goldfish. Plasma POE, levels in the reproductive stage significantly increased as compared with those in non reproductive stages. Also, both plasma Ca and TRAP increased in the reproductive stage. Significant positive correlations were recognized between plasma Ca and the gonad somatic index (r=0.81, p&lt;0.001), plasma Ca and plasma PGE(2) levels (r=0.635, p&lt;0.05), and plasma Ca and plasma TRAP activities (r=0.584, p&lt;0.05) from the analysis using samples of both reproductive and non reproductive stages. Taking these data into consideration, we suggested that PGE(2) acts on osteoclasts and increases plasma Ca as a result of osteoclastic bone resorption, and we concluded that PGE(2), is an important hormone in Ca metabolism during fish reproduction.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.cbpa.2013.04.023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblastic and osteoclastic activities were measured. Osteoblastic activity significantly increased under 2- to 4-G loading by both centrifugation and vibration. In contrast, we found that osteoclastic activity significantly decreased under 2- and 3-G loading in response to both centrifugation and vibration. Under 4-G loading, osteoclastic activity also decreased on centrifugation, but significantly increased under 4-G loading by vibration, concomitant with markedly increased osteoblastic activity. Expression of the receptor activator of the NF-kappa B ligand (RANKL), an activation factor of osteoclasts expressed in osteoblasts, increased significantly under 4-G loading by vibration but was unchanged by centrifugal loading. A protein sequence similar to osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, was found in medaka using our sequence analysis. The ratio of RANKL/OPG-like mRNAs in the vibration-loaded scales was significantly higher than that in the control scales, although there was no difference between centrifugal loaded scales and the control scales. Accordingly, medaka scales provide a useful model by which to analyze bone metabolism in response to physical strain.

DOI： 10.2108/zsj.30.217

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.11223/jard.11.27

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Language：English  

DOI： 10.1016/j.ygcen.2012.07.027

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Language：English   Publisher：(公社)日本動物学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Complementary DNA of osteoblast-specific genes (dlx5, runx2a, runx2b, osterix, RANKL, type I collagen, ALP, and osteocalcin) was cloned from goldfish (Carassius auratus) scale. Messenger RNA expressions were analyzed during spontaneous scale regeneration. Dlx5 had an early peak of expression on day 7, whereas osterix was constantly expressed during days 7-21. Runx2, a major osteoblastic transcription factor in mammalian bone, did not show any significant expression. The expressions of two functional genes, type I collagen and ALP, continually increased after day 7, while that of osteocalcin increased on day 14. As for osteoclastic markers, in addition to the cloning of two functional genes, TRAP and cathepsin K, in our previous study, we here cloned the transcription factor NFATc1 to use as an early osteoclastic marker. Using these bone markers, we investigate the signal key that controls the onset of scale resorption and regeneration by performing intra-scale-pocket autotransplantation of five groups of modified scales, namely, 1) methanol-fixed scale, 2) proteinase K-treated cell-free scale, 3) polarity reversal (upside-down) scale, 4) U-shape trimmed scale, and 5) circular-hole perforated scale. In this autotransplantation, each ontogenic scale was pulled out, modified, and then re-inserted into the same scale pocket. At post-transplant, inside the pockets of all modified transplant groups, new regenerating scales formed, attaching to the ongoing resorbed transplants. Autotransplantation of methanol-fixed scale, proteinase K-treated cell-free scale, and polarity reversal (upside-down) scale triggered scale resorption and scale regeneration. These two processes of scale resorption and regeneration occurred in accordance with osteoclastic and osteoblastic marker gene expressions. These results were microscopically confirmed using TRAP and ALP staining. Regarding the autotransplantation of U-shape trimmed and circular-hole perforated scales, new scales regenerated and grew at the trimmed/perforated part of each transplant, while scale resorption occurred apparently only around the trimmed/perforated area In contrast, no scale resorption or regeneration was detected in sham transplantations. Our finding suggests that loss of correct cell-to-cell contact between the scale-pocket lining cells and the scale cortex cells is the key to switch on the onset of scale resorption and regeneration. Overall, the present study shows that goldfish scale regeneration shares similarities in gene expression with intramembranous bone regeneration. Improved understanding of goldfish scale regeneration will help elucidate the process of intramembranous bone regeneration and make goldfish scale a possible new tool to study bone regeneration. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2012.03.021

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Language：English  

DOI： 10.1016/j.bone.2012.02.309

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2012.03.042

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The effect of fugu parathyroid hormone 1 (fugu PTH1) on osteoblasts and osteoclasts in teleosts was examined with an assay system using teleost scale and the following markers: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Synthetic fugu PTH1 (1-34) (100 pg/m1-10 ng/ml) significantly increased ALP activity at 6 h of incubation. High-dose (10 ng/ml) fugu PTH1 significantly increased ALP activity even after 18 h of incubation. In the case of TRAP activity, fugu PTH1 did not change at 6 h of incubation, but fugu PTH1 (100 pg/m1-10 ng/ml) significantly increased TRAP activity at 18 h. Similar results were obtained for human PTH (1-34), but there was an even greater response with fugu PTH1 than with human PTH. In vitro, we demonstrated that both the receptor activator of the NF-kappa B ligand in osteoblasts and the receptor activator NF-kappa B mRNA expression in osteoclasts increased significantly by fugu PTH1 treatment. In an in vivo experiment, fugu PTH1 induced hypercalcemia resulted from the increase of both osteoblastic and osteoclastic activities in the scale as well as the decrease of scale calcium contents after fugu PTH1 injection. In addition, an in vitro experiment with intramuscular autotransplanted scale indicated that the ratio of multinucleated osteoclasts/mononucleated osteoclasts in PTH-treated scales was significantly higher than that in the control scales. Thus, we concluded that PTH acts on osteoblasts and osteoclasts in the scales and regulates calcium metabolism in goldfish. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2011.02.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KYUSHU UNIV, FACULTY AGRICULTURAL PUBLICATIONS  

The fish scales are the major source of internal calcium requirement due to having a higher internal calcium reservoir than the body skeleton during the periods of drastic calcium demand, such as sexual maturation. Therefore, we developed original in vitro assay system using goldfish scales that contain osteoclasts and osteoblasts, and examined the direct effect of inorganic mercury (HgCl2) on osteoclasts and osteoblasts. In this assay system, we measured the activities of tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as respective indicators of each activity in osteoclasts and osteoblasts. TRAP activity in the scales significantly decreased by the treatment of HgCl2 (10(-6)to 10(-3)M) during 6 hrs of incubation. In addition, mRNA expressions of osteoclastic markers: TRAP and cathepsin K significantly decreased compared with control. In our knowledge, this is the first report of a direct effect of inorganic mercury on osteoclasts. On the other hand, ALP activity decreased after exposures of HgCl2, at a concentration of 10(-6), 10(-5) or 10(-4)M for 36 and 64 hrs, although its activity did not change after 6 and 18 hrs. The mRNA expression of metallothionein (MT) which is a metal binding protein that protects the organism from heavy metal, significantly increased by HgCl2 (10(-4)M) although insulin like growth factor-I (osteoblastic marker) was less than those of control scales by treatment with HgCl2 (10(-4)M). These results suggests that osteoblasts may synthesize MT and protect from mercury until 18 hrs incubation. Thus, the scale in vitro assay system would be a useful means for analysis of heavy metal on bone metabolism.

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1365-2826.2010.02081.x

CiNii Article

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The adaptive response of bone to mechanical loading in teleosts is not well understood. We recently developed a new assay system using teleost scales, which consists of osteoblasts, osteoclasts, and bone matrix protein. In this system, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) were used as markers of osteoblasts and osteoclasts, respectively. Using this assay system, we examined the effects of mechanical loading on ALP and TRAP activity in goldfish scales. ALP activity in the scales was significantly elevated (p&lt;0.01) by ultrasound stimuli (1 MHz, 50% duty factor, 0.5 Hz pulse repetition frequency, 60 mW/cm(2) [I(SATA)] and 6 min) after both 18 h and 24 h of incubation while TRAP activity remained unchanged. In addition, mRNA expression of both insulin-like growth factor-I (IGF-I) and estrogen receptors (ER) increased significantly, as did ALP activity. After the goldfish had been swimming for 3 days (speed: 2 body lengths/second, duration: 3 h/day), the scales&apos; ALP activity increased significantly (p&lt;0.01) but TRAP activity did not change. These in vitro and in vivo results strongly suggest that osteoblasts in the goldfish scale respond sensitively to mechanical stress and may be important in promoting bone formation. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpa.2010.03.002

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DOI： 10.1210/en.2009-0908

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.neulet.2009.06.076

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Language：English  

DOI： 10.1016/j.bone.2009.01.347

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Aims: We previously demonstrated that monohydroxylated polycyclic aromatic hydrocarbons (OHPAHs) bound to a human estrogen receptor (ER) by a yeast two-hybrid assay, but polycyclic aromatic hydrocarbons did not have a binding activity. Therefore, the direct effect of 3-hydroxybenz[a]anthracene (3-OHBaA) and 4-hydroxybenz[a]anthracene (4-OHBaA) on osteoclasts and osteoblasts in teleosts was examined. As a negative control, 1 -hydroxypyrene (1-OHPy), which has no binding activity to human ER, was used.
Main methods: The effect of CHPAHs on osteoclasts and osteoblasts was examined by an assay system using teleost scale as each marker: tartrate-resistant acid phosphatase for osteoclasts and alkaline phosphatase for osteoblasts. Changes in cathepsin K (an osteoclastic marker) and insulin-like growth factor-1 (IGF-1) (an osteoblastic marker) mRNA expressions in 4-OHBaA-treated goldfish scales were examined by using a reverse transcription-polymerase chain reaction.
Key findings: In both goldfish (a freshwater teleost) and wrasse (a marine teleost), the osteoclastic activity in the scales was significantly suppressed by 3-OHBaA and 4-OHBaA, although 1-OHPy did not affect the osteoclastic activity. In reference to osteoblasts, the osteoblastic activity decreased with both 3-CHBaA and 4-CHBaA and did not change with the 1-OHPy treatment. However, 17 beta-estradiol (E(2)) significantly increased both the osteoclastic and osteoblastic activities in the scales of both goldfish and wrasse. The mRNA expressions of both cathepsin K and IGF-1 decreased in the 4-OHBaA-treated scales but increased in the E(2)-treated scales.
Significance: The current data are the first to demonstrate that 3-OHBaA and 4-OHBaA inhibited both osteoclasts and osteoblasts and disrupted the bone metabolism in teleosts. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2009.01.008

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Language：English  

DOI： 10.1016/j.cbpa.2008.09.030

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1600-079X.2007.00549.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The teleost scale is a calcified tissue that contains osteoclasts, osteoblasts, and bone matrix, all of which are similar to those found in mammalian membrane bone. Using the goldfish scale, we recently developed a new in vitro assay system and previously demonstrated that melatonin suppressed both osteoclastic and osteoblastic activities in this assay system. In mammals, 2-bromomelatonin possesses a higher affinity for the melatonin receptor than does melatonin. Using a newly developed synthetic method, we synthesized 2-bromomelatonin, 2,4,6-tribromomelatonin and novel bromomelatonin derivatives (1-allyl-2,4,6-tribromomelatonin, 1-propargyl-2,4,6-tribromomelatonin, 1-benzyl-2,4,6-tribromomelatonin, and 2,4,6,7-tetrabromomelatonin) and then examined the effects of these chemicals on osteoclasts and osteoblasts. All bromomelatonin derivatives, as well as melatonin, had an inhibitory action on osteoclasts. In particular, 1-benzyl-2,4,6-tribromomelatonin (benzyl-tribromomelatonin) possessed a stronger activity than melatonin. At an in vitro concentration of 10(-10) M, benzyl-tribromomelatonin still suppressed osteoclastic activity after 6 hr of incubation. In reference to osteoblasts, all bromomelatonin derivatives had a stimulatory action, although melatonin inhibited osteoblastic activity. In addition, estrogen receptor mRNA expression (an osteoblastic marker) was increased in benzyl-tribromomelatonin (10(-7) M)-treated scales.Taken together the present,. results strongly suggest that these novel melatonin derivatives have significant potential for use as beneficial drug for bone diseases such as osteoporosis.

DOI： 10.1111/j.1600-079X.2007.00533.x

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DOI： 10.2187/bss.22.3

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DOI： 10.2108/zsj.25.739

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Other Link： http://orcid.org/0000-0003-0283-9910

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/j.1365-2826.2007.01603.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Complementary DNAs encoding two major osteoclastic markers, tartrate-resistant acid phosphatase (TRAP) and cathepsin K (Cath K) were cloned from the scales of a teleost, the goldfish. This is the first report of the full coding sequence of TRAP and Cath K molecules in fish. In the goldfish scale both TRAP and Cath K mRNAs were expressed in the multinucleate osteoclasts, which showed large numbers of mitochondria and lysosomes, and a well developed ruffled border. These characteristic features of osteoclasts in the scales are similar to those in mammals. Most teleosts use the scale as an internal calcium reservoir during the reproductive season. The expression of TRAP and Cath K mRNAs in the scale significantly increased in April, which is a reproductive season, compared with that in October, a non-reproductive season. Thus, both of these molecular markers should be useful for the study of osteoclasts in the teleost scale. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.08.010

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：English   Publisher：The Japan Society of Sonochemistry  

DOI： 10.20577/pamjss.2007.suppl.0_54

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Language：English  

DOI： 10.1016/j.asr.2007.04.104

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

According to the definition of the ideal synthetic method, an example aimed at the leads for an alpha(2)-blocker, an inhibitor of platelet aggregation, and an anti-osteoporosis agent is established starting from tryptamine. The originality rate, the intellectual property, and the application potential factors of the method are 71, 54, and 100, respectively. The method employs only conventional reagents and reaction conditions without using any protecting groups.

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Language：Japanese   Publisher：聖マリアンナ医科大学医学会  

骨感染症における骨形成能の低下や病的骨吸収は、細菌由来の因子や炎症性サイトカインなどの生体側の因子が作用して生じるとされているが、そのメカニズムは複雑で不明な点が多い。開放骨折部には皮膚の常在細菌である表皮ブドウ球菌が付着することがある。しかし、表皮ブドウ球菌が骨形成過程に及ぼす影響を検討した報告はみられない。そこで、本研究では、骨器官培養モデルとしてキンギョの再生ウロコを用い、皮膚の常在細菌である表皮ブドウ球菌由来の膜タンパク質が、骨折における骨形成過程に重要である線維芽細胞成長因子-2(FGF-2)に対して及ぼす影響を調べることを目的とした。再生ウロコの器官培養系を用い、FGF-2(1.0×10-7M)単独投与群と、FGF-2(1.0×10-7M)およびリチウムクロライドで抽出した表皮ブドウ球菌構成膜タンパク質の同時投与群を作製した。そして、骨芽細胞マーカーとしてアルカリフォスファターゼ(ALP)活性を、破骨細胞のマーカーとして酒石酸抵抗性酸フォスファターゼ(TRAP)活性を測定した。ALP活性は、FGF-2単独投与群では、コントロール群に対して有意に高値(p<0.05)を示した。FGF-2と表皮ブドウ球菌膜タンパク質の同時投与群では、FGF-2単独投与群に比べ、ALP活性は有意に低値を示した。一方、TRAP活性においては、FGF-2単独投与群とFGF-2と表皮ブドウ球菌膜タンパク質の同時投与群の間で、有意な差は認めなかった。さらにFGF抗体を用いた免疫染色により、再生ウロコにFGF-2の発現を認めた。以上の結果より、キンギョのウロコには、哺乳類と同等なFGF-2への反応性を有する骨芽細胞、および破骨細胞が存在することが明らかとなった。また、皮膚の常在菌である表皮ブドウ球菌を構成する膜タンパク質が、仮骨形成初期に発現するFGF-2の作用を明らかに抑制することが示された。すなわち、開放骨折において感染が持続していなくても、骨癒合が遷延化する可能性が示唆された。(著者抄録)

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Other Link： https://search-tp.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2006&ichushi_jid=J00746&link_issn=&doc_id=20061205140018&doc_link_id=%2Feq5maria%2F2006%2F003404%2F018%2F0395-0404%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Feq5maria%2F2006%2F003404%2F018%2F0395-0404%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：Japanese   Publisher：The Japan Society of Sonochemistry  

We recently developed a new in vitro assay system for the evaluation of bone metabolism using goldfish scale. This system can simultaneously detect the activities of both scale osteoblasts (formation cells) and osteoclasts (resorption cells) with alkaline phosphatase and tartrate-resistant acid phosphatase as markers. Using this scale in vitro assay system, in the present study, we analyzed the effect of ultrasound (US) stimulation on osteoblastic and osteoclastic activities. The osteoblastic activity significantly increased by pulsed low-density US (1MHz, 60mW/cm^2 I_<SATA>, 50% duty factor at 0.5Hz, 180 pulses) in 18 hrs of incubation at 15℃ after US treatment but not in shorter incubation periods, while the osteoclastic activity did not change in the same incubation period. To examine the mechanism of US in osteoblasts, the estrogen receptor (ER) and insulin-like growth factor-I (IGF-I) mRNA expressions in the cultured scales were analyzed by RT-PCR. ER mRNA expression was found to be higher in the US-treated scales than in the control scales in 18 hrs of incubation at 15℃ after treatment, although ER mRNA expression did not change in 3 hrs of incubation. On the other hand, IGF-I mRNA expression increased in 3 hrs of incubation at 15℃ after US treatment. Therefore, IGF-I mRNA expression was more rapid to respond to US than ER mRNA expression, and IGF-I may have an important function in the activation of osteoblasts by US treatment. We found that both osteoblastic and osteoclastic activities increased in the remaining ontogenic scales of the left side at 3 days after the removal of all scales in the right side. Using these scales, we examined the effects of US stimulation on osteoblasts and osteoclasts under the same conditions described above. In 18 hrs of incubation at 15℃ after US treatment, the osteoblastic activity increased by US treatment in the ontogenic scale as well as the normal scale. Furthermore, we indicated that the osteoclastic activity in the ontogenic scale decreased by US treatment. The conditions of osteoblasts and osteoclasts in the ontogenic scale are similar to those in the human osteoporosis, indicating that US stimulation may contribute to the cure of osteoporosis.

DOI： 10.20577/pamjss.15.0_3

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DOI： 10.1016/j.lfs.2005.10.004

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Other Link： http://orcid.org/0000-0003-0283-9910

Language：English  

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Language：English   Publishing type：Research paper (scientific journal)  

Effects of nucleotides and cations on 2-[125I]iodomelatonin binding sites in the goldfish brain were examined. Nucleotides (10–6–10–3 M) dose-dependently inhibited the specific binding with the following order of potency: guanosine 5''-O-(3-thiotriphosphate) (GTPγS) &gt
GTP = GDP &gt
GMP = ATP &gt
cyclic GMP. Cyclic AMP was ineffective. The treatment of membranes with GTPγS induced rapid dissociation of 2-[125I]iodomelatonin from membranes when added at the steady state, increased the Kd and decreased the Bmax values as revealed by saturation analysis, and increased the IC50 value of melatonin to inhibit the specific binding. The treatment decreased the specific binding to membrane preparations obtained from six brain regions as well. Inorganic salts (5–200 mM) dose-dependently inhibited the specific binding with the following order of potency: CaCl2 &gt
MgCl2 &gt
LiCl &gt
NaCl &gt
choline chloride &gt
KCl, except for 5 mM MgCl2, which enhanced the specific binding. Saturation experiments demonstrated that 75 mM CaCl2,100 mM MgCl2 and 200 mM NaCl increased the Kd and decreased the Bmax while 5 mM MgCl2 increased the Bmax value. These results imply that G protein and physiological concentrations of cations are involved in the regulation of melatonin binding sites in the goldfish brain. © 1997 S. Karger AG, Basel.

DOI： 10.1159/000109106

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DOI： 10.1006/gcen.1997.6940

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Other Link： http://search.jamas.or.jp/link/ui/2017246704

Language：English   Publishing type：Other   Publisher：SPRINGER  

DOI： 10.1007/s12020-016-1153-9

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Language：English  

OBJECTIVE: Low-intensity pulsed ultrasound (LIPUS) provides noninvasive therapeutic treatment to accelerate fracture repair and distraction osteogenesis. However, most studies concerning the influence of LIPUS on bone metabolism have been conducted in vivo systems using osteoblastic cells. Therefore, details of the direct effect of LIPUS on osteoclasts are not yet fully understood. Teleost scale is a calcified tissue that contains osteoclasts and osteoblasts. Its bone matrix consists of type I collagen and hydroxyapatite, and is similar to that of mammalian bone. Therefore, we examined the effect of LIPUS on the osteoclasts and osteoblasts of zebrafish and goldfish scales, as a model of the bone matrix simplified to its bare bones. METHODS: Ultrasound was generated with the Sonic Accelerated Fracture Healing System (SAFHS 4000J; Teijin Pharma, Ltd). Scales were collected from zebrafish under anesthesia; they were then treated with LIPUS for 20 minutes, incubated at 15°C for 3, 6, and 18 hours in L-15 medium, and subjected to measurement of the mRNA expression. Following the osteoclast induction by the autotransplantation of goldfish scales, we further examined the number of apoptotic osteoclasts after LIPUS treatment. RESULTS AND DISCUSSION: At 3 hours of incubation after LIPUS treatment, osteoclastic marker expression decreased while osteoblastic markers increased. Using GeneChip analysis of zebrafish scales treated by LIPUS, we found that cell death-related genes were up-regulated by LIPUS treatment. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was elevated by treatment with LIPUS at 3 hours of incubation. Thus, we conclude that LIPUS promotes apoptosis in osteoclasts shortly after exposure.

DOI： 10.1097/01.bot.0000489982.40329.52

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J-GLOBAL

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Language：Japanese  

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Other Link： http://search.jamas.or.jp/link/ui/2015015043

Language：Japanese  

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Other Link： http://search.jamas.or.jp/link/ui/2014143533

Language：Japanese  

DOI： 10.11223/jard.12.11

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J-GLOBAL

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J-GLOBAL

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Language：Japanese  

CiNii Article

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Language：Japanese   Publisher：Japan Society for Comparative Endocrinology  

DOI： 10.5983/nl2008jsce.37.194

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Language：Japanese   Publisher：日本比較内分泌学会  

DOI： 10.5983/nl2008jsce.36.106

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Other Link： http://search.jamas.or.jp/link/ui/2010234144

Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：WILEY-BLACKWELL  

Several reports indicate that melatonin is involved in the regulation of bone metabolism. To examine the direct effect of melatonin on osteoclasts and osteoblasts, we developed an in vitro assay using fish scales that contain osteoclasts, osteoblasts, and bone matrix, all of which are similar to those found in mammalian membrane bone. Using the assay, we demonstrated that melatonin suppressed osteoclastic and osteoblastic activities. These findings are in agreement with the reports from in vivo studies in mice and rats. In an attempt to develop molecules that increase bone mass, novel bromomelatonin derivatives were synthesized, and the effects of these chemicals on osteoclasts and osteoblasts using the scale assay were examined. As a result, novel bromomelatonin derivatives with the ability to possibly increase bone formation were identified. In scale osteoclasts, particularly, 1-benzyl-2,4,6-tribromomelatonin had a more potent activity than melatonin. In reference to osteoblasts, this agent (10(-9)-10(-6) M) significantly activated osteoblasts. The effect of 1-benzyl-2,4,6-tribromomelatonin on bone formation was confirmed in ovariectomized rats. Thus, the oral administration of 1-benzyl-2,4,6-tribromomelatonin augmented the total bone mineral density of the femoral metaphysis of ovariectomized rats. The stress-strain index of the diaphysis in 1-benzyl-2,4,6-tribromomelatonin-treated rats significantly increased in comparison with that in ovariectomized rats. In rats fed a low-calcium diet, the total bone mineral density of the femoral metaphysis significantly increased following the oral administration of 1-benzyl-2,4,6-tribromomelatonin. These studies identified a melatonin derivative that may have potential use in the treatment of bone diseases, such as osteoporosis.

DOI： 10.1111/j.1600-079X.2008.00623.x

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Language：Japanese   Publisher：ニュー・サイエンス社  

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Other Link： http://search.jamas.or.jp/link/ui/2007135425

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：Japanese   Publisher：メディカルレビュー社  

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Other Link： http://search.jamas.or.jp/link/ui/2006140136

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Osteogenesis in the teleost was morphologically observed using regenerating scales of goldfish. Histological observations indicated that osteoblasts around the regenerating scales on days 7 to 10 were greater in size and number than those at other stages. Therefore, further experiments were carried out to examine the activity of osteoblasts in the regenerating period. To quantify their osteoblastic activities, scales on the left side of the body were taken, and the regenerating scales were then used to measure the activities of alkaline phosphatase (ALP), a marker of osteoblasts, on days 7, 10, and 15. The ontogenic scales on the right side of the body were also collected and used to measure ALP activity on the same days. Osteoblasts at all stages of regenerating scales were more active than those in the remaining ontogenic scales. The regenerating scales on day 10 had the highest activity. Furthermore, we found that estrogen receptor (ER) mRNA was expressed in the regenerating scales because estrogen participates in osteoblastic growth and differentiation in mammals. Therefore, using a scale culture system reported previously, the estrogenic response was examined in the ontogenic and regenerating scales on day 10. The reactivity was much higher in regenerating scales, although estrogen treatment significantly activated the osteoblastic activities in both scales. We are the first to demonstrate that ER is expressed in regenerating scales and that estrogen participates in osteogenesis as it does in mammalian bone. Our findings strongly suggest that regenerating scales can be used as a model of osteogenesis in vertebrates. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2004.10.063

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

Daily and circadian variations of melatonin contents in the diencephalic region containing the pineal organ, the lateral eyes, and plasma were studied in a urodele amphibian, the Japanese newt (Cynops pyrrhogaster), to investigate the possible roles of melatonin in the circadian system. Melatonin levels in the pineal region and the lateral eyes exhibited daily variations with higher levels during the dark phase than during the light phase under a light-dark cycle of 12 h light and 12 h darkness (LD12:12). These rhythms persisted even under constant darkness but the phase of the rhythm was different from each other. Melatonin levels in the plasma also exhibited significant day-night changes with higher values at mid-dark than at mid-light under LID 12:12. The day-night changes in plasma melatonin levels were abolished in the pinealectomized (Px), ophthalmectomized (Ex), and Px+Ex newts but not in the sham-operated newts. These results indicate that in the Japanese newts, melatonin production in the pineal organ and the lateral eyes were regulated by both environmental light-dark cycles and endogenous circadian clocks, probably located in the pineal organ and the retina, respectively, and that both the pineal organ and the lateral eyes are required to maintain the daily variations of circulating melatonin levels.

DOI： 10.2108/zsj.22.65

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publisher：SPRINGER TOKYO  

To examine the effects of heavy metals such as cadmium and mercury on calcium homeostasis, plasma calcium and calcitonin were measured in goldfish. Cadmium induced hypocalcemia both at 4 and at 8 days. In methylmercury-treated goldfish, the plasma calcium level increased at 2 days and then decreased at 8 days. The plasma calcitonin level increased in correspondence with the increased plasma calcium by methylmercury treatment, although cadmium did not cause a significant change. To elucidate the mechanism in detail, fish scales, which have both osteoclasts and osteoblasts and are similar to mammalian membrane bone, were used in the present study. We measured tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) activity as respective indicators of activity in both types of cells. TRAP activity in the scales decreased by treatment of cadmium and methylmercury at 6 h incubation. Particularly, cadmium (even at 10(-13) M) significantly suppressed TRAP activity, suggesting that this system is utilized as an acute biosensor for cadmium. ALP activity decreased after exposures of 64 and 96 h, although the activity did not change after 6, 18, and 36 h. In addition, mRNA expression of the estrogen receptor and insulin-like growth factor 1, which participate in osteoblastic growth and differentiation, was less than the control values by treatment with both metals. This study demonstrates that mercury directly acts on the bone cells and influences calcium homeostasis and indicates that, in a short-term exposure, mercury has a different action from that of cadmium and induces hypercalcemia.

DOI： 10.1007/s00774-004-0505-3

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The direct effect of bisphenol A on osteoclasts and osteoblasts was examined using a culture system of goldfish scales. Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were used as markers of osteoclasts and osteoblasts, respectively. Bisphenol A (10(-5) M) significantly suppressed both TRAP and ALP activities. These data were reproduced in an in vivo experiment. From an analysis of a reverse transcription-polymerase chain reaction in the in vitro-cultured scales, it was demonstrated that the insulin-like growth factor (IGF)-1 mRNA expression decreased by a bisphenol A treatment. On the other hand, 17beta-estradiol (E-2) stimulated both TRAP and ALP activities and did not change IGF-1 mRNA expression, suggesting that bisphenol A has a different effect from E-2 on bone metabolism. This study is the first to demonstrate that bisphenol A functions to suppress directly osteoblasts and osteoclasts among vertebrates, which strongly suggests that this scale in an in vitro assay system can be utilized for the evaluation of the effects of endocrine disrupters on bone cells. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S0024-3205(03)00603-9

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

In teleosts, it is well known that plasma calcium levels increase as a result of treatment with estrogen for at least during 2 weeks and that calcitonin secretion is induced by estrogen. The present study examined the influence of bisphenol A on calcium homeostasis in goldfish and compared the above known estrogenic action. In goldfish kept in water containing bisphenol A (10(-6) M) the plasma calcium concentration increased significantly (P&lt;0.001) at 4 days but decreased significantly (P&lt;0.05) at 8 days. By the treatment of bisphenol A, calcitonin secretion was not induced until 4 days. At 8 days, however, plasma calcitonin, as well as calcium, decreased significantly (P&lt;0.05), although vitellogenin was detected in the plasma. Therefore, bisphenol A influences plasma calcium levels, but its action is different from that of estrogen, which indicates that bisphenol A affects the calcium homeostasis and might bring about abnormal conditions in teleosts.

DOI： 10.2108/zsj.20.745

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Language：English   Publisher：Zoological Society of Japan  

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Other Link： http://id.nii.ac.jp/1141/00038257/

Language：English   Publisher：BLACKWELL MUNKSGAARD  

The effects of melatonin on osteoclastic and osteoblastic cells were examined using a culture system of the goldfish scale. Tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) were used as markers of osteoclastic and osteoblastic cells, respectively. In Earle's minimum essential medium containing melatonin (10(-9) to 10(-5) M, activities of both enzymes in scales were significantly suppressed at 6 hr after incubation (TRACP: 10(- 8), 10(-6), 10(-5) M ALP: 10(-7) to 10(-5) M, but at 18 hr only ALP activity was significantly lowered (10(-8), 10(-7) M. Estradiol-17beta (E-2) enhanced both activities, which were significantly inhibited and brought down to the level of the controls when co-incubated with E-2 and melatonin (TRACP at 6 hr: 10(-9) to 10(-5) M ALP at 6 hr: 10(-7) M ALP at 18 hr: 10(-8) M). Moreover, using reverse-transcription polymerase chain reaction, the mRNA expression of the estrogen receptor (ER) and insulin-like growth factor (IGF)-1, which are related to osteoblastic growth and differentiation, was decreased in the melatonin-treated scales. These results suggest that melatonin acts directly on the scale osteoclastic and osteoblastic cells where it suppresses the ALP activity via down-regulation of ER and IGF-1 mRNAs expression. This is the first report on the function of melatonin in osteoclasts and on the suppressive effect of melatonin in osteoblasts among vertebrates.

DOI： 10.1034/j.1600-079X.2002.02953.x

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Language：English   Publisher：Zoological Society of Japan  

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Other Link： http://id.nii.ac.jp/1141/00037298/

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Other Link： http://id.nii.ac.jp/1141/00037299/

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

DOI： 10.1016/S0168-8278(99)80329-8

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Background. It is well known that an unbalanced diet induces various changes in the pituitary gland. However, little attention has been paid to the molecular aspects of this perturbation. We studied the influence of a low-protein diet (LPD) on the prolactin (PRL) and growth hormone (GH) cells in the rat pituitary gland using immunohistochemical staining and in situ hybridization.
Materials: Rats aged 20 days were fed a diet containing 27% protein or one with 8% protein (LPD) for 30 days. Pituitary glands were obtained and subjected to either immunohistochemistry or in situ hybridization. Quantitative morphological analysis was then conducted to determine cell number and area as well as the percentage of cells stained by the respective antisera and/or cDNA probe in each experimental group.
Results: The average sectional areas of both PRL-and GH-producing cells in the LPD group were smaller in size than those in the controls. The cell numbers per unit area (mm(2)) of PRL-positive cells and PRL mRNA-positive cells were 3,596.5 and 3,948.6, respectively, in the LPD group, and 3,179.6 and 4,888.5, respectively, in the controls. The numbers per unit area of GH-positive cells and GH mRNA-positive cells in the LPD group were similar (2,252.3 and 2,224.4), as compared to 2,161.3 and 1,684.2, respectively, in the well-fed rats. Whereas PRL-positive cells comprised about 27% of the total number of cells in both animal groups, those given the LPD contained a lower percentage (29%) of PRL mRNA-positive cells as compared to the controls (44%). On the other hand, GH mRNA-positive cells numbered about 15% of the total cell population both animal groups; however, the malnourished rats contained a lower percentage (16%) of GH-positive cells than did their well-fed counterparts (20%).
Conclusions: Taken together, these results indicate that in the rat pituitary gland, administration of an LPD reduced the size of PRL- and GH-positive cells as well as differentially affecting a subpopulation of the PRL mRNA-positive cells and the GH-positive cells. (C) 1998 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-0185(199805)251:1<37::AID-AR7>3.0.CO;2-B

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Language：English   Publisher：SAGE PUBLICATIONS INC  

Ocular melatonin rhythms in the goldfish were studied and compared to those in the pineal organ and plasma. Under Light:dark (LD) of 12 h light:12 h dark, melatonin contents in the eye as well as the pineal organ and plasma exhibited clear day-night changes with higher levels at mid-dark than at mid-light. However, melatonin contents in the eye at mid-light and mid-dark were approximately 100 and 9 times greater than those in the pineal organ, respectively. Day-night changes of ocular melatonin persisted after pinealectomy, which abolished those in plasma melatonin under LD 12:12. Ocular melatonin contents in the pinealectomized fish at mid-light were significantly higher than those in the sham-operated control. Under constant darkness (DD), circadian melatonin rhythms were observed in the eye but damped on the 3rd day, whereas plasma melatonin rhythms generated by the pineal organ persisted for at least 3 days. Under constant light, ocular melatonin contents exhibited a significant fluctuation with a smaller amplitude than that under DD, whereas plasma melatonin remained at low levels. These results indicate the involvement of LD cycles, a circadian clock, and the pineal organ in the regulation of ocular melatonin rhythms in the goldfish.

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Language：English   Publisher：ELSEVIER SCIENCE BV  

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole-O-methyltransferase (HIOMT; S-adenosyl-L-methionine:N-acetylserotonin-O-methyltransferase EC 2.1.1.4), which catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyI-L-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit far melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.

DOI： 10.1016/S0378-4347(96)00503-8

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Language：Japanese   Publisher：公益社団法人 日本化学会  

DOI： 10.20665/kakyoshi.45.5_256

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Language：English   Publisher：KARGER  

Effects of nucleotides and cations on 2-[I-125]iodomelatonin binding sites in the goldfish brain were examined. Nucleotides (10(-6)-10(-3) M) dose-dependently inhibited the specific binding with the following order of potency: guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) &gt; GTP = GDP &gt; GMP = ATP &gt; cyclic GMP. Cyclic AMP was ineffective. The treatment of membranes with GTP gamma S induced rapid dissociation of 2-[I-125]iodomelatonin from membranes when added at the steady state, increased the K-d and decreased the B-max values as revealed by saturation analysis, and increased the IC50 value of melatonin to inhibit the specific binding. The treatment decreased the specific binding to membrane preparations obtained from six brain regions as well. Inorganic salts (5-200 mM) dose-dependently inhibited the specific binding with the following order of potency: CaCl2 &gt; MgCl2 &gt; LiCl &gt; NaCl &gt; choline chloride &gt; KCl, except for 5 mM MgCl2, which enhanced the specific binding. Saturation experiments demonstrated that 75 mM CaCl2, 100 mM MgCl2 and 200 mM NaCl increased the K-d and decreased the B-max while 5 mM MgCl2 increased the B-max value. These results imply that G protein and physiological concentrations of cations are involved in the regulation of melatonin binding sites in the goldfish brain.

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Relation between retinal melatonin and corneal mitotic rhythms in the Japanese quail was investigated in experiments manipulating the ocular physiology by treatments with formoguanamine hydrochloride (FG) and eye-lid suture. In experiment 1, we investigated the effects of FG, which is known to induce photoreceptor degeneration, on retinal melatonin and corneal mitotic rhythms. FG-treatment completely abolished the retinal melatonin rhythms in both LD 12:12 and constant darkness (DD), but the corneal mitotic rhythm was maintained with high mitotic rate in darkness under a LD cycle and subjective night under DD. The result suggests that 1) the photoreceptor cells in the retina are the site for melatonin production and/or for the oscillator which drives the circadian rhythm in retinal melatonin, and 2) melatonin is not involved in generation of the corneal mitotic rhythm. In experiment 2, we investigated the effects of eye-lid suture, which is known to induce eye enlargement and bulgy cornea, on the retinal melatonin and corneal mitotic rhythms. Eye-lid suture abolished the corneal mitotic rhythm in both LD and DD, with a high mitotic rate being maintained throughout 24 hr. But retinal melatonin maintained its rhythm with high levels in darkness under a LD cycle and in subjective night under DD. The result suggests that 1) bulgy cornea in the sutured eye was induced by the increase in mitotic rate in the light period, and 2) disappearance of the corneal mitotic rhythm does not have a relation to retinal melatonin. These results suggest that retinal melatonin is not involved in generation of the corneal mitotic rhythm and that there are two circadian clock systems in the eye.

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Language：English   Publisher：BLACKWELL SCIENCE LTD  

OBJECTIVE Previous studies of Pit-1 expression in human pituitary tumours have produced conflicting results, We have studied expression of Pit-1 mRNA in human pituitary adenomas, as well as in normal human and rat pituitaries, and results were compared with clinical, histological, and immunohistochemical features, In addition, expression of GH, PRL, and TSH-beta mRNA was also studied and compared with Pit-1 gene expression.
MATERIAL AND METHODS The adenomas consisted of 13 GH cell adenomas, 7 PRL cell adenomas, 3 TSH cell adenomas, 4 ACTH cell adenomas, and 10 clinically non-functioning adenomas. Expression of the Pit-1, its isoforms, and each hormone, was studied using the reverse transcription polymerase chain reaction.
RESULTS Pit-1 mRNA was expressed not only in normal human and rat pituitaries, but also in all GH, PRL and TSH cell adenomas. There was no correlation between Pit-1 transcripts and biological behaviour or histological findings in these three types of adenoma, suggesting that Pit-1 is generally required for the determination of cell phenotype but is insufficient for the regulation of hormonal activity and tumour growth in these pituitary adenomas. In addition, Pit-1 was also expressed in some ACTH cell (2/4) and non-functioning adenomas (7/10), Although there were no GH, PRL or TSH-beta transcripts in Pit-1 mRNA-negative ACTH cell and non-functioning adenomas, PRL mRNA was detected in all Pit-1 mRNA-positive ACTH cell adenomas and GH, PRL and/or TSH-beta mRNA were found in four of seven Pit-1 mRNA-positive non-functioning adenomas, In contrast, Pit-1 mRNA was expressed without any GH, PRL, or TSH-beta transcripts in only three nonfunctioning adenomas, These data suggest that expression of Pit-1 mRNA in these two types of adenomas can be mainly attributed to the presence of GH, PRL and/or TSH-beta mRNA expressing cells and that true Pit-1 transcripts found in non-functioning adenomas may be rare event, Moreover, there were two cases which expressed Pit-1 alpha mRNA, but failed to show other Pit-1 isoform mRNA, There were, however, no clinical or histological differences between these two adenomas showing only Pit-1 alpha mRNA and the others expressing both Pit-1 alpha mRNA and other Pit-1 isoform mRNA.
CONCLUSIONS Pit-1 mRNA was expressed not only in GH, PRL and TSH cell adenomas but also in other types of adenoma, However, it is suggested that expression of Pit-1 mRNA in most ACTH cell and non-functioning adenomas can be attributed to GH, PRL and/or TSH-beta mRNA expressing cells, Further studies are necessary to elucidate the role of Pit-1 transcripts in the three nonfunctioning adenomas without GH, PRL and/or TSH-beta mRNA expression.

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Language：English   Publisher：ELSEVIER SCI PUBL IRELAND LTD  

Melatonin (N-acetyl-5-methoxytryptamine) was identified in the head and hemolymph of the silkworm, Bombyx mori, using reversed-phase high-performance liquid chromatography coupled with fluorometric detection and radioimmunoassay. In addition, evidence of arylalkylamine (serotonin) N-acetyltransferase (NAT) a key enzyme controlling the synthesis of melatonin in vertebrates, was found in the head of the silkworm. Melatonin levels in the head acid hemolymph and the NAT activity in the head were significantly higher during the dark period than during the light period of a 12-h light/12-h dark cycle. The day-night changes persisted in constant darkness but were suppressed by constant light. The results suggest that the synthesis and release of melatonin in the silkworm head occur as a circadian rhythm that is entrained by environmental light/dark cycles, as it is in the pineal gland of vertebrates. Melatonin in the silkworm head may function as a neurochemical mediator of photoperiodic control of developmental events such as molting, eclosion and diapause.

DOI： 10.1016/0303-7207(95)03670-3

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Effects of pinealectomy and constant light exposure on day-night changes of melatonin binding sites in the goldfish brain were examined. The density and affinity of binding sites were higher at mid-day than at mid-night in sham-pinealectomized goldfish under light-dark cycles. The rhythms disappeared after pinealectomy, or constant light exposure both of which abolish plasma melatonin rhythms. The effects of pinealectomy and constant light exposure were not additive. These results indicate that diel changes of melatonin binding sites in the goldfish brain are regulated by endogenous melatonin of pineal origin.

DOI： 10.1016/0304-3940(95)11903-A

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Language：English   Publisher：MUNKSGAARD INT PUBL LTD  

Day-night levels of melatonin (N-acetyl-5-methoxytryptamine) were de termined in different organs of adult female crickets (Gryllus bimaculatus) exposed to a 12/12 light/dark cycle, using reversed-phase high performance liquid chromatography coupled with fluorometric detection. Melatonin levels in the compound eye, brain, and palp were significantly higher during the dark period than during the light period, suggesting that a diurnal rhythm of melatonin levels exists in these organs of crickets, with a peak during the dark period. Conversely, melatonin levels were significantly higher during the light period than the dark period in the cercus, ovipositor, antenna, hind-leg and ovary. No significant day-night difference was found in the fore- and mid-legs, Malpighian tube, and digestive tube. Thus, these organs may have different melatonin-metabolizing systems compared to those found in the compound eye, brain, and palp. Differences in the phasing of the melatonin rhythm in various organs of the cricket suggest possible differences in melatonin function in these organs.

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Language：English   Publisher：ACADEMIC PRESS AUST  

Twenty-four edible plants were investigated for the presence of melatonin, heretofore considered to be a molecule found only in the animal kingdom. The amount of melatonin in different plants varied greatly with highest melatonin being present in plants of the rice family. Melatonin was identified by radioimmunoassay and verified by high performance liquid chromatography with fluorescence detection. Feeding a diet containing plant products rich in melatonin to chicks increased radioimmunoassayable levels of melatonin in their blood. Likewise, melatonin extracted from plants inhibited binding of [I-125]iodomelatonin to rabbit brain. Thus, melatonin ingested in foodstuffs enters the blood and is capable of binding to melatonin binding sites in the brain of mammals.

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The in vitro effect of melatonin on the release of luteinizing hormone (LH) and follicle stimulating hormone (FSK) from fetal rat pituitary cells was investigated. A significant inhibition of LH release induced by 10(-9) M luteinizing hormone releasing hormone (LHRH) was seen when cells were incubated with 10(-9) M melatonin. FSH release was unaffected by either LHRH alone or LHRH in combination with melatonin. In addition, the significant inhibitory effect of melatonin was reduced by pretreatment of the pituitary cells with 10(-10) M melatonin. These findings indicate that melatonin can act directly on the fetal pituitary gland to suppress LHRH-induced release of LH perhaps by a mechanism which eventually involves down-regulation of the melatonin receptors.

DOI： 10.1016/0304-3940(94)11181-H

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

The possible presence of melatonin (N-acetyl-5-methoxytryptamine) was investigated in different tissues and organs of adult crickets by the use of reversed-phase high-performance liquid chromatography with fluorometric detection and radioimmunoassay. Melatonin was detected in the compound eye, antenna, ovipositor, palp, cercus, leg, wing, brain, ovary, digestive tube and Malpighian tube of the crickets, indicating that melatonin was very widely distributed in the crickets. The results suggest that melatonin in these organs might be involved in the local control of various aspects of rhythmic activity.

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Melatonin binding sites in the goldfish brain were characterized by radioreceptor assay using 2-[I-125]iodomelatonin as the radioligand. Specific binding of 2-[I-125]iodomelatonin was rapid, stable, saturable and reversible. Saturation experiments demonstrated that 2-[I-125]iodomelatonin binds to a single class of receptor site with an affinity constant (K-d) of 29.8 +/- 0.7 pM and a total binding capacity (B-max) of 11.47 +/- 0.33 fmol/mg protein at mid-light. At mid-dark, the B-max value decreased significantly to 7.90 +/- 0.23 fmol/mg protein (P &lt; 0.01) with no significant variation in the Kb value(33.8 +/- 1.5 pM). Competition experiments revealed the following order of pharmacological affinities: 2-iodomelatonin &gt; melatonin &gt; 6-hydroxymelatonin &gt; N-acetyl-5-hydroxytryptamine &gt; 5-methoxytryptamine &gt; 5-methoxytryptophol &gt; 5-methoxyindole-3-acetic acid. 5-Hydroxytryptamine, 5-hydroxytryptophol, 5-hydroxyindole-3-acetic acid, norepinephrine and acetylcholine exhibited no inhibition. Subcellular distribution of melatonin binding sites was demonstrated to be greatest in the P-2 and P-3 fractions as compared with the P-1 fraction. Localization of melatonin binding sites in discrete brain areas was determined to be highest in the optic tectum-thalamus and hypothalamus, intermediate in the telencephalon, cerebellum and medulla oblongata, and lowest in the olfactory bulbs and pituitary gland. These results suggest that characteristics of melatonin receptors are highly conserved during evolution and that in this species melatonin plays neuromodulatory roles in the central nervous system through specific receptors.

DOI： 10.1016/0006-8993(94)91682-9

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Language：English   Publisher：MUNKSGAARD INT PUBL LTD  

The daytime and nighttime levels of pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin were measured in adult male and female valley pocket gophers, Thomomys bottae. This species was chosen for study because it is a subterranean rodent that inhabits burrows whose openings to the surface are closed. Therefore, under field conditions it is estimated that the pocket gopher spends roughly 99% of its time in absolute darkness in underground burrows. When wild captured pocket gophers were maintained under a light:dark cycle (light intensity during the day of roughly 140 mu W/cm(2)), nighttime levels of pineal NAT activity and melatonin content were higher than values measured during the day; on the other hand, HIOMT activity in the pineal gland was similar in the day and at night. When pocket gophers were exposed to an extended light period (220 mu W/cm(2)) 4 hr into the night, the rise in melatonin synthesis normally associated with darkness onset was not inhibited. Also, when gophers were acutely exposed to a light intensity of 400 mu W/cm(2) for 1 hr beginning 4 hr after darkness onset, neither high nocturnal levels of pineal NAT nor pineal melatonin contents were reduced. Finally, when pocket gophers were exposed to a 600 mu W/cm(2) light intensity at either 4 hr or 8 hr into the dark period, pineal melatonin synthesis remained elevated at a level comparable to that measured in dark-exposed controls. The results show that under controlled laboratory conditions the pineal gland of the valley pocket gopher, a species that in its natural habitat spends about 99% of its time in absolute darkness, exhibits higher melatonin synthesis during night than during the day. While the rhythm in pineal melatonin production in the pocket gopher is clearly synchronized by the prevailing light:dark cycle, high nighttime pineal melatonin synthesis is not readily inhibited by light in the intensity range of 220 to 600 mu W/cm(2). In terms of its relative insensitivity to light at night, the pineal gland of the valley pocket gopher resembles that of other diurnally active rodents.

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Publisher：社団法人日本産科婦人科学会  

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Language：English   Publisher：HUMANA PRESS INC  

Effects of testosterone (T) and LHRH on the occurrence of immunoreactive ubiquitin in the nucleus of rat LH cells were investigated in cell culture. The proportion of LH cells with ubiquitin-immunoreactive nuclei (%LH-nucUb) was significantly higher in cells from castrated rats than in those from intact rats. The increase in %LH-nucUb after castration was suppressed by T (1 x 10(-8) M) treatment for three days in vitro. Short repeated exposure to LHRH (10(-9) M for 10 min x 3) for 3 h did not affect the %LH-nucUb in either intact or castrated groups. In contrast, alternating prolonged exposure to LHRH (10(-9) M for 1 h and 10(-11) M for 3 h) for 60 h increased the %LH-nucUb in cells from intact rats, but not in cells from castrated rats. These results indicate that T decreases the occurrence of ubiquitin in the nucleus of activated LH cells, whereas LHRH increases it in less-activated LH cells, suggesting the involvement of ubiquitin in activation of LH cells. The present study also indicates that T and LHRH directly affect pituitary cells on the occurrence of ubiquitin in the nucleus of LH cells.

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Language：English   Publisher：MUNKSGAARD INT PUBL LTD  

The purpose of this study was to examine the day/night levels of pineal melatonin and its rate limiting enzyme N-acetyltransferase (NAT) in relationship to the ratio of 11-cis- to all-trans-retinal. Three-week-old chicks were placed in 12:12 light:dark (LD 12:12) cycle for one week, pineals were collected during the light phase at 1500 (i.e., after 10 hr light), during the dark phase at 1900 (i.e., 2 hr after dark), at 2100 (i.e., 4 hr after dark), and at 2300 (i.e., 6 hr after dark) and after light extension to 1900. The results show that light-sensitive 11-cis-retinal in the chick pineal has the same diurnal rhythm as NAT and melatonin; all constituents increased within 2, hr of darkness onset (at 1900) and reached their peak after 4 hr of dark. All values were lowest during the light phase at 1500. Low values for 11-cis-retinal, NAT, and melatonin were also seen in the group of chicks which experienced light extension to 1900. The data indicate that in vivo light plays a major role in triggering rhodopsin-bound 11-cis-retinal production within 2-4 hr after darkness onset; this change likely serves as the signal for the subsequent formation of the hormonal product of the pineal gland, melatonin.

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Language：English   Publisher：MUNKSGAARD INT PUBL LTD  

The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650 muW/cm2) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.

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Language：English   Publisher：WILEY-LISS  

A quantitative analysis of the pituitary gland was conducted to ascertain the effects of protein-calorie malnutrition on the morphology of the somatotrophs, gonadotrophs, thyrotrophs, and corticotrophs. Male rats were fed a low, 8% protein diet from 20 to 50 days of age, while their age-matched controls were given a diet containing 27% protein. The hypophyses were then processed for light microscopic immunocytochemical staining using antibodies to growth hormone, the beta subunits of luteinizing hormone and thyroid stimulating hormone, and adrenal corticotrophic hormone. The number of each cell type along with an evaluation of the cell, cytoplasmic, and nuclear areas was conducted using a computerized image analyzer. All of these parameters were reduced significantly in the somatotrophs as a result of the low protein diet, while in the gonadotrophs, the cell, cytoplasmic, and nuclear areas were similarly affected. Smaller cell number, cell area, and nuclear area were noted in the corticotrophs of the malnourished animals, while in the thyrotrophs, only the cell and nuclear areas were reduced. The data demonstrate that each pituitary cell type responds in a unique manner to undernutrition.

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Language：English   Publisher：ENDOCRINE SOC  

Immunocytochemical detection of ubiquitin in the nucleus of rat LH cells and the effects of castration and testosterone replacement on the occurrence of immunoreactive ubiquitin in the nucleus were investigated. Immunoreactive ubiquitin occurred in certain nuclei, mostly belonging to identified LH cells. The concentration of testosterone in blood was altered by castration and implantation of testosterone into castrated rats, and the occurrence of ubiquitin was examined weekly for the following 4 weeks. In castrated rats, the proportion of LH cells with ubiquitin-immunoreactive nuclei was high throughout the experiment. In castrated rats implanted with testosterone, on the contrary, the proportion remained significantly lower. Ubiquitin may be involved in the cellular activity of LH cells in the rat pituitary.

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Language：Japanese   Publisher：聖マリアンナ医科大学医学会  

1個の細胞からのホルモン放出量を経時的に測定することができるreverse hemolytic plaque assayの変法(continuous plaque assayとsequential plaque assay)を用い,LHRH刺激後のLH放出動態を個々の細胞で調べた。用いた動物は60日齢のWistar-Imamichi系の雄ラットで,摘出した下垂体前葉を酵素によりsingle cellsに解離し,24時間培養後,10-9MのLHRHを添加して2種類のassayを行った。大きく分類すると4種類の放出パターンが観察された。すなわち,第1は,LHRH刺激後急速にLHを放出し,その後ほとんど放出しない細胞群,第2は,徐々にLHの放出量を増加させる細胞群,第3は,LHRHで刺激しているにもかかわらず一定時間が経過しないと放出を開始しない細胞群,第4は,培養時間(7時間)内ではまったくLHを放出しない細胞群である。以上の結果より,同じLH細胞といっても個々の細胞でLHRHに対する反応性が異なることが明らかになった

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Language：English   Publisher：GUSTAV FISCHER VERLAG  

In this study, 3 GH-producing adenomas were studied as materials to clarify the usefulness of electron microscopic investigation of plaque-forming single adenoma cells, and ultrastructural morphometric comparisons were made to determine whether some differences exist at the cytoplasmic organelle level between small plaque-forming cells and large plaque-forming cells. In two cases, the relative cytoplasmic volume density of Golgi apparatus and lysosomes were significantly greater and the diameter of seretory granules were significantly smaller in large plaque-forming cells compared to small plaque-forming cells. Moreover, in one of them, not only the diameter but also the cytoplasmic volume density of secretory granules were significantly smaller in large plaque-forming cells. These findings suggest a greater secretory granule synthesis and internal granule destruction, and faster granules secretion at an early stage of formation in large plaque-forming cells compared with small plaque-forming cells. It became apparent through this study that electron microscopic investigation of plaque-forming single cells is practically useful since plasma membrane any cytoplasmic organelles were preserved well after processing for EM in most plaque-forming cells.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-LISS  

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DOI： 10.11477/mf.2425905204

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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Language：English   Publisher：J ENDOCRINOLOGY LTD  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

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Language：English   Publisher：Gunma University  

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Language：Japanese   Publisher：学会出版センタ-  

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Other Link： http://search.jamas.or.jp/link/ui/1984081453

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：フジテレビ１階マルチシアター  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京  

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Symposium, workshop panel (public)  

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Language：English   Presentation type：Symposium, workshop panel (public)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：大阪  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：富山  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：富山  

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Language：Japanese  

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Language：Japanese   Presentation type：Poster presentation  

Venue：パシフィコ横浜  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福井県立恐竜博物館  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京国際フォーラム, 東京  

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Language：Japanese   Presentation type：Poster presentation  

Venue：長崎大学  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京海洋大学品川キャンパス，東京都  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Ishikawa, Japan  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Ishikawa, Japan  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Ishikawa, Japan  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Ishikawa, Japan  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北里大学、神奈川  

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Language：Japanese   Presentation type：Poster presentation  

Venue：北里大学, 神奈川県  

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Language：English   Presentation type：Poster presentation  

Venue：Okinawa, Japan  

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Language：English   Presentation type：Poster presentation  

Venue：Okinawa, Japan  

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Language：English   Presentation type：Poster presentation  

Venue：Okinawa Convention Center, Okinawa, Japan  

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Language：English   Presentation type：Poster presentation  

Venue：Okinawa Convention Center, Okinawa, Japan  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

Venue：Okinawa Convention Center, Okinawa, Japan  

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Language：English   Presentation type：Poster presentation  

Venue：Okinawa Convention Center, Okinawa, Japan  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：札幌  

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