スギの雄性不稔の原因遺伝子MALE STERILITY 1 (MS1) の簡易判定法を確立するため、PCR産物を目視で確認できるsingle-tag hybridization chromatographic printed array strip (STH-PAS) を用いてPCR後の操作の省力化を試みた。タグまたはビオチンを付加したプライマーを用いてPCR を行い、増幅産物を相補タグが固相化された棒状のメンブレンに展開した。およそ15分で検出ラインが呈色し、MS1遺伝子型が判定できた。判定は電気泳動結果と一致し、STH-PASが判定に必要な時間と労力を大幅に削減可能なことが示された。

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　骨格系の代謝の基本は破骨細胞による骨吸収と骨芽細胞による骨形成の繰り返しである。これらの細胞は全身性因子の統御の下，様々な局所因子を分泌しauto-crine/paracrine 的に互いの機能調整を行っている。
　本稿では，その広範な生理的役割が注目されている一酸化窒素 (NO) が骨組織における強力な局所メディエータであることを支持する最近の研究成果を紹介した。とくに，成長期ラットの骨発育においてinducible型のNO合成酵素活性が重要な役割を果たすことを証明したわれわれの研究について詳述した。最後に，NOの骨代謝調節機構を対象とする研究の将来の方向性に触れた。

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We evaluated inhibitory effects of plastoquinones (1, 2) and chromene derivative (3) from the methanol extract of Sargassum micracanthum on the differentiation of osteoclast progenitors into osteoclast-like cells (OCLs), bone-resorbing activity and the survival of OCLs. When OCL formation was investigated using osteoclast progenitor cells obtained from macrophage-colony stimulating factor (M-CSF)-treated mouse bone marrow, 1—3 inhibited dose-dependently OCL formation at the concentrations of 3—10 μM, the order of inhibitory potency being 1>3>2. In addition, they suppressed dose-dependently the pit formation induced by OCL on dentine slices, the order of inhibitory potency being 1>3>2. The survival of OCLs was inhibited by about 35, 45 and 60% in the presence of 6 μM of 2, 3 and 1, respectively. These results suggest that the inhibitory effects of the three compounds on the differentiation, pit formation and the survival of OCLs might contribute to the suppression of bone resorption.

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Steroid-induced osteoporosis is the most common form of secondary osteoporosis. The mechanisms of glucocorticoid (GC)-induced osteoporosis may be divided into indirect and direct effects. A putative indirect mechanism of GC-induced osteoporosis is thought to be secondary hyperparathyroidism or estrogen deficiency. Recent studies, however, suggest that the direct action of GC on bone metabolism is more important. GC stimulates the expression of the receptor activator of NF-κB ligand (RANKL) and inhibits osteoprotegerin (OPG) expression in a dose-dependent manner in human osteoblasts. The resultant increase in the RANKL:OPG ratio results in the induction of osteoclastogenesis. GC predominantly inhibits human osteoblast proliferation and enhances the differentiation of human-pre-osteoblasts. GC also acts directly on human osteoblasts to up-regulate the expression of M-CSF which is an essential cytokine for the survival of osteoclast precursors. The addition of GC to the culture of human peripheral blood mononuclear cells (PBMC) results in a marked increase in the formation of osteoclasts and an increase in lacunar resorption. Moreover, GC has been shown to inhibit apoptosis in mouse osteoclasts. In addition, the direct effect of dexamethasone (Dex) upon human osteoclastogenesis from PBMC is mediated via the balance between RANKL and IFN-γ in activated CD4 T cells. Dex induced human osteoclastogenesis without adding osteoblasts or soluble RANKL. Dex dose-dependently increased the ratio of RANKL-positive cells to IFN-γ positive cells (RANKL:IFN-γ ratio) only by reducing IFN-γ-positive cells, suggesting that Dex stimulates osteoclast differentiation by elevating the RANKL:IFN-γ ratio in CD4⁺ T cells. The balance between RANKL and OPG (RANKL:OPG) or RANKL and IFN-γ (RANKL:IFN-γ) on osteoblasts and T cells may have an important role in GC-induced osteoclastogenesis. Thus, therapeutic modulation of the expression of RANKL, OPG and IFN-γ by osteoblasts and T cells represents a novel strategy to prevent GC-induced osteoporosis.

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The bee pollen Cistus ladaniferus (C. ladaniferus) extract has an anabolic effect on bone metabolism. The effects of the fractionated extracts obtained from bee pollen on bone calcium content and osteoclast-like cell formation in vitro were investigated. Rat femoral-diaphyseal and -metaphyseal tissues were cultured for 48 hr in a medium containing either vehicle or a water-solubilized extract with the membrane fractions obtained from bee pollen. The active component of bee pollen in increasing calcium content in diaphyseal tissues was seen in the fraction of molecular weight (MW) of less than 1000, and it was not observed in fractions of greater than MW 1000. Culture with the fractionated bee pollen extract (25 or 50 μg/ml of medium) of less than MW 1000 caused a significant increase in calcium content in the diaphyseal or metaphyseal tissues. The parathyroid hormone (PTH; 10^-6 M)-induced decrease in diaphyseal calcium content was significantly prevented in the presence of the fractionated bee pollen extracts (10 μg/ml) of less than MW 1000 or greater than MW 1000. Mouse marrow cells were cultured for 7 days in a medium containing PTH (10^-6 M) in the presence or absence of the fractionated bee pollen extract (10 or 50 μg/ml). The PTH-induced increase in osteoclast-like cell formation was markedly suppressed in the presence of extracts of less than MW 1000 as compared with that in the presence of fractions of greater than MW 1000. The effects of the fractionated bee pollen extracts of less than MW 1000 in increasing diaphyseal calcium content and in inhibiting PTH-induced osteoclastic cell formation were significantly decreased upon heat treatment for 20 and 60 min at 80°C. This study demonstrates that the active component of bee pollen C. ladaniferus extract, which stimulates bone formation and inhibits osteoclastic bone resorption, is the fraction with MW less than 1000.

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A vector material is defined as the one to manipulate surrounding inorganic and organic compounds, microorganisms, and tissues using a force irradiated locally and continuously from the material itself. The effect given by a vector material is ca materials are of unparalleled importance under the circumstance of the limited use of external forces such as in vivo. We have recently experimentally demonstrated the effectiveness of electrically polarized hydroxyapatite (HA) electrets as vector ceramics, which can independently irradiate an electrostatic force to the surrounding constituents; crystal growth from a simulated body fluid was accelerated or decelerated and proliferation of a microorganism was controlled on the surfaces of polarized HA, depending upon the electric signs of surface charges. Prior to our study, certain types of vector effects have already been employed in biomedical applications; the so-called 45S5-type bioactive glass (Bioglass^®), developed by Hench et al., has long been recognized to be bioactive because of its solubility in a aqueous medium. Radioactive ceramics irradiate β-ray to and work on surrounding cancer cells. These are also vector materials. In addition to these examples, various kinds of vector effects and ceramics are now under development. This advanced report presents the concept of vector effects and reviews the examples of vector ceramics.

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Endothelin-1 (ET-1) localization in dental and skeletal tissues was examined immunocytochemically using a biotin-streptoavidin-horseradish peroxidase method in paraffin sections. Mouse anti-ET-1 monoclonal antibody was used as the primary antibody. In teeth and periodontal tissues, intense immunostaining was observable mainly in periodontal ligament cells, i.e. fibroblasts, osteoblasts, osteoclasts, and vascular endothelial cells. Dental pulp, dentine, cementum, bone matrix, and gingival connective tissues were nonimmunoreactive. In the gingival epithelium, cells in the basal, prickle, and glanular layers stained intensely, whereas those of the keratinized layer were devoid of immunostaining. In metaphyseal bone marrow, intense immunostaining was observed over osteoclasts, osteoblasts, young osteocytes, and vascular endothelial cells. Bone and cartilage matrices, as well as chondrocytes in resting and proliferating zones, were, however, negative for immunoreaction. These results indicate that ET-1 is present in the cells of dental, periodontal, and bone tissues. Its wide distribution may reflect its involvement in various cellular functions in these tissues.

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Ipriflavone (7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one) is a synthetic flavonoid that has been shown to stimulate the activity of osteoblasts. We show here that ipriflavone also promotes the deposition of calcium and the formation of mineralized nodules by newborn rat calvarial osteoblast-like (ROB) cells as well as the activity of alkaline phosphatase. We reported previously that endothelin-1 inhibits the differentiation of ROB cells [Y. Hiruma et al. (1998) J. Cardiovasc. Pharmacol. 31, S521-S523]. Therefore, we examined the effects of ipriflavone on the expression of endothelin receptors in ROB cells by polymerase chain reaction-Southern blot analysis and in binding assays with ¹²⁵I-labeled endothelin-1. Ipriflavone reduced levels of endothelin ET_A receptors (to 48% of the control level) in ROB cells around day 7 in our standard cultures, while it had no apparent effect on the expression of the mRNA for the endothelin ET_A receptor. By contrast, treatment with 10^-7M endothelin-1 on days 6 through 9 alone suppressed mineralization by ROB cells. Ipriflavone also reduced the ability of endothelin-1 to inhibit mineralization by ROB cells. These results suggest that the acceleration of osteoblastic differentiation by ipriflavone might be due, at least in part, to a time-specific down-regulation of endothelin receptors.

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  Aging induces a decrease in bone mass, and osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public health problem. Bone loss with increasing age may be due to decreased bone formation and increased bone resorption. Pharmacologic and nutritional factors may prevent bone loss with aging, although chemical compounds in food and plants which act on bone metabolism are poorly understood. We have found that isoflavones (including genistein and daidzein), which are contained in soybeans, have a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption, thereby increasing bone mass. Menaquinone-7, an analogue of vitamin K₂ which is abundant in fermented soybeans, has been demonstrated to stimulate osteoblastic bone formation and to inhibit osteoclastic bone resorption. Of various carotenoids, β-cryptoxanthin, which is abundant in Satsuma mandarin (Citrus unchiu MARC), has a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. The supplementation of these factors has a preventive effect on bone loss induced by ovariectomy in rats, which are an animal model of osteoporosis, and their intake has been shown to have a stimulatory effect on bone mass in humans. Factors with an anabolic effect on bone metabolism were found in extracts obtained from wasabi leafstalk (Wasabi japonica MATSUM), the marine alga Sargassum horneri, and bee pollen Cistus ladaniferus. Phytocomponent p-hydroxycinnamic acid was also found to have an anabolic effect on bone metabolism. Food chemical factors thus play a role in bone health and may be important in the prevention of bone loss with increasing age.

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Some filoviruses such as ebolaviruses and marburgviruses, cause hemorrhagic fever in humans and nonhuman primates. Pigs are suggested to play a potential role in the filovirus ecology. We investigated the seroprevalence of filovirus infection in pigs in Ghana. Using a viral glycoprotein (GP)-based enzyme-linked immunosorbent assay, we detected filovirus-specific immunoglobulin G antibodies in 5 of 139 samples. These positive sera showed specificities to four different filovirus species. Particularly, two of the positive sera reacted to GPs of two African ebolaviruses (i.e., Ebola virus and Taï Forest virus) in Western blotting. Our results suggest that these Ghanaian pigs were exposed to multiple filoviruses and emphasize the importance of continuous monitoring of filovirus infection in pig populations in West African countries.

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　ポリオーマウイルスはポリオーマウイルス科に属する哺乳類動物由来のOrthopolyomavirusとWukipolyomavirus，及び鳥類由来のAvipolyomavirusに分類されている．我々は疫学研究を通じて新規のポリオーマウイルスであるMastomys Polyoamvirus (MasPyV)及びVervet monkey Polyoamvirus-1 (VmPyV-1)を単離し，そのゲノムがコードするウイルスタンパク質の機能解析を実施した．更に，ヒトポリオーマウイルスであるJC polyomavirs (JCPyV)についての基礎研究を推進し，最近，早期タンパク質であるLarge T抗原の感染細胞における機能解析，後期タンパク質であるVP1のシステイン残基の粒子形成への影響，及び後期タンパク質であるAgnoのウイルス粒子の細胞外放出機構に対する影響について知見を得たのでその内容を紹介する．

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It was examined histomorphometrically whether or not and how chronic treatment with nasal salmon calcitonin (SCT) of rats could prevent ovariectomy (OVX)-induced osteoporotic changes in the trabecular bone of tibia. During 7 weeks on a synthetic low Ca diet after OVX, rats developed type 1 osteopenia which was defined by strut analysis as resulting mainly from loss in the connectivity of strut but in the thickness. An intermittent dosing regimen of nasal SCT (10 or 40U/rat/d, 3d/week, for 7 weeks) was able to retard development of osteopenia in a dose-dependent manner. The results indicate that the nasal route would be usable for chronic treatment of experimental osteoporosis with SCT and possibly other peptide anti-osteoporotics.

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　新型コロナウイルス（SARS-CoV-2）を含む人獣共通感染症原因ウイルスの多くは野生動物を起源とする．今後も発生することが予想される新たなウイルス性人獣共通感染症への備えとして，さまざまなウイルスの自然宿主，自然界における存続機構と伝播経路に関する知見を蓄積することが重要である．筆者らは，アフリカおよびインドネシアの研究者と共同で，現地のコウモリ，げっ歯類動物，霊長類動物および節足動物が保有するウイルスを対象とした研究を実施している．これまでに，上記の動物から，人に病気を起こすウイルスやこれまで知られていない新規ウイルスを多数見出した．本稿では，パルボウイルスとロタウイルスに関する成果を中心として紹介したい．

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In fura-2-loaded human gingival fibroblasts, 1-50 μM MnCl₂ evoked an increase in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in a dose-dependent manner. This increase was abolished in the absence of extracellular Ca²⁺. MnCl₂ induced an increase in [Ca²⁺]_i provoked prostaglandin E₂ release in human gingival fibroblasts in time- and dose-dependent manners. MnCl₂ failed to evoke prostaglandin E₂ release in the absence of extracellular Ca²⁺. A protein kinase C activator and a tyrosine kinase inhibitor inhibited the increase in [Ca²⁺]_i induced by MnCl₂. These results suggest that Mn²⁺ evokes Ca²⁺ entry and prostaglandin E₂ release in human gingival fibroblasts, which are regulated by protein kinase C and tyrosine kinase activities.

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The effects of bee pollen extract on osteoclastic bone resorption in vitro were investigated. The water-solubilized extracts were obtained from the bee pollen of Cistus ladaniferus. Femoral-diaphyseal or -metaphyseal tissues of rats were cultured for 48 hr in medium containing either vehicle, bone-resorbing factor, or bone-resorbing factor plus bee pollen extracts (10, 100, or 1000 μg/ml of medium). Culture with parathyroid hormone [human (1-34) PTH; 10^-7 M], prostaglandin E₂ (PGE₂; 10^-5 M), or 1,25-dihydroxyvitamin D₃ (10^-6 M) caused a significant decrease in calcium content in the diaphyseal or metaphyseal tissues. These decreases were completely prevented in the presence of bee pollen extracts (10, 100, or 1000 μg/ml). The presence of PTH (10^-7 M) caused a significant increase in medium glucose consumption and lactic acid production in the diaphyseal or metaphyseal tissues. These increases were significantly inhibited by culture with bee pollen extracts (10, 100, or 1000 μg/ml). Mouse bone marrow cells were cultured for 7 days in the presence of bone-resorbing factor in vitro. Culture with PTH (10^-7 M), PGE₂ (10^-5 M), tumor necrosis factor-α(10 ng/ml of medium), or lypopolysaccharide (10 μg/ml of medium) caused a significant increase in osteoclast-like cell formation. These increases were significantly inhibited in the presence of bee pollen extracts (10, 50, or 100 μg/ml of medium). This study demonstrates that bee pollen extract has inhibitory effects on osteoclastic bone resorption in vitro.

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