迅速・高感度・簡便な新規骨粗鬆症モデル系の開発と予防法への応用

金沢大学医薬保健研究域保健学系[研究目的]魚類のウロコには骨形成をする骨芽細胞、骨吸収をする破骨細胞およびコラーゲンを中心とする骨基質タンパクからなり、健常骨モデル、骨代謝亢進モデルおよび骨吸収亢進モデル等の骨のモデルが作成できることを既に報告している。昨年は、これらの骨モデルに機械的刺激である低強度超音波を照射しその効果を調べた。その結果、健常骨モデルおよび骨代謝亢進モデルでは、骨形成が増加し、骨代謝亢進モデルでは、さらに骨吸収が抑制される効果も明らかとなった。一方、炎症性の骨吸収亢進モデルでは、骨形成および骨吸収作用に変化がないことが明らかとなった。今年度は、ウロコモデルで確認された低強度超音波の効果をマウスの頭蓋骨を使い、哺乳類の骨代謝に対する低強度超音波の効果を調べた。[研究成果]マウス新生仔をエーテル過剰吸入麻酔後、断頭し、頭蓋骨を取り出し左右を2等分した2骨片を対照群と曝露群とした。ウロコの系で決定した強度の低強度超音波を曝露群に照射後、対照群と同様に24時間組織培養し、超音波破砕し上清の酵素活性を測定した。その結果、骨形成をする骨芽細胞活性および骨吸収をする破骨細胞活性のどちらも変化しなかった。哺乳類の骨細胞は、骨基質内に存在しているため超音波が反射され届かないためと思われた。できるだけ骨組織が薄く柔らかいマウスの新生仔頭蓋骨を使ったが、細胞が表面にいるウロコとはやはり大きく異なることが明らかとなった。したがって、哺乳類の骨代謝を改善するためには、骨基質内にある細胞にも到達しうる機械的刺激が必要なことが示唆された。また、超音波の骨代謝への効果を調べるには、骨基質の表面に骨芽細胞と破骨細胞が共存するウロコが適したモデルとなることが明らかとなった。研究課題/領域番号:18650197, 研究期間(年度):2006 – 2008出典：「迅速・高感度・簡便な新規骨粗鬆症モデル系の開発と予防法への応用」研究成果報告書　課題番号18650197（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-18650197/)を加工して作

A detailed protocol for perspiration monitoring using a novel, small, wireless device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity. Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick\u27s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e. transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss. As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example. © 2016 Journal of Visualized Experiments.Embargo Period 24 month

Development of a small wireless device for perspiration monitoring

A small and wireless device that can capture the temporal pattern of perspiration by a novel structure of water vapor collection combined with reusable desiccant has been developed. The novel device consists of a small cylindrical case with a temperature/relative humidity sensor, battery-driven data logger, and silica gel (desiccant). Water vapor of perspiration was detected by the change in relative humidity and then adsorbed by silica gel, allowing continuous recording of perspiration within a closed and wireless chamber, which has not been previously achieved. By comparative experiments using the commercially-available perspiration monitoring device, the developed device could measure perspiration as efficiently as the conventional one, with a normalized cross coefficient of 0.738 with 6 s delay and the interclass correlation coefficient [ICC(2, 1)] of 0.84. These results imply a good agreement between the conventional and developed devices, and thus suggest the applicability of the developed device for perspiration monitoring. © 2015 IPEM.Embargo Period 12 month

ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline

H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition

Effects of low-intensity pulsed ultrasound on osteoclasts: Analysis with goldfish scales as a model of bone

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. © 2017 Biomedical Research Foundation. All rights reserved

Low-intensity pulsed ultrasound induces apoptosis in osteoclasts: Fish scales are a suitable model for the analysis of bone metabolism by ultrasound

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. © 2016 Elsevier Inc.Embargo Period 12 month

Effects of inorganic mercury and methylmercury on osteoclasts and osteoblasts in the scales of the marine teleost as a model system of bone

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. © 2014 Zoological Society of Japan

Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish

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. © 2017 Elsevier Inc.Embargo Period 12 month

Static and dynamic hypergravity responses of osteoblasts and osteoclasts in medaka scales

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-αB 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. © 2013 Zoological Society of Japan