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Osteoclasts are the only bone-resorbing cells in organisms and understanding their differentiation mechanism is crucial for the treatment of osteoporosis. In the present study, we investigated the effect of Thiamet G, an O-GlcNAcase specific inhibitor, on osteoclastogenic differentiation. Thiamet G treatment increased global O-GlcNAcylation in murine RAW264 cells and suppressed receptor activator of nuclear factor-κB ligand (RANKL)-dependent formation in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, thereby suppressing the upregulation of osteoclast specific genes. Meanwhile, knockdown of O-linked N-acetylglucosamine (O-GlcNAc) transferase promoted the formation TRAP-positive multinuclear cells. Thiamet G treatment also suppressed RANKL and macrophage colony-stimulating factor (M-CSF) dependent osteoclast formation and bone-resorbing activity in mouse primary bone marrow cells and human peripheral blood mononuclear cells. These results indicate that the promotion of O-GlcNAc modification specifically suppresses osteoclast formation and its activity and suggest that chemicals affecting O-GlcNAc modification might potentially be useful in the prevention or treatment of osteoporosis in future.

Background In order to investigate the combination effect of anticancer drugs and X-ray irradiation on neurotoxic side-effects (neurotoxicity), a method that provides homogeneously X-ray-irradiated cells was newly established. Materials and methods PC12 cell suspension was irradiated by X-ray (0.5 Gy) in serum-supplemented medium, immediately inoculated into 96-microwell plates and incubated overnight. The medium was replaced with fresh serum-depleted medium containing 50 ng/ml nerve growth factor to induce differentiation toward nerve-like cells with characteristic neurites according to the overlay method without changing the medium. The differentiated cells were treated by anticancer drugs as well as antioxidants, oxaliplatin or bortezomib, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Results Antioxidants and anticancer drugs were cytotoxic to differentiating PC12 cells. Combination of anticancer drugs and X-ray irradiation slightly reduced cell viability. Conclusion The present 'population irradiation method' may be useful for the investigation of the combination effect of X-ray irradiation and any pharmaceutical drug.

Capsaicinoids are unique compounds that give chili pepper fruits their pungent taste. Capsaicinoid levels vary widely among pungent cultivars, which range from low pungency to extremely pungent. However, the molecular mechanisms underlying this quantitative variation have not been elucidated. Our previous study identified various loss-of-function alleles of the pAMT gene which led to low pungency. The mutations in these alleles are commonly defined by Tcc transposon insertion and its footprint. In this study, we identified two leaky pamt alleles (pamtL1 and pamtL2 ) with different levels of putative aminotransferase (pAMT) activity. Notably, both alleles had a Tcc transposon insertion in intron 3, but the locations of the insertions within the intron were different. Genetic analysis revealed that pamtL1 , pamtL2 and a loss-of-function pamt allele reduced capsaicinoid levels to about 50%, 10% and less than 1%, respectively. pamtL1 and pamtL2 encoded functional pAMT proteins, but they exhibited lower transcript levels than the functional type. RNA sequencing analysis showed that intronic transposons disrupted splicing in intron 3, which resulted in simultaneous expression of functional pAMT mRNA and non-functional splice variants containing partial sequences of Tcc. The non-functional splice variants were more dominant in pamtL2 than in pamtL1 . This suggested that the difference in position of the intronic transposons could alter splicing efficiency, leading to different pAMT activities and reducing capsaicinoid content to different levels. Our results provide a striking example of allelic variations caused by intronic transposons; these variations contribute to quantitative differences in secondary metabolite contents.

It has been reported that the intake of milk basic protein (MBP) increases bone density by promoting bone formation and suppressing bone resorption. However, few studies have been done on MBP in cartilage, the tissue adjacent to bone. We therefore investigated the effect of MBP on a chondrocyte cell line, ATDC5. In a proliferative assay using the WST-1 method, the addition of 10, 100, and 1,000 µg/mL of MBP to ATDC5 cells significantly increased the cell number by about 1.2-, 1.5-, and 1.7-fold, respectively, compared with the control cells. The cell cycle analysis using flow-cytometry revealed that the proportion of S- and G2/M-phase cells was increased but that of G0/G1 phase was decreased in a dose-dependent manner with MBP addition. We measured the alkaline phosphatase activity of MBP-treated ATDC5 cells to examine the differentiation stage of the cells. Alkaline phosphatase activity was suppressed in a dose-dependent manner with MBP addition and was especially drastic at higher doses of MBP (100 and 1,000 µg/mL). The Alizarin Red S staining intensity, the indicator for calcification of cells, was lower in the MBP-treated (100 µg/mL) cells than in nontreated control cells. In the reverse-transcription PCR experiment, the mRNA level of SRY-box containing gene 9 (Sox9) and type II collagen (Col2) was significantly increased in the MBP-treated cells compared with the control cells. A significant decrease of the mRNA level of runt-related transcription factor 2 (Runx2) and type X collagen (Col10) was also observed in the MBP-treated cells. These results suggested that MBP promoted the proliferation of chondrocytes by suppressing their differentiation toward calcification.

Osteoclasts are the only bone-resorbing cells in organisms and understanding their differentiation mechanism is crucial for the treatment of osteoporosis. In the present study, we investigated the effect of Thiamet G, an O-GlcNAcase specific inhibitor, on osteoclastogenic differentiation. Thiamet G treatment increased global O-GlcNAcylation in murine RAW264 cells and suppressed receptor activator of nuclear factor-κB ligand (RANKL)-dependent formation in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, thereby suppressing the upregulation of osteoclast specific genes. Meanwhile, knockdown of O-linked N-acetylglucosamine (O-GlcNAc) transferase promoted the formation TRAP-positive multinuclear cells. Thiamet G treatment also suppressed RANKL and macrophage colony-stimulating factor (M-CSF) dependent osteoclast formation and bone-resorbing activity in mouse primary bone marrow cells and human peripheral blood mononuclear cells. These results indicate that the promotion of O-GlcNAc modification specifically suppresses osteoclast formation and its activity and suggest that chemicals affecting O-GlcNAc modification might potentially be useful in the prevention or treatment of osteoporosis in future.

Background/Aim: Chitosan-coated iron oxide nanoparticles (Chi-NP) have gained attention because of their biocompatibility, biodegradability, low toxicity and targetability under magnetic field. In this study, we investigated various biological properties of Chi-NP. Materials and Methods: Chi-NP was prepared by mixing magnetic NP with chitosan FL-80. Particle size was determined by scanning and transmission electron microscopes, cell viability by MTT assay, cell cycle distribution by cell sorter, synergism with anticancer drugs by combination index, PGE(2) production in human gingival fibroblast was assayed by ELISA. Results: The synthetic process of Chi-NP from FL-80 and magnetic NP increased the affinity to cells, up to the level attained by nanofibers. Upon contact with the culture medium, Chi-NP instantly formed aggregates and interfered with intracellular uptake. Aggregated Chi-NP did not show cytotoxicity, synergism with anticancer drugs, induce apoptosis (accumulation of subG1 cell population), protect the cells from X-ray-induced damage, nor affected both basal and IL-1β-induced PGE(2) production. Conclusion: Chi-NP is biologically inert and shows high affinity to cells, further confirming its superiority as a scaffold for drug delivery.

Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3‑E1 osteoblastic cells and osteoclast‑like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST‑1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription‑polymerase chain reaction (RT‑PCR) demonstrated that mangiferin significantly increased the mRNA level of runt‑related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate‑resistant acid phosphatase‑positive multinuclear cells. RT‑PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast‑associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3‑E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes.

Osteoclasts represent the only bone resorbing cells in an organism. In this study, we investigated the effect of glucosamine (GlcN), a nutrient used to prevent joint pain and bone loss, on the osteoclastogenesis of murine macrophage-like RAW264 cells. GlcN supplementation suppressed the upregulation of osteoclast-specific genes (tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metallopeptidase 9, and nuclear factor of activated T cell c1 (NFATc1)), receptor activator of nuclear factor-κB ligand (RANKL)-dependent upregulation of TRAP enzyme activity, and the formation of TRAP-positive multinuclear cells more effectively than N-acetylglucosamine (GlcNAc), which we have previously shown to inhibit osteoclast differentiation. To clarify the mechanism by which GlcN suppresses osteoclastogenesis, we further investigated the effect of GlcN on O-GlcNAcylation by Western blotting and on other types of glycosylation by lectin blotting. We found that, upon addition of GlcN, the O-GlcNAcylation of cellular proteins was increased whereas α2,6-linked sialic acid modification was decreased. Therefore, these glycan modifications in cellular proteins may contribute to the suppression of osteoclastogenesis.

In Japan, there is a system in place called 'Foods with Function Claims'. This system basically requires that if a food company wants to make a claim about the specific benefits of a foodstuff, it must be supported by scientific evidence. This is an important initiative for a wide variety of reasons. For instance, people taking foodstuffs that are not supported by scientific evidence might well decide not to take supplements with genuine benefits because they believe they are already taking adequate steps. To comply with the 'Foods with Function Claims' system, companies rely on the work of researchers to determine whether what they are claiming is supported by scientific evidence. Dr Sachie Nakatani leads a team based at Josai University in Japan, which is investigating a wide range of different foodstuffs to determine whether they have genuine health benefits., Impact is published under a CC-BY Creative Commons licence.PDFタイトル：Science-Based Benefits; The Potential Benefits of Collagen Hydrolysate

Osteoclasts represent the only bone resorbing cells in an organism. In this study, we investigated the effect of glucosamine (GlcN), a nutrient used to prevent joint pain and bone loss, on the osteoclastogenesis of murine macrophage-like RAW264 cells. GlcN supplementation suppressed the upregulation of osteoclast-specific genes (tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metallopeptidase 9, and nuclear factor of activated T cell c1 (NFATc1)), receptor activator of nuclear factor-κB ligand (RANKL)-dependent upregulation of TRAP enzyme activity, and the formation of TRAP-positive multinuclear cells more effectively than N-acetylglucosamine (GlcNAc), which we have previously shown to inhibit osteoclast differentiation. To clarify the mechanism by which GlcN suppresses osteoclastogenesis, we further investigated the effect of GlcN on O-GlcNAcylation by Western blotting and on other types of glycosylation by lectin blotting. We found that, upon addition of GlcN, the O-GlcNAcylation of cellular proteins was increased whereas α2,6-linked sialic acid modification was decreased. Therefore, these glycan modifications in cellular proteins may contribute to the suppression of osteoclastogenesis.

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

Salacia reticulata is a native plant of Sri Lanka. In the traditional medicine of Sri Lanka and India, Salacia reticulata bark is considered orally effective in the treatment of rheumatism, gonorrhea, skin disease and diabetes. We have investigated, both in vivo and in vitro, whether the leaf of Salacia reticulata (SRL) can ameliorate collagen antibody-induced arthritis (CAIA) in mice as the rheumatoid arthritis (RA) model. The mice were fed a lard containing chow diet (AIN-93G) or the same diet containing 1% (w/w) SRL powder. All mice were bred for 23 days. On day 7 or 14 after LPS injection, mice were killed, and tissue and blood samples were collected. Histological analysis was performed, and serum levels of inflammatory mediators and the mRNA levels of inflammation-related genes and osteoclast-related genes were measured. SRL treatment ameliorated the rapid initial paw swelling, inflammatory cells infiltration, skeletal tissues damage, osteoclast activation and the mRNA levels for osteoclast-related genes compared with the CAIA mice. However, the serum and mRNA levels of inflammatory mediators did not differ between the CAIA mice and the SRL-treated mice. SRL might reduce the inflammatory cells induction and skeletal tissue degradation by CAIA by the regulating osteoclastogenesis.

Ethnopharmacological relevance Kothala himbutu (Salacia reticulata) is a medicinal plant that has been used in Ayurvedic system of Indian and Sri Lankan traditional medicine to treat diabetes. Aim of the study This study aimed to clarify the mechanism(s) by which aqueous extracts of Kothala himbutu (KTE) stems decreases fasting blood glucose levels. Materials and methods Gene expression profiles were assessed by DNA microarray and RT-PCR analyses of RNA from the liver of KK-Ay diabetic mice administered KTE or control distilled water for 4 weeks, and from cultured liver cells treated with freeze-dried KTE (KTED) or selected phenolic compounds. Results DNA microarray and RT-PCR analyses revealed that gluconeogenic fructose-1,6-bisphosphatase (FBP) was decreased compared with the control in KTE-treated KK-Ay mice. RT-PCR analysis using cultured liver cells treated with KTED and/or actinomycin D or cycloheximide, revealed that KTED directly decreased FBP mRNA levels via destabilization of the mRNA. One compound in KTE, mangiferin, was demonstrated to dose-dependently down-regulate FBP mRNA. Conclusions These findings suggest that the mangiferin in KTE acts directly on liver cells and down-regulates the gluconeogenic pathway through regulation of FBP expression, thereby decreasing fasting blood glucose levels in mice. Our results demonstrate that gluconeogenic gene regulation is one possible mechanism by which KT exerts its effects in traditional diabetic medicine.

Kothala himbutu is a traditional Ayurvedic medicinal plant used to treat diabetes. We aimed to evaluate the safety of an aqueous extract of Kothala himbutu stem (KTE) in normal mice. The mice were divided into two groups: one was administered KTE and the other distilled water for 3 weeks. During the test period, the groups showed no significant differences in body weight gain or plasma parameters, such as fasting blood glucose level, oral glucose tolerance test, or aspartate transaminase (AST) or alanine transaminase (ALT) activity. DNA microarray analysis revealed that expression of genes of known function, such as those for the stress response, ribosomal proteins, transcription, cell function, the inflammatory/immune response, and metabolism (xenobiotic, glutathione, etc.) remained largely unaffected by KTE. However some genes such as catechol-o-methyltransferase and succinyl-CoA synthetase were regulated by KTE, indicating that KTE is not toxic to normal mice and might be effective as a functional food.

Kothala himbutu (KT) is a traditional medicinal plant used in treating diabetes in Ayurvedic medicine. We investigated the effect of the aqueous extract of KT stems (KTE) on energy expenditure in normal mice. Male C57BL/6J mice (n = 28) were divided into 4 groups depending on the type of diet they were fed for 9 weeks: normal (N) diet group (N: 13.8% energy in the form of fat), high-fat (HF) diet group (HF: 53.0% energy in the form of fat), 0.1% freeze-dried KTE (KTED)-supplemented N diet group (N+KTED), and 0.1% KTED-supplemented HF diet group (HF+KTED). KTED intake significantly reduced body weight gain in mice in the N and HF groups. Although it did not affect the plasma levels of glucose, triglyceride, and nonesterified fatty acid, KTED significantly decreased the HF diet-induced increased plasma insulin level. The epididymal and perirenal white adipose tissue (WAT) weights were significantly lower in the HF+KTED group than in the HF group. The oxygen consumption (VO2), measured by indirect calorimetry, of the mice in th eK TED-supplemented groups was significantly higher than that of the mice in the N and HF control groups. Moreover, KTED significantly reduced the size of epididymal WAT adi pocytes in the N and HF groups. Thus, KTED promoted VO2 and suppressed WAT accumulation in the mice on the N and HF diets. Therefore, KTE is beneficial in reducing N diet- and HF diet-induced obesity, which may be partly attributable to the stimulation of whole body energy metabolism.

Osteoarthritis (OA) is a slowly progressing chronic joint disease. Glucosamine (GlcN) is a saccharide that is widely used to relieve symptoms associated with OA. However, the mechanism of the effects of GlcN on articular cartilage remains unclear. We studied the effects of GlcN and its analogues, including chitin derivatives included in health supplements containing GlcN, on a chondrogenic cell line, ATDC5. We examined the effects of these saccharides on the proliferation and differentiation of ATDC5 cells. Glucosamine analogues, such as N-acetyl glucosamine and chitobiose, did not affect the proliferation or differentiation of ATDC5 cells. While GlcN did not affect the proliferation of ATDC5 cells, it inhibited their differentiation. Next, we examined whether GlcN affects mineralization and glycosaminoglycan (GAG) production by ATDC5 cells. Mineralization was markedly inhibited by addition of GlcN to the cell culture medium. Moreover, GlcN induced the formation of sulfated GAG in ATDC5. We also analyzed the mRNA levels in ATDC5 cells. GlcN reduced the mRNA levels of Smad2, Smad4 and MGP. GlcN might inhibit expression of MGP mRNA and induce the production of chondroitin sulfate in ATDC5 cells. The mechanism by which GlcN inhibits mineralization may be by regulating the expression of mRNA for the Smad2 and Smad4 chondrogenic master genes.

The present study discussed the effects of electrolytes and pore surface area and/or oxygen functional groups of activated carbon fiber (ACF) electrodes on their electric capacity. The electric capacity was affected by the electro-conductivity of electrolytes and increased with increase in oxygen functional groups. It depended more on oxygen functional groups than on BET surface area. However, ACFs which have higher acidity exhibited more remarkable decrease in capacity with current density, due to the increase of internal resistance. At the greatest 30% of increase in electric capacity was obtained by using ACFs with different amount of oxygen functional groups between the positive electrode and the negative one.

Background : Animal experiment studies have revealed a positive association between intake of citrus fruits and bone health. Nomilin, a limonoid present in citrus fruits, is reported to have many biological activities in mammalian systems, but the mechanism of nomilin on bone metabolism regulation is currently unclear. Purpose : To reveal the mechanism of nomilin on osteoclastic differentiation of mouse primary bone marrow-derived macrophages (BMMs) and the mouse RAW 264.7 macrophage cell line into osteoclasts. Study design : Controlled laboratory study. Effects of nomilin on osteoclastic differentiation were studied in in vitro cell cultures. Methods : Cell viability of RAW 264.7 cells and BMMs was measured with the Cell Counting Kit. TRAP-positive multinucleated cells were counted as osteoclast cell numbers. The number and area of resorption pits were measured as bone-resorbing activity. Osteoclast-specific genes expression was evaluated by quantitative real-time PCR; and proteins expression was evaluated by western blot. Results : Nomilin significantly decreased TRAP-positive multinucleated cell numbers compared with the control, and exhibited no cytotoxicity. Nomilin decreased bone resorption activity. Nomilin downregulated osteoclast-specific genes, NFATc1 and TRAP mRNA levels. Furthermore, nomilin suppressed MAPK signaling pathways. Conclusion : This study demonstrates clearly that nomilin has inhibitory effects on osteoclastic differentiation in vitro. These findings indicate that nomilin-containing herbal preparations have potential utility for the prevention of bone metabolic diseases.

An IF5 cDNA was isolated by expression cloning from a mouse oocyte cDNA library. It encoded a protein of 250 amino acids, and the region of it encoding amino acids 1?137 showed 86.8% alignment with the anti-proliferative domain of BTG/TOB family genes. This gene is also termed BTG4 or PC3B. Transiently expressed IF5/BTG4 induced alkaline phosphatase activity in human embryonic kidney (HEK293T) and 2T3 cells. IF5/BTG4 mRNA was detected by reverse transcription polymerase chain reaction in pharynx, larynx, trachea, oviduct, ovary, caput epididymis, and testis, but not in lung, intestine, or liver. Immunohistochemistry showed the IF5/BTG4 protein to be present in epithelial cells of the tongue, palate, pharynx, internal nose, and trachea. Both protein and mRNA levels of IF5/BTG4 were reduced by aging when comparing 4-week-old mice with 48-week-old mice. Our findings suggest that IF5/BTG4 may be an aging-related gene in epithelial cells. IF5/BTG4 was shown to be an aging-related gene, whose expression decreases with aging in the oral and respiratory cavities. Furthermore, IF5/BTG4 regulates cell differentiation., Original Article: The online platform for Taylor & Francis Online.

Long-chain N-vanillyl-acylamides (LCNVAs) were generated from plant oils and vanillylamine (VA) by nucleophilic amidation without any catalytic reagents. The resulting LCNVAs varied according to the fatty acid composition of the plant oil used. Therefore, the LCNVAs contained in Capsicum oleoresins were products that were spontaneously generated from the oleoresin during storage., "This is an Author's Original Manuscript of an article published by Taylor & Francis in Bioscience, Biotechnology and Biochemistry on 28/05/2014, available online at http://www.tandfonline.com/doi/abs/10.1080/09168451.2014.912118."

The IF3 gene was isolated by expression cloning from a cDNA library of mouse oocytes. This gene was revealed to have no homology to any known gene and its cDNA encodes a 202-amino acid protein that contains a signal-peptide sequence. Moreover, an IF3 isoform, IF3 2 , was expressed in both liver and ovary. Its cDNA encoded a 92-amino acid protein contains a signal-peptide sequence, which may be an alternative splice and frameshift form of IF3. The mRNA of IF3s was expressed in oocytes, ovary, and liver. Moreover, the gene expression of IF3s was regulated in a development-dependent manner in preimplantation-embryo and liver. Both IF3 1 and IF3 2 isoforms induced the differentiation of 2T3 and ATDC5 cells to the osteogenic and chondrogenic phenotype, respectively, suggesting that IF3s may modulate the differentiation status. Our findings suggest that IF3 may be one of the secreted factors that regulate oogenesis and certain liver functions.