Roles of mitochondrial alternative oxidase in photosynthetic electron transport in illuminated leaves of Arabidopsis thaliana at low temperature.

ATP-uncoupling alternative oxidase (AOX) in the plant respiratory chain is often induced under stress conditions such as low temperature (LT). The importance of AOX in photosynthesis has been examined, and leaves having larger amounts of AOX tended to show larger decrease in photosynthetic electron transport rate (ETR) by AOX inhibition. However, the details were not clarified. Here, we used three ecotypes of Arabidopsis thaliana which differed in AOX amounts and their responses to LT, and examined whether AOX amount was related to the degree of decrease in ETR by AOX inhibition. In Tiv-0, which originates from a warmer site, grown at high temperature (HT), AOX inhibition decreased ETR, but not in the other ecotypes. LT treatment significantly increased ETR and AOX, especially in Bur-0, but AOX inhibition did not decrease ETR in LT plants of any ecotype. AOX inhibition significantly increased the non-regulated energy dissipation in photosystem II (PSII), Y(NO), and decreased the maximal quantum yield of PSII, Fv/Fm, especially in LT plants. Since AOX inhibition did not affect the parameters of PSI, AOX inhibition may directly affect the reaction center of PSII in LT plants.

Bacteriologically Determined De Novo Tuberculosis during Tumor Necrosis Factor-α Inhibitor Therapy.

A 58-year-old man with Crohn's disease received adalimumab for 13 months after screening results for tuberculosis were found to be negative. He was diagnosed with de novo mediastinal lymph-node tuberculosis, which was proved to be bacteriologically identical to that of an individual with smear positive lung tuberculosis by a variable number of tandem repeat analyses. After initiating anti-tuberculosis therapy, the patient developed immune reconstitution syndrome, which was improved by the re-administration of adalimumab. Even in countries with an intermediate tuberculosis burden, including Japan, we need to be alert for de novo tuberculosis as well as its reactivation during tumor necrosis factor-α inhibitor therapy.

Quantitation of sevoflurane in whole blood and aqueous solutions by volatile organic compound sensing.

INTRODUCTION: It is difficult to quantify poorly soluble volatile anesthetics in aqueous solutions; this necessitates the development of alternative prompt methods to analyze the in vivo blood concentrations of anesthetics for the clinical assessment of anesthesia depth. In this study, we demonstrated that the difficulties can be overcome by using volatile organic compound (VOC) sensors, which allow the levels of vaporized VOCs to be quantified in several seconds and obviate the need for conventional techniques such as gas chromatography or nuclear magnetic resonance (NMR). METHODS: The concentrations of a volatile general anesthetic (sevoflurane) in aqueous solutions containing human blood components and rabbit blood were measured using a VOC sensor and those in distilled water and phosphatidylcholine suspension were compared to those determined by NMR. RESULTS: For all aqueous solutions with concentrations of up to 5 mM, the relationship between the VOC content and sevoflurane concentration was represented by a straight line passing through the origin. The concentration of sevoflurane determined by VOC sensing was well correlated with the values obtained by NMR at <1 mM, which is within the clinically relevant concentration levels. DISCUSSION: Considering the results from this study, we can conclude that VOC sensing may be useful for measuring intraoperative blood anesthetic concentrations.

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro.

Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24­well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume­dependent manner. A co­culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate­resistant acid phosphatase (TRAP)­positive multinuclear cells (≥2 nuclei), whereas the formation of large osteoclastic TRAP­positive multinuclear cells (≥8 nuclei) was increased, in a media volume­dependent manner. There were also no differences in receptor activator of nuclear factor­κB ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume­dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems.

Release from optimal compressive force suppresses osteoclast differentiation.

Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate­resistant acid phosphatase (TRAP)­positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription­quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast­specific genes, TRAP, RANK, matrix metalloproteinase­9, cathepsin­K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell­specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC­STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments.

Stereoselective Construction of 2,7-Disubstituted fused-Bis Tetrahydrofuran Skeletons: Biomimetic-Type Synthesis and Biological Evaluation of (±)- and (-)-Aplysiallene and Their Derivatives.

A series of trans/trans and cis/cis fused-bis tetrahydrofuran compounds have been obtained stereoselectively in high yields via a one-pot operation involving the intramolecular haloetherification of (Z,Z)-diene diol 19a and (E,E)-diene disilylether 19d, respectively. This method was subsequently applied to the biomimetic-type synthesis of (±)- and (-)-aplysiallene. The inhibitory activities of these compounds and their bromodiene isomers toward Na(+)/K(+) ATPase were determined in vitro, and gave IC50 values of approximately 15 µM in all cases.

Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells.

Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast­associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied.

Rosmarinic acid and arbutin suppress osteoclast differentiation by inhibiting superoxide and NFATc1 downregulation in RAW 264.7 cells.

The present study investigated the effect of the natural polyphenols, rosmarinic acid and arbutin, on osteoclast differentiation in RAW 264.7 cells. Rosmarinic acid and arbutin suppressed osteoclast differentiation and had no cytotoxic effect on osteoclast precursor cells. Rosmarinic acid and arbutin inhibited superoxide production in a dose-dependent manner. mRNA expression of the master regulator of osteoclastogenesis, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and the osteoclast marker genes, matrix metalloproteinase-9, tartrate-resistant acid phosphatase and cathepsin-K, decreased following treatments with rosmarinic acid and arbutin. Furthermore, resorption activity decreased with the number of osteoclasts. These results suggest that rosmarinic acid and arbutin may be useful for the prevention and treatment of bone diseases, such as osteoporosis, through mechanisms involving inhibition of superoxide and downregulation of NFATc1.

An easy-to-use excimer fluorescence derivatization reagent, 2-chloro-4-methoxy-6-(4-(pyren-4-yl)butoxy)-1,3,5-triazine, for use in the highly sensitive and selective liquid chromatography analysis of histamine in Japanese soy sauces.

In this study, a novel pre-column excimer fluorescence derivatization reagent, 2-chloro-4-methoxy-6-(4-(pyren-4-yl)butoxy)-1,3,5-triazine (CMPT), was developed for polyamines, specifically histamine. By CMPT derivatization, the polyamines, histamine and tyramine were converted to polypyrene derivatives, and emitted intra-molecular excimer fluorescence at 475nm. This could clearly be distinguished from the normal fluorescence emitted from reagent blanks at 375 nm. Unlike conventional excimer fluorescence derivatization reagents, CMPT is chemically stable and its reactivity sustained over at least 36 days even in solution state. We successfully applied this reagent to the sensitive and selective analysis of histamine in different kinds of Japanese commercial soy sauces. The detection and quantification limits of histamine were 15 and 50 µg L(-1), respectively, equating to 1.35 pmol and 4.5 pmol for a 6 µL injection. This sensitivity helped the direct analysis of soy sauce samples only treated by one-step liquid-liquid extraction without concentration. The histamine levels of commercial soy sauce samples (koikuchi, usukuchi and saishikomi) investigated were 1.24-768.5 mg L(-1).

Simple and sensitive analysis of histamine and tyramine in Japanese soy sauces and their intermediates using the stable isotope dilution HILIC-MS/MS method.

We established a simple, sensitive, and reproducible method to analyze the histamine and tyramine levels in Japanese soy sauce and its mash (called moromi) using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Histamine and tyramine quantification was performed using their stable isotopes for electrospray ionization-tandem mass spectrometry in the selected reaction monitoring mode. The sample pretreatment process was a simple, one-step liquid-liquid extraction. HILIC separation was performed with a gradient elution of aqueous ammonium formate and acetonitrile. Because of validation tests, the linearity, the accuracies, and precisions were sufficient. The limit of detection and the limit of quantification were 0.09 and 0.29 ppm for histamine and 0.13 and 0.42 ppm for tyramine, respectively. We successfully applied this method to histamine and tyramine determination in four kinds of commercial Japanese soy sauces and also in moromi samples during soy sauce production.

Short-term mechanical stress inhibits osteoclastogenesis via suppression of DC-STAMP in RAW264.7 cells.

Mechanical stress is an important factor in bone homeostasis, which is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. However, little is known about the effects of mechanical stress on osteoclast differentiation. In this study, we examined the effects of short-term mechanical stress on osteoclastogenesis by applying tensile force to RAW264.7 cells stimulated with receptor activator of nuclear factor-κB ligand (RANKL) using a Flexercell tension system. We counted the number of osteoclasts that were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (two or more nuclei) with or without application of mechanical stress for 24 h. Osteoclast number was lower after mechanical stress compared with no mechanical stress. Furthermore, mechanical stress for up to 24 h caused downregulation of osteoclast-specific gene expression and fusion-related molecule [dendritic cell specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), E-cadherin, Integrin αV and Integrin ß3] mRNA levels. Protein expression of DC-STAMP decreased with mechanical stress for 24 h compared to the control without mechanical stress, whereas the expression of E-cadherin, Integrin αV and Integrin ß3 was slightly decreased. Nuclear factor of activated T cells c1 (NFATc1) mRNA levels were decreased at 6 h and increased at 12 and 24 h compared with the control. The levels of NFATc2, NFATc3 mRNA did not change compared with the control group. By contrast, mechanical stress for 24 h significantly enhanced NFAT transcriptional activity compared with the control, despite a decrease in DC-STAMP mRNA and protein levels. These results suggest that short-term mechanical stress strongly inhibits osteoclastogenesis through the downregulation of DC-STAMP and other fusion-related molecules and that short-term mechanical stress induces a negative regulatory mechanism that cancels the enhancement of NFAT transcriptional activity.

Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells.

Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.

Toll like receptor 5 ligand induces monocyte chemoattractant protein-1 in mouse osteoblastic cells.

Flagellin, the ligand of Toll like receptor 5, is the major subunit of bacterial flagella. Flagellin stimulates various cells to release chemokines. Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC chemokine family that is involved in monocyte infiltration in inflammatory diseases. It has been reported that serum MCP-1 levels increase proportionally with the severity of periodontal disease. Inflammatory mediators induce MCP-1 production in various cells, including osteoblasts. However, it remains unclear whether MCP-1 is released from osteoblasts in response to flagellin. In the present study, we investigated the effects of flagellin on the expression of MCP-1 in the mouse osteoblastic cell line, MC3T3-E1 (E1) cells. Flagellin markedly increased MCP-1 mRNA level in a dose-dependent manner. The effect of flagellin on MCP-1 mRNA expression in E1 cells was transient, with a peak at 1 h. Concomitant with MCP-1 mRNA expression, MCP-1 protein levels were clearly elevated at 3 h after flagellin exposure. In addition, we revealed that JNK and MEK-ERK1/2 are involved in flagellin-induced MCP-1 expression in E1 cells. These results indicated that bacterial flagellin may play an important role in the progression of periodontitis. Results of further studies will provide more clues to the prevention of periodontal diseases.

Platinum nanoparticles suppress osteoclastogenesis through scavenging of reactive oxygen species produced in RAW264.7 cells.

Recent research has shown that platinum nanoparticles (nano-Pt) efficiently quench reactive oxygen species (ROS) as a reducing catalyst. ROS have been suggested to regulate receptor activator of NF-κB ligand (RANKL)-stimulated osteoclast differentiation. In the present study, we examined the direct effects of platinum nano-Pt on RANKL-induced osteoclast differentiation of murine pre-osteoclastic RAW 264.7 cells. The effect of the nano-Pt on the number of osteoclasts was measured and their effect on the mRNA expression for osteoclast differentiation was assayed using real-time PCR. Nano-Pt appeared to have a ROS-scavenging activity. Nano-Pt decreased the number of osteoclasts (2+ nuclei) and large osteoclasts (8+ nuclei) in a dose-dependent manner without affecting cell viability. In addition, this agent significantly blocked RANKL-induced mRNA expression of osteoclastic differentiation genes such as c-fms, NFATc1, NFATc2, and DC-STAMP as well as that of osteoclast-specific marker genes including MMP-9, Cath-K, CLC7, ATP6i, CTR, and TRAP. Although nano-Pt attenuated expression of the ROS-producing NOX-family oxidases, Nox1 and Nox4, they up-regulated expression of Nox2, the major Nox enzyme in macrophages. These findings suggest that the nano-Pt inhibit RANKL-stimulated osteoclast differentiation via their ROS scavenging property. The use of nano-Pt as scavengers of ROS that is generated by RANKL may be a novel and innovative therapy for bone diseases.

[Pulmonary nocardiosis with elevation of serum beta-D-glucan in a patient with polymyositis].

A 73-year-old woman with polymyositis, who had received corticosteroids and immune-suppressive agents, was admitted to our hospital because of general fatigue and severe cough. Chest X-ray film and CT scan showed a large tumor shadow in the left upper lobe and several ground-glass opacities (GGOs) scattered in both lungs. As the white blood cell and C-reactive protein levels were elevated, pnueumonia was suspected and antibiotics were administered. Subsequently, Nocardia spp. was cultured from the sputum and pulmonary nocardiosis was established. She gradually recovered after sulfamethoxazole-trimethoprim (ST) administration. The pretreatment serum beta-D-glucan level was highly elevated and decreased in parallel with clinical feature. In general, ST should be administered for 6 months to treat pulmonary nocardiosis in a compromised host. It is possible that P3-D-glucan may be a useful marker to treat pulmonary nocardiosis in patients with polymyositis.

Tannin-fluoride preparation attenuates prostaglandin E2 production by dental pulp cells.

Glass ionomer cements (GICs) are widely used for the operative restoration of dental caries. However, it has been reported that the components of GICs cause pulpal inflammatory responses. Recently, GICs containing tannin-fluoride preparation (HY agent) were developed. In this study, we investigated the effect of HY agent on prostaglandin E2 (PGE2) release from GIC-stimulated rat dental pulp cells (RPC-C2A). Extracts derived from GIC disks were used with HY(+) and without HY(-) agent. After treatment with GIC extracts, ATP contents, COX-2 mRNA and protein expression in RPC-C2A cells, and PGE2 production in culture media were analyzed. HY agent suppressed HY(-)-stimulated PGE2 release from RPC-C2A cells, as well as COX-2 mRNA and protein expression. Moreover, tannic acid attenuated COX-2 mRNA induced by HY(-) extract in a dose-dependent manner. Taken together, these results suggest that tannic acid in HY agent may suppress GIC-induced production of PGE2 by inhibition of COX-2 expression in dental pulp cells.

Effect of the release from mechanical stress on osteoclastogenesis in RAW264.7 cells.

The effects of mechanical stress release on osteoclastogenesis may be as important as those of mechanical stress application. However, the direct effects of mechanical stress on the behavior of osteoclasts has not been thoroughly investigated and there is limited information on the results of the release from mechanical stress. In this study, the effects of mechanical stress application and its release on osteoclast differentiation were examined. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells were measured and the expression of osteoclast differentiation genes, which was altered in response to the release from mechanical stress according to the Flexercell tension system was evaluated by real-time PCR. Osteoclast differentiation and fusion were suppressed by mechanical stress application and were rapidly induced after mechanical stress release. The mRNA expression of the osteoclast specific genes, TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin-K (cath-k), calcitonin receptor (CTR), ATPase H+ transporting vacuolar proton pump member I (ATP6i), chloride channel-7 (ClC7) and dendritic cell-specific transmembrane protein (DC-STAMP) was decreased with mechanical stress application, and increased up to 48 h after the release from it. These alterations in gene mRNA expression were associated with the number of osteoclasts and large osteoclasts. Inducible nitric oxide synthetase (iNOS) mRNA was increased with mechanical stress and decreased after its release. Nitric oxide (NO) production was increased with mechanical stress. Nuclear factor of activated T cells cytoplasmic (NFATc) family mRNAs were not altered with mechanical stress, but were up-regulated up to 48 h after the release from it. These findings indicate that the suppression of osteoclast differentiation and fusion induced by mechanical stress is the result of NO increase via iNOS, and that the promotion of osteoclast differentiation and fusion after the release from mechanical stress is related to the NFATc family genes, whose expression remained constant during mechanical stress but was up-regulated after the release from mechanical stress.

Effect of N-acetylcysteine on rat dental pulp cells cultured on mineral trioxide aggregate.

INTRODUCTION: The purpose of this study was to evaluate the cytotoxicity of mineral trioxide aggregate (MTA) and its potential detoxification by an antioxidant amino acid, N-acetylcysteine (NAC). METHODS: Rat dental pulp cells extracted from rat maxillary incisors were directly cultured on MTA with or without NAC in culture medium. The number of cells and their spreading behavior were both assessed 24 hours after seeding. The intracellular levels of reactive oxygen species (ROS) and glutathione (GSH) were also assessed after 24 hours of culture. RESULTS: The number of cells attached to MTA was 60% greater when NAC was added to the culture medium. In addition, the area and perimeter of the cells were found to be 2-fold greater in the culture containing NAC. Cells cultured on MTA alone showed large ROS concentrations, which disappeared when the medium was supplemented with NAC. The intracellular GSH level, however, increased 3.5-fold with NAC addition. CONCLUSIONS: This study demonstrated that the presence of NAC in environments can substantially improve attachment and spreading behaviors of dental pulp cells on MTA. This biological effect was associated with an improvement in the cellular redox system by NAC and warrants further exploration of NAC for determining its therapeutic value in improving the biocompatibility of MTA.

Amino acid derivative-mediated detoxification and functionalization of dual cure dental restorative material for dental pulp cell mineralization.

Current dental restorative materials are only used to fill the defect of hard tissues, such as dentin and enamel, because of their cytotoxicity. Therefore, exposed dental pulp tissues in deep cavities must be first covered by a pulp capping material like calcium hydroxide to form a layer of mineralized tissue. However, this tissue mineralization is based on pathological reaction and triggers long-lasting inflammation, often causing clinical problems. This study tested the ability of N-acetyl cysteine (NAC), amino acid derivative, to reduce cytotoxicity and induce mineralized tissue conductivity in resin-modified glass ionomer (RMGI), a widely used dental restorative material having dual cure mechanism. Rat dental pulp cells were cultured on untreated or NAC-supplemented RMGI. NAC supplementation substantially increased the percentage of viable cells from 46.7 to 73.3% after 24-h incubation. Cell attachment, spreading, proliferative activity, and odontoblast-related gene and protein expressions increased significantly on NAC-supplemented RMGI. The mineralization capability of cells, which was nearly suppressed on untreated RMGI, was induced on NAC-supplemented RMGI. These improved behaviors and functions of dental pulp cells on NAC-supplemented RMGI were associated with a considerable reduction in the production of intracellular reactive oxygen species and with the increased level of intracellular glutathione reserves. These results demonstrated that NAC could detoxify and functionalize RMGIs via two different mechanisms involving in situ material detoxification and antioxidant cell protection. We believe that this study provides a new approach for developing dental restorative materials that enables mineralized tissue regeneration.