Novel Osteogenic Behaviors around Hydrophilic and Radical-Free 4-META/MMA-TBB: Implications of an Osseointegrating Bone Cement.

Poly(methyl methacrylate) (PMMA)-based bone cement, which is widely used to affix orthopedic metallic implants, is considered bio-tolerant but lacks osteoconductivity and is cytotoxic. Implant loosening and toxic complications are significant and recognized problems. Here we devised two strategies to improve PMMA-based bone cement: (1) adding 4-methacryloyloxylethyl trimellitate anhydride (4-META) to MMA monomer to render it hydrophilic; and (2) using tri-n-butyl borane (TBB) as a polymerization initiator instead of benzoyl peroxide (BPO) to reduce free radical production. Rat bone marrow-derived osteoblasts were cultured on PMMA-BPO, common bone cement ingredients, and 4-META/MMA-TBB, newly formulated ingredients. After 24 h of incubation, more cells survived on 4-META/MMA-TBB than on PMMA-BPO. The mineralized area was 20-times greater on 4-META/MMA-TBB than PMMA-BPO at the later culture stage and was accompanied by upregulated osteogenic gene expression. The strength of bone-to-cement integration in rat femurs was 4- and 7-times greater for 4-META/MMA-TBB than PMMA-BPO during early- and late-stage healing, respectively. MicroCT and histomorphometric analyses revealed contact osteogenesis exclusively around 4-META/MMA-TBB, with minimal soft tissue interposition. Hydrophilicity of 4-META/MMA-TBB was sustained for 24 h, particularly under wet conditions, whereas PMMA-BPO was hydrophobic immediately after mixing and was unaffected by time or condition. Electron spin resonance (ESR) spectroscopy revealed that the free radical production for 4-META/MMA-TBB was 1/10 to 1/20 that of PMMA-BPO within 24 h, and the substantial difference persisted for at least 10 days. The compromised ability of PMMA-BPO in recruiting cells was substantially alleviated by adding free radical-scavenging amino-acid N-acetyl cysteine (NAC) into the material, whereas adding NAC did not affect the ability of 4-META/MMA-TBB. These results suggest that 4-META/MMA-TBB shows significantly reduced cytotoxicity compared to PMMA-BPO and induces osteoconductivity due to uniquely created hydrophilic and radical-free interface. Further pre-clinical and clinical validations are warranted.

Bactericidal effect of hydroxyl radicals generated by the sonolysis and photolysis of hydrogen peroxide for endodontic applications.

The aim of endodontic root canal treatment is the elimination of bacteria and their products from an infected tooth root canal. To effectively disinfect a root canal, an ultrasonic irrigation system, in which hydroxyl radicals (HO·) generated artificially by sonolysis of H2O2, was developed previously for endodontic applications and was demonstrated to have bactericidal efficacy against Enterococcus faecalis. To improve this system, we examined the in vitro bactericidal effects of HO· generated from H2O2, activated by simultaneous irradiation with ultrasound for sonolysis and dental LED light for photolysis with a peak wavelength of 405 nm. Regarding the LED irradiation, two methods were used: (i) 'ideal' experimental conditions (irradiation close to the glass tube), and (ii) simulated endodontic conditions (more distant irradiation of a masked glass tube). In these conditions, HO· generation from H2O2 was detected by electron spin resonance (ESR) spectroscopy, and bactericidal efficacy against E. faecalis was assessed by measuring the colony forming units (CFU)/mL. The results indicated that HO· generation by ESR measurements and the bactericidal effect on E. faecalis by viable count using CFU/mL were enhanced significantly in a time-dependent manner in both conditions. In a comparison of these conditions, bactericidal activity under 'ideal' experimental conditions was similar to that under simulated endodontic conditions. Moreover, the irradiation time for effective killing of E. faecalis through the sonolysis and photolysis of H2O2 under simulated endodontic conditions was shorter than that with sonolysis alone. These results demonstrate that H2O2 activated by ultrasound and LED light may be a safe and effective disinfection technique for endodontic root canal treatment.

Porphyromonas gingivalis infection modifies oral microcirculation and aortic vascular function in the stroke-prone spontaneously hypertensive rat (SHRSP).

The functional modulation of vascular endothelial cells associated with stroke and periodontal disease has not yet been clarified. The objective of this study is to analyze the vascular endothelial function of periodontitis and stroke animal models. We examined endothelial function and gingival blood flow in oral microcirculation in vivo and measured the isometric tension in vitro of the aorta in animal models for lifestyle-related diseases, such as periodontitis and stroke. Gingival reactive hyperemia (GRH) was measured using laser Doppler flowmetry. Wistar Kyoto rats (WKY) were used as control animals; Porphyromonas gingivalis (P. gingivalis) infected WKY (WKY + Pg) as the periodontitis model; stroke-prone spontaneously hypertensive rat (SHRSP) as the stroke model; and a final group consisting of P. gingivalis infected SHRSP (SHRSP + Pg). Furthermore, for each group, the relaxation of descending aortic ring preparations was measured using a force transducer. The GRH was estimated by maximum response (peak), time taken for the maximum response to fall to one half (T1/2), and increased total amount of blood flow (mass). The relative change in T1/2 and mass increased in SHRSP + Pg compared to WKY. However, mass significantly increased in WKY (758.59 ± 88.21 ml/min/100 g s to 1755.55 ± 226.10 ml/min/100 g s) and SHRSP (1214.87 ± 141.61 ml/min/100 g s to 2674.32 ± 675.48 ml/min/100 g s) after treatment with acetylcholine. In addition, T1/2 and mass significantly increased in WKY + Pg (624.18 ± 96.36 ml/min/100 g s to 2629.90 ± 612.01 ml/min/100 g s) and SHRSP + Pg (1116.36 ± 206.24 ml/min/100 g s to 1952.76 ± 217.39 ml/min/100 g s) after treatment with nitroglycerin. Furthermore, the endothelium-dependent relaxation of ring preparations, evoked by acetylcholine, was attenuated in SHRSP compared with WKY, but not in SHRSP + Pg. This attenuation effect in SHRSP could be prevented by superoxide dismutase pretreatment. Our results suggest altered endothelial function may occur in gingival tissue in animal models experiencing both periodontitis and stroke. Therefore, these results indicate the disruption of vascular function in oral microcirculation may be caused by the interaction between the oxidative stress induced by periodontitis and nitric oxide in periodontitis, similar to the interactions present in stroke cases.

Protective effects of (6R)-5,6,7,8-tetrahydro-l-biopterin on local ischemia/reperfusion-induced suppression of reactive hyperemia in rat gingiva.

We herein investigated the regulatory mechanism in the circulation responsible for rat gingival reactive hyperemia (RH) associated with ischemia/reperfusion (I/R). RH was analyzed using a laser Doppler flowmeter. RH and I/R were elicited by gingival compression and release with a laser Doppler probe. RH increased in a time-dependent manner when the duration of compression was between 30 s and 20 min. This increase was significantly suppressed by N (ω)-nitro-l-arginine-methyl-ester (l-NAME), 7-nitroindazole (7-NI), and 2,4-diamino-6-hydroxypyrimidine (DAHP). However, RH was markedly inhibited following 60 min of compression. This inhibition was significantly decreased by treatments with superoxide dismutase (SOD), (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4), and sepiapterin. The luminescent intensity of superoxide anion (O2 (•-))-induced 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-[1,2-a] pyrazine-3-one (MCLA) was markedly decreased by SOD and BH4, but only slightly by sepiapterin. BH4 significantly decreased O2 (•-) scavenging activity in a time-dependent manner. These results suggested that nitric oxide (NO) secreted by the nitrergic nerve played a role in regulating local circulation in rat gingiva. This NO-related regulation of local circulation was temporarily inhibited in the gingiva by the I/R treatment. The decrease observed in the production of NO, which was caused by suppression of NO synthase (NOS) activity subsequent to depletion of the NOS co-factor BH4 by O2 (•-), played a partial role in this inhibition.

Relationship among salivary antioxidant activity, cytokines, and periodontitis: the Nagasaki Island study.

AIM: Antioxidant activities and cytokine levels in human body fluids are considered to be strongly associated with periodontitis. The aim of this study was to elucidate the relationship between salivary antioxidant activities against superoxide or hydroxyl radical, cytokines, and periodontal conditions through a community-based cross-sectional study conducted in Goto city, Japan. MATERIALS AND METHODS: Saliva samples were analysed for superoxide or hydroxyl radical scavenging activities and cytokine levels from 160 participants. We demonstrated that saliva contained superoxide and hydroxyl radical scavenging activities by using electron spin resonance with a spin-trapping agent. The concentrations of eight cytokines were measured using multiplex bead assays. RESULTS: There were significant differences in salivary superoxide or hydroxyl radical scavenging activity, and the levels of Interleukin-1ß, Interleukin-6, and Interleukin-8 between periodontitis classifications. Multivariate stepwise logistic regression model showed that salivary superoxide and hydroxyl radical scavenging activities were significantly associated with the classification of periodontitis. In addition, salivary superoxide scavenging activity was found to have significant association with all periodontal parameters using multiple linear regression analysis. CONCLUSIONS: These findings suggest that the evaluation of salivary antioxidant activities, as assessed by electron spin resonance, are associated with periodontitis and various clinical variables in community-dwelling participants (ClinicalTrials.gov number NCT01742728).

Inhibitory effects of French pine bark extract, Pycnogenol®, on alveolar bone resorption and on the osteoclast differentiation.

Pycnogenol(®) (PYC) is a standardized bark extract from French maritime pine (Pinus pinaster Aiton). We examined the inhibitory effects of PYC on alveolar bone resorption, which is a characteristic feature of periodontitis, induced by Porphyromonas gingivalis (P. gingivalis) and osteoclast differentiation. In rat periodontitis model, rats were divided into four groups: group A served as the non-infected control, group B was infected orally with P. gingivalis ATCC 33277, group C was administered PYC in the diet (0.025%: w/w), and group D was infected with P. gingivalis and administered PYC. Administration of PYC along with P. gingivalis infection significantly reduced alveolar bone resorption. Treatment of P. gingivalis with 1 µg/ml PYC reduced the number of viable bacterial cells. Addition of PYC to epithelial cells inhibited adhesion and invasion by P. gingivalis. The effect of PYC on osteoclast formation was confirmed by tartrate-resistant acid phosphatase staining. PYC treatment significantly inhibited osteoclast formation. Addition of PYC (1-100 µg/ml) to purified osteoclasts culture induced cell apoptosis. These results suggest that PYC may prevent alveolar bone resorption through its antibacterial activity against P. gingivalis and by suppressing osteoclastogenesis. Therefore, PYC may be useful as a therapeutic and preventative agent for bone diseases such as periodontitis.

Biocompatibility of 4-META/MMA-TBB resin used as a dental luting agent.

STATEMENT OF PROBLEM: The bonding and biological properties of currently used luting/cementing materials need to be improved. 4-Acryloyloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butylborane (4-META/MMA-TBB) resin is primarily used for splinting mobile teeth or treating fractured teeth. It undergoes moisture-resistant polymerization and bonds strongly to dentin and metals. PURPOSE: The purpose of this in vitro study was to compare the biological and biochemical properties META/MMA-TBB resin with those of conventional polymethyl methacrylate (PMMA)-MMA resin and other currently used luting materials in order to determine whether it may be a viable dental luting agent. MATERIAL AND METHODS: The degree of polymerization of 4-META/MMA-TBB resin, PMMA-MMA autopolymerizing resin, 10-methacryloyloxydecyl dihydrogen phosphate-dimethacrylate (MDP-DMA) adhesive resin, and a glass ionomer cement was measured by Fourier-transformed infrared spectroscopy. Free radical production during setting was evaluated by electron spin resonance (ESR) spectroscopy. Rat dental pulp cells cultured on these materials were examined for cell viability, attachment, proliferation, and functional phenotype. RESULTS: The degree of polymerization of 4-META/MMA-TBB resin was 82% thirty minutes after preparation, compared to 66% for PMMA-MMA autopolymerizing resin. ESR spectroscopy revealed free radical production from 4-META/MMA-TBB resin and glass ionomer cement was equivalent 24 hours after preparation, with no spike in radical generation observed. In contrast, free radical production from PMMA-MMA and MDP-DMA adhesive resins was rapid and sustained and 10 to 20 times greater than that from 4-META/MMA-TBB. The percentage of viable dental pulp cells 24 hours after seeding was considerably higher on MDP-DMA and 4-META/MMA-TBB resin than on glass ionomer cement. Cell number, proliferation, and alkaline phosphatase activity were highest on 4-META/MMA-TBB resin and lowest on the glass ionomer cement. CONCLUSIONS: 4-META/MMA-TBB resin is at least as biocompatible, and perhaps even more biocompatible, than other current luting materials, with fast, favorable, and nontoxic polymerization properties. Further in vivo and human studies of 4-META/MMA-TBB resin as a dental luting agent are warranted.

Medical-grade collagen peptide in injectables provides antioxidant protection.

Medical-grade collagen peptide is used as an additive agent in pharmaceutical formulations; however, it is unknown as to whether the compound exerts antioxidant effects in vitro. In this study, we evaluated the antioxidant effects of medical-grade collagen peptide on reactive oxygen species such as hydroxyl radical, superoxide anion radical and singlet oxygen using electron spin resonance and spin trapping. We confirmed that medical-grade collagen peptide directly inhibited hydroxyl radical generated by the Fenton reaction or by ultraviolet irradiation of hydrogen peroxide, and singlet oxygen. In addition, an antioxidant effect of medical-grade collagen peptide on singlet oxygen was observed in peptide fractions 12-22. The total amount of antioxidant amino acids (Gly, Hyp, Glu, Ala, Cys, Met and His) constituted more than half of the total amino acids in these fractions. These results suggest that the observed antioxidant properties of medical-grade collagen peptide are due to the compound containing antioxidant amino acids. Medical-grade collagen peptide, which is used in pharmaceuticals, and especially in injectables, could provide useful antioxidant properties to protect the active ingredient from oxidation.

Contribution of nitrergic nerve in canine gingival reactive hyperemia.

Reactive hyperemia reflects a compensatory vasodilation response of the local vasculature in ischemic tissue. The purpose of this study is to clarify the mechanism of regulation of this response in gingival circulation by using pharmacological analysis of reactive hyperemia and histochemical analysis of gingival tissue. Application of pressure to the gingiva was used to create temporary ischemia, and gingival blood flow was measured after pressure release. Reactive hyperemia increased in proportion to the duration of pressure. Systemic hemodynamics remained unaffected by the stimulus; therefore, the gingival reactive hyperemia reflected a local adjustment in circulation. Gingival reactive hyperemia was significantly suppressed by nitric oxide (NO) synthase inhibitors, especially the neural NO synthase-selective antagonist 7-nitroindazole, but not by anticholinergic drugs, ß-blockers, or antihistaminergic drugs. Moreover, immunohistochemical staining for neural NO synthase and histochemical staining for NADPH diaphorase activity were both positive in the gingival perivascular region. These histochemical and pharmacological analyses show that reactive hyperemia following pressure release is mediated by NO-induced vasodilation. Furthermore, histochemical analysis strongly suggests that NO originates from nitrergic nerves. Therefore, NO may play an important role in the neural regulation of local circulation in gingival tissue ischemia.

Measurement and characterization of superoxide generation from xanthine dehydrogenase: a redox-regulated pathway of radical generation in ischemic tissues.

The enzyme xanthine oxidoreductase (XOR) is an important source of oxygen free radicals and related postischemic injury. Xanthine dehydrogenase (XDH), the major form of XOR in tissues, can be converted to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. The conversion of XDH to XO has been assumed to be required for radical generation and tissue injury. It is also possible that XDH could generate significant quantities of superoxide, •O2⁻, for cellular signaling or injury; however, this possibility and its potential ramifications have not been previously considered. To unambiguously determine if XDH can be a significant source of •O2⁻, experiments were performed to measure and characterize •O²â» generation using XDH from chicken liver that is locked in the dehydrogenase conformation. Electron paramagnetic resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide demonstrated that XDH in the presence of xanthine produces significant amounts of •O2⁻. NAD⁺ and NADH inhibited the generation of •O2⁻ from XDH in a dose-dependent manner, with NAD⁺ exhibiting stronger inhibition than NADH at low physiological concentrations. Decreased amounts of NAD⁺ and NADH, which occur during and following tissue ischemia, enhanced the generation of •O2⁻ from XDH in the presence of xanthine. It was observed that XDH-mediated oxygen radical generation markedly depressed Ca²âº-ATPase activity of isolated sarcoplasmic reticulum vesicles from cardiac muscle, and this was modulated by NAD⁺ and NADH. Thus, XDH can be an important redox-regulated source of •O2⁻ generation in ischemic tissue, and conversion to XO is not required to activate radical formation and subsequent tissue injury.

Antimicrobial photodynamic therapy suppresses dental plaque formation in healthy adults: a randomized controlled clinical trial.

BACKGROUND: Oral care is important for oral and systemic health, especially for elderly institutionalized individuals and compromised patients. However, conventional mechanical plaque control is often difficult for these patients because of the pain or the risk of aspiration. Although antimicrobial photodynamic therapy (aPDT), which is considered an alternative or adjunct to mechanical approaches, has potential application as a less stressful method of daily plaque control, no clinical application of this technique has been reported. METHODS: We investigated the inhibitory effect of a combination of toluidine blue O (TBO), and a red light-emitting diode (LED) on dental plaque formation in healthy volunteers. The optimal concentration of TBO was determined in preliminary in vitro experiments to evaluate the bactericidal effect of aPDT on Streptococcus oralis and to clarify its safety in fibroblast cells. To survey the mechanism of TBO-mediated aPDT, the quality and quantity of reactive oxygen species (ROS) generated during aPDT were also examined using electron spin resonance (ESR) spectroscopy. Subsequently, the inhibitory effect of aPDT on dental plaque formation was investigated in eleven subjects as a clinical pilot study. The right or left mandibular premolars were randomly assigned to the treatment (with aPDT) or control (without aPDT) groups. In total, aPDT was applied six times (twice per day) to the teeth in the test group over a period of four days. On the fourth day, the study concluded and the analyses were performed. RESULTS: A combination of 500 or 1000 µg/ml TBO and LED irradiation for 20 s significantly decreased the number of colony forming units of Streptococcus oralis. The cytotoxicity of aPDT was comparable to that of standard antiseptics used in the oral cavity. Hydroxyl radicals were detected by ESR analysis, but singlet oxygen was not. A randomized controlled trial demonstrated that aPDT with 1000 µg/ml TBO and red LED irradiation significantly suppressed dental plaque formation without harming teeth or the surrounding tissues. CONCLUSIONS: aPDT has the potential to be a promising novel technical modality for dental plaque control. TRIAL REGISTRATION: This trial was registered with University Hospital Medical Information Network Clinical Trials Registry (number UMIN000012504).

Bactericidal effect of hydroxyl radicals generated from a low concentration hydrogen peroxide with ultrasound in endodontic treatment.

One approach to enhance the disinfection of root canals in endodontic treatment is ultrasonic irrigation with sodium hypochlorite. Reactive oxygen species, such as hydroxyl radical, are generated by biological defense systems to kill invading bacteria. Ultrasonic irrigation with hydrogen peroxide may be a promising option to increase hydroxyl radical generation. We examined the bactericidal effects of hydroxyl radical generated from low concentration hydrogen peroxide with ultrasound in vitro. An ultrasonic tip was submerged in 0.5 or 1.0 M hydrogen peroxide in a microfuge tube. hydrogen peroxide was irradiated with the ultrasound, the tip of which was maintained centered in the tube to mimic ultrasonic irrigation. Hydroxyl radical generation was assessed by electron spin resonance spectroscopy. Subsequently, Enterococcus faecalis suspension in hydrogen peroxide was prepared and irradiated as described above. Bactericidal effects were assessed by viable counting. Electron spin resonance measurements showed that hydroxyl radical generation increased significantly in a time- and dose-dependent manner (two-way analysis of variance and Tukey's test, p<0.05). Moreover, the bactericidal effects of hydrogen peroxide against Enterococcus faecalis were enhanced by ultrasonic irradiation in a time- and dose-dependent manner. These results suggest that ultrasonic irrigation in the presence of low concentration hydrogen peroxide can serve as a disinfection strategy in endodontic treatment.

Transplantation of side population cells restores the function of damaged exocrine glands through clusterin.

Stem cell-based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation-induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31-positive endothelial-like cells. In addition, we clarified that endothelial cell-derived clusterin, a secretory protein, was an essential factor for SP cell-mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin-deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress-induced cell damage. Thus, endothelial cell-derived clusterin possibly inhibit oxidative stress-induced hypofunction of these glands.

Assessment of oxidative stress and antioxidant property using electron spin resonance (ESR) spectroscopy.

The pathophysiology of hypertension or stroke is associated with an excess of ROS generation in the vascular system, and results in induction of various pathological cascades of cerebrovascular damage. We have demonstrated that electron spin resonance methods using a spin trap or spin probe will be useful for understanding redox status under conditions of oxidative stress in the spontaneously hypertensive rat or stroke-prone spontaneously hypertensive rat brain. We have used electron spin resonance imaging and noninvasive L-band electron spin resonance to characterize the higher degree of brain oxidative stress in the stroke-prone spontaneously hypertensive rat and spontaneously hypertensive rat than in the Wistar-Kyoto rat brain, and the lower extent of oxidative stress in the spontaneously hypertensive rat than in the stroke-prone spontaneously hypertensive rat brain. Indeed, we may be able to confirm propofol medium-chain triglyceride/long-chain triglyceride (MCT/LCT) as neuroprotective anesthesia and crocetin as antioxidant food factor against human stroke after screening for antioxidant properties in stroke models such as stroke-prone spontaneously hypertensive rat. Thus, our electron spin resonance biomedical application suggests that it could be used to assess antioxidant effects on oxidative stress in the brain using spontaneously hypertensive rat and stroke-prone spontaneously hypertensive rat. We hope that further advances in the instrumentation used for electron spin resonance imaging and the development of optimized nontoxic spin probes will make this technology even more promising for novel clinical prediction or noninvasive diagnosis of human stroke. After screening drugs or foods for antioxidant property using in vitro or in vivo electron spin resonance assessment, it will be possible to find and develop novel drugs or food factors with such properties for the prevention of stroke in the near future.

Calcium-calmodulin signaling induced by epithelial cell differentiation upregulates BRAK/CXCL14 expression via the binding of SP1 to the BRAK promoter region.

The chemokine BRAK/CXCL14 (BRAK) is expressed in normal squamous epithelium, but is not expressed or is expressed at negligible levels in head and neck squamous cell carcinoma. Malignant cells are known to be dedifferentiated compared with normal epithelial cells, suggesting a role for differentiation cues in the expression of BRAK. Thus, we examined the relationship between BRAK expression and stages of differentiation level in epithelial cells. Immunohistochemical analysis showed that BRAK protein was expressed in cells above the spinous cell layer in normal epithelia. In HSC-3 cells in culture, expression of BRAK mRNA was significantly upregulated by cell contact in a cell density-dependent manner, and mRNA expression of cell differentiation markers such as involucrin, cystatin-A, TGM1, TGM3, and TGM5 was concomitantly augmented. Furthermore, the upregulation of BRAK induced by cell contact was suppressed by chlorpromazine, a specific inhibitor of calmodulin. We previously reported that GC boxes and a TATA-like sequence in the BRAK promoter region are associated with the expression of BRAK. Using a promoter assay and ChIP, we demonstrated that binding of the stimulating protein-1 (SP1) transcription factor to a GC box upstream of the BRAK transcription start site was necessary for cell density-dependent upregulation of BRAK. These results indicated that upregulation of BRAK was accompanied by differentiation of epithelial cells induced by calcium/calmodulin signaling, and that SP1 binding to the BRAK promoter region played an important role in this signaling.

Fasudil suppresses fibrosarcoma growth by stimulating secretion of the chemokine CXCL14/BRAK.

We previously reported that chemokine CXCL14/BRAK (BRAK) has antitumor activity in several carcinoma cells indicating that BRAK secretion suppresses carcinoma cells. Ras-homologous small GTPase (RhoA) and Rho-associated coiled-coil-containing protein kinase (ROCK) are important regulators of secretory processes, and activation of the RhoA/ROCK signaling pathway stimulates tumor invasion and metastasis. We investigated the effects of fasudil, a specific ROCK inhibitor, on BRAK secretion and tumor progression in mesenchymal fibrosarcoma cells (MC57). We demonstrated the antitumor activity of secreted BRAK using MC57 transplantation of BRAK in overexpressed transgenic mice. Further, to eliminate the influence of change in the mRNA expression of endogenous BRAK, we produced stable MC57 cell lines expressing BRAK (MC57-BRAK) or mock vector (MC57-MOCK). Fasudil significantly increased BRAK secretion by MC57-BRAK cells in a dose-dependent manner. To determine the effect of fasudil on tumor growth, MC57-BRAK and MC57-MOCK cells were transplanted into wild-type mice. Fasudil treatment suppressed tumor growth only in mice that had received MC57-BRAK cell transplants. These results indicate that fasudil inhibits fibrosarcoma growth by stimulating BRAK secretion and suggests that fasudil therapy might have clinical efficacy.

Direct assessments of the antioxidant effects of the novel collagen peptide on reactive oxygen species using electron spin resonance spectroscopy.

In the present study, we evaluated the antioxidant effects of a pepsin-treated novel collagen peptide (P-NCP) on reactive oxygen species (ROS) such as hydroxyl radical (HO(•)), superoxide anion radical (O(2)(•-)), and singlet oxygen ((1)O(2)), and the effects on cell viability after ultraviolet ray (UV) irradiation of human fibroblasts. We confirmed, using electron spin resonance, that P-NCP directly inhibited HO(•) and (1)O(2). Furthermore, addition of P-NCP to fibroblasts inhibited cell death induced by UVA (400-315 nm) irradiation in a dose-dependent manner. In addition, the antioxidant effect on (1)O(2) was observed in the peptide fractions rich in Gly, Pro, Hyp, Glu, Ala, and Arg. We found that Gly, Hyp, Glu, and Ala directly scavenged (1)O(2). These results indicated that a peptide sequence including Gly, Hyp, Glu, and Ala could play a key role in the antioxidant effects of P-NCP on (1)O(2). It was suggested that P-NCP can inhibit photo-aging related to ROS owing to its antioxidant effects.

Direct assessment of the antioxidant properties of midazolam by electron spin resonance spectroscopy.

Some antioxidant anesthetics directly inhibit lipid peroxidation mediated via the generation of reactive oxygen species (ROS). To date, the scavenging effects of midazolam on ROS have not been directly assessed. We investigated the inhibitory effect of midazolam on ROS [hydroxyl radical (HO(·)) and superoxide (O (2) (·-) )] by in vitro X-band electron spin resonance with the spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide. Our results indicated that HO(·) and O (2) (·-) were not affected by midazolam at clinically relevant concentrations, but were directly scavenged by midazolam at high concentrations (i.e., >4.6 and >1.5 mM, respectively).

Association between Antimicrobial Peptide Histatin 5 Levels and Prevalence of Candida in Saliva of Patients with Down Syndrome.

There are no studies on Candida colonization and micropeptides of saliva in any patient. Therefore, we studied the effects of the salivary antimicrobial peptide histatin 5 on oral fungal colonization; subjects were subdivided into Down syndrome (D) and normal (N) groups by age: N-1 and D-1, age <20 years; N-2 and D-2, age >40 years. Histatin 5 concentration in saliva was measured by enzyme-linked immunosorbent assay. Oral Candida species were identified using CHROMagar Candida. Candida colonization was significantly enhanced in the D-1 and D-2 groups compared to the N-1 and N-2 groups. There was no predominant difference in salivary histatin 5 concentration between the D-1 and N-1 groups, but it was significantly lower in the D-2 group than in the N-2 group. Only in the N-2 group was there a correlation between the concentration of histatin 5 and total protein, while no correlation was found in the other groups. In elderly patients with Down syndrome, the decrease in histatin 5 shown in this study may lead to oral Candida colony formation. Therefore, the results of this study suggest that a deficiency of the antimicrobial peptide histatin 5 could possibly induce oral Candida infection in DS.

The effects of bathing in neutral bicarbonate ion water.

Percutaneously absorbed carbon dioxide enhances blood flow. The mechanism by which it does so is unclear, but we hypothesized that it involves bicarbonate ions. BALB/c mice were bathed in neutral bicarbonate ionized water (NBIW) and showed increased blood bicarbonate levels and blood flow via phosphorylation of peripheral vascular endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO). Phosphorylation of eNOS and NO production were also increased in human umbilical vein endothelial cells cultured in medium containing NBIW, and NBIW showed reactive oxygen species scavenging activity. In a double-blind, randomized study in men and women aged 30 to 59 years with subjective cold intolerance, bathing in NBIW elevated body temperature faster than bathing in a control solution and improved chills and sleep quality. Taken together, our results show that percutaneously absorbed carbon dioxide changes to bicarbonate ions, which act directly on endothelial cells to increase NO production by phosphorylation of eNOS and thus improve blood flow.