Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH 3) and hydrogen peroxide (H 2O 2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode. © 2012 Elsevier Inc.

Occurrence of sensitivity during at-home and in-office tooth bleaching therapies with or without use of light sources

The aim of this study was to evaluate the effect of tooth bleaching with 10% carbamide peroxide (CP) or 35% hydrogen peroxide (HP), with or without quartz-tungsten-halogen light or hybrid source LED/infrared laser exposition on the occurrence duration, intensity and location of tooth sensitivity Forty patients were selected and randomly divided into four groups: GI--home bleaching with CP for 4 hours a day, over the course of 3 weeks; GII--three sessions of HP with three 10-minute applications at each session and no light source; GIII--the same procedure as GII with quartz-tungsten-halogen light irradiation; GIV--the same procedure as GII with LED/laser light irradiation. The evaluation included an appointment with each patient before and after each HP bleaching session or each weekly CP bleaching and 7, 30 and 180 days after the end of treatment. The Kruskal-Wallis test revealed that the duration and intensity of post-treatment sensitivity were significantly higher for HP than for CP (p< 0.05), and symptoms were located predominantly in anterior teeth. All bleaching methods generated sensitivity, which was more frequent in anterior teeth. However, treatment with CP generated lower sensitivity than treatment with HP independently of the light sources.

Bleaching gels containing calcium and fluoride: Effect on enamel erosion susceptibility

This in vitro study evaluated the effect of 35 hydrogen peroxide (HP) bleaching gel modified or not by the addition of calcium and fluoride on enamel susceptibility to erosion. Bovine enamel samples (3 mm in diameter) were divided into four groups (n = 15) according to the bleaching agent: control-without bleaching (C); 35 hydrogen peroxide (HP); 35 HP with the addition of 2 calcium gluconate (HP + Ca); 35 HP with the addition of 0.6 sodium fluoride (HP + F). The bleaching gels were applied on the enamel surface for 40 min, and the specimens were subjected to erosive challenge with Sprite Zero and remineralization with artificial saliva for 5 days. Enamel wear was assessed using profilometry. The data were analyzed by ANOVA/ Tukey's test (P 0.05). There were significant differences among the groups (P = 0.009). The most enamel wear was seen for C (3.37 ± 0.80 μm), followed by HP (2.89 ± 0.98 μm) and HP + F (2.72 ± 0.64 μm). HP + Ca (2.31 ± 0.92 μm) was the only group able to significantly reduce enamel erosion compared to C. The application of HP bleaching agent did not increase the enamel susceptibility to erosion. However, the addition of calcium gluconate to the HP gel resulted in reduced susceptibility of the enamel to erosion. © 2012 Alessandra B. Borges et al.

Efficacy and cytotoxicity of a bleaching gel after short application times on dental enamel

Objectives: This study aimed to evaluate and correlate the efficacy and cytotoxicity of a 35 % hydrogen peroxide (HP) bleaching gel after different application times on dental enamel. Materials and methods: Enamel/dentin disks in artificial pulp chambers were placed in wells containing culture medium. The following groups were formed: G1, control (no bleaching); G2 and G3, three or one 15-min bleaching applications, respectively; and G4 and G5, three or one 5-min bleaching applications, respectively. Extracts (culture medium with bleaching gel components) were applied for 60 min on cultured odontoblast-like MDPC-23 cells. Cell metabolism (methyl tetrazolium assay) (Kruskal-Wallis/Mann-Whitney; α = 5 %) and cell morphology (scanning electron microscopy) were analyzed immediately after the bleaching procedures and the trans-enamel and trans-dentinal HP diffusion quantified (one-way analysis of variance/Tukey's test; α = 5 %). The alkaline phosphatase (ALP) activity was evaluated 24 h after the contact time of the extracts with the cells (Kruskal-Wallis/Mann-Whitney; α = 5 %). Tooth color was analyzed before and 24 h after bleaching using a spectrophotometer according to the Commission Internationale de l'Eclairage L*a*b* system (Kruskal-Wallis/Mann-Whitney; α = 0.05). Results: Significant difference (p < 0.05) in cell metabolism occurred only between G1 (control, 100 %) and G2 (60.6 %). A significant decrease (p < 0.05) in ALP activity was observed between G2, G3, and G4 in comparison with G1. Alterations on cell morphology were observed in all bleached groups. The highest values of HP diffusion and color alterations were observed for G2, with significant difference among all experimental groups (p < 0.05). G3 and G4 presented intermediate color change and HP diffusion values with no statistically significant differences between them (p > 0.05). The lowest amount of HP diffusion was observed in G5 (p < 0.05), which also exhibited no significant color alteration compared to the control group (p > 0.05). Conclusions: HP diffusion through dental tissues and its cytotoxic effects were proportional to the contact time of the bleaching gel with enamel. However, shorter bleaching times reduced bleaching efficacy. Clinical relevance: Shortening the in-office tooth bleaching time could be an alternative to minimize the cytotoxic effects of this clinical procedure to pulp tissue. However, the reduced time of bleaching agent application on enamel may not provide adequate esthetic outcome. © 2012 Springer-Verlag Berlin Heidelberg.

Ultrasonic activation of internal bleaching agents

Aim: To evaluate the effectiveness of ultrasonic activation of bleaching agents during ex vivo internal bleaching. Methodology: Fifty canine human teeth were artificially stained, root filled and divided into five groups (n = 10) that received SP - sodium perborate plus deionized water (control group), CP - 37% carbamide peroxide gel, CPUS - 37% carbamide peroxide gel plus ultrasonic application, HP - 35% hydrogen peroxide gel or HPUS - 35% hydrogen peroxide gel plus ultrasonic application. In groups CP and HP, the bleaching agent was left inside the pulp chamber for three applications of 10 min. In groups CPUS and HPUS, the same process was performed, but ultrasonic vibration was applied to the bleaching agent by an alloy tip for 30 s, with 30 s intervals. Two sessions were performed. The colour was measured initially and after each session by an intraoral dental spectrophotometer. The variation (Δ) of the colour parameters based on the CIELab system L*, a* and b*, and the colour alteration ΔE* were calculated after first and second section. Data were analysed by one-way anova and Tukey's test. Results: There was no significant difference amongst groups for ΔL*, Δa* and ΔE*, but there was a significant difference for Δb* in the first and second sessions (P = 0.0006 and 0.0016, respectively). After the first session, Δb* was significantly greater for groups HP and HPUS, without a significant difference between them. For the second session, group HPUS had the greatest Δb* values, but they were similar to groups HP and SP; group CP had the lowest values, which were similar to groups CPUS and SP. Conclusion: Ultrasonic activation of bleaching agents during ex vivo internal bleaching was no more effective than conventional internal bleaching procedures, without activation. © 2012 International Endodontic Journal.

Efficacy of mouth rinses and toothpaste on tooth whitening.

People increasingly desire tooth whitening. Considering the wide range of whitening products on the market, this study evaluated the efficacy of whitening toothpastes and mouth rinses compared with the 10% carbamide peroxide (CP) whitening gel. We obtained 120 cylindrical specimens from bovine teeth, which were darkened for 24 hours in a coffee solution. The color measurement was performed by a spectrophotometer using the CIE L*a*b* system, and specimens were divided into six groups according to the use of the following agents: group 1, conventional fluoridated toothpaste; group 2, Close Up White Now; group 3, Listerine Whitening; group 4, Colgate Plax Whitening; group 5, experimental mouth rinse with Plasdone; and group 6, 10% CP Whiteness Perfect. After the simulation of 12 weeks of treatment for groups 1 to 5 and 14 days of treatment for group 6, the specimens were subjected to a new color reading. Data were subjected to one-way analysis of variance (α=0.05), which showed significant differences among groups after 12 weeks for ΔE (p=0.001). Results of the Tukey test revealed that groups 3, 4, and 6 presented significantly higher color alteration than groups 1, 2, and 5. The whitening toothpaste Close Up White Now and the experimental mouth rinse with Plasdone showed similar color alteration as conventional toothpaste after a 12-week treatment simulation. These groups presented significantly lower color alteration compared with whitening mouth rinses Listerine and Colgate Plax Whitening, which showed similar results to those observed after 14 days of bleaching with 10% CP treatment.

Dentin hypersensitivity after teeth bleaching with in-office systems. randomized clinical trial

Purpose: To comparatively and prospectively compare in a randomized clinical trial, dentin hypersensitivity after treatment with three in-office bleaching systems, based on hydrogen peroxide at different concentrations, with and without light source activation. Methods: 88 individuals were included according to inclusion and exclusion criteria. Subjects were randomly divided into the following three treatment groups: Group 1 was treated with three 15-minute applications of hydrogen peroxide at 15% with titanium dioxide (Lase Peroxide Lite) that was light-activated (Light Plus Whitening Lase) with five cycles of 1 minute and 30 seconds each cycle, giving a total treatment time of 45 minutes; Group 2 was treated with three 10-minute applications of hydrogen peroxide at 35% (Lase Peroxide Sensy), activated by light (LPWL) same activation cycles than Group 1, with a total treatment time of 30 minutes; Group 3 was treated with only one application for 45 minutes of hydrogen peroxide at 35% (Whitegold Office) without light activation. Each subject underwent one session of bleaching on the anterior teeth according to the manufacturers' instructions. Dentin sensitivity was recorded with a visual analogue scale (VAS) at baseline, immediately after, and at 7 and 30 days after treatment using a stimulus of an evaporative blowing triple syringe for 3 seconds on the upper central incisors from a distance of 1 cm. A Kruskal-Wallis test followed by Mann-Whitney test was performed for statistical analysis. Results: All groups showed increased sensitivity immediately after treatment. Group 1 displayed less changes relative to baseline with no significant differences (P= 0.104). At 7 and 30 days after treatment, a comparison of VAS values indicated no significant differences between all groups (P= 0.598 and 0.489, respectively).

Recovery of mutans streptococci on MSB, SB-20 and SB-20M agar media

Objective: The recovery of mutans streptococci in saliva and dental biofilm samples depends, in part, on the culture medium used. In this study, we compared (i) the culture media Sucrose-Bacitracin agar (SB-20), Modified SB-20 (SB-20M) and Mitis Salivarius Bacitracin agar (MSB) in the count of colony forming units (cfu) of mutans streptococci and (ii) in the morphological and biochemical differentiation between Streptococcus mutans and Streptococcus sobrinus. Design: Samples of non-stimulated saliva from 20 children were plated on SB-20, SB-20M and MSB, and incubated in microaerophilia at 37 °C for 72 h. Identification of microorganisms was based on analysis of colony morphology under stereomicroscopy. The biochemical identification of colonies was done by biochemical tests using sugar fermentation, resistance to bacitracin and hydrogen peroxide production. Results: There was no significant difference (p > 0.05) in the number of cfu of mutans streptococci recovered on SB-20 and SB-20M agar. Comparing the media, SB-20 and SB-20M yielded a larger number of mutans streptococci colonies (p < 0.05) and were more effective than MSB in the identification of S. sobrinus (p < 0.05), but not of S. mutans (p > 0.05). Conclusion: There was no significant difference between SB-20 and SB-20M culture media in the count of mutans streptococci, demonstrating that the replacement of sucrose by coarse granular cane sugar did not alter the efficacy of the medium. Compared with MSB, SB-20 and SB-20M allowed counting a larger number of mutans streptococci colonies and a more effective morphological identification of S. sobrinus. © 2012 Elsevier Ltd.

Assessment of the chemopreventive effect of casearin B, a clerodane diterpene extracted from Casearia sylvestris (Salicaceae)

Studies have shown that Casearia sylvestris compounds protect DNA from damage both in vitro and in vivo. Complementarily, the aim of the present study was to assess the chemopreventive effect of casearin B (CASB) against DNA damage using the Ames test, the comet assay and the DCFDA antioxidant assay. The genotoxicity was assessed by the comet assay in HepG2 cells. CASB was genotoxic at concentrations higher than 0.30μM when incubated with the FPG (formamidopyrimidine-DNA glycosylase) enzyme. For the antigenotoxicity comet assay, CASB protected the DNA from damage caused by H2O2 in the HepG2 cell line in concentrations above 0.04μM with post-treatment, and above 0.08μM with pre-treatment. CASB was not mutagenic (Ames test) in TA 98 and TA 102. In the antimutagenicity assays, the compound was a strong inhibitor against aflatoxin B1 (AFB) in TA 98 (>88.8%), whereas it was moderate (42.7-59.4%) inhibitor against mytomicin C (MMC) in TA 102. Additionally, in the antioxidant assay using DCFDA, CASB reduced reactive oxygen species (ROS) generated by H2O2. In conclusion, CASB was genotoxic to HepG2 cells at high concentrations; was protective of DNA at low concentrations, as shown by the Ames test and comet assay; and was also antioxidant. © 2012 Elsevier Ltd.

Apocynin: Chemical and biophysical properties of a NADPH oxidase inhibitor

Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (ε275nm = 1.1 × 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H 2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M -1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

Silibinin attenuates oxidative metabolism and cytokine production by monocytes from preeclamptic women.

Silibinin is a polyphenolic plant flavonoid with anti-inflammatory properties. The present study investigated the effect of silibinin on oxidative metabolism and cytokine production - tumor necrosis factor-alpha (TNF-α), interleukin (IL)12, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-10, and transforming growth factor beta (TGF-β1) - by peripheral blood monocytes (PBM) from preeclamptic pregnant women. It is a case-controlled study involving women with preeclampsia (PE, n = 30) compared with normotensive pregnant (NT, n = 30) and with non-pregnant (NP, n = 30) women. Monocytes were obtained and cultured with or without silibinin (5 μM or 50 μM) for 18 h. Superoxide anion (O2-) and hydrogen peroxide (H2O2) release were determined by specific assays, and cytokine levels were determined by immunoenzymatic assays (ELISA). Monocytes from preeclamptic women cultured without stimulus released higher levels of O22, H2O2 and TNF-α, and lower levels of IL-10 and TGF-β1 than did monocytes from NT and NP women. Treatment in vitro with silibinin significantly inhibited spontaneous O2- and H2O2 release and TNF-α production by monocytes from preeclamptic women. The main effect of silibinin was obtained at 50 μM concentration. Thus, silibinin exerts anti-oxidative and anti-inflammatory effects on monocytes from preeclamptic pregnant women by inhibiting the in vitro endogenous release of reactive oxygen species and TNF-α production.

Effects of LED-laser hybrid light on bleaching effectiveness and tooth sensitivity: A randomized clinical study

The study evaluated the effectiveness and the sensitivity of in-office tooth bleaching with the use of a hybrid photo-activation system composed by LEDs and lasers. 40 patients, both genders, aged 18 through 25 years, were randomly distributed into two treatment groups: group I, 35% hydrogen peroxide, with a total bleaching time of 135 min divided into three sessions, and group II, 35% hydrogen peroxide and photo-thermal catalysis by an LED-laser system (300 mW cm-2), for a total bleaching time of 72 min divided into three sessions. The treatment efficiency was measured by reflectance spectroscopy and sensitivity by a visual analog scale (VAS). The final luminosity value (ΔL), color variation (ΔE) and sensitivity (S) resulting from the treatments were analyzed by the generalized estimating equations method (GEEs), and Bonferroni post hoc multiple comparisons at 5% significance. The two groups presented similar colors (ΔE) and luminosities (ΔL) after treatment. Group I presented a greater sensitivity index (37.6 ± 5.9%) compared to group II (11.1 ± 3.3%), statistically significant at p < 0.05. The use of LED-laser hybrid light, as a catalyst of the bleaching agents, showed a significant decrease of provoked tooth sensitivity and a treatment time reduced by 53%, with the same aesthetic results as without a light source. © 2013 Astro Ltd.