Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Protective action of indole-3-acetic acid on induced hepatocarcinoma in mice

In this study, we report the protective effects of IAA on diethylnitrosamine (DEN)-induced hepatocarcinogenesis. BALB/c mice received daily IAA at 50 (T(50)), 250 (T(250)), and 500 (T(500)) mg K(-1) per body mass by gavage for 15 days. At day 15, animals were administered DEN and sacrificed 4 h later. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed in sera. In addition., hepatomorphologic alterations, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), gene expression of antioxidant enzymes and DNA integrity were evaluated in the liver. IAA administration did not show any alterations in any of the parameters available, except for a reduction of the gene expression for antioxidant enzyrries by 55, 56, 27, and 28% for SOD, CAT, GPx, and GR upon T(500). respectively compared with the control. Several hepatic alterations were observed by DEN exposure. Moreover, IAA administration at 3 doses was shown to provide a total prevention of the active reduction of CAT and GR induced by DEN exposure compared with the control. IAA at T5(00) was shown to give partial protection (87, 71, 57, and 90% for respectively SOD, CAT. GPx. and GR) on the down-regulation of the enzymes induced by DEN and this auxin showed a partial protection (50%) on DEN-induced DNA fragmentation for both parameters when compared to DEN alone. This work showed IAA hepatocarcinogenesis protection for the first time by means of a DEN-protective effect on CAT and GR activity. and by affecting antioxidant gene expression and DNA fragmentation. Copyright (C) 2008 John Wiley & Sons, Ltd.

Influence of Ascorbic Acid and Glutathione Antioxidants on Frozen-Thawed Canine Semen

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 mM and Glu 5 mM. In study II, semen was collected twice and divided into three aliquots; control, AA 50 mu M and AA 250 mu M. Initial sperm motility was significantly higher in sperm diluted with AA 50 mu M; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 mM.

In vitro chemosensitivity of canine mast cell tumors grades II and III to all-trans-retinoic acid (ATRA).

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect the skin and soft tissue of dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. While retinoids are well recognized as promising antitumor agents, there have been only a few reports about retinoids` effect on canine cancers. The aim of this study was to investigate the chemosensitivity of MCT grades II and III to all-trans retinoic acid (ATRA). Immediately after surgical resection, MCT were prepared for primary culture. Samples of MCTs were also fixed in formalin for histopathology and grading according to the classification of Patnaik et al. (Veterinary Pathology 21(5):469-474, 1984). The best results were obtained when neoplastic mast cells were co-cultivated with fibroblasts. Cultured mast cells were, then, treated with concentrations of 10(-4) to 10(-7) M of ATRA, in order to evaluate their chemosensitivity to this retinoid. MTT assay was performed to estimate cell growth and death. The highest level of mast cell chemosensivity was obtained at the dose of 10(-4) M (p < 0,002). MCT of grades II or III were equally susceptible to the treatment with ATRA. Cell death was observed on the first 24 h until 48 h. According to these results, ATRA may be a potential chemotherapeutic agent for the treatment of canine MCT.

Acid-Base Changes in Canine Neonates Following Normal Birth or Dystocia

There are limited data concerning blood gas parameters in neonatal dogs. Knowledge of the normal physiology may facilitate effective therapeutic intervention and potentially reduce neonatal mortality. This study examined acid-base parameters in pups born at normal parturition (n = 27) compared with those born after obstetrical assistance or caesarean operation (n = 13) and those born following oxytocin (OXY) administration for treatment of uterine inertia (n = 11). Pups were subjected to an objective scoring method of neonatal health adapted from use in humans (the Apgar score) at birth and again at 5 and 60 min after birth. Venous blood samples were collected at 5 and 60 min after birth for evaluation of blood gas parameters. At birth, all pups had low Apgar scores and a mixed acidosis. The base excess was lowest for pups delivered after OXY administration. The Apgar score improved for all pups after 5 min of birth and there was an improvement in carbon dioxide tension, base excess and venous blood pH at 1 h, although in all pups a metabolic acidosis persisted. These data provide an important insight into neonatal physiology and the variability of blood gas parameters in pups born at normal and abnormal parturition and provide the basis for clinical decision making following dystocia.

Performance of Petrifilm Aerobic Count plates on enumeration of lactic acid bacteria in fermented milks

This study aimed to compare Petrifilm Aerobic Count (AC) plates and the conventional pour plate methodology using the de Man-Rogosa-Sharpe (MRS) agar for the enumeration of lactic acid bacteria (LAB) in fermented milks (FMs), with different starter cultures added. FM samples (n = 66) were collected and plated on both methodologies, with incubation under anaerobic conditions at 35C for 48 h. The count results were compared by analysis of variance (P <= 0.05) and regression analysis. No differences between the mean counts obtained by both methodologies were observed, even when distinct FMs were compared. Considering all samples, a high correlation level was obtained between Petrifilm AC and MRS agar (r = 0.92), but these indexes were lower in FMs with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (r = 0.90) and Lactobacillus fortis (r = 0.81). Despite some slight interferences, Petrifilm AC has proven to be a convenient methodology on enumerating LAB in FM.

Prevention of toothbrushing abrasion of acid-softened enamel by CO(2) laser irradiation

Objective: The aim of the present study was to evaluate the effect of CO(2) laser irradiation (10.6 mu m) at 0.3 J/cm(2) (0.5 mu s; 226 Hz) on the resistance of softened enamel to toothbrushing abrasion, in vitro. Methods: Sixty human enamel samples were obtained, polished with silicon carbide papers and randomly divided into five groups (n = 12), receiving 5 different surface treatments: laser irradiation (L), fluoride (AmF/NaF gel) application (F), laser prior to fluoride (LF), fluoride prior to laser (FL), non-treated control (C). After surface treatment they were submitted to a 25-day erosive-abrasive cycle in 100 ml sprite light (90 s) and brushed twice daily with an electric toothbrush. Between the demineralization periods samples were immersed in supersaturated mineral solution. At the end of the experiments enamel surface loss was determined using a contact profilometer and morphological analysis was performed using scanning electron microscopy (SEM). For SEM analysis of demineralization pattern, cross-sectional cuts of cycled samples were prepared. The data were statistically analysed by one-way ANOVA model with subsequent pairwise comparison of treatments. Results: Abrasive surface loss was significantly lower in all laser groups compared to both control and fluoride groups (p < 0.0001 in all cases). Amongst the laser groups no significant difference was observed. Softened enamel layer underneath lesions was less pronounced in laser-irradiated samples. Conclusion: Irradiation of dental enamel with a CO(2) laser at 0.3 J/cm(2) (5 mu s, 226 Hz) either alone or in combination with amine fluoride gel significantly decreases toothbrushing abrasion of softened-enamel, in vitro. (C) 2011 Elsevier Ltd. All rights reserved.

Influence of Final Rinse Technique on Ability of Ethylenediaminetetraacetic Acid of Removing Smear Layer

Introduction: There is ongoing debate regarding the ideal sequence, volume, and concentration of irrigants, length of time for irrigation, and irrigation technique to achieve debridement of the root canal system. The aim of this study was to verify the impact of the final rinse technique on smear layer removal ability of 17% ethylenediaminetetraacetic acid (EDTA). Methods: Sixteen single-rooted human teeth were instrumented and divided into 2 groups at the final rinse step according to the following final rinse techniques used: continuous rinse group, continuous rinse with EDTA during 3 minutes, and rinse and soaking group, rinse with 1 mL of EDTA, soaking of the canal for 2 minutes and 30 seconds, and rinse completion with the remaining 4 mL for 30 seconds. The specimens were split lengthwise and observed under scanning electron microscope. Results: Data were analyzed with Kruskal-Wallis and Dunn tests. The continuous rinse group presented more debris-free surfaces when compared with the rinse and soaking group (P <. 01). When the root canal areas were compared within the groups, no statistical differences were found (P > .05). Conclusions: It can be concluded that a continuous rinse with 5 mL of EDTA for 3 minutes can more efficiently remove the smear layer from root canal walls. (J Endod 2010;36:512-514)

Fluoride uptake and acid resistance of enamel irradiated with Er : YAG laser

This study evaluated the resistance to demineralization and fluoride incorporation of enamel irradiated with Er:YAG. A total of 110 bovine teeth were selected and divided into eight groups: unlased, 37% phosphoric acid, and samples irradiated with the Er:YAG laser at several fluences (31.84 J/cm(2), 25.47 J/cm(2), 19.10 J/cm(2), 2.08 J/cm(2), 1.8 J/cm(2), and 0.9 J/cm(2)). The application of acidulated phosphate fluoride was performed after treatments. All samples were immersed in 2 ml of 2.0 M acetic-acetate acid solution at pH 4.5 for 8 h, and fluoride, calcium, and phosphorus ions dissolved were analyzed by atomic absorption spectrometry and spectrophotometry. The phosphoric acid and 31.84 J/cm(2) groups presented the lowest dissolution of calcium and phosphorus ions. Higher fluoride incorporation was observed on 1.8 J/cm(2) and 0.9 J/cm(2) groups. Based on these results, Er:YAG laser was able to decrease acid dissolution and increase fluoride uptake and can be a promissory alternative for preventive dentistry.

Rehardening of acid-softened enamel and prevention of enamel softening through CO(2) laser irradiation

Objectives: The aims of the present study were to investigate whether irradiation with a CO(2) laser could prevent surface softening (i) in sound and (ii) in already softened enamel in vitro. Methods: 130 human enamel samples were obtained and polished with silicon carbide papers. They were divided into 10 groups (n = 13) receiving 5 different surface treatments: laser irradiation (L), fluoride (AmF/NaF gel) application (F), laser prior to fluoride (LF), fluoride prior to laser (FL), non-treated control (C); and submitted to 2 different procedures: half of the groups was acid-softened before surface treatment and the other half after. Immersion in 1% citric acid was the acid challenge. Surface microhardness (SMH) was measured at baseline, after softening and after treatment. Additionally, fluoride uptake in the enamel was quantified. The data were statistically analysed by two-way repeated measurements ANOVA and post hoc comparisons at 5% significance level. Results: When softening was performed either before or after laser treatment, the L group presented at the end of the experiments SMH means that were not significantly different from baseline (p = 0.8432, p = 0.4620). Treatment after softening resulted for all laser groups in statistically significant increase in SMH means as compared to values after softening (p < 0.0001). Enamel fluoride uptake was significantly higher for combined laser-fluoride treatment than in control (p < 0.0001). Conclusion: Irradiation of dental enamel with a CO(2) laser at 0.3J/cm(2) (5 mu s, 226 Hz) not only significantly decreased erosive mineral loss (97%) but also rehardened previously softened enamel in vitro. (C) 2011 Elsevier Ltd. All rights reserved.

Expression of Homeobox Genes in Oral Squamous Cell Carcinoma Cell Lines Treated With All-Trans Retinoic Acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All-trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA-mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA-sensitive SCC-25 cells compared to atRA-resistant SCC-9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC-25 cells but not in SCC-9 cells. Gene expression levels were confirmed for seven of these genes by RT-qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC-25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA-dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on clay 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437-1444, 2010. (C) 2010 Wiley-Liss, Inc.

Alpha lipoic acid in burning mouth syndrome - a randomized double-blind placebo-controlled trial

The burning mouth syndrome (BMS) is a chronic condition characterized by oral burning pain in the absence of clinical abnormalities and without established therapy. The purpose of this study was to evaluate the effectiveness of alpha lipoic acid (ALA) in the management of BMS symptoms through a randomized double-blind placebo-controlled trial. Thirty-eight patients (34 women and four men, median age 62.9 years, range 36-78) were included and 31 completed the study. The patients were randomized into two cycles of treatment: one with alpha lipoic acid and one with placebo both administered in identical capsules. These cycles were separated by a washout period of 20 days. The oral symptoms and the treatment response were assessed using a 100-mm visual analog scale before and after each cycle and the global perceived effect score, using a 5-point scale after each treatment cycle. The level of reduction on burning was significant for both treatments (paired t-test: P < 0.05; rp = 0.011; ral < 0.001). Considering the two cycles together, 22 patients reported at least some improvement after ALA use and 23 patients after placebo. Comparison of the oral assessment scores of the two cycles failed to demonstrate the effectiveness of ALA over placebo (t-test: P > 0.05; r = 0.75).

Acid phosphatase and cathepsin D are active expressed enzymes in the placenta of the cat

Enzymes are crucial for the metabolism of macromolecular substrates. In the great majority of cells, most enzymes are constitutive. Nevertheless, inducible enzymes can predominate, determining specialized cell functions. Within this context, histochemistry/immunohistochemistry and biochemistry were used to investigate expression of peroxidase and reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-oxidase, as well as the expression and activity of cathepsin D and acid phosphatase, in trophoblast cells within the endotheliochorial labyrinth and marginal hematoma of the term cat placenta. In the marginal hematoma, elevated Cathepsin D expression and activity was accompanied by erythrophagocytosis. In contrast, acid phosphatase activity was much more intense in the labyrinth, where metabolic exchanges occur. Peroxidase and NAD(P)H-oxidase were predominantly active in trophoblast cells within endosomal vesicles of different placental compartments, indicating that, although reactive oxygen species might participate in endosomal/lysosomal processes, they are not territorially specific or functional markers. These findings highlight differential characteristics of cathepsin D and acid phosphatase activity within each placental compartment, thereby contributing to the comprehension of the territorial role played by the placenta and facilitating future metabolic studies. (C) 2007 Elsevier Ltd. All rights reserved.

Six-month evaluation of adhesives interface created by a hydrophobic adhesive to acid-etched ethanol-wet bonded dentine with simplified dehydration protocols

Objectives: To evaluate the efficacy of simplified dehydration protocols, in the absence of tubular occlusion, on bond strength and interfacial nanoleakage of a hydrophobic experimental adhesive blend to acid-etched, ethanol-dehydrated dentine immediately and after 6 months. Methods: Molars were randomly assigned to 6 treatment groups (n = 5). Under pulpal pressure simulation, dentine crowns were acid-etched with 35% H(3)PO(4) and rinsed with water. Adper Scotchbond Multi-Purpose was used for the control group. The remaining groups had their dentine surface dehydrated with ethanol solutions: group 1 = 50%, 70%, 80%, 95% and 3 x 100%, 30 s for each application; group 2 the same ethanol sequence with 15 s for each solution; groups 3, 4 and 5 used 100% ethanol only, applied in seven, three or one 30 s step, respectively. After dehydration, a primer (50% BisGMA + TEGDMA, 50% ethanol) was used, followed by the neat comonomer adhesive application. Resin composite build-ups were then prepared using an incremental technique. Specimens were stored for 24 h, sectioned into beams and stressed to failure after 24 h or after 6 months of artificial ageing. Interfacial silver leakage evaluation was performed for both storage periods (n = 5 per subgroup). Results: Group 1 showed higher bond strengths at 24 h or after 6 months of ageing (45.6 +/- 5.9(a)/43.1 +/- 3.2(a) MPa) and lower silver impregnation. Bond strength results were statistically similar to control group (41.2 +/- 3.3(ab)/38.3 +/- 4.0(ab) MPa), group 2 (40.0 +/- 3.1(ab)/38.6 +/- 3.2(ab) MPa), and group 3 at 24 h (35.5 +/- 4.3(ab) MPa). Groups 4 (34.6 +/- 5.7(bc)/25.9 +/- 4.1(c) MPa) and 5 (24.7 +/- 4.9(c)/18.2 +/- 4.2(c) MPa) resulted in lower bond strengths, extensive interfacial nanoleakage and more prominent reductions (up to 25%) in bond strengths after 6 months of ageing. Conclusions: Simplified dehydration protocols using one or three 100% ethanol applications should be avoided for the ethanol-wet bonding technique in the absence of tubular occlusion, as they showed decreased bond strength, more severe nanoleakage and reduced bond stability over time. (C) 2009 Elsevier Ltd. All rights reserved.