Synthesis of chalcones and their antimicrobial and drug potentiating activities.

The increased resistance of microorganisms to antimicrobial drugs makes it necessary to search for new active compounds, such as chalcones. Their simple chemical structure makes them molecules easy to synthesize. Therefore, the aim of this study was to evaluate the antimicrobial and potentiating activity of antibiotics and antifungals by synthetic chalcones against strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Candida tropicalis. The synthesis of chalcones was carried out by Claisen-Schimidt aldol condensation. Nuclear Magnetic Resonance (NMR) and Gas Chromatography Coupled to Mass Spectrometry (GC/MS) were also performed. Microbiological tests were performed by the broth microdilution method, using gentamicin, norfloxacin and penicillin as standard drugs for the antibacterial assay, and fluconazole for the antifungal assay. Three chalcones were obtained (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one (DB-Acetone), (1E,3E,6E,8E)-1,9-diphenylnone-1,3,6,8-tetraen-5-one (DB-CNM), (1E,4E)-1,5-bis (4-methoxyphenyl) penta-1,4-dien-3-one (DB-Anisal). The compound DB-Acetone was able to inhibit P. aeruginosa ATCC 9027 at a concentration of 1.4 × 102 µM (32 µg/mL), while DB-CNM and DB-Anisal inhibited the growth of S. aureus ATCC 25923 at 17.88 × 102 µM and 2.71 × 101 µM (512 µg/mL and 8 µg/mL) respectively. In the combined activity, DB-Anisal was able to potentiate the effect of the three antibacterial drugs tested against E. coli 06, norfloxacin (128 for 4 µg/mL ±1) against P. aeruginosa 24 and penicillin (1,024 for 16 µg/mL ±1) against S. aureus 10. In antifungal assays, chalcones were not able to inhibit the growth of fungal strains tested. However, both showed potentiating activity with fluconazole, ranging from 8.17 x 10-1 µM (0.4909 µg/mL) to 2.35 µM (13.96 µg/mL). It is concluded that synthetic chalcones have antimicrobial potential, demonstrating good intrinsic activity against fungi and bacteria, in addition to potentiating the antibiotics and antifungal tested. Further studies are needed addressing the mechanisms of action responsible for the results found in this work.

Production of acetone, butanol, and ethanol by electro-fermentation with Clostridium saccharoperbutylacetonicum N1-4.

This manuscript describes the effect of altering the extracellular redox potential during the production of acetone, butanol, and ethanol on a dual chamber H-type microbial fuel cell by fermenting glucose with Clostridium saccharoperbutylacetonicum N1-4. Extracellular redox potential modification was achieved by either supplementing the microbial broth with the redox agent NADH or by poising the cathode potential at -600 mV vs. Ag/AgCl. The addition of NADH was found to foment the production of acetone via fermentation of glucose. The addition of 200 mM of NADH to the catholyte rendered the highest production of acetone (2.4 g L-1), thus outperforming the production of acetone by conventional fermentation means (control treatment) by a factor of 2.2. The experimental evidence gathered here, indicates that cathodic electro-fermentation of glucose favors the production of butanol. When poising the cathode potential at -600 mV vs Ag/AgCl (electro-fermentation), the largest production of butanol was achieved (5.8 g L-1), outperforming the control treatment by a factor of 1.5. The production of ABE solvents and the electrochemical measurements demonstrate the electroactive properties of C. saccharoperbutylacetonicum N1-4 and illustrates the usefulness of bio-electrochemical systems to improve conventional fermentative processes.

Cleaning and microstructural effects of amyl acetate on pulp chamber dentin impregnated with epoxy resin-based endodontic sealer.

OBJECTIVE: To evaluate the cleaning potential of 95% ethanol, acetone, and amyl acetate solutions used solely or in association, to remove epoxy resin-based sealer residues from pulp chamber dentin and their microstructural effects. MATERIALS AND METHODS: One hundred and eighty bovine incisor specimens were divided into nine groups according to the cleaning protocol: ET (ethanol); AC (acetone); AA (amyl acetate); E1: AA+AC; E2: AA+ET; E3: AC+ET; E4: AA+AC+ET; PC (positive control), and NC (negative control). All groups were impregnated with epoxy resin-sealer, except NC. Ninety specimens were divided into groups (n = 10) for evaluation of persistence of residues and amount of open dentinal tubules by SEM analysis and evaluation of chemical compounds on the dentin surface after cleaning with electron dispersive spectroscopy. The others 90 specimens were submitted to Knoop microhardness evaluation. Persistence of residues data were submitted to the Kruskal Wallis and Dunn tests (α = 0.05). Open dentinal tubules and microhardness data were submitted to one-way ANOVA and Mann Whitney tests (α = 0.05). RESULTS: AA and E4 protocols showed the lowest persistence of residues. E4 group had the highest incidence of open dentinal tubules. E3 and E4 groups showed no changes in the atomic ratio Ca/P, which was similar to NC group. E4 group did not present W, an element presents in all the other groups. ET and E4 protocols showed the smallest reduction in dentin microhardness. CONCLUSIONS: The combination of amyl acetate, acetone and ethanol is the most effective and safe protocol to remove epoxy sealer residues on pulp chamber dentin. Moreover, it has the lowest microhardness reduction. CLINICAL SIGNIFICANCE: The combined use of amyl acetate, acetone, and ethanol enhanced the cleaning of pulp chamber dentin with minimal microstructural damage.

Protective effect of kavain in meristematic cells of Allium cepa L.

Kavain is one of the main kavalactones of Piper methysticum (Piperaceae) with anxiolytic, analgesic, and antioxidant activities. Therefore, the aim of the study was to evaluate the cytotoxic, mutagenic, and antimutagenic potential of kavain in Allium cepa cells. Roots of A. cepa were transferred to the negative (2% acetone) and positive (10 µg/mL of Methylmethanesulfonate, MMS) controls and to the concentrations of kavain (32, 64 and 128 µg/mL) for 48 h. A total of 5,000 meristematic cells were analyzed under an optical microscope to determine the mitotic index, mean number of chromosomal alterations and percentage of damage reduction. Data were analyzed by Kruskal-Wallis test (p <0.05). All concentrations of kavain were not cytotoxic and did not show significant chromosomal changes when compared to 2% acetone. Kavain showed a cytoprotective effect in the pre (128 µg/mL) and in the post-treatment (32 and 64 µg/mL) and reduced damage against the mutagenic action of MMS in all concentrations of the pre and simultaneous and at the highest of post (128 µg/mL). Kavain promoted a significant reduction in micronuclei, nuclear buds and chromosomal losses in relation to MMS. The observed data indicate the importance of kavain for the inhibition of damage and chemoprevention.

Effect of Collagen Matrix Saturation on the Surface Free Energy of Dentin using Different Agents.

INTRODUCTION: The surface free energy of conditioned-dentin is one of the factors that interfere with monomeric infiltration of the interfibrillar spaces. Saturation of the tooth matrix with different substances may modulate this energy and, consequently, the wettability of the dentin. AIM: To evaluate the influence of different substances used to saturate conditioned-dentin on surface free energy (SFE) of this substrate. MATERIALS AND METHODS: Dentin blocks (4 × 7 × 1 mm, n = 6/ group), obtained from the roots of bovine incisors, were etched using phosphoric acid for 15 seconds, rinsed and gently dried. The surfaces were treated for 60 seconds with: ultra-purified water (H20-control); ethanol (EtOH), acetone (ACT), chlorhexidine (CHX), ethylenediaminetetraacetic acid (EDTA); or sodium hypochlorite (NaOCl). The tooth surfaces were once again dried with absorbent paper and prepared for SFE evaluation using three standards: water, formamide and bromonaphthalene. Analysis of variance (ANOVA) and Dunnet's tests (a = 0.05) were applied to the data. RESULTS: Ethylenediaminetetraacetic acid was the only substance that caused a change to the contact angle for the standards water and formamide, while only EtOH influenced the angles formed between formamide and the dentin surface. None of the substances exerted a significant effect for bromonaphtha-lene. In comparison to the control, only EDTA and NaOCl altered both polar components of the SFE. Total SFE was increased by saturation of the collagen matrix by EDTA and reduced when NaOCl was used. CONCLUSION: Saturation of the collagen matrix by EDTA and EtOH changed the surface free energy of the dentin. In addition, the use of NaOCl negatively interfered with the properties evaluated. CLINICAL SIGNIFICANCE: The increase of surface free energy and wettability of the dentin surface would allow higher penetration of the the adhesive system, which would be of importance to the clinical success of resin-dentin union.

Synthesis and antimalarial activity of dihydroperoxides and tetraoxanes conjugated with bis(benzyl)acetone derivatives.

Dihydroperoxides and tetraoxanes derived from symmetrically substituted bis(arylmethyl)acetones were synthesized in modest to good yields using several methods. Three of these compounds exhibit an important in vitro antimalarial activity (1.0 µm ≤ IC(50) ≤ 5.0 µm) against blood forms of the human malaria parasite Plasmodium falciparum.

Changes in host-seeking behavior of Puerto Rican Aedes aegypti after colonization.

The effects of colonization on host-seeking behavior of mosquitoes was examined by comparing attraction responses of newly colonized Aedes aegypti (L.) from field-collected eggs in Puerto Rico to that of the Gainesville (Florida) strain, originally from Orlando (Florida) and in colony since 1952. Females from the Orlando and the F0 through F10 generations of the Puerto Rico strain were evaluated using attractant odors in a triple-cage dual-port olfactometer. Two attractant sources were used: odors from the hand of a volunteer and a standard blend of L-lactic acid, acetone, and dimethyl disulfide. Convergence of the percentage of attraction responses occurred around the F4-F6 generations of the Puerto Rico strain. Both the Orlando and Puerto Rico strains exhibited similar responses for tests with the remaining F7-F10 generations. A temporal effect on mosquito responses was observed for both strains regardless of the attractant blend used in tests. This study indicates that Ae. aegypti host-seeking behavior changes significantly over the first four to six generations after introduction into the laboratory, whereas the field-collected strain increases in attraction response until it stabilizes at a new level.

Antimicrobial efficacy of fruit extracts of two piper species against selected bacterial and oral fungal pathogens

Aim: To assess the antimicrobial efficacy of five solvent extracts of two Piper species commonly used in diet and traditional medicine, P. cubeba and P. longum, against selected bacterial and oral fungal pathogens i.e. Streptococcus mutans, Staphylococcus aureus, Candida albicans and Saccharomyces cerevisiae. Methods: The antimicrobial activity of five extracts of cubeb berries and Indian long pepper fruits was determined by the agar well diffusion method. The minimum inhibitory concentration (MIC) for the acetonic, methanolic and ethanolic extracts was determined by the modified agar well diffusion method. Results: Of the 5 fruit extracts evaluated, acetone, ethanol and methanol extracts of both the Piper spp. were found to have variable antimicrobial activities against all the four oral pathogens. The acetonic fruit extract of P. cubeba was the most effective against both the yeasts with the highest zone of inhibition (15.31 mm) against C. albicans followed by the methanolic (12.31 mm) and ethanolic (11.94 mm) extracts. C. albicans was found to be most sensitive pathogen, which survived up to 6.25 mg/mL in the acetonic extract (MIC = 12.5 mg/mL) followed by the methanolic and ethanolic extracts (MIC = 25 mg/mL). The acetonic, methanolic and ethanolic extracts of P. longum fruits showed almost equal inhibition zones of both yeasts, ranging between 10.64 and 14 mm. C. albicans survived up to 12.5 mg/mL (MIC= 25 mg/mL) while S.cerevisiae survived up to 25 mg/mL (MIC = 50 mg/mL). Conclusions: The crude extracts obtained from the fruits of the two Piper spp. may be used to treat oral fungal species, especially C. albicans, as they produced larger inhibition zones than antifungal drugs often used to treat these pathogens.

Lipid peroxidation and antioxidant system in rats acutely treated with acetone.

Cascades of metabolic changes leading to acetone production are induced in states of energy catabolism such as starvation or the use of a ketogenic diet. The reduced capacity for cell detoxification or the increased generation of free radicals is responsible for the toxic effect of acetone. The objective of the present study was to determine the effects of acute treatment (AT) with acetone on the oxidative and metabolic status of rats. The AT group (n=16) was treated by gavage with a single administration of 7.0 g acetone/kg body weight at a concentration of 25% (m/v). Eight rats were euthanized 6 h later (AT6) and eight 24 h later (AT24). Acetone levels were determined in blood and urine and oxidative parameters were analyzed by determining thiobarbituric acid reactive species (TBARS, indicators of lipid peroxidation) and reduced glutathione (GSH) and vitamin E as antioxidant parameters. Serum glucose, blood cholesterol and triglycerieds and hepatic fat were also determined. The results indicated a significant difference in the hepatic oxidative parameters, serum glucose and in plasma triglycerides between the groups. Thus, we conclude that the administration of acute acetone doses can promote changes in some biochemical parameters and in the hepatic oxidative profile.

Cytokine production from human primary teeth pulp fibroblasts stimulated by different pulpotomy agents.

PURPOSE: After exposing the pulp tissue, cytokines are produced that regulate the pulp inflammatory response. The dental literature, however, lacks information on the participation of primary tooth fibroblasts in this process. The purpose of this study was to verify the participation of human primary tooth pulp fibroblasts in the inflammatory process, evaluate the production of interleukin 1 beta (IL-l beta) and interleukin 8 (IL-8) from these cells. METHODS: Pulpotomy agents were applied as conditioned media on cell cultures in the following groups: (1) negative control; (2) positive control (Lipopolysaccharide -LPS); (3) calcium hydroxide (powder); (4) mineral trioxide aggregate (MTA); (5) adhesive resin; and (6) formocresol. After 24 hours in contact with the cells, the conditioned media were removed, the proteins were extracted from the cells and IL-l beta and IL-8 were quantified by ELISA (Enzyme linked immuno-sorbent assay). RESULTS: Data were analyzed by analysis of variance (P<0.05) and Tukey's test (P<0.05). It was observed that calcium hydroxide has stimulated the production of IL-l beta, without stimulating IL-8. Conversely, the adhesive resin and formocresol stimulated the production of IL-8, and did not stimulate IL-l beta. MTA stimulated both cytokines in an intermediate level when compared to the other materials. CONCLUSION: Primary tooth fibroblasts can respond immunologically, and different pulp capping materials can help in this process.

A quantitative resazurin assay to determinate the viability of Trichomonas vaginalis and the cytotoxicity of organic solvents and surfactant agents.

Trichomonas vaginalis causes trichomonosis, the most common, non-viral sexually transmitted disease. To test anti-Trichomonas agents, usually many with low water solubility, organic solvents and surfactant agents should be used. Therefore, the minimal inhibitory concentration (MIC) of acetone, methanol, ethanol, isopropanol, DMSO, Tween 20, Tween 80, and Triton X-100 was determined against T. vaginalis isolates using the quantitative resazurin method. Our results showed that solvents and surfactant agents can be employed as vehicles to test bioactive compounds at lower concentrations than MIC values and we suggest acetone and DMSO as preferential. Moreover, a new methodology is established to substitute or to complement the counting of viable trophozoites. The amount of resazurin reduced by T. vaginalis can be quantified by fluorescence spectroscopy, making the test a quantitative determination of cell viability. These results contribute for pharmacological investigations of bioactive compounds that need the use of solvents as solubilization vehicles to test anti-Trichomonas activity.

In vitro antibacterial activity of adhesive systems on Streptococcus mutans.

PURPOSE: To evaluate the antibacterial activity of three adhesive systems -- Prime & Bond 2.1 (PB), Clearfil SE Bond (CS) and One Up Bond F (OU) -- on Streptococcus mutans in vitro. MATERIALS AND METHODS: Adherence and agar disk-diffusion tests were performed. For the adherence testing, 40 human enamel specimens (4 mm2) were sterilized and the adhesive sytems were applied (n = 10). The control group did not receive the application of any adhesive system. Specimens were immersed in brain heart infusion broth (BHI) inoculated with S. mutans standardized suspension (10(6) cells/ml) for 48 h at 37 degrees C and 5% CO2. The number of S. mutans cells adhered to each specimen was evaluated by the plating method on BHI agar. For agar disk-diffusion testing, adhesive disks and disks soaked in distilled water (negative control) or 0.2% chlorexidine (positive control) were incubated with S. mutans for 48 h. The diameters of the zones of bacterial inhibition were measured. Adherence data were transformed in logarithms of base 10 (log10). Data were submitted to Kruskal-Wallis and Student-Neuman-Keuls tests at the 5% level of significance. RESULTS: The results of the adherence test showed that One Up Bond F (OU) and Clearfil SE Bond (CS) did not differ significantly from one another, but allowed significantly less adherence than Prime & Bond 2.1 (PB) and control [mean log10 (standard deviation) values: PB 6.10 (0.19); CS primer 4.55 (0.98); OU 4.65 (0.54); control group 6.34 (0.27)]. The disk-diffusion test showed no significant difference between OU (diameter in mm: 3.02 +/- 0.13) and CS (3.0 +/- 0.12), but both were significantly more effective in inhibiting bacterial growth than PB (1.0 +/- 0.10). CONCLUSION: The self-etching systems Clearfil SE Bond and One Up Bond F presented a greater inhibitory effect against S. mutans, also in terms of adherence, than did the conventional system, Prime & Bond 2.1.

Using acetone as solvent to study removal of anthracene in soil inhibits microbial activity and alters nitrogen dynamics.

Acetone is often used as a carrier to contaminate soil with polycyclic aromatic hydrocarbons (PAHs) and then to study the factors that control their removal. Acetone is an organic solvent that might affect soil processes. An alkaline saline (Texcoco soil) and an agricultural soil (Acolman soil) were amended with or without acetone, nitrogen + phosphorus (NP), and contaminated with anthracene at 520 mg/kg soil while emissions of CO2 and N2O and concentrations of NH4+, NO2(-) and NO3(-) were monitored. The CO2 emission rate decreased greater than 10 times in the soils amended with acetone. Emission of N2O decreased 70 times in the Acolman soil amended with acetone and NP and 5 times in the Texcoco soil. The concentration of NH4+ decreased in the unamended Acolman and Texcoco soil but increased when acetone was added in the first and remained constant in the latter. Acetone inhibited the increase in the amount of NO3(-) in the Acolman soil but not in the Texcoco soil. It was found that microbial activity as evidenced by the emission of CO2, nitrification, and production of N2O were inhibited by acetone. The amount of acetone used as solvent should thus be kept to a minimum, but it can be assumed that its effect on soil processes will be temporary, as microorganisms are known to repopulate soil quickly.

Aminoacetone, a putative endogenous source of methylglyoxal, causes oxidative stress and death to insulin-producing RINm5f cells.

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4(+) ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O2(*-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 microM) and Fe(3+) ion (100 microM) addition to the cell cultures had no effect, whereas Cu(2+) (5.0-100 microM) significantly increased cell death. Supplementation of the AA- and Cu(2+)-containing culture medium with antioxidants, such as catalase (5.0 microM), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu(2+) ions (10-50 microM) relative to control cultures. This may imply higher activity of antioxidant enzymes in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) plus Cu(2+) (100 microM) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

Effects of solvents and surfactant agents on the female and larvae of cattle tick Boophilus microplus.

Many natural compounds have low water solubility and need to be dissolved in organic solvents, or surfactant agents must be used before addition into experimental systems. Therefore, it is necessary to determine their toxicity. Experiments were performed aiming to select solvents to be used in the bioassays, searching new acaricide agents from plants. Laboratory tests were carried out on larvae and adults of the cattle tick Boophilus microplus to determine the sensibility of B. microplus female and larvae to different solvents (acetone, methanol, ethanol and 1% dimethyl sulfoxide) and surfactant agents (1% Tween 80 and 5% Triton X-100) using the larval immersion test (LIT) and adult immersion test (AIT). In the AIT, the effect of the treatments on engorged females was assessed by measuring egg production and hatching rate. Acetone was toxic to the adults promoting mortality of 100%. Methanol and ethanol caused 45.3 and 14.2% of mortality, respectively. The other tested substances were not toxic to the engorged females of B. microplus. In the LIT, it was observed that the larvae were more resistant; after 48 h, about 100% of the larvae were alive in all the treatments except with acetone that caused a mortality of 10%.

Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent?

The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked beta-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked alpha-fucose and one or two nonsulfated, 4-linked beta-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.

Purification of xylitol from fermented hemicellulosic hydrolyzate using liquid-liquid extraction and precipitation techniques.

Xylitol was produced by Candida guilliermondii by fermentation of sugarcane bagasse hemicellulosic hydrolysate. Undesirable impurities were extracted from the broth using either ethyl acetate, chloroform or dichloromethane. The best results on clarification of the broth without xylitol loss were obtained with ethyl acetate. When ethanol, acetone or tetrahydrofuran were used for precipitation of impurities, only tetrahydrofuran clarified the fermented broth, but a high xylitol loss (approximately 30%) was observed.

Effect of solvent type on microtensile bond strength of a total-etch one-bottle adhesive system to moist or dry dentin.

This study evaluated the effect of organic solvent (acetone or ethanol) on the microtensile bond strengths (MTBS) of an adhesive system applied to dry and moist dentin. Sixteen extracted human third molars were ground to expose a flat occlusal dentin surface and acid etched for 20 seconds (20% phosphoric acid gel, Gluma Etch 20 Gel, Heraeus/Kulzer). After rinsing the acid etchant, an ethanol-based one-bottle adhesive system was applied to the mesial half of the occlusal dentin surface. An acetone-based, one-bottle adhesive system was applied to the distal half of the ground dentin surface. The teeth were randomly assigned to groups. In Group 1, the etched dentin was thoroughly air dried and an ethanol-based one-bottle adhesive system was applied (Gluma Comfort Bond, Heraeus/Kulzer) (GCB). In Group 2, the etched dentin was thoroughly air dried and an acetone-based one-bottle adhesive system was applied (Gluma One Bond, Heraeus/Kulzer)(GOB). In Group 3, excess moisture was removed after acid etching, leaving a moist dentin surface and a one-bottle ethanol-based adhesive was applied (Gluma Comfort Bond). In Group 4, excess moisture was removed after acid etching, leaving a moist dentin surface and an acetone-based adhesive was applied (Gluma One Bond). A hybrid resin composite (Venus, Heraeus/Kulzer) was applied to the bonded surface in four 1-mm increments and light cured according to manufacturer's directions. The specimens were then sectioned with a slow-speed diamond saw in two perpendicular directions to obtain sticks with a cross-section of 0.5 +/- 0.05 mm2. The microtensile bond strength (MTBS) test was performed with a Bencor device in an Instron machine at a crosshead speed of 0.5 mm/minute. The data were subjected to a two-way ANOVA and Scheffé Post hoc test (p < 0.05). The experimental MTBS measured for dry dentin were Group 1 = 37.0 +/- 10.6 and Group 2 = 34.7 +/- 9.0 in MPa (mean +/- SD); and on moist dentin, Group 3 = 50.7 +/- 11.0 and Group 4 = 38.5 +/- 10.5 in MPa (mean +/- SD). The ethanol based adhesives resulted in higher MTBS than acetone-based adhesive (p < 0.008) and bonding to moist dentin resulted in higher MTBS (p < 0.001). GCB applied on moist dentin resulted in statistically higher bond strengths than the other groups. The highest MTBS were achieved with the use of an ethanol-based adhesive to moist dentin.

Effects of solvents on the early stage stiffening rate of demineralized dentin matrix.

OBJECTIVES: To monitor the stiffening rate of demineralized dentin matrix at the early stages after exposure to different neat solvents. METHODS: Dentin beams approximately 0.8x0.7x8.0 mm were obtained from human third molars. After covering their ends with resin composite, the middle exposed length of 4.0mm (gauge-length) was demineralized in 0.5 M EDTA (pH 7.0) for 7 days. The specimens were gripped by a testing machine, pre-loaded to 10 g and cyclically stressed in tension to 5% strain, for 30 repeated cycles (total 20 min) at 0.6 mm/min while immersed in water (control). Then, water was replaced by either 100% acetone, methanol, ethanol, propanol, HEMA or air and the specimens subjected to the same cyclic protocol. The maximum apparent modulus of elasticity (E(Max)) was calculated for every cycle, plotted as a function of time and subjected to regression analysis. Stiffening rate was calculated as changes in E (min). Regression analysis examined the relationship between E and time for each solvent. Data were analyzed by one-way ANOVA and Student-Newman-Keuls test at alpha=0.05. RESULTS: Regression analysis showed that E increased significantly with time in all water-free solvents (R2=0.8-0.99). Stiffening rate was higher for acetone (0.9 MPa/min) and ethanol (0.8 MPa/min), intermediate for air (0.7 MPa/min), methanol (0.6 MPa/min) and propanol (0.5 MPa/min), lower for HEMA (0.2 MPa/min) and practically none for water (0.07 MPa/min) with p<0.05. CONCLUSIONS: The solvent-induced stiffening rate of demineralized dentin matrix is both time and solvent-dependent. The ability of solvents to promptly stiffen the demineralized dentin matrix may be important in maintaining the resin-infiltrated matrix expanded during the solvent evaporation stage of resin bonding.

Inactivation of yeast inorganic pyrophosphatase by organic solvents.

A number of application for enzymes in organic solvents have been developed in chemical processing, food related conversions and analyses. The only unsolved problem related to nonaqueous enzymology is the notion that enzymes in organic solvent are mostly far less active than in water. Therefore, studies concerning the mechanisms by which enzymes are inactivated by organic solvents would reveal a clear understanding of the structure-function relationship of this phenomenon. Here we analyzed the effects of a series of alcohols (methanol, ethanol, 1-propanol and 2-propanol) and acetone on the activity of yeast inorganic pyrophosphatase. We observed that solvents inactivated the enzyme in a dose-dependent manner. This inactivation is also dependent on the hydrophobicity of the solvent, where the most hydrophobic solvent is also the most effective one. The I50 for inactivation by n-alcohols are 5.9 +/- 4, 2.7 +/- 1 and 2.5 +/- 1 M for methanol, ethanol and 1-propanol, respectively. Inactivation was less effective at 37 degrees C than at 5 degrees C, when the I50 for inactivation by methanol, ethanol and 1-propanol are 4.5 +/- 2, 2.1 +/- 2 and 1.7 +/- 1 M, respectively. Our proposal is that solvent binds to the enzyme structure promoting the inactivation by stabilizing an unfolded structure, and that this binding is through the hydrophobic regions of either the protein or the solvent.