Effect of power densities and irradiation times on the degree of conversion and temperature increase of a microhybrid dental composite resin

The different parameters used for the photoactivation process provide changes in the degree of conversion (DC%) and temperature rise (TR) of the composite resins. Thus, the purpose of this study was to evaluate the DC (%) and TR of the microhybrid composite resin photoactivated by a new generation LED. For the KBr pellet technique, the composite resin was placed into a metallic mould (1-mm thickness and 4-mm diameter) and photoactivated as follows: continuous LED LCU with different power density values (50-1000 mW/cm(2)). The measurements for the DC (%) were made in a FTIR Spectrometer Bomen (model MB-102, Quebec-Canada). The spectroscopy (FTIR) spectra for both uncured and cured samples were analyzed using an accessory for the diffuse reflectance. The measurements were recorded in the absorbance operating under the following conditions: 32 scans, 4-cm(-1) resolution, and a 300 to 4000-cm(-1) wavelength. The percentage of unreacted carbon-carbon double bonds (% C=C) was determined from the ratio of the absorbance intensities of aliphatic C=C (peak at 1638 cm(-1)) against an internal standard before and after the curing of the specimen: aromatic C-C (peak at 1608 cm-1). For the TR, the samples were made in a metallic mould (2-mm thickness and 4-mm diameter) and photoactivated during 5, 10, and 20 s. The thermocouple was attached to the multimeter to allow the temperature readings. The DC (%) and TR were calculated by the standard technique and submitted to ANOVA and Tukey`s test (p < 0.05). The degree of conversion values varied from 35.0 (+/- 1.3) to 45.0 (+/- 2.4) for 5 s, 45.0 (+/- 1.3) to 55.0 (+/- 2.4) for 10 s, and 47.0 (+/- 1.3) to 52.0 (+/- 2.4) for 20 s. For the TR, the values ranged from 0.3 (+/- 0.01) to 5.4 (+/- 0.11)degrees C for 5 s, from 0.5 (+/- 0.02) to 9.3 (+/- 0.28)degrees C for 10 s, and from 1.0 (+/- 0.06) to 15.0 (+/- 0.95)degrees C for 20 s. The power densities and irradiation times showed a significant effect on the degree of conversion and temperature rise.

Enzyme immobilisation on electroactive nanostructured membranes (ENM): Optimised architectures for biosensing

Electroactive nanostructured membranes have been produced by the layer-by-layer (LbL) technique, and used to make electrochemical enzyme biosensors for glucose by modification with cobalt hexacyanoferrate redox mediator and immobilisation of glucose oxidase enzyme. Indium tin oxide (ITO) glass electrodes were modified with up to three bilayers of polyamidoamine (PAMAM) dendrimers containing gold nanoparticles and poly(vinylsulfonate) (PVS). The gold nanoparticles were covered with cobalt hexacyanoferrate that functioned as a redox mediator, allowing the modified electrode to be used to detect H(2)O(2), the product of the oxidase enzymatic reaction, at 0.0 V vs. SCE. Enzyme was then immobilised by cross-linking with glutaraldehyde. Several parameters for optimisation of the glucose biosensor were investigated, including the number of deposited bilayers, the enzyme immobilisation protocol and the concentrations of immobilised enzyme and of the protein that was crosslinked with PAMAM. The latter was used to provide glucose oxidase with a friendly environment, in order to preserve its bioactivity. The optimised biosensor, with three bilayers, has high sensitivity and operational stability, with a detection limit of 6.1 mu M and an apparent Michaelis-Menten constant of 0.20 mM. It showed good selectivity against interferents and is suitable for glucose measurements in natural samples. (C) 2008 Elsevier B.V. All rights reserved.

Continuous and gradual photo-activation methods: influence on degree of conversion and crosslink density of composite resins

Thermal properties and degree of conversion (DC%) of two composite resins (microhybrid and nanocomposite) and two photo-activation methods (continuous and gradual) displayed by the light-emitting diode (LED) light-curing units (LCUs) were investigated in this study. Differential scanning calorimetry (DSC) thermal analysis technique was used to investigate the glass transition temperature (T(g)) and degradation temperature. The DC% was determined by Fourier transform infrared spectroscopy (FT-IR). The results showed that the microhybrid composite resin presented the highest T(g) and degradation temperature values, i.e., the best thermal stability. Gradual photo-activation methods showed higher or similar T(g) and degradation temperature values when compared to continuous method. The Elipar Freelight 2 (TM) LCU showed the lowest T(g) values. With respect to the DC%, the photo-activation method did not influence the final conversion of composite resins. However, Elipar Freelight 2 (TM) LCU and microhybrid resin showed the lowest DC% values. Thus, the presented results suggest that gradual method photo-activation with LED LCUs provides adequate degree of conversion without promoting changes in the polymer chain of composite resins. However, the thermal properties and final conversion of composite resins can be influenced by the kind of composite resin and LCU.

Influence of light guide tip used in the photo-activation on degree of conversion and hardness of one nanofilled dental composite

The aim of this study was to evaluate the degree of conversion and hardness of a dental composite resin Filtek (TM) Z-350 (3M ESPE, Dental Products St. Paul, MN) photo-activated for 20 s of irradiation time with two different light guide tips, metal and polymer, coupled on blue LED Ultraled LCU (Dabi Atlante, SP, Brazil). With the metal light tip, power density was of 352 and with the polymer was of 456 mW/cm(2), respectively. Five samples (4 mm in diameter and 2mm in thickness-ISO 4049), were made for each Group evaluated. The measurements for DC (%) were made in a Nexus-470 FT-IR, Thermo Nicolet, E.U.A. Spectroscopy (FTIR). Spectra for both uncured and cured samples were analyzed using an accessory of reflectance diffuse. The measurements were recorded in absorbance operating under the following conditions: 32 scans, 4 cm(-1) resolution, 300-4000 cm(-1) wavelength. The percentage of unreacted carbon double bonds (% C=C) was determined from the ratio of absorbance intensities of aliphatic C=C (peak at 1637 cm(-1)) against internal standard before and after curing of the sample: aromatic C-C (peak at 1610 cm(-1)). The Vickers hardness measurements (top and bottom surfaces) were performed in a universal testing machine (Buehler MMT-3 digital microhardness tester Lake Bluff, Illinois USA). A 50 gf load was used and the indenter with a dwell time of 30 s. The data were submitted to the test t Student at significance level of 5%. The mean values of degree of conversion for the polymer and metal light guide tip no were statistically different (p = 0.8389). The hardness mean values were no statistically significant different among the light guide tips (p = 0.6244), however, there was difference between top and bottom surfaces (p < 0.001). The results show that so much the polymer light tip as the metal light tip can be used for the photo-activation, probably for the low quality of the light guide tip metal.

Enzyme Activity of Catalase Immobilized in Langmuir-Blodgett Films of Phospholipids

A major challenge for producing low cost biosensors based on nanostructured films with control of molecular architectures is to preserve the catalytic activity of the immobilized biomolecules. In this study, we show that catalase (HRP) keeps its activity if immobilized in Langmuir-Blodgett (LB) films of dipalmitoyl phosphatidylglycerol (DPPG). The incorporation of catalase into a DPPG monolayer at the at interface was demonstrated with surface pressure and surface potential isotherms, in addition to polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). According to the PM-IRRAS data. catalase was not denatured upon adsorption on a preformed DPPG monolayer and could be transferred onto a solid substrate. The catalytic activity of catalase in a mixed LB film with DPPG was ca. 13% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allows catalase-containing LB films to be used in sensing hydrogen peroxide.

Influence from the sulfonate group (RSO(3)(-)) on the conversion process and emission efficiency of poly(p-phenylene vinylene)

The discovery of an alternative route to convert poly(xylyliden tetrahydrothiophenium chloride) (PTHT) into poly(p-phenylene vinylene) (PPV) using dodecylbenzenesulfonate (DBS) has allowed the formation of ultrathin films with unprecedented control of architecture and emission properties. In this work, we show that this route may be performed with several sufonated compounds where RSO(3)(-) replaces the counter-ion (Cl(-)) of PTHT, some of which are even more efficient than DBS. Spin-coating films were produced from PTHT and azo-dye molecules, an azo-polymer and organic salts as counter-ions of PTHT. The effects of the thermal annealing step of PTHT/RSO(3)(-) films at 110 and 230 degrees C were monitored by measuring the absorption and emission spectra. The results indicate that the exchange of the counterion Cl(-) of PTHT by a linear long chain with RSO(3)(-) group is a general procedure to obtain PPV polymer at lower conversion temperature (ca. 110 degrees C) with significant increase in the emission efficiency, regardless of the chemical position and the number of sulfonate groups. With the enhanced emission caused by Congo Red and Tinopal as counter-ions, it is demonstrated that the new synthetic route is entirely generic, which may allow accurate control of conversion and emission properties. (C) 2010 Elsevier B.V. All rights reserved.

Role of cis-cis muconic acid in the catalysis of Pseudomonas putida chlorocatechol 1,2-dioxygenase

Chlorocatechol 1,2-dioxygenase (1,2-CCD) is a non-heme iron protein involved in the intradiol cleavage of aromatic compounds that are recalcitrant to biodegradation. In particular, 1,2-CCD catalyzes the conversion of catechol and its halogenated derivatives to cis-cis muconic acid. In this study we describe a series of experiments concerning the interaction of chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp1,2-CCD) with cis-cis muconic acid. We used single-injection ITC to show that the reaction product inhibits enzyme kinetics. DSC and EPR measurements probed whether this was accomplished by a direct binding of the product to the enzyme active site. DSC shows that cis-cis muconic acid affects the thermal unfolding of the protein and allowed us to estimate a binding constant. Furthermore, EPR spectra of the Fe(III) center demonstrate that, upon product binding, a significant decrease in resonance intensity is observed, indicating that cis-cis muconic acid binds directly to the active site. Based on the increasing interest for understanding dioxygenases mechanism of action and, moreover, how to control such process, our data indicate that the product of the reaction does play a relevant role in the catalysis and should therefore be taken into account when one thinks about ways of regulating enzyme activity. (C) 2010 Elsevier B.V. All rights reserved.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Novel ruthenium complexes as potential drugs for Chagas`s disease: enzyme inhibition and in vitro/in vivo trypanocidal activity

Background and purpose: The discovery of the pharmacological functions of nitric oxide has led to the development of NO donor compounds as therapeutic agents. A new generation of ruthenium NO donors, cis-[Ru(NO)(bpy)(2)L]X(n) , has been developed, and our aim was to show that these complexes are able to lyse Trypanosoma cruzi in vitro and in vivo. Experimental approach: NO donors were incubated with T. cruzi and their anti-T. cruzi activities evaluated as the percentage of lysed parasites compared to the negative control. In vivo, trypanocidal activity was evaluated by observing the levels of parasitaemia, survival rate and elimination of amastigotes in mouse myocardial tissue. The inhibition of GAPDH was monitored by the biochemical reduction of NAD+ to NADH. Key results: The NO donors cis-[Ru(NO)(bpy)(2)L]X(n) presented inhibitory effects on T. cruzi GAPDH (IC(50) ranging from 89 to 153 mu M). The crystal structure of the enzyme shows that the inhibitory mechanism is compatible with S-nitrosylation of the active cysteine (cys166) site. Compounds cis-[Ru(NO)(bpy)(2)imN](PF(6))(3) and cis-[Ru(NO)(bpy)(2)SO(3)]PF(6), at a dose of 385 nmol center dot kg-1, yielded survival rates of 80 and 60%, respectively, in infected mice, and eradicated any amastigotes from their myocardial tissue. Conclusions and implications: The ruthenium compounds exhibited potent in vitro and in vivo trypanocidal activities at doses up to 1000-fold lower than the clinical dose for benznidazole. Furthermore, one mechanism of action of these compounds is via the S-nitrosylation of Cys166 of T. cruzi GAPDH. Thus, these compounds show huge potential as candidates for the development of new drugs for the treatment of Chagas`s disease. This article is commented on by Machado et al., pp. 258-259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00662.x and to view a related paper in this issue by Guedes et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00576.x.

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bouhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH(2) (RGE) and IVYYPDRGETGL-NH(2) (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein. (C) 2009 Elsevier Ltd. All rights reserved.

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R(free)=21.2%) at 1.70 angstrom resolution and 15.81% (R(free) = 19.2%) at 1.85 angstrom resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly beta-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity. (C) 2008 Elsevier Ltd. All rights reserved.

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of similar to 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. (C) 2008 Elsevier Inc. All rights reserved.

Peritrophic membrane role in enhancing digestive efficiency Theoretical and experimental models

The pentrophic membrane (PM) is an anatomical structure surrounding the food bolus in most insects. Rejecting the idea that PM has evolved from coating mucus to play the same protective role as it, novel functions were proposed and experimentally tested. The theoretical principles underlying the digestive enzyme recycling mechanism were described and used to develop an algorithm to calculate enzyme distributions along the midgut and to infer secretory and absorptive sites. The activity of a Spodoptera frugiperda microvillar aminopeptidase decreases by 50% if placed in the presence of midgut contents. S. frugiperda trypsin preparations placed into dialysis bags in stirred and unstirred media have activities of 210 and 160%, respectively, over the activities of samples in a test tube. The ectoperitrophic fluid (EF) present in the midgut caeca of Rhynchosciara americana may be collected. If the enzymes restricted to this fluid are assayed in the presence of PM contents (PMC) their activities decrease by at least 58%. The lack of PM caused by calcofluor feeding impairs growth due to an increase in the metabolic cost associated with the conversion of food into body mass. This probably results from an increase in digestive enzyme excretion and useless homeostatic attempt to reestablish destroyed midgut gradients. The experimental models support the view that PM enhances digestive efficiency by: (a) prevention of non-specific binding of undigested material onto cell Surface; (b) prevention of excretion by allowing enzyme recycling powered by an ectoperitrophic counterflux of fluid; (c) removal from inside PM of the oligomeric molecules that may inhibit the enzymes involved in initial digestion; (d) restriction of oligomer hydrolases to ectoperitrophic space (ECS) to avoid probable partial inhibition by non-dispersed undigested food. Finally,PM functions are discussed regarding insects feeding on any diet. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta)

The enzyme nitrate reductase (NR) responsible for the conversion of nitrate to nitrite is considered to be the rate-limiting step in nitrogen assimilation. The economically important marine macroalga Gracilaria tenuistipitata presents a circadian oscillation in NR protein content and activity. In order to identify if the regulation of NR in G. tenuistipitata happens at transcriptional levels, the NR cDNA and gene were sequenced and the NR mRNA expression was studied. Analysis of the sequenced gene revealed absence of introns which is unusual for NR genes. The transcriptional profiling revealed a circadian rhythm for NR; furthermore, a rhythm was observed in constant light condition, suggesting a possible regulation by the biological clock at the mRNA levels for NR in G. tenuistipitata.

Electrochemical Behavior of biosensor by determination the catechol and phenolic compounds using the enzyme PPO

An electrochemical biosensor using poly-phenol oxidasa (PPO) was constructed for the determination of phenolic compounds. The PPO employed with enzyme, it was obtained from Archontophoenix Cunninghamiana. The biosensor showed range of linearity in the range of 1 x 10(-3) to 1 x 10(-4) mol/L and a detection limit of 1 x 10(-4) mol/L. The optimal pH was 6,7 in medium phosphate buffer. The lifetime of the biosensors was 1 months, stored in phosphate buffer solution 0.1 mol/L to ambient temperature.