Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid

Lys49-Phospholipase A(2) (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region pert-nits quaternary structural transitions between open and closed membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure [1]. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78Angstrom, (ii) MjTX-II/STE complex at a resolution of 1.8 Angstrom and (W) BthTX-I/DMPC complex at 2.72Angstrom. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (W) and using using a Synchrotron Radiation Source (Laboratorio Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles

A partial pseudo-ternary phase diagram has been studied for the cethyltrimethylammonium bromide/isooctane:hexanol:butanol/potassium phosphate buffer system, where the two-phase diagram consisting of the reverse micelle phase (L-2) in equilibrium with the solvent is indicated. Based on these diagrams two-phase systems of reverse micelles were prepared with different compositions of the compounds and used for extraction and recovery of two enzymes, and the percentage of enzyme recovery yield monitored. The enzymes glucose-6-phosphate dehydrogenase (G6PD) and xylose redutase (XR) obtained from Candida guilliermondii yeast were used in the extraction procedures. The recovery yield data indicate that micelles having different composition give selective extraction of enzymes. The method can thus be used to optimize enzyme extraction processes. (c) 2007 Elsevier B.V. All rights reserved.

Spontaneous vesicles of sodium dihexadecylphosphate in HEPES buffer

The formation of spontaneous vesicles of dihexadecylphosphate (DHP) in a HEPES buffered solution at pH 7.4, the size, morphology and melting temperature, obtained by cryogenic transmission electron microscopy (cryo-TEM) and differential scanning calorimetry (DSC), are reported. The vesicles were formed by simply mixing a 5.0 mM lipid-solvent mixture at a temperature (75 degrees C) safely above the higher melting temperature T-m = 70.4 degrees C of DHP. The vesicle diameter ranges from 100 to 332 nm and their geometry is spherical, faceted or oblong. T-m increases from 66.8 to 70.4 as DHP concentration is raised from 0.6 to 5.0 mM. (c) 2008 Elsevier B.V. All rights reserved.

Peroxide penetration from the pulp chamber to the external root surface after internal bleaching

Purpose: To quantify the amount of peroxide penetration from the pulp chamber to the external surface of teeth during the walking bleaching technique. Methods: Seventy-two bovine lateral incisors were randomly divided over five experimental groups and one control (n = 12 per group): (1) 35% hydrogen peroxide (HP); (2) 35% carbamide peroxide (CP); (3) sodium perborate (SP); (4) (HP+SP); (5) (CP+SP) and (6) Control (CG), deionized water. All groups were treated according to the walking bleach technique. After 7 days at 37 degrees C in an acetate buffer solution, 100 mu l violet leukocrystal coloring and 50 mu l peroxidase was added, producing a blue stain that could be measured in a spectrophotometer and then converted into peroxide mu g/ml. Results: G5 exhibited the greatest penetration, while G2 and G3 produced the lowest values. All bleaching agents penetrated from the pulp chamber to the external root surface. There was a direct correlation between the presence of oxidative agents and penetration potential. Sodium perborate in distilled water was less oxidative and appeared to be the least aggressive bleaching agent. (Am J Dent 2010;23:171-174).

BONE SPREADER TECHNIQUE: A PRELIMINARY 3-YEAR STUDY

The purpose of this study was to observe the clinical outcome of bone spreading and standardized dilation of horizontally resorbed bone during immediate implant placement using a "screw-type" configuration of expansion and threadformers. Fifty-three patients were included in this study, and 41 edentulous areas in anterior and posterior maxillas were treated. Sixty-eight implants were placed using an insertion torque of at least 40 Ncm. Abutments were delivered 4 to 6 months after implant placement. The overall failure percentage was 4.41% (3 failures). A retrieved analysis of I implant removed at 3 years after placement demonstrated bone resorption down to the level of the third thread. The bone spreader technique is different from Summers' osteotome, both in clinical use and in armamentarium. The main advantage of the crest-expanding technique is that it is a less invasive procedure; the facial wall expands after the medullary bone is compressed against the cortical wall. Within the limits of this preliminary study, the cumulative survival rate for this method of implant placement is 95.58% at 3 years. This study confirms that a bone spreader used in the maxilla shows an unusually low failure rate after 3 years.

Effect of Caries Infiltration Technique and Fluoride Therapy on Microhardness of Enamel Carious Lesions

Enamel white spot subsurface lesions compromise esthetics and precede cavitation; therefore, they must be halted. The aim of this study was to evaluate the effect of a caries infiltration technique and fluoride therapy on the microhardness of enamel carious lesions. Subsurface carious lesions were produced in 60 bovine specimens with polished enamel surfaces. The specimens were divided into four groups (n=15), according to the treatment used: CON, control immersion in artificial saliva; DF, daily 0.05% fluoride solution; WF, weekly 2% fluoride gel; and IC, resin infiltration (Icon). The specimens were kept in artificial saliva and evaluated for microhardness at five points: baseline, after caries production, after four and eight weeks of treatment, and a final evaluation after being submitted to a new acid challenge. The repeated-measures analysis of variance showed significant differences according to the type of treatment (TREAT; p=0.001) and time of evaluation (EV; p=0.001). The results of the Tukey test were TREAT: CON = 45.18 (+/- 29.17)a, DF = 107.75 (+/- 67.38)b, WF = 83.25 (+/- 51.17)c, and IC = 160.83 (+/- 91.11)d. Analysis of correlation between the TREAT and EV factors showed no significant differences for DF (138.63 +/- 38.94) and IC (160.99 +/- 46.13) after the new acid challenge. The microhardness results in decreasing order after eight weeks were IC > DF > WF > CON. It was concluded that the microhardness of carious lesions increased with the infiltration of resin, while the final microhardness after a new acid challenge was similar for DF and IC.

Effects of Self-Etching Adhesive Systems Used in the Dental Modelling Technique on the Cohesive Strength of Composite Resin

This study evaluated the cohesive strength of composite using self-etching adhesive systems (SE) in the lubrication of instruments between layers of composite. The specimens were made by using a Teflon (R) device. SE were used at the interface to lubricate the instruments: Group 1(G1) - control group, no lubricant was used; Group 2(G2) -Futurabond (R) M; Group 3(G3) - Optibond (R) All-In-One; Group 4(G4) - Clearfil (R) SE Bond; Group 5(G5) - Futurabond (R) NR; Group 6(G6) - Adper (R) SE Plus; Group 7(G7) - One Up Bond (R) F. Specimens were submitted to the tensile test to evaluate the cohesive strength. Data were submitted to the ANOVA and Tukey tests. ANOVA showed a value of p = 0.00. The average means (SD): G2 = 11.33(+/-3.44) a, G3 = 15.36(+/-4.06) ab, G4 = 18.9(+/-4.72) bc, G7 = 19.62(+/-4.46) bc, G5 = 21.02(+/-5.09) bc, G6 = 23.39(+/-4.17) cd, and G1 = 28.49(+/-2.89) d. All SE decreased the cohesive strength of the composite, except for Adper (R) SE Plus.