Effects of cyclic temperature changes on water sorption and solubility of composite restoratives.

This study investigated the effects of cyclic temperature changes on the water sorption and solubility of four commercial composite resins (Silux Plus, Z100, Ariston pHc and Surefil). The methodology was based upon ISO 4049 procedures with modifications for specimen dimension and thermal-cycling. Eighteen disc specimens (10 +/- 1 mm diameter and 1 +/- 0.1 mm thick) were made for each composite and randomly divided into three groups. The specimens were stored in a desiccator maintained at 35 +/- 1 degrees C until a constant mass was achieved and treated as follows: Group 1--stored in distilled water at 356 degrees C for 178 hrs; Group 2--stored in distilled water at 35 degrees C for 173 hours and subjected to five hours of thermal-cycling with an upper temperature of 45 degrees C; and Group 3--stored in distilled water at 35 degrees C for 173 hours and subjected to five hours of thermal-cycling with an upper temperature of 60 degrees C. Mass after treatment was measured and specimens were re-conditioned to constant mass. The volume of the specimens was obtained and water sorption/solubility calculated. Data was analyzed using factorial ANOVA/Scheffe's post-hoc test at significance level 0.05. The effects of thermal-cycling on water sorption was material dependent. Thermal-cycling at an upper temperature of 60 degrees C significantly increased water sorption of Silux Plus. A significant increase in water sorption was also observed when Z100 was thermal-cycled at an upper temperature of 45 degrees C. The water sorption of Ariston pHc and Surefil was not affected by thermal-cycling. Thermal-cycling did not affect the solubility of all composites. For all treatment groups, Surefil had significantly lower water sorption than the other composites evaluated. The water sorption of Z100 and Surefil was significantly lower than Silux Plus and Ariston pHc.

Effects of cyclic temperature changes on hardness of composite restoratives.

The clinical durability of some composite restorative materials may be significantly affected by cyclic temperature changes. This study investigated the effects of cyclic temperature changes on surface hardness of four commercial composite resins (Silux, Z100, Ariston and Surefil). Eighteen specimens of each material were divided into three treatment groups comprising a control and two different thermal cycling regimes. Control specimens were stored in distilled water at 35 degrees C for 178 hours. Thermal cycled specimens were stored in distilled water at 35 degrees C for 173 hours and subjected to five hours (300 cycles) of a thermal cycling regime consisting of the cycle ABAC, where A and B represent the fixed temperatures of 35 degrees C (28 seconds) and 15 degrees C (two seconds) and C, depending on the treatment group, either 45 degrees C or 60 degrees C (two seconds). All specimens were subsequently subjected to hardness testing (KHN) using a digital microhardness tester (load = 500 gf; dwell time = 15 seconds). Results were analyzed using ANOVA/Scheffe's test (p<0.05). The effect of thermal cycling on hardness was material-dependent. While thermal cycling significantly increased the surface hardness of Z100 and Surefil, it significantly decreased the hardness of Ariston. The hardness of Silux was not significantly affected by cyclic temperature changes. For all treatment groups, Z100 was significantly harder than the other composite resins evaluated and Surefil was significantly harder than Silux and Ariston. For both thermal cycled groups, Silux was significantly harder than Ariston.

Influence of thermal cycling on OCA wear of composite restoratives.

This study investigated the effects of thermal cycling on wear of four commercial composite resins (Silux, Z100, Ariston and Surefil). Specimens of each material were divided into three treatment groups comprising a control and two different thermal cycling regimes. Control specimens were stored in distilled water at 35 degrees C for 178 hours. Thermal cycled specimens were stored in distilled water at 35 degrees C for 173 hours and subjected to five hours (300 cycles) of a thermal cycling regime consisting of the cycle ABAC, where A and B represent the fixed temperatures of 35 degrees C (28 seconds) and 15 degrees C (two seconds) and C, depending on the treatment group, was either 45 degrees C or 60 degrees C (two seconds). All specimens were subsequently subjected to wear testing at 20 MPa contact stress against SS304 counterbodies with distilled water as the lubricant. Wear depth (microm; n=6) was measured using profilometry every 2,000 cycles up to 10,000 cycles. Results were analyzed using ANOVA/Scheffe's test (p<0.05). The effect of thermal cycling on wear was material-dependent. The wear of Silux and Z100 were not significantly affected by thermal cycling. Thermal cycling of Ariston at an upper temperature of 60 degrees C significantly decreased wear resistance. Thermal cycling affected only the early wear resistance of Surefil.

Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization.

The mdm2 gene product is an important regulator of p53 function and stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed with individual components and the p53-Ub((n)) is qualitatively similar to p53-Ub((n)) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.

Deficiency of current methods in assaying endochitinase activity.

Since chitin is degraded by a combination of both endo- and exochitinases, it is likely that both enzymes will be present in a crude extract. Currently used substrates for detecting endochitinase activity suffer from the fact that they could easily be digested by the contaminating exochitinase, thus giving either a false-positive or an inaccurate reading of the endochitinase activity. Using Photorhabdus luminescens, a bacterium symbiotically associated with insect-parasitic nematode Heterorhabditis bacteriophora as an exemplary system, we have identified these two chitinases by a simple "fluorimetric zymography" procedure. The exochitinase is a metalloenzyme and its activity is inhibited by 1,10-phenanthroline. Once the exochitinase activity is detected in a crude extract, its contribution must be eliminated before accurate determination of the endochitinase activity can be carried out. Specific properties of these enzymes including the pH activity profile, the requirement of metal ions for activity, and the molecular weight of the enzymes are among the factors to be considered in developing assaying procedures for endochitinase.

Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate.

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).