Repolishing Resin Composites After Bleaching Treatments: Effects on Color Stability and Smoothness.

This study aimed to evaluate the effect of repolishing after bleaching on color stability and smoothness of two resin composites aged in a high-staining beverage. Fifty-six disc-shaped specimens (8×2 mm) of each resin composite were fabricated (Filtek Z250, 3M ESPE, microhybrid, and Filtek Z350 XT, 3M ESPE, nanofilled) and then divided according to treatment: bleached or nonbleached. After treatment application, groups were subdivided according to the surface treatment: repolished or unrepolished. A new subdivision was performed according to aging conditions: immersion in red wine for 15 min/d or in artificial saliva for 24 h/d during 30 days. Color (CIE L*a*b* system) and roughness (Ra) were assessed at baseline (P0), after bleaching procedures (P1), after surface treatment (P2), and after aging (P3). Color change (ΔE00) was calculated through the CIEDE2000 formula. Statistical analysis was performed using repeated measures analysis of variance and the Tukey post hoc test. Bleached repolished groups presented lower color alteration than the bleached unrepolished groups from both resin composites when aged in red wine. Repolishing (P1 vs P2) promoted a slight decrease in roughness values of almost all groups. Nanofilled composite presented greater ΔE00 values than microhybrid composite when aged in red wine.

CAD/CAM machining Vs pre-sintering in-lab fabrication techniques of Y-TZP ceramic specimens: Effects on their mechanical fatigue behavior.

This study evaluated the effects of different pre-sintering fabrication processing techniques of Y-TZP ceramic (CAD/CAM Vs. in-lab), considering surface characteristics and mechanical performance outcomes. Pre-sintered discs of Y-TZP ceramic (IPS e.max ZirCAD, Ivoclar Vivadent) were produced using different pre-sintering fabrication processing techniques: Machined- milling with a CAD/CAM system; Polished- fabrication using a cutting device followed by polishing (600 and 1200 SiC papers); Xfine- fabrication using a cutting machine followed by grinding with extra-fine diamond bur (grit size 30 µm); Fine- fabrication using a cutting machine followed by grinding with fine diamond bur (grit size 46 µm); SiC- fabrication using a cutting machine followed by grinding with 220 SiC paper. Afterwards, the discs were sintered and submitted to roughness (n=35), surface topography (n=2), phase transformation (n=2), biaxial flexural strength (n=20), and biaxial flexural fatigue strength (fatigue limit) (n=15) analyses. No monoclinic-phase content was observed in all processing techniques. It can be observed that obtaining a surface with similar characteristics to CAD/CAM milling is essential for the observation of similar mechanical performance. On this sense, grinding with fine diamond bur before sintering (Fine group) was the best mimic protocol in comparison to the CAD/CAM milling.

Efficacy and safety of acetaminophen and naproxen in the treatment of tension-type headache. A randomized, double-blind, placebo-controlled trial.

The objective of this study was to evaluate and compare the efficacy and safety of single doses of acetaminophen (paracetamol) 1000 mg and naproxen 375 mg vs. placebo over a six-hour period in the treatment of tension-type headache. The treatments were compared in a randomized, double-blind, multicentre, placebo-controlled study. Efficacy was evaluated using four standard analgesic summary endpoints (the sum of pain intensity differences from baseline, the maximum pain intensity from baseline, the sum of the pain relief scores, and the maximum pain relief score). Both acetaminophen 1000 mg and naproxen 375 mg were significantly superior to placebo (Por=0.498) for these four endpoints. For example, the mean sum of pain intensity differences from baseline was 9.14+/-0.34 for acetaminophen 1000 mg and 8.81+/-0.35 for naproxen 375 mg compared with 7.42+/-0.34 for placebo. Other efficacy endpoints (percentage of responders (pain reduced to none) at two hours, onset of meaningful relief, time to use of rescue medication and subject's overall impression of study medication) showed similar trends. A significantly larger mean pain intensity difference from baseline was observed for acetaminophen 1000 mg (1.13) than for naproxen 375 mg (0.95) (P=0.036) at one hour after treatment. There was no significant difference among the treatment groups in the incidence of adverse events (P=0.730). In summary, the results of this well-controlled, double-blind study demonstrate that over-the-counter acetaminophen 1000 mg and prescription naproxen 375 mg are effective and well tolerated in the treatment of tough (moderate-to-severe) tension-type headache.

Involvement of protein kinase C and protein kinase A in the muscarinic receptor signalling pathways mediating phospholipase C activation, arachidonic acid release and calcium mobilisation.

The involvement of protein kinase C (PKC) and protein kinase A (PKA) in cholinergic signalling in CHO cells expressing the M3 subtype of the muscarinic acetylcholine receptor was examined. Muscarinic signalling was assessed by measuring carbachol-induced activation of phospholipase C (PLC), arachidonic acid release, and calcium mobilisation. Carbachol activation of PLC was not altered by inhibition of PKC with chelerythrine chloride, bisindolylmaleimide or chronic treatment with phorbol myristate acetate (PMA). Activation of PKC by acute treatment with PMA was similarly without effect. In contrast, inhibition of PKC blocked carbachol stimulation of arachidonic acid release. Likewise, PKC inhibition resulted in a decreased ability of carbachol to mobilise calcium, whereas PKC activation potentiated calcium mobilisation. Inhibition of PKA with H89 or Rp-cAMP did not alter the ability of carbachol to activate PLC. Similarly, PKA activation with Sp-cAMP or forskolin had no effect on PLC stimulation by carbachol. Carbachol-mediated release of arachidonic acid was decreased by H89 but only slightly increased by forskolin. Forskolin also increased calcium mobilisation by carbachol. These results suggest a function for PKC and PKA in M3 stimulation of arachidonic acid release and calcium mobilisation but not in PLC activation.

M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms.

Although muscarinic acetylcholine receptors (mAChR) regulate the activity of smooth muscle myosin, the effects of mAChR activation on cytoplasmic myosin have not been characterized. We found that activation of transfected human M3 mAChR induces the phosphorylation of myosin light chains (MLC) and the formation of myosin-containing stress fibers in Chinese hamster ovary (CHO-m3) cells. Direct activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain phosphorylation and myosin reorganization in CHO-m3 cells. Conventional (alpha), novel (delta), and atypical (iota) PKC isoforms are activated by mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC translocation or degradation. mAChR-mediated myosin reorganization is abolished by inhibiting conventional PKC isoforms with Go6976 (IC50 = 0.4 microM), calphostin C (IC50 = 2.4 microM), or chelerythrine (IC50 = 8.0 microM). Stable expression of dominant negative RhoAAsn-19 diminishes, but does not abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells. Similarly, mAChR-mediated myosin reorganization is diminished, but not abolished, in CHO-m3 cells which are multi-nucleate due to inactivation of Rho with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does not affect PMA-induced myosin reorganization. These findings indicate that the PKC-mediated pathway of myosin reorganization (induced either by M3 mAChR activation or PMA treatment) can continue to operate even when RhoA activity is diminished in CHO-m3 cells. Conventional PKC isoforms and RhoA may participate in separate but parallel pathways induced by M3 mAChR activation to regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by M3 mAChR activation may contribute to the unique ability of these receptors to regulate cell morphology, adhesion, and proliferation.

Ethanol disrupts carbamylcholine-stimulated release of arachidonic acid from Chinese hamster ovary cells expressing different subtypes of human muscarinic receptor.

Ethanol disrupts signal transduction mediated by a variety of G-protein coupled receptors. We examined the effects of ethanol on arachidonic acid release mediated by muscarinic acetylcholine receptors. Chinese hamster ovary (CHO) cells transfected with the different subtypes of human muscarinic receptors (M1 to M5) were incubated with [3H]arachidonic acid ([3H]AA) for 18 hr, washed, and exposed to the cholinergic agonist carbamylcholine for 15 min. Carbamylcholine induced [3H]AA release from CHO cells expressing M1, M3, or M5, but not M2 or M4, muscarinic receptors. Dose response curves revealed that carbamylcholine stimulated [3H]AA release by up to 12-fold with an ECo of approximately 0.4 microM; maximal responses were obtained with 10 microM carbamylcholine. Exposure of M1-, M3-, or M5-expressing cells to ethanol for 5 min before stimulating with carbamylcholine reduced [3H]AA release by 40 to 65%; 50% of the maximal inhibition was obtained with an ethanol concentration of 30 to 50 mM. Ethanol did not affect basal [3H]AA release measured in the absence of carbamylcholine. Dose response curves suggest that ethanol acts as a noncompetitive inhibitor of muscarinic receptor-induced [3H]AA release insofar as maximal [3H]AA release was depressed in the presence of ethanol with no apparent change in the EC50 for stimulation by carbamylcholine. Exposure of CHO cells to 38 mM ethanol for 48 hr increased [3H]AA release induced by carbamylcholine without affecting basal [3H]AA release or altering the EC50 for carbamylcholine. These results indicate that ethanol acutely inhibits muscarinic receptor signaling through the arachidonic acid pathway in a noncompetitive manner, but chronically enhances muscarinic signaling through the same pathway.

Influence of acute and chronic ethanol treatment on muscarinic responses and receptor expression in Chinese hamster ovary cells.

The influence of ethanol on the muscarinic receptor-mediated release of inositol phosphate from Chinese hamster ovary (CHO) cells stably transfected with one of the five subtypes of muscarinic acetylcholine receptor was determined. In CHO cells expressing M3 muscarinic receptors (CHO-M3), carbamylcholine increased muscarinic receptor-induced release of inositol phosphate by 150-350% following a 15-min incubation with an EC50 of approximately 30 microM. Maximal responses were obtained with 1 mM carbamylcholine, while responses to 10 mM carbamylcholine were somewhat less than maximal. Preincubation with atropine for 10 min inhibited the response with an IC50 of approximately 30 nM. CHO cells transfected with M1, M3, and M5 receptors displayed a similar pattern of activity; CHO cells transfected with M2 and M4, as well as untransfected cells, were unresponsive to carbamylcholine. Ethanol acutely inhibited the response of CHO-M3 cells to carbamylcholine by 15% at 18 mM and by 47% at 180 mM (the highest concentration examined). CHO-M3 cells were incubated with 50 mM ethanol for 48 hr. This treatment did not affect the number of cells or their protein content (113 pg/cell). The expression of M3 muscarinic receptors (determined using [3H]N-methylscopolamine) increased from 1.34 +/- 0.23 to 1.75 +/- 0.16 pmol/mg protein (P < 0.05). In contrast, carbamylcholine-stimulated release of inositol phosphate was depressed by 40-70% in four experiments. Concentration-response analyses indicated a non-competitive inhibitory mechanism. This dissociation of muscarinic receptor expression and muscarinic signaling suggests a compensatory increase in receptor expression in response to chronic inhibition of muscarinic signaling by ethanol.

Parathyroid hormone uses both adenylate cyclase and protein kinase C to regulate acid production in osteoclasts.

Osteoclasts, isolated from the endosteum of 2.5- to 3-week-old chickens, were treated with acridine orange, a hydrogen ion concentration-sensitive fluorescent dye, in order to monitor changes in acid production. The adenylate cyclase inhibitor, alloxan, blocked parathyroid hormone (PTH)-stimulated acid production. Dibutyryl cyclic adenosine monophosphate, a membrane-permeant form of cyclic adenosine monophosphate, mimicked the PTH effect. Bisindolylmaleimide, a specific inhibitor of protein kinase C (PKC), blocked the initial stimulation (15, 30, and 60 min) of acid production by PTH but had no effect on long-term stimulation (120 min). Confocal microscopy of osteoclasts stained with fluorescein-conjugated bisindolylmateimide revealed a shift in location of PKC from the cytoplasm to the plasma membrane region after treatment with parathyroid hormone. The results of these studies support the hypothesis that PTH regulation of acid production in osteoclasts involves both adenylate cyclase and PKC as effectors.

Multiple G-protein involvement in parathyroid hormone regulation of acid production by osteoclasts.

The involvement of multiple G-proteins in parathyroid hormone regulation of acid production was demonstrated in a highly enriched osteoclast population. Osteoclasts were isolated from the endosteum of 2.5 to 3-week-old chicken tibia using sequential enzymatic digestion. Single cell analysis of acid production was accomplished using microscope photometry and vital staining with acridine orange, a hydrogen ion concentration sensitive fluorescent dye. Lithium chloride, an uncoupler of G-proteins from their respective receptors, blocked parathyroid hormone stimulated production of acid. Cholera toxin, which permanently activates Gs-proteins, mimicked PTH stimulation. Pertussis toxin, which prevents receptor interaction with Gi- and Go-proteins, blocked both 10(-8) M and 10(-11) M PTH stimulated acid production, suggesting that the pertussis toxin-sensitive G-protein is utilized at both PTH concentrations. Immunoblots of osteoclast plasma membrane proteins, using a panel of antibodies generated against specific G-protein alpha subunits, revealed a 48 kDa Gs alpha, a 41 kDa Go alpha, a 34 kDa Gi alpha-3, and a unique 68 kDa G alpha subunit, with the 41 kDa and 34 kDa bands being the most intense. Immunoblots of osteoblast plasma membrane proteins had a substantially different profile with the most intense bands being a Gs alpha (48 kDa) and a Go alpha (36 and 38 kDa). The studies suggest the utilization of at least two different G-proteins in the parathyroid hormone regulation of acid formation by osteoclasts, a Gs and a pertussis toxin-sensitive G-protein (Go and/or Gi alpha-3).

Development of a new method for obtaining osteoclasts from endosteal surfaces.

Techniques for the isolation of a highly pure population of viable osteoclasts are limited. For this reason, we developed an isolation procedure that results in a high yield of osteoclast-like cells, up to 92% pure, from 3-wk-old chicken tibias. The unique feature of the method is the migration of cells from marrow-free endosteal surfaces to vitronectin-coated plates. The cells retain the osteoclast phenotype and remain viable in culture for a minimum of 1 wk. The cells were characterized and compared to two populations of authentic avian osteoclasts, which were isolated on the basis of association with fibronectin-coated plates. The cells contained substantial amounts of tartrate-resistant acid phosphatase. Alkaline phosphatase levels were negligible, suggesting little contamination by osteoblasts. Response to parathyroid hormone, dibutyryl cyclic adenosine monophosphate, calcitonin, acetazolamide, 17 beta-estradiol, and prostaglandin E2 was evident, as detected by measuring acid production. The vitronectin-associating cells contained numerous mitochondria, had the ability to resorb bone in an in vitro bone slice assay, and specifically bound biotinylated vitronectin. At 5 d of culture, the cells demonstrated marginal multinuclearity, having two to three nuclei. A large number (approximately 1 x 10(6) cells/tibia) of viable cells that exhibit characteristics of authentic osteoclasts can be obtained by the method described. Potentially, this method could be applied to other species.