Porosity of flowable resin composites is not influenced by applicator tip diameter.

PURPOSE: To assess the influence of applicator tip diameter on the inclusion of porosities in three different flowable resin composites. METHODS: The initial porosity of three syringes [Filtek Supreme XTE Flowable (XTE), Grandio Flow (GRF), Gradia Direct Flo (GDF)] was determined by 3D tomography. 25 samples per syringe, i.e. 75 samples in total, were prepared using five applicator tips of different diameters (n= 5). The porosity of the 75 samples was assessed by 3D tomography. RESULTS: For each of the materials, the applicator tips, irrespective of type, all generated an increase in the average porosity percentage compared to the initial porosity of the syringes. For XTE and GRF, the applicator tips, irrespective of type, all generated a decrease in the average porosity volume compared to the initial average porosity volume in their respective syringes. Conversely, for GDF the average porosity volume of the samples was increased. Furthermore, for each of the materials, varying the diameter of the applicator tips had no significant influence on the porosity percentage and volume. Using the present study conditions, the applicator tip generated a variation in the initial porosity of the materials; however, the diameter of the tip had no influence on said variation. CLINICAL SIGNIFICANCE: It appears that practitioners can choose an applicator tip with a diameter that best suits the size and shape of the cavity to be filled using a syringe of flowable resin composite without this having any impact on the percentage and volume of porosities in the final filling of the cavity.

Dynamic thermo-mechanical properties of various flowable resin composites.

BACKGROUND: This study compared the storage modulus (E'), the loss modulus (E'') and the loss tangent (tan δ) of various flowable resin composites. MATERIAL AND METHODS: Grandio Flow (GRF), GrandioSo Heavy Flow (GHF), Filtek Supreme XTE (XTE) and Filtek Bulk Fill (BUL) flowable resins and Clinpro Sealant (CLI) ultra-flowable pit and fissure sealant resin were used. 25 samples were tested using a dynamical mechanical thermal analysis system in bending mode. Measurements were taken within a temperature range of 10 to 55°C. The results were statistically analyzed using mixed-effect and repeated-measure analysis of variance followed by paired multiple comparisons. RESULTS: For all the materials, the E' values decrease with temperature, whereas the tan δ values increase. Irrespective of the temperature, GHF and GRF present E' and E'' values significantly higher than all the other materials and CLI presents values significantly lower than all the other materials. Observation of the values for all the materials reveals a linear progression of the tan δ values with temperature. CONCLUSIONS: A variation in temperature within a physiological range generates modifications in mechanical properties without damaging the material, however. Filler content in volume terms appears to be the crucial parameter in the mechanical behavior of tested materials. Key words:Dynamic mechanical thermal analysis, elastic modulus, filler content, flowable resin composites, loss modulus, loss tangent.

c-Src mediated tyrosine phosphorylation of plakophilin 3 as a new mechanism to control desmosome composition in cells exposed to oxidative stress.

Plakophilins (PKP1 to PKP3) are essential for the structure and function of desmosomal junctions as demonstrated by the severe skin defects observed as a result of loss-of-function mutations in mice and men. PKPs play additional roles in cell signaling processes, such as those controlling the cellular stress response and cell proliferation. A key post-translational process controlling PKP function is phosphorylation. We have discovered that reactive oxygen species (ROS) trigger the c-Src kinase-mediated tyrosine (Tyr)-195 phosphorylation of PKP3. This modification is associated with a change in the subcellular distribution of the protein. Specifically, PKP3 bearing phospho-Tyr-195 is released from the desmosomes, suggesting that phospho-Tyr-195 is relevant for the control of desmosome disassembly and function, at least in cells exposed to ROS. Tyr-195 phosphorylation is transient under normal physiological conditions and seems to be strictly regulated, as the activation of particular growth factor receptors results in a modification at this site only when tyrosine phosphatases are inactivated by pervanadate. We have identified Tyr-195 of PKP3 as a phosphorylation target of epidermal growth factor receptor signaling. Interestingly, this PKP3 phosphorylation also occurs in certain poorly differentiated adenocarcinomas of the prostate, suggesting a possible role in tumor progression. Our study thus identifies a new mechanism controlling PKP3 and hence desmosome function in epithelial cells.

Differential expression of desmosomal plakophilins in various types of carcinomas: correlation with cell type and differentiation.

Plakophilins (PKPs) are a set of 3 constitutive armadillo repeat proteins of the desmosomal plaque, termed PKP 1, PKP 2, and PKP 3, which have been shown to be functionally relevant for desmosomal adhesion. We have performed a systematic immunohistochemical study of the 3 PKPs in oral and pharyngeal squamous cell carcinomas (SqCCs; n = 40); colorectal, pancreatic, and prostate adenocarcinomas (n = 31), and hepatocellular carcinomas (HCCs; n = 8). In SqCCs, PKP 1 and PKP 3 revealed common desmosome-type immunostaining, their expression level being inversely correlated with the degree of malignancy. Instead, staining for PKP 2 was limited. In contrast, all adenocarcinomas contained PKP 2 and-often abundantly-PKP 3 in desmosome-typical pattern, whereas PKP 1 was expressed only in prostate tumors. The presence of PKP 3 in adenocarcinomas was confirmed by immunoblotting. In HCCs, only PKP 2 was detected. Under certain staining conditions, focal nuclear immunoreactivity for PKP 1 was observed in some SqCCs and HCCs. Our results, which are inconsistent with previously published data to some extent, indicate a principal preservation of the cell type and differentiation-related expression patterns of PKPs in normal epithelia. For PKP 1, a suppressor function of malignant behavior seems conceivable, whereas the putative functional significance of its occurrence in tumor cell nuclei requires further studies.

Plakophilins--hard work in the desmosome, recreation in the nucleus?

The linkage of the different types of cytoskeletal proteins to cell adhesion structures at the cytoplasmic membrane and the connection of these contact sites to corresponding sites of adjacent cells is a prerequisite for integrity and stability of cells and tissues. The structurally most prominent types of such cell-cell adhesion complexes are the desmosomes (maculae adhaerentes), which are found in all epithelia and certain non-epithelial tissues. As an element of the cytoskeleton, intermediate filaments are connected to the adhesive desmosomal transmembrane proteins by the cytoplasmic desmosomal plaque proteins. At least three different types of proteins are found in the desmosomal plaque, one of which is represented by the plakophilins, a recently described sub-family of sequence-related armadillo-repeat proteins. Consisting of three isoforms, plakophilins (plakophilin 1 to 3, PKP 1 to 3) are located in all desmosomes in a differentiation-dependent manner. While PKP 2 and PKP 3 are part of almost all desmosome-bearing cell types (PKP 2 except for differentiated cells of stratified epithelia and PKP 3 for hepatocytes and cardiomyocytes), PKP 1 is restricted to desmosomes of cells of stratified and complex epithelia. Besides the architectural function that plakophilins seem to fulfill in the desmosomes, at least PKP 1 and 2 are also localized in the nucleus independently of any differentiation-related processes and with an up to now enigmatic function in this compartment. In the following article we want to summarize the current knowledge concerning structure, function and regulation of the plakophilins that has been achieved during the last decade.

Exoribonuclease R interacts with endoribonuclease E and an RNA helicase in the psychrotrophic bacterium Pseudomonas syringae Lz4W.

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.

Composition and activity of the Rhodobacter capsulatus degradosome vary under different oxygen concentrations.

The influence of changes in temperature or oxygen tension during growth of Rhodobacter capsulatus on the composition and activity of the degradosome, an RNA-processing protein complex, was investigated. Only minor differences in the amount of specific proteins of the complex were observed after a decrease or increase of the temperature, but dramatic variations were detectable during growth at different oxygen concentrations. In particular, the amount of the transcription factor Rho, which was previously shown to be associated with the R. capsulatus degradosome, was strongly increased under aerobic conditions. Remarkably, oxygen tension oppositely affected the levels of the two helicases associated with the degradosome. RNase E and the degradosome from aerobically grown cultures degraded a transcript which represents part of the puf operon encoding proteins of the photosynthetic apparatus faster than did the degradosome from semiaerobically grown cultures.

Temperature-dependent processing of the cspA mRNA in Rhodobacter capsulatus.

The expression of genes for cold-shock proteins is proposed to be regulated primarily at the post-transcriptional level by increase of mRNA stability after transition to low temperatures. Destabilization of the Escherichia coli cold-induced cspA transcript at 37 degrees C as well as stabilization upon cold shock is known to depend on the unusually long (159 nt) 5'-untranslated region. Determination of the cspA mRNA 5'-end from Rhodobacter capsulatus revealed a shorter distance between the start of transcription and the start codon for translation. The cspA mRNA of R. capsulatus was shown to be stabilized at low temperatures to a greater extent than other investigated transcripts. To address the mechanism of decay of the cspA transcript, it was incubated with purified degradosome of R. capsulatus. Endoribonucleolytic in vitro cleavage in the 5'-untranslated region as reported for the cspA transcript of E. coli in vivo was not observed. Instead, the data indicated that the cspA mRNA decay in R. capsulatus is mediated by endoribonucleolytic cleavages within the cspA coding region.

CIRCE is not involved in heat-dependent transcription of groESL but in stabilization of the mRNA 5'-end in Rhodobacter capsulatus.

The CIRCE element, an inverted DNA repeat, is known to be involved in the temperature-dependent regulation of genes for heat shock proteins in a variety of organisms. The CIRCE element was identified as the target for the HrcA protein, which represses transcription of heat shock genes under normal growth temperature. Our data reveal that the CIRCE element is not involved in the temperature-dependent transcription of the groESL genes in Rhodobacter capsulatus. Apparently, R.capsulatus does not harbour an HrcA protein. The mechanisms of heat shock regulation of the groESL genes in R.capsulatus therefore diverge significantly from the regulatory pathway identified in other organisms. A structural analysis of the CIRCE RNA element revealed a stem of 11 nt pairs and a loop of only 5 nt. This folding differs from a structure with a 9 nt loop suggested previously on the basis of computer analysis. The RNA structure leads to a slight stabilization of the groESL mRNA that is more pronounced at normal growth temperature than under heat shock conditions.