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The aim of this study was to evaluate the fracture resistance of 2 different types of all-ceramic crown using immediate dentin sealing (IDS), obtained using a CAD/CAM system on molars with different preparations. Forty extracted lower molars were endodontically treated and divided into four groups (n = 10) according to the dental preparation. Group 1 (SP0) was prepared without filling the pulp chamber and crown-root junction was located at the cementoenamel junction (CEJ). Group 2 (SP1) was prepared without filling the pulp chamber and crown-root junction was located 1-mm above the CEJ. Groups 3 and 4 contained a flat preparation surface with no axial wall height. Group 3 (CP0) was made IDS with complete filling of the pulp chamber with composite resin and crown-root junction was located at the CEJ. Group 4 (CP1) was prepared with complete filling of the pulp chamber and crown-root junction was located 1-mm above the CEJ. All groups were restored with CAD/CAM lithium disilicate ceramic crowns. Specimens were subjected to the fracture test and statistically analyzed using analysis of variance (ANOVA). Fracture mode was determined using a stereoscopic microscope, classified as repairable or nonrepairable, and analyzed using Fischer's exact test. Results indicated that there were no significant differences between the groups in terms of fracture resistance or fracture pattern (p >0.05). Fracture resistance was the lowest in the SP0 group, followed by the SP1 group (1634.38 N) of CP0 (1821.50 N), and it was the highest in the CP1 group. There was a predominance of nonrepairable fractures and there were no significant differences in the fracture resistance and fracture mode of CAD/CAM lithium disilicate molar all-ceramic crowns. Endodontically treated molars teeth might be restored with endocrowns or all-ceramic crowns on flat preparation; however tooth fracture failures that affect reliability of these types of restorations should be considered.

The platelet-extracellular matrix interaction in platelet rich plasma (PRP) through thrombospondin receptor-CD36 induces the secretion of growth factors responsible for cellular proliferation and differentiation during the repair process. Since CD36 also acts as a class B-scavenger-receptor for development of foam-like cells and mitogen-activated kinases, such as Erk1/2 and p38α/β, are important proteins activated by platelet growth factor, the aim of this study was to evaluate the immunohistochemical presence of CD36, Erk1/2, p38α/β during the bone repair treated and non-treated with PRP and to compare these results with the histomorphometry of repair. Simultaneously, the immunopresence of adiponectin was analyzed, which may contribute to osteogenesis at the same time it inhibits fibrosis and impairs adipogenesis and foam cell formation in the medullary area. An artificial bone defect measuring 5×1 mm was produced in the calvaria of 56 Wistar rats. The defects were randomly treated with autograft, autograft+PRP, PRP alone and sham. The animals were euthanized at 2 and 6 weeks post-surgery. Data were analyzed by ANOVA followed by non-parametric test Student Newman-Keuls (p<0.05) for histomorphometric and immunohistochemical interpretation. The results revealed that in specimens that received PRP the immunopositivity for Erk1/2, p38α/β and CD36 proteins increased significantly while the immunohistochemical expression of adiponectin decreased simultaneously. There was also an accentuated reduction of bone matrix deposition and increase of the medullary area represented by fibrosis and/or presence of foam-like cells, which exhibited immunophenotype CD36+adiponectin. The findings of this study suggest that PRP acted as an inhibitor of osteogenesis during the craniofacial bone repair and induced a pathological condition that mimics an atherofibrotic condition.

A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188 +/- 41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 microns diameter, each potentially bearing one or more approximately 80-nm-wide coated evaginations. These coated evaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated evaginations pinch off to form a second group of coated vesicles (90 +/- 17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early endosomes.

Dynamin was discovered in bovine brain tissue as a nucleotide-sensitive microtubule-binding protein of relative molecular mass 100,000. It was found to cross-link microtubules into highly ordered bundles, and appeared to have a role in intermicrotubule sliding in vitro. Cloning and sequencing of rat brain dynamin complementary DNA identified an N-terminal region of about 300 amino acids which contained the three consensus elements characteristic of GTP-binding proteins. Extensive homology was found between this domain and the mammalian Mx proteins which are involved in interferon-induced viral resistance, and with the product of the VPS1 locus in Saccharomyces cerevisiae, which has been implicated both in membrane protein sorting, and in meiotic spindle pole separation. Dynamin-containing microtubule bundles were not observed in an immunofluorescence study of cultured mammalian cells, but a role for a GTP-requiring protein in intermicrotubule sliding during mitosis in plants has been reported. We report here that Drosophila melanogaster contains multiple tissue-specific and developmentally-regulated forms of dynamin, which are products of the shibire locus previously implicated in endocytic protein sorting.

Cytoplasmic microtubule-based motility in Paramecium was investigated using video-enhanced contrast microscopy, the quick-freeze, deep-etch technique, and biochemical isolations. Three distinct vesicle populations were found to be transported unidirectionally along the cytopharyngeal microtubular ribbons. This minus-end-directed movement exhibited unique in vivo features in that the vesicle transport was nonsaltatory, rapid, and predominantly along one side of the microtubular ribbons. To identify candidate motor proteins which may participate in vesicle transport, we prepared cytosolic extracts of Paramecium and used bovine brain microtubules as an affinity matrix. These preparations were found to contain a microtubule-stimulated ATPase which supported microtubule gliding in vitro. This protein was verified as a cytoplasmic dynein based upon its relative molecular mass, sedimentation coefficient of 16S, susceptibility to vanadate photocleavage, elevated CTPase/ATPase ratio, and its typical two-headed dynein morphology. This dynein was directly compared with the axonemal dyneins from Paramecium and found to differ by five criteria: morphology, sedimentation coefficient, CTPase/ATPase ratio, vanadate cleavage patterns, and polypeptide composition. The cytoplasmic dynein is therefore not an axonemal dynein precursor, but rather it represents a candidate for supporting the microtubule-based vesicle transport which proceeds along the microtubular ribbons.

Physical fixation by rapid freezing followed by freeze-fracture and deep-etching has provided the means for potentially seeing the three-dimensional arrangement in the native state of particles on mitochondrial inner membranes. We have used these techniques to study the tubular cristae of Paramecium in the hope of determining the arrangement of F1 complexes, their abundance, and location in the membranes. We also sought information regarding other respiratory complexes in these membranes. Our results, supported by stereo pairs, show that F1 complexes are arranged as a double row of particles spaced at 12 nm along each row as a zipper following the full length of the outer curve of the helically shaped tubular cristae. There are an average of 1,500 highly ordered F1 complexes per micrometer squared of 50-nm tubular cristae surface. The F1 complexes definitely lie outside the membranes in their native state. Other particle subsets, also nonrandomly arrayed, were seen. One such population located along the inner helical curve consisted of large 13-nm-wide particles that were spaced at 30 nm center-to-center. Such particles, because of their large size and relative abundance when compared to F1 units, resemble complex I of the respiratory complexes. Any models attempting to understand the coupling of respiratory complexes with F0F1 ATPase in Paramecium must take into account a relatively high degree of order and potential immobility of at least some of these integral membrane complexes.

The compartments of the Paramecium digestive system were investigated with wheat germ agglutinin (WGA). By use of cryosectioning or Lowicryl K4M embedding combined with pulse-chase studies and WGA-gold labeling, WGA binding sites were located on membranes of the phagosome-lysosome system, including all four stages of digestive vacuoles, the discoidal vesicles, acidosomes, and lysosomes. In addition, the contents of lysosomes, cisternae at the trans face of Golgi stacks, and coated and uncoated blebs and vesicles at the putative trans Golgi network bind to WGA. Crystal-containing vacuoles characteristic of mid-log to stationary-phase cultures are enclosed by heavily labeled membranes. Alveoli underlying the plasma membrane sometimes contain binding sites, particularly on their outer membranes. Ciliary membranes previously shown to be labeled with WGA-FITC are negative in frozen thin and Lowicryl K4M sections. The presence of WGA binding sites on the trans face of the Golgi stack is the first indication in ciliated protozoa, such as Paramecium, of probable Golgi complex involvement in glycosylation similar to that in higher organisms. WGA-labeled coated vesicles in the endoplasm apparently lose their coats and coalesce to form lysosomes. Our study shows that WGA can be used as a specific intracellular marker of all digestive system membranes and of lysosomal content. These results support and extend our published scheme of membrane flow and recycling in Paramecium by providing another means of demonstrating membrane relationships.