Academic literature on the topic 'In vivo capping'

Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on-rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.

ABSTRACT The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase–CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, whilesrb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1–null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1’s ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their “actin phenotype.”

Conventional direct pulp capping, such as calcium hydroxide (Ca(OH)2) or silicate products, usually induces an inflammatory reaction to provoke pulp regeneration. Phosphophoryn (PP) and dentin sialoprotein (DSP), the two most abundant non-collagenous proteins in the dentin matrix, are responsible for dentin mineralization, pulp cell migration, and differentiation. Here we examined the PP and combined DSP/PP as bio-inductive pulp capping materials by in vitro and in vivo tests. Firstly, the effects of the PP dose on pulp cell migration and matrix protein expression were examined by an agarose bead test. Secondly, the role of recombinant DSP (recDSP) and recDSP/PP on stimulating DSP-PP transcript expression was examined by RT-PCR. DSPP mRNA was also knocked down by RNA interference (RNAi) to examine their functions on dentin matrix mineralization. Finally, we used ferret animal models to test PP and recDSP/PP acting as capping agents on in vivo pulp responses and reparative dentin formation. The result showed that intermediate-dose PP was the most effective to enhance cell migration and differentiation. RecDSP/PP strongly enhanced the DSP-PP transcript expression, while inhibition of DSPP mRNA expression by siRNAs partially or completely affected dental pulp cell mineralization. The in vivo results showed that intermediate-dose PP and recDSP/PP proteins induced less pulp inflammation and promoted reparative dentin formation. Contrarily, conventional calcium hydroxide induced severe pulp inflammation. With these findings, DSP and PP could serve as capping agents for pulp capping therapy.

A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution.

Background : Perforation in normal pulp could be happened anytime. In sterile condition, direct pulp capping was the right therapy to maintain the vitality and pulp function. Direct pulp capping agent must contact with the pulp tissue. Generally, body had an immunologic respond with foreign body that contact with tissue. The respond could be inflammatory reaction. The used direct pulp capping agents were calcium hydroxide, and Mineral Trioxide Aggregate (MTA) which known as better agent but relatively expensive and difficult to obtain. So that, the alternative of MTA, Portland cement which was the same essential of MTA, was being developed. The aim of this study, to analized inflammatory reaction of the pulp tissue with direct pulpcapping agents calcium hydroxide, MTA, and Portland cement.Methods: Free caries M. Nemestrina’s posterior teeth were prepared to formpin point perforations on buccal surface of the crowns. The teeth were appliedwith one of the three capping agents. Teeth were filled and extracted. Theextracted teeth were proceed into histopatological preparation slides to evaluatethe inflammatic reaction.Results: The result showed no statistically significant differences of pulp inflammatory reaction between calcium hydroxide, MTA and Portland cement in7, 14, 42, and 90 days.Conclusion: The inflammatory reaction of pulp tissue of the three pulp cappingagent (calcium hydroxide, MTA, and PC) were insignificant different.

Objective: To do a clinical and radiographic evaluation of the effectiveness of MTA when used as a direct pulp capping material in primary molars. Study design: Clinical and radiographic follow-up was performed on 30 primary molars with deep caries lesions in 30 patients from 3 to 9.75 years of age. Pulps exposed during cavity preparation were treated by direct pulp capping with MTA. The follow-up clinical and radiographic examinations were carried out at different time intervals: 6, 7–12, 13–18, 19–24, and >24 months after treatment. Results: Twenty-four teeth were evaluated during the entire observation period. Positive clinical and radiographic outcomes were achieved in 19 teeth (13 teeth were saved and 6 were exfoliated). In five teeth, complications were observed only in children under 7 years old. Conclusion: Based on these clinical and radiographic results, MTA was found to be successful when used as a direct pulp capping material in primary teeth.

ABSTRACTObjectives:This study aimed to evaluate in vivo the occurrence of secondary caries and dentin characteristics in permanent molars after traditional amalgam restorations, by means of clinical visual examination, radiographs and laser-induced fluorescence (LF) (DIAGNOdent).Methods:Thirty first permanent molars of 30 schoolchildren in the 7 to14 year-old age group were included. Caries was removed by hand. Thus, indirect pulp capping was performed with glassionomer cement (GIC), the cavity was varnished and amalgam filled. LF was measured before and after cavity preparation and after a 12-month observation period. Dentin color after cavity preparation and after the 12-month observation period was recorded. Recurrent caries was also investigated by visual clinical and radiographic examinations, in addition to dentin thickness between pulp and indirect GIC pulp capping. Data was analyzed by ANOVA for repeated measurements, paired “t” test and descriptive statistic.Results:There were statistically significant differences (P<.05) among LF scores for dentin in all periods evaluated, with the lowest scores shown after 12 month of observation. There was no statistical difference between dentin color after cavity preparation and following 12 months of observation. Moreover, there was no recurrent caries attack at 12-month follow-up; dentin thickness between pulp and indirect GIC pulp capping was similar between baseline and final observation periods. It was concluded that the clinical restorative procedure using hand caries removal, indirect pulp capping with GIC, varnishing and amalgam filling the cavity did not provide secondary caries and increased dentin mineral content after 12 months. (Eur J Dent 2012;6:263-269)

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

Orientador: Carlos Alberto de Souza Costa
Banca: Osmir Batista de Oliveira Júnior
Banca: Maria Salete Machado Cândido
Banca: Alcebíades Nunes Barbosa
Banca: Adair Luiz Stefanello Busato
Resumo: Este estudo teve como objetivo avaliar a capacidade de formação de barreira de tecido duro quando da realização de pulpotomias, onde as feridas pulpares foram capeadas com três diferentes materiais. Hidróxido de cálcio, Agregado de Trióxido Mineral (MTA), e PROROOT® MTA foram estudados quanto à morfologia e localização da barreira de tecido reparador formada, e também quanto à extensão do reparo. Três métodos de avaliação (microscopia eletrônica de varredura, lupa estereoscópica e microscópio de luz) foram empregados para analisar as imagens da barreira de tecido duro, e comparados entre si para avaliar a abrangência do espectro de leitura de cada método. Cinco cães Beagle, com idade de doze meses foram utilizados, sendo as pulpotomias realizadas nos segundos, terceiros e quartos pré-molares. Os dentes foram capeados com os materiais experimentais e controle e as cavidades restauradas com amálgama de prata. Após o período mínimo de 90 dias e máximo de 105 os animais foram sacrificados e os dentes submetidos ao processamento laboratorial para avaliação em microscopia eletrônica de varredura. Um programa analisador de imagens foi empregado para delimitar e determinar as áreas dos condutos e compará-las com as áreas das barreiras de tecido duro formadas no local. Os dados da microscopia eletrônica de varredura foram utilizados para verificar a morfologia da barreira. A localização e extensão desta estrutura reparadora foi também avaliada com auxílio de lupa esteroscópica e microscópio de luz. Os resultados obtidos permitiram demonstrar que nos espécimes pertencentes ao grupo PROROOT® ocorreu maior número de reparações completas, caracterizadas pela deposição de barreira de tecido duro, a qual apresentava característica tubular, quando comparado ao grupo hidróxido de cálcio.... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This in vivo study was carried out to evaluate the reparative potential of pulps mechanically exposed following pulpotomy with three different capping agents. Calcium hydroxide, Mineral Trioxide Aggregate (MTA), and PROROOT® MTA were evaluated regarding the morphology, location, and extension of the reparative hardbarrier deposition. Three methods were used to evaluate the images from the reparative area (scanning electron microscope - SEM, stereo microscope - Lupe, and optical microscope). The methods were compared to evaluate the reading spectrum of each applied method. Five Beagle dogs, twelve months-old were used. Pulpotomies were performed in the second, third and forth premolars. The exposed pulps were capped with the selected experimental or control materials and the cavities restored with amalgam. After 90 to 106 days the animals were sacrificed and the teeth surgically removed in bloc were processed for SEM assessment. An image analyzer was used to measure the total area of the exposition and compare it to the area occupied by the hard-barrier. Data from SEM were used to evaluate morphology and location of the hard-barrier formation as well as the percentage of pulpal wound obliteration, which was also measured by stereo microscope and optical microscope. Larger number of samples in which PROROOT® was applied as pulp-capping agent exhibited complete hard-barrier formation when compared to calcium hydroxide and MTA. For most of PROROOT® samples, the hardbarrier exhibited several dentinal tubules. On the other hand, calcium hydroxide samples presented the lowest number of total repairs and the hard-barrier observed in a few samples exhibited amorphous histological characteristics. Finally, it was demonstrated that SEM evaluation does not allow detailed assessment of the hard-barrier formation such as its extension and location... (Complete abstract, click electronic address below)
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Este estudo teve como objetivo avaliar a capacidade de formação de barreira de tecido duro quando da realização de pulpotomias, onde as feridas pulpares foram capeadas com três diferentes materiais. Hidróxido de cálcio, Agregado de Trióxido Mineral (MTA), e PROROOT® MTA foram estudados quanto à morfologia e localização da barreira de tecido reparador formada, e também quanto à extensão do reparo. Três métodos de avaliação (microscopia eletrônica de varredura, lupa estereoscópica e microscópio de luz) foram empregados para analisar as imagens da barreira de tecido duro, e comparados entre si para avaliar a abrangência do espectro de leitura de cada método. Cinco cães Beagle, com idade de doze meses foram utilizados, sendo as pulpotomias realizadas nos segundos, terceiros e quartos pré-molares. Os dentes foram capeados com os materiais experimentais e controle e as cavidades restauradas com amálgama de prata. Após o período mínimo de 90 dias e máximo de 105 os animais foram sacrificados e os dentes submetidos ao processamento laboratorial para avaliação em microscopia eletrônica de varredura. Um programa analisador de imagens foi empregado para delimitar e determinar as áreas dos condutos e compará-las com as áreas das barreiras de tecido duro formadas no local. Os dados da microscopia eletrônica de varredura foram utilizados para verificar a morfologia da barreira. A localização e extensão desta estrutura reparadora foi também avaliada com auxílio de lupa esteroscópica e microscópio de luz. Os resultados obtidos permitiram demonstrar que nos espécimes pertencentes ao grupo PROROOT® ocorreu maior número de reparações completas, caracterizadas pela deposição de barreira de tecido duro, a qual apresentava característica tubular, quando comparado ao grupo hidróxido de cálcio....
This in vivo study was carried out to evaluate the reparative potential of pulps mechanically exposed following pulpotomy with three different capping agents. Calcium hydroxide, Mineral Trioxide Aggregate (MTA), and PROROOT® MTA were evaluated regarding the morphology, location, and extension of the reparative hardbarrier deposition. Three methods were used to evaluate the images from the reparative area (scanning electron microscope - SEM, stereo microscope - Lupe, and optical microscope). The methods were compared to evaluate the reading spectrum of each applied method. Five Beagle dogs, twelve months-old were used. Pulpotomies were performed in the second, third and forth premolars. The exposed pulps were capped with the selected experimental or control materials and the cavities restored with amalgam. After 90 to 106 days the animals were sacrificed and the teeth surgically removed in bloc were processed for SEM assessment. An image analyzer was used to measure the total area of the exposition and compare it to the area occupied by the hard-barrier. Data from SEM were used to evaluate morphology and location of the hard-barrier formation as well as the percentage of pulpal wound obliteration, which was also measured by stereo microscope and optical microscope. Larger number of samples in which PROROOT® was applied as pulp-capping agent exhibited complete hard-barrier formation when compared to calcium hydroxide and MTA. For most of PROROOT® samples, the hardbarrier exhibited several dentinal tubules. On the other hand, calcium hydroxide samples presented the lowest number of total repairs and the hard-barrier observed in a few samples exhibited amorphous histological characteristics. Finally, it was demonstrated that SEM evaluation does not allow detailed assessment of the hard-barrier formation such as its extension and location... (Complete abstract, click electronic address below)