Journal articles on the topic 'In vivo capping'
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Schafer, D. A., P. B. Jennings, and J. A. Cooper. "Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides." Journal of Cell Biology 135, no. 1 (October 1, 1996): 169–79. http://dx.doi.org/10.1083/jcb.135.1.169.
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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.
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Rodriguez, Christine R., Eun-Jung Cho, Michael-C. Keogh, Claire L. Moore, Arno L. Greenleaf, and Stephen Buratowski. "Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II." Molecular and Cellular Biology 20, no. 1 (January 1, 2000): 104–12. http://dx.doi.org/10.1128/mcb.20.1.104-112.2000.
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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.
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Jung, Goeh, Christopher J. Alexander, Xufeng S. Wu, Grzegorz Piszczek, Bi-Chang Chen, Eric Betzig, and John A. Hammer. "V-1 regulates capping protein activity in vivo." Proceedings of the National Academy of Sciences 113, no. 43 (October 10, 2016): E6610—E6619. http://dx.doi.org/10.1073/pnas.1605350113.
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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.”
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Chuang, Shu-Fen, Yu-Hsuan Chen, Peter X. Ma, and Helena H. Ritchie. "Dentin Sialoprotein/Phosphophoryn (DSP/PP) as Bio-Inductive Materials for Direct Pulp Capping." Polymers 14, no. 17 (September 3, 2022): 3656. http://dx.doi.org/10.3390/polym14173656.
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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.
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DiNubile, M. J., L. Cassimeris, M. Joyce, and S. H. Zigmond. "Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2." Molecular Biology of the Cell 6, no. 12 (December 1995): 1659–71. http://dx.doi.org/10.1091/mbc.6.12.1659.
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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.
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Lien Nghiem, Thi Ha, Thi Tuyen Nguyen, Emmanuel Fort, Thanh Phuong Nguyen, Thi My Nhung Hoang, Thi Quy Nguyen, and Hong Nhung Tran. "Capping and in vivo toxicity studies of gold nanoparticles." Advances in Natural Sciences: Nanoscience and Nanotechnology 3, no. 1 (February 9, 2012): 015002. http://dx.doi.org/10.1088/2043-6262/3/1/015002.
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Witasari, Jos Erry Hesti, Usman Munyati, and Anggraini Dewi. "THE INFLAMMATORY REACTION OF PULP TISSUE WITH CALCIUM HYDROXIDE, MTA AND PORTLAND CEMENT (In Vivo)." ODONTO : Dental Journal 4, no. 2 (December 1, 2017): 114. http://dx.doi.org/10.30659/odj.4.2.114-121.
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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.
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Luczaj-Cepowicz, Elzbieta, Grazyna Marczuk-Kolada, Malgorzata Pawinska, and Marta Obidzinska. "Direct Pulp Capping in Primary Molars using Mineral Trioxide Aggregate: An in Vivo Study." Journal of Clinical Pediatric Dentistry 41, no. 6 (January 1, 2017): 446–49. http://dx.doi.org/10.17796/1053-4628-41.6.5.
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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.
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Pinheiro, Isauremi Vieira de Assunção, Boniek Castillo Dutra Borges, and Kenio Costa de Lima. "In vivo assessment of secondary caries and dentin characteristics after traditional amalgam restorations." European Journal of Dentistry 06, no. 03 (July 2012): 263–69. http://dx.doi.org/10.1055/s-0039-1698960.
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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)
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Revenu, Céline, Matthieu Courtois, Alphée Michelot, Cécile Sykes, Daniel Louvard, and Sylvie Robine. "Villin Severing Activity Enhances Actin-based Motility In Vivo." Molecular Biology of the Cell 18, no. 3 (March 2007): 827–38. http://dx.doi.org/10.1091/mbc.e06-05-0423.
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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.
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Muruganandhan, Jayanandan, Govindarajan Sujatha, Saravanan Poorni, Manali Ramakrishnan Srinivasan, Nezar Boreak, Ahmed Al-Kahtani, Mohammed Mashyakhy, et al. "Comparison of Four Dental Pulp-Capping Agents by Cone-Beam Computed Tomography and Histological Techniques—A Split-Mouth Design Ex Vivo Study." Applied Sciences 11, no. 7 (March 29, 2021): 3045. http://dx.doi.org/10.3390/app11073045.
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Dental pulp-capping is done to preserve vital teeth when the pulp is exposed due to caries, trauma or instrumentation. Various materials are used as pulp-capping agents. The introduction of newer materials requires scientific studies to assess their clinical efficacy. The study was designed as a split-mouth randomized analysis of four pulp-capping agents (calcium hydroxide, mineral trioxide aggregate (MTA), Biodentine and EndoSequence root repair material (ERRM)). Based on selection criteria, 15 orthodontic patients requiring the extraction of four premolars (60 teeth total) were included in the study. After pulp-capping, the teeth were extracted after 8 weeks. We analyzed the extracted teeth using cone-beam computed tomography (CBCT) and histological sections to determine the quality of the dentinal bridge and the pulpal response. Ordinal scores were given based on the completeness of the dentinal bridge, the type of bridge and the degree of pulpal inflammation. Results were analyzed using a Kruskal–Wallis test (p < 0.05) with post hoc Conover values being used when applicable. All four pulp-capping materials elicited dentinal bridge formation (60/60). MTA had the highest scores (10/15) in dentinal bridge formation followed by ERRM (8/15). Both materials showed more samples with complete dentinal bridges (9/15 each) and a favorable pulpal response (15/15). Teeth capped with calcium hydroxide showed more cases of incomplete bridge formation (9/15) and pulpal inflammation. These differences in dentinal bridge formation and pulpal inflammation were statistically significant (p 0.001 and p 0.00005, respectively), with post hoc tests revealing no significant differences between MTA and ERRM (p 0.49 and p 0.71, respectively). MTA and ERRM performed better than the other pulp-capping materials but did not differ significantly from each other. The individual preference for a pulp-capping material may be based on clinical efficacy and handling characteristics.
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Ho, C. Kiong, Alexandra Martins, and Stewart Shuman. "A Yeast-Based Genetic System for Functional Analysis of Viral mRNA Capping Enzymes." Journal of Virology 74, no. 12 (June 15, 2000): 5486–94. http://dx.doi.org/10.1128/jvi.74.12.5486-5494.2000.
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ABSTRACT Virus-encoded mRNA capping enzymes are attractive targets for antiviral therapy, but functional studies have been limited by the lack of genetically tractable in vivo systems that focus exclusively on the RNA-processing activities of the viral proteins. Here we have developed such a system by engineering a viral capping enzyme—vaccinia virus D1(1-545)p, an RNA triphosphatase and RNA guanylyltransferase—to function in the budding yeast Saccharomyces cerevisiae in lieu of the endogenous fungal triphosphatase (Cet1p) and guanylyltransferase (Ceg1p). This was accomplished by fusion of D1(1-545)p to the C-terminal guanylyltransferase domain of mammalian capping enzyme, Mce1(211-597)p, which serves as a vehicle to target the viral capping enzyme to the RNA polymerase II elongation complex. An inactivating mutation (K294A) of the mammalian guanylyltransferase active site in the fusion protein had no impact on genetic complementation of cet1Δceg1Δ cells, thus proving that (i) the viral guanylyltransferase was active in vivo and (ii) the mammalian domain can serve purely as a chaperone to direct other proteins to the transcription complex. Alanine scanning had identified five amino acids of vaccinia virus capping enzyme—Glu37, Glu39, Arg77, Glu192, and Glu194—that are essential for γ phosphate cleavage in vitro. Here we show that the introduction of mutation E37A, R77A, or E192A into the fusion protein abrogates RNA triphosphatase function in vivo. The essential residues are located within three motifs that define a family of viral and fungal metal-dependent phosphohydrolases with a distinctive capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates in the presence of manganese or cobalt. The acidic residues Glu37, Glu39, and Glu192 likely comprise the metal-binding site of vaccinia virus triphosphatase, insofar as their replacement by glutamine abolishes the RNA triphosphatase and ATPase activities.
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Takase, Yasutaka, Toshimitsu Takagi, Philip B. Komarnitsky, and Stephen Buratowski. "The Essential Interaction between Yeast mRNA Capping Enzyme Subunits Is Not Required for Triphosphatase Function In Vivo." Molecular and Cellular Biology 20, no. 24 (December 15, 2000): 9307–16. http://dx.doi.org/10.1128/mcb.20.24.9307-9316.2000.
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ABSTRACT The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (Cet1) and an mRNA guanylyltransferase (Ceg1). In yeast, the capping enzyme is recruited to the RNA polymerase II (Pol II) transcription complex via an interaction between Ceg1 and the phosphorylated carboxy-terminal domain of the Pol II largest subunit. Previous in vitro experiments showed that the Cet1 carboxy-terminal region (amino acids 265 to 549) carries RNA triphosphatase activity, while the region containing amino acids 205 to 265 of Cet1 has two functions: it mediates dimerization with Ceg1, but it also allosterically activates Ceg1 guanylyltransferase activity in the context of Pol II binding. Here we characterize several Cet1 mutants in vivo. Mutations or deletions of Cet1 that disrupt interaction with Ceg1 are lethal, showing that this interaction is essential for proper capping enzyme function in vivo. Remarkably, the interaction region of Ceg1 becomes completely dispensable when Ceg1 is substituted by the mouse guanylyltransferase, which does not require allosteric activation by Cet1. Although no interaction between Cet1 and mouse guanylyltransferase is detectable, both proteins are present at yeast promoters in vivo. These results strongly suggest that the primary physiological role of the Ceg1-Cet1 interaction is to allosterically activate Ceg1, rather than to recruit Cet1 to the Pol II complex.
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Berro, Julien, and Thomas D. Pollard. "Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin-mediated endocytosis and cell polarization in fission yeast." Molecular Biology of the Cell 25, no. 22 (November 5, 2014): 3515–27. http://dx.doi.org/10.1091/mbc.e13-01-0005.
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Aip1p cooperates with actin-depolymerizing factor (ADF)/cofilin to disassemble actin filaments in vitro and in vivo, and is proposed to cap actin filament barbed ends. We address the synergies between Aip1p and the capping protein heterodimer Acp1p/Acp2p during clathrin-mediated endocytosis in fission yeast. Using quantitative microscopy and new methods we have developed for data alignment and analysis, we show that heterodimeric capping protein can replace Aip1p, but Aip1p cannot replace capping protein in endocytic patches. Our quantitative analysis reveals that the actin meshwork is organized radially and is compacted by the cross-linker fimbrin before the endocytic vesicle is released from the plasma membrane. Capping protein and Aip1p help maintain the high density of actin filaments in meshwork by keeping actin filaments close enough for cross-linking. Our experiments also reveal new cellular functions for Acp1p and Acp2p independent of their capping activity. We identified two independent pathways that control polarization of endocytic sites, one depending on acp2+ and aip1+ during interphase and the other independent of acp1+, acp2+, and aip1+ during mitosis.
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Saha, Nayanendu, Stewart Shuman, and Beate Schwer. "Yeast-Based Genetic System for Functional Analysis of Poxvirus mRNA Cap Methyltransferase." Journal of Virology 77, no. 13 (July 1, 2003): 7300–7307. http://dx.doi.org/10.1128/jvi.77.13.7300-7307.2003.
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ABSTRACT Structural differences between poxvirus and human mRNA capping enzymes recommend cap formation as a target for antipoxviral drug discovery. Genetic and pharmacologic analysis of the poxvirus capping enzymes requires in vivo assays in which the readout depends on the capacity of the viral enzyme to catalyze cap synthesis. Here we have used the budding yeast Saccharomyces cerevisiae as a genetic model for the study of poxvirus cap guanine-N7 methyltransferase. The S. cerevisiae capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components. All three activities are essential for cell growth. We report that the methyltransferase domain of vaccinia virus capping enzyme (composed of catalytic vD1-C and stimulatory vD12 subunits) can function in lieu of yeast Abd1. Coexpression of both vaccinia virus subunits is required for complementation of the growth of abd1Δ cells. Previously described mutations of vD1-C and vD12 that eliminate or reduce methyltransferase activity in vitro either abolish abd1Δ complementation or elicit conditional growth defects. We have used the yeast complementation assay as the primary screen in a new round of alanine scanning of the catalytic subunit. We thereby identified several new amino acids that are critical for cap methylation activity in vivo. Studies of recombinant proteins show that the lethal vD1-C mutations do not preclude heterodimerization with vD12 but either eliminate or reduce cap methyltransferase activity in vitro.
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Sizonenko, G. I., T. S. Karpova, D. J. Gattermeir, and J. A. Cooper. "Mutational analysis of capping protein function in Saccharomyces cerevisiae." Molecular Biology of the Cell 7, no. 1 (January 1996): 1–15. http://dx.doi.org/10.1091/mbc.7.1.1.
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To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.
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Kim, Kyoungtae, Atsuko Yamashita, Martin A. Wear, Yuichiro Maéda, and John A. Cooper. "Capping protein binding to actin in yeast." Journal of Cell Biology 164, no. 4 (February 9, 2004): 567–80. http://dx.doi.org/10.1083/jcb.200308061.
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The mechanism by which capping protein (CP) binds barbed ends of actin filaments is not understood, and the physiological significance of CP binding to actin is not defined. The CP crystal structure suggests that the COOH-terminal regions of the CP α and β subunits bind to the barbed end. Using purified recombinant mutant yeast CP, we tested this model. CP lacking both COOH-terminal regions did not bind actin. The α COOH-terminal region was more important than that of β. The significance of CP's actin-binding activity in vivo was tested by determining how well CP actin-binding mutants rescued null mutant phenotypes. Rescue correlated well with capping activity, as did localization of CP to actin patches, indicating that capping is a physiological function for CP. Actin filaments of patches appear to be nucleated first, then capped with CP. The binding constants of yeast CP for actin suggest that actin capping in yeast is more dynamic than in vertebrates.
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Takagi, Toshimitsu, Eun-Jung Cho, Rozmin T. K. Janoo, Vladimir Polodny, Yasutaka Takase, Michael-C. Keogh, Sue-Ann Woo, Lucille D. Fresco-Cohen, Charles S. Hoffman, and Stephen Buratowski. "Divergent Subunit Interactions among Fungal mRNA 5′-Capping Machineries." Eukaryotic Cell 1, no. 3 (June 2002): 448–57. http://dx.doi.org/10.1128/ec.1.3.448-457.2002.
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ABSTRACT The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans. Surprisingly, the S. pombe capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The S. pombe RTPase gene (pct1 +) is essential for viability. Pct1 can replace the S. cerevisiae RTPase when GTase activity is supplied by the S. pombe or mouse enzymes but not by the S. cerevisiae GTase. The C. albicans capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.
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Agrawal, Navin, Vimmi Singh, Ashish Shrestha, Mehul Jaisani, Iccha Kumar Maharjan, and Neetu Jain. "Evaluation of Mineral Trioxide Aggregate and Biodentine as Direct Pulp-capping Agents in Human Teeth: An Ex-Vivo Study." Journal of Nepalese Association of Pediatric Dentistry 3, no. 1 (December 12, 2022): 14–19. http://dx.doi.org/10.3126/jnapd.v3i1.50059.
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Introduction: Biodentine (Tricalcium Silicate based) is a modern bioactive cement that is comparable to the broadly utilized mineral trioxide aggregate (MTA). It has dentin-like mechanical properties, which can be considered appropriate material for diseased dentin-pulp complex recovery such as direct pulp capping. The present study was to compare the reaction of the pulp-dentin complex in human teeth after direct pulp capping with use of MTA and Biodentine. Objective: To assess the histomorphologic response of human dental pulps on direct pulp capping with MTA and Biodentine. Methods: Pulps of 34 maxillary permanent intact human third molars planned for extraction were mechanically exposed with sterile rotary round bur and allotted to one of the two exploratory groups, MTA or Biodentine. After four weeks, the teeth were extracted, stained with hematoxylin- eosin, and categorized by employing a preset histologic scoring framework. The histopathologic evaluations scored data were recorded and statically analyzed using SPSS where Mann-Whitney U test was used and p<0.05 was considered statistically significant. Results: No inflammatory pulp reaction seen in 34 (100%) teeth whereas 19 (55.9 %) showed complete dentinal bridge arrangement. Layers of well-arranged odontoblast and odontoblast-like cells were found to make tubular dentin beneath the osteodentin. Inferential statistics using showed insignificance between the MTA and Biodentine test groups amid the perception period, showing similar outcomes. Conclusions: Biodentine had comparative adequacy in the clinical setting and may be considered an appropriate alternative to MTA as the pulp- capping agent.
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LaPointe, Autumn T., and Kevin J. Sokoloski. "Noncapped Genomic RNA Are Critical for Alphaviral Infection and Pathogenicity." Proceedings 50, no. 1 (June 13, 2020): 44. http://dx.doi.org/10.3390/proceedings2020050044.
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Alphaviruses are positive-sense RNA arthropod-borne viruses that represent a significant threat to public health. During alphaviral replication, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced and packaged into viral particles, despite the fact that the noncapped genomes cannot be translated and are essentially noninfectious. Previously, we have reported that the capping efficiency of nsP1, the alphaviral capping enzyme, of Sindbis virus (SINV) could be modulated via point mutation. It was found that increasing RNA capping efficiency led to decreased viral growth kinetics via decreased particle production, despite increased innate immune evasion, whereas decreasing capping efficiency led to wild-type growth kinetics and particle production. This led to the conclusion that the noncapped viral RNAs meaningfully contribute to the biology of alphaviral infections at the molecular level. To determine the importance of the noncapped viral RNAs in vivo, we characterized the impact of altered capping efficiency in a murine model of infection utilizing a neurovirulent strain of SINV. Mice infected with the nsP1 mutant with decreased capping exhibited wild-type rates of mortality, weight loss, and neurological symptoms. Conversely, the mice infected with the increased capping nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with the wild-type virus. Interestingly, viral titers in the ankle, serum, and brain were equivalent between the wild-type virus and the two mutant viruses. Importantly, examination of the brain tissue revealed that mice infected with the increased capping mutant had significantly reduced immune cell infiltration and expression of proinflammatory cytokines compared to the decreased capping mutant and wild-type virus. Collectively, these data indicate that the noncapped viral RNAs have important roles during the early and late stages of alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aids in viral pathogenesis.
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Sood, Akanksha, Nisha Garg, Ajay Chhabra, Ankita Sood, Suvaani Kataria, and Vandana Chhabra. "Outcome of direct pulp capping in young permanent molars with three different pulp capping agents: An in vivo study." Baba Farid University Dental Journal 12, no. 1 (2022): 34–37. http://dx.doi.org/10.5958/2230-7273.2022.00004.7.
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Silva, Julianne Coelho, Tainah Oliveira Rifane, Antônio Ernando Ferreira-Junior, Ana Paula Alves, Richard Miron, Yufeng Zhang, Pierre Basílio Almeida Fechine, Elayne Valério Carvalho, and Victor Pinheiro Feitosa. "In Vitro and In Vivo Efficacy of New Composite for Direct Pulp Capping." BioMed Research International 2021 (November 25, 2021): 1–10. http://dx.doi.org/10.1155/2021/8414577.
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Objectives. To investigate physicochemical properties, dentin bonding, cytotoxicity, and in vivo pulp response of experimental self-adhesive composites tailored to direct pulp capping. Materials and Methods. Experimental composites were prepared with beta-tricalcium phosphate and hydroxyapatite nanoparticles adsorbed with simvastatin and glutathione added at 0% (control resin), 1 wt% (Res 1%), and 10 wt% (Res 10%). A commercial light-curable calcium hydroxide (Ca(OH)2) (Ultra-Blend Plus) was used as control material. The physicochemical properties investigated were flexural strength and modulus, calcium release, and degree of conversion. Dentin bonding was assessed by the push-out test. Proliferation and cell counting assays were performed to evaluate in vitro cytotoxicity using fluorescence microscopy. In vivo pulp capping was performed on molars of Wistar rats, which were euthanized after 14 days and evaluated by histological analysis. Results. No statistical difference was observed in flexural strength and cell viability ( p > 0.05 ). Res 10% presented higher modulus than control resin and Ca(OH)2. Also, Res 10% attained statistically higher degree of conversion when compared to other experimental composites. Ca(OH)2 showed higher calcium release after 28 and 45 days of storage, with no statistical difference at 45 days to Res 10%. All experimental composites achieved significantly higher bond strength when compared to Ca(OH)2. While no significant difference was observed in the cell proliferation rates, resins at lower concentrations showed higher cell viability. In vivo evaluation of pulp response demonstrated no pulp damage with experimental composites. Conclusions. The experimental composite investigated in this study achieved adequate physicochemical properties with minor in vivo pulpal inflammation and proved to be a valuable alternative for direct pulp capping.
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Whitehouse, LLE, NH Thomson, T. Do, and GA Feichtinger. "Bioactive molecules for regenerative pulp capping." European Cells and Materials 42 (November 29, 2021): 415–37. http://dx.doi.org/10.22203/ecm.v042a26.
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Since the discovery of bioactive molecules sequestered in dentine, researchers have been exploring ways to harness their activities for dental regeneration. One specific area, discussed in this review, is that of dental-pulp capping. Dental-pulp caps are placed when the dental pulp is exposed due to decay or trauma in an attempt to enhance tertiary dentine deposition. Several materials are used for dental-pulp capping; however, natural biomimetic scaffolds may offer advantages over manufactured materials such as improved aesthetic, biocompatibility and success rate. The present review discusses and appraises the current evidence surrounding biomimetic dental-pulp capping, with a focus on bioactive molecules sequestered in dentine. Molecules covered most extensively in the literature include transforming growth factors (TGF-βs, specifically TGF-β1) and bone morphogenetic proteins (BMPs, specifically BMP-2 and BMP-7). Further studies would need to explore the synergistic use of multiple peptides together with the development of a tailored scaffold carrier. The roles of some of the molecules identified in dentine need to be explored before they can be considered as potential bioactive molecules in a biomimetic scaffold for dental-pulp capping. Future in vivo work needs to consider the inflammatory environment of the dental pulp in pulpal exposures and compare pulp-capping materials.
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Suprenant, K. A., and W. L. Dentler. "Release of intact microtubule-capping structures from Tetrahymena cilia." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2259–69. http://dx.doi.org/10.1083/jcb.107.6.2259.
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The distal ends of ciliary microtubules are attached to the membrane by microtubule-capping structures. The capping structures are located at the sites of tubulin addition and loss in vivo and may be part of the regulatory system that directs ciliary and flagellar microtubule assembly. This study describes conditions for the release and stabilization of microtubule capping structures as a first step in their purification. Two types of capping structures, the distal filaments and the central microtubule caps, are selectively and independently released from the axoneme by CaCl2 and MgCl2 but not by MgSO4, ZnCl2, NaCl, KCl, or KI. The release of the caps and filaments is specific for Ca+2, Mg+2, and Cl- and is not simply a function of ionic strength. The capping structures are released without major disruption of the axonemal structure. In addition to providing a means to purify and identify the cap and filament components, these results suggest ways in which their binding to the axoneme may be modulated during periods of microtubule growth or shortening. This report also reveals that the distal filaments are composed of two separable components, a small bead inserted into the end of each A-tubule and a "Y"-shaped plug and filament that slips through the bead.
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Kunert, Marta, and Monika Lukomska-Szymanska. "Bio-Inductive Materials in Direct and Indirect Pulp Capping—A Review Article." Materials 13, no. 5 (March 7, 2020): 1204. http://dx.doi.org/10.3390/ma13051204.
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The article is aimed at analyzing the available research and comparing the properties of bio-inductive materials in direct and indirect pulp capping procedures. The properties and clinical performances of four calcium-silicate cements (ProRoot MTA, MTA Angelus, RetroMTA, Biodentine), a light-cured calcium silicate-based material (TheraCal LC) and an enhanced resin-modified glass-ionomer (ACTIVA BioACTIVE) are widely discussed. A correlation of in vitro and in vivo data revealed that, currently, the most validated material for pulp capping procedures is still MTA. Despite Biodentine’s superiority in relatively easier manipulation, competitive pricing and predictable clinical outcome, more long-term clinical studies on Biodentine as a pulp capping agent are needed. According to available research, there is also insufficient evidence to support the use of TheraCal LC or ACTIVA BioACTIVE BASE/LINER in vital pulp therapy.
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Widjiastuti, Ira, Ari Subiyanto, Evri Kusumah Ningtyas, Rendy Popyandra, Michael Golden Kurniawan, and Fauziah Diajeng Retnaningsih. "Propolis extract as pulp capping material enhances odontoblast-like cell thickness and type 1 collagen expression (in vivo)." Dental Journal (Majalah Kedokteran Gigi) 53, no. 1 (March 31, 2020): 1. http://dx.doi.org/10.20473/j.djmkg.v53.i1.p1-5.
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Background: Propolis is a natural biocompatible material that has been widely studied in dentistry because of its inflammatory, anti-microbial and immunomodulatory properties. One of the active components is caffeic acid phenethyl ester (CAPE). CAPE is effective in stimulating collagen as well as inhibiting the inflammation and degeneration of dental pulp. Purpose: To investigate the post-administration of propolis extract as pulp capping material enhances odontoblast-like cell thickness and type 1 collagen expression in Wistar rats (Rattus Norvegicus) Methods: This research was a true experimental design with a posttest-only control group design. Sixty-three Wistar rats were randomly divided into three groups, with each group consisting of 21 rats: Group I: Positive control; no capping material was administered; Group II: CAPE was administered; Group III: 11% of the propolis extract was administered. All samples were filled with glass ionomer cement. Seven rats from each group were sacrificed after days 7, 14 and 28 of post-pulp capping administration, and their afflicted teeth were subsequently extracted for histologic analysis. Results: No significant difference was seen in odontoblast-like cell thickness after the application of CAPE and propolis on days 7 and 14 (p > 0.05). However, a significant difference was noticed on day 28 (p < 0.05), with the thickness of odontoblast-like cell in CAPE being thinner than that in propolis. A significant difference in the expression of type 1 collagen was observed on days 7, 14 and 28 after the application of the propolis extract compared with CAPE (p < 0.05). Conclusion: The post-administration of propolis extract as a pulp capping material could enhance odontoblast-like cell thickness and type 1 collagen expression in Wistar rats.
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Ho, C. Kiong, Beate Schwer, and Stewart Shuman. "Genetic, Physical, and Functional Interactions between the Triphosphatase and Guanylyltransferase Components of the Yeast mRNA Capping Apparatus." Molecular and Cellular Biology 18, no. 9 (September 1, 1998): 5189–98. http://dx.doi.org/10.1128/mcb.18.9.5189.
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ABSTRACT We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme). CES5 is identical toCET1, which encodes the RNA triphosphatase component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes hydrolysis of the γ phosphate of triphosphate-terminated RNA at a rate of 1 s−1. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the result of increased affinity for the GTP substrate. A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary. The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth. This work highlights the potential for regulating mRNA cap formation through protein-protein interactions.
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Hsiao, Susan J., Marc F. Poitras, Brandoch D. Cook, Yie Liu, and Susan Smith. "Tankyrase 2 Poly(ADP-Ribose) Polymerase Domain-Deleted Mice Exhibit Growth Defects but Have Normal Telomere Length and Capping." Molecular and Cellular Biology 26, no. 6 (March 15, 2006): 2044–54. http://dx.doi.org/10.1128/mcb.26.6.2044-2054.2006.
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ABSTRACT Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardation phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement for Tnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together, these results suggest that Tnks2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.
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Karpova, T. S., K. Tatchell, and J. A. Cooper. "Actin filaments in yeast are unstable in the absence of capping protein or fimbrin." Journal of Cell Biology 131, no. 6 (December 15, 1995): 1483–93. http://dx.doi.org/10.1083/jcb.131.6.1483.
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Many actin-binding proteins affect filament assembly in vitro and localize with actin in vivo, but how their molecular actions contribute to filament assembly in vivo is not understood well. We report here that capping protein (CP) and fimbrin are both important for actin filament assembly in vivo in Saccharomyces cerevisiae, based on finding decreased actin filament assembly in CP and fimbrin mutants. We have also identified mutations in actin that enhance the CP phenotype and find that those mutants also have decreased actin filament assembly in vivo. In vitro, actin purified from some of these mutants is defective in polymerization or binding fimbrin. These findings support the conclusion that CP acts to stabilize actin filaments in vivo. This conclusion is particularly remarkable because it is the opposite of the conclusion drawn from recent studies in Dictyostelium (Hug, C., P.Y. Jay, I. Reddy, J.G. McNally, P.C. Bridgman, E.L. Elson, and J.A. Cooper. 1995. Cell. 81:591-600). In addition, we find that the unpolymerized pool of actin in yeast is very small relative to that found in higher cells, which suggests that actin filament assembly is less dynamic in yeast than higher cells.
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Hart, Marilyn C., and John A. Cooper. "Vertebrate Isoforms of Actin Capping Protein β Have Distinct Functions in Vivo." Journal of Cell Biology 147, no. 6 (December 13, 1999): 1287–98. http://dx.doi.org/10.1083/jcb.147.6.1287.
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Actin capping protein (CP) binds barbed ends of actin filaments to regulate actin assembly. CP is an α/β heterodimer. Vertebrates have conserved isoforms of each subunit. Muscle cells contain two β isoforms. β1 is at the Z-line; β2 is at the intercalated disc and cell periphery in general. To investigate the functions of the isoforms, we replaced one isoform with another using expression in hearts of transgenic mice. Mice expressing β2 had a severe phenotype with juvenile lethality. Myofibril architecture was severely disrupted. The β2 did not localize to the Z-line. Therefore, β1 has a distinct function that includes interactions at the Z-line. Mice expressing β1 showed altered morphology of the intercalated disc, without the lethality or myofibril disruption of the β2-expressing mice. The in vivo function of CP is presumed to involve binding barbed ends of actin filaments. To test this hypothesis, we expressed a β1 mutant that poorly binds actin. These mice showed both myofibril disruption and intercalated disc remodeling, as predicted. Therefore, CPβ1 and CPβ2 each have a distinct function that cannot be provided by the other isoform. CPβ1 attaches actin filaments to the Z-line, and CPβ2 organizes the actin at the intercalated discs.
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Moya-Ramírez, Ignacio, Clement Bouton, Cleo Kontoravdi, and Karen Polizzi. "High resolution biosensor to test the capping level and integrity of mRNAs." Nucleic Acids Research 48, no. 22 (November 5, 2020): e129-e129. http://dx.doi.org/10.1093/nar/gkaa955.
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Abstract 5′ Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing, translation initiation and stability. Here we describe a biosensor designed to detect the presence of cap structures on mRNAs that is also sensitive to mRNA degradation, so uncapped or degraded mRNAs can be detected in a single step. The biosensor is based on a chimeric protein that combines the recognition and transduction roles in a single molecule. The main feature of this sensor is its simplicity, enabling semi-quantitative analyses of capping levels with minimal instrumentation. The biosensor was demonstrated to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range remained constant with RNAs ranging in size from 250 nt to approximately 2700 nt and the biosensor was able to detect variations in the capping level in increments of at least 20%, with a limit of detection of 2.4 pmol. Remarkably, it also can be applied to more complex analytes, such mRNA vaccines and mRNAs transcribed in vivo. This biosensor is an innovative example of a technology able to detect analytically challenging structures such as mRNA caps. It could find application in a variety of scenarios, from quality analysis of mRNA-based products such as vaccines to optimization of in vitro capping reactions.
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Kim, Kyoungtae, Brian J. Galletta, Kevin O. Schmidt, Fanny S. Chang, Kendall J. Blumer, and John A. Cooper. "Actin-based Motility during Endocytosis in Budding Yeast." Molecular Biology of the Cell 17, no. 3 (March 2006): 1354–63. http://dx.doi.org/10.1091/mbc.e05-10-0925.
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Actin assembly nucleated by Arp2/3 complex has been implicated in the formation and movement of endocytic vesicles. The dendritic nucleation model has been proposed to account for Arp2/3-mediated actin assembly and movement. Here, we explored the model by examining the role of capping protein in vivo, with quantitative tracking analysis of fluorescence markers for different stages of endocytosis in yeast. Capping protein was most important for the initial movement of endocytic vesicles away from the plasma membrane, which presumably corresponds to vesicle scission and release. The next phase of endosome movement away from the plasma membrane was also affected, but less so. The results are consistent with the dendritic nucleation model's prediction of capping protein as important for efficient actin assembly and force production. In contrast, the movement of late-stage endocytic vesicles, traveling through the cytoplasm en route to the vacuole, did not depend on capping protein. The movement of these vesicles was found previously to depend on Lsb6, a WASp interactor, whereas Lsb6 was found here to be dispensable for early endosome movement. Thus, the molecular requirements for Arp2/3-based actin assembly differ in early versus later stages of endocytosis. Finally, acute loss of actin cables led to increased patch motility.
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Arandi, Naji Ziad, and Tarek Rabi. "TheraCal LC: From Biochemical and Bioactive Properties to Clinical Applications." International Journal of Dentistry 2018 (2018): 1–6. http://dx.doi.org/10.1155/2018/3484653.
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Background. Direct pulp capping is a popular treatment modality among dentists. TheraCal LC is a calcium silicate-based material that is designed as a direct/indirect pulp capping material. The material might be very attractive for clinicians because of its ease of handling. Unlike other calcium silicate-based materials, TheraCal LC is resin-based and does not require any conditioning of the dentine surface. The material can be bonded with different types of adhesives directly after application. There has been considerable research performed on this material since its launching; however, there are no review articles that collates information and data obtained from these studies. This review discusses the various characteristics of the material with the aim of establishing a better understanding for its clinical use. Methods. A search was conducted using search engines (PubMed and Cochrane databases) in addition to reference mining of the articles that was used to locate other papers. The process of searching for the relevant studies was performed using the keywords pulp protection, pulp capping, TheraCal, and calcium silicates. Only articles in English published in peer-reviewed journals were included in the review. Conclusion. This review underlines the fact that further in vitro and in vivo studies are required before TheraCal LC can be used as a direct pulp capping material.
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Casella, J. F., S. W. Craig, D. J. Maack, and A. E. Brown. "Cap Z(36/32), a barbed end actin-capping protein, is a component of the Z-line of skeletal muscle." Journal of Cell Biology 105, no. 1 (July 1, 1987): 371–79. http://dx.doi.org/10.1083/jcb.105.1.371.
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Various biological activities have been attributed to actin-capping proteins based on their in vitro effects on actin filaments. However, there is little direct evidence for their in vivo activities. In this paper, we show that Cap Z(36/32), a barbed end, actin-capping protein isolated from muscle (Casella, J. F., D. J. Maack, and S. Lin, 1986, J. Biol. Chem., 261:10915-10921) is localized to the barbed ends of actin filaments by electron microscopy and to the Z-line of chicken skeletal muscle by indirect immunofluorescence and electron microscopy. Since actin filaments associate with the Z-line at their barbed ends, these findings suggest that Cap Z(36/32) may play a role in regulating length, orienting, or attaching actin filaments to Z-discs.
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Abdel Sameia, MonaM, AbeerM Darrag, and WalaaM Ghoneim. "Two calcium silicate-based materials used in direct pulp capping (in-vivo study)." Tanta Dental Journal 17, no. 2 (2020): 78. http://dx.doi.org/10.4103/tdj.tdj_5_20.
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Pei, Yi, Hongyan Du, Juliet Singer, Courtney St. Amour, Selena Granitto, Stewart Shuman, and Robert P. Fisher. "Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo." Molecular and Cellular Biology 26, no. 3 (February 1, 2006): 777–88. http://dx.doi.org/10.1128/mcb.26.3.777-788.2006.
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ABSTRACT Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK—Mcs6, the kinase component of transcription factor IIH (TFIIH)—cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1 + cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1Δ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1—the third essential CDK-cyclin complex described in fission yeast—helps to target the capping apparatus to the transcriptional elongation complex.
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Gnanadhas, Divya Prakash, Midhun Ben Thomas, Rony Thomas, Ashok M. Raichur, and Dipshikha Chakravortty. "Interaction of Silver Nanoparticles with Serum Proteins Affects Their Antimicrobial ActivityIn Vivo." Antimicrobial Agents and Chemotherapy 57, no. 10 (July 22, 2013): 4945–55. http://dx.doi.org/10.1128/aac.00152-13.
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ABSTRACTThe emergence of multidrug-resistant bacteria is a global threat for human society. There exist recorded data that silver was used as an antimicrobial agent by the ancient Greeks and Romans during the 8th century. Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities, with minimal cytotoxic effects on the cells. However, very few reports have shown the usage of AgNPs for antibacterial therapyin vivo. In this study, we deciphered the importance of the chosen methods for synthesis and capping of AgNPs for their improved activityin vivo. The interaction of AgNPs with serum albumin has a significant effect on their antibacterial activity. It was observed that uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins, due to the interaction with bovine serum albumin (BSA), which was confirmed by UV-Vis spectroscopy. However, capped AgNPs [with citrate or poly(vinylpyrrolidone)] exhibited antibacterial properties due to minimized interactions with serum proteins. The damage in the bacterial membrane was assessed by flow cytometry, which also showed that only capped AgNPs exhibited antibacterial properties, even in the presence of BSA. In order to understand thein vivorelevance of the antibacterial activities of different AgNPs, a murine salmonellosis model was used. It was conclusively proved that AgNPs capped with citrate or PVP exhibited significant antibacterial activitiesin vivoagainstSalmonellainfection compared to uncapped AgNPs. These results clearly demonstrate the importance of capping agents and the synthesis method for AgNPs in their use as antimicrobial agents for therapeutic purposes.
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DuBois, Michelle L., Zara W. Haimberger, Martin W. McIntosh, and Daniel E. Gottschling. "A Quantitative Assay for Telomere Protection in Saccharomyces cerevisiae." Genetics 161, no. 3 (July 1, 2002): 995–1013. http://dx.doi.org/10.1093/genetics/161.3.995.
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Abstract Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This “chromosomal cap” assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected.
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Casagrande, Luciano, Letícia Westphalen Bento, Simone Ossok Rerin, Èvelin de Resende Lucas, Débora Martini Dalpian, and Fernando Borba de Araujo. "In vivo outcomes of Indirect Pulp Treatment using a Self-etching Primer versus Calcium Hydroxide over the Demineralized Dentin in Primary Molars." Journal of Clinical Pediatric Dentistry 33, no. 2 (December 1, 2008): 131–36. http://dx.doi.org/10.17796/jcpd.33.2.82r1tp71x75m5345.
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Objective: To evaluate the clinical and radiographic outcomes (24 months) of indirect pulp treatment (IPT)in primary teeth when a self-etching primer or a calcium hydroxide layer was used over the remaining carious dentin. Study design: Primary molar teeth with deep carious lesions without signs and symptoms of irreversible pulpitis were divided into two groups, according to the capping material: Experimental group(1): self-etching adhesive system (Clearfill SE Bond); and Control group (2): calcium hydroxide liner(Dycal). Both groups were followed up after application of a resin restoration (3M - Z250). Results: After 2 years of clinical and radiographic follow-up, no statistical difference was found between groups (p=1). The overall success rate reached 87%. Conclusion: These results demonstrate that IPT has a high clinical and radiographic performance in primary teeth and is not dependent on the capping material used over the demineralized dentin.
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O’Reilly, Erin K., Zhaohui Wang, Roy French, and C. Cheng Kao. "Interactions between the Structural Domains of the RNA Replication Proteins of Plant-Infecting RNA Viruses." Journal of Virology 72, no. 9 (September 1, 1998): 7160–69. http://dx.doi.org/10.1128/jvi.72.9.7160-7169.1998.
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ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.
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Adams, A. E., J. A. Cooper, and D. G. Drubin. "Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast." Molecular Biology of the Cell 4, no. 5 (May 1993): 459–68. http://dx.doi.org/10.1091/mbc.4.5.459.
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To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.
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Pedano, Mariano S., Xin Li, Kumiko Yoshihara, Kirsten Van Landuyt, and Bart Van Meerbeek. "Cytotoxicity and Bioactivity of Dental Pulp-Capping Agents towards Human Tooth-Pulp Cells: A Systematic Review of In-Vitro Studies and Meta-Analysis of Randomized and Controlled Clinical Trials." Materials 13, no. 12 (June 12, 2020): 2670. http://dx.doi.org/10.3390/ma13122670.
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Background. In the era of biology-driven endodontics, vital pulp therapies are regaining popularity as a valid clinical option to postpone root-canal treatment. In this sense, many different materials are available in the market for pulp-capping purposes. Objectives. The main aim of this systematic review and meta-analysis was to examine literature regarding cytotoxicity and bioactivity of pulp-capping agents by exposure of human dental pulp cells of primary origin to these materials. A secondary objective was to evaluate the inflammatory reaction and reparative dentin-bridge formation induced by the different pulp-capping agents on human pulp tissue. Data sources. A literature search strategy was carried out on PubMed, EMBASE and the Web of Science databases. The last search was done on 1 May 2020. No filters or language restrictions were initially applied. Two researchers independently selected the studies and extracted the data. Study selection included eligibility criteria, participants and interventions, study appraisal and synthesis methods. In vitro studies were included when human dental pulp cells of primary origin were (in) directly exposed to pulp-capping agents. Parallel or split-mouth randomized or controlled clinical trials (RCT or CCT) were selected to investigate the effects of different pulp-capping agents on the inflammation and reparative bridge-formation capacity of human pulp tissue. Data were synthesized via odds ratios (95% confidence interval) with fixed or random effects models, depending on the homogeneity of the studies. The relative risks (95% confidence interval) were presented for the sake of interpretation. Results. In total, 26 in vitro and 30 in vivo studies were included in the systematic review and meta-analysis, respectively. The qualitative analysis of in vitro data suggested that resin-free hydraulic calcium-silicate cements promote cell viability and bioactivity towards human dental pulp cells better than resin-based calcium-silicate cements, glass ionomers and calcium-hydroxide cements. The meta-analysis of the in vivo studies indicated that calcium-hydroxide powder/saline promotes reparative bridge formation better than the popular commercial resin-free calcium-silicate cement Pro-Root MTA (Dentsply-Sirona), although the difference was borderline non-significant (p = 0.06), and better than calcium-hydroxide cements (p < 0.0001). Moreover, resin-free pulp-capping agents fostered the formation of a complete reparative bridge better than resin-based materials (p < 0.001). On the other hand, no difference was found among the different materials tested regarding the inflammatory effect provoked at human pulp tissue. Conclusions. Calcium-hydroxide (CH) powder and Pro-Root MTA (Dentsply-Sirona) have shown excellent biocompatibility in vitro and in vivo when tested on human cells and teeth. Their use after many years of research and clinical experience seems safe and proven for vital pulp therapy in healthy individuals, given that an aseptic environment (rubber dam isolation) is provided. Although in vitro evidence suggests that most modern hydraulic calcium-silicate cements promote bioactivity when exposed to human dental pulp cells, care should be taken when these new materials are clinically applied in patients, as small changes in their composition might have big consequences on their clinical efficacy. Key findings (clinical significance). Pure calcium-hydroxide powder/saline and the commercial resin-free hydraulic calcium-silicate cement Pro-Root MTA (Dentsply-Sirona) are the best options to provide a complete reparative bridge upon vital pulp therapy. Systematic review registration number. PROSPERO registration number: CRD42020164374.
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Gassmann, Martin, Kurt E. Amrein, and Paul Burn. "CD4: p56lckAssociation Studied in vivo Using Antibody-Induced Capping and Double Indirect Immunofluorescence Microscopy." Journal of Receptor Research 13, no. 1-4 (January 1993): 711–24. http://dx.doi.org/10.3109/10799899309073688.
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Liu, Siyi, Sainan Wang, and Yanmei Dong. "Evaluation of a Bioceramic as a Pulp Capping Agent In Vitro and In Vivo." Journal of Endodontics 41, no. 5 (May 2015): 652–57. http://dx.doi.org/10.1016/j.joen.2014.12.009.
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Hopmann, Roberta, and Kathryn G. Miller. "A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle." Molecular Biology of the Cell 14, no. 1 (January 2003): 118–28. http://dx.doi.org/10.1091/mbc.e02-05-0300.
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Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of thecpb loss-of-function phenotype. The interaction betweencpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.
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Swarup, SJ, A. Rao, K. Boaz, N. Srikant, and R. Shenoy. "Pulpal Response to Nano Hydroxyapatite, Mineral Trioxide Aggregate and Calcium Hydroxide when Used as a Direct Pulp Capping Agent: An in Vivo study." Journal of Clinical Pediatric Dentistry 38, no. 3 (April 1, 2014): 201–6. http://dx.doi.org/10.17796/jcpd.38.3.83121661121g6773.
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Nano hydroxyapatite (Nano-HA) and Mineral Trioxide Aggregate (MTA) because of its better qualities can be used as an alternative to calcium hydroxide in direct pulp capping procedures. The aim of the study was to compare the response of exposed human pulp to Nano-HA, Mineral Trioxide Aggregate and calcium hydroxide. Study design: The study was done on 30 premolars, ranging from patients between 11-15 years. Intentional pulp capping was done using one of the experimental materials. The extracted teeth were then subjected to staining procedure and evaluated for dentin bridge and pulpal response after 15 and 30 days. Intragroup comparisons of the observed values were analyzed using Chi-square test. Results: Nano-HA and MTA produced continuous dentin bridges. Dentin bridge that was formed in MTA group had regular pattern of dentinal tubules but no tubules were seen in the nano-HA group. Dentin bridge was not observed in Dycal group for the 15 days period in majority of the sample and by 30 days dentin bridge was observed that were both continuous and interrupted in equal number of samples. The initial inflammatory response and necrosis was more with Nano-HA and calcium hydroxide which reduced with time. Conclusion: MTA showed no inflammatory changes in majority of the samples in both the study periods. Necrosis was least observed in MTA group followed by Nano-HA. Vascularity increased in Nano-HA group in the initial periods which reduced with increasing time. Based on the ability of nano-HA to produce complete dentinal bridges, favorable cellular and vascular response, the material could be considered as an substitute and could be tried used as a direct pulp capping agent.
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Constantin, B., K. Meerschaert, J. Vandekerckhove, and J. Gettemans. "Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions." Journal of Cell Science 111, no. 12 (June 15, 1998): 1695–706. http://dx.doi.org/10.1242/jcs.111.12.1695.
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Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actin-fragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.
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Jung, Goeh, Kirsten Remmert, Xufeng Wu, Joanne M. Volosky, and John A. Hammer. "The Dictyostelium Carmil Protein Links Capping Protein and the Arp2/3 Complex to Type I Myosins through Their Sh3 Domains." Journal of Cell Biology 153, no. 7 (June 25, 2001): 1479–98. http://dx.doi.org/10.1083/jcb.153.7.1479.
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Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the α and β subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end–directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.
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Huang, Haiyan, Linjuan Luo, Lefeng Li, Yun Guan, Yanhong Yan, Zhen Jiang, and Beizhan Jiang. "Calcium Phosphate Cement Promotes Odontoblastic Differentiation of Dental Pulp Cells In Vitro and In Vivo." Coatings 12, no. 4 (April 18, 2022): 543. http://dx.doi.org/10.3390/coatings12040543.
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In the case of pulp injury, odontoblastic differentiation of dental pulp cells (DPCs) at the site of the exposed pulp is necessary for a successful direct pulp capping treatment. Calcium phosphate cement (CPC), a kind of hydroxyapatite-like bone cement, exhibits therapeutic potential in osteogenesis by regulating cell cycle progression and promoting osteoblastic differentiation. Based on the similar biological process of osteo/odontoblastic differentiation, the present study evaluated the effects of CPC on odontoblastic differentiation of DPCs in vitro and in vivo, respectively. The morphology of CPC was observed by scanning electron microscopy. Colony-forming units were used to assess the antibacterial activity. The effects of CPC on cell proliferation and odontoblastic differentiation of human dental pulp cells (hDPCs) were also measured. Histological staining was performed to observe the reparative dentin formation in rat molars. In vitro, results of antibacterial studies showed that CPC significantly inhibited the growth of Streptococcus mutans. The appropriate concentration of CPC extract showed low cytotoxicity on hDPCs. Furthermore, CPC extract also promoted odontoblastic differentiation and mineralization compared with the control group, as shown by a dynamic increase in the expression of odontogenic marker genes and the increased number of mineralized nodules at 21 days. The pulpotomy models with CPC facilitated the formation of dentin bridge with the highly expressed dentin matrix protein 1 (DMP1) in odontoblast-like cells. In conclusion, the favorable biocompatibility, antibacterial property and bio-inductivity of CPC suggest that CPC can be used as a promising direct pulp capping material.
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Ray, Suhita, Linda Chee, Nicholas T. Woods, and Kyle J. Hewitt. "Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis." Blood 138, Supplement 1 (November 5, 2021): 288. http://dx.doi.org/10.1182/blood-2021-152898.
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Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p<0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.