Background:The functional importance of apolipoprotein A-I (apoA-I) nitration at tyrosine 166 (Tyr 166 ) in vivo is controversial. Results: Nitrotyrosine 166-apoA-I accounts for 8% of apoA-I within human atheroma, is not HDL-associated, and is functionally impaired. Conclusion: Buoyant density ultracentrifugation of HDL can lead to erroneous results, particularly with modified apoA-I forms. Significance: Detection and quantification of nitrotyrosine 166-apoA-I may provide insights into a pathophysiological process within the artery wall.
Cholesterol from peripheral tissues, such as within the artery wall, is transported to the liver by high density lipoprotein (HDL) particles, which are continuously remodeled in a process called reverse cholesterol transport ( 1, 2 ). HDL particles are highly heterogeneous, varying in protein and lipid content ( 3 ). Apolipoprotein A1 (apoA1) is Abstract Small-angle neutron scattering (SANS) with contrast variation was used to obtain the low-resolution structure of nascent HDL (nHDL) reconstituted with dimyristoyl phosphatidylcholine (DMPC) in the absence and presence of cholesterol, [apoA1:DMPC (1:80, mol:mol) and apoA1:DMPC:cholesterol (1:86:9, mol:mol:mol)]. The overall shape of both particles is discoidal with the low-resolution structure of apoA1 visualized as an open, contorted, and out of plane conformation with three arms in nascent HDL/dimyristoyl phosphatidylcholine without cholesterol (nHDL DMPC ) and two arms in nascent HDL/dimyristoyl phosphatidylcholine with cholesterol (nHDL DMPC+Chol ). The low-resolution shape of the lipid phase in both nHDL DMPC and nHDL DMPC+Chol were oblate ellipsoids, and fi t well within their respective protein shapes. Modeling studies indicate that apoA1 is folded onto itself in nHDL DMPC , making a large hairpin, which was also confi rmed independently by both cross-linking mass spectrometry and hydrogen-deuterium exchange (HDX) mass spectrometry analyses. In nHDL DMPC+Chol , the lipid was expanded and no hairpin was visible. Importantly, despite the overall discoidal shape of the whole particle in both nHDL DMPC Press, January 23, 2013 DOI 10.1194 The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion Abbreviations: DMPC, dimyristoyl phosphatidylcholine; DSH, double super helix; ESR, electron spin resonance; FRET, fl uorescence resonance energy transfer; HDX, hydrogen-deuterium exchange; LUV, large unilamellar vesicle; nHDL, nascent HDL; nHDL DMPC , nascent HDL/dimyristoyl phosphatidylcholine without cholesterol; nHDL DMPC+Chol , nascent HDL/dimyristoyl phosphatidylcholine with cholesterol; nHDL POPC , nascent HDL/POPC and cholesterol; PC, phosphatidyl choline ; POPC, 1-palmitoyl-2-oleoyl-sn -glycero-3-phosphocholine; q , range of momentum transfer; SANS, small-angle neutron scattering; SCX, strong-cation exchange. Published, JLR Papers in
The goal of endodontic treatment is to remove all the vital and necrotic tissues, microorganisms and microbial byproducts from root canal system. This goal can be achieved through chemical and mechanical debridement of root canals. This article narrates the specifics and requirements of the irrigation solutions. Sodium hypochlorite is proposed as the primary irrigant by virtue of its organic tissue dissolution capacity and broad antimicrobial properties. On the other hand, chelation solutions are recommended as auxiliary solutions to remove the smear layer or to hinder its formation on dentin surface. Thus, it's hoped that sealers and root canal fillers can penetrate to dentin tubules and obturate the canals hermetically. There are new studies on traditional irrigants especially on some irrigants that can replace sodium hypoclorite. This article reviews the new irrigants which can be used in future endodontic practice, and their advantages and limitations. Moreover, actions and interactions of recently used irrigants are adverted.
Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases and Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 and Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH--transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems.
Isolation of high density lipoproteins (HDL) for structural and functional studies typically relies on ultracentrifugation techniques, which are time-consuming and difficult to scale. With emerging interest in the clinical relevance of HDL structure and function to cardiovascular disease, a significant gap exists between current and desirable sample preparation throughput. To enable proteomic studies of HDL with large clinical cohorts, we have developed an affinity enrichment approach that relies on the association of histidine-tagged, lipid free ApoA-I with HDL followed by standard metal chelate chromatography. Characterization of the resulting affinity-enriched ApoA-I associated lipoprotein (AALP) pool using biochemical, electrophoretic, and proteomic analysis demonstrates that the isolated material is closely related in structural features, lipid content, protein complement, and relative protein distribution to HDL isolated by ultracentrifugation using sequential density adjustment. The simplicity of the method provides avenues for high-throughput analysis of HDL associated proteins.
Quantitative analysis of Lp-PLA concentration and activity by LC-MS/MS assays provided key insight into resolving the well-documented discordance between Lp-PLA concentration (determined by immunoassay) and activity. Quantitative detection of Lp-PLA by immunoassay appears to be strongly inhibited by interaction of Lp-PLA with lipoprotein. Together, the results illustrate the advantages of quantitative LC-MS/MS for measurement of Lp-PLA concentration (by SISCAPA) and activity (by direct product detection).
Introduction: The purpose of this study is to examine the effect of different chelating solutions on microhardness and surface roughness of root canal dentin. Methodology: The crowns of sixty recently extracted maxillary central incisors were separated by diamond burs from the cemento-enamel junction. Roots are divided longitudinally into two pieces with diamond saw. The obtained samples were embedded in autopolymeric-acrylic and the dentin-surfaces of the teeth were grounded. Micro-hardness and surface-roughness measurements were carried out before the chelating processes. First group, the specimens were treated with gel-formed 17% Ethylenediaminetetraacetic acid [EDTA] (JE), second group, specimens were treated with 17% Liquid-formed EDTA (LE), third group, specimens were treated with 20% Citric acid (CA), and the last group specimens were treated with 7% Maleic acid (MA) for 120 seconds. After application of the chelating agents, micro-hardness and surface-roughness measurements of all samples were performed again. The difference between the initial and final measurements was calculated and statistically analyzed. One Way Analysis of Variance ANOVA was used for statistical-analysis and statistically significant difference was observed between the groups (p <0.05). Results: According to the test results, the microhardness of LE was statistically equal to that of JE and caused the least decrease in micro-hardness, SA decreased micro-hardness more than LE and JE, and MA decreased dentin micro-hardness more than all other solutions. And surface-roughness results are: MA increased surface-roughness more than other groups, SA and JE less roughened the dentin surface than MA, and there was no significant difference between the two solutions, LE caused less increase in dentin surface roughness compared to all solutions. Conclusions: Besides the use of EDTA and SA, the clinical use of MA can be considered, and it should also be noted that EDTA differs in terms of surface roughness between liquid and gel forms. How to cite this article: Topbaş C, Adıgüzel Ö, Çölgeçen Ö. Investigation of the effects of different chelating solutions on the microhardness and surface roughness of root canal dentin. Int Dent Res 2019;9(1):22-9. Linguistic Revision: The English in this manuscript has been checked by at least two professional editors, both native speakers of English.
Background: The functional importance of apolipoprotein A-I (apoA-I) nitration at tyrosine 166 (Tyr 166) in vivo is controversial. Results: Nitrotyrosine 166-apoA-I accounts for 8% of apoA-I within human atheroma, is not HDL-associated, and is functionally impaired. Conclusion: Buoyant density ultracentrifugation of HDL can lead to erroneous results, particularly with modified apoA-I forms. Significance: Detection and quantification of nitrotyrosine 166-apoA-I may provide insights into a pathophysiological process within the artery wall. We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr 166), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr 166 nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO 2-Tyr 166-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNA CUA pair for functional studies. Studies with mAb 4G11.2 showed that NO 2-Tyr 166-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ϳ8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO 2-Tyr 166-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d ؍ 1.063-1.21). NO 2-Tyr 166-apoA-I in plasma showed a similar distribution. Recovery of NO 2-Tyr 166-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ؎ 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr 166 is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall. Post-translational modification (PTM) 4 of proteins through multiple distinct oxidative processes is a common occurrence * This work was supported, in whole or in part, by National Institutes of Health Grants P01HL098055, P01HL076491, and HL17964. This work was also supported by a grant from the LeDucq Fondation. Dr. Tang has previously received research grant support from Abbott Laboratories. Drs. Hazen and Smith report being listed as co-inventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diag...
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