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The role of cross-linking structures to the formation of one-dimensional nano-organized polyaniline and their Raman fingerprint

NASCIMENTO, Gustavo M. do; SILVA, Claudio H. B.; IZUMI, Celly M. S.; TEMPERINI, Marcia L. A.
Fonte: PERGAMON-ELSEVIER SCIENCE LTD Publicador: PERGAMON-ELSEVIER SCIENCE LTD
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
66.1%
In the present work. the resonance Raman. UV-vis-NIR and scanning electron microscopic (SEM) data of nanorods (about similar to 300 rim in diameter) and nanofibers (about similar to 93 nm in diameter) of PANI are presented and compared. The PANI samples were synthesized in aqueous media with dodecybenzenesulfonic acid (DBSA) and beta-naphtalenesulfonic acid (beta-NSA) as dopants, respectively. The presence of hands at 578, 1400 and 1632cm(-1) in the Raman spectra of PANI-NSA and PANI-DBSA shows that the formation of cross-linking structures is a general feature of the PANI chains prepared in micellar media. It is proposed that these structures are responsible for the one-dimensional PANI morphology formation. In addition, the Raman band at 609cm(-1) of PANI fibers is correlated with the extended PANI chain coil formation. (C) 2008 Elsevier B.V. All rights reserved.; FAPESP (Brazilian agency); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); CNPq; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Peripheral Sterile Corneal Ring Infiltrate After Riboflavin-UVA Collagen Cross-Linking in Keratoconus

Ghanem, Ramon C.; Netto, Marcelo V.; Ghanem, Vinicius C.; Santhiago, Marcony R.; Wilson, Steven E.
Fonte: LIPPINCOTT WILLIAMS & WILKINS; PHILADELPHIA Publicador: LIPPINCOTT WILLIAMS & WILKINS; PHILADELPHIA
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
66.1%
Purpose: To present 7 cases of peripheral sterile corneal infiltrates that occurred after corneal cross-linking (CXL) for progressive keratectasia. Methods: Seven patients who had their progressive keratoconus documented underwent corneal deepithelization and subsequently CXL, which was performed with the application of 0.1% riboflavin with 20% dextran, and exposure to UVA light (370 nm, 2.9-3.1 mW/cm(2)) for 30 minutes. Results: Nearly a week after the procedure, the patients presented with peripheral stromal infiltrates. The ring-like infiltrates were superficial and were present at the 9.0-mm zone. Sterile infiltration was diagnosed. Patients were treated with topical corticosteroids, and complete resolution was achieved after a few weeks of treatment. Conclusions: We hypothesize that the phototoxic effect on the corneal stroma may be the main mechanism that triggers these infiltrates. Alternatively, alterations in antigenicity that occur in native proteins after CXL could result in patients recognizing the proteins as nonself and mounting immune responses.

Corneal Absorption of a New Riboflavin-Nanostructured System for Transepithelial Collagen Cross-Linking

Bottos, Katia M.; Oliveira, Anselmo Gomes de; Bersanetti, Patrícia A.; Nogueira, Regina F.; Lima-Filho, Acácio A. S.; Cardillo, José A.; Schor, Paulo; Chamon, Wallace
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
66.19%
Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes...

Utilização de espectrometria de massas, ligação cruzada (cross-linking) e footprinting no estudo de interações proteina-proteina; Use of mass spectrometry, cross-linking and footprinting in the study of protein-protein interactions

Amadeu Hoshi Iglesias
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 03/04/2009 PT
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56.26%
A Espectrometria de Massas (MS) é hoje a principal técnica de caracterização de estrutura primária de proteínas devido às vantagens intrínsecas da técnica. As técnicas de cross-linking e footprinting visam desfrutar de tais vantagens para obtenção de informações estruturais de complexos protéicos. Nesse projeto foram realizados estudos de fragmentação de peptídeos contendo como cross-linker o DSS, reagente mais utilizado para esse propósito. Os mecanismos de fragmentação foram propostos baseados na dissociação de peptídeos modelos sintetizados para gerar espécies que mimetizassem experimentos com proteínas. Esses estudos permitiram a identificação de íons marcadores, que foram posteriormente utilizados em experimentos de Varredura de Íons Precursores para auxiliar na detecção de peptídeos modificados. Para realização dos experimentos de footprinting, foi desenvolvida uma linha de luz no LNLS. Posteriormente, foi proposto um novo método de quantificação de cinética de oxidação baseado nos dados de LC-MS, levando em consideração todos os produtos de oxidação formados. Esses métodos foram utilizados no estudo de interação das proteínas Tif34 e Tif35 do fator de iniciação de tradução de Saccharomyces cerevisiae. Os resultados obtidos indicam que Tif34 apresenta dois possíveis sítios de interação para a proteína Tif35; Mass Spectrometry (MS) is the most important tool for analyses related to protein primary structure. Cross-Linking and footprinting aim to bring those advantages to gain insights in the spatial structure of protein complexes. In this work the fragmentation of peptides containing DSS...

Uso de espectrometria de massas e mobilidade iônica na caracterização de peptídeos provenientes de experimentos de ligação cruzada; Mass spectrometry and ion mobility in the characterization and of peptides from protein cross-linking experiments

Luiz Fernando Arruda Santos
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 10/03/2011 PT
Relevância na Pesquisa
66.28%
A função de uma proteína está diretamente relacionada com sua estrutura e conhecendo a estrutura de uma proteína é possível verificar onde estão localizados e como agem seus sítios ativos e de interação. Dentre os métodos empregados para análise estrutural de proteínas por espectrometria de massas (MS), experimentos de ligação cruzada vêm recebendo grande destaque. Nestes experimentos, após digestão enzimática três tipos de peptídeos contendo agentes de ligação cruzada (ALCs) podem ser formados: espécies intramoleculares, quando o ALC está ligado entre dois resíduos do mesmo peptídeo; espécies intermoleculares, quando o ALC une dois peptídeos distintos e dead-end, quando o ALC liga-se apenas a um resíduo, sendo que o outro lado de sua cadeia normalmente sofre hidrólise. A etapa crucial em experimento de ligação cruzada consiste na correta atribuição e identificação dos peptídeos modificados por ALC, etapa que pode ser facilitada conhecendo a fragmentação destes peptídeos. Desse modo, o foco principal deste trabalho foi realizar experimentos de fragmentação das diferentes espécies de peptídeos modificados por ALCs utilizando metodologias baseadas na técnica de espectrometria de massas, como dissociação induzida por colisão (CID)...

Corneal thickness changes during corneal collagen cross-linking with UV-A irradiation and hypo-osmolar riboflavin in thin corneas

Nassaralla,Belquiz Amaral; Vieira,Diogo Mafia; Machado,Márcia Leite; Figueiredo,Marisa Novaes Faleiro Chaves de; Nassaralla Jr,João Jorge
Fonte: Conselho Brasileiro de Oftalmologia Publicador: Conselho Brasileiro de Oftalmologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2013 EN
Relevância na Pesquisa
66.1%
PURPOSE: To evaluate the thinnest corneal thickness changes during and after corneal collagen cross-linking treatment with ultraviolet-A irradiation, using hypo-osmolar riboflavin solution in thin corneas. METHODS: Eighteen eyes of 18 patients were included in this study. After epithelium removal, iso-osmolar 0.1% riboflavin solution was instilled to the cornea every 3 minutes for 30 minutes. Hypo-osmolar 0.1% riboflavin solution was then applied every 20 seconds for 5 minutes or until the thinnest corneal thickness reached 400 µm. Ultraviolet-A irradiation was performed for 30 minutes. During irradiation, iso-osmolar 0.1% riboflavin drops were applied every 5 minutes. Ultrasound pachymetry was performed at approximately the thinnest point of the cornea preoperatively, after epithelial removal, after iso-osmolar riboflavin instillation, after hypo-osmolar riboflavin instillation, after ultraviolet-A irradiation, and at 1, 6 and 12 months after treatment. RESULTS: Mean preoperative thinnest corneal thickness was 380 ± 11 µm. After epithelial removal it decreased to 341 ± 11 µm, and after 30 minutes of iso-osmolar 0.1% riboflavin drops, to 330 ± 7.6 µm. After hypo-osmolar 0.1% riboflavin drops, mean thinnest corneal thickness increased to 418 ± 11 µm. After UVA irradiation...

Changes in corneal sensitivity following cross-linking for progressive early-stage keratoconus

Lago,Anelise de Medeiros; Nassaralla,Belquiz R. do Amaral; Stival,Larissa Rossana Souza; Nassaralla Junior,João Jorge
Fonte: Conselho Brasileiro de Oftalmologia Publicador: Conselho Brasileiro de Oftalmologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2014 EN
Relevância na Pesquisa
66.1%
Purpose: To evaluate changes in corneal sensitivity following corneal cross-linking (CXL) in patients with progressive earlier stage keratoconus. Methods: Thirty-eight eyes of 19 patients (11 women, 8 men) were included in a prospective, nonrandomized clinical study. The mean patient age was 22 years (range, 18-26 years). Inclusion criteria were early stage bilateral progressive keratoconus, a transparent cornea, and a thickness of ≥440 µm in the thinnest area of the cornea. Using the Cochet-Bonnet esthesiometer, central corneal sensitivity was measured before surgery, 7 days after surgery, and once a month thereafter until recovery of baseline preoperative levels. Central corneal sensitivity >40 mm was considered normal. Results: Corneal sensitivity gradually returned to preoperative levels in all treated eyes. The mean central corneal sensitivity was 52.2, 24.0, 38.2, 42.5, 50.0, and 52.5 mm before surgery, 7 days after surgery, and at 1, 2, 3, and 4 months after surgery, respectively. Normal levels of corneal sensation, but not baseline (preoperative) levels, were observed 2 months after surgery. The preoperative levels were observed 3 months after surgery. Conclusions: Our results suggest that central corneal sensitivity can be decreased for as long as 3 months after CXL for progressive earlier stage keratoconus.

Induction of corneal collagen cross-linking in experimental corneal alkali burns in rabbits

Colombo-Barboza,Marcello; Colombo-Barboza,Guilherme; Felberg,Sergio; Dantas,Paulo Elias Corrêa; Sato,Elcio Hideo
Fonte: Conselho Brasileiro de Oftalmologia Publicador: Conselho Brasileiro de Oftalmologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/10/2014 EN
Relevância na Pesquisa
66.15%
Objective: To evaluate the effect of riboflavin-ultraviolet-A-induced cross-linking (CXL) following corneal alkali burns in rabbits. Methods: The right corneas and limbi of ten rabbits were burned using a 1N solution of NaOH and the animals were then divided into two groups: a control group submitted to clinical treatment alone and an experimental group that was treated 1 h after injury with CXL, followed by the same clinical treatment as administered to the controls. Clinical parameters were evaluated post-injury at 1, 7, 15, and 30 days by two independent observers. Following this evaluation, the corneas were excised and examined histologically. Results: There were no statistically significant differences in clinical parameters, such as hyperemia, corneal edema, ciliary injection, limbal ischemia, secretion, corneal neovascularization, symblepharon, or blepharospasm, at any of the time-points evaluated. However, the size of the epithelial defect was significantly smaller in the CXL group (p<0.05) (day 15: p=0.008 and day 30: p=0.008) and the extent of the corneal injury (opacity lesion) was also smaller (day 30: p=0.021). Histopathology showed the presence of collagen bridges linking the collagen fibers in only the CXL group. Conclusions: These results suggest that the use of CXL may improve the prognosis of acute corneal alkali burns.

Preparation, characterization and properties of films obtained from cross-linked guar gum

Banegas,Rodrigo S.; Zornio,Clarice F.; Borges,Adriana de M. G.; Porto,Ledilege C.; Soldi,Valdir
Fonte: Associação Brasileira de Polímeros Publicador: Associação Brasileira de Polímeros
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2013 EN
Relevância na Pesquisa
56.27%
In this study the viability of using guar gum to form films was investigated along with the effectiveness of the cross-linking process employing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as the cross-linking agent. In addition, the cross-linked films were evaluated considering the water absorption, thermal stability and mechanical properties. The cross-linking process of guar gum films was confirmed by the low solubility in water and through infrared analysis. The results shown that the properties evaluated were affected by the cross-linking process due to changes in the polysaccharide structure. For example, the swelling behavior and water vapor absorption decreased with an increase in the amount of EDC. The EDC content (10-30%) also affected the polymer structure and hydrogen bond formation, reducing the thermal stability of the system.

Spray-dried chitosan microspheres cross-linked with d, l-glyceraldehyde as a potential drug delivery system: preparation and characterization

Oliveira,B. F; Santana,M. H. A.; Ré,M. I.
Fonte: Brazilian Society of Chemical Engineering Publicador: Brazilian Society of Chemical Engineering
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2005 EN
Relevância na Pesquisa
56.36%
Chitosan microspheres of a small particle size and with good sphericity were prepared by a spray-drying method followed by treatment with a cross-linking agent. Owing to restrictions on the use of cross-linked chitosan microspheres in the pharmaceutical field, d,l-glyceraldehyde, a biocompatible reatant was used. The parameters studied affecting extent of cross-linking were cross-linking time and concentration of the cross-linking agent. Glutaraldehyde, the aldehyde most frequently employed as chemical cross-linking agent for proteins, was also used as a control. The cross-linked spray-dried chitosan microspheres were analyzed with respect to their morphological aspects, particle size, zeta potential and water uptake capacity. It was found that an increase either in d,l-glyceraldehyde concentration or in duration of cross-linking caused a decrease in both the swelling capacity and the zeta potential of the chitosan microspheres. Compared to glutaraldehyde, d,l-glyceraldehyde appears to be a good cross-linking agent for chitosan microspheres with the advantage that it is nontoxic.

Efeito terapêutico do cross-linking corneal em portadores de ceratopatia bolhosa sintomática

Nery Benevides Gadelha, Diego; Teixeira Brandt, Carlos (Orientador)
Fonte: Universidade Federal de Pernambuco Publicador: Universidade Federal de Pernambuco
Tipo: Outros
PT_BR
Relevância na Pesquisa
66.19%
Objetivo: Avaliar o efeito do cross-linking corneal (CCL) nos portadores de ceratopatia bolhosa sintomática, e seu impacto na acuidade visual, espessura corneal e sintomatologia dolorosa. Métodos: Doze pacientes com ceratopatia bolhosa sintomática foram incluídos. Exame clínico com questionário específico para a pesquisa com escala de dor (escala visual analógica numérica), acuidade visual e mensuração da espessura corneal foi realizada pré CCL, 7, 30, 60, 180 e 365 dias após. Em todos os pacientes o tratamento com UVA-crosslinking foi realizado após abrasão do epitélio corneal em lâmpada de fenda e instilação de solução de riboflavina 0,1% a cada cinco minutos por 30 minutos. Após esse período, o paciente foi submetido à exposição à luz UVA, utilizando riboflavina e anestesia tópica a cada cinco minutos por 30 minutos. Os pacientes utilizaram colírios de ofloxacina 0,3% e lágrima artificial até completa reepitelização. O teste de Friedman foi usado para comparar as médias das freqüências da acuidade visual, sintomatologia dolorosa e espessura central da córnea. p <0.05 foi utilizado para rejeitar a hipótese nula. Resultados: Doze olhos de 12 pacientes com erosões epiteliais recorrentes foram tratados. O tempo de seguimento foi de um ano. Foi observada redução significante da dor (p<0...

Analysis of Shigella flexneri Wzz (Rol) function by mutagenesis and cross-linking: Wzz is able to oligomerise.

Daniels, C.; Morona, R.
Fonte: BLACKWELL SCIENCE LTD Publicador: BLACKWELL SCIENCE LTD
Tipo: Artigo de Revista Científica
Publicado em //1999 EN
Relevância na Pesquisa
66.19%
The modal length or degree of polymerization (dp) of the Shigella flexneri O-antigen is determined in an unknown manner by the Wzz/Rol protein. The Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32-52; TM2 amino acids 295-315) with the central loop of the protein located in the periplasm. Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S. flexneri Wzz (WzzSF) and Salmonella typhimurium Wzz (WzzST). These imparted O-antigen modal chain lengths that implied that the carboxy-terminal region of Wzz was involved in chain length determination. Site-directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino-/carboxy-terminal domains of WzzSF. Some of the WzzSF variants resulted in O-antigen modal chain lengths much shorter than those of wild-type WzzSF, whereas other mutants inactivated WzzSF function entirely and a third class had a longer O-antigen chain length distribution. The data indicate that amino acids throughout the length of the WzzSF protein are important in determination of O-antigen modal chain length. In vivo cross-linking experiments were performed to investigate the interactions between Wzz proteins. The experiments indicated that the WzzSF protein is able to form dimers and oligomers of at least six WzzSF proteins. A carboxy-terminal-truncated WzzSF protein having the amino terminal 194 amino acids was able to oligomerize...

Hydralazine inhibits rapid acrolein-induced protein oligomerization: Role of aldehyde scavenging and adduct trapping in cross-link blocking and cytoprotection

Burcham, P.; Pyke, S.
Fonte: Amer Soc Pharmacology Experimental Therapeutics Publicador: Amer Soc Pharmacology Experimental Therapeutics
Tipo: Artigo de Revista Científica
Publicado em //2006 EN
Relevância na Pesquisa
56.27%
Hydralazine strongly suppresses the toxicity of acrolein, a reactive aldehyde that contributes to numerous health disorders. At least two mechanisms may underlie the cytoprotection, both of which involve the nucleophilic hydrazine possessed by hydralazine. Under the simplest scenario, hydralazine directly scavenges free acrolein, decreasing intracellular acrolein availability and thereby suppressing macromolecular adduction. In a second "adduct-trapping" mechanism, the drug forms hydrazones with acrolein-derived Michael adducts in cell proteins, preventing secondary reactions of adducted proteins that may trigger cell death. To identify the most important mechanism, we explored these two pathways in mouse hepatocytes poisoned with the acrolein precursor allyl alcohol. Intense concentration-dependent adduct-trapping in cell proteins accompanied the suppression of toxicity by hydralazine. However, protective concentrations of hydralazine did not alter extracellular free acrolein levels, cellular glutathione loss, or protein carbonylation, suggesting that the cytoprotection is not due to minimization of intracellular acrolein availability. To explore ways whereby adduct-trapping might confer cytoprotection, the effect of hydralazine on acrolein-induced protein cross-linking was examined. Using bovine pancreas ribonuclease A as a model protein...

A novel tetrafunctional reagent for simultaneous intramolecular and intermolecular cross-linking of bovine hemoglobin

Yun, Q.; He, M.; Xing, W.; Bi, J.; Ma, G.; Su, Z.
Fonte: Kluwer Academic Publ Publicador: Kluwer Academic Publ
Tipo: Artigo de Revista Científica
Publicado em //2004 EN
Relevância na Pesquisa
66.24%
A novel tetrafunctional reagent, α, γ, α, γ-tetra-succinimidyl-hexanediamide-di-glutamate ester (HDG(OSu)4), was successfully synthesized, and a well-defined cross-linked bovine hemoglobin (mainly 128 kDa) was prepared with this reagent. Due to the spatial structure of this cross-linking reagent, the intramolecular and intermolecular cross-linking of bovine hemoglobin was formed simultaneously in one reaction. Although the cross-linked bovine hemoglobin showed a slight decrease in half-saturated O2 pressure value (P50, from 28.1 mm Hg to 21.7 mm Hg) and Hill coefficient (from 2.5 to 2), due to the cross-linkage, it still performed well for O2 delivery.; Qiang Yun, Minglei He, Wenchao Xing, Jingxiu Bi, Guanghui Ma & Zhiguo Su; The definitive version may be found at http://www.springerlink.com/

A negative ion mass spectrometry approach to identify cross-linked peptides utilizing characteristic disulfide fragmentations

Calabrese, A.; Good, N.; Wang, T.; He, J.; Bowie, J.; Pukala, T.
Fonte: Elsevier Science Inc Publicador: Elsevier Science Inc
Tipo: Artigo de Revista Científica
Publicado em //2012 EN
Relevância na Pesquisa
56.31%
Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.; Antonio N. Calabrese...

Negative ion fragmentations of disulfide-containing cross-linking reagents are competitive with aspartic acid side-chain-induced cleavages

Calabrese, A.; Wang, T.; Bowie, J.; Pukala, T.
Fonte: John Wiley & Sons Ltd Publicador: John Wiley & Sons Ltd
Tipo: Artigo de Revista Científica
Publicado em //2013 EN
Relevância na Pesquisa
66.22%
RATIONALE: It has been shown that the disulfide moiety in the chemical cross-linking reagent dithiobis(succinimidyl)propionate (DSP), which is similar in structure to the natural cystine disulfide, cleaves preferentially to the peptide backbone in the negative ion mode. However, the tandem mass (MS/MS) spectra of peptides in the negative ion mode are often dominated by products arising from low-energy, side-chain-induced processes, which may compete with any facile cross-linker fragmentations and complicate identification of chemical cross-links in a complex mixture. METHODS: Two disulfide-containing crosslinking reagents similar to DSP, but with varying spacer arm lengths, were synthesized and the MS/MS spectra of several model peptides cross-linked with these reagents were investigated. Theoretical calculations were used to describe the energetics of the cross-linker fragmentations as well as several low-energy side-chain-induced fragmentations which compete with disulfide cleavages. RESULTS: Altering the spacer arm length of the cross-linker, such that there is one methylene group less than in DSP, results in a more facile cleavage process, whilst the opposite is true when a methylene group is added. Of the low-energy side-chain-induced fragmentations studied...

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

Rahimi, Farid; Maiti, Panchanan; Bitan, Gal
Fonte: MyJove Corporation Publicador: MyJove Corporation
Tipo: Artigo de Revista Científica
Publicado em 12/01/2009 EN
Relevância na Pesquisa
66.34%
The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule...

Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking

Mouradov, Dmitri; Craven, Ari; Forwood, Jade; Flanagan, Jack U; Garcia-Castellanos, Raquel; Gomis-Ruth, F. Xavier; Hume, D A; Martin, Jennifer Louise; Kobe, Bostjan; Huber, Thomas
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
66.21%
We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were

Protein Structure Determination Using a Combination of Cross-Linking, Mass Spectrometry, and Molecular Modeling

Mouradov, Dmitri; King, Gordon J.; Ross, Ian R.; Forwood, Jade; Hume, D A; Sinz, Andrea; Martin, Jennifer Louise; Kobe, Bostjan; Huber, Thomas
Fonte: Humana Press Inc. Publicador: Humana Press Inc.
Tipo: Parte de Livro
Relevância na Pesquisa
66.14%
Cross-linking in combination with mass spectrometry can be used as a tool for structural modeling of protein complexes and multidomain proteins. Although cross-links represent only weak structural constraints, the combination of a limited set of experimen

XL-MS: Protein cross-linking coupled with mass spectrometry

Holding, Andrew N.
Fonte: Elsevier Publicador: Elsevier
Tipo: Article; published version
EN
Relevância na Pesquisa
66.1%
This is the final version. It was first published by Elsevier at http://dx.doi.org/10.1016/j.ymeth.2015.06.010; With the continuing trend to study larger and more complex systems, the application of protein cross-linking coupled with mass spectrometry (XL-MS) provides a varied toolkit perfectly suited to achieve these goals. By freezing the transient interactions through the formation of covalent bonds, XL-MS provides a vital insight into both the structure and organization of proteins in a wide variety of conditions. This review covers some of the established methods that underpin the field alongside the more recent developments that hold promise to further realize its potential in new directions.; This work was support by the Medical Research Council ? United Kingdom.