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[PMID]:29353670
[Au] Autor:Liu Y; Zhu P; Huang Z; Zhou L; Shi P
[Ad] Endereço:State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China.
[Ti] Título:Simultaneous detection of 5-fluorocytosine and 5-fluorouracil in human cells carrying CD/5-FC suicide gene system by using capillary zone electrophoresis.
[So] Source:J Chromatogr B Analyt Technol Biomed Life Sci;1076:1-7, 2018 Feb 15.
[Is] ISSN:1873-376X
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:A well-known suicide gene therapy approach, cytosine deaminase (CD) in combination with prodrug 5-flurocytosine (5-FC), has become an effective strategy of tumor treatment. However, there are short of simple and convenient detection methods to evaluate the efficiency of 5-FC conversion to 5-fluorouracil (5-FU) in human cells carrying various CD/5-FC systems. In this study, we developed an effective capillary zone electrophoresis (CZE) method to simultaneously measure 5-FC and 5-FU in cells carrying CD/5-FC suicide gene system. Under the condition of 60 mM borate buffer (pH 9.5) and 25 kV separation voltage with 0.5 psi × 15 s injection in 210 nm, the separation of 5-FC and 5-FU could be completely achieved within 15 min. The linearity of the calibration curve of standard 5-FC and 5-FU was in the range from 1 to 1000 µM (r > 0.999) and their recoveries were 98.4% and 96.0%, respectively. Due to the simple sample preparation and easy detection, this method is suitable for the study of the conversion efficiency of CD/5-FC suicide gene system. It aims to intuitively evaluate CD/5-FC systems and helps to guide the improvement of more effective CD/5-FC suicide gene systems.
[Mh] Termos MeSH primário: Eletroforese Capilar/métodos
Flucitosina/análise
Fluoruracila/análise
Genes Transgênicos Suicidas
[Mh] Termos MeSH secundário: Flucitosina/metabolismo
Fluoruracila/metabolismo
Células HEK293
Seres Humanos
Limite de Detecção
Modelos Lineares
Reprodutibilidade dos Testes
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
D83282DT06 (Flucytosine); U3P01618RT (Fluorouracil)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180307
[Lr] Data última revisão:
180307
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180123
[St] Status:MEDLINE


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[PMID]:29334632
[Au] Autor:MacLennan MS; Kok MGM; Soliman L; So A; Hurtado-Coll A; Chen DDY
[Ad] Endereço:University of British Columbia, Department of Chemistry, Vancouver, BC V6T 1Z1, Canada.
[Ti] Título:Capillary electrophoresis-mass spectrometry for targeted and untargeted analysis of the sub-5 kDa urine metabolome of patients with prostate or bladder cancer: A feasibility study.
[So] Source:J Chromatogr B Analyt Technol Biomed Life Sci;1074-1075:79-85, 2018 Feb 01.
[Is] ISSN:1873-376X
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:Targeted and untargeted analyses of the sub-5 kDa urine metabolome of genitourinary cancer patients (prostate and/or bladder) were performed without chemical derivatization using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). For targeted analysis, endogenous levels of sarcosine and 5 other amino acid metabolites implicated in the progression of prostate cancer were quantified in four patients and in a pooled urine sample from healthy volunteers. An untargeted analysis (m/z 50 to 850) of patient urine was performed using the same CE-ESI-MS system identifying over 400 distinct molecular features per patient. All patient urine samples were collected at prostatectomy/cystectomy via catheter. Patient urine samples were filtered by centrifugation, with endogenous sarcosine enriched by solid-phase extraction, and the processed samples loaded onto CE-ESI-MS for analysis. Diagnostic information, digital pathological slides, and tissue samples were collected and stored in a comprehensive biobanking database. The introduction of urine sample collection into the surgery workflow was facile and is a promising strategy for addressing the translational research challenge of moving smoothly from "chromatogram to nomogram".
[Mh] Termos MeSH primário: Biomarcadores Tumorais
Eletroforese Capilar/métodos
Metaboloma
Neoplasias da Próstata
Neoplasias da Bexiga Urinária
[Mh] Termos MeSH secundário: Adulto
Biomarcadores Tumorais/metabolismo
Biomarcadores Tumorais/urina
Estudos de Viabilidade
Seres Humanos
Masculino
Metabolômica/métodos
Neoplasias da Próstata/metabolismo
Neoplasias da Próstata/urina
Reprodutibilidade dos Testes
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
Neoplasias da Bexiga Urinária/metabolismo
Neoplasias da Bexiga Urinária/urina
Adulto Jovem
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Biomarkers, Tumor)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180305
[Lr] Data última revisão:
180305
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180116
[St] Status:MEDLINE


  3 / 16547 MEDLINE  
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[PMID]:29287247
[Au] Autor:Beloborodov SS; Bao J; Krylova SM; Shala-Lawrence A; Johnson PE; Krylov SN
[Ad] Endereço:Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada.
[Ti] Título:Aptamer facilitated purification of functional proteins.
[So] Source:J Chromatogr B Analyt Technol Biomed Life Sci;1073:201-206, 2018 Jan 15.
[Is] ISSN:1873-376X
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies.
[Mh] Termos MeSH primário: Aptâmeros de Nucleotídeos/química
Cromatografia de Afinidade/métodos
Ácidos Nucleicos Imobilizados/química
Proteínas Recombinantes/isolamento & purificação
[Mh] Termos MeSH secundário: Enzimas AlkB/química
Enzimas AlkB/genética
Enzimas AlkB/isolamento & purificação
Enzimas AlkB/metabolismo
Aptâmeros de Nucleotídeos/metabolismo
Eletroforese Capilar
Eletroforese em Gel de Poliacrilamida
Escherichia coli/genética
Ácidos Nucleicos Imobilizados/metabolismo
Metilação
Proteína MutS de Ligação de DNA com Erro de Pareamento/química
Proteína MutS de Ligação de DNA com Erro de Pareamento/genética
Proteína MutS de Ligação de DNA com Erro de Pareamento/isolamento & purificação
Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo
Ligação Proteica
Proteínas Recombinantes/química
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Aptamers, Nucleotide); 0 (Immobilized Nucleic Acids); 0 (Recombinant Proteins); EC 1.14.11.33 (AlkB Enzymes); EC 3.6.1.3 (MutS DNA Mismatch-Binding Protein)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180302
[Lr] Data última revisão:
180302
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171230
[St] Status:MEDLINE


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[PMID]:29188309
[Au] Autor:Gibson-Daw G; Crenshaw K; McCord B
[Ad] Endereço:Department of Chemistry and Biochemistry and International Forensic Research Institute, Florida International University, University Park, Miami, FL, 33199, USA.
[Ti] Título:Optimization of ultrahigh-speed multiplex PCR for forensic analysis.
[So] Source:Anal Bioanal Chem;410(1):235-245, 2018 Jan.
[Is] ISSN:1618-2650
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.
[Mh] Termos MeSH primário: Genética Forense/métodos
Técnicas de Genotipagem/métodos
Reação em Cadeia da Polimerase Multiplex/métodos
[Mh] Termos MeSH secundário: DNA/genética
Eletroforese Capilar
Genética Forense/economia
Genótipo
Técnicas de Genotipagem/economia
Seres Humanos
Dispositivos Lab-On-A-Chip
Repetições de Microssatélites
Reação em Cadeia da Polimerase Multiplex/economia
Fatores de Tempo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
9007-49-2 (DNA)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171201
[St] Status:MEDLINE
[do] DOI:10.1007/s00216-017-0715-x


  5 / 16547 MEDLINE  
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[PMID]:27774906
[Au] Autor:Tylkowski B; Nowak M; Tsibranska I; Trojanowska A; Marciniak L; Valls RG; Gumi T; Giamberini M; Jastrzab R
[Ad] Endereço:Centre Tecnològic de la Química de Catalunya, Carrer de Marcel·lí Domingo, 43007 Tarragona, Spain.
[Ti] Título:Concentration and Fractionation of Polyphenols by Membrane Operations.
[So] Source:Curr Pharm Des;23(2):231-241, 2017.
[Is] ISSN:1873-4286
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: This review aims to present the relevant background information and current research status in concentration of polyphenols using membrane technologies. The potential implementation of membrane separation to bioactive compounds like soluble phenolics from aqueous and organic solvent solutions is gaining increasing interest in the recent years. This review does not pretend to cover the abundant published literature on the subject, but to be representative for the observed tendencies in membrane processes applications for concentration of polyphenols derived from natural products. The first part of the article includes general information regarding the polyphenols and the traditional methods for their separation (such as: thin layer chromatography; paper chromatography; gas chromatography; high performance liquid chromatography; capillary electrophoresis), while the second part presents a review of different membrane processes applied for concentration of polyphenols. Three main sources for such implementations are discussed: (1) aqueous or organic solvent extracts from plant material, (2) fruits, and (3) recovery of polyphenols from industrial waste liquids. A diversity of membrane processes are considered in a large scope of implementations ranging from lab-scale studies to pilot and semiindustrial scale operations. CONCLUSION: Membrane technology is an excellent candidate to make a paradigm shift in biological active compounds fractionation/separation processes. Presented results clearly demonstrate that membrane processes are of great advantages over traditionally used methods; however, characterization of separated polyphenols has to be improved. Most of citied authors concentrated their investigation only on the total amount of polyphenols determination. Exhaustive studies including: antioxidant activities, retention index, total soluble solids, or volume reduction factor, have been only carried out by a few authors.
[Mh] Termos MeSH primário: Fracionamento Químico/métodos
Membranas Artificiais
Polifenóis/isolamento & purificação
[Mh] Termos MeSH secundário: Cromatografia Gasosa
Cromatografia Líquida de Alta Pressão
Cromatografia em Papel
Cromatografia em Camada Delgada
Eletroforese Capilar
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Membranes, Artificial); 0 (Polyphenols)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180227
[Lr] Data última revisão:
180227
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161025
[St] Status:MEDLINE
[do] DOI:10.2174/1381612822666161021124358


  6 / 16547 MEDLINE  
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[PMID]:29202360
[Au] Autor:Woo N; Kim SK; Sun Y; Kang SH
[Ad] Endereço:Department of Chemistry, Graduate School, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea.
[Ti] Título:Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR.
[So] Source:J Chromatogr B Analyt Technol Biomed Life Sci;1072:290-299, 2018 Jan 01.
[Is] ISSN:1873-376X
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer's disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer's. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120-180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4-62.0pM, which were approximately 100-100,000 times more sensitive than previous Alzheimer's diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer's patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer's with high sensitivity and short analysis time even with direct use of whole blood.
[Mh] Termos MeSH primário: Doença de Alzheimer/diagnóstico
Doença de Alzheimer/genética
Apolipoproteínas E/genética
Eletroforese Capilar/métodos
Técnicas de Genotipagem/métodos
Reação em Cadeia da Polimerase Multiplex/métodos
[Mh] Termos MeSH secundário: Doença de Alzheimer/sangue
Genótipo
Seres Humanos
Limite de Detecção
Modelos Lineares
Reprodutibilidade dos Testes
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (ApoE protein, human); 0 (Apolipoproteins E)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180221
[Lr] Data última revisão:
180221
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171205
[St] Status:MEDLINE


  7 / 16547 MEDLINE  
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[PMID]:29346414
[Au] Autor:Harada S; Hirayama A; Chan Q; Kurihara A; Fukai K; Iida M; Kato S; Sugiyama D; Kuwabara K; Takeuchi A; Akiyama M; Okamura T; Ebbels TMD; Elliott P; Tomita M; Sato A; Suzuki C; Sugimoto M; Soga T; Takebayashi T
[Ad] Endereço:Department of Preventive Medicine and Public Health, Keio University School of Medicine, Tokyo, Japan.
[Ti] Título:Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry.
[So] Source:PLoS One;13(1):e0191230, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Cohort studies with metabolomics data are becoming more widespread, however, large-scale studies involving 10,000s of participants are still limited, especially in Asian populations. Therefore, we started the Tsuruoka Metabolomics Cohort Study enrolling 11,002 community-dwelling adults in Japan, and using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry. The CE-MS method is highly amenable to absolute quantification of polar metabolites, however, its reliability for large-scale measurement is unclear. The aim of this study is to examine reproducibility and validity of large-scale CE-MS measurements. In addition, the study presents absolute concentrations of polar metabolites in human plasma, which can be used in future as reference ranges in a Japanese population. METHODS: Metabolomic profiling of 8,413 fasting plasma samples were completed using CE-MS, and 94 polar metabolites were structurally identified and quantified. Quality control (QC) samples were injected every ten samples and assessed throughout the analysis. Inter- and intra-batch coefficients of variation of QC and participant samples, and technical intraclass correlation coefficients were estimated. Passing-Bablok regression of plasma concentrations by CE-MS on serum concentrations by standard clinical chemistry assays was conducted for creatinine and uric acid. RESULTS AND CONCLUSIONS: In QC samples, coefficient of variation was less than 20% for 64 metabolites, and less than 30% for 80 metabolites out of the 94 metabolites. Inter-batch coefficient of variation was less than 20% for 81 metabolites. Estimated technical intraclass correlation coefficient was above 0.75 for 67 metabolites. The slope of Passing-Bablok regression was estimated as 0.97 (95% confidence interval: 0.95, 0.98) for creatinine and 0.95 (0.92, 0.96) for uric acid. Compared to published data from other large cohort measurement platforms, reproducibility of metabolites common to the platforms was similar to or better than in the other studies. These results show that our CE-MS platform is suitable for conducting large-scale epidemiological studies.
[Mh] Termos MeSH primário: Eletroforese Capilar/métodos
Espectrometria de Massas/métodos
Metabolômica/métodos
Plasma/metabolismo
[Mh] Termos MeSH secundário: Adulto
Idoso
Análise de Variância
Grupo com Ancestrais do Continente Asiático
Biomarcadores/sangue
Estudos de Coortes
Feminino
Seres Humanos
Japão
Masculino
Metaboloma
Metabolômica/normas
Meia-Idade
Controle de Qualidade
Valores de Referência
Reprodutibilidade dos Testes
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Biomarkers)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180220
[Lr] Data última revisão:
180220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180119
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0191230


  8 / 16547 MEDLINE  
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[PMID]:29284038
[Au] Autor:Bauer RJ; Zhelkovsky A; Bilotti K; Crowell LE; Evans TC; McReynolds LA; Lohman GJS
[Ad] Endereço:Research Division, New England Biolabs, Inc., Ipswich, MA, United States of America.
[Ti] Título:Comparative analysis of the end-joining activity of several DNA ligases.
[So] Source:PLoS One;12(12):e0190062, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, Paramecium bursaria chlorella virus 1 (PBCV1) DNA ligase, human DNA ligase 3, and Escherichia coli DNA ligase) were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3'- and 5'- single base overhangs, and 5'-two base overhangs). This analysis revealed that T4 DNA ligase, the most common enzyme utilized for in vitro ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5'-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3'-single base overhangs and 2-base overhangs effectively with little blunt or 5'- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5'-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain) were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.
[Mh] Termos MeSH primário: DNA Ligases/metabolismo
[Mh] Termos MeSH secundário: Quebras de DNA de Cadeia Dupla
Eletroforese Capilar
Seres Humanos
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
EC 6.5.1.- (DNA Ligases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180215
[Lr] Data última revisão:
180215
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171229
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0190062


  9 / 16547 MEDLINE  
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[PMID]:29244942
[Au] Autor:Ivanov AV; Kuchukova MY; Virus ED; Zurina IM; Luzyanin BP; Kubatiev AA
[Ti] Título:Application of the capillary electrophoresis method for the study of plasma proteins homocysteinylation.
[So] Source:Patol Fiziol Eksp Ter;60(4):178-84, 2016 Oct-Dec.
[Is] ISSN:0031-2991
[Cp] País de publicação:Russia (Federation)
[La] Idioma:eng
[Ab] Resumo:Purpose: Purpose. This article describes the use of capillary electrophoresis with UV detection to determine the ratio of protein-bound homocysteine and cysteine concentrations in human plasma. Methods: Plasma samples were reduced with dithiothreitol and derivatized by thiocarbonyldiimidazole before being filtered again for purification of proteins. The pre-concentration of analytes was carried out directly in the capillary (48.5 cm in length and an inner diameter of 50 mkm) by NaOH post-injection. The eletrophoretic separation of analytes was carried out using 0.2 M ammonium acetate with 25 mM hexadecyltrimethylammonium bromide. Results: Limit of quantitation for homocysteine was 0.8 mkM, reproducible ratio of cysteine/homocysteine <5%, full analysis time 15 min. Conclusion: The ratio of bound cysteine to homocysteine is characterized by the same regularity as the ratio of their total content. It has a fairly high degree of correlation with the level of bound homocysteine and it is characterized by less variability than the level of total homocysteine. This has the advantage of use the bound cysteine/homocysteine ratio for assessing the risk of cardiovascular disease complications.
[Mh] Termos MeSH primário: Proteínas Sanguíneas/metabolismo
Homocisteína/metabolismo
Processamento de Proteína Pós-Traducional
[Mh] Termos MeSH secundário: Adulto
Eletroforese Capilar/métodos
Feminino
Seres Humanos
Meia-Idade
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Blood Proteins); 0LVT1QZ0BA (Homocysteine)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180118
[Lr] Data última revisão:
180118
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171216
[St] Status:MEDLINE


  10 / 16547 MEDLINE  
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[PMID]:27776640
[Au] Autor:Moreno-Gordaliza E; van der Lee SJ; Demirkan A; van Duijn CM; Kuiper J; Lindenburg PW; Hankemeier T
[Ad] Endereço:Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Faculty of Science, Universiteit Leiden, Einsteinweg 55, 2300 RA Leiden, The Netherlands. Electronic address: emorenog@ucm.es.
[Ti] Título:A novel method for serum lipoprotein profiling using high performance capillary isotachophoresis.
[So] Source:Anal Chim Acta;944:57-69, 2016 Nov 09.
[Is] ISSN:1873-4324
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:A new capillary isotachophoresis (cITP) method for lipoprotein profiling with superior lipoprotein coverage compared to previous methods has been developed, resolving twice as many lipoprotein species (18 peaks/fractions) in serum or plasma in less than 9.5 min. For this, a novel mixture of 24 spacers, including amino acids, dipeptides and sulfonic acids, was developed and fine-tuned, using predictive software (PeakMaster) and testing of spiked serum samples. Lipoprotein peaks were identified by serum-spiking with reference lipoproteins. Compatibility with common lipophilic stains for selective lipoprotein detection with either UV/Vis or laser-induced fluorescence was demonstrated. A special new capillary with a neutral coating (combining water-compatible OV1701-OH deactivation and methylation) was used for the first time for electrodriven separations, allowing very stable separations in a pH 8.8-9.4 gradient system, being functional for more than 100 injections. Excellent reproducibility was achieved, with coefficients of variation lower than 2.6% for absolute migration times. Comparison was performed with human plasma samples analyzed by NMR, leading to similar results with cITP after multivariate statistics, regarding group-clustering and lipoprotein species correlation. The new cITP method was applied to the analysis of serum samples from a LDL receptor knock-out mice model fed either a normal diet or a western-type diet. Differences in the lipoprotein levels and in the sublipoprotein types were detected, showing a shift to more atherogenic particles due to the high cholesterol diet. In summary, this novel method will allow more detailed and informative profiling of lipoprotein particle subtypes for cardiovascular disease research.
[Mh] Termos MeSH primário: Análise Química do Sangue/métodos
Eletroforese Capilar/métodos
Isotacoforese/métodos
Lipoproteínas/sangue
Lipoproteínas/isolamento & purificação
[Mh] Termos MeSH secundário: Animais
Técnicas de Inativação de Genes
Seres Humanos
Camundongos
Receptores de LDL/deficiência
Receptores de LDL/genética
Software
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Lipoproteins); 0 (Receptors, LDL)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180116
[Lr] Data última revisão:
180116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161026
[St] Status:MEDLINE



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