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[PMID]:26370251
[Au] Autor:Shin Y; Lim SY; Lee TY; Park MK
[Ad] Endereço:Institute of Microelectronics, A*STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, Singapore 117685.
[Ti] Título:Dimethyl adipimidate/Thin film Sample processing (DTS); A simple, low-cost, and versatile nucleic acid extraction assay for downstream analysis.
[So] Source:Sci Rep;5:14127, 2015 Sep 15.
[Is] ISSN:2045-2322
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Sample processing, especially that involving nucleic acid extraction, is a prerequisite step for the isolation of high quantities of relatively pure DNA for downstream analyses in many life science and biomedical engineering studies. However, existing methods still have major problems, including labor-intensive time-consuming methods and high costs, as well as requirements for a centrifuge and the complex fabrication of filters and membranes. Here, we first report a versatile Dimethyl adipimidate/Thin film based Sample processing (DTS) procedure without the limitations of existing methods. This procedure is useful for the extraction of DNA from a variety of sources, including 6 eukaryotic cells, 6 bacteria cells, and 2 body fluids in a single step. Specifically, the DTS procedure does not require a centrifuge and has improved time efficiency (30 min), affordability, and sensitivity in downstream analysis. We validated the DTS procedure for the extraction of DNA from human body fluids, as well as confirmed that the quality and quantity of the extracted DNA were sufficient to allow robust detection of genetic and epigenetic biomarkers in downstream analysis.
[Mh] Termos MeSH primário: DNA/isolamento & purificação
Dimetil Adipimidato/química
[Mh] Termos MeSH secundário: Animais
Líquidos Corporais
Células Eucarióticas
Seres Humanos
Dispositivos Lab-On-A-Chip
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
13139-70-3 (Dimethyl Adipimidate); 9007-49-2 (DNA)
[Em] Mês de entrada:1608
[Cu] Atualização por classe:170220
[Lr] Data última revisão:
170220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:150916
[St] Status:MEDLINE
[do] DOI:10.1038/srep14127


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[PMID]:24263404
[Au] Autor:Shin Y; Perera AP; Wong CC; Park MK
[Ad] Endereço:Institute of Microelectronics, A*STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II, Singapore 117685. parkmk@ime.a-star.edu.sg.
[Ti] Título:Solid phase nucleic acid extraction technique in a microfluidic chip using a novel non-chaotropic agent: dimethyl adipimidate.
[So] Source:Lab Chip;14(2):359-68, 2014 Jan 21.
[Is] ISSN:1473-0189
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Here, we present a silicon microfluidic system for the purification and extraction of nucleic acids from human body fluid samples utilizing a dimethyl adipimidate (DMA)-based solid-phase extraction method. We propose DMA, which has been used as an amino-reactive cross-linking agent within cells and proteins, as a non-chaotropic reagent for the capture of nucleic acids to overcome the limitations of existing chaotropic and non-chaotropic techniques such as low binding efficiency, PCR inhibition and so on. DMA contains bi-functional imidoesters that form reversible cross-linking structures with DNA therefore providing a high surface-area to volume ratio for capturing DNA without structurally modifying microfluidic channels. In this work, we have first demonstrated highly efficient capture and purification of genomic DNA (T24 cell line) with DMA using a label-free silicon microring resonator sensor device. In addition, we observed the improvement of the DNA amplification efficiency by using the proposed technique for both the genetic (HRAS) and epigenetic (RARß) analysis of DNA biomarkers. Particularly, we confirmed that the DMA-based solid-phase extraction technique can be applied for the extraction of genomic DNA with higher purity (p < 0.001) using human body fluids (blood and urine) in silicon microfluidic devices compared to other chaotropic methods. Therefore, the proposed technique would be able to harmonize with a micro-total analysis system platform for the analysis of genetic and epigenetic DNA biomarkers related to human diseases in the field of point-of-care (POC) diagnostic applications.
[Mh] Termos MeSH primário: Dimetil Adipimidato/química
Técnicas Analíticas Microfluídicas/instrumentação
Ácidos Nucleicos/isolamento & purificação
[Mh] Termos MeSH secundário: Sequência de Bases
Primers do DNA
Epigênese Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (DNA Primers); 0 (Nucleic Acids); 13139-70-3 (Dimethyl Adipimidate)
[Em] Mês de entrada:1408
[Cu] Atualização por classe:131211
[Lr] Data última revisão:
131211
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:131123
[St] Status:MEDLINE
[do] DOI:10.1039/c3lc51035b


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[PMID]:23468006
[Au] Autor:Ismaya WT; Hasan K; Kardi I; Zainuri A; Rahmawaty RI; Permanahadi S; El Viera BV; Harinanto G; Gaffar S; Natalia D; Subroto T; Soemitro S
[Ad] Endereço:Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jalan Singaperbangsa No. 2, 40133 Bandung, Indonesia. wangsatirta@gmail.com
[Ti] Título:Chemical modification of Saccharomycopsis fibuligera R64 α-amylase to improve its stability against thermal, chelator, and proteolytic inactivation.
[So] Source:Appl Biochem Biotechnol;170(1):44-57, 2013 May.
[Is] ISSN:1559-0291
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:α-Amylase catalyzes hydrolysis of starch to oligosaccharides, which are further degraded to simple sugars. The enzyme has been widely used in food and textile industries and recently, in generation of renewable energy. An α-amylase from yeast Saccharomycopsis fibuligera R64 (Sfamy) is active at 50 °C and capable of degrading raw starch, making it attractive for the aforementioned applications. To improve its characteristics as well as to provide information for structural study ab initio, the enzyme was chemically modified by acid anhydrides (nonpolar groups), glyoxylic acid (GA) (polar group), dimethyl adipimidate (DMA) (cross-linking), and polyethylene glycol (PEG) (hydrophilization). Introduction of nonpolar groups increased enzyme stability up to 18 times, while modification by a cross-linking agent resulted in protection of the calcium ion, which is essential for enzyme activity and integrity. The hydrophilization with PEG resulted in protection against tryptic digestion. The chemical modification of Sfamy by various modifiers has thereby resulted in improvement of its characteristics and provided systematic information beneficial for structural study of the enzyme. An in silico structural study of the enzyme improved the interpretation of the results.
[Mh] Termos MeSH primário: Proteínas Fúngicas/química
Engenharia de Proteínas/métodos
Saccharomycopsis/química
alfa-Amilases/química
[Mh] Termos MeSH secundário: Anidridos Acéticos/química
Sequência de Aminoácidos
Quelantes/química
Reagentes para Ligações Cruzadas/química
Dimetil Adipimidato/química
Estabilidade Enzimática
Glioxilatos/química
Temperatura Alta
Hidrólise
Modelos Moleculares
Dados de Sequência Molecular
Polietilenoglicóis/química
Proteólise
Saccharomycopsis/enzimologia
Amido/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Acetic Anhydrides); 0 (Chelating Agents); 0 (Cross-Linking Reagents); 0 (Fungal Proteins); 0 (Glyoxylates); 13139-70-3 (Dimethyl Adipimidate); 30IQX730WE (Polyethylene Glycols); 9005-25-8 (Starch); EC 3.2.1.1 (alpha-Amylases); JQ39C92HH6 (glyoxylic acid)
[Em] Mês de entrada:1310
[Cu] Atualização por classe:151119
[Lr] Data última revisão:
151119
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:130308
[St] Status:MEDLINE
[do] DOI:10.1007/s12010-013-0164-8


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[PMID]:16543728
[Au] Autor:Muzyamba MC; Campbell EH; Gibson JS
[Ad] Endereço:Department of Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, UK.
[Ti] Título:Effect of intracellular magnesium and oxygen tension on K+-Cl- cotransport in normal and sickle human red cells.
[So] Source:Cell Physiol Biochem;17(3-4):121-8, 2006.
[Is] ISSN:1015-8987
[Cp] País de publicação:Switzerland
[La] Idioma:eng
[Ab] Resumo:In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb.
[Mh] Termos MeSH primário: Eritrócitos Anormais/metabolismo
Eritrócitos/metabolismo
Magnésio/sangue
Oxigênio/fisiologia
Simportadores/metabolismo
[Mh] Termos MeSH secundário: Anemia Falciforme/sangue
Antidrepanocíticos/farmacologia
Dimetil Adipimidato/farmacologia
Inibidores Enzimáticos/farmacologia
Etilmaleimida/farmacologia
Seres Humanos
Manometria
Oxazóis/farmacologia
Oxigênio/sangue
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Antisickling Agents); 0 (Enzyme Inhibitors); 0 (Oxazoles); 0 (Symporters); 0 (potassium-chloride symporters); 13139-70-3 (Dimethyl Adipimidate); 7D07U14TK3 (calyculin A); I38ZP9992A (Magnesium); O3C74ACM9V (Ethylmaleimide); S88TT14065 (Oxygen)
[Em] Mês de entrada:0605
[Cu] Atualização por classe:170922
[Lr] Data última revisão:
170922
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:060318
[St] Status:MEDLINE


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[PMID]:16321965
[Au] Autor:McCabe CD; Innis JW
[Ad] Endereço:Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA.
[Ti] Título:A genomic approach to the identification and characterization of HOXA13 functional binding elements.
[So] Source:Nucleic Acids Res;33(21):6782-94, 2005.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:HOX proteins are important transcriptional regulators in mammalian embryonic development and are dysregulated in human cancers. However, there are few known direct HOX target genes and their mechanisms of regulation are incompletely understood. To isolate and characterize gene segments through which HOX proteins regulate transcription we used cesium chloride centrifugation-based chromatin purification and immunoprecipitation (ChIP). From NIH 3T3-derived HOXA13-FLAG expressing cells, 33% of randomly selected, ChIP clones were reproducibly enriched. Hox-enriched fragments (HEFs) were more AT-rich compared with cloned fragments that failed reproducible ChIP. All HEFs augmented transcription of a heterologous promoter upon coexpression with HOXA13. One HEF was from intron 2 of Enpp2, a gene highly upregulated in these cells and has been implicated in cell motility. Using Enpp2 as a candidate direct target, we identified three additional HEFs upstream of the transcription start site. HOXA13 upregulated transcription from an Enpp2 promoter construct containing these sites, and each site was necessary for full HOXA13-induced expression. Lastly, given that HOX proteins have been demonstrated to interact with histone deacetylases and/or CBP, we explored whether histone acetylation changed at Enpp2 upon HOXA13-induced activation. No change in the general histone acetylation state was observed. Our results support models in which occupation of multiple HOX binding sites is associated with highly activated genes.
[Mh] Termos MeSH primário: Proteínas de Homeodomínio/metabolismo
Elementos de Resposta
Ativação Transcricional
[Mh] Termos MeSH secundário: Acetilação
Animais
Sítios de Ligação
Césio/química
Cloretos/química
Cromatina/isolamento & purificação
Imunoprecipitação da Cromatina
Reagentes para Ligações Cruzadas/química
Dimetil Adipimidato/química
Genes Reporter
Genômica
Histonas/metabolismo
Camundongos
Complexos Multienzimáticos/genética
Células NIH 3T3
Fosfodiesterase I/genética
Diester Fosfórico Hidrolases
Pirofosfatases/genética
Regulação para Cima
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL
[Nm] Nome de substância:
0 (Chlorides); 0 (Chromatin); 0 (Cross-Linking Reagents); 0 (Histones); 0 (Homeodomain Proteins); 0 (Multienzyme Complexes); 0 (homeobox protein HOXA13); 13139-70-3 (Dimethyl Adipimidate); 1KSV9V4Y4I (Cesium); EC 3.1.4.- (Phosphoric Diester Hydrolases); EC 3.1.4.1 (Phosphodiesterase I); EC 3.1.4.39 (alkylglycerophosphoethanolamine phosphodiesterase); EC 3.6.1.- (Pyrophosphatases); GNR9HML8BA (cesium chloride)
[Em] Mês de entrada:0512
[Cu] Atualização por classe:161019
[Lr] Data última revisão:
161019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:051203
[St] Status:MEDLINE


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[PMID]:12614227
[Au] Autor:Turner EJ; Jarvis HG; Chetty MC; Landon G; Rowley PS; Ho MM; Stewart GW
[Ad] Endereço:Department of Medicine, University College London, Rayne Institute, London, UK.
[Ti] Título:ATP-dependent vesiculation in red cell membranes from different hereditary stomatocytosis variants.
[So] Source:Br J Haematol;120(5):894-902, 2003 Mar.
[Is] ISSN:0007-1048
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The hereditary stomatocytoses are a group of dominant haemolytic anaemias that show two main features: invaginated, 'stomatocytic' morphology; and a membrane leak to the univalent cations Na and K. A patient with the most severe variant of these conditions was reported to show a defect in an in vitro process of ATP-dependent endocytic vesiculation (ADEV), which is found in normal red cells. We have examined this endocytosis process in 11 leaky red cell pedigrees available to us in the UK. ADEV in broken membranes was absent only in the two most severely affected, 'overhydrated' pedigrees studied, both of which showed a deficiency in the membrane raft protein, stomatin. The process was present, although typically diminished by about 10-20% compared with normal red cells, in all others. The cross-linker dimethyl adipimate (DMA), which could correct the cation leak in some of these patients, also corrected the ADEV defect in the same patients. In those patients in whom DMA had no effect on the ion leak, ADEV was not absent. In normal cells, this process of vesiculation was inhibited by inhibitors of membrane 'raft' function, by an antistomatin antibody and by vanadate and N-ethyl maleimide, but not by inhibitors of a number of kinases. These data highlight the heterogeneity of these conditions. A mechanism is discussed by which a defect in raft-based endocytosis could lead to the exaggerated surface exposure of an ion channel, which could then function constitutively, i.e. 'leak'.
[Mh] Termos MeSH primário: Trifosfato de Adenosina/metabolismo
Anemia Hemolítica/genética
Membrana Eritrocítica/metabolismo
[Mh] Termos MeSH secundário: Anemia Hemolítica/sangue
Cátions
Vesículas Citoplasmáticas
Dimetil Adipimidato/farmacologia
Relação Dose-Resposta a Droga
Endocitose/genética
Seres Humanos
Indicadores e Reagentes/farmacologia
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cations); 0 (Indicators and Reagents); 13139-70-3 (Dimethyl Adipimidate); 8L70Q75FXE (Adenosine Triphosphate)
[Em] Mês de entrada:0305
[Cu] Atualização por classe:131121
[Lr] Data última revisão:
131121
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:030305
[St] Status:MEDLINE


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[PMID]:12051752
[Au] Autor:Hu T; Su Z
[Ad] Endereço:National Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China. hutao1975@yahoo.com
[Ti] Título:Preparation of well-defined bovine polyhemoglobin based on dimethyl adipimidate and glutaraldebyde cross-linkage.
[So] Source:Biochem Biophys Res Commun;293(3):958-61, 2002 May 10.
[Is] ISSN:0006-291X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:A well-defined bovine polyhemoglobin was prepared by dimethyl adipimidate (DMA) and glutaraldehyde double cross-linkage method. DMA was used to block some amino groups of hemoglobin, followed by further polymerization with glutaraldehyde. The amino modification degree of hemoglobin was 32% when DMA reacted with hemoglobin at the molar ratio of 200. The bovine polyhemoglobin with narrow molecular weight distribution (mainly 128 kDa) was obtained when glutaraldehyde reacted with DMA-modified hemoglobin. The P(50) and the Hill coefficient for DMA-modified hemoglobin were 19.4 mm Hg and 2.28, respectively, while those for the bovine polyhemoglobin were 15.1 mm Hg and 1.70, respectively. The number of Bohr protons released for DMA-modified hemoglobin and the polyhemoglobin was 0.86 and 0.56 H/tetramer, respectively.
[Mh] Termos MeSH primário: Substitutos Sanguíneos/química
Reagentes para Ligações Cruzadas/química
Dimetil Adipimidato/química
Glutaral/química
Hemoglobinas/química
[Mh] Termos MeSH secundário: Animais
Substitutos Sanguíneos/metabolismo
Bovinos
Eletroforese em Gel de Poliacrilamida
Hemoglobinas/metabolismo
Concentração de Íons de Hidrogênio
Oxigênio/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Blood Substitutes); 0 (Cross-Linking Reagents); 0 (Hemoglobins); 0 (polyhemoglobin); 13139-70-3 (Dimethyl Adipimidate); S88TT14065 (Oxygen); T3C89M417N (Glutaral)
[Em] Mês de entrada:0206
[Cu] Atualização por classe:131121
[Lr] Data última revisão:
131121
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:020608
[St] Status:MEDLINE


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[PMID]:11034324
[Au] Autor:Gibson JS; Stewart GW; Ellory JC
[Ad] Endereço:Department of Physiology, St George's Hospital Medical School, University of London, Cranmer Terrace, UK. jsgibson@sghms.ac.uk
[Ti] Título:Effect of dimethyl adipimidate on K+ transport and shape change in red blood cells from sickle cell patients.
[So] Source:FEBS Lett;480(2-3):179-83, 2000 Sep 01.
[Is] ISSN:0014-5793
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Dimethyl adipimidate (DMA) reduces K+ loss from, and dehydration of, red cells containing haemoglobin S (HbS cells). Three membrane transporters may contribute to these processes: the deoxygenation-induced cation-selective channel (Psickle), the Ca2+-activated K+ channel (or Gardos channel) and the K+-CI- cotransporter (KCC). We show that DMA inhibited all three pathways in deoxygenated HbS cells. The Gardos channel could be activated following Ca2+ loading. Considerable KCC activity was present in oxygenated HbS cells, showing a selective action of DMA on the transporter in deoxygenated cells. Inhibition of sickling correlated strongly with that of Psickle and moderately with that of KCC activity. We conclude that DMA does not inhibit the K+ pathways directly, but acts mainly by preventing HbS polymerisation and sickling. These findings are relevant to the development of novel chemotherapeutic agents for amelioration of sickle cell disease.
[Mh] Termos MeSH primário: Anemia Falciforme/sangue
Dimetil Adipimidato/farmacologia
Eritrócitos/efeitos dos fármacos
Potássio/metabolismo
Simportadores
[Mh] Termos MeSH secundário: Transporte Biológico
Proteínas de Transporte/metabolismo
Cátions Monovalentes
Tamanho Celular/efeitos dos fármacos
Células Cultivadas
Eritrócitos/citologia
Eritrócitos/metabolismo
Hemoglobina Falciforme
Seres Humanos
Oxigênio/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Carrier Proteins); 0 (Cations, Monovalent); 0 (Hemoglobin, Sickle); 0 (Symporters); 0 (potassium-chloride symporters); 13139-70-3 (Dimethyl Adipimidate); RWP5GA015D (Potassium); S88TT14065 (Oxygen)
[Em] Mês de entrada:0011
[Cu] Atualização por classe:131121
[Lr] Data última revisão:
131121
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:001018
[St] Status:MEDLINE


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[PMID]:10438143
[Au] Autor:Ouimet PM; Kapoor M
[Ad] Endereço:Department of Biological Sciences, University of Calgary, Canada.
[Ti] Título:Nucleotide binding and hydrolysis properties of Neurospora crassa cytosolic molecular chaperones, Hsp70 and Hsp80, heat-inducible members of the eukaryotic stress-70 and stress-90 families.
[So] Source:Biochem Cell Biol;77(2):89-99, 1999.
[Is] ISSN:0829-8211
[Cp] País de publicação:Canada
[La] Idioma:eng
[Ab] Resumo:Formation of a hetero-oligomeric complex between Hsp70 and Hsp80 of Neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (ELISA). The present study documents the effect of nucleotides on the subunit structure of Hsp70 and Hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). The inter-protomer crosslinking of Hsp80 with DMA and DMS was suppressed by ATP and to a lesser extent by ADP, CTP, and NAD. Crosslinking of purified Hsp70 by glutaraldehyde yielded dimers and higher order oligomers. Binding of ATP, ADP, CTP, and NAD, but not NADH, led to a marked reduction in the yield of oligomers. Similarly, crosslinking by DMA and DMS was suppressed by ADP, ATP, and CTP. Both Hsp70 and Hsp80 exhibited intrinsic ATPase activity. Interestingly, ATP levels exceeding 25 microM resulted in pronounced inhibition of the ATPase activity of Hsp80 and 0.5 mM and 0.25 mM ATP led to a prolonged lag in the reaction. Addition of NAD resulted in the abolition of the lag period. The binding of 2-p-toluidinylnapthalene-6-sulfonate (TNS) to Hsp70 and its displacement by ATP and other nucleotides demonstrated the hydrophobic nature of the nucleotide-binding region.
[Mh] Termos MeSH primário: Proteínas Fúngicas/metabolismo
Proteínas de Choque Térmico HSP70/metabolismo
Proteínas de Choque Térmico/metabolismo
Neurospora crassa/metabolismo
Nucleotídeos/metabolismo
[Mh] Termos MeSH secundário: Adenosina Trifosfatases/metabolismo
Reagentes para Ligações Cruzadas
Citosol/metabolismo
Dimetil Adipimidato
Dimetil Suberimidato
Corantes Fluorescentes/metabolismo
Glutaral
Calefação
Hidrólise
Naftalenossulfonatos/metabolismo
Especificidade por Substrato
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cross-Linking Reagents); 0 (Fluorescent Dyes); 0 (Fungal Proteins); 0 (HSP70 Heat-Shock Proteins); 0 (Heat-Shock Proteins); 0 (Naphthalenesulfonates); 0 (Nucleotides); 13139-70-3 (Dimethyl Adipimidate); 29878-26-0 (Dimethyl Suberimidate); 7724-15-4 (2-(4-toluidino)-6-naphthalenesulfonic acid); EC 3.6.1.- (Adenosine Triphosphatases); T3C89M417N (Glutaral)
[Em] Mês de entrada:9910
[Cu] Atualização por classe:131121
[Lr] Data última revisão:
131121
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:990807
[St] Status:MEDLINE


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[PMID]:9729623
[Au] Autor:Duszyk M; Shu Y; Ho AK; Man SF
[Ad] Endereço:Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. mduszyk@physio.med.ualberta.ca
[Ti] Título:Activation of bovine tracheal chloride channels by amino group-specific reagents.
[So] Source:J Physiol;512 ( Pt 1):129-36, 1998 Oct 01.
[Is] ISSN:0022-3751
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:1. The predominant Cl- channel in bovine tracheal epithelial cells has a conductance of approximately 71 pS and accounts for more than 80 % of the total chloride conductance. We examined the effects of protein-modifying reagents on channel function and found that amino groups are critically involved in gating. 2. Patch clamp studies showed that lysine-specific reagents, such as dimethyl adipimidate (DMA), significantly increased the channel open probability, but not its conductance. This suggests that modified residues are involved in the gating mechanism, but are distant from the channel permeation pathway. 3. Kinetic analysis of channel activity showed that histograms of open and closed durations could be well fitted by double exponential distributions, suggesting that the channel has at least two open and two closed states. DMA did not change the number of open or closed states, but increased channel mean open time. 4. Since membrane impermeant reagents were effective only from the extracellular side, we conclude that lysine residues in the extracellular domain of the channel are critically involved in gating. These residues may present an important target for site-directed mutagenesis and pharmacological activation of Cl- channels in epithelial cells.
[Mh] Termos MeSH primário: Canais de Cloreto/fisiologia
Dimetil Adipimidato/farmacologia
Traqueia/fisiologia
[Mh] Termos MeSH secundário: Animais
Bovinos
Membrana Celular/fisiologia
Canais de Cloreto/efeitos dos fármacos
Condutividade Elétrica
Células Epiteliais/efeitos dos fármacos
Células Epiteliais/fisiologia
Ativação do Canal Iônico/fisiologia
Cinética
Cadeias de Markov
Proteínas de Membrana/metabolismo
Técnicas de Patch-Clamp
Fosforilação
Espectrometria de Fluorescência
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Chloride Channels); 0 (Membrane Proteins); 13139-70-3 (Dimethyl Adipimidate)
[Em] Mês de entrada:9812
[Cu] Atualização por classe:140617
[Lr] Data última revisão:
140617
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:980908
[St] Status:MEDLINE



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