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  1 / 1906 MEDLINE  
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[PMID]:28947300
[Au] Autor:Prokopowicz M; Gren B; Ciesla J; Kierdaszuk B
[Ad] Endereço:Inter-Faculty Interdisciplinary Doctoral Studies in Natural Sciences and Mathematics, University of Warsaw, Stefana Banacha 2C, Warsaw 02-097, Poland; Department of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, Warsaw 02-089, Poland. Ele
[Ti] Título:Towards understanding the E. coli PNP binding mechanism and FRET absence between E. coli PNP and formycin A.
[So] Source:Biophys Chem;230:99-108, 2017 Nov.
[Is] ISSN:1873-4200
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The aim of this study is threefold: (1) augmentation of the knowledge of the E. coli PNP binding mechanism; (2) explanation of the previously observed 'lack of FRET' phenomenon and (3) an introduction of the correction (modified method) for FRET efficiency calculation in the PNP-FA complexes. We present fluorescence studies of the two E. coli PNP mutants (F159Y and F159A) with formycin A (FA), that indicate that the aromatic amino acid is indispensable in the nucleotide binding, additional hydroxyl group at position 159 probably enhances the strength of binding and that the amino acids pair 159-160 has a great impact on the spectroscopic properties of the enzyme. The experiments were carried out in hepes and phosphate buffers, at pH7 and 8.3. Two methods, a conventional and a modified one, that utilizes the dissociation constant, for calculations of the energy transfer efficiency (E) and the acceptor-to-donor distance (r) between FA and the Tyr (energy donor) were employed. Total difference spectra were calculated for emission spectra (λ 280nm, 295nm, 305nm and 313nm) for all studied systems. Time-resolved techniques allowed to conclude the existence of a specific structure formed by amino acids at positions 159 and 160. The results showed an unexpected pattern change of FRET in the mutants, when compared to the wild type enzyme and a probable presence of a structure created between 159 and 160 residue, that might influence the binding efficiency. Additionally, we confirmed the indispensable role of the modification of the FRET efficiency (E) calculation on the fraction of enzyme saturation in PNP-FA systems.
[Mh] Termos MeSH primário: Proteínas de Escherichia coli/metabolismo
Escherichia coli/enzimologia
Formicinas/metabolismo
Purina-Núcleosídeo Fosforilase/metabolismo
[Mh] Termos MeSH secundário: Proteínas de Escherichia coli/química
Proteínas de Escherichia coli/genética
Transferência Ressonante de Energia de Fluorescência
Formicinas/química
Concentração de Íons de Hidrogênio
Cinética
Mutagênese Sítio-Dirigida
Ligação Proteica
Estrutura Terciária de Proteína
Purina-Núcleosídeo Fosforilase/química
Purina-Núcleosídeo Fosforilase/genética
Espectrometria de Fluorescência
Temperatura Ambiente
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Escherichia coli Proteins); 0 (Formycins); 6742-12-7 (formycin); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171030
[Lr] Data última revisão:
171030
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170927
[St] Status:MEDLINE


  2 / 1906 MEDLINE  
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[PMID]:28836767
[Au] Autor:Namanja-Magliano HA; Evans GB; Harijan RK; Tyler PC; Schramm VL
[Ad] Endereço:Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
[Ti] Título:Transition State Analogue Inhibitors of 5'-Deoxyadenosine/5'-Methylthioadenosine Nucleosidase from Mycobacterium tuberculosis.
[So] Source:Biochemistry;56(38):5090-5098, 2017 Sep 26.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Mycobacterium tuberculosis 5'-deoxyadenosine/5'-methylthioadenosine nucleosidase (Rv0091) catalyzes the N-riboside hydrolysis of its substrates 5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'-dAdo). 5'-dAdo is the preferred substrate, a product of radical S-adenosylmethionine-dependent enzyme reactions. Rv0091 is characterized by a ribocation-like transition state, with low N-ribosidic bond order, an N7-protonated adenine leaving group, and an activated but weakly bonded water nucleophile. DADMe-Immucillins incorporating 5'-substituents of the substrates 5'-dAdo and MTA were synthesized and characterized as inhibitors of Rv0091. 5'-Deoxy-DADMe-Immucillin-A was the most potent among the 5'-dAdo transition state analogues with a dissociation constant of 640 pM. Among the 5'-thio substituents, hexylthio-DADMe-Immucillin-A was the best inhibitor at 87 pM. The specificity of Rv0091 for the Immucillin transition state analogues differs from those of other bacterial homologues because of an altered hydrophobic tunnel accepting the 5'-substituents. Inhibitors of Rv0091 had weak cell growth effects on M. tuberculosis or Mycobacterium smegmatis but were lethal toward Helicobacter pylori, where the 5'-methylthioadenosine nucleosidase is essential in menaquinone biosynthesis. We propose that Rv0091 plays a role in 5'-deoxyadenosine recycling but is not essential for growth in these Mycobacteria.
[Mh] Termos MeSH primário: Antibacterianos/farmacologia
Proteínas de Bactérias/antagonistas & inibidores
Inibidores Enzimáticos/farmacologia
Mycobacterium tuberculosis/enzimologia
Purina-Núcleosídeo Fosforilase/antagonistas & inibidores
[Mh] Termos MeSH secundário: Adenina/análogos & derivados
Adenina/química
Adenina/farmacologia
Antibacterianos/síntese química
Antibacterianos/química
Proteínas de Bactérias/química
Proteínas de Bactérias/metabolismo
Técnicas de Química Sintética
Inibidores Enzimáticos/síntese química
Inibidores Enzimáticos/química
Helicobacter pylori/efeitos dos fármacos
Testes de Sensibilidade Microbiana
Modelos Moleculares
Mycobacterium tuberculosis/efeitos dos fármacos
Mycobacterium tuberculosis/genética
Purina-Núcleosídeo Fosforilase/química
Purina-Núcleosídeo Fosforilase/metabolismo
Pirrolidinas/química
Pirrolidinas/farmacologia
Homologia Estrutural de Proteína
Relação Estrutura-Atividade
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (4'-deaza-1'-aza-2'-deoxy-1'-(9-methylene)-immucillin A); 0 (Anti-Bacterial Agents); 0 (Bacterial Proteins); 0 (Enzyme Inhibitors); 0 (Pyrrolidines); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase); EC 2.4.2.28 (5'-methylthioadenosine phosphorylase); JAC85A2161 (Adenine)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171010
[Lr] Data última revisão:
171010
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170825
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.7b00576


  3 / 1906 MEDLINE  
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[PMID]:28794158
[Au] Autor:Gebre ST; Cameron SA; Li L; Babu YS; Schramm VL
[Ad] Endereço:From the Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 and.
[Ti] Título:Intracellular rebinding of transition-state analogues provides extended inhibition lifetimes on human purine nucleoside phosphorylase.
[So] Source:J Biol Chem;292(38):15907-15915, 2017 Sep 22.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Purine nucleoside phosphorylase (PNP) is part of the human purine salvage pathway. Its deficiency triggers apoptosis of activated T-cells, making it a target for T-cell proliferative disorders. Transition-state analogues of PNP bind with picomolar (pm) dissociation constants. Tight-binding PNP inhibitors show exceptionally long lifetimes on the target enzyme. We solve the mechanism of the target residence time by comparing functional off-rates and We report PNP-inhibitor dissociation rates ( ) from 3 to 31 min for seven Immucillins with dissociation constants of 115 to 6 pm Treatment of human erythrocytes with DADMe-Immucillin-H (DADMe-ImmH, 22 pm) causes complete inhibition of PNP. Loss of [ C]DADMe-ImmH from erythrocytes during multiple washes is slow and biphasic, resulting from inhibitor release and rebinding to PNP catalytic sites. The slow phase gave a of 84 h. Loss of [ C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a of 1.6 h by preventing rebinding. Thus, in human erythrocytes, rebinding of DADMe-ImmH is 50-fold more likely than diffusional loss of the inhibitor from the erythrocyte. Humans treated with a single oral dose of DADMe-ImmH in phase 1 clinical trials exhibit regain of PNP activity with a of 59 days, corresponding to the erythropoiesis rate in humans. Thus, the PNP catalytic site recapture of DADMe-ImmH is highly favored We conclude that transition-state analogues with picomolar dissociation constants exhibit long lifetimes on their targets because the probability of the target enzyme recapturing inhibitor molecules is greater than diffusional loss to the extracellular space.
[Mh] Termos MeSH primário: Inibidores Enzimáticos/metabolismo
Inibidores Enzimáticos/farmacologia
Espaço Intracelular/metabolismo
Purina-Núcleosídeo Fosforilase/antagonistas & inibidores
[Mh] Termos MeSH secundário: Transporte Biológico
Ensaios Clínicos Fase I como Assunto
Enzimas
Eritrócitos/efeitos dos fármacos
Eritrócitos/enzimologia
Seres Humanos
Espaço Intracelular/efeitos dos fármacos
Ligação Proteica
Purina-Núcleosídeo Fosforilase/metabolismo
Pirrolidinas/metabolismo
Pirrolidinas/farmacologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Enzyme Inhibitors); 0 (Enzymes); 0 (Pyrrolidines); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171009
[Lr] Data última revisão:
171009
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170811
[St] Status:MEDLINE
[do] DOI:10.1074/jbc.M117.801779


  4 / 1906 MEDLINE  
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[PMID]:28720843
[Au] Autor:Barrett MT; Deiotte R; Lenkiewicz E; Malasi S; Holley T; Evers L; Posner RG; Jones T; Han H; Sausen M; Velculescu VE; Drebin J; O'Dwyer P; Jameson G; Ramanathan RK; Von Hoff DD
[Ad] Endereço:Mayo Clinic in Arizona, Scottsdale, AZ 85259, USA.
[Ti] Título:Clinical study of genomic drivers in pancreatic ductal adenocarcinoma.
[So] Source:Br J Cancer;117(4):572-582, 2017 Aug 08.
[Is] ISSN:1532-1827
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer with complex genomes and dense fibrotic stroma. This study was designed to identify clinically relevant somatic aberrations in pancreatic cancer genomes of patients with primary and metastatic disease enrolled and treated in two clinical trials. METHODS: Tumour nuclei were flow sorted prior to whole genome copy number variant (CNV) analysis. Targeted or whole exome sequencing was performed on most samples. We profiled biopsies from 68 patients enrolled in two Stand Up to Cancer (SU2C)-sponsored clinical trials. These included 38 resected chemoradiation naïve tumours (SU2C 20206-003) and metastases from 30 patients who progressed on prior therapies (SU2C 20206-001). Patient outcomes including progression-free survival (PFS) and overall survival (OS) were observed. RESULTS: We defined: (a) CDKN2A homozygous deletions that included the adjacent MTAP gene, only its' 3' region, or excluded MTAP; (b) SMAD4 homozygous deletions that included ME2; (c) a pancreas-specific MYC super-enhancer region; (d) DNA repair-deficient genomes; and (e) copy number aberrations present in PDA patients with long-term (⩾ 40 months) and short-term (⩽ 12 months) survival after surgical resection. CONCLUSIONS: We provide a clinically relevant framework for genomic drivers of PDA and for advancing novel treatments.
[Mh] Termos MeSH primário: Sequência de Bases
Carcinoma Ductal Pancreático/genética
Neoplasias Pancreáticas/genética
Deleção de Sequência
[Mh] Termos MeSH secundário: Adulto
Idoso
Idoso de 80 Anos ou mais
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico
Biópsia
Carcinoma Ductal Pancreático/tratamento farmacológico
Carcinoma Ductal Pancreático/secundário
Inibidor de Quinase Dependente de Ciclina p18/genética
Variações do Número de Cópias de DNA
Análise Mutacional de DNA
Reparo do DNA/genética
Intervalo Livre de Doença
Elementos Facilitadores Genéticos
Exoma
Feminino
Genes myc
Homozigoto
Seres Humanos
Malato Desidrogenase/genética
Masculino
Proteínas Associadas aos Microtúbulos/genética
Meia-Idade
Pâncreas/patologia
Neoplasias Pancreáticas/tratamento farmacológico
Neoplasias Pancreáticas/patologia
Proteínas Proto-Oncogênicas p21(ras)/genética
Purina-Núcleosídeo Fosforilase/genética
Proteína Smad4/genética
Taxa de Sobrevida
Proteína Supressora de Tumor p53/genética
[Pt] Tipo de publicação:CLINICAL STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (CDKN2A protein, human); 0 (Cyclin-Dependent Kinase Inhibitor p18); 0 (KRAS protein, human); 0 (Microtubule-Associated Proteins); 0 (SMAD4 protein, human); 0 (Smad4 Protein); 0 (Tumor Suppressor Protein p53); EC 1.1.1.37 (Malate Dehydrogenase); EC 1.1.1.37 (malic enzyme 2; human); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase); EC 2.4.2.28 (5'-methylthioadenosine phosphorylase); EC 3.6.5.2 (Proto-Oncogene Proteins p21(ras))
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170907
[Lr] Data última revisão:
170907
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170720
[St] Status:MEDLINE
[do] DOI:10.1038/bjc.2017.209


  5 / 1906 MEDLINE  
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[PMID]:28393254
[Au] Autor:Wang X; Sun L; Sun X; Yu J; Wang K; Wu Y; Gao Q; Zheng J
[Ad] Endereço:Department of Oncology, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China.
[Ti] Título:Antitumor effects of a dual-specific lentiviral vector carrying the Escherichia coli purine nucleoside phosphorylase gene.
[So] Source:Int J Oncol;50(5):1612-1622, 2017 May.
[Is] ISSN:1791-2423
[Cp] País de publicação:Greece
[La] Idioma:eng
[Ab] Resumo:The Escherichia coli purine nucleoside phosphorylase/Fludarabine phosphate (ePNP/Fludara) suicide system has several drawbacks, such as side-effects and the low efficiency of ePNP expression. In this study, we evaluated the antitumor effects of the dual-specific 8HSEs-hTERTp-ePNP/Fludara suicide system under hyperthermia in vitro and in vivo. Luciferase activities from the 8HSEs­hTERT and CMV promoters were compared using the dual luciferase assay in SW480 (high hTERT expression) and MKN74 cells (hTERT-negative) in the presence and absence of hyperthermia. Then, we investigated the effects of overexpressing the suicide gene ePNP using 8HSEs­hTERT-driven lentiviral vectors with Fludara on in vitro cell viability, side-effects, apoptosis, cycle distribution, colony formation and in vivo xenograft tumor growth. At 43˚C, luciferase activity from the 8HSEs­hTERT promoter was significantly increased in SW480 cells, but not in MKN74 cells. Importantly, luciferase activities from the 8HSEs­hTERT promoter were much higher than from the CMV promoter in hTERT-expressing SW480 cells under heated conditions. The in vitro quantitative analysis showed a 4-fold higher ePNP protein expression from the 8HSEs­hTERT promoter at 43˚C than at 37˚C in SW480 cells and the ePNP mRNA expression in SW480 cells at 43˚C was also higher than at 37˚C. Conversely, ePNP mRNA and protein expression were low, almost absent, in hTERT-negative MKN74 cells with or without hyperthermia. After Fludara addition, cell cytotoxicity assays showed that the significant inhibitory effect of the 8HSEs­hTERTp-ePNP on SW480 cells was dose- and time-dependent with hyperthermia. The 8HSEs­hTERTp-ePNP/Fludara suicide system significantly inhibited SW480 cell viability, colony formation, cell cycle progression and induced apoptosis in vitro, but also induced significant bystander effects, especially under the heated conditions. At the protein level, the suicide system significantly promoted Bax, caspase-3 and p53 expression and suppressed Bcl-2 expression. In sections from mouse xenografts, TUNEL assays showed that the suicide system reduced xenograft growth and induced SW480 apoptosis. These results indicated that the combinatorial cancer- and heat-specific promoter system has great potential for improving the efficacy of cancer treatment with hyperthermia. The 8HSEs­hTERTp-ePNP/Fludara system may serve as a powerful strategy for cancer gene therapy combined with hyperthermia.
[Mh] Termos MeSH primário: Terapia Genética
Vetores Genéticos/uso terapêutico
Neoplasias/genética
Neoplasias/terapia
Purina-Núcleosídeo Fosforilase/genética
[Mh] Termos MeSH secundário: Animais
Apoptose/genética
Linhagem Celular Tumoral
Escherichia coli/genética
Regulação Enzimológica da Expressão Gênica
Genes Transgênicos Suicidas/genética
Vetores Genéticos/genética
Proteínas de Fluorescência Verde
Seres Humanos
Lentivirus/genética
Camundongos
Neoplasias/patologia
Purina-Núcleosídeo Fosforilase/biossíntese
Purina-Núcleosídeo Fosforilase/uso terapêutico
Telomerase/genética
Telomerase/uso terapêutico
Ensaios Antitumorais Modelo de Xenoenxerto
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
147336-22-9 (Green Fluorescent Proteins); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase); EC 2.7.7.49 (TERT protein, human); EC 2.7.7.49 (Telomerase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170726
[Lr] Data última revisão:
170726
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170411
[St] Status:MEDLINE
[do] DOI:10.3892/ijo.2017.3949


  6 / 1906 MEDLINE  
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[PMID]:28251649
[Au] Autor:Giuliani P; Zuccarini M; Buccella S; Peña-Altamira LE; Polazzi E; Virgili M; Monti B; Poli A; Rathbone MP; Di Iorio P; Ciccarelli R; Caciagli F
[Ad] Endereço:Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara, Chieti, Italy.
[Ti] Título:Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells.
[So] Source:J Neurochem;141(2):208-221, 2017 04.
[Is] ISSN:1471-4159
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Intracellular purine turnover is mainly oriented to preserving the level of triphosphate nucleotides, fundamental molecules in vital cell functions that, when released outside cells, act as receptor signals. Conversely, high levels of purine bases and uric acid are found in the extracellular milieu, even in resting conditions. These compounds could derive from nucleosides/bases that, having escaped to cell reuptake, are metabolized by extracellular enzymes similar to the cytosolic ones. Focusing on purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of purine (deoxy)-nucleosides/bases, we found that it is constitutively released from cultured rat C6 glioma cells into the medium, and has a molecular weight and enzyme activity similar to the cytosolic enzyme. Cell exposure to 10 µM ATP or guanosine triphosphate (GTP) increased the extracellular amount of all corresponding purines without modifying the levels/activity of released PNP, whereas selective activation of ATP P2Y or adenosine A metabotropic receptors increased PNP release and purine base formation. The reduction to 1% in oxygen supply (2 h) to cells decreased the levels of released PNP, leading to an increased presence of extracellular nucleosides and to a reduced formation of xanthine and uric acid. Conversely, 2 h cell re-oxygenation enhanced the extracellular amounts of both PNP and purine bases. Thus, hypoxia and re-oxygenation modulated in opposite manner the PNP release/activity and, thereby, the extracellular formation of purine metabolism end-products. In conclusion, extracellular PNP and likely other enzymes deputed to purine base metabolism are released from cells, contributing to the purinergic system homeostasis and exhibiting an important pathophysiological role.
[Mh] Termos MeSH primário: Glioma/enzimologia
Purina-Núcleosídeo Fosforilase/secreção
[Mh] Termos MeSH secundário: Animais
Linhagem Celular Tumoral
Ratos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
EC 2.4.2.1 (Purine-Nucleoside Phosphorylase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170713
[Lr] Data última revisão:
170713
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170303
[St] Status:MEDLINE
[do] DOI:10.1111/jnc.14004


  7 / 1906 MEDLINE  
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[PMID]:28213009
[Au] Autor:Hida T; Hamasaki M; Matsumoto S; Sato A; Tsujimura T; Kawahara K; Iwasaki A; Okamoto T; Oda Y; Honda H; Nabeshima K
[Ad] Endereço:Department of Pathology, Fukuoka University Hospital and School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan; Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; Department of Clinical Radio
[Ti] Título:Immunohistochemical detection of MTAP and BAP1 protein loss for mesothelioma diagnosis: Comparison with 9p21 FISH and BAP1 immunohistochemistry.
[So] Source:Lung Cancer;104:98-105, 2017 Feb.
[Is] ISSN:1872-8332
[Cp] País de publicação:Ireland
[La] Idioma:eng
[Ab] Resumo:OBJECTIVES: Differentiating malignant pleural mesothelioma (MPM) from reactive mesothelial hyperplasia (RMH) is still challenging. Detection of homozygous deletion (HD) of 9p21 region including p16 (p16) by fluorescence in situ hybridization (FISH) and immunohistochemical detection of loss of BRCA1 associated protein 1 (BAP1), are reliable markers for MPM diagnosis. However, not all laboratories are equipped to perform 9p21 FISH; immunohistochemistry (IHC) is a more common and feasible technique. Thus, we sought to develop a IHC-based method that could predict the deletion of p16 in MPM in concordance with 9p21 FISH. MATERIALS AND METHODS: We examined the expression of the 9p21.3-related proteins (p14, p15, p16, and methylthioadenosine phosphorylase (MTAP)) and BAP1 using IHC in 51 MPM and 25 RMH cases, and assessed their correlation with HD of p16 detected by FISH. The diagnostic usefulness of IHC of the 9p21.3-related proteins and BAP1 and their combinations was assessed using the cut-off values set by receiver operating characteristic (ROC) analysis. RESULTS: Among the 9p21.3-related proteins, MTAP IHC findings showed best concordance with 9p21 FISH results (kappa coefficient of 0.69) and a specificity of 100%. We also examined the combinations of MTAP IHC with the other products. The loss of p16 and MTAP had better concordance (kappa coefficient of 0.71), although lower specificity (85%). For differentiating MPM from RMH, only MTAP showed 100% specificity among the 9p21.3-related proteins, as did BAP1 IHC and 9p21 FISH. Among BAP1 combinations, only that of BAP1 with MTAP showed 100% specificity. Its sensitivity was 76.5%, which was lower than BAP1 IHC and 9p21 FISH combination (84.3%), but higher than BAP1 IHC alone (60.8%) or 9p21 FISH alone (60.8%). CONCLUSIONS: A combination of MTAP or BAP1 loss detected by IHC can likely detect MPM with good sensitivity and 100% specificity, and serve as useful ancillary IHC for discriminating MPM from RMH.
[Mh] Termos MeSH primário: Proteína BRCA1/metabolismo
Genes p16
Imuno-Histoquímica
Mesotelioma/diagnóstico
Neoplasias Pleurais/diagnóstico
Purina-Núcleosídeo Fosforilase/metabolismo
[Mh] Termos MeSH secundário: Adulto
Idoso
Idoso de 80 Anos ou mais
Linhagem Celular Tumoral/metabolismo
Linhagem Celular Tumoral/patologia
Diagnóstico Diferencial
Feminino
Seres Humanos
Hiperplasia/patologia
Hibridização in Situ Fluorescente
Neoplasias Pulmonares/diagnóstico
Neoplasias Pulmonares/metabolismo
Masculino
Mesotelioma/metabolismo
Meia-Idade
Neoplasias Pleurais/metabolismo
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (BRCA1 Protein); 0 (BRCA1 protein, human); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase); EC 2.4.2.28 (5'-methylthioadenosine phosphorylase)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171108
[Lr] Data última revisão:
171108
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170219
[St] Status:MEDLINE


  8 / 1906 MEDLINE  
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[PMID]:28026167
[Au] Autor:Firestone RS; Cameron SA; Karp JM; Arcus VL; Schramm VL
[Ad] Endereço:Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
[Ti] Título:Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase.
[So] Source:ACS Chem Biol;12(2):464-473, 2017 Feb 17.
[Is] ISSN:1554-8937
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Human 5'-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5'-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (-ΔC ), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔC of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔC . Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔC for binding. In addition, ΔC values are not correlated with K values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (-ΔC ). A comparison of the ΔC for MTAP presteady state chemistry and ΔC for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP-inhibitor complexes indicate remarkable protein thermal stability (to T = 99 °C) in complexes with transition-state analogues.
[Mh] Termos MeSH primário: Temperatura Alta
Purina-Núcleosídeo Fosforilase/metabolismo
[Mh] Termos MeSH secundário: Calorimetria
Catálise
Inibidores Enzimáticos/metabolismo
Seres Humanos
Interações Hidrofóbicas e Hidrofílicas
Cinética
Simulação de Dinâmica Molecular
Conformação Proteica
Termodinâmica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Enzyme Inhibitors); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase); EC 2.4.2.28 (5'-methylthioadenosine phosphorylase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:171014
[Lr] Data última revisão:
171014
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161228
[St] Status:MEDLINE
[do] DOI:10.1021/acschembio.6b00885


  9 / 1906 MEDLINE  
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[PMID]:27976868
[Au] Autor:Isaksen GV; Åqvist J; Brandsdal BO
[Ad] Endereço:The Centre for Theoretical and Computational Chemistry, Department of Chemistry, University of Tromsø , NO-9037 Tromsø, Norway.
[Ti] Título:Thermodynamics of the Purine Nucleoside Phosphorylase Reaction Revealed by Computer Simulations.
[So] Source:Biochemistry;56(1):306-312, 2017 Jan 10.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Enzymes are able to catalyze chemical reactions by reducing the activation free energy, yielding significant increases in the reaction rates. This can thermodynamically be accomplished by either reducing the activation enthalpy or increasing the activation entropy. The effect of remote mutations on the thermodynamic activation parameters of human purine nucleoside phosphorylase is examined using extensive molecular dynamics and free energy simulations. More than 2700 independent reaction free energy profiles for six different temperatures have been calculated to obtain high-precision computational Arrhenius plots. On the basis of these, the activation enthalpies and entropies were computed from linear regression of the plots with ΔG as a function of 1/T, and the obtained thermodynamic activation parameters are in very good agreement with those from experiments. The Arrhenius plots immediately show that the 6-oxopurines (INO and GUO) have identical slopes, whereas the 6-aminopurine (ADO) has a significantly different slope, indicating that the substrate specificity is related to the difference in thermodynamic activation parameters. Furthermore, the calculations show that the human PNP specificity for 6-oxopurines over 6-aminopurines originates from significant differences in electrostatic preorganization. The effect of the remote double mutation, K22E and H104R (E:R), has also been examined, as it alters human PNP toward the bovine PNP. These residues are situated on the protein surface, 28-35 Å from the active site, and the mutation alters the enthalpy-entropy balance with little effect on the catalytic rates. It is thus quite remarkable that the empirical valence bond method can reproduce the enthalpies and entropies induced by these long-range mutations.
[Mh] Termos MeSH primário: Simulação de Dinâmica Molecular
Domínios Proteicos
Purina-Núcleosídeo Fosforilase/química
Termodinâmica
[Mh] Termos MeSH secundário: Adenosina/química
Adenosina/metabolismo
Animais
Biocatálise
Domínio Catalítico
Bovinos
Guanosina/química
Guanosina/metabolismo
Seres Humanos
Inosina/química
Inosina/metabolismo
Cinética
Modelos Lineares
Estrutura Molecular
Mutação
Ligação Proteica
Purina-Núcleosídeo Fosforilase/genética
Purina-Núcleosídeo Fosforilase/metabolismo
Eletricidade Estática
Especificidade por Substrato
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
12133JR80S (Guanosine); 5A614L51CT (Inosine); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase); K72T3FS567 (Adenosine)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170522
[Lr] Data última revisão:
170522
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161216
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b00967


  10 / 1906 MEDLINE  
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[PMID]:27935959
[Au] Autor:Torini JR; Brandão-Neto J; DeMarco R; Pereira HD
[Ad] Endereço:Laboratório de Biologia Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
[Ti] Título:Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5'-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway.
[So] Source:PLoS Negl Trop Dis;10(12):e0005178, 2016 Dec.
[Is] ISSN:1935-2735
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Schistosoma mansoni do not have de novo purine pathways and rely on purine salvage for their purine supply. It has been demonstrated that, unlike humans, the S. mansoni is able to produce adenine directly from adenosine, although the enzyme responsible for this activity was unknown. In the present work we show that S. mansoni 5´-deoxy-5´-methylthioadenosine phosphorylase (MTAP, E.C. 2.4.2.28) is capable of use adenosine as a substrate to the production of adenine. Through kinetics assays, we show that the Schistosoma mansoni MTAP (SmMTAP), unlike the mammalian MTAP, uses adenosine substrate with the same efficiency as MTA phosphorolysis, which suggests that this enzyme is part of the purine pathway salvage in S. mansoni and could be a promising target for anti-schistosoma therapies. Here, we present 13 SmMTAP structures from the wild type (WT), including three single and one double mutant, and generate a solid structural framework for structure description. These crystal structures of SmMTAP reveal that the active site contains three substitutions within and near the active site when compared to it mammalian counterpart, thus opening up the possibility of developing specific inhibitors to the parasite MTAP. The structural and kinetic data for 5 substrates reveal the structural basis for this interaction, providing substract for inteligent design of new compounds for block this enzyme activity.
[Mh] Termos MeSH primário: Adenosina/metabolismo
Modelos Moleculares
Purina-Núcleosídeo Fosforilase/química
Purina-Núcleosídeo Fosforilase/metabolismo
Schistosoma mansoni/enzimologia
[Mh] Termos MeSH secundário: Animais
Domínio Catalítico
Cristalização
Cristalografia por Raios X
Seres Humanos
Cinética
Redes e Vias Metabólicas
Mutação
Purina-Núcleosídeo Fosforilase/genética
Purina-Núcleosídeo Fosforilase/isolamento & purificação
Purinas/metabolismo
Schistosoma mansoni/genética
Alinhamento de Sequência
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Purines); EC 2.4.2.- (adenosine phosphorylase); EC 2.4.2.1 (Purine-Nucleoside Phosphorylase); EC 2.4.2.28 (5'-methylthioadenosine phosphorylase); K72T3FS567 (Adenosine); W60KTZ3IZY (purine)
[Em] Mês de entrada:1706
[Cu] Atualização por classe:170613
[Lr] Data última revisão:
170613
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161210
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pntd.0005178



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