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[PMID]:29254920
[Au] Autor:Ma SY; Xia QY
[Ad] Endereço:State Key Laboratory of Silkworm Genome Biology, Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing 400716, China.
[Ti] Título:Genetic breeding of silkworms: from traditional hybridization to molecular design.
[So] Source:Yi Chuan;39(11):1025-1032, 2017 Nov 20.
[Is] ISSN:0253-9772
[Cp] País de publicação:China
[La] Idioma:eng
[Ab] Resumo:Sericulture is one of the great inventions of the Chinese people and has become an important cultural feature of China. As China is the long-lasting center of silk production, genetic breeding of silkworm was highly developed historically, and has formed a comprehensive system for breeding and preservation of new varieties. However, silkworm breeding reached a bottleneck recently, because most of the traditional genetic resources have been utilized and silkworm strains have become homogeneous. Meanwhile, sericulture in China meets huge challenges in the 21 century. In recent years, with the development and rapid application of molecular biology, genomics, transgene and genome editing, silkworm genetic breeding has entered a new era. In this review, we summarize the development of silkworm genetic breeding, especially the progress and perspective of transgene and genome editing in genetic engineering of silkworms. We also discuss the future development of silkworm genetic breeding.
[Mh] Termos MeSH primário: Bombyx/genética
Cruzamento
[Mh] Termos MeSH secundário: Animais
Edição de Genes
Hibridização Genética
Transgenes
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180306
[Lr] Data última revisão:
180306
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171220
[St] Status:MEDLINE
[do] DOI:10.16288/j.yczz.17-103


  2 / 16674 MEDLINE  
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[PMID]:28452714
[Au] Autor:Li D; Palanca AMS; Won SY; Gao L; Feng Y; Vashisht AA; Liu L; Zhao Y; Liu X; Wu X; Li S; Le B; Kim YJ; Yang G; Li S; Liu J; Wohlschlegel JA; Guo H; Mo B; Chen X; Law JA
[Ad] Endereço:Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, United States.
[Ti] Título:The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status.
[So] Source:Elife;6, 2017 04 28.
[Is] ISSN:2050-084X
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in that suppresses the transcriptional silencing of two ( reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing.
[Mh] Termos MeSH primário: Arabidopsis
Metilação de DNA
Proteínas de Ligação a DNA/metabolismo
Regulação da Expressão Gênica de Plantas
Proteínas de Plantas/metabolismo
[Mh] Termos MeSH secundário: Genes Reporter
Luciferases/análise
Transgenes
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T; RESEARCH SUPPORT, N.I.H., EXTRAMURAL
[Nm] Nome de substância:
0 (DNA-Binding Proteins); 0 (Plant Proteins); EC 1.13.12.- (Luciferases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180305
[Lr] Data última revisão:
180305
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170429
[St] Status:MEDLINE


  3 / 16674 MEDLINE  
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[PMID]:28743796
[Au] Autor:Roberts SL; Dun XP; Doddrell RDS; Mindos T; Drake LK; Onaitis MW; Florio F; Quattrini A; Lloyd AC; D'Antonio M; Parkinson DB
[Ad] Endereço:Plymouth University Peninsula Schools of Medicine and Dentistry, John Bull Building, Plymouth Science Park, Plymouth PL6 8BU, UK.
[Ti] Título:Sox2 expression in Schwann cells inhibits myelination and induces influx of macrophages to the nerve.
[So] Source:Development;144(17):3114-3125, 2017 09 01.
[Is] ISSN:1477-9129
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Correct myelination is crucial for the function of the peripheral nervous system. Both positive and negative regulators within the axon and Schwann cell function to ensure the correct onset and progression of myelination during both development and following peripheral nerve injury and repair. The Sox2 transcription factor is well known for its roles in the development and maintenance of progenitor and stem cell populations, but has also been proposed as a negative regulator of myelination in Schwann cells. We wished to test fully whether Sox2 regulates myelination and show here that, in mice, sustained Sox2 expression blocks myelination in the peripheral nerves and maintains Schwann cells in a proliferative non-differentiated state, which is also associated with increased inflammation within the nerve. The plasticity of Schwann cells allows them to re-myelinate regenerated axons following injury and we show that re-myelination is also blocked by Sox2 expression in Schwann cells. These findings identify Sox2 as a physiological regulator of Schwann cell myelination and its potential to play a role in disorders of myelination in the peripheral nervous system.
[Mh] Termos MeSH primário: Macrófagos/metabolismo
Bainha de Mielina/metabolismo
Nervos Periféricos/metabolismo
Fatores de Transcrição SOXB1/metabolismo
Células de Schwann/metabolismo
[Mh] Termos MeSH secundário: Animais
Biomarcadores/metabolismo
Caderinas/metabolismo
Proliferação Celular
Proteína 2 de Resposta de Crescimento Precoce/metabolismo
Proteínas de Fluorescência Verde/metabolismo
Camundongos Transgênicos
Atividade Motora
Condução Nervosa
Traumatismos dos Nervos Periféricos/metabolismo
Traumatismos dos Nervos Periféricos/patologia
Nervos Periféricos/patologia
Nervos Periféricos/ultraestrutura
Proteínas Proto-Oncogênicas c-jun/metabolismo
Ratos
Recuperação de Função Fisiológica
Células de Schwann/patologia
Transgenes
beta Catenina/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Biomarkers); 0 (Cadherins); 0 (Early Growth Response Protein 2); 0 (Proto-Oncogene Proteins c-jun); 0 (SOXB1 Transcription Factors); 0 (beta Catenin); 147336-22-9 (Green Fluorescent Proteins)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:180302
[Lr] Data última revisão:
180302
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170727
[St] Status:MEDLINE
[do] DOI:10.1242/dev.150656


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[PMID]:29306121
[Au] Autor:Akoum N
[Ad] Endereço:Atrial Fibrillation Program, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195, United States. Electronic address: nakoum@cardiology.washington.edu.
[Ti] Título:CREM-transgene mice: A step towards understanding thrombogenesis in atrial fibrillation.
[So] Source:Thromb Res;162:60-61, 2018 02.
[Is] ISSN:1879-2472
[Cp] País de publicação:United States
[La] Idioma:eng
[Mh] Termos MeSH primário: Fibrilação Atrial/genética
Transgenes
[Mh] Termos MeSH secundário: Animais
Camundongos
Camundongos Transgênicos
[Pt] Tipo de publicação:EDITORIAL; COMMENT
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180228
[Lr] Data última revisão:
180228
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180107
[St] Status:MEDLINE


  5 / 16674 MEDLINE  
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[PMID]:29385169
[Au] Autor:Bissa M; Forlani G; Zanotto C; Tosi G; De Giuli Morghen C; Accolla RS; Radaelli A
[Ad] Endereço:Department of Pharmacological and Biomolecular Sciences, University of Milan, via Balzaretti 9, Milan, Italy.
[Ti] Título:Fowlpoxvirus recombinants coding for the CIITA gene increase the expression of endogenous MHC-II and Fowlpox Gag/Pro and Env SIV transgenes.
[So] Source:PLoS One;13(1):e0190869, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:A complete eradication of an HIV infection has never been achieved by vaccination and the search for new immunogens that can induce long-lasting protective responses is ongoing. Avipoxvirus recombinants are host-restricted for replication to avian species and they do not have the undesired side effects induced by vaccinia recombinants. In particular, Fowlpox (FP) recombinants can express transgenes over long periods and can induce protective immunity in mammals, mainly due to CD4-dependent CD8+ T cells. In this context, the class II transactivator (CIITA) has a pivotal role in triggering the adaptive immune response through induction of the expression of class-II major histocompatibility complex molecule (MHC-II), that can present antigens to CD4+ T helper cells. Here, we report on construction of novel FPgp and FPenv recombinants that express the highly immunogenic SIV Gag-pro and Env structural antigens. Several FP-based recombinants, with single or dual genes, were also developed that express CIITA, driven from H6 or SP promoters. These recombinants were used to infect CEF and Vero cells in vitro and determine transgene expression, which was evaluated by real-time PCR and Western blotting. Subcellular localisation of the different proteins was evaluated by confocal microscopy, whereas HLA-DR or MHC-II expression was measured by flow cytometry. Fowlpox recombinants were also used to infect syngeneic T/SA tumour cells, then injected into Balb/c mice to elicit MHC-II immune response and define the presentation of the SIV transgene products in the presence or absence of FPCIITA. Antibodies to Env were measured by ELISA. Our data show that the H6 promoter was more efficient than SP to drive CIITA expression and that CIITA can enhance the levels of the gag/pro and env gene products only when infection is performed by FP single recombinants. Also, CIITA expression is higher when carried by FP single recombinants than when combined with FPgp or FPenv constructs and can induce HLA-DR cell surface expression. However, in-vivo experiments did not show any significant increase in the humoral response. As CIITA already proved to elicit immunogenicity by improving antigen presentation, further in-vivo experiments should be performed to increase the immune responses. The use of prime/boost immunisation protocols and the oral administration route of the recombinants may enhance the immunogenicity of Env peptides presented by MHC-II and provide CD4+ T-cell stimulation.
[Mh] Termos MeSH primário: Genes Virais
Complexo Principal de Histocompatibilidade/genética
Proteínas Nucleares/genética
Poxviridae/genética
Recombinação Genética
Vírus da Imunodeficiência Símia/genética
Transativadores/genética
Transgenes
[Mh] Termos MeSH secundário: Vacinas contra a AIDS/genética
Vacinas contra a AIDS/imunologia
Animais
Western Blotting
Linfócitos T CD4-Positivos/imunologia
Linfócitos T CD8-Positivos/imunologia
Linhagem Celular
Embrião de Galinha
Ensaio de Imunoadsorção Enzimática
Infecções por HIV/imunologia
Infecções por HIV/prevenção & controle
Seres Humanos
Camundongos
Camundongos Endogâmicos BALB C
Microscopia Confocal
Regiões Promotoras Genéticas
Reação em Cadeia da Polimerase em Tempo Real
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (AIDS Vaccines); 0 (MHC class II transactivator protein); 0 (Nuclear Proteins); 0 (Trans-Activators)
[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:180201
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0190869


  6 / 16674 MEDLINE  
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[PMID]:28935584
[Au] Autor:Liu C; Jia X; Zou Z; Wang X; Wang Y; Zhang Z
[Ad] Endereço:Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen 361021, China.
[Ti] Título:VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1.
[So] Source:Gen Comp Endocrinol;255:1-11, 2018 Jan 01.
[Is] ISSN:1095-6840
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Vitellogenesis-inhibiting hormone (VIH) is known to regulate ovarian maturation by suppressing the synthesis of vitellogenin (Vtg) in crustaceans, which belongs to a member of crustacean hyperglycemic hormone (CHH) family synthesized and secreted from the X-organ/sinus gland complex of eyestalks. In this study, the cDNA, genomic DNA (gDNA) and the 5'-upstream regulatory (promoter region) sequences of VIH gene were obtained by conventional PCR, genome walker and tail-PCR techniques according to our transcriptomic database of Scylla paramamosain. The full-length cDNA of SpVIH is 634bp including 105bp 5'UTR, 151bp 3'UTR and 378bp ORF that encodes a peptide of 125 amino acids. The full length gDNA of SpVIH is 790bp containing two exons and one intron. The 5'-flanking promoter regions of SpVIH we isolated are 3070bp from the translation initiation (ATG) and 2398bp from the predicted transcription initiation (A), which consists of putative core promoter region and multiple potential transcription factor binding sites. SpVIH was only expressed in eyestalk. The expression level of SpVIH in eyestalk of female crab decreased gradually along with the development of ovary. As there is not cell line of crabs available, we chose the mature transfection system HEK293FT cell lines to explore the mechanism of transcription regulation of SpVIH in crabs. Sequential deletion assays using luciferase reporter gene in HEK293FT cells revealed that the possible promoter activity regions (including positive and negative transcription factors binding sites simultaneously) presented between pSpVIH-4 and pSpVIH-6. In order to further identify the crucial transcription factors binding site in this region, the site-directed mutagenesis of Sox9/Oct4/Oct1 binding site of pSpVIH-4 was created. The results demonstrated that the transcriptional activity of pSpVIH-4â–³ decreased significantly (p<0.05). Thus, it is reasonable to deduce that the Sox9/Oct4/Oct1 may be the essential positive transcription factors which regulate the expression of SpVIH.
[Mh] Termos MeSH primário: Braquiúros/metabolismo
Proteínas de Transporte/metabolismo
Olho/metabolismo
Hormônios de Invertebrado/metabolismo
Fator 1 de Transcrição de Octâmero/metabolismo
Fator 3 de Transcrição de Octâmero/metabolismo
Fatores de Transcrição SOX9/metabolismo
Transativadores/metabolismo
[Mh] Termos MeSH secundário: Região 5'-Flanqueadora/genética
Sequência de Aminoácidos
Animais
Sequência de Bases
Proteínas de Transporte/química
Proteínas de Transporte/genética
DNA Complementar/genética
Feminino
Regulação da Expressão Gênica no Desenvolvimento
Células HEK293
Seres Humanos
Hormônios de Invertebrado/química
Hormônios de Invertebrado/genética
Mutação/genética
Ovário/embriologia
Ovário/metabolismo
Filogenia
Regiões Promotoras Genéticas/genética
Análise de Sequência de DNA
Transgenes
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Carrier Proteins); 0 (DNA, Complementary); 0 (Invertebrate Hormones); 0 (Octamer Transcription Factor-1); 0 (Octamer Transcription Factor-3); 0 (SOX9 Transcription Factor); 0 (Trans-Activators); 138360-48-2 (vitellogenesis inhibiting hormone)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180216
[Lr] Data última revisão:
180216
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170923
[St] Status:MEDLINE


  7 / 16674 MEDLINE  
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[PMID]:28467906
[Au] Autor:Muñoz S; Búa S; Rodríguez-Acebes S; Megías D; Ortega S; de Martino A; Méndez J
[Ad] Endereço:DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernández Almagro, 28029 Madrid, Spain.
[Ti] Título:In Vivo DNA Re-replication Elicits Lethal Tissue Dysplasias.
[So] Source:Cell Rep;19(5):928-938, 2017 May 02.
[Is] ISSN:2211-1247
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Mammalian DNA replication origins are "licensed" by the loading of DNA helicases, a reaction that is mediated by CDC6 and CDT1 proteins. After initiation of DNA synthesis, CDC6 and CDT1 are inhibited to prevent origin reactivation and DNA overreplication before cell division. CDC6 and CDT1 are highly expressed in many types of cancer cells, but the impact of their deregulated expression had not been investigated in vivo. Here, we have generated mice strains that allow the conditional overexpression of both proteins. Adult mice were unharmed by the individual overexpression of either CDC6 or CDT1, but their combined deregulation led to DNA re-replication in progenitor cells and lethal tissue dysplasias. This study offers mechanistic insights into the necessary cooperation between CDC6 and CDT1 for facilitation of origin reactivation and describes the physiological consequences of DNA overreplication.
[Mh] Termos MeSH primário: Replicação do DNA
Diarreia Infantil/genética
Mucosa Intestinal/metabolismo
Síndromes de Malabsorção/genética
[Mh] Termos MeSH secundário: Animais
Proteínas de Ciclo Celular/genética
Proteínas de Ciclo Celular/metabolismo
Proteínas de Ligação a DNA/genética
Proteínas de Ligação a DNA/metabolismo
Diarreia Infantil/metabolismo
Feminino
Mucosa Intestinal/patologia
Síndromes de Malabsorção/metabolismo
Masculino
Camundongos
Proteínas Nucleares/genética
Proteínas Nucleares/metabolismo
Transgenes
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (CDC6 protein, mouse); 0 (Cell Cycle Proteins); 0 (DNA-Binding Proteins); 0 (Nuclear Proteins); 0 (Ris2 protein, mouse)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180213
[Lr] Data última revisão:
180213
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170504
[St] Status:MEDLINE


  8 / 16674 MEDLINE  
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Arruda, Valder R
Registro de Ensaios Clínicos
Registro de Ensaios Clínicos
Texto completo
[PMID]:29211678
[Au] Autor:George LA; Sullivan SK; Giermasz A; Rasko JEJ; Samelson-Jones BJ; Ducore J; Cuker A; Sullivan LM; Majumdar S; Teitel J; McGuinn CE; Ragni MV; Luk AY; Hui D; Wright JF; Chen Y; Liu Y; Wachtel K; Winters A; Tiefenbacher S; Arruda VR; van der Loo JCM; Zelenaia O; Takefman D; Carr ME; Couto LB; Anguela XM; High KA
[Ad] Endereço:From the Division of Hematology (L.A.G., B.J.S.-J., A.W., V.R.A.) and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics (L.A.G., B.J.S.-J., A.W., V.R.A., J.C.M.L., O.Z.), Children's Hospital of Philadelphia, the Departments of Pediatrics (L.A.G., B.J.S.-J., V.R.A.) and Medicine
[Ti] Título:Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant.
[So] Source:N Engl J Med;377(23):2215-2227, 2017 12 07.
[Is] ISSN:1533-4406
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. METHODS: We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX-R338L) transgene at a dose of 5×10 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. RESULTS: No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady-state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P=0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P=0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. CONCLUSIONS: We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092 .).
[Mh] Termos MeSH primário: Fator IX/genética
Terapia Genética/métodos
Vetores Genéticos
Hemofilia B/terapia
Transgenes
[Mh] Termos MeSH secundário: Adolescente
Adulto
Dependovirus/imunologia
Fator IX/metabolismo
Fator IX/uso terapêutico
Vetores Genéticos/administração & dosagem
Hemofilia B/genética
Hemofilia B/metabolismo
Hemorragia/prevenção & controle
Seres Humanos
Masculino
Meia-Idade
Adulto Jovem
[Pt] Tipo de publicação:CLINICAL TRIAL, PHASE I; CLINICAL TRIAL, PHASE II; JOURNAL ARTICLE; MULTICENTER STUDY; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
9001-28-9 (Factor IX)
[Em] Mês de entrada:1712
[Cu] Atualização por classe:180213
[Lr] Data última revisão:
180213
[Sb] Subgrupo de revista:AIM; IM
[Da] Data de entrada para processamento:171207
[Cl] Clinical Trial:ClinicalTrial
[St] Status:MEDLINE
[do] DOI:10.1056/NEJMoa1708538


  9 / 16674 MEDLINE  
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[PMID]:28870733
[Au] Autor:Stenger B; Gerber A; Bernhardt R; Hannemann F
[Ad] Endereço:Institute of Biochemistry, Saarland University, Saarbrücken, Germany.
[Ti] Título:Functionalized poly(3-hydroxybutyric acid) bodies as new in vitro biocatalysts.
[So] Source:Biochim Biophys Acta;1866(1):52-59, 2018 01.
[Is] ISSN:0006-3002
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:Cytochromes P450 play a key role in the drug and steroid metabolism in the human body. This leads to a high interest in this class of proteins. Mammalian cytochromes P450 are rather delicate. Due to their localization in the mitochondrial or microsomal membrane, they tend to aggregate during expression and purification and to convert to an inactive form so that they have to be purified and stored in complex buffers. The complex buffers and low storage temperatures, however, limit the feasibility of fast, automated screening of the corresponding cytochrome P450-effector interactions, which are necessary to study substrate-protein and inhibitor-protein interactions. Here, we present the production and isolation of functionalized poly(3-hydroxybutyrate) granules (PHB bodies) from Bacillus megaterium MS941 strain. In contrast to the expression in Escherichia coli, where mammalian cytochromes P450 are associated to the cell membrane, when CYP11A1 is heterologously expressed in Bacillus megaterium, it is located on the PHB bodies. The surface of these particles provides a matrix for immobilization and stabilization of the CYP11A1 during the storage of the protein and substrate conversion. It was demonstrated that the PHB polymer basis is inert concerning the performed conversion. Immobilization of the CYP11A1 onto the PHB bodies allows freeze-drying of the complex without significant decrease of the CYP11A1 activity. This is the first lyophilization of a mammalian cytochrome P450, which allows storage over more than 18days at 4°C instead of storage at -80°C. In addition, we were able to immobilize the cytochrome P450 on the PHB bodies in vitro. In this case the expression of the protein is separated from the production of the immobilization matrix, which widens the application of this method. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
[Mh] Termos MeSH primário: Ácido 3-Hidroxibutírico/química
Bacillus megaterium/genética
Biotecnologia/métodos
Enzima de Clivagem da Cadeia Lateral do Colesterol/química
Proteínas Imobilizadas/biossíntese
Proteínas Mitocondriais/biossíntese
[Mh] Termos MeSH secundário: Ácido 3-Hidroxibutírico/biossíntese
Animais
Bacillus megaterium/enzimologia
Biocatálise
Bovinos
Colesterol/química
Colesterol/metabolismo
Enzima de Clivagem da Cadeia Lateral do Colesterol/genética
Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo
Grânulos Citoplasmáticos/química
Liofilização
Expressão Gênica
Proteínas Imobilizadas/química
Proteínas Imobilizadas/genética
Proteínas Mitocondriais/química
Proteínas Mitocondriais/genética
Pregnenolona/biossíntese
Pregnenolona/química
Refrigeração
Transgenes
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Immobilized Proteins); 0 (Mitochondrial Proteins); 73R90F7MQ8 (Pregnenolone); 97C5T2UQ7J (Cholesterol); EC 1.14.15.6 (Cholesterol Side-Chain Cleavage Enzyme); TZP1275679 (3-Hydroxybutyric Acid)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180208
[Lr] Data última revisão:
180208
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170906
[St] Status:MEDLINE


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[PMID]:29368492
[Au] Autor:Marenkova TV; Deineko EV
[Ti] Título:[Hybridological analysis of inheritance of mosaic nptII gene expression in transgenic tobacco plants].
[So] Source:Genetika;52(6):641-9, 2016 Jun.
[Is] ISSN:0016-6758
[Cp] País de publicação:Russia (Federation)
[La] Idioma:rus
[Ab] Resumo:On the basis of the results of hybridological analysis, it was established that significant differences in the stability of manifestation of the nptII gene expression are observed between the Nu5 and Nu6 lines obtained from the same initial Nu21 transformant (in spite of the identical genetic environment). Relatively stable expression of the marker gene is registered in the Nu5 line; the frequencies of detection of mosaic descendants are not high. The Nu6 line is characterized by a high frequency of the appearance of mosaic plants (up to 100%), indicating an increase in the marker gene inactivation in this line. When combining the nptII gene alleles in the hybrid genome, the allele coming from the Nu6 line was manifested as semidominant and had a suppressing effect on the allele coming from the Nu5 line. No transinactivation phenomena at the level of phenotype were detected during the interaction of the nptII gene alleles from the Nu5 and Nu6 lines in diheterozygote with the alleles of homologous genes inactivated at the transcriptional or post-transcriptional levels. During segregation to F2, separation of the Nu21 line progeny into two independent groups with preservation of the different character of the marker gene expression (with a moderate level of appearance of mosaic plants for the Nu5 line and with high level for the Nu6 line) was again registered. Further studies are directed to detection of the mechanisms leading to the mosaic type of the studied gene manifestation in transgenic plants of the Nu5 and Nu6 lines.
[Mh] Termos MeSH primário: Regulação da Expressão Gênica de Plantas
Mosaicismo
Proteínas de Plantas
Plantas Geneticamente Modificadas
Tabaco
Transgenes
[Mh] Termos MeSH secundário: Proteínas de Plantas/biossíntese
Proteínas de Plantas/genética
Plantas Geneticamente Modificadas/genética
Plantas Geneticamente Modificadas/metabolismo
Tabaco/genética
Tabaco/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Plant Proteins)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180205
[Lr] Data última revisão:
180205
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180126
[St] Status:MEDLINE



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