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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(5): 1469-1475, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31607300

RESUMO

OBJECTIVE: To investigate the effect of LNK gene silencing and overexpression on the expression of STAT3 gene in human monocytic leukemia cells (THP-1). METHODS: THP-1 cells were cultured, and the lentivirus was used as a vector to silence and overexpres the LNK gene stably. After transfection for 72 hours, the GFP expression levels were observed by inverted fluorescence microscopy. The lentiviral transfection efficiencies were detected by flow cytometry. The effects of LNK silencing and overexpression were confirmed, and the expression of STAT3 mRNA was detected by RT-PCR. The protein levels of LNK and STAT3 were detected by Western blotting. RESULTS: The GFP expression level of THP-1 cells reached more than 85% after transfection with lentivirus for 72 hours, and the transfection efficiency of cells was above 99%. mRNA expressions levels of LNK and STAT3 in LNK silencing group were signifycantly lower than those in control group, while LNK and STAT3 mRNA levels in the LNK overexpression group was significantly higher than those in control group (P<0.05). The protein expression levels of LNK and STAT3 in LNK silencing group were significantly lower than those in control group, while that in LNK overexpression group was significantly higher than that in control group (P<0.05). CONCLUSION: The THP-1 cell line with LNK gene silencing and overexpression has been successfully established. The LNK gene silencing resulted in decrease of STAT3 expression; LNK gene overexpression and leads to inereases of STAT3 expression indicating that LNK participates in the regulation of STAT3.


Assuntos
Fator de Transcrição STAT3/metabolismo , Linhagem Celular Tumoral , Inativação Gênica , Vetores Genéticos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lentivirus , Proteínas , RNA Interferente Pequeno , Células THP-1 , Transfecção
2.
Cancer Immunol Immunother ; 68(11): 1891-1899, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31628525

RESUMO

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and overall survival remains poor. Chemotherapy is the standard of care for intensive induction therapy. Patients who achieve a complete remission require post-remission therapies to prevent relapse. There is no standard of care for patients with minimal residual disease (MRD), and stem cell transplantation is a salvage therapy. Considering the AML genetic heterogeneity and the leukemia immune-suppressive properties, novel cellular immune therapies to effectively harness immunological responses to prevent relapse are needed. We developed a novel modality of immune therapy consisting of monocytes reprogrammed with lentiviral vectors expressing GM-CSF, IFN-α and antigens. Preclinical studies in humanized mice showed that the reprogrammed monocytes self-differentiated into highly viable induced dendritic cells (iDCs) in vivo which migrated effectively to lymph nodes, producing remarkable effects in the de novo regeneration of T and B cell responses. For the first-in-man clinical trial, the patient's monocytes will be transduced with an integrase-defective tricistronic lentiviral vector expressing GM-CSF, IFN-α and a truncated WT1 antigen. For transplanted patients, pre-clinical development of iDCs co-expressing cytomegalovirus antigens is ongoing. To simplify the product chain for a de-centralized supply model, we are currently exploring a closed automated system for a short two-day manufacturing of iDCs. A phase I clinical trial study is in preparation for immune therapy of AML patients with MRD. The proposed cell therapy can fill an important gap in the current and foreseeable future immunotherapies of AML.


Assuntos
Antígenos de Neoplasias/metabolismo , Vetores Genéticos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunoterapia , Interferon-alfa/metabolismo , Leucemia Mieloide Aguda/terapia , Monócitos/imunologia , Células Dendríticas/imunologia , Humanos , Lentivirus/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Medicina de Precisão , Transplante de Células-Tronco
3.
Nucleic Acids Res ; 47(16): 8874-8887, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616952

RESUMO

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein-protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis ß-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.


Assuntos
Bacteriófago lambda/genética , Cromossomos Bacterianos/química , DNA Nucleotidiltransferases/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/química , Proteínas Virais/química , Sítio Alostérico , Bacteriófago lambda/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/genética , Fator Proteico para Inversão de Estimulação/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparo de DNA por Recombinação , Alinhamento de Sequência , Termodinâmica , Proteínas Virais/genética , Proteínas Virais/metabolismo
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(9): 1017-1022, 2019 Sep 30.
Artigo em Chinês | MEDLINE | ID: mdl-31640958

RESUMO

OBJECTIVE: To generate a new strain of HBeAg transgenic mice using CRISPR/Cas9 technique. METHODS: Hepatitis B virus (HBV) HBeAg gene was cloned and inserted in the pliver-HBeAg expression frame at the site of Rosa26 gene using CRISPR/Cas9 and homologous recombination techniques to construct the pliver-HBeAg expression vector containing HBeAg gene. The linear DNA fragment containing HBeAg gene was obtained by enzyme digestion. Cas9 mRNA, gRNA and the donor vector were microinjected into fertilized eggs of C57BL/6J mice, which were then transplanted into the uterus of C57BL/6J female surrogate mice to obtain F0 generation mice. The F0 generation mice were identified by long fragment PCR to obtain F0 transgenic mice with HBeAg gene. The positive F0 generation mice were bred with wild-type C57BL/6J mice to produce the F1 mice, which were identified by PCR and sequencing. The positive F1 transgenic mice carrying HBeAg gene were backcrossed until the homozygous offspring transgenic mice were obtained. The genotypes of the offspring mice were identified. The expressions of HBeAg and HBeAb in the heterozygous and homozygous HBeAg transgenic mice were detected by automatic chemiluminescence immunoassay, immune colloidal gold technique and immunohistochemistry method. RESULTS: A total of 56 F0 mice were obtained, and 2 of them carried homologous recombined HBeAg gene. Six positive F1 mice were obtained, from which 22 homozygous and 29 heterozygous F2 generation HBeAg transgenic mice were obtained. High concentration of HBeAg protein was detected in the peripheral blood of all the positive HBeAg transgenic mice without HBeAb expression. HBeAg expression was detected in the hepatocytes of HBeAg transgenic mice. CONCLUSIONS: We obtained a new strain of HBeAg transgenic mice with stable expression of HBeAg in the hepatocytes and immune tolerance to HBeAg using CRISPR/Cas9 technique, which provide a new animal model for studying HBV.


Assuntos
Sistemas CRISPR-Cas , Antígenos E da Hepatite B/genética , Camundongos Transgênicos , Animais , Feminino , Vetores Genéticos , Vírus da Hepatite B , Camundongos , Camundongos Endogâmicos C57BL
5.
Zhonghua Gan Zang Bing Za Zhi ; 27(9): 687-692, 2019 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-31594093

RESUMO

Objective: To construct the recombinant adenoviral containing fructose 1, 6-biphosphatase 1 (FBP1), and to investigate whether FBP1 has effect on autophagy and proliferation in liver cancer cells (HepG2). Methods: FBP1 cDNA sequence was amplified by PCR and cloned in adenovirus vector pAdTrack-TO4, and then recombinant adenovirus plasmid pAdTrack-FBP1 was constructed. The recombinant adenovirus plasmid was transfected into HEK293 cells by Lipofectamine 3000. High-titer of recombinant adenovirus AdFBP1 was obtained by packaging and amplification. HepG2 cells were infected with recombinant adenovirus AdFBP1, and the Mock and AdGFP group were set at the same time. Western blot and confocal laser scanning microscopy were used to observe the effect of FBP1 on the level of autophagy in hepatocellular carcinoma cells, and the effect of FBP1on the proliferation was observed by MTS and colony formation assay. A t-test and one-way ANOVA were used to compare the mean between group. Results: A high-titer recombinant adenovirus FBP1 was successfully constructed. Western blot and confocal laser scanning microscopy showed that the level of autophagy in AdFBP1 group was significantly lower than that in AdGFP group. Western blot results showed that LC3-II protein expression level in AdGFP was 1.10 ± 0.10 and 0.30 ± 0.01 in AdFBP1 group, F = 90.36, P < 0.01. Confocal laser scanning microscopy analysis showed that the average number of autophages in AdGFP was 28.33 ± 1.53 and 12.33 ± 1.53 in AdFBP1group, F = 97.40, P < 0.01. In addition, the results of colony formation assay and MTS assay showed that the proliferation of liver cancer cells in the AdFBP1 group was significantly inhibited compared with the AdGFP group. The results of colony formation showed that the cell clones in the AdGFP group was 65.66 ± 2.57 and 34.00 ± 2.00 in AdFBP1 group, F = 141.50, P < 0.01. MTS results showed that the absorbance of AdGFP group at 96h was 39.13 ± 2.21 and 30.61 ± 3.33 in AdFBP1 group, F = 7.80, P < 0.05. Conclusion: FBP1 inhibited the autophagy and proliferation in liver cancer cells (HepG2).


Assuntos
Autofagia , Proliferação de Células , Frutose-Bifosfatase/metabolismo , Neoplasias Hepáticas/patologia , Adenoviridae , Vetores Genéticos , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimologia , Transfecção
7.
Nat Med ; 25(9): 1396-1401, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501599

RESUMO

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date1,2. Mutations in FANCA account for more than 60% of FA cases worldwide3,4. Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70-80% of patients with FA during the first decade of life5,6. In this clinical study (ClinicalTrials.gov, NCT03157804 ; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder.


Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Adolescente , Adulto , Células da Medula Óssea/citologia , Criança , Pré-Escolar , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatologia , Feminino , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Lentivirus/genética , Masculino , Mutação/genética , Espanha/epidemiologia , Reparo Gênico Alvo-Dirigido , Transdução Genética , Adulto Jovem
8.
Adv Exp Med Biol ; 1148: 25-54, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482493

RESUMO

Since ERT for several LSDs treatment has emerged at the beginning of the 1980s with Orphan Drug approval, patients' expectancy and life quality have been improved. Most LSDs treatment are based on the replaced of mutated or deficient protein with the natural or recombinant protein.One of the main ERT drawback is the high drug prices. Therefore, different strategies trying to optimize the global ERT biotherapeutic production have been proposed. LVs, a gene delivery tool, can be proposed as an alternative method to generate stable cell lines in manufacturing of recombinant proteins. Since LVs have been used in human gene therapy, clinical trials, safety testing assays and procedures have been developed. Moreover, one of the main advantages of LVs strategy to obtain manufacturing cell line is the short period required as well as the high protein levels achieved.In this chapter, we will focus on LVs as a recombinant protein production platform and we will present a case study that employs LVs to express in a manufacturing cell line, alpha-Galactosidase A (rhαGAL), which is used as ERT for Fabry disease treatment.


Assuntos
Enzimas/biossíntese , Técnicas de Transferência de Genes , Lentivirus , Enzimas/farmacologia , Doença de Fabry/terapia , Vetores Genéticos , Humanos , alfa-Galactosidase/biossíntese , alfa-Galactosidase/farmacologia
9.
Anticancer Res ; 39(9): 4643-4652, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519562

RESUMO

BACKGROUND/AIM: Adenoviral-mediated expression of CD40 ligand (CD40L) on dendritic cells (DCs) activates immune check point CD40/CD40L, enhancing the immunostimulation of DCs and effector cells against human renal carcinoma cells (RCC) and inducing tumor cell apoptosis in vitro. MATERIALS AND METHODS: DCs, isolated from buffy coats from healthy donors, were transduced with adenoviruses carrying human CD40L (Ad-hCD40L). Subsequently maturation marker and cytokine expression were analyzed by fluorescence-activated cell sorting and enzyme-linked immunosorbent assay. RESULTS: Adenoviral transduction induced high expression of soluble CD40L and membrane-bound CD40L, leading to a strong CD40-CD40L interaction in DCs. Interestingly, a T-helper cell type 1 shift of expressed cytokines/chemokines was observed due to the expression of membrane-bound CD40L rather than due to soIuble CD40L alone, which significantly reduced immunoactivation of DCs. However, supernatants of Ad-hCD40L-transduced DCs induced apoptosis of RCC cells. Co-culture of Ad-hCD40L DCs with cytokine-induced killer cells led to a significant stimulation of tumor-specific cytokine-induced killer cells, with increased proliferation and cytotoxicity. CONCLUSION: Use of Ad-hCD40L-transduced DCs is a promising approach to treating RCC.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Carcinoma de Células Renais/metabolismo , Células Dendríticas/metabolismo , Imunomodulação , Neoplasias Renais/metabolismo , Adenoviridae/genética , Biomarcadores , Ligante de CD40/genética , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Neoplasias Renais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 653-658, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537250

RESUMO

Objective To produce rabbit polyclonal antibodies against human retinol-binding protein (RBP). Methods RBP cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) and then the amplified products were inserted into prokaryotic expression vector pET-28a(+) to construct recombinant plasmid pET-28a(+)-RBP. The established plasmid was then transformed into E. coli. Isopropylthio-ß-D-thiogalactoside (IPTG) was used to induce the expression of recombinant protein His-RBP in E. coli. The expression products were identified by SDS-PAGE from different clones of E. coli to screen positive bacteria, followed by amplifying culture. His-RBP protein was purified from the expression products of positive clones. The purified recombinant His-RBP was used to immunize New Zealand white rabbits. Antisera were acquired after four times of booster immunization. The prepared purified polyclonal antibodies were identified by SDS-PAGE, ELISA and Western blotting. Results We successfully constructed the recombinant plasmid pET-28a(+)-RBP, and acquired recombinant protein His-RBP of high purity. ELISA showed that the antibody titer reached 1:512 000. Conclusion The rabbit polyclonal antibodies against human RBP have been successfully prepared.


Assuntos
Anticorpos/metabolismo , Escherichia coli , Proteínas de Ligação ao Retinol/biossíntese , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Humanos , Plasmídeos , Coelhos
11.
BMC Plant Biol ; 19(1): 371, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438856

RESUMO

BACKGROUND: Propamocarb (PM) is one of the main pesticides used for controlling cucumber downy mildew. However, due to its volatility and internal absorption, PM can easily form pesticide residues on cucumber fruits that seriously endanger human health and pollute the environment. The breeding of new cucumber varieties with a low abundance of PM residues via genetic methods constitutes an effective strategy for reducing pesticide residues and improving cucumber safety and quality. To help elucidate the molecular mechanism resulting in a low PM residue abundance in cucumber, we used the cucumber cultivar 'D0351' (which has the lowest PM residue content) as the test material and identified genes related to low PM residue abundance through high-throughput tag-sequencing (Tag-Seq). RESULTS: CsMAPEG was constitutively expressed and showed both varietal and organizational differences. This gene was strongly expressed in 'D0351'. The expression levels of CsMAPEG in different cucumber tissues under PM stress were as follows: fruit>leaf>stem>root. CsMAPEG can respond to salicylic acid (SA), gibberellin (GA) and Corynespora cassiicola Wei (Cor) stress and thus plays an important regulatory role in plant responses to abiotic and biological stresses. The PM residue abundance in the fruits of CsMAPEG-overexpressing plants was lower than those found in antisense CsMAPEG plants and wild-type plants at all tested time points. The results revealed that CsMAPEG played a positive role in reducing the PM residue abundance. A CsMAPEG sense construct increased the contents of SOD, POD and GST in cucumber fruits, enhanced the degradation and metabolism of PM in cucumber, and thus effectively reduced the pesticide residue abundance in cucumber fruits. CONCLUSIONS: The expression patterns of CsMAPEG in cucumber cultivars with high and low pesticide residue abundances and a transgenic verification analysis showed that CsMAPEG can actively respond to PM stress and effectively reduce the PM residue abundance in cucumber fruits. The results of this study will help researchers further elucidate the mechanism responsible for a low PM residue abundance in cucumber and lay a foundation for the breeding of new agricultural cucumber varieties with low pesticide residue abundances.


Assuntos
Carbamatos/farmacologia , Cucumis sativus/genética , Fungicidas Industriais/farmacologia , Genes de Plantas , Resíduos de Praguicidas , Clonagem Molecular , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/enzimologia , Cucumis sativus/fisiologia , Perfilação da Expressão Gênica , Vetores Genéticos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transformação Genética
12.
Nucleic Acids Res ; 47(16): 8888-8898, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372631

RESUMO

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.


Assuntos
Apoproteínas/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
13.
Nucleic Acids Res ; 47(16): 8913-8925, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392336

RESUMO

The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.


Assuntos
DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Arch Virol ; 164(10): 2505-2513, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377888

RESUMO

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.


Assuntos
Expressão Gênica , Vetores Genéticos , Vírus da Necrose Hematopoética Infecciosa/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Reação em Cadeia da Polimerase em Tempo Real , Genética Reversa
17.
Cancer Radiother ; 23(5): 449-465, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400956

RESUMO

Nowadays, ionizing radiations have numerous applications, especially in medicine for diagnosis and therapy. Pharmacological radioprotection aims at increasing detoxification of free radicals. Radiomitigation aims at improving survival and proliferation of damaged cells. Both strategies are essential research area, as non-contained radiation can lead to harmful effects. Some advances allowing the comprehension of normal tissue injury mechanisms, and the discovery of related predictive biomarkers, have led to developing several highly promising radioprotector or radiomitigator drugs. Next to these drugs, a growing interest does exist for biotherapy in this field, including gene therapy and cell therapy through mesenchymal stem cells. In this review article, we provide an overview of the management of radiation damages to healthy tissues via gene or cell therapy in the context of radiotherapy. The early management aims at preventing the occurrence of these damages before exposure or just after exposure. The late management offers promises in the reversion of constituted late damages following irradiation.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Genética/métodos , Lesões por Radiação/prevenção & controle , Proteção Radiológica/métodos , Amifostina/uso terapêutico , Animais , Antioxidantes/uso terapêutico , Ensaios Clínicos como Assunto , Fracionamento da Dose de Radiação , Edição de Genes , Vetores Genéticos/uso terapêutico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Transplante de Células-Tronco Mesenquimais , Camundongos , Oxirredutases/genética , Oxirredutases/uso terapêutico , Lesões por Radiação/etiologia , Lesões por Radiação/terapia , Lesões Experimentais por Radiação/prevenção & controle , Lesões Experimentais por Radiação/terapia , Protetores contra Radiação/farmacologia , Protetores contra Radiação/uso terapêutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Fatores de Tempo
18.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1463-1468, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441617

RESUMO

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.


Assuntos
Produtos do Gene tat , Membrana Celular , Primers do DNA , Escherichia coli , Expressão Gênica , Vetores Genéticos , Proteínas Recombinantes de Fusão
19.
Nucleic Acids Res ; 47(15): 7767-7780, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31329919

RESUMO

A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA/química , Oligonucleotídeos/química , Marcadores de Spin/síntese química , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
20.
Nucleic Acids Res ; 47(16): 8675-8692, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31329932

RESUMO

The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-activating miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5' end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme's active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.


Assuntos
Metiltransferases/química , Capuzes de RNA/química , Proteínas de Ligação a RNA/química , S-Adenosil-Homocisteína/química , S-Adenosilmetionina/química , Regulação Alostérica , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , Transcrição Genética
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