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1.
Chem Rec ; 17(6): 555-568, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28387472

RESUMO

Magnetic resonance imaging (MRI) has become a prominent non- or low-invasive imaging technique, providing high-resolution, three-dimensional images as well as physiological information about tissues. Low-molecular-weight Gd-MRI contrast agents (CAs), such as Gd-DTPA (DTPA: diethylenetriaminepentaacetic acid), are commonly used in the clinical diagnosis, while macromolecular Gd-MRI CAs have several advantages over low-molecular-weight Gd-MRI CAs, which help minimize the dose of CAs and the risk of side effects. Accordingly, we developed chiral dendrimer Gd-MRI CAs, which showed high r1 values. The association constant values (Ka ) of S-isomeric dendrimer CAs to bovine serum albumin (BSA) were higher than those of R-isomeric dendrimer CAs. Besides, based on a totally new concept, we developed 13 C/15 N-enriched multiple-resonance NMR/MRI probes, which realized highly selective observation of the probes and analysis of metabolic reactions of interest. This account summarizes our recent study on developing both chiral dendrimer Gd-MRI CAs, and self-traceable 13 C/15 N-enriched phosphorylcholine polymer probes for early detection of tumors.


Assuntos
Imageamento por Ressonância Magnética , Neoplasias/diagnóstico por imagem , Polímeros/química , Animais , Meios de Contraste/química , Dendrímeros/química , Gadolínio DTPA/química , Humanos , Fosforilcolina/química
2.
J Am Chem Soc ; 137(2): 799-806, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25560796

RESUMO

Polymers are concentration-amplified with respect to the monomeric units. We show here that a phosphorylcholine polymer enriched with (13)C/(15)N at the methyl groups is self-traceable by multiple-resonance (heteronuclear-correlation) NMR in tumor-bearing mice inoculated with the mouse rectal cancer cell line (colon 26). Preliminary measurements indicated that the present polymeric nanoprobe was satisfactorily distinguished from lipids and detectable with far sub-micromolar spectroscopic and far sub-millimolar imaging sensitivities. Detailed ex vivo and in vivo studies for the tumor-bearing mice administered the probe with a mean molecular weight of 63,000 and a mean size of 13 nm, revealed the following: (1) this probe accumulates in the tumor highly selectively (besides renal excretion) and efficiently (up to 30% of the injected dose), (2) the tumor can thus be clearly in vivo imaged, the lowest clearly imageable dose of the probe being 100 mg/kg or 2.0 mg/20-g mouse, and (3) the competition between renal excretion and tumor accumulation is size-controlled; that is, the larger (higher molecular-weight) and smaller (lower molecular-weight) portions of the probe undergo tumor accumulation and renal excretion, respectively. The observed size dependence suggests that the efficient tumor-targeting of the present probe is stimulated primarily by the so-called enhanced permeability and retention (EPR) effect, that is, size-allowed invasion of the probe into the tumor tissue via defective vascular wall. Self-traceable polymers thus open an important area of magnetic resonance imaging (MRI) of tumors and may provide a highly potential tool to visualize various delivery/localization processes using synthetic polymers.


Assuntos
Neoplasias do Colo/diagnóstico , Neoplasias do Colo/metabolismo , Imageamento por Ressonância Magnética , Fosforilcolina/química , Fosforilcolina/metabolismo , Polímeros/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos
3.
Bioorg Med Chem Lett ; 25(13): 2675-8, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25958246

RESUMO

A (13)C-enriched phosphorylcholine polymer ((13)C-PMPC) as a self-traceable MR (magnetic resonance) tag was conjugated with a fragment (scFv) of Herceptin, a clinical antibody against antigen Her2. When injected in model mice bearing Her2(+) (gastric) and Her2(-) (pancreatic) tumors, the antibody-tag conjugate (13)C-PMPC-scFv selectively accumulated in the Her2(+) tumor with a rapid build-up/decay (accumulation/clearance) profile and, with the use of the (1)H-(13)C double-resonance (heteronuclear correlation) technique, the Her2(+) gastric tumor was clearly MR imaged.


Assuntos
Imunoconjugados/farmacocinética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Gástricas/diagnóstico , Animais , Isótopos de Carbono/farmacocinética , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/química , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacocinética , Ácidos Polimetacrílicos/farmacocinética , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Anticorpos de Cadeia Única/farmacocinética , Neoplasias Gástricas/metabolismo
4.
Biochem Biophys Res Commun ; 414(4): 733-6, 2011 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-22001928

RESUMO

We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation. Because sandwiched ZFNs can recognize and cleave a variety of long (≥ 26-bp) target DNAs, they may not only expand the utility of ZFNs for construction of recombinant plasmids, but also serve as useful meganucleases for synthesis of artificial genomes.


Assuntos
Clonagem Molecular/métodos , Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/química , Dedos de Zinco , DNA/química , DNA/genética , Plasmídeos/química , Plasmídeos/genética
5.
BMC Res Notes ; 14(1): 237, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162412

RESUMO

OBJECTIVE: Tomato yellow leaf curl virus (TYLCV) is one of the pathogens severely damaging tomato crops. Therefore, methods to treat or prevent TYLCV infection need to be developed. For this purpose, a method to conveniently and quickly assess infection of tomatoes by TYLCV is desired. In the present study, we established a quick method to evaluate TYLCV infection using cotyledons of Micro-Tom, a miniature tomato cultivar. RESULTS: First, we constructed a binary plasmid harboring 1.5 copies of the TYLCV genome and transformed Agrobacterium with the plasmid. By injecting agroinoculum from the resulting transformant into the branches of Micro-Tom, we confirmed the susceptibility of Micro-Tom to TYLCV. To shorten the evaluation process of TYLCV infection further, we agroinoculated cotyledons of Micro-Tom 10 days after sowing seeds. We consistently observed typical symptoms of TYLCV infection on true leaves 10 days after agroinoculation. Molecular analysis detected TYLCV progeny DNA in all leaves demonstrating symptoms 6 days after agroinoculation. Therefore, our new protocol enabled assessment of TYLCV infection within 20 days after sowing seeds. Thus, agroinoculation of Micro-Tom cotyledons will accelerate the process of screening TYLCV-resistant Micro-Toms and enable screening of larger numbers of plants more quickly, contributing to the development of TYLCV-resistant tomatoes.


Assuntos
Begomovirus , Solanum lycopersicum , Begomovirus/genética , Cotilédone/genética , Doenças das Plantas
6.
Bioorg Med Chem Lett ; 20(12): 3479-81, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20529678

RESUMO

Zinc-finger-based artificial transcription factors (ATFs) have been used to regulate expression of target genes both in vitro and in vivo. However, if we develop ATF expression further, target gene regulation by ATFs may be more effective. Here, we report a new transcriptional regulation system in which an ATF that is designed to upregulate a target gene also activates itself. To construct the system, we inserted tandem copies of the ATF-binding sites upstream of a promoter for ATF expression. Using the endogenous human VEGF-A gene, we demonstrated that the new expression system amplified ATF expression in a manner dependent on the number of copies of the ATF-binding site, and that the 'self-propagating ATF' upregulated VEGF-A gene expression more efficiently than a control promoter with no ATF-binding site.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Marcação de Genes/métodos , Engenharia de Proteínas/métodos , Fatores de Transcrição/síntese química , Fatores de Transcrição/genética , Regulação para Cima/genética , Sítios de Ligação/genética , Humanos , Regiões Promotoras Genéticas/genética , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Dedos de Zinco
7.
Sci Rep ; 10(1): 19363, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168875

RESUMO

Three-dimensional (3D) representation of a tumor with respect to its size, shape, location, and boundaries is still a challenge in photoacoustic (PA) imaging using artificial contrast agents as probes. We carried out PA imaging of tumors in mice using 800RS-PMPC, which was obtained by coupling of 800RS, a near-infrared cyanine dye, with PMPC, a highly selective tumor-targeting methacrylate polymer having phosphorylcholine side chains, as a probe. The conjugate 800RS-PMPC forms compact nanoparticles (dDLS = 14.3 nm), retains the biocompatibility of the parent polymer (PMPC) and exhibits unprecedented PA performance. When applied to mice bearing a 6 × 3 × 3 mm3 tumor buried 6 mm beneath the skin, the probe 800RS-PMPC selectively accumulates in the tumor and emits PA signals that are strong enough to be unambiguously distinguished from noise signals of endogenous blood/hemoglobin. The PA image thus obtained under high-threshold conditions allows 3D characterization of the tumor in terms of its size, shape, location, and boundaries.


Assuntos
Neoplasias do Colo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Verde de Indocianina/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Técnicas Fotoacústicas/métodos , Animais , Materiais Biocompatíveis , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Sistemas de Liberação de Medicamentos , Feminino , Hemoglobinas/química , Processamento de Imagem Assistida por Computador , Luz , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Polímeros/química , Espalhamento de Radiação , Espectroscopia de Luz Próxima ao Infravermelho
8.
Biochem Biophys Res Commun ; 390(3): 845-8, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19836352

RESUMO

Activation of vascular endothelial growth factor A (VEGF-A) is an attractive approach to treatment of ischemic diseases. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expression of ATFs upregulated the endogenous VEGF-A gene expression, activation of VEGF-A specifically in ischemic tissues is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-specific activation of human VEGF-A gene by hypoxia-driven expression of the ATF. We constructed a hypoxia-driven promoter for the ATF expression and placed it upstream of the ATF-encoding regions. The resulting hypoxia-driven expression plasmid induced the ATF expression in hypoxia but not in normoxia, and the hypoxia-specific expression of the ATF activated the VEGF-A expression specifically in hypoxia. Thus, the engineered expression system of ATFs may enable activation of VEGF-A expression specifically in ischemic tissues without affecting normal, healthy tissues in vivo.


Assuntos
Hipóxia/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/genética , Células Cultivadas , Engenharia Genética , Humanos , Hipóxia/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Regulação para Cima , Dedos de Zinco
9.
Biochem Biophys Res Commun ; 390(3): 694-7, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19825368

RESUMO

Zinc-finger nucleases (ZFNs) are a powerful tool for manipulation of genomic DNA. Recently, we reported a new ZFN composed of one artificial zinc-finger protein (AZP) and a single-chain FokI dimer (scFokI) that refines ZFN technology. While AZP-scFokI cleaved DNA specifically around the AZP-target site, several nucleotide positions were cleaved due to the mobility of the scFokI domain. In the present study, we aimed to improve the DNA-cleavage specificity at the nucleotide level. To this end, we sandwiched a scFokI domain between two AZPs to reduce the mobility of the scFokI moiety when bound to DNA. We demonstrated that the AZP-sandwiched scFokI cleaved DNA at a single nucleotide position of a target plasmid, in which two AZP-binding sites were connected with a 6-bp spacer, with multiple turnovers. Further improvement of AZP-sandwiched scFokI will lead to development of ideal artificial meganucleases.


Assuntos
Clivagem do DNA , DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Dedos de Zinco , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Engenharia de Proteínas , Multimerização Proteica
10.
Bioorg Med Chem ; 17(6): 2381-7, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19261481

RESUMO

We have synthesized a series of 5'-phosphorylated and 5'-cytidylyl-(3'-5')-cytidylyl-(3'-5')-puromycin derivatives that have backbone-elongated substrates. All the synthesized puromycin derivatives showed good solubility in water and were applied to translation inhibitory assay in a reconstituted Escherichia coli translation system.


Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/química , Puromicina/farmacologia , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Fosforilação , Puromicina/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Biochemistry ; 47(47): 12257-9, 2008 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-18980382

RESUMO

To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.


Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , DNA/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Staphylococcus/enzimologia , Dedos de Zinco , Sequência de Bases , Sítios de Ligação , DNA/genética , Ligação Proteica
12.
Chem Commun (Camb) ; (33): 3858-60, 2008 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-18726014

RESUMO

The "light-up" RNA aptamer-Hoechst pair can be used as a fluorescent tag to monitor transcription processes.


Assuntos
Aptâmeros de Nucleotídeos , Transcrição Gênica , Aptâmeros de Nucleotídeos/química , Fluorescência , Conformação de Ácido Nucleico
13.
Medicine (Baltimore) ; 96(28): e7494, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28700496

RESUMO

BACKGROUND: Information received subconsciously can influence exercise performance; however, it remains unclear whether subliminal or supraliminal reward is more effective in improving standing balance ability when priming stimuli are subconsciously delivered. The present study aimed to compare the effects of subliminal priming-plus-subliminal reward stimuli (experimental) with subliminal priming-plus-supraliminal reward stimuli (control) on standing balance ability. METHODS: This was a single-blind (outcome assessor), parallel-group, randomized controlled trial involving healthy young adults recruited from a university in Japan. Assessments were conducted at baseline and immediately after intervention. The primary outcome was the functional reach test (FRT) measurement. The secondary outcome was one-leg standing time (OLST) with eyes closed. Of the 52 participants screened, 25 were randomly assigned to experimental and control groups each. RESULTS: Both interventions were effective for improving the FRT between the baseline and intervention; however, smaller improvements were observed in the experimental group. We found a large between-groups effect size immediately after the intervention for the FRT (d = -0.92). In contrast, there were no differences in improvements in OLST between the 2 groups (d = -0.06); furthermore, neither intervention was found to be effective for this parameter. CONCLUSION: We concluded that subliminal priming with conscious reward stimuli results in improvements in immediate-term forward reach ability, which is superior to that achieved by subliminal priming with subconscious reward stimuli.


Assuntos
Equilíbrio Postural , Priming de Repetição , Recompensa , Estimulação Subliminar , Adolescente , Braço , Humanos , Perna (Membro) , Atividade Motora , Método Simples-Cego , Adulto Jovem
14.
J Control Release ; 116(2): 107-14, 2006 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-16905218

RESUMO

We investigated the transfection properties of singly and triply lactose-functionalized derivatives, Lac(1) and Lac(3), and non-glycosylated one, Lac(0), of calix[4]resorcarene-based amphiphilic octaamine 1 in light of those previously reported for more extensively glycosylated compounds Lac(5) and Lac(8). They all strongly bind to the luciferase-encoding plasmid DNA pCMVluc (7040 base-pairs) with a saturation stoichiometry of Lac(n)/P approximately =0.5 (n=0, 1, or 3) or 0.7 (n=5 or 8), where P stands for a phosphate moiety of the plasmid DNA. The resulting carrier-DNA conjugates are positively charged and monomeric (monomolecular with respect to DNA) as such when n=0 or 1, neutral and monomeric when n=3, or neutral and aggregated when n=5 or 8. Transfection of HeLa (uterine) and HepG2 (hepatic) cell lines shows a general trend of decreasing luciferase expression efficiencies (E) in lively cells as well as cytotoxicities with increasing n's. The cell selectivities for HepG2 over HeLa sharply increase with increasing n's; E(HepG2)/E(HeLa)=0.3, 0.6, 7, 14, and 120 for Lac(0), Lac(1), Lac(3), Lac(5), and Lac(8), respectively, as a result of specific receptor pathway involving the asialoglycoprotein receptors on the hepatic (HepG2) cell surfaces and clustering lactose moieties of the carrier-DNA conjugates. The toxicity-corrected, overall efficiency of gene delivery to hepatocytes is optimized at Lac(3), which forms compactly packed (approximately 40 nm), charge-masked (xi approximately = 0 mV), and less toxic virus-like particles capable of receptor-mediated hepatocyte targeting.


Assuntos
Receptor de Asialoglicoproteína/metabolismo , DNA/metabolismo , Hepatócitos/metabolismo , Lactose/química , Compostos Macrocíclicos/metabolismo , Poliaminas/metabolismo , Transfecção/métodos , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , Feminino , Genes Reporter , Terapia Genética/métodos , Glicosilação , Células HeLa , Humanos , Luciferases , Compostos Macrocíclicos/química , Compostos Macrocíclicos/toxicidade , Tamanho da Partícula , Plasmídeos , Poliaminas/química , Poliaminas/toxicidade , Útero/citologia , Útero/metabolismo
15.
ChemistryOpen ; 5(2): 125-8, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-27308224

RESUMO

In an attempt to monitor µm-level trace constituents, we applied here (1)H-{(13)C-(15)N} triple-resonance nuclear magnetic resonance (NMR) to (13)C/(15)N-enriched l-Dopa as the inevitable precursor of the neurotransmitter dopamine in the brain. The perfect selectivity (to render endogenous components silent) and µm-level sensitivity (700 MHz spectrometer equipped with a cryogenic probe) of triple-resonance allowed the unambiguous and quantitative metabolic and pharmacokinetic analyses of administered l-Dopa/dopamine in the brain and liver of mice. The level of dopamine generated in the brain (within the range 7-76 µm, which covers the typical stimulated level of ∼30 µm) could be clearly monitored ex vivo, but was slightly short of the detection limit of a 7 T MR machine for small animals. This work suggests that µm-level trace constituents are potential targets of ex vivo monitoring as long as they contain N atom(s) and their appropriate (13)C/(15)N-enrichment is synthetically accessible.

16.
FEBS Lett ; 553(3): 271-6, 2003 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-14572636

RESUMO

Molecular chaperone-like activity for protein refolding was investigated using nanogels of self-assembly of cholesterol-bearing pullulan. Nanogels effectively prevented protein aggregation (i.e. carbonic anhydrase and citrate synthase) during protein refolding from GdmCl denaturation. Enzyme activity recovered in high yields upon dissociation of the gel structure in which the proteins were trapped, by the addition of cyclodextrins. The nanogels assisted protein refolding in a manner similar to the mechanism of molecular chaperones, namely by catching and releasing proteins. The nanogels acted as a host for the trapping of refolded intermediate proteins. Cyclodextrin is an effector molecule that controls the binding ability of these host nanogels to proteins. The present nanogel system was also effective at the renaturation of inclusion body of a recombinant protein of the serine protease family.


Assuntos
Anidrases Carbônicas/química , Citrato (si)-Sintase/química , Hidrogéis/química , Chaperonas Moleculares/química , Renaturação Proteica , Animais , Anidrases Carbônicas/metabolismo , Bovinos , Citrato (si)-Sintase/metabolismo , Ciclodextrinas/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glucanos/química , Guanidina/química , Corpos de Inclusão/química , Corpos de Inclusão/genética , Cinética , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Suínos
18.
Mol Biotechnol ; 56(8): 731-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24682726

RESUMO

Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.


Assuntos
Replicação do DNA/genética , DNA Viral/genética , DNA Viral/metabolismo , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Sítios de Ligação/genética , Biotecnologia , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/toxicidade , Feminino , Células HEK293 , Papillomavirus Humano 18/patogenicidade , Humanos , Nuclease do Micrococo/genética , Nuclease do Micrococo/toxicidade , Reação em Cadeia da Polimerase , Especificidade por Substrato , Transfecção , Dedos de Zinco/genética
19.
PLoS One ; 8(2): e56633, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23437192

RESUMO

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.


Assuntos
Replicação do DNA/efeitos dos fármacos , Nuclease do Micrococo/farmacologia , Papillomaviridae/efeitos dos fármacos , Replicação Viral/genética , Replicação do DNA/genética , DNA Viral/efeitos dos fármacos , DNA Viral/genética , Genoma Viral , Papillomavirus Humano 18/efeitos dos fármacos , Papillomavirus Humano 18/genética , Humanos , Nuclease do Micrococo/síntese química , Nuclease do Micrococo/genética , Papillomaviridae/genética , Origem de Replicação , Replicação Viral/efeitos dos fármacos , Dedos de Zinco/genética
20.
Mol Biotechnol ; 54(2): 198-203, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22576255

RESUMO

Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5'-ATCGGTGT ATCGGTGT-3' in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays. We then constructed a 6-finger AZP that bound to a 19-bp DNA including the Rep-binding site. We demonstrated that the binding affinity of the AZP was >1,000-fold greater than that of Rep and that the AZP inhibited Rep binding completely in vitro. Because the binding capability of the AZP was same as that of the AZP previously designed for geminivirus-resistant Arabidopsis thaliana, we predict that the present AZP will prevent TYLCV infection in vivo.


Assuntos
Begomovirus/genética , Ligação Proteica/genética , Origem de Replicação/genética , Solanum lycopersicum/virologia , Proteínas Virais/genética , Dedos de Zinco/genética , Begomovirus/metabolismo , Sítios de Ligação/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/metabolismo
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