RESUMEN
MicroRNA (miRNA) has been widely used as an effective gene drug for tumor therapy, but its chemical instability limited its therapeutic application in vivo. In this research, we fabricate an efficient miRNA nano-delivery system using zeolitic imidazolate framework-8 (ZIF-8) coated with bacterial outer membrane vesicles (OMVs), aimed for cancer treatment. The acid-sensitive ZIF-8 core enables this system to encapsulate miRNA and release them from lysosome quickly and efficiently in the target cells. The OMVs engineered to display programmed death receptor 1 (PD1) on the surface provides a specific tumor-targeting capability. Using a murine breast cancer model, we show that this system has high miRNA delivery efficiency and accurate tumor targeting. Moreover, the miR-34a payloads in carriers can further synergize with immune activation and checkpoint inhibition triggered by OMV-PD1 to enhance tumor therapeutic efficacy. Overall, this biomimetic nano-delivery platform provides a powerful tool for the intracellular delivery of miRNA and has great potential in RNA-based cancer therapeutic applications.
Asunto(s)
MicroARNs , Nanopartículas , Zeolitas , Animales , Ratones , Membrana Externa Bacteriana , Portadores de Fármacos , MicroARNs/genéticaRESUMEN
Outer membrane vesicles (OMVs) of Escherichia coli as nanoscale spherical vesicles have been recently used in cancer therapy as drug carriers. However, most of them need complicated methods to load cargos. Herein, we proposed an inexpensive and potentially mass-produced method for the preparation of OMV engineered with over-expressed pre-miRNA. In this work, we found that OMV can be released and inherit over-expressed tRNALys-pre-miRNA from mother E. coli that directly used for the tumor therapy. The eukaryotic cells infection experiments revealed that the over-expressed pre-miRNA inside OMV could be released and processed into mature miRNAs with the aid of the camouflage of "tRNA scaffold". Moreover, the group in vivo treated with targeted OMVtRNA-pre-miR-126 obviously inhibited the expression of target oncogenic CXCR4, and significantly restrain the proliferation of breast cancer tissues. Together, these findings indicated that the OMV-based platform is a versatile and powerful strategy for personalized tumor therapy directly and specificity.
Asunto(s)
Vesículas Extracelulares , MicroARNs , Neoplasias , Proteínas de la Membrana Bacteriana Externa , Portadores de Fármacos/metabolismo , Escherichia coli/genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias/tratamiento farmacológicoRESUMEN
DAL-1/4.1B is frequently absent in lung cancer tissues, which is significantly related to the occurrence and development of lung cancer. In this research, we found that DAL-1/4.1B affected the uptake of exosomes by lung cancer cells. When the expression of DAL-1/4.1B increased and decreased, the ability of exosome uptake enhanced and attenuated correspondingly. And we found that when cells were treated with different vesicles uptake inhibitors (chlorpromazine, methyl-ß-cyclodextrin (MßCD), cytochalasin D, chloroquine and heparin) and heparinase (HSPE), only heparin and HSPE counteracted the uptake enhancement effect caused by DAL-1/4.1B. Therefore, we speculated that DAL-1/4.1B might promote the uptake of exosomes through the heparan sulfate proteoglycans (HSPGs) pathway. After screening the expression of HSPGs and HSPE in H292 cells, the expression of heparan sulfate proteoglycan 2 (HSPG2) increased with overexpression of DAL-1/4.1B and decreased with knockdown of DAL-1/4.1B. Meanwhile, exosome uptake decreased with HSPG2 knockdown in H292 and DAL-1/4.1B-overexpressing H292 cells. Moreover, knockdown of DAL-1/4.1B and HSPG2 in lung cancer A549 cells resulted in a similar decrease in exosome uptake, and the expression of HSPG2 was also decreased with DAL-1/4.1B knockdown. These results indicated that HSPG2 directly affected the uptake of exosomes, while DAL-1/4.1B positively affected the expression of HSPG2. Therefore, DAL-1/4.1B may promote cellular adhesion and inhibit migration in cancer cells.
Asunto(s)
Exosomas/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Células A549 , Exosomas/genética , Proteoglicanos de Heparán Sulfato/genética , Humanos , Neoplasias Pulmonares/genética , Células MCF-7 , Proteínas de Microfilamentos/genética , Proteínas de Neoplasias/genéticaRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.6312.].
RESUMEN
In this study, we developed a multi-signal mitochondria-targeted fluorescent probe (NIR-Cys) for simultaneous detection of Cys and its metabolite, SO2. In the design of the probe, the acrylate group and the C[double bond, length as m-dash]C of the coumarin ring were used as the recognizing moiety for Cys and SO2, respectively. The probe exhibited high sensitivity, excellent specificity, and fast response. NIR-Cys was found to precisely target and visualize Cys metabolism in mitochondria of living cells with a multi-fluorescence signal. This probe is expected to be a useful tool for understanding Cys metabolism.
Asunto(s)
Acrilatos/química , Cromanos/química , Cumarinas/química , Cisteína/análisis , Colorantes Fluorescentes/química , Mitocondrias/metabolismo , Dióxido de Azufre/análisis , Acrilatos/síntesis química , Acrilatos/metabolismo , Acrilatos/toxicidad , Animales , Línea Celular Tumoral , Cromanos/síntesis química , Cromanos/metabolismo , Cromanos/toxicidad , Cumarinas/síntesis química , Cumarinas/metabolismo , Cumarinas/toxicidad , Cisteína/metabolismo , Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/toxicidad , Humanos , Límite de Detección , Hígado/metabolismo , Ratones , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Sulfitos/análisis , Sulfitos/química , Dióxido de Azufre/metabolismo , Pez CebraRESUMEN
Liver tumor-initiating cells (TICs), the drivers for liver tumorigenesis, accounts for liver tumor initiation, metastasis, drug resistance and relapse. Wnt/ß-catenin signaling pathway emerges as a critical modulator in liver TIC self-renewal. However, the molecular mechanism of Wnt/ß-catenin initiation in liver tumorigenesis and liver TICs is still elusive. Here, we examined the expression pattern of 10 Wnt receptors (FZD1-FZD10), and found only FZD6 is overexpressed along with liver tumorigenesis. What's more, a divergent lncRNA of FZD6, termed lncFZD6, is also highly expressed in liver cancer and liver TICs. LncFZD6 drives liver TIC self-renewal and tumor initiation capacity through FZD6-dependent manner. LncFZD6 interacts with BRG1-embedded SWI/SNF complex and recruits it to FZD6 promoter, and thus drives the transcriptional initiation of FZD6 by chromatin remodeling. WNT5A, a ligand of FZD6, is highly expressed in liver non-TICs and drives the self-renewal of liver TICs through lncFZD6-BRG1-FZD6-dependent manner. Through FZD6 transcriptional regulation in cis, lncFZD6 activates Wnt/ß-catenin signaling in liver TICs. LncFZD6-BRG1-Wnt5A/ß-catenin pathway can serve as a target for liver TIC elimination. Altogether, lncFZD6 promotes Wnt/ß-catenin activation and liver TIC self-renewal through BRG1-dependent FZD6 expression.
Asunto(s)
ADN Helicasas/metabolismo , Receptores Frizzled/genética , Neoplasias Hepáticas/patología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteína Wnt-5a/metabolismo , beta Catenina/metabolismo , Anciano , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , ADN Helicasas/genética , Femenino , Receptores Frizzled/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad , Proteínas Nucleares/genética , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Activación Transcripcional , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The membrane skeleton forms a scaffold on the cytoplasmic side of the plasma membrane. The erythrocyte membrane represents an archetype of such structural organization. It has been documented that a similar membrane skeleton also exits in the Golgi complex. It has been previously shown that ßII spectrin and ankyrin G are localized at the lateral membrane of human bronchial epithelial cells. Here we show that protein 4.1N is also located at the lateral membrane where it associates E-cadherin, ß-catenin and ßII spectrin. Importantly, depletion of 4.1N by RNAi in human bronchial epithelial cells resulted in decreased height of lateral membrane, which was reversed following re-expression of mouse 4.1N. Furthermore, although the initial phase of lateral membrane biogenesis proceeded normally in 4.1N-depleted cells, the final height of the lateral membrane of 4.1N-depleted cells was shorter compared to that of control cells. Our findings together with previous findings imply that 4.1N, ßII spectrin and ankyrin G are structural components of the lateral membrane skeleton and that this skeleton plays an essential role in the assembly of a fully functional lateral membrane.
Asunto(s)
Bronquios/metabolismo , Proteínas del Citoesqueleto/fisiología , Células Epiteliales/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/fisiología , Neuropéptidos/fisiología , Mucosa Respiratoria/metabolismo , Animales , Bronquios/citología , Bronquios/ultraestructura , Comunicación Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/ultraestructura , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Neuropéptidos/química , Neuropéptidos/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/ultraestructuraRESUMEN
OBJECTIVES: To investigate the effect of the linker region (LR) on the enzymatic activity, stability, and flexibility of Bacillus subtilis cellulase CelI15, six mutants were constructed that contained increasing numbers of the LR. RESULTS: The CelI15 mutant with three copies of the LR (approx. 57 amino acids) showed the highest activity, which was almost 20 % greater than that of wild type CelI15. The stability of the mutant enzymes increased as the copy number of the LR decreased. However, the substrate affinity of the mutant enzymes increased as the LR copy number increased, and the mutant with four copies of the LR exhibited the highest substrate affinity. Additionally, the flexibility of the CelI15 mutants increased as the LR copy number increased from zero to four copies, although it decreased sharply for the mutant with five copies of the LR. CONCLUSION: The activity of CelI15 was increased by increasing the LR copy number, which could be a potential way to improve its enzymatic properties.
Asunto(s)
Bacillus subtilis/enzimología , Celulasa/metabolismo , Celulasa/genética , MutaciónRESUMEN
Protein 4.1G is a membrane skeletal protein that can serve as an adapter between transmembrane proteins and the underlying membrane skeleton. The function of 4.1G remains largely unexplored. Here, using 4.1G knockout mouse embryonic fibroblasts (MEFs) as a model system, we explored the function of 4.1G in motile cells. We show that the adhesion, spreading, and migration of 4.1G(-/-) MEF cells are impaired significantly. We further show that, although the total cellular expression of ß1 integrin is unchanged, the surface expression of ß1 integrin and its active form are decreased significantly in 4.1G(-/-) MEF cells. Moreover, the phosphorylation of focal adhesion kinase, a downstream component of the integrin-mediated signal transduction pathway, is suppressed in 4.1G(-/-) MEF cells. Co-immunoprecipitation experiments and in vitro binding assays showed that 4.1G binds directly to ß1 integrin via its membrane-binding domain. These findings identified a novel role of 4.1G in cell adhesion, spreading, and migration in MEF cells by modulating the surface expression of ß1 integrin and subsequent downstream signal transduction.
Asunto(s)
Fibroblastos/metabolismo , Regulación de la Expresión Génica , Integrina beta1/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Adhesión Celular , Movimiento Celular , Citoesqueleto/metabolismo , Citometría de Flujo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Glutatión Transferasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Protein 4.1N is a member of protein 4.1 family and has been recognized as a potential tumor suppressor in solid tumors. Here, we aimed to investigate the role and mechanisms of 4.1N in non-small cell lung cancer (NSCLC). We confirmed that the expression level of 4.1N was inversely correlated with the metastatic properties of NSCLC cell lines and histological grade of clinical NSCLC tissues. Specific knockdown of 4.1N promoted tumor cell proliferation, migration and adhesion in vitro, and tumor growth and metastasis in mouse xenograft models. Furthermore, we identified PP1 as a novel 4.1N-interacting molecule, and the FERM domain of 4.1N mediated the interaction between 4.1N and PP1. Further, ectopic expression of 4.1N could inactivate JNK-c-Jun signaling pathway through enhancing PP1 activity and interaction between PP1 and p-JNK. Correspondingly, expression of potential downstream metastasis targets (ezrin and MMP9) and cell cycle targets (p53, p21 and p19) of JNK-c-Jun pathway were also regulated by 4.1N. Our data suggest that down-regulation of 4.1N expression is a critical step for NSCLC development and that repression of JNK-c-Jun signaling through PP1 is one of the key anti-tumor mechanisms of 4.1N.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pulmonares/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Neuropéptidos/metabolismo , Receptores de Neuropéptido Y/metabolismo , Adulto , Anciano , Animales , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/genética , Proteínas del Citoesqueleto/genética , Femenino , Humanos , Inmunohistoquímica , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Persona de Mediana Edad , Neuropéptidos/genética , Unión Proteica , Interferencia de ARN , Receptores de Neuropéptido Y/genética , Trasplante Heterólogo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Adulto JovenRESUMEN
The malaria parasite Plasmodium falciparum exports a large number of proteins into the erythrocyte cytoplasm during the asexual intraerythrocytic stage of its life cycle. A subset of these proteins interacts with erythrocyte membrane skeletal proteins and grossly alters the structure and function of the membrane. Several of the exported proteins, such as PfEMP1, PfEMP3, RESA and KAHRP, interact with the preponderant erythrocyte skeleton protein, spectrin. Here we have searched for possible interaction of these four malaria proteins with another major erythrocyte skeleton protein, ankyrin R. We have shown that KAHRP, but none of the other three, binds to ankyrin R. We have mapped the binding site for ankyrin R to a 79-residue segment of the KAHRP sequence, and the reciprocal binding site for KAHRP in ankyrin R to a subdomain (D3) of the 89kDa ankyrin R membrane-binding domain. Interaction of intact ankyrin R with KAHRP was inhibited by the free D3 subdomain. When, moreover, red cells loaded with the soluble D3 subdomain were infected with P. falciparum, KAHRP secreted by the intraerythrocytic parasite no longer migrated to the host cell membrane, but remained diffusely distributed throughout the cytosol. Our findings suggest a potentially important role for interaction of KAHRP with red cell membrane skeleton in promoting the adhesion of malaria-infected red cells to endothelial surfaces, a central element in the pathophysiology of malaria.
Asunto(s)
Ancirinas/química , Eritrocitos/parasitología , Oligopéptidos/química , Péptidos/química , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Ancirinas/genética , Ancirinas/metabolismo , Sitios de Unión , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Interacciones Huésped-Parásitos , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Péptidos/genética , Péptidos/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Protein 4.1G is a member of the protein 4.1 family, which in general serves as adaptors linking transmembrane proteins to the cytoskeleton. 4.1G is thought to be widely expressed in many cells and tissues, but its function remains largely unknown. To explore the function of 4.1G in vivo, we generated 4.1G(-/-) mice and bred the mice in two backgrounds: C57BL/6 (B6) and 129/Sv (129) hybrids (B6-129) and inbred B6. Although the B6 4.1G(-/-) mice showed no obvious abnormalities, deficiency of 4.1G in B6-129 hybrids was associated with male infertility. Histological examinations of these 4.1G(-/-) mice revealed atrophy, impaired cell-cell contact and sloughing off of spermatogenic cells in seminiferous epithelium, and lack of mature spermatids in the epididymis. Ultrastructural examination revealed enlarged intercellular spaces between spermatogenic and Sertoli cells as well as the spermatid deformities. At the molecular level, 4.1G is associated with the nectin-like 4 (NECL4) adhesion molecule. Importantly, the expression of NECL4 was decreased, and the localization of NECL4 was altered in 4.1G(-/-) testis. Thus, our findings imply that 4.1G plays a role in spermatogenesis by mediating cell-cell adhesion between spermatogenic and Sertoli cells through its interaction with NECL4 on Sertoli cells. Additionally, the finding that infertility is present in B6-129 but not on the B6 background suggests the presence of a major modifier gene(s) that influences 4.1G function and is associated with male infertility.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Germinativas/metabolismo , Inmunoglobulinas/metabolismo , Infertilidad Masculina/metabolismo , Proteínas de Microfilamentos/fisiología , Células de Sertoli/metabolismo , Espermatogénesis , Testículo/metabolismo , Animales , Adhesión Celular , Expresión Génica , Immunoblotting , Infertilidad Masculina/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas , Células de Sertoli/citología , Testículo/ultraestructuraRESUMEN
An efficient vector, designated as pCAGX, was designed for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells. It relied on the well-known TA-cloning principle, and utilized the CMV enhancer/chicken beta-actin/rabbit beta-globin (CAG) hybrid promoter instead of the classical CMV promoter to drive more efficient transgene expression in wider host cells. The specially designed cassette under CAG hybrid promoter contained two tandemly arrayed XcmI sites which were spaced by an additional EcoRV site. For direct cloning and expressing PCR-amplified ORFs, the T-vector was prepared by further digesting the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3'-ends, which could efficiently minimize the non-recombinant background of T-vector and eliminate the necessity of selective marker genes such as LacZ that allowed blue/white screening. Various PCR fragments in length were prepared to verify the cloning efficiency by ligation with this vector, and GFP gene expression under control of the CAG hybrid promoter in different host cells was assayed by flow cytometry. The results indicated that this vector was higher efficient, especially suitable for cloning and expressing a number of interesting ORFs in parallel, and higher-level transgene expression in different mammalian cells was obtained than the reported vectors using the CMV promoter.
Asunto(s)
Clonación Molecular/métodos , Vectores Genéticos/genética , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Línea Celular , Eritrocitos/citología , Eritrocitos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Intrones/genética , Ratones , Datos de Secuencia Molecular , Plásmidos/genética , Receptores de IgG/genética , TransfecciónRESUMEN
A novel bacterial strain with high cellulase activity (2.82 U/ml) was isolated, and then identified by its morphological character and 16S rRNA sequence, and named Bacillus subtilis strain I15. The extracellular thermostable cellulase exhibited the maximum activity at 60 degrees C and pH 6.0. It was very stable since more than 90% of original CMCase activity was maintained at 65 degrees C after incubation for 2 h. The cellulase gene, celI15, was cloned and extracellularly expressed by Escherichia coli BL21 (DE3), which encoded the extracellular protein of about 52 kDa. The extracellular activity of CelI15 from E. coli BL21 was up to about 6.78 U/ml, and all the other properties were almost the same as that from the wild-type strain.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/aislamiento & purificación , Celulasa/genética , Celulasa/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/genética , Celulasa/química , Celulasa/aislamiento & purificación , Clonación Molecular , Secuencia Conservada , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
A novel vector for direct PCR fragments cloning by positive selection, pBN, was constructed based on the lethal barnase from Bacillus amyloliquefaciens. Barnase was modified by inserting an additional insert at a pivotal Ile-54 site, which could take crucial affect on protein structure and absolute activity. The lacZ' expressing cassette of pUC19 was replaced by the modified barnase under the NptIII promoter. This novel vector could exist in large quantities as pUC19 in E. coli hosts. For the direct cloning PCR fragments, the positive selective vector was prepared by linearizing pBN with EcoRV to cut off the additional insert. PCR fragments with different length were prepared to verify this vector by ligation with this vector. The results showed that this positive selective vector for PCR fragment cloning was higher efficient and more convenient in manipulation than previous positive vectors.
Asunto(s)
Vectores Genéticos/genética , Reacción en Cadena de la Polimerasa/métodos , Ribonucleasas/genética , Selección Genética , Proteínas Bacterianas , Clonación Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Estructura Secundaria de ProteínaRESUMEN
The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.
Asunto(s)
Aspergillus/enzimología , Aspergillus/metabolismo , Celulasas/biosíntesis , Zea mays/metabolismo , Aspergillus/genética , Aspergillus/aislamiento & purificación , Carbono/metabolismo , Celulasas/química , Celulosa/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Estabilidad de Enzimas , Fermentación , Concentración de Iones de Hidrógeno , Hidrólisis , Nitrógeno/metabolismo , Filogenia , Saccharomyces cerevisiae/metabolismo , Temperatura , Factores de Tiempo , beta-Glucosidasa/biosíntesisRESUMEN
An endo-beta-1,4-glucanase from a thermoacidophilic fungus, Aspergillus terreus M11, was purified 18-fold with 14% yield and a specific activity of 67 U mg(-1) protein. The optimal pH was 2 and the cellulase was stable from pH 2 to 5. The cellulase had a temperature optimum of 60 degrees C measured over 30 min and retained more than 60% of its activity after heating at 70 degrees C for 1 h. The molecular mass of the cellulase was about 25 kDa. Its activity was inhibited by 77% by Hg(2+) (2 mM) and by 59% by Cu(2+) (2 mM).
Asunto(s)
Aspergillus/enzimología , Celulasa/química , Celulasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
A novel practical binary vector to get marker-free transgenic plant was constructed. The estrogen-inducible Cre/loxP DNA recombination system was adopted in this system. All non-target genes located between two identical orientation loxP sites could be excised from the transgenic genome by the Cre expression. In order to analyze this system, the target gene, GUS expression box (CaMV35s: :GUS), was inserted in the MCS outside the region franked by two loxP sites. Then it was introduced into the tobaccos. Results showed that the high-efficiency DNA recombination had take place and the target gene was working order after DNA excitation.
Asunto(s)
Vectores Genéticos/genética , Plantas Modificadas Genéticamente/genética , Recombinación Genética , Secuencia de Bases , Integrasas/metabolismo , Datos de Secuencia Molecular , Nicotiana/genéticaRESUMEN
With the development of transgenic crops, regulations to label the genetically modified organisms (GMOs) and their derived products have been issued in many countries. Polymerase chain reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods are generally needed to amplify the transgene and compare the amplified results with that of a corresponding reference gene to get the reliable results. Specific primers were developed for the rapeseed (Brassica napus), high-mobility-group protein I/Y(HMG-I/Y) single-copy gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 15 different rapeseed varieties, and identical amplified products were obtained with all of them. No amplification was observed when templates were the DNA samples from the other species of Brassica genus or other species, such as broccoli, stem mustard, cauliflower, Chinese cabbage, cabbage, sprouts, Arabidopsis thaliana, carrot, tobacco, soybean, mung bean, tomato, pepper, eggplant, plum, wheat, maize, barley, rice, lupine, and sunflower. This system was specific for rapeseed. Limits of detection and quantitation in qualitative and quantitative PCR systems were about 13 pg DNA (about 10 haploid genomes) and about 1.3 pg DNA (about 1 haploid genome), respectively. To further test the feasibility of this HMG-I/Y gene as an endogenous reference gene, samples containing transgenic rapeseed GT73 with the inserted glyphosate oxidoreductase (GOX) gene were quantitated. These demonstrated that the endogenous PCR detection systems were applicable to the qualitative and quantitative detection of transgenic rapeseed.