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1.
Electron. j. biotechnol ; 30: 1-5, nov. 2017. ilus, tab, graf
Artigo em Inglês | LILACS | ID: biblio-1021034

RESUMO

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.


Assuntos
Sementes/enzimologia , Hordeum/enzimologia , Thermococcus/metabolismo , alfa-Amilases/metabolismo , Sementes/genética , Sementes/microbiologia , Transformação Genética , Hordeum/genética , Hordeum/microbiologia , Cerveja , Estabilidade Enzimática , Plantas Geneticamente Modificadas/enzimologia , Clonagem Molecular , Técnicas de Transferência de Genes , alfa-Amilases/genética , Fermentação , Termotolerância , Temperatura Alta , Concentração de Íons de Hidrogênio
2.
Clin. biomed. res ; 37(4): 330-333, 2017. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-876699

RESUMO

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by deficiency of alpha-L-iduronidase (IDUA). Limitations such as the need for weekly injections, high morbidity and mortality, and high cost of current treatments show that new approaches to treat this disease are required. In this study, we aimed to correct fibroblasts from a patient with MPS I using non-viral gene therapy. Using a plasmid encoding the human IDUA cDNA, we achieved stable high IDUA levels in transfected fibroblasts up to 6 months of treatment. These results serve as proof of concept that a non-viral approach can correct the enzyme deficiency in cells of patients with lysosomal storage disorders, which can be used as a research tool for a series of disease aspects. Future studies should focus on showing if this approach can be useful in small animals and clinical trials (AU)


Assuntos
Humanos , Fibroblastos/enzimologia , Técnicas de Transferência de Genes , Vetores Genéticos , Iduronidase/metabolismo , Mucopolissacaridose I/terapia , DNA Complementar , Terapia Genética/métodos , Iduronidase/genética , Mucopolissacaridose I/genética , Plasmídeos/genética , Transfecção/métodos
3.
Rev. chil. pediatr ; 86(4): 251-256, ago. 2015. tab
Artigo em Espanhol | LILACS | ID: lil-764081

RESUMO

Introducción: Autoconcepto es el conjunto de ideas y actitudes que se tiene acerca de sí mismo. Nuestro objetivo fue evaluar si existen diferencias en el nivel de autoconcepto de niños de 8-12 años con y sin secuelas de quemaduras, e identificar variables predictoras del autoconcepto en los niños con secuelas. Pacientes y método: Estudio comparativo, transversal de 109 niños con secuelas de quemaduras de 8 a 12 años de edad, con 109 niños sin secuelas de quemaduras, del mismo grupo de edad y nivel socioeconómico. Se utilizó la escala de autoconcepto de Piers-Harris, que entrega medida de autoconcepto general y dimensiones: conductual, estatus intelectual y escolar, apariencia y atributos físicos, ansiedad, popularidad, felicidad y satisfacción. Resultados: No hubo diferencias significativas en el nivel de autoconcepto general ni en sus dimensiones al comparar ambos grupos (p > 0,05). Dentro del grupo con secuelas de quemaduras la variable número de secuelas resultó ser un factor protector para las dimensiones ansiedad, popularidad, felicidad-satisfacción y autoconcepto general. La variable localización surgió como factor de riesgo para la dimensión conductual. Discusión: La ausencia de diferencias en autoconcepto entre niños con secuelas de quemaduras y sin ellas es similar a lo reportado por la literatura. El hallazgo en factores de riesgo y protectores motiva a continuar investigando, incorporando antecedentes premórbidos y familiares.


Introduction: Self-concept is the set of ideas and attitudes that a person has about him/herself. Objective: To evaluate whether there are differences in the level of self-concept in children 8-12 years old with and without burns sequelae. To identify predictive variables of self-concept in children with sequelae. Patients and method: A comparative cross-sectional study of self-concept in 109 children with burns sequelae, from 8 to 12 years old, with 109 children without burns sequelae, and of the same age and socioeconomic status. The Piers-Harris self-concept scale is used, which provides a general measurement of self-concept and behavioural, intellectual and school status, appearance, and physical attributes, anxiety, popularity, happiness and satisfaction dimensions. Results: There were no significant differences in the level of general self-concept or their dimensions (P > .05). In the group with burns sequelae, the protective factor was the variable number of sequels was associated with the dimensions of anxiety, popularity, happiness-satisfaction and general self-concept. The location variable emerged as a risk factor for the behavioural dimension. Discussion: The absence of differences in self-concept between children with burns sequelae and children without them is similar to that reported in the literature. The finding in the risk and protective factors encourages to further research, and perhaps incorporating pre-morbidity and family background.


Assuntos
Humanos , DNA , Compostos de Amônio Quaternário/química , Serina/química , Tensoativos/química , Amidas/química , Aminas/química , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Ésteres/química , Técnicas de Transferência de Genes , Terapia Genética/métodos , Células HeLa , Lipídeos/química , Transfecção/métodos
4.
Rev. chil. pediatr ; 86(4): 287-290, ago. 2015. ilus, graf
Artigo em Espanhol | LILACS | ID: lil-764087

RESUMO

Introducción: La telorragia es un síntoma poco frecuente en pacientes pediátricos, la causa más frecuente en esta población es la ectasia ductal mamaria (EDM), que es una afección benigna y autolimitada, caracterizada por la dilatación del conducto mamario, fibrosis e inflamación periductal. Objetivo: Presentar un caso de EDM, para facilitar el rápido reconocimiento por parte de los médicos, y evitar estudios y tratamientos agresivos. Caso clínico: Lactante de sexo masculino de 6 meses de edad, sano, alimentado por lactancia materna exclusiva; consultó por un nódulo retroareolar derecho y telorragia unilateral. Se realizó una ecografía Doppler que mostró una lesión multiquística, sugerente de una EDM. Se planteó tratamiento expectante y acudió a control a los 6 meses con excelente evolución. Conclusiones: La EDM es la principal causa de telorragia en niños, corresponde a una afección benigna, y la resolución generalmente es espontánea, antes de los 9 meses. Por lo que su conocimiento es de gran relevancia para el adecuado diagnóstico y manejo de estos pacientes.


Introduction: Bloody nipple discharge is an infrequent symptom during childhood. The most common cause in this population is mammary duct ectasia (MDE), which is a benign and self-limiting condition, that is characterized by dilatation of the mammary ducts, fibrosis and periductal inflammation. Objective: Report of a case of MDE in order to improve physicians’ diagnosis accuracy and avoid aggressive studies and treatments. Case report: Six-months old male healthy infant, exclusively breastfeeded, that visited our clinic with a lump beneath his right nipple and bloody discharge from the same nipple. An ultrasound was performed which showed a multicystic lesion suggestive of MDE. Watchful waiting was decided as treatment, with good evolution after six months of follow up. Conclusions: The MDE is the leading cause of bloody discharge in pediatric population, being a benign condition that resolves spontaneously before nine months. The knowledge of this condition is essential so as to accurately diagnose and treat it.


Assuntos
Humanos , Cátions/química , Indicadores e Reagentes/química , Lipídeos/química , Polienos/química , RNA Interferente Pequeno/química , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células HeLa , Lipossomos/química , Luciferases/química , Fosfolipídeos/química , RNA Interferente Pequeno/genética , Transfecção/métodos
5.
Einstein (Säo Paulo) ; 13(1): 89-95, Jan-Mar/2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-745883

RESUMO

Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. .


Objetivo Avaliar os efeitos da transferência gênica do VEGF165 no processo de remodelamento da matriz extracelular após infarto agudo do miocárdio. Métodos Ratos Wistar foram submetidos ao infarto do miocárdio por ligação da artéria coronária descendente esquerda, e a fração de ejeção de ventrículo esquerdo foi utilizada para classificar os infartos em grandes e pequenos. Os animais foram divididos em grupos de dez animais, de acordo com o tamanho do infarto (grande ou pequeno), e receberam ou não tratamento com o VEGF165. A avaliação dos diferentes marcadores foi realizada por imuno-histoquímica e quantificação digital. Os anticorpos primários utilizados foram antifibronectina, antivimentina, anti- CD44, anti-E-caderina, anti-CD24, anti-alfa-1-actina e anti-PCNA. Os resultados foram representados como média e erro padrão, e analisados por ANOVA, sendo considerado estatisticamente significativo se p≤0,05. Resultados Houve aumento significativo da expressão de marcadores de células indiferenciadas, como fibronectina (proteína presente na matriz extracelular) e CD44 (glicoproteína presente nas células endoteliais). Entretanto, houve diminuição da expressão de vimentina e PCNA, indicando possível diminuição do processo de proliferação celular após o tratamento com VEGF165. Os marcadores de células diferenciadas, E-caderina (proteína de adesão entre as células do miocárdio), CD24 (proteína presente nos vasos sanguíneos) e alfa-1-actina (marcador especifico de miócitos) também apresentaram maior expressão nos grupos submetidos à terapia gênica, comparativamente com o grupo não tratado. O valor obtido pela relação entre alfa-1-actina e vimentina foi aproximadamente três vezes maior nos grupos tratados com VEGF165, indicando maior diferenciação tecidual. Conclusão O papel dos miócitos se mostrou importante no processo de remodelamento tecidual, confirmando que o VEGF165 parece conferir um efeito protetor no tratamento do infarto agudo do miocárdio. .


Assuntos
Animais , Feminino , Matriz Extracelular/fisiologia , Técnicas de Transferência de Genes , Infarto do Miocárdio/terapia , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Actinas/análise , /análise , /análise , Caderinas/análise , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Fibronectinas/análise , Terapia Genética/métodos , Imuno-Histoquímica , Miócitos Cardíacos/fisiologia , Ratos Wistar , Reprodutibilidade dos Testes , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/genética , Vimentina/análise
6.
Rev. Assoc. Med. Bras. (1992) ; 60(6): 520-524, Nov-Dec/2014.
Artigo em Inglês | LILACS | ID: lil-736322

RESUMO

Methods and techniques employed in gene therapy are reviewed in parallel with pertinent ethical conflicts. Clinical interventions based on gene therapy techniques preferentially use vectors for the transportation of therapeutic genes, however little is known about the potential risks and damages to the patient. Thus, attending carefully to the clinical complications arising as well as to security is essential. Despite the scientific and technological advances, there are still many uncertainties about the side effects of gene therapy. Moreover, there is a need, above all, to understand the principles of bioethics as both science and ethics, in accordance with its socioecological responsibility, in order to prioritize the health and welfare of man and nature, using properly natural resources and technology. Therefore, it is hard to determine objective results and to which extent the insertion of genes can affect the organism, as well as the ethical implication.


Métodos e técnicas empregadas na terapia gênica são revisados em paralelo a conflitos éticos pertinentes. Intervenções clínicas com base em técnicas de terapia gênica são usadas preferencialmente em vetores para o transporte de genes terapêuticos; porém, pouco se sabe sobre os possíveis riscos e danos para o paciente, sendo necessário atender cuidadosamente às complicações clínicas resultantes, bem como à segurança. Apesar dos avanços científicos e tecnológicos relacionados à terapia gênica, ainda há muitas incertezas sobre os efeitos colaterais do uso dessa terapia. Além disso, é necessário, acima de tudo, compreender os princípios da bioética como uma ética da ciência para com a responsabilidade socioecológica, a fim de priorizar a saúde e o bem-estar do homem e da natureza, utilizando adequadamente recursos naturais e tecnologia. Portanto, é difícil afirmar qual é o rendimento real, bem como os resultados do aumento da genética inserida no organismo e as implicações éticas.


Assuntos
Humanos , Temas Bioéticos , Técnicas de Transferência de Genes , Terapia Genética , Atitude do Pessoal de Saúde , Segurança do Paciente , Medição de Risco/normas , Responsabilidade Social
7.
Braz. j. microbiol ; 45(3): 785-789, July-Sept. 2014. tab
Artigo em Inglês | LILACS | ID: lil-727003

RESUMO

Streptococcus agalactiae (GBS) is a major source of human perinatal diseases and bovine mastitis. Erythromycin (Ery) and tetracycline (Tet) are usually employed for preventing human and bovine infections although resistance to such agents has become common among GBS strains. Ery and Tet resistance genes are usually carried by conjugative transposons (CTns) belonging to the Tn916 family, but their presence and transferability among GBS strains have not been totally explored. Here we evaluated the presence of Tet resistance genes (tetM and tetO) and CTns among Ery-resistant (Ery-R) and Ery-susceptible (Ery-S) GBS strains isolated from human and bovine sources; and analyzed the ability for transferring resistance determinants between strains from both origins. Tet resistance and int-Tn genes were more common among Ery-R when compared to Ery-S isolates. Conjugative transfer of all resistance genes detected among the GBS strains included in this study (ermA, ermB, mef, tetM and tetO), in frequencies between 1.10-7 and 9.10-7, was possible from bovine donor strains to human recipient strain, but not the other way around. This is, to our knowledge, the first report of in vitro conjugation of Ery and Tet resistance genes among GBS strains recovered from different hosts.


Assuntos
Animais , Bovinos , Humanos , Conjugação Genética , Técnicas de Transferência de Genes , Streptococcus agalactiae/genética , Antibacterianos/farmacologia , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana , Eritromicina/farmacologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/isolamento & purificação , Tetraciclina/farmacologia
8.
Braz. j. med. biol. res ; 46(9): 752-757, 19/set. 2013. graf
Artigo em Inglês | LILACS | ID: lil-686571

RESUMO

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.


Assuntos
Animais , Embrião de Galinha , Eletroporação/economia , Eletroporação/instrumentação , Eletroporação/métodos , Regulação da Expressão Gênica/genética , Técnicas de Transferência de Genes/instrumentação , Eletrodos , Desenho de Equipamento , Proteínas de Fluorescência Verde
9.
Braz. j. med. biol. res ; 46(9): 809-814, 19/set. 2013. graf
Artigo em Inglês | LILACS | ID: lil-686578

RESUMO

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.


Assuntos
Animais , Adenoviridae/metabolismo , Células da Medula Óssea/citologia , /metabolismo , Diferenciação Celular/fisiologia , /metabolismo , Osteogênese/fisiologia , Células-Tronco/citologia , Análise de Variância , Adenoviridae/genética , Fosfatase Alcalina/metabolismo , Sequência de Bases , Células da Medula Óssea/virologia , /genética , Centrifugação com Gradiente de Concentração , Ensaio de Imunoadsorção Enzimática , /genética , Técnicas de Transferência de Genes , Cabras , Vetores Genéticos/metabolismo , Imuno-Histoquímica , Osteoblastos/citologia , Cultura Primária de Células , Proteínas Recombinantes/genética , Células-Tronco/virologia
10.
Acta bioquím. clín. latinoam ; 47(2): 399-406, abr.-jun. 2013. ilus, graf, tab
Artigo em Espanhol | LILACS | ID: lil-694562

RESUMO

Las nanopartículas magnéticas (MNP) complejadas con vectores génicos pueden, en presencia de un campo magnético externo, amplificar sustancialmente la eficiencia de la transferencia génica. Esta técnica, denominada magnetofección, es de gran interés en el campo de la terapia génica. En este estudio se caracterizó la mejora de transferencia génica en células gliales B92 utilizando complejos constituidos por diferentes proporciones de MNP asociadas a dos vectores adenovirales, a saber: los complejos entre las MNP denominadas PEI-Mag2 asociadas al adenovector RAd-GFP que expresa la proteína fluorescente verde GFP o al adenovector RAd-DsRed que expresa la proteína fluorescente roja DsRed2. Se demostró que para ambos vectores, a medida que la relación MNP/partícula viral física (PVF) va aumentando, la amplificación de la transfección también aumenta hasta que se llega a una relación MNP/PVF a partir de la cual el factor de amplificación alcanza un plateau. Se determinó que para el complejo PEI-Mag2/RAd-GFP la relación a partir de la cual se alcanza el plateau es de aproximadamente 0,5 fg Fe/PVF mientras que para el complejo PEI-Mag2/RAd-DsRed, esta relación corresponde a aproximadamente 71 fg Fe/PVF. Se concluye que los dos complejos magnéticos estudiados representan promisorias herramientas para mejorar la eficiencia en la terapia génica en células cerebrales.


It is known that certain types of magnetic nanoparticles (MNPs) complexed to gene vectors can, in the presence of an external magnetic field, greatly enhance gene transfer into cells. This technique, called magnetofection, is of great relevance to gene therapy. In the present study the ability of MNP/adenovector complexes to enhance gene transfer to B92 glial cells was assessed. Two complexes were assessed, namely PEI-Mag2/RAd-GFP and PEI-Mag2/RAd-DsRed, which are constituted by the MNP PEI-Mag2 complexed to the adenovector RAd-GFP (expressing the green fluorescent protein GFP) and RAd-DsRed (expressing the red fluorescent protein DsRed2), respectively. It was shown that for both vectors, an increase in the ratio MNP/PVP (physical viral particle) is paralleled by an increase in transduction efficiency, up to a certain threshold value at which an efficiency plateau is reached. This threshold value was 0.5 fg Fe/PVP for the RAd-GFP complex and about 71 fg Fe/PVP for the RAd-DsRed complex. It can be concluded that both magnetic complexes assessed in this study represent promising tools for enhancing the efficiency of gene therapy in brain cells.


As nanopartículas magnéticas (MNPs) complexadas com vetores de genes podem, em presença de um campo magnético externo, aumentar consideravelmente a eficiência da transferência gênica. Esta técnica, chamada magnetofecção, é de grande relevância para a terapia genética. No presente estudo, foi caracterizada a melhoria de transferência de genes em células gliais B92 utilizando complexos constituídos por diferentes proporções de MNP associadas a dois vetores adenovirais, a saber: os complexos entre as MNP denominadas PEI-Mag2 associadas ao adenovetor RAd-GFP que expressa a proteína fluorescente verde GFP ou ao adenovetor RAd-DsRed que expressa a proteína fluorescente vermelha DsRed2. Foi demonstrado que para ambos os vetores, enquanto a relação MNP/partícula viral física (PVF) vai aumentando, a amplificação da transfecção também aumenta até que se chega a uma relação MNP/PVF a partir da qual o fator de amplificação alcança um limiar. Determinou-se que para o complexo PEI-Mag2/RAd-GFP a relação a partir da qual se atinge o limiar é de aproximadamente 0,5 fg Fe/PVF ao passo que para o complexo PEI-Mag2/RAd-DsRed, esta relação corresponde a aproximadamente 71 fg Fe/PVF. Conclui-se que os dois complexos magnéticos estudados representam promissoras ferramentas para melhorar a eficiência na terapia de genes em células cerebrais.


Assuntos
Animais , Ratos , Glioma/líquido cefalorraquidiano , Nanopartículas de Magnetita , Neoplasias/líquido cefalorraquidiano , Técnicas de Transferência de Genes , Sistema Nervoso , Neuroglia
11.
Biol. Res ; 46(4): 373-382, 2013. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-700400

RESUMO

Cyanobacteria display a large diversity of cellular forms ranging from unicellular to complex multicellular filaments or aggregates. Species in the group present a wide range of metabolic characteristics including the fixation of atmospheric nitrogen, resistance to extreme environments, production of hydrogen, secondary metabolites and exopolysaccharides. These characteristics led to the growing interest in cyanobacteria across the fields of ecology, evolution, cell biology and biotechnology. The number of available cyanobacterial genome sequences has increased considerably in recent years, with more than 140 fully sequenced genomes to date. Genetic engineering of cyanobacteria is widely applied to the model unicellular strains Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. However the establishment of transformation protocols in many other cyanobacterial strains is challenging. One obstacle to the development of these novel model organisms is that many species have doubling times of 48 h or more, much longer than the bacterial models E. coli or B. subtilis. Furthermore, cyanobacterial defense mechanisms against foreign DNA pose a physical and biochemical barrier to DNA insertion in most strains. Here we review the various barriers to DNA uptake in the context of lateral gene transfer among microbes and the various mechanisms for DNA acquisition within the prokaryotic domain. Understanding the cyanobacterial defense mechanisms is expected to assist in the development and establishment of novel transformation protocols that are specifically suitable for this group.


Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , DNA Bacteriano/genética , Sequência de Aminoácidos , Sequência de Bases , Técnicas de Transferência de Genes , Filogenia
12.
Braz. j. med. biol. res ; 45(12): 1141-1149, Dec. 2012. ilus, tab
Artigo em Inglês | LILACS | ID: lil-659634

RESUMO

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.


Assuntos
Feminino , Humanos , Neoplasias da Mama/metabolismo , Chlorella vulgaris/enzimologia , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ácidos Graxos Dessaturases/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Células Tumorais Cultivadas
14.
Braz. j. med. biol. res ; 44(12): 1194-1201, Dec. 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-606537

RESUMO

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such ‘gene doping’, exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.


Assuntos
Humanos , Desempenho Atlético , Doping nos Esportes/métodos , Técnicas de Transferência de Genes , Melhoramento Genético/métodos , Temas Bioéticos , Doping nos Esportes , Endorfinas/genética , Endorfinas/farmacologia , Eritropoetina/genética , Eritropoetina/farmacologia , Melhoramento Genético , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/farmacologia , Miostatina/genética , Miostatina/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia
15.
Braz. j. med. biol. res ; 44(11): 1097-1104, Nov. 2011. ilus
Artigo em Inglês | LILACS | ID: lil-604280

RESUMO

Gene therapy is an alternative treatment for genetic lung disease, especially monogenic disorders such as cystic fibrosis. Cystic fibrosis is a severe autosomal recessive disease affecting one in 2500 live births in the white population, caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). The disease is classically characterized by pancreatic enzyme insufficiency, an increased concentration of chloride in sweat, and varying severity of chronic obstructive lung disease. Currently, the greatest challenge for gene therapy is finding an ideal vector to deliver the transgene (CFTR) to the affected organ (lung). Adeno-associated virus is the most promising viral vector system for the treatment of respiratory disease because it has natural tropism for airway epithelial cells and does not cause any human disease. This review focuses on the basic properties of adeno-associated virus and its use as a vector for cystic fibrosis gene therapy.


Assuntos
Humanos , Adenovírus Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Adenovírus Humanos/classificação , Técnicas de Transferência de Genes
16.
Mem. Inst. Oswaldo Cruz ; 106(7): 785-793, Nov. 2011. ilus
Artigo em Inglês | LILACS | ID: lil-606640

RESUMO

Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.


Assuntos
Animais , Humanos , Camundongos , Técnicas de Transferência de Genes , Genoma Helmíntico/genética , Schistosoma japonicum/genética , Schistosoma mansoni/genética , Animais Geneticamente Modificados , Cromossomos/genética , Cromossomos/virologia , Elementos de DNA Transponíveis , DNA de Helmintos/genética , DNA Viral/genética , DNA Viral/isolamento & purificação , Vetores Genéticos , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/isolamento & purificação , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Interferência de RNA , Schistosoma japonicum/virologia , Schistosoma mansoni/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação
17.
Braz. j. microbiol ; 42(1): 330-339, Jan.-Mar. 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-571407

RESUMO

Streptococcus mutans (S. mutans), harboring biofilm formation, considered as a main aetiological factor of dental caries. Gtf genes play an important role in S. mutans biofilm formation. The purpose of this study was to investigate the effect of Lactobacillus acidophilus-derived biosurfactant on S. mutans biofilm formation and gtfB/C expression level (S. mutans standard strain ATCC35668 and isolated S. mutans strain (22) from dental plaque). The Lactobacillus acidophilus (L. acidophilus) DSM 20079 was selected as a probiotic strain to produce biosurfactant. The FTIR analysis of its biosurfactant showed that it appears to have a protein-like component. Due to the release of such biosurfactants, L. acidophilus was able to interfere in the adhesion and biofilm formation of the S. mutans to glass slide. It also could make streptococcal chains shorter. Using realtime RT-PCR quantitation method made it clear that gtfB and gtfC gene expression were decreased in the presence of L. acidophilus-derived biosurfactant fraction. Several properties of S. mutans cells (the surface properties, biofilm formation, adhesion ability and gene expression) were changed after L. acidophilus-derived biosurfactant treatment. It is also concluded that biosurfacant treatment can provide an optional way to control biofilm development. On the basis of our findings, we can suggest that the prepared biosurfactant may interfere with adhesion processes of S. mutans to teeth surfaces, provided additional evaluation produce satisfactory results.


Assuntos
Humanos , Biofilmes , Cárie Dentária , Expressão Gênica , Técnicas de Transferência de Genes , Técnicas In Vitro , Lactobacillus acidophilus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Streptococcus mutans/isolamento & purificação , Técnicas e Procedimentos Diagnósticos , Métodos , Fenótipo
18.
Biol. Res ; 44(3): 229-234, 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-608618

RESUMO

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.


Assuntos
Animais , Feminino , Camundongos , Dimetil Sulfóxido/farmacologia , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/administração & dosagem , Camundongos Transgênicos/genética , Testículo , Transgenes , Animais Geneticamente Modificados , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Lipossomos/farmacologia , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Testículo/efeitos dos fármacos , Testículo/patologia , Transfecção/métodos
19.
Electron. j. biotechnol ; 13(5): 9-10, Sept. 2010. ilus, tab
Artigo em Inglês | LILACS | ID: lil-591891

RESUMO

Although Agrobacterium-mediated transformation protocols for many economically important plant species have been well established, protocol for a number of flowering plants including Anthurium andraeanum remains challenging. In this study, we report success in generating transgenic Anthurium andraeanum cv Arizona using Agrobacterium GV3101 strain harboring a binary vector carrying gfp as a reporter gene. The possibility of facilitating the screening process for transgenic plants expressing functional proteins using gfp marker was explored. In order to realize high transformation efficiency, different explant sources including undifferentiated callus pieces and petioles were compared for their regeneration efficiency and susceptibility to Agrobacterium-mediated transformation. We also optimized the concentration of AS added to co-cultivation media. Genomic PCR revealed that 11 of the 22 resistant plantlets regenerated on selective medium were successfully transformed. Green fluorescence was observed using a fluorescence microscope in 7 of the 11 PCR-positive plants, indicating GFP was expressed stably in the transformed Anthurium andraeanum. The highest transformation efficiency obtained in this study was 1.71 percent (percentage of explants with transgenic shoots in total explants) when callus explants were used as starting material and 125 umol l-1 AS was added during the co-cultivation process.


Assuntos
Araceae/genética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Rhizobium/genética , DNA de Plantas/isolamento & purificação , Técnicas de Cocultura , Genes Reporter , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Plantas Geneticamente Modificadas/genética , Regeneração , Transformação Genética
20.
Arch. cardiol. Méx ; 79(2): 147-156, abr.-jun. 2009. tab
Artigo em Espanhol | LILACS | ID: lil-565717

RESUMO

Gene therapy as a therapeutic strategy for Heart Failure, is an area that within the last 10 years has experienced an important increase in research, becoming one of the most promising areas to obtain a successful therapy for heart failure due to the possibility of correcting the basic defects observed at the cellular level in this pathology. One of the first things to consider on the use of this therapy is the way to deliver the genetic material, Adenovirus, and Adeno-associated virus, have shown the best capabilities in the myocardium; the delivery by local means has shown best results when compared with peripheral administration. Multiple physiopathological mechanisms susceptible of modifying by gene therapy have been identified, including the regulation of Ca2+ fluxes during excitation-contraction coupling, altered intracellular signalling, and adrenergic system, blockade of apoptosis and angiogenesis. The objective of this review, is to made a recount about the status of the literature and analyze future perspectives for gene therapy.


Assuntos
Humanos , Terapia Genética , Insuficiência Cardíaca , Técnicas de Transferência de Genes , Vetores Genéticos , Terapia Genética/métodos
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