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Chinese Journal of Biotechnology ; (12): 2915-2923, 2021.
Article in Chinese | WPRIM | ID: wpr-887853


Antimicrobial peptides are the most promising alternatives to antibiotics. However, the strategy of producing antimicrobial peptides by recombinant technology is complicated and expensive, which is not conducive to the large-scale production. Oxysterlin 1 is a novel type of cecropin antimicrobial peptide mainly targeting on Gram-negative bacteria and is of low cytotoxicity. In this study, a simple and cost-effective method was developed to produce Oxysterlin 1 in Escherichia coli. The Oxysterlin 1 gene was cloned into a plasmid containing elastin-like polypeptide (ELP) and protein splicing elements (intein) to construct the recombinant expression plasmid (pET-ELP-I-Oxysterlin 1). The recombinant protein was mainly expressed in soluble form in E. coli, and then the target peptide can be purified with a simple salting out method followed by pH changing. The final yield of Oxysterlin 1 was about 1.2 mg/L, and the subsequent antimicrobial experiment showed the expected antimicrobial activity. This study holds promise for large-scale production of antimicrobial peptides and the in-depth study of its antimicrobial mechanism.

Elastin , Escherichia coli/genetics , Inteins , Peptides/pharmacology , Pore Forming Cytotoxic Proteins , Recombinant Fusion Proteins/genetics
Chinese Journal of Biotechnology ; (12): 4277-4292, 2021.
Article in Chinese | WPRIM | ID: wpr-921505


The influence of different affinity tags on enzyme characteristics varies. The (S)-carbonyl reductase 2 (SCR2) from Candida parapsilosis can reduce 2-hydroxyacetophenone, which is a valuable prochiral ketones. Different affinity tags, i.e. his-tag, strep-tag and MBP-tag, were attached to the N terminus of SCR2. These tagged SCR2 enzymes, i.e. his6-SCR2, strep-SCR2 and MBP-SCR2, were heterologously expressed in Escherichia coli and purified to study their characteristics towards 2-hydroxyacetophenone reduction. Affinity tags did affect the characteristics of the recombinant SCR2 enzymes. Specifically, affinity tags affect the stability of recombinant SCR2 enzymes: 1) At pH 6.0, the remaining enzyme activities of his6-SCR2 and strep-SCR2 were only 95.2% and 90.0% of the untagged SCR2, while that of MBP-SCR2 was 1.2 times of the untagged SCR2 after incubating for 13 h at 30 °C. 2) The half-life of MBP-SCR2 at 50 °C was 26.6%-48.8% longer than those of strep-SCR2, his6-SCR2 and untagged SCR2. 3) The kcat of MBP-SCR2 was about 1.25-1.45 times of that of small affinity-tagged and untagged SCR2 after storing at -80 °C for 60 d. Structural informatics indicated that the α-helices at the C terminus of MBP-SCR2 contributed to the stability of the N terminus of fusion protein of SCR2. Data from circular dichroism showed that the MBP-tag has some influence on the secondary structure of SCR2, while melting temperature analysis demonstrated that the Tm of the recombinant MBP-SCR2 was about 5 °C higher than that of the untagged SCR2. This study obtained an efficient and stable recombinant SCR2, i.e. the MBP-SCR2. Moreover, this study could serve as a reference for other researchers to evaluate and select appropriate affinity tags for their research.

Alcohol Oxidoreductases , Escherichia coli/genetics , Recombinant Fusion Proteins/genetics
Chinese Journal of Biotechnology ; (12): 2868-2876, 2020.
Article in Chinese | WPRIM | ID: wpr-878536


Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.

Humans , Cloning, Molecular , Escherichia coli/genetics , Recombinant Fusion Proteins/genetics , Recombinant Proteins , Tissue Inhibitor of Metalloproteinase-2/genetics
Electron. j. biotechnol ; 28: 14-19, July. 2017. ilus, graf
Article in English | LILACS | ID: biblio-1015726


Background: Gain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy. Results: In this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 µg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis. Conclusion: These results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.

Urinary Bladder Neoplasms/metabolism , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/metabolism , Recombinant Fusion Proteins/genetics , Protamines/metabolism , Inclusion Bodies , Cloning, Molecular , Apoptosis , RNA, Small Interfering , Escherichia coli/metabolism , Receptor, Fibroblast Growth Factor, Type 3 , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/genetics , Flow Cytometry
Braz. j. microbiol ; 48(1): 151-158, Jan.-Mar. 2017. graf
Article in English | LILACS | ID: biblio-839342


Abstract Although infections with NonTuberculous Mycobacteria have become less common in AIDS patients, they are important opportunistic infections after surgical procedures, likely because they are ubiquitous and not efficiently killed by many commonly used disinfectants. In Venezuela there have recently been many non-tuberculous mycobacteria soft tissue infections after minor surgical procedures, some apparently related to the use of a commercial disinfectant based on a Quaternary Ammonium Compound. We studied the activity of this and other quaternary ammonium compounds on different non-tuberculous mycobacteria by transforming the mycobacteria with a dnaA-gfp fusion and then monitoring fluorescence to gauge the capacity of different quaternary ammonium compounds to inhibit bacterial growth. The minimum inhibitory concentration varied for the different quaternary ammonium compounds, but M. chelonae and M. abscessus were consistently more resistant than M. smegmatis, and M. terrae more resistant than M. bovis BCG.

Gene Expression , Green Fluorescent Proteins , Disinfectants/pharmacology , Quaternary Ammonium Compounds/pharmacology , Anti-Bacterial Agents/pharmacology , Nontuberculous Mycobacteria/drug effects , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Microbial Sensitivity Tests , Green Fluorescent Proteins/genetics , Dose-Response Relationship, Drug , Nontuberculous Mycobacteria/classification , Nontuberculous Mycobacteria/genetics
Braz. j. microbiol ; 47(2): 518-526, Apr.-June 2016. graf
Article in English | LILACS | ID: lil-780835


Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

Bacterial Proteins/genetics , Xanthomonas/genetics , Recombinant Fusion Proteins/genetics , Plant Diseases/microbiology , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Xanthomonas/metabolism , Xanthomonas/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Open Reading Frames , Citrus/microbiology , Genetic Vectors/genetics , Genetic Vectors/metabolism
ABCD arq. bras. cir. dig ; 28(1): 28-31, 2015. graf
Article in English | LILACS | ID: lil-742748


BACKGROUND: The surgical treatment of advanced megaesophagus has no consensus, being esophagectomy the more commonly used method. Since it has high morbimortality - inconvenient for benign disease -, in recent years an alternative has been introduced: the esophageal mucosal resection. AIM: To compare early and late results of the two techniques evaluating the operative time, length of ICU stay; postoperative hospitalization; total hospitalization; intra- and postoperative complication rates; mortality; and long-term results. METHODS: Were evaluated retrospectively 40 charts, 23 esophagectomies and 17 mucosectomies. In assessing postoperative results, interviews were conducted by using a specific questionnaire. RESULTS: Comparing the means of esophagectomy and mucosal resection, respectively, the data were: 1) surgical time - 310.2 min and 279.7 min (p> 0.05); 2) length of stay in ICU - 5 days and 2.53 days (p <0.05); 3) total time of hospitalization - 24.25 days and 20.76 days (p> 0.05); 4) length of hospital stay after surgery - 19.05 days and 14.94 days (p> 0.05); 5) presence of intraoperative complications - 65% and 18% (p <0.05); 6) the presence of postoperative complications - 65% and 35% (p> 0.05). In the assessment of late postoperative score (range 0-10) esophagectomy (n = 5) obtained 8.8 points and 8.8 points also got mucosal resection (n = 5). CONCLUSIONS: Esophageal mucosal resection proved to be good alternative for surgical treatment of megaesophagus. It was advantageous in the immediate postoperative period by presenting a lower average time in operation, the total hospitalization, ICU staying and complications rate. In the late postoperative period, the result was excellent and good in both operations. .

RACIONAL: O tratamento cirúrgico do megaesôfago avançado não é consensual sendo mais comumente usada a esofagectomia. Por tratar-se de técnica que apresenta maior morbimortalidade e empregada em doença benigna, foi introduzida nos últimos anos, como alternativa, a mucosectomia esofágica. OBJETIVO: Comparar os resultados imediatos e tardios das duas técnicas avaliando-se os tempos operatório, de internação em UTI, de internação do pós-operatório, de internação total; taxas de complicações intra-operatórias e pós-operatórias; taxa de mortalidade; e resultados a longo prazo. MÉTODOS: Foram avaliados 40 prontuários, retrospectivamente, sendo 23 esofagectomias e 17 mucosectomias. Na avaliação dos resultados pós-operatórios, foram realizadas entrevistas, mediante uso de questionário específico. RESULTADOS: Comparando-se as médias da esofagectomia e mucosectomia, respectivamente, os dados foram: 1) tempo cirúrgico - 310,2 min e 279,7 min (p>0,05); 2) tempo de internação em UTI - 5 dias e 2,53 dias (p<0,05); 3) tempo de internação total - 24,25 dias e 20,76 dias (p>0,05); 4) tempo de internação após a operação - 19,05 dias e 14,94 dias (p>0,05); 5) presença de complicações intra-operatórias - 65% e 18% (p<0,05); 6) presença de complicações pós-operatórias imediatas - 65% e 35% (p>0,05). Na avaliação do escore pós-operatório tardio (escala 0-10) a esofagectomia (n=5) obteve 8,8 pontos e também 8,8 pontos obteve a mucosectomia (n=5). CONCLUSÕES: A mucosectomia esofágica mostrou-se boa alternativa no tratamento cirúrgico do megaesôfago avançado. Foi vantajosa no pós-operatório imediato por apresentar menor média de tempo na operação, na internação total, na UTI e no índice de complicações. No pós-operatório tardio, o resultado foi excelente e bom nas duas operações. .

Animals , Male , Mice , Energy Metabolism , /metabolism , Insulin/metabolism , Myocardial Contraction/physiology , Myocytes, Cardiac/metabolism , Signal Transduction/physiology , Hypoxia/metabolism , Cells, Cultured , Clathrin/metabolism , /genetics , Mice, Transgenic , Myocytes, Cardiac/cytology , Protein Transport/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sarcolemma/metabolism , Sarcolemma/ultrastructure
Salud pública Méx ; 56(6): 660-665, nov.-dic. 2014.
Article in Spanish | LILACS | ID: lil-733346


Este artículo fue concebido para analizar la función de la Escuela de Salud Pública de México (ESPM) desde el año 2000 hasta el presente. Uno de sus puntos centrales es el análisis del proceso de reorientación de la labor educativa de la escuela con la finalidad de responder a los retos en materia de salud y educación surgidos a finales del siglo XX. Para exponer cómo ha evolucionado dicho proceso, retomamos tres ejes rectores que caracterizan la labor de la escuela en la actualidad: el cambio de modelo pedagógico, la incorporación de las tecnologías de la información y las comunicaciones, y la profesionalización de la docencia. Con la exposición de este tema, y a través del contraste entre el pasado y el presente, buscamos completar la historia de trabajo ininterrumpido de la Escuela durante sus 92 años de existencia, que ha trascendido los confines del país.

This article was conceived to analyze the work of the School of Public Health of Mexico (ESPM for is acronym in Spanish) from the year 2000 to the present day. One of the highlights that we will examine is the reorientation of the educational work of the school in order to meet the challenges in health and education that emerged during the end of the twentieth century. In order to explain the evolution of this process, we will describe the three main guiding principles that characterize the present work of the school: the pedagogical model's change, the incorporation of the information and communication technologies, and the professionalization in teaching. The purpose of this work is to define those guiding principles, and to expose, through the contrast between past and present, the complete history of uninterrupted work of the School of Public Health of Mexico during its ninety-two years of existence, that has gone beyond the boundaries of the country.

Animals , Female , Humans , Mice , Cysteine Endopeptidases/metabolism , Mengovirus/enzymology , Viral Proteins , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Antibodies, Viral/metabolism , Capsid/metabolism , Chlorides/pharmacology , Cysteine Endopeptidases/genetics , Enzyme Inhibitors/pharmacology , Ethylmaleimide/pharmacology , HeLa Cells , Iodoacetamide/pharmacology , Leucine/analogs & derivatives , Leucine/pharmacology , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/metabolism , Protein Precursors/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Zinc Compounds/pharmacology
Rev. Esc. Enferm. USP ; 48(spe): 74-79, 08/2014. tab
Article in English | LILACS, BDENF | ID: lil-731303


Affective, cognitive and behavioral components affect nurses´ attitudes to include families in the care processes. The purpose of this study was to investigate the attitudes of nurses about the importance of including families in nursing care. Data collection was performed in pediatric and maternal-child unit of a Brazilian university hospital. A sample of 50 nurses completed the Portuguese version of the instrument Families’Importance in Nursing Care-Nurses’ Attitudes (FINC-NA). The results indicated that nurses have supportive attitudes regarding families participation in nursing care. Attitudes of lower support for involving families in nursing care were found among nurses with older age, more time in the profession and who had no previous contact with contents related to Family Nursing. The application of the instrument in other contexts of assistance may help to illuminate important aspects of the challenges to implementing a family-centered approach in clinical practice.


El propósito de este estudio fue identificar las actitudes de los enfermeros sobre la importancia de incluir a las familias en el cuidado de enfermería. La recolección de datos se llevó a cabo en las unidades de pediatría y materno-infantil de un hospital universitario brasileño. Una muestra de 50 enfermeras completó la versión en portugués del el instrumento Families’ Importance in Nursing Care-Nurses’ Attitudes (FINC-NA). Los resultados indicaron las puntuaciones más altas en dimensiones indicativas de actitudes de apoyo a la participación de las familias en el cuidado. Enfermeras con más tiempo en la profesión y que no tenían conocimiento previo de enfermería de familia tuvieron puntuaciones que indican actitudes de menor apoyo para involucrar a las familias en el cuidado de enfermería. La aplicación de este instrumento en otro tipo de asistencia contextos puede ayudar a iluminar aspectos importantes de los desafíos para la implementación de un enfoque centrado en la familia, en la práctica clínica.

Objetivo Identificar as atitudes dos enfermeiros sobre a importância de incluir as famílias nos cuidados de enfermagem. Método Estudo de abordagem quantitativa descritiva, cuja coleta de dados foi realizada em unidades de pediatria e materno-infantil de um hospital universitário brasileiro. Uma amostra de 50 enfermeiros completou a versão em português da escala Families Importance in Nursing Care-Nurses Attitudes (FINC-NA). Resultados Indicaram escores mais elevados em dimensões indicativas de atitudes de apoio sobre a participação das famílias no cuidado de enfermagem. Enfermeiros com mais tempo na profissão e que não tiveram conhecimento prévio de enfermagem da família apresentaram escores indicativos de atitudes de menor apoio para envolver as famílias no cuidado de enfermagem. Conclusão A aplicação desse instrumento em outros contextos de assistência poderá contribuir para iluminar importantes aspectos relacionados aos desafios para a implementação de uma abordagem centrada na família na prática clínica e subsidiar o desenvolvimento de pesquisas mais amplas.

Azoarcus/metabolism , Coenzyme A Ligases/genetics , Phenylacetates/metabolism , Aerobiosis , Azoarcus/genetics , Coenzyme A Ligases/metabolism , Immunoblotting , Mutation , Recombinant Fusion Proteins/genetics , Sequence Analysis, DNA , beta-Galactosidase/genetics
Rev. Esc. Enferm. USP ; 48(spe): 16-22, 08/2014. tab
Article in English | LILACS, BDENF | ID: lil-731304


Objective To analyze the determinants of emergency contraception non-use among women in unplanned and ambivalent pregnancies. Method Cross-sectional study with a probabilistic sample of 366 pregnant women from 12 primary health care units in the city of São Paulo, Brazil. A multinomial logistic regression was performed, comparing three groups: women who used emergency contraception to prevent ongoing pregnancies (reference); women who made no use of emergency contraception, but used other contraceptive methods; and women who made no use of any contraceptive methods at all. Results Cohabitation with a partner was the common determinant of emergency contraception non-use. No pregnancy risk awareness, ambivalent pregnancies and no previous use of emergency contraception also contributed to emergency contraception non-use. Conclusion Apart from what is pointed out in the literature, knowledge of emergency contraception and the fertile period were not associated to its use. .

Objetivo Analizar los determinantes del no uso de la anticoncepción de emergencia entre las mujeres con embarazo no planeado o ambivalente. Método Estudio transversal en una muestra probabilística de 366 mujeres embarazadas de 12 Unidades Básicas de Salud de São Paulo. Mediante regresión logística multinomial, se comparó tres grupos de mujeres: aquellas que usaron la anticoncepción de emergencia para prevenir el embarazo en curso (referencia), aquellas que usaron algún método anticonceptivo, pero no la anticoncepción de emergência; y aquellas que no usaron ningún método. Resultados Los hallazgos mostraron que vivir com la pareja fue el determinante común del no uso de la anticoncepción de emergencia. No tener conciencia del riesgo de embarazo, estar en un embarazo ambivalente y nunca tener utilizado la anticoncepción de emergencia también fueron associados con su no uso para prevenir el embarazo en curso. Conclusión Contrariamente a lo que reporta la literatura, el conocimiento de la anticoncepción de emergencia y el período fértil no mostró asociación con el no uso. .

Objetivo Analisar os determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente. Método Estudo transversal com amostra probabilística de 366 gestantes de 12 Unidades Básicas de Saúde da cidade de São Paulo. Por meio de regressão logística multinomial, compararam-se três grupos de mulheres: as que usaram anticoncepção de emergência para prevenir a gravidez em curso (referência); as que usaram algum método contraceptivo, mas não anticoncepção de emergência; e as que não usaram nenhum método. Resultados Os achados mostraram que morar com o parceiro foi o determinante comum do não uso da anticoncepção de emergência. Não ter consciência do risco de engravidar, estar em uma gravidez ambivalente e nunca ter usado anticoncepção de emergência também foram associados ao seu não uso para prevenir a gravidez em curso. Conclusão Diferentemente do que relata a literatura, o conhecimento sobre anticoncepção de emergência e sobre o período fértil não mostrou qualquer associação ao não uso. .

DNA-Binding Proteins , Escherichia coli/genetics , Protein Interaction Mapping/methods , Two-Hybrid System Techniques , Bacteriophage lambda/genetics , DNA, Bacterial/genetics , DNA-Directed RNA Polymerases/biosynthesis , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/physiology , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Escherichia coli Proteins/physiology , Escherichia coli/enzymology , Genes, Reporter/genetics , Phosphorylation , Plasmids/biosynthesis , Plasmids/genetics , Promoter Regions, Genetic/genetics , RNA, Bacterial/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiology , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Repressor Proteins/physiology , Transcription, Genetic/genetics , Transcription, Genetic/physiology , Viral Proteins/biosynthesis , Viral Proteins/genetics , Viral Proteins/physiology , Viral Regulatory and Accessory Proteins , beta-Galactosidase/biosynthesis , beta-Lactamases/biosynthesis
Biomédica (Bogotá) ; 34(supl.1): 41-49, abr. 2014. ilus, tab
Article in English | LILACS | ID: lil-712420


Introduction: Aminoglycosides like streptomycin are well-known for binding at specific regions of ribosome RNA and then acting as translation inhibitors. Nowadays, several pathogens have been detected to acquire an undefined strategy involving mutation at non structural ribosome genes like those acting as RNA methylases. rsmG is one of those genes which encodes an AdoMet-dependent methyltransferase responsible for the synthesis of m 7 G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m 7 G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning. Objectives: After taking into account genetic information indicating that some clinical isolates of human pathogens show streptomycin resistance associated with mutations at rsmG , we decided to explore new hot spots for mutation capable of impairing the RsmG in vivo function and of promoting low-level streptomycin resistance. Materials and methods: To gain insights into the molecular and genetic mechanism of acquiring this aminoglycoside resistance phenotype and the emergence of high-level streptomycin resistance in rsmG mutants, we mutated Escherichia coli rsmG and also performed a genotyping study on rpsL from several isolates showing the ability to grow at higher streptomycin concentrations than parental strains. Results: We found that the mutations at rpsL were preferentially present in these mutants, and we observed a clear synergy between rsmG and rpsL genes to induce streptomycin resistance. Conclusion: We contribute to understand a common mechanism that is probably transferable to other ribosome RNA methylase genes responsible for modifications at central sites for ribosome function.

Introducción. Los aminoglucósidos son moléculas antibióticas capaces de inhibir la síntesis de proteínas bacterianas tras su unión al ribosoma procariota. La resistencia a aminoglucósidos está clásicamente asociada a mutaciones en genes estructurales del ribosoma bacteriano; sin embargo, varios estudios recientes han demostrado, de forma recurrente, la presencia de un nuevo mecanismo dependiente de mutación que no involucra genes estructurales. El gen rsmG es uno de ellos y se caracteriza por codificar una metiltransferasa que sintetiza el nucleósido m 7 G527 localizado en el loop 530 del ribosoma bacteriano, este último caracterizado como sitio preferencial al cual se une la estreptomicina. Objetivo. Partiendo de las recientes asociaciones clínicas entre las mutaciones en el gen rsmG y la resistencia a estreptomicina, este estudio se propuso la caracterización de nuevos puntos calientes de mutación en este gen que puedan causar resistencia a estreptomicina usando Escherichia coli como modelo de estudio. Materiales y métodos. Se indagó sobre el mecanismo genético y molecular por el cual se adquiere la resistencia a estreptomicina y su transición a la resistencia a altas dosis mediante mutagénesis dirigida del gen rsmG y genotipificación del gen rpsL . Resultados. Se encontró que la mutación N39A en rsmG inactiva la proteína y se reportó un nuevo conjunto de mutaciones en rpsL que confieren resistencia a altas dosis de estreptomicina. Conclusiones. Aunque los mecanismos genéticos subyacentes permanecen sin esclarecer, se concluyó que dichos patrones secuenciales de mutación podrían tener lugar en otros genes modificadores del ARN bacteriano debido a la conservación evolutiva y al papel crítico que juegan tales modificaciones en la síntesis de proteínas.

Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Escherichia coli Proteins/genetics , Mutation, Missense , Methyltransferases/genetics , Point Mutation , RNA Processing, Post-Transcriptional/genetics , RNA, Bacterial/metabolism , /metabolism , Streptomycin/pharmacology , Amino Acid Sequence , Binding Sites/genetics , Catalytic Domain/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Methylation , Models, Molecular , Molecular Sequence Data , Methyltransferases/chemistry , Methyltransferases/metabolism , Phylogeny , Protein Conformation , RNA, Bacterial/genetics , /genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , S-Adenosylmethionine/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Deletion , Sequence Homology, Amino Acid
Article in English | WPRIM | ID: wpr-191848


Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

Animals , Female , Mice , Antibodies, Viral/blood , Chickens , Chimera/genetics , Coronavirus Infections/prevention & control , Immunity, Innate , Infectious bronchitis virus/genetics , Influenza A Virus, H5N1 Subtype/genetics , Injections, Intramuscular/veterinary , Mice, Inbred BALB C , Neuraminidase/genetics , Poultry Diseases/prevention & control , Recombinant Fusion Proteins/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Virus-Like Particle/administration & dosage , Viral Proteins/genetics
Article in English | WPRIM | ID: wpr-103945


Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10(9)TCID50/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-gamma and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.

Animals , Mice , Adenoviridae/genetics , Antibodies, Fungal/blood , Antigens, Fungal/genetics , Drug Carriers , Fungal Proteins/genetics , Fungal Vaccines/administration & dosage , Immunoglobulin G/blood , Interferon-gamma/blood , Interleukin-4/blood , Mice, Inbred BALB C , Neospora/genetics , Recombinant Fusion Proteins/genetics , Vaccines, Synthetic/administration & dosage
Indian J Biochem Biophys ; 2011 June; 48(3): 141-147
Article in English | IMSEAR | ID: sea-135311


This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). The fusion proteins were constructed in the form of chimeric toxins, aiming to obtain bifunctional analgesic and antitumor activity. The fusion proteins consisted of luteinizing hormone-releasing hormone (LHRH), three different types of flexible linkers (L1, Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met; L2, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser; L3, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser), and BmKAGAP. The genes coding three fusion proteins were cloned and expressed in E. coli in soluble form. Following two successive column chromatographic separations, purified fusion proteins were obtained. These fusion proteins exhibited analgesic activity in mice and were cytotoxic to a hepatocellular carcinoma cell line Hep3B.

Analgesics/administration & dosage , Analgesics/isolation & purification , Analgesics/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Liver Neoplasms, Experimental/drug therapy , Mice , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacology , Scorpion Venoms/administration & dosage , Scorpion Venoms/biosynthesis , Scorpion Venoms/chemistry , Scorpion Venoms/isolation & purification , Scorpion Venoms/pharmacology , Scorpions
Indian J Hum Genet ; 2009 Sept; 15(3): 98-102
Article in English | IMSEAR | ID: sea-138880


Systemic lupus erythematosus (SLE) is an autoimmune disorder affecting multiple organ systems. It is characterized by the presence of autoantibodies reactive against various self-antigens. Susceptibility to SLE is found to be associated with many major histocompatibility complex (MHC) and non-MHC genes, one of which is APO-1/Fas gene, which is present on chromosome 10 in humans. The APO-1/Fas promoter contains consensus sequences for binding of several transcription factors that affect the intensity of Fas expression in cells. The mutations in the APO-1/Fas promoter are associated with risk and severity in various autoimmune diseases and other malignancies. The APO-1/Fas receptor is expressed by many cell types. Two forms of APO-1/Fas protein that are involved in regulation of apoptosis have been identified. Fas receptor-mediated apoptosis plays a physiological and pathological role in killing of infected cell targets. In this review, we have focused on APO-1/Fas gene structure, promoter variants and its association with SLE and other autoimmune diseases. Functional aspects of Fas receptor in apoptosis are also discussed.

Adolescent , Adult , fas Receptor/genetics , Apoptosis/genetics , Autoimmune Diseases/genetics , Chromosomes, Human, Pair 10/genetics , Female , Humans , Lupus Erythematosus, Systemic/epidemiology , Lupus Erythematosus, Systemic/genetics , Recombinant Fusion Proteins/genetics
Article in English | IMSEAR | ID: sea-135861


Background & objectives: An inability or decreased ability of spermatozoa to bind to the zona pellucida (ZP), an extracellular glycoproteinaceous matrix surrounding egg, is one of the plausible causes of idiopathic infertility. It will be clinically useful to distinguish this condition from other causes of infertility. An assay system, investigating binding of human sperm with ZP glycoprotein may prove useful in this regard. We attempted to develop a simple assay system to analyse the binding of capacitated human spermatozoa to human zona pellucida glycoprotein-3 (ZP3) using baculovirus-expressed recombinant human ZP3 coated beads. Methods: Recombinant baculovirus-expressed ZP3 was purified, labelled with biotin and coated on streptavidin sepharose beads. An in vitro assay system was optimized to study binding of capacitated human sperm to ZP3 coated beads. Results: A higher percentage of baculovirus-expressed recombinant human ZP3 coated beads showed significant (P<0.05) binding of capacitated human sperm as compared to beads coated with fetuin. An inhibition in the binding of sperm to ZP3 coated beads was observed in presence of cold recombinant human ZP3. Further, prior incubation of ZP3 coated beads with monoclonal antibodies (MAbs) against ZP3 but not against ZP2 resulted in the decrease in number of sperm bound to bead. Interpretation & conclusion: An in vitro assay system to study the binding of human sperm to ZP3- primary sperm receptor was established, which may be useful to determine the functional competence of spermatozoa.

Egg Proteins/genetics , Egg Proteins/metabolism , Humans , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Protein Binding , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sperm Capacitation/physiology , Spermatozoa/cytology , Spermatozoa/metabolism , Zona Pellucida/metabolism , alpha-Fetoproteins/metabolism
Indian J Biochem Biophys ; 2008 Dec; 45(6): 374-8
Article in English | IMSEAR | ID: sea-27413


The expression of glutathione-S-transferase (GST) fusion protein is extensively utilized in the study of protein-protein interactions. In the commonly used purification method, the overexpressed GST fusion protein is bound to the glutathione (GSH)-coupled resins via affinity chromatography, and then eluted by an excessive quantity of reduced GSH. However, this technique has certain limitations, such as low product purity, retention of GSH in the sample, as well as relatively high cost. To overcome these limitations, in this study, elution buffer containing 2% formic acid was utilized rather than GSH to elute the GST-fusion protein, and thereafter the acidic samples were neutralized using collecting buffer. By using this method, highly purified GST-cyclophilin A (CypA) fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities. Moreover, the procedure is also cost-effective, due to the low cost of formic acid as compared with GSH.

Animals , Cloning, Molecular , Cyclophilin A/genetics , Escherichia coli/enzymology , Formates/chemistry , Glutathione Transferase/genetics , Molecular Chaperones/genetics , Protein Binding , Protein Folding , Rats , Recombinant Fusion Proteins/genetics
Indian J Exp Biol ; 2008 Apr; 46(4): 207-11
Article in English | IMSEAR | ID: sea-62548


Tissue specific expression of transgenes in plant species has several advantages over constitutive expression. Identification of ovule specific promoters would be useful in genetic engineering of plants with a variety of desirable traits such as genetically engineered parthenocarpy, female sterile plants or seedless fruits. Relative inaccessibility and difficulty in harvesting adequate amounts of tissue at known developmental stages has impeded the progress in cloning of promoters involved in ovule development. In the present study an ovule specific promoter was cloned from Arabidopsis AGL11 gene and used to express GUS (beta-glucuronidase) gene in transgenic Arabidopsis. Histochemical staining of GUS appeared in the center of young ovary (ovules), but no detectable GUS activity was observed in vegetative plant tissues, sepals, petals and androecium. AGL11 gene promoter can be useful to modify the developmental path of plants by expressing either plant hormones or lethal genes for agronomic purpose.

AGAMOUS Protein, Arabidopsis/genetics , Arabidopsis/enzymology , Base Sequence , Cloning, Molecular , Conserved Sequence , Flowers/enzymology , Gene Expression Regulation, Plant , Glucuronidase/genetics , Homeodomain Proteins/genetics , Molecular Sequence Data , Plant Proteins/genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Recombinant Fusion Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transgenes
Genet. mol. res. (Online) ; 7(2): 314-325, 2008. tab, ilus
Article in English | LILACS | ID: lil-641008


We explored the potential of fusion of hepatic locus control region 1 (HCR-1) with HCR-2 to express B-domain-deleted human factor VIII (FVIII) in four cell lines. B-domain-deleted human FVIII expression was controlled by HCR-1/HCR-2, followed by liver specific and ubiquitous promoters. Chimera enhancer HCR-1/HCR-2, followed by cytomegalovirus (CMV) promoter, gave 2-fold more FVIII expression in all cell lines (105.6 ± 2.8 for Hek-293, 68.8 ± 3.8 for HepG2, 34.8 ± 1.3 for CHO, and 27.2 ± 1.6 ng-mL-1-106 cells-1 for L.N.) when compared to the vector with CMV alone (54.8 ± 3.3 for Hek-293, 32.4 ± 1.2 for HepG2, 18.6 ± 1.1 for CHO, and 10.1 ± 1.7 ng-mL-1-106 cells-1 for L.N.). Elongation factor 1-α gene and human CMV promoters were more efficient than the promoters from the human α-1-antitrypsin gene, and fviii was less efficient in hepatic cell lines. HCR-1/HCR-2, followed by strong promoters, increases FVIII expression in vitro. Our results underscore the importance of cis sequences for enhancing in vitro FVIII expression; this may be helpful for designing new strategies to improve heterologous expression systems.

Humans , Animals , Enhancer Elements, Genetic/genetics , Factor VIII/genetics , Promoter Regions, Genetic/genetics , Genetic Vectors/genetics , Cell Line , Cell Line, Tumor , CHO Cells , Cricetinae , Cricetulus , Cytomegalovirus/genetics , Factor VIII/metabolism , Immunohistochemistry , Microscopy, Fluorescence , Plasmids , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction
Genet. mol. res. (Online) ; 7(2): 305-313, 2008. ilus
Article in English | LILACS | ID: lil-641009


Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Animals , Antigens, Protozoan/genetics , Membrane Proteins/genetics , Protozoan Proteins/genetics , Toxoplasma/genetics , Antigens, Protozoan/immunology , Antigens, Protozoan/metabolism , Blotting, Western , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Oligonucleotide Array Sequence Analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Membrane Proteins/immunology , Membrane Proteins/metabolism , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Toxoplasma/immunology , Toxoplasma/metabolism