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1.
Electron. j. biotechnol ; 52: 85-92, July. 2021. graf, tab
Artigo em Inglês | LILACS | ID: biblio-1283600

RESUMO

BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.


Assuntos
Peptídeo Sintases/metabolismo , Reatores Biológicos/microbiologia , Escherichia coli , Temperatura , Biotecnologia , Carbono/metabolismo , Modelos Estatísticos , Eletroforese em Gel de Poliacrilamida , Bioengenharia , Concentração de Íons de Hidrogênio
2.
Arq. Inst. Biol ; 87: e0142020, 2020. tab
Artigo em Inglês | VETINDEX, LILACS | ID: biblio-1130108

RESUMO

The genus Streptomyces is associated with the ability to produce and excrete a variety of bioactive compounds, such as antibiotic, antifungal and antiviral. Biological active polyketide and peptide compounds with applications in medicine, agriculture and biochemical research are synthesized by PKS-I and NRPS genes. The evaluation of the presence of these genes associated with the biosynthesis of secondary metabolites in different phytopathogenic Streptomyces strains were performed using degenerated primers. The positive signal was observed in 58/63 Streptomyces strains for NRPS gene, 43/63 for PKS-I, and for PKS-II all the 63 strains showed positive signal of amplification. These strains also were tested with double layer agar-well technique against bacterial with clinical importance, and it was possible to observe the Streptomyces spp. strains were able to inhibit the growth of 14, 20, 13 and 3 isolates Gram-positive and Gram-negative bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 11775) respectively. The Streptomyces sp. strains IBSBF 2019 and IBSBF 2397 showed antibacterial activity against all four bacteria-target tested.(AU)


O gênero Streptomyces apresenta alta capacidade de produzir e excretar uma grande variedade de compostos biologicamente ativos, como antibióticos, antifúngicos e antivirais. Compostos biologicamente ativos de policetídeos e peptídeos com aplicações na medicina, agricultura e pesquisas bioquímicas são sintetizados pelos genes PKS-I e NRPS. A avaliação da presença desses genes associados à biossíntese de metabólitos secundários em diferentes linhagens de Streptomyces fitopatogênicas foi realizada através do uso de primers degenerados. O sinal positivo foi observado em 58/63 linhagens de Streptomyces para o gene NRPS, 43/63 para o gene PKS-I e, para o gene PKS-II, todas as 63 linhagens apesentaram o sinal positivo de amplificação. Essas linhagens também foram testadas através da técnica de dupla camada contra bactérias de importância clínica e foi possível observar que as linhagens de Streptomyces spp. foram capazes de inibir o crescimento de 14, 20, 13 e 3 isolados de bactérias Gram-positivas e Gram-negativas, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) e Escherichia coli (ATCC 11775), respectivamente. As linhagens de Streptomyces sp. ISBSF 2019 e 2397 apresentaram atividade antibacteriana contra todas as bactérias-alvo testadas.(AU)


Assuntos
Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Streptomyces/metabolismo , Bacillus cereus/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Antibacterianos/metabolismo , Peptídeo Sintases/genética , Streptomyces/genética , Amplificação de Genes , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Primers do DNA , Policetídeo Sintases/genética , Antibacterianos/farmacologia
3.
Biol. Res ; 51: 24, 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-950907

RESUMO

BACKGROUND: Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an enzyme required for de novo purine biosynthesis, is associated with and involved in tumorigenesis. This study aimed to evaluate the role of PAICS in human breast cancer, which remains the most frequently diagnosed cancer and the leading cause of cancer-related death among women in less developed countries. RESULTS: Lentivirus-based short hairpin RNA targeting PAICS specifically depleted its endogenous expression in ZR-75-30 and MDA-MB-231 breast cancer cells. Depletion of PAICS led to a significant decrease in cell viability and proliferation. To ascertain the mechanisms through which PAICS modulates cell proliferation, flow cytometry was performed, and it was confirmed that G1-S transition was blocked in ZR-75-30 cells through PAICS knockdown. This might have occurred partly through the suppression of Cyclin E and the upregulation of Cyclin D1, P21, and CDK4. Moreover, PAICS knockdown obviously promoted cell apoptosis in ZR-75-30 cells through the activation of PARP and caspase 3 and downregulation of Bcl-2 and Bcl-xl expression in ZR-75-30 cells. CONCLUSIONS: These findings demonstrate that PAICS plays an essential role in breast cancer proliferation in vitro, which provides a new opportunity for discovering and identifying novel effective treatment strategies.


Assuntos
Humanos , Feminino , Peptídeo Sintases/fisiologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Carboxiliases/biossíntese , Biomarcadores Tumorais/fisiologia , Proliferação de Células , Peptídeo Sintases/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Citometria de Fluxo
4.
Mem. Inst. Oswaldo Cruz ; 112(10): 655-663, Oct. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-894834

RESUMO

BACKGROUND The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes. OBJECTIVES To analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development. METHODS We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses. FINDINGS The mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence. MAIN CONCLUSIONS We have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development.


Assuntos
Humanos , Peptídeo Sintases , Vírus Sinciciais Respiratórios , Variação Genética , Proteínas Virais/genética , Genótipo , Filogenia , Testes Imunológicos
5.
Genet. mol. res. (Online) ; 5(3): 503-512, 2006. ilus, tab, graf
Artigo em Inglês | LILACS | ID: lil-441046

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is the major pathogen involved in nosocomial infections, leading to high rates of morbidity and mortality in hospitals worldwide. The methicillin resistance occurs due to the presence of an additional penicillin-binding protein, PBP2a, which has low affinity for b-lactam antibiotics. In the past few years, vancomycin has been the only antibiotic option for treatment of infections caused by multiresistant MRSA; however, reports of vancomycin-resistant strains have generated great concerns regarding the treatment to overcome these infections. In the present study, we report preliminary results regarding the humoral immune response generated in BALB/c mice by two different doses of naked DNA vaccine containing an internal region, comprising the serine-protease domain, of the PBP2a of MRSA. The immunization procedure consisted of four immunizations given intramuscularly within 15-day intervals. Blood was collect weekly and anti-PBP2a-specific antibodies were screened by ELISA. BALB/c mice immunized with DNA vaccine anti-PBP2a have shown higher antibody titers mainly after the fourth immunization, and intriguingly, no correlation between the humoral immune response and DNA dose was observed. Our results suggest that the DNA vaccine anti-PBP2a induced an immune response by production of specific antibodies anti-MRSA in a non-dose-dependent manner, and it could represent a new and valuable approach to produce specific antibodies for passive immunization to overcome MRSA infections.


Assuntos
Humanos , Animais , Camundongos , Anticorpos Antibacterianos/biossíntese , Resistência a Meticilina/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/imunologia , Peptídeo Sintases/imunologia , Vacinas Antiestafilocócicas/administração & dosagem , Staphylococcus aureus/imunologia , Vacinas de DNA/administração & dosagem , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Resistência a Meticilina/imunologia , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Vacinas Antiestafilocócicas/imunologia , Vacinas de DNA/imunologia
6.
Bol. malariol. salud ambient ; 46(1): 1-13, 2006.
Artigo em Espanhol | LILACS | ID: lil-503745

RESUMO

La teniasis es la infección parasitaria producida por el adulto de Taenia solium y T. saginata, mientras que la cisticercosis es causada por el estadío larvario (cisticerco) de estos ténidos enhospedadores intermediarios; el hombre puede de forma accidental adquirir la cisticercosis. El binomio teniasis/cisticercosis causa graves problemas de salud pública y económicos en las zonas endémicas de África, Asia, y Latinoamérica, además de otras áreas como consecuencia de los viajes y las migraciónes. La neurocisticercosis es la enfermedad parasitaria más importante del sistema nervioso central. El diagnóstico de la teniasis se logra generalmente mediante examenes coprológicos, mientras que el diagnóstico de la cisticercosis se lleva a cabo por métodos parasitológicos, por técnicas de imágenes y una amplia variedad de ensayos inmunológicos. Los métodos de diagnóstico inmunológico convencional presentan graves limitaciones, baja sensibilidad y especificidad, no estandarizados convenientemente y basados en la utilización como antígeno del siempre escaso material parasitario. Actualmente se están utilizando nuevas herramientas y técnicas que permiten un mejor diagnóstico de estas enfermedades, por ejemplo, anticuerpos monoclonales, antígenos recombinantes, péptidos sintéticos, PCR, cuya manipulación es de fácil estandarización e independientes de las fuentes del siempre preciado material parasitario.


Assuntos
Humanos , Masculino , Feminino , Cisticercose/diagnóstico , Doenças Parasitárias , Peptídeo Sintases , Teníase/diagnóstico , Parasitologia , Saúde Pública , Venezuela
7.
Cuad. Hosp. Clín ; 48(1): 87-96, 2003. ilus
Artigo em Espanhol | LILACS | ID: lil-344367

RESUMO

En las últimad décadas el óxido nítrico ha pasado de ser un contaminante ambiental a una mólecula implicada en múltiples funciones fisiológicas, actualmente se han multiplicado las investigaciones en torno a sus funciones. El óxido nítrico juega un papel imortnate en la regulación de una serie de procesos fisiológicos como ser: el tono vasomotor, la motilidad intestinal, actúa como un neurotransmisor central y periférico, desemeña un papel fundamental en la funciones del sistema inmune y la fisiologia del os procesos de agregación plaquetaria y leucocitaria. Sus acciones fisioatológiocas incluyen un rol determinante en la génesis del Shock séptico, en el daño tisular resultado de la inflamación, en el envejecimiento motocondrial, en el desarrollo dela rtrosis y la artritis reumatoidea, enfermedades degenerativas dels istema nervioso central y en el daño producido por la isquemia miocárdica y cerebral. Demostrando múltiples aplicaciones terapéuticas en el tratamiento de varias patologías.


Assuntos
Poluição Ambiental/efeitos adversos , Poluição Ambiental/estatística & dados numéricos , Poluição Ambiental/prevenção & controle , Óxido Nítrico/efeitos adversos , Peptídeo Sintases , Substâncias Tóxicas
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