45 research outputs found
Extração, purificação e caracterização físico-química da proteína verde fluorescente recombinante (GFPuv) expressa em Escherichia coli
O aumento do uso da proteína verde fluorescente (GFPuv) como ferramenta de pesquisas biotecnológicas requer um estudo mais cuidadoso das propriedades bioquímicas e físicas da molécula de GFPuv. Este trabalho teve como objetivo a aplicação de métodos físicos e químicos para o isolamento, a extração da GFPuv de células de Escherichia coli DH5-±, purificação da proteína, e o estudo da estabilidade desta em diferentes valores de pH. Células de E. coli expressando GFPuv foram submetidas a quatro ciclos sucessivos de congelamento e descongelamento intercalados por sonicação (CDS), para promover a permeação seletiva da GFPuv. Os permeados foram submetidos à extração por partição em três fases (TPP) e posterior purificação por eluição da proteína em coluna cromatográfica de interação hidrofóbica (HIC).Obteve-se rendimento semelhante em GFPuv no 1º ciclo de permeação seletiva (CDS) e por extração (TPP) associada à purificação (HIC) para os quais impurezas não foram visualizadas por eletroforese. As estruturas moleculares da GFPuv extraída e purificada mostraram-se inalteradas em valores de pH entre 6,0 e 9,8, e foram confirmadas nos espectros de emissão e de excitação.The recombinant green fluorescent protein (GFPuv) has been used as a marker in several research fields. The purpose of the present work was to evaluate the influence of the selective physical permeation procedure applied to the cells of Escherichia coli for the extraction of GFPuv in relation to the chemical procedures of extraction and purification. Transformed cells (0.92-1.44 mg/mL) of E. coli DH5-a expressing GFPuv were submitted to four cycles (1º, 2º, 3º, 4º) of freezing (-20 ºC/ 0.83 ºC/ min/thawing interlaid by sonication (3 pulses/6 s/25 vibrations). The intracellular permeate with GFPuv in buffer solution (Tris-HCl 25 mM pH 8.0 + b-mercaptoethanol (1 mM) + PMSF (0.1 mM)) was submitted to the three-phase partitioning (TPP) method and subsequent purification through hydrophobic interaction chromatography column (HIC). The results showed that the first selective permeation cycle applied to the cells was more efficient in the release of the protein from the cell fragments without subsequent extraction and purification in relation to the second, third and fourth permeation procedures. The permeated, extracted and purified GFPuv showed similarity to the standard GFPuv characteristics of fluorescence, stability and solubility
Thermal processing validation of a canned vegetal protein product
A validação de processos nas indústrias alimentícia e farmacêutica é uma das principais ferramentas da garantia de qualidade, tornando os produtos seguros, eficazes e confiáveis.Realizou-se a validação física e biológica da esterilização terminal de um produto protéico constituído de proteína texturizada de soja, proteína de trigo (glúten), gordura vegetal hidrogenada e condimentos, envasado em latas metálicas de 850 gramas.A validação física do autoclave foi executada em três ciclos na câmara com 12 sensores de temperatura. Confirmou-se para os pontos geometricamente distribuídos no interior do autoclave que a diferença máxima de temperatura entre um ponto e outro foi de 1,0 ºC em relação à temperatura média da câmara (121 ºC). Nos ciclos realizados com carga máxima, o menor valor F121ºC10ºC (64,86 minutos) garantiu S.A.L.(Sterility Assurance Level ou nível de garantia de esterilidade) de pelo menos 10-12 em relação ao indicador biológico de Bacillus stearothermophilus ATCC 7953 (valor D121ºC de 3,68 minutos e população média de 1,00 x 10(6) esporos por fita).Para todas as operações de esterilização a presença de esporos sobreviventes no indicador biológico não foi detectada.Apesar de não existir margem de segurança definida para esterilização de alimentos enlatados, a validação periódica de um autoclave proporcionou maior confiabilidade na avaliação da esterilidade, que o simples teste de incubação por amostragem de produtos acabados, recomendado pelo Ministério da Saúde do Brasil.Process validation in the Food and Pharmaceutics Industries is one of the most important tools meant to guarantee quality, making safe and trustworthy products. Physical and biological validations were performed in the terminal sterilization of a canned vegetal protein product. Physical validation of the autoclave has been proceeded in three empty chamber cycles and 12 temperature sensors. It has been confirmed to the geometrically distributed spots inside the autoclave that the maximum temperature difference between one and another spot has been 1.0 ºC concerning the average chamber temperature (122 ºC). Sterility Assurance Level (S.A.L.) of at least 10-12, in relation to biological indicator of B. stearothermophilus with D121ºC value of 3.68 minutes and average spore population of 1,00 x 10(6) per strip, was obtained. Despite the absence of a margin of safety for canned products sterilization, periodical validation of an autoclave has provided greater trust on the sterility evaluation than the simple incubation test of some samples, recommended by the Brazilian Ministry of Health
Expression of green fluorescent protein (GFPuv) in Escherichia coli DH5-a, under different growth conditions
The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by a fractional factorial (24-1) design at two levels: (i) the effect of storing (24-48 h) the seeded broth at 4oC prior to incubation at 37oC; (ii) the effect of agitation speed (100-200 rpm); (iii) the final concentration (0.05-0.5 mM) of IPTG (isopropyl–β-D-thiogalactopyranoside); and (iv) the addition of IPTG at set cell densities (OD660 0.01-0.8). GFPuv was extracted from cells by the three phase partitioning method (TPP) and further purified with a methyl HIC column. The cultures grown at 37oC/24 h provided the highest yields of GFPuv under the conditions: (i) pre-storage at 4oC/24 h; (ii) agitation speed at 100 rpm; (iii) 0.5 mM IPTG; (iv) IPTG addition at OD660=~0.01. On the other hand, at 37oC/ 8 h, GFPuv expression was dependent upon agitation of broth cultures at 200 rpm and the IPTG addition at the beginning of the growth exponential phase.
Key Words: Green fluorescent protein (GFPuv), Escherichia coli DH5-α, growth kinetic parameters, expressed GFPuv kinetic parameters, three phase partitioning extraction (TPP).
African Journal of Biotechnology Vol.3(1) 2004: 105-11
Tratamento químico de hortaliças poluídas
The effectiveness of chemicals as desinfectants for reducing the parasite contamination of lettuce leaves (Lactuca sativa, L.) was investigated. Potassium permanganate at 1: 5000 and 1: 10000 was employed, while sodium hypochlorite compound was used in con centrations of 10, 15, 20, 25, 30 and 40 ppm. In all experiments the contact time was 5,10, 20 and 40 minutes. The expousure of lettuce leaves to sodium hypochlorite 40 ppm of free cloro for 10 minutes was found to be highly effective.Das amostras de hortaliças em folhas coletadas no período de abril a agosto de 1981, no Entreposto Comercial da Cidade de São Paulo, SP (Brasil), 75% e 57% apresentaram contagens de coliformes fecais e de Escherichia coli, por grama de produto, superiores a 2 x 10² respectivamente; e em 85% constatou-se a presença de parasitas protozoários ou/e helmintos. O tratamento químico das folhas de alface (Lactuca sativa), com solução de hipoclorito de sódio, por um período de exposição de 10 min à concentração de 40 ppm de cloro livre, mostrou-se eficaz do ponto de vista parasitológico
Determination of decimal reduction time (D-value) of chemical agents used in hospital disinfection
Stability of furosemide and aminophylline in parenteral solutions
Parenteral solutions (PS) are one of the most commonly used drug delivery vehicles. Interactions among the drug, components in the drug's formulation, and the PS can result in the formation of inactive complexes that limit efficacy or increase side effects. The aim of this work was to evaluate possible interactions between the drugs and PS, assess drug stability and to identify degradation products after 20 h at room temperature. Furosemide (FSM) and Aminophylline (APN) were added to PS containing either 20% mannitol or 0.9% NaCl at pH 6.5-7.5 and 10-11. Their behavior was studied individually and as an admixture, after 1 h oxidation with H2O2, using a spectrophotometer and HPLC. Individually, FSM and APN added to 20% mannitol and 0.9% NaCl solutions had the highest stability at pH 10-11. When FSM and APN were combined, the behavior of FSM was similar to the behavior observed for the drug individually in the same solutions. With the drugs combined in 20% mannitol pH 10-11, HPLC showed that both drugs were stable after a 20 h period yielding two distinct peaks; in oxidized samples, the elution profile showed four peaks with retention times unrelated to the untreated samples.Soluções parenterais de grande volume são frequentemente utilizadas no ambiente hospitalar para a veiculação de fármacos. No entanto, possíveis incompatibilidades entre as estruturas dos fármacos, em diferentes veículos de administração, podem gerar possíveis associações antagônicas ou sinérgicas, resultando em alterações das propriedades físico-químicas, consequentemente, dos efeitos farmacológicos e das respostas clínicas esperadas. Este artigo avaliou a estabilidade e a possível formação de produtos de degradação entre os fármacos furosemida e aminofilina quando estes foram veiculados em soluções parenterais, após o preparo e após o período de 20 h. Furosemida e aminofilina foram adicionadas às soluções de 20% manitol e 0,9% NaCl nos valores de pH 6,5-7,5 e 10-11. A estabilidade dos fármacos foi avaliada individualmente, combinada e após degradação com peróxido de hidrogênio através de espectrofotometria de UV e HPLC. Furosemida e aminofilina individualmente avaliadas mostraram alta estabilidade em ambas as soluções estudadas nos valores de pH 10-11. Quando os fármacos foram combinados o comportamento da furosemida foi similar ao observado na ausência de aminofilina. Os fármacos combinados em 20% manitol pH10-11 por HPLC foram estáveis após o período de 20 h. Após degradação o perfil de cromatograma encontrado foi diferente do observado na ausência de degradação mostrando que o método é indicativo de estabilidade.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Pesquisa (CNPq)Coordenadoria de Apoio a Pesquisa (CAPES
Biological and physicochemical stability of ceftazidime and aminophylline on glucose parenteral solution
Ceftazidima é um antimicrobiano administrado por via parenteral, que apresenta amplo espectro de ação, principalmente contra Pseudomonas aeruginosa. O tempo em que a concentração sérica de ceftazidima permanece acima da concentração mínima inibitória (MIC) é um importante parâmetro farmacodinâmico para a determinação da eficácia antimicrobiana e pode ser potencializado através da utilização de infusão contínua em soluções parenterais (PS). Este artigo visa a avaliar a estabilidade da ceftazidima em solução de glicose 5%, na presença e na ausência do fármaco aminofilina, através de cromatografia líquida de alta eficiência HPLC e MIC durante o período de 24 horas. Os microorganismos selecionados para a determinação do MIC foram Escherichia coli e Pseudomonas aeruginosa. Os ensaios em cromatógrafo líquido confirmaram a estabilidade dos fármacos ceftazidima e aminofilina quando são individualmente associados em PS, enquanto os valores de MIC ficaram maiores que os valores encontrados na literatura. Quando ambos os fármacos foram associados na mesma solução parenteral a concentração de ceftazidima obtida por HPLC diminuiu 25% depois de 24 horas. Os valores de MIC mostraram maior decaimento da atividade antimicrobiana neste mesmo período e também valores de MIC alterados nas soluções preparadas no tempo zero, decaimento este que não foi detectado em HPLC. Os resultados indicaram incompatibilidade na associação dos fármacos em PS, enfatizando a importância dos resultados de MIC para interações de fármacos.Ceftazidime is a broad spectrum antibiotic administered mainly by the parenteral route, and it is especially effective against Pseudomonas aeruginosa. The period of time in which serum levels exceed the Minimum Inhibitory Concentration (MIC) is an important pharmacodynamic parameter for its efficacy. One of the forms to extend this period is to administer the antibiotic by continuous infusion, after prior dilution in a Parenteral Solution (PS). The present work assessed the stability of ceftazidime in 5% glucose PS for 24 hours, combined or not with aminophylline, through High Performance Liquid Chromatography (HPLC). The physicochemical evaluation was accompanied by in vitro antimicrobial activity compared MIC test in the 24-hour period. Escherichia coli and Pseudomonas aeruginosa were the microorganisms chosen for the MIC comparison. The HPLC analysis confirmed ceftazidime and aminophylline individual stability on PS, while the MIC values were slightly higher than the mean described in the literature. When both drugs were associated in the same PS, the ceftazidime concentration by HPLC decreased 25% after 24 hours. Not only did the MIC values show high loss of antibiotic activity within the same period, but also altered MIC values immediately after the preparation, which was not detected by HPLC. Our results indicate that this drug combination is not compatible, even if used right away, and that PS might not be the best vehicle for ceftazidime, emphasizing the importance of the MIC evaluation for drug interactions
Evaluation of the sterilization efficacy of domestic electric drills used in orthopaedic surgeries
Methods of endotoxin removal from biological preparations : a review
ABSTRACTPURPOSE: Endotoxins, also called lipopolysaccharides (LPS), are major contaminants found in commercially available proteins or biologically active substances, which often complicate study of the biological effects of the main ingredient. The presence of small amounts of endotoxin in recombinant protein preparations can cause side effects in host organism such as endotoxin shock, tissue injury, and even death. Due to these reactions, it is essential to remove endotoxins from drugs, injectables, and other biological and pharmaceutical products. An overview of this subject is provided by this article. METHODS: An extensive review of literature with regard to methods for removal of endotoxin from biotechnological preparations was carried out. RESULTS: A short history of endotoxin is presented first. This is followed by a review of chemical and physical properties of endotoxin and its pathophysiological effects when the body is exposed to LPS excessively or systemically. The techniques of endotoxin determination and interaction of endotoxin with proteins is also presented, taking into consideration the established techniques as well as the state of the art technology in this field. A review of techniques of endotoxin mentioned with relatively high protein recoveries; however, special attention is given to two-phase aqueous micellar systems, which are valuable tools for endotoxin removal from pharmaceutical proteins on a small scale because they provide a mild environment for biological materials. CONCLUSIONS: Efficient and cost-effective removal of endotoxins from pharmaceutical and biotechnology preparations is challenging. Despite development of novel methods, such as the two- phase aqueous micellar systems, in recent years, more research is needed in this field
Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system
BACKGROUND: A typical purification system that provides purified water which meets ionic and organic chemical standards, must be protected from microbial proliferation to minimize cross-contamination for use in cleaning and preparations in pharmaceutical industries and in health environments. METHODOLOGY: Samples of water were taken directly from the public distribution water tank at twelve different stages of a typical purification system were analyzed for the identification of isolated bacteria. Two miniature kits were used: (i) identification system (api 20 NE, Bio-Mérieux) for non-enteric and non-fermenting gram-negative rods; and (ii) identification system (BBL crystal, Becton and Dickson) for enteric and non-fermenting gram-negative rods. The efficiency of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was evaluated by the minimum inhibitory concentration (MIC) method. RESULTS: The 78 isolated colonies were identified as the following bacteria genera: Pseudomonas, Flavobacterium and Acinetobacter. According to the miniature kits used in the identification, there was a prevalence of isolation of P. aeruginosa 32.05%, P. picketti (Ralstonia picketti) 23.08%, P. vesiculares 12.82%,P. diminuta 11.54%, F. aureum 6.42%, P. fluorescens 5.13%, A. lwoffi 2.56%, P. putida 2.56%, P. alcaligenes 1.28%, P. paucimobilis 1.28%, and F. multivorum 1.28%. CONCLUSIONS: We found that research was required for the identification of gram-negative non-fermenting bacteria, which were isolated from drinking water and water purification systems, since Pseudomonas genera represents opportunistic pathogens which disperse and adhere easily to surfaces, forming a biofilm which interferes with the cleaning and disinfection procedures in hospital and industrial environments