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1.
Electron. j. biotechnol ; 27: 37-43, May. 2017. tab, ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1010283

RESUMO

Background: ß-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of ß-galactosidase, but the properties of this enzyme are incompletely studied. Results: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative ß-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal ß-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4­5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All ß-galactosidases performed transgalactosylation activity optimally in an acidic environment. Conclusions: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three ß-galactosidase family members with remarkably different biochemical properties.


Assuntos
Aspergillus niger/enzimologia , beta-Galactosidase/química , Especificidade por Substrato , Cinética , Sequência de Aminoácidos , Clonagem Molecular , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
2.
Acta sci., Biol. sci ; 38(2): 149-155, abr.-jun. 2016.
Artigo em Inglês | LILACS | ID: biblio-2531

RESUMO

The permeabilization was used to transform microorganisms in cell biocatalysts with high enzymatic activity. The Saccharomyces fragilis IZ 275 yeast cells were permeabilized with ethanol, as permeabilizing agent. To optimize the permeabilization conditions were used the design of Box-Behnken 15 trials (3 central points). The independent variables and their levels were ethanol (29, 32 and 35%), temperature (15, 20 and 25°C) and time (15, 20 and 25 min). The answer (Y) function has beta-galactosidase activity (U mg-1). The optimum conditions for obtaining a high enzymatic activity were observed in 35% ethanol concentration, temperature 15ºC and 20 min. treatment time. The maximum activity of the enzyme beta-galactosidase obtained was 10.59 U mg-1. The permeabilization of the S. fragilis IZ 275 cells was efficient.


A permeabilização foi usada para transformar células de microrganismos em biocatalisadores com alta atividade enzimática. As células de levedura de Saccharomyces fragilis IZ 275 foram permeabilizadas com etanol, como agente permeabilizante. Para otimizar as condições de permeabilização foi utilizado o delineamento de Box-Behnken com 15 ensaios (3 repetições no ponto central) . As variáveis independentes e seus níveis foram etanol (29, 32 e 35%), temperatura (15, 20 e 25ºC) e tempo (15, 20 e 25 min.). A função resposta (Y) foi atividade de beta-galactosidase (U mg-1). As condições ótimas para a obtenção de uma alta atividade enzimática foram observadas em 35% de concentração de etanol, temperatura de 15°C e tempo de tratamento de 20 minutos. A máxima atividade da enzima beta-galactosidase obtida foi de 10.59 U mg-1. A permeabilização das células de S. fragilis IZ 275 foi eficiente.


Assuntos
beta-Galactosidase , Saccharomyces , Biocatálise , Biotecnologia , Hidrólise , Lactose , Permeabilidade , Saccharomyces , Leveduras
3.
Braz. j. microbiol ; 45(3): 873-883, July-Sept. 2014. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-727016

RESUMO

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.


Assuntos
Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Hexoquinase/metabolismo , Penicilinas/biossíntese , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Hexoquinase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transformação Genética , beta-Galactosidase/biossíntese
4.
Rev. Esc. Enferm. USP ; 48(spe): 74-79, 08/2014. tab
Artigo em Inglês | LILACS | ID: lil-731303

RESUMO

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.
 .


Assuntos
Azoarcus/metabolismo , Coenzima A Ligases/genética , Fenilacetatos/metabolismo , Aerobiose , Azoarcus/genética , Coenzima A Ligases/metabolismo , Immunoblotting , Mutação , Proteínas Recombinantes de Fusão/genética , Análise de Sequência de DNA , beta-Galactosidase/genética
5.
Rev. Esc. Enferm. USP ; 48(spe): 16-22, 08/2014. tab
Artigo em Inglês | LILACS | ID: lil-731304

RESUMO

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. .


Assuntos
Proteínas de Ligação a DNA , Escherichia coli/genética , Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido , Bacteriófago lambda/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Genes Reporter/genética , Fosforilação , Plasmídeos/biossíntese , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Transcrição Genética/genética , Transcrição Genética/fisiologia , Proteínas Virais Reguladoras e Acessórias , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/fisiologia , beta-Galactosidase/biossíntese , beta-Lactamases/biossíntese
6.
Rev. cuba. pediatr ; 86(1): 103-107, abr.-jun. 2014.
Artigo em Espanhol | LILACS | ID: lil-709199

RESUMO

La gangliosidosis generalizada tipo 1 es una enfermedad de acúmulo lisosomal producida por mutaciones en el gen de la enzima b-galactosidasa, caracterizada fundamentalmente por toma del sistema nervioso central, la visceromegalia, disostosis ósea y dimorfismo facial. Se presenta el caso de un lactante varón, hijo de padres no consanguíneos, de 5 meses de edad, Apgar 6/8 debido a hipoxia neonatal, con historia de múltiples ingresos por enfermedad diarreica e infecciones respiratorias. Es remitido a la Consulta de Genética Clínica por retardo del desarrollo psicomotor, macrocráneo y hepatomegalia, además de máculas hipercrómicas en piel. En el examen físico se encontraron evidencias de una posible afectación por enfermedad metabólica lisosomal. Entre las enfermedades a descartar estaban la galactosialidosis, de características clínicas similares, y la enfermedad de Morquio, con diferente presentación clínica pero idéntico defecto enzimático


Generalized or GM 1 gangliosidosis is a lysosomal storage disease caused by mutations in the enzyme b-galactosidase gene, mainly characterized by affecting the central nervous system, visceromegalia, osseous dysostosis and facial dimorphism. This is the case of a male nursling born to non-consanguineous parents, 5 months of age, Apgar index of 6/8 due to neonatal hypoxia, with a history of several admissions to hospital because of diarrheal disease and respiratory infections. He was referred to the clinical genetic service since he presented with retarded psychomotor development, macrocrania and hepatomegalia, in addition to hyperchromic skin spots. The physical exam found evidence of possible effects by lysosomal metabolic disease. Among the diseases to be ruled out for the diagnosis were galactosialidosis of similar clinical characteristics and Morquio B disease with different clinical presentation but identical enzymatic deficiency


Assuntos
Humanos , Masculino , Lactente , Doenças por Armazenamento dos Lisossomos/complicações , Gangliosidose GM1 , beta-Galactosidase/genética , Relatos de Casos
7.
Braz. j. microbiol ; 45(2): 595-601, Apr.-June 2014. tab
Artigo em Inglês | LILACS | ID: lil-723123

RESUMO

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively.Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity .The method was found to be sensitive enough to determine 1.7 x 10(6) CFU mL-1 in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.


Assuntos
Azotobacter/isolamento & purificação , Carga Bacteriana/métodos , Genes Reporter , beta-Galactosidase/análise , Brassica rapa/microbiologia , Sensibilidade e Especificidade , beta-Galactosidase/genética
8.
Braz. j. microbiol ; 44(4): 1067-1074, Oct.-Dec. 2013. graf, tab
Artigo em Inglês | LILACS | ID: lil-705252

RESUMO

The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L-1 oNP min-1 g-1 was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry.


Assuntos
Membrana Celular/efeitos dos fármacos , Etanol/toxicidade , Kluyveromyces/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Membrana Celular/fisiologia , Kluyveromyces/fisiologia , Modelos Estatísticos , Temperatura , Fatores de Tempo , beta-Galactosidase/análise
9.
Braz. j. med. biol. res ; 45(12): 1135-1140, Dec. 2012. ilus, tab
Artigo em Inglês | LILACS | ID: lil-659653

RESUMO

Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.


Assuntos
Trifosfato de Adenosina/metabolismo , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/metabolismo , Ácidos Cetoglutáricos/metabolismo , Fatores de Transcrição/metabolismo , beta-Galactosidase/metabolismo , Azospirillum brasilense/metabolismo , Vetores Genéticos , Plasmídeos
10.
Clinics ; 67(2): 135-143, 2012. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-614637

RESUMO

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. RESULTS: The cell cycle was arrested in the G0/G1 phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G0/G1 phase and increased cell populations in the G2/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.


Assuntos
Humanos , Antioxidantes/farmacologia , Senescência Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Cromanos/farmacologia , Fibroblastos/efeitos dos fármacos , Vitamina E/análogos & derivados , beta-Galactosidase/análise , Análise de Variância , Biomarcadores/análise , Células Cultivadas , Senescência Celular/genética , Ciclo Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Diploide , Fibroblastos/citologia , Fibroblastos/metabolismo , /genética , /metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Regulação para Cima/efeitos dos fármacos , Vitamina E/farmacologia , beta-Galactosidase/metabolismo
11.
Electron. j. biotechnol ; 14(6): 9-9, Nov. 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-640526

RESUMO

Whey is a co-product of processes for the production of cheese and casein that retains most of the lactose content in milk. World production of whey is estimated around 200 million tons per year with an increase rate of about 2 percent/per year. Milk production is seasonal, so surplus whey is unavoidable. Traditionally, whey producers have considered it as a nuisance and strategies of whey handling have been mostly oriented to their more convenient disposal. This vision has been steadily evolving because of the upgrading potential of whey major components (lactose and whey proteins), but also because of more stringent regulations of waste disposal. Only the big cheese manufacturing companies are in the position of implementing technologies for their recovery and upgrading, so there is a major challenge in incorporating medium and small size producers to a platform of whey utilization, conciliating industrial interest with environmental protection within the framework of sustainable development. Within this context, among the many technological options for whey upgrading, transformation of whey components by enzyme biocatalysis appears as prominent. In fact, enzymes are green catalysts that can perform a myriad of transformation reactions under mild conditions and with strict specificity, so reducing production costs and environmental burden. This review pretends to highlight the impact of biocatalysis within a platform of whey upgrading. Technological options are shortly reviewed and then an in-depth and critical appraisal of enzyme technologies for whey upgrading is presented, with a special focus on newly developed enzymatic processes of organic synthesis, where the added value is high, being then a powerful driving force for industrial implementation.


Assuntos
Lactose , Leite/enzimologia , Oligossacarídeos/metabolismo , Prebióticos , beta-Galactosidase/metabolismo , Biocatálise , Esterificação , Enzimas/metabolismo
12.
Hig. aliment ; 25(194/195): 79-85, mar.-abr. 2011. tab
Artigo em Português | LILACS | ID: lil-607072

RESUMO

O mercado de produtos com baixo teor de lactose ainda é pouco explorado no Brasil, porém muitas pessoas sofrem de intolerância à lactose e muitos produtos lácteos cristalizam-se com maior facilidade devido à baixa solubilidade desse dissacarídeo. A hidrólise da lactose é um processo promissor para a indústria de alimentos porque possibilita o desenvolvimento de novos produtos sem lactose em suas composições. Esta operação oferece certas vantagens uma vez que ela diminui os riscos de cristalização em derivados lácteos e aumenta o poder adoçante. O objetivo deste trabalho foi fazer uma revisão de como utilizar a enzima ß – galactosidase no processo de redução do teor de lactose do leite a ser utilizado para a produção de doce de leite, aumentando o seu tempo de armazenamento, através da redução da arenosidade causada pela cristalização da lactose no processo de concentração.


Assuntos
beta-Galactosidase , Doces , Laticínios , Conservação de Alimentos , Armazenamento de Alimentos , Substitutos do Leite Humano
14.
Rev. ciênc. farm. básica apl ; 31(3)set.-dez. 2010.
Artigo em Inglês | LILACS | ID: lil-570160

RESUMO

Este estudo demonstra como a Beta-galactosidase pode ser desativada e reativada usando EDTA e íons metálicos divalentes. A enzima foi desativada após 20 minutos na presença de EDTA. Desativação máxima para a menor concentração de EDTA (10-3 mol.L-1) ocorreu na presença do tampão Tris-HCl. A enzima recuperou 50% de sua atividade inicial após 10 minutos na presença de Mg2+ em concentrações superiores a 0,1mmol.L-1.Concentrações de 10-4 e 10-3 mol.L-1 de Mn2+ e Co2+ foram suficientes para reativar a enzima em 300% comparado ao controle de íons Mn2+ e aproximadamente 100% para íons Co2+. A enzima perdeu gradualmente a sua atividade quando a concentração foi de 10-2 mol.L-1. Ni2+ e Zn2+ foram incapazes de restabelecer a atividade catalítica. Km app e Vmax app foram 1,95 ± 0,05 mmol.L-1 e 5,40 ± 0,86 x 10-2 mmol.min-1.mg-1. A temperatura e pH ótimos foram 34ºC e 7,5. A meia vida da holoenzima foi de 17,5 min a 30ºC e para a apoenzima foi de 11,0 min a 30ºC. Quanto à variação de pH, a apoenzima provou ser mais sensível que a holoenzima.


In this study, it was demonstrated that Beta-galactosidase can be deactivated and reactivated with EDTA and divalent metal ions. The enzyme was deactivated after 20 minutes in EDTA solution. Maximal deactivation at the lowest EDTA concentration (10-3 mol.L-1) occurred in the presence of Tris-HCl buffer (pH 7.0). The enzyme recovered 50% of its initial activity after 10 minutes at Mg2+concentrations higher than 0.1 mmol.L-1. Experimental concentrations of 0.1 mmol.L-1 Mn2+ and 1.0 mmol.L-1 Co2+ were sufficient to reactivate the enzyme to around 300% of the control activity for the Mn2+ ion and nearly 100% for the Co2+ ion. The enzyme gradually lost its activity when the Co2+ concentration was 10-2 mol.L-1. Ni2+ and Zn2+ were unable to restore the catalytic activity. Km app and Vmax app were 1.95 ± 0.05 mmol.L-1 and 5.40 ± 0.86x10-2 mmol.min-1.mg-1, with o-NPG as substrate. Optimal temperature and pH were 34oC and 7.5. The half-life (t1/2) at 30ºC was 17.5 min for the holoenzyme and 11.0 min for the apoenzyme. With respect to pH variation, the apoenzyme proved to be more sensitive than the holoenzyme.


Assuntos
Humanos , Ácido Edético , Kluyveromyces , beta-Galactosidase/isolamento & purificação , Ativação Enzimática
15.
Braz. j. microbiol ; 41(3): 596-602, Oct. 2010. ilus, tab
Artigo em Inglês | LILACS | ID: lil-549400

RESUMO

AmpC â-lactamases are cephalosporinases that hydrolyze cephamycins as well as other extended-spectrum cephalosporins and are poorly inhibited by clavulanic acid. Although reported with increasing frequency, the true rate of occurrence of AmpC â-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Pondicherry state, South India. A total of 235 Gram negative clinical isolates were tested for resistance to cefoxitin, third generation cephalosporin (3GC) antibiotics, ampicillin, amikacin, co-trimoxazole, gentamicin, meropenem and tetracycline by disc diffusion method. Isolates found resistant to 3GC and cefoxitin were tested for the production of AmpC â -lactamases by three dimensional extraction method and AmpC disc method. Isolates found to sensitive to 3GC were subjected to disc antagonism test for inducible AmpC production. One hundred and thirty four (57 percent) strains were resistant to 3GC, among which 63(47 percent) were positive for plasmid-mediated AmpC beta lactamases production. Among the 101 strains sensitive to 3GC, 23 (22.7 percent) revealed the presence of inducible AmpC beta lactamases by disc approximation test. A total of 80.9 percent (51/63) of screen positive isolates were detected by Amp C disc test and 93.6 percent (59/63) by three dimensional extraction method. Out of the 86 AmpC producers, 67 (77.9 percent) were cefoxitin resistant .Inducible AmpC was not found in Esch.coli and Klebsiella spp. The AmpC producers also concurrently showed multidrug resistance pattern. AmpC producers were found to be prevalent in our hospital and though three dimensional extraction test detects AmpC better, the disk test is easier to perform routinely and is user- friendly.


Assuntos
Humanos , Antibacterianos , Ácido Clavulânico/análise , Ensaios Enzimáticos Clínicos , Cefalosporinas/análise , Resistência a Medicamentos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , beta-Galactosidase/análise , beta-Galactosidase/isolamento & purificação , Métodos , Técnicas
16.
Invest. clín ; 51(3): 351-367, Sept. 2010. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-574452

RESUMO

La actividad de la ß-galactosidasa refleja la tasa de envejecimiento celular in vitro. Mediante quimioluminiscencia se cuantificó dicha actividad a pH 6 en células epiteliales ováricas provenientes de 28 donantes sin antecedentes de cáncer. Las células fueron cultivadas en forma seriada hasta alcanzar el estado de detención permanente de crecimiento. Durante la fase de crecimiento exponencial, todos los cultivos mostraron un patrón semejante de crecimiento y una baja actividad ß-galactosidasa. Sin embargo, el inicio de la disminución de la capacidad replicativa que caracteriza el final de dicha fase, así como el inicio de la fase estacionaria o senescente presentaron un aumento significativo en la actividad enzimática. Nuestros resultados mostraron que la actividad ß-galactosidasa puede ser considerada como marcador de senescencia replicativa del epitelio superficial del ovario a pH 6.


ß-galactosidase activity reflects the rate of cellular aging in vitro. Such activity was quantified at pH 6 in ovarian epithelial cells from 28 donors without a history of cancer, by the chemoluminiscent method. The cells were serially cultured until they achieved the state of permanent growth arrest. During the exponential growth phase, all cultures showed a similar pattern of growth and low ß-galactosidase activity. However, both in the onset of decrease replicative activity, as well as in the onset of the stationary phase, there was a significant rise in the enzyme activity. Our results showed that ß-galactosidase activity can be considered as a replicative senescence marker of the ovarian surface epithelium at pH 6.


Assuntos
Humanos , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Senescência Celular , Células Epitelioides , Ovário/citologia , beta-Galactosidase/administração & dosagem , Biomarcadores/análise
17.
Dermatol. pediatr. latinoam. (Impr.) ; 8(2): 5-15, mayo-ago. 2010. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-600309

RESUMO

Los angioqueratomas son lesiones vasculares relativamente infrecuentes que consisten en pápulas hiperqueratósicas rojo-violáceas. Éstas pueden ser únicas o múltiples, estar localizadas en un único segmento corporal o ser generalizadas y estar o no asociadas a otras enfermedades subyacentes. El presente trabajo abordará en profundidad los angioqueratomas generalizados complementando la primera parte en la que se trataron los angioqueratomas localizados.


Angiokeratomas are a relatively non-frequent group of vascular lesions that consist on hyperkeratotic red-violaceous papules. Lesions can be solitary or multiple, localized or generalized and may be associated or not with a systemic disease. The present work is a throughout review on generalized angiokeratomas and it is a complement of the first part in which localized angiokeratomas have been discussed.


Assuntos
Humanos , alfa-Manosidose , Angioceratoma , Aspartilglucosilaminase , beta-Galactosidase , beta-Manosidose , Doença de Fabry , Fucosidose , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso , Diagnóstico Diferencial
18.
Braz. j. microbiol ; 41(2): 333-344, Apr.-June 2010. tab, ilus
Artigo em Inglês | LILACS | ID: lil-545339

RESUMO

A total of 187 isolates from 470 clinical specimens were collected from three hospitals in El-Minia governorate and identified as 132 Staphylococcus aureus strains and 55 coagulase-negative staphylococci (CoNS) strains. Susceptibility of isolates to antimicrobial agents was tested by the agar dilution method. The isolated S. aureus strains showed low resistance to vancomycin (1.5 percent), amikacin (2.3 percent) and gatifloxacin (3.8 percent). Vancomycin was the most effective antibiotic against CoNS. The ampicillin-resistant isolates were tested for â-lactamase production where, 61.7 percent of S. aureus and 42.9 percent of CoNS were positive for â-lactamase enzyme. Beta-lactamase producing strains were screened for their plasmid profile using alkaline lysis method. Some of these strains carried at least one plasmid suggesting plasmid-mediated antibiotic resistance. When cells of these strains were exposed to curing agent ethidium bromide, the production of the â-lactamase was lost. Resistance by efflux was studied by a modified fluorometric assay. Addition of uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased norfloxacin accumulation in quinolone resistant S. aureus strains, suggesting endogenous energy-dependent efflux. Combinations of ciprofloxacin with four antimicrobial agents against methicillin resistant S.aureus (MRSA) strains were investigated using decimal assay for additivity (DAA) technique. Synergistic interaction was observed between ciprofloxacin and oxacillin. ciprofloxacin plus cefepime and gentamicin appeared to be additive, while ciprofloxacin plus erythromycin was antagonistic.


Assuntos
Humanos , Coagulase , Suscetibilidade a Doenças , Resistência Microbiana a Medicamentos , Infecções Estafilocócicas , Staphylococcus aureus/enzimologia , Staphylococcus aureus/isolamento & purificação , beta-Galactosidase/isolamento & purificação , Técnicas e Procedimentos Diagnósticos , Ativação Enzimática , Fluorometria , Métodos
19.
Braz. j. med. biol. res ; 43(2): 195-200, Feb. 2010. graf
Artigo em Inglês | LILACS | ID: lil-538230

RESUMO

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERá and hERâ and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the â-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the â-galactosidase activity (EC50) of the tuber extracts in relation to 17â-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERá and hERâ, respectively, with a higher relative estrogenic potency with hERâ than with hERá. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17â-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.


Assuntos
Animais , Ratos , Receptor alfa de Estrogênio/análise , Receptor beta de Estrogênio/análise , Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pueraria/química , Bioensaio , Cromatografia Líquida de Alta Pressão , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Isoflavonas/análise , Isoflavonas/metabolismo , Fígado/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , /metabolismo , beta-Galactosidase/análise , beta-Galactosidase/antagonistas & inibidores
20.
Electron. j. biotechnol ; 12(2): 12-13, Apr. 2009. ilus, tab
Artigo em Inglês | LILACS | ID: lil-551373

RESUMO

The b-Galactosidase activity at pH 6 is used as a cellular marker to identify senescent cell cultures. The classic method to identify this enzymatic activity is using cytochemical staining with X-Gal after 16 hrs. In this work, a differential pH sensor was used to measure b-Galactosidase activity at pH 6. The measurement is easy and only takes 3 min.


Assuntos
Concentração de Íons de Hidrogênio , beta-Galactosidase/análise , Senescência Celular , Ativação Enzimática , Hexoquinase/metabolismo
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