Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
1.
Lab Chip ; 4(6): 563-9, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15570366

RESUMO

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).


Assuntos
Células Dendríticas/citologia , Análise de Injeção de Fluxo/instrumentação , Imunoensaio de Fluorescência por Polarização/instrumentação , Microquímica/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos/imunologia , Células Dendríticas/metabolismo , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/métodos , Imunoensaio de Fluorescência por Polarização/métodos , Humanos , Microquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Miniaturização , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/imunologia
2.
Proteomics ; 4(10): 2849-76, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15378759

RESUMO

The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for its application in microbial physiology. DNA arrays were used to calculate the number of genes transcribed in growing cells. From the 4100 B. subtilis genes, 2515 were actively transcribed in cells grown under standard conditions. From these genes 1544 proteins should be covered by our standard gel system pI 4-7. Using this standard gel system and supplementary zoom gels (pI 5.5-6.7, 5-6, 4.5-5.5, and 4-5) 693 proteins which are expressed in growing cells were detected that cover more than 40% of the vegetative proteome predicted for this region. Particularly broad coverage and thus comprehensive monitoring will be possible for central carbohydrate metabolism (glycolysis, pentose phosphate shunt, and citric acid cycle), amino acid synthesis pathways, purine and pyrimidine metabolism, fatty acid metabolism, and main cellular functions like replication, transcription, translation, and cell wall synthesis. Comparing the theoretical pI and Mr values with those experimentally determined a reasonable correlation was found for the majority of protein spots. By a color code outliers with dramatic deviations in charge or mass were visualized that may indicate post-translational modifications. In addition to the cytosolic neutral and alkaline proteins, 130 membrane proteins were found relying on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) techniques. The vegetative proteome containing 876 proteins in total is now ready for physiological applications. Two main proteome fractions (pI 4-7 and zoom gel pI 4.5-5.5) should be sufficient for such high-throughput physiological proteomics.


Assuntos
Bacillus subtilis/metabolismo , Proteoma , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Western Blotting , Metabolismo dos Carboidratos , Biologia Computacional , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Espectrometria de Massas , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transcrição Gênica
3.
Eur J Biochem ; 271(1): 195-204, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14686932

RESUMO

H2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) is an unusual hydrogenase present in many methanogenic archaea. The homodimeric enzyme dubbed 'metal-free' hydrogenase does not contain iron-sulfur clusters or nickel and thus differs from [Ni-Fe] and [Fe-Fe] hydrogenases, which are all iron-sulfur proteins. Hmd preparations were found to contain up to 1 mol iron per 40 kDa subunit, but the iron was considered to be a contaminant as none of the catalytic and spectroscopic properties of the enzyme indicated that it was an essential component. Hmd does, however, harbour a low molecular mass cofactor of yet unknown structure. We report here that the iron found in Hmd is most probably functional after all. Further investigation was initiated by the discovery that Hmd is inactivated upon exposure to UV-A (320-400 nm) or blue-light (400-500 nm). Enzyme purified in the dark exhibited an absorption spectrum with a maximum at approximately 360 nm and which mirrored its sensitivity towards light. In UV-A/blue-light the enzyme was bleached. The cofactor extracted from active Hmd was also light sensitive. It showed an UV/visible spectrum similar to that of the active enzyme and was bleached upon exposure to light. Photobleached cofactor no longer had the ability to reconstitute active Hmd from the apoenzyme. When purified in the dark, Hmd consistently contained per monomer about one Fe, which was tightly bound to the cofactor. The iron was released from the enzyme and from the cofactor upon light inactivation. Hmd activity was inhibited by high concentrations of CO and CO protected the enzyme from light inactivation indicating that the iron in Hmd is of functional importance. Therefore, reference to Hmd as 'metal-free' hydrogenase is no longer appropriate.


Assuntos
Hidrogenase/efeitos da radiação , Methanobacteriaceae/enzimologia , Methanobacterium/enzimologia , Raios Ultravioleta , Proteínas Arqueais/antagonistas & inibidores , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/efeitos da radiação , Cromatografia em Gel , Hidrogenase/antagonistas & inibidores , Hidrogenase/isolamento & purificação , Ferro/análise , Cinética , Luz , Espectrofotometria
4.
J Am Chem Soc ; 125(44): 13308-9, 2003 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-14582997

RESUMO

Using transferred cross-correlated relaxation and DFT calculations, the conformation of the relevant conformation of N5,N10-methylenetetrahydromethanopterin, a reaction intermediate bound to the 80 kD H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase is determined. The conformation of the intermediate differs from the free form in solution and makes the reaction mechanism plausible.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Methanobacteriaceae/enzimologia , Modelos Químicos , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Conformação Proteica , Pterinas/química , Pterinas/metabolismo
5.
Angew Chem Int Ed Engl ; 37(23): 3300-3303, 1998 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-29711416

RESUMO

By activation of the hydrogen acceptor, the metal-free hydrogenase from methanogenic archaea catalyzes the reduction of methenyl tetrahydromethanopterin with H2 . According to NMR spectroscopic analysis of the conformation of the hydrogen acceptor in solution and of the stereospecificity of the catalyzed and noncatalyzed reaction, in the enzyme-catalyzed reaction the hydrogenation product is formed in a constraint conformation which relaxes upon dissociation from the enzyme. This exergonic conformational change could help to avoid product inhibition of the enzyme.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA