Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Biotecnología/normas , Técnicas de Cultivo/normas , Citocinas/normas , Proteínas Recombinantes/normas , United States Food and Drug Administration , Vacunas Sintéticas/biosíntesis , Biotecnología/métodos , Técnicas de Cultivo/métodos , Citocinas/biosíntesis , Humanos , Modelos Teóricos , Proteínas Recombinantes/biosíntesis , Estados UnidosRESUMEN
The Center for Biologics Evaluation and Research, whose regulatory authority includes monoclonal antibodies, cytokines, vaccines, toxins and somatic cellular therapies, communicates to sponsors issues for consideration in the development of biological products through the publication of "Points to Consider" and "Guideline" documents. This paper summarizes the available "Points to Consider" and "Guideline" documents and outlines recommendations from these documents for characterizing the cells used to produce biological products.
Asunto(s)
Productos Biológicos/biosíntesis , Biotecnología/legislación & jurisprudencia , Animales , Línea Celular , Humanos , Muridae , Retroviridae/aislamiento & purificación , Estados UnidosRESUMEN
Catalytic antibodies are introduced as an important new class of biomolecules for molecular recognition in biosensors in which the binding sites are continually regenerated by the catalytic reaction of the substrate. Consequently, molecular recognition by catalytic antibodies can yield reversible immunoblosensors. In this example, a prototype potentiometric biosensor is described in which a micro-pH electrode is modified with a catalytic antibody that catalyzes the hydrolysis of phenyl acetate, producing hydrogen ions that can be monitored by the electrode. The reversible response is linear with the log of substrate concentration over a range of 20-500 microM with a detection limit of 5 microM under the conditions of this study. Alternative applications of catalytic antibodies in other biosensor configurations are discussed.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Técnicas Biosensibles , Concentración de Iones de Hidrógeno , Hidrólisis , Potenciometría/métodosRESUMEN
The redox centers in the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum were examined by potentiometric titration and electron paramagnetic resonance spectroscopy. At low temperature two overlapping iron-sulfur signals which correlated with enzymatic activity were observed with formal potentials near -400 mV vs. SHE. Based on their temperature dependences, one signal is assigned to a reduced Fe2S2 cluster and one to a reduced Fe4S4 cluster. Quantitation of signal intensity suggests two Fe2S2 and two Fe4S4 clusters per formate dehydrogenase molecule. Another signal (g = 2.101, 1.980, 1.950) present in low concentrations at more negative potentials was observable up to 200 degrees K and is not attributed to any iron-sulfur cluster. The possible origin of this signal is analyzed using ligand field theory, and the redox behavior is considered with respect to possible ligation at the active site.
Asunto(s)
Aldehído Oxidorreductasas , Clostridium/enzimología , Formiato Deshidrogenasas , Tungsteno/análisis , Aldehído Oxidorreductasas/análisis , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Formiato Deshidrogenasas/análisis , Oxidación-Reducción , TemperaturaRESUMEN
Investigation of lumiflavin and several other isoalloxazine ring derivatives has been carried out by geometry-optimized molecular orbital calculations. The results have provided insight into the flexibility of the flavin cofactor in the reduced and oxidized states, the regions of the fused three-ring system that should play an important role in flavin electron transfers, and the structural and functional role of the xylene and heteroatomic portions of the flavin system. The significance of these results is reviewed in relation to the experimentally identified chemical and biochemical properties of the flavin nucleotide coenzymes.
Asunto(s)
Flavinas , Fenómenos Químicos , Química , Coenzimas , Transporte de Electrón , Flavinas/metabolismo , Conformación Molecular , Oxidación-ReducciónRESUMEN
Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme. FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively. Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase. The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Formiato Deshidrogenasas/metabolismo , Nitrato Reductasas/metabolismo , Azotobacter/enzimología , Clostridium/enzimología , Activación Enzimática , Neurospora crassa/enzimología , Nitrogenasa/metabolismoRESUMEN
Formate dehydrogenase (FDH) (EC 1.2.1.43) from C. thermoaceticum has been purified in two forms. One contains tungsten (W), and the other is enriched in molybdenum (Mo). The W-FDH is clearly active, while the Mo results are ambiguous with enzymatic activities generally lower in the Mo-enriched samples. Spectroscopic studies (EPR, absorption, and CD) on W-FDH and Mo-FDH demonstrate that no signal correlates to the group VI metal active site in the dithionite-reduced enzyme. This lack of a W(V) EPR signal is in contrast to the results observed for tungsten-substituted sulfite oxidase which is inactive.