RESUMO
Carboxypeptidase E (CPE) is involved in the biosynthesis of peptide hormones and neurotransmitters. To determine whether a recently reported Aplysia californica cDNA encodes a CPE-like enzyme, this cDNA was expressed in the baculovirus system. The Aplysia CPE is optimal at pH 5.5-6.5 and is inhibited by chelating agents and by the sulfhydryl reagent p-chloromercuriphenyl sulfonate. The effect of divalent cations and active site-directed inhibitors on enzyme activity are generally similar for Aplysia and rat CPE. Western blot analysis using antisera to the N- and C-terminal regions of the Aplysia CPE show that the Aplysia CPE is present in atrial glands and ovotestis. This Aplysia CPE is purified on a p-aminobenzoyl-Arg Sepharose affinity column under conditions that selectively purify rat CPE. Taken together, these results suggest that the previously cloned cDNA represents a CPE-like enzyme that is expressed in Aplysia tissue.
Assuntos
Aplysia/enzimologia , Carboxipeptidases/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Western Blotting , Carboxipeptidase H , Carboxipeptidases/genética , Carboxipeptidases/isolamento & purificação , Cátions Bivalentes/farmacologia , DNA Complementar , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Knowledge-based image analysis and interpretation of radiological images is of significant interest for several reasons including a means to identify and label each part of the image for further automated diagnostic analysis. Also, there is a need to develop a knowledge-based biomedical image analysis system which can analyze and interpret the anatomical images (such as those obtained from X-ray computed tomography (CT) scanning) in order to help analysis of functional images (such as those obtained from positron emission tomography (PET) scanning) of the organ of the same patient. This paper deals with the design and implementation of a knowledge-based system to analyze and interpret CT anatomical images of the human chest. In the approach presented here, the emphasis has been on the development of a strong low-level analysis system with the capability of analyzing in both bottom-up and top-down modes; and on the use of hierarchical relational, spatial, and structural knowledge of human anatomy in the process of high-level analysis and recognition.
Assuntos
Inteligência Artificial , Interpretação de Imagem Assistida por Computador/métodos , Humanos , Processamento de Imagem Assistida por Computador , Intensificação de Imagem Radiográfica/métodos , Radiografia Torácica , Valores de Referência , Tomografia Computadorizada de Emissão/métodos , Tomografia Computadorizada por Raios X/métodosRESUMO
P-glycoprotein is an integral membrane protein that functions as a cytotoxic drug-efflux pump. Studies suggest that the transporter exists in the membrane as a dimer and possibly higher order structures. We report the bacterial expression of the linker region (amino acids 621-688) of murine mdr1b P-glycoprotein and demonstrate that this region, which serves to link the two homologous halves of the transporter, has the potential to serve as a dimerization domain. The recombinant peptide (8742 daltons) eluted from a gel filtration column at a position corresponding to a dimer (i.e. 17,500 daltons). A dimer:monomer equilibrium, as a function of peptide concentration, was confirmed by large zone gel filtration chromatography. The dissociation constant (KD) for the dimer:monomer equilibrium was 350 nM. It was possible to dissociate the dimer by low pH (3.0) or high ionic strength (2.5 M NaCl). Dimerization was not affected by an alkaline pH of 10 or 5 mM EDTA. Studies with a truncated linker peptide indicated that the N-terminal 48 amino acids were sufficient for dimerization.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/isolamento & purificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia em Gel , Clonagem Molecular , Primers do DNA , Dimerização , Escherichia coli , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The mRNA for organic anion transport protein (oatp) was previously shown to be present in abundance in liver and kidney, and in small amounts in brain. Data obtained from experiments with reverse transcriptase-PCR techniques and in situ hybridization analysis showed that the oatp mRNA is present within the brain, localized to the choroid plexus. A sequence-specific antibody to the oatp polypeptide demonstrated the presence of the expected polypeptide with a molecular weight of 80,000 plus an immunoreactive species with a higher molecular weight in preparations of choroid plexus membranes. Examination of the choroid plexus by fluorescence confocal microscopy revealed that immunoreactive oatp polypeptide is localized to the apical surface of the choroid plexus epithelial cells, which contacts the cerebrospinal fluid. This localization of oatp is consistent with previous experiments showing vectorial transport of organic anions between the choroid plexus and the cerebrospinal fluid.