RESUMO
Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage PhiX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics.
Assuntos
Anticorpos Biespecíficos/imunologia , Patógenos Transmitidos pelo Sangue , Modelos Animais de Doenças , Receptores de Complemento/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Bacteriófago phi X 174/imunologia , Relação Dose-Resposta Imunológica , Eritrócitos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , PapioRESUMO
PAS domains sense oxygen, redox potential and light, and are implicated in behaviour, circadian rhythmicity, development and metabolic regulation. Although PAS domains are widespread in archaea, bacteria and eukaryota, the mechanism of signal transduction has been elucidated only for the bacterial photo sensor PYP and oxygen sensor FixL. We investigated the signalling mechanism in the PAS domain of Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Forty-two residues in Aer were substituted using cysteine-replacement mutagenesis. Eight mutations resulted in a null phenotype for aerotaxis, the behavioural response to oxygen. Four of them also led to the loss of the non-covalently bound FAD cofactor. Three mutant Aer proteins, N34C, F66C and N85C, transmitted a constant signal-on bias. One mutation, Y111C, inverted signalling by the transducer so that positive stimuli produced negative signals and vice versa. Residues critical for signalling were mapped onto a three-dimensional model of the Aer PAS domain, and an FAD-binding site and 'active site' for signal transduction are proposed.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Mutagênese , Oxirredução , Estrutura Terciária de ProteínaAssuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Plasmídeos/genética , Sequência de Bases , Clonagem Molecular , Metilação de DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Especificidade por SubstratoRESUMO
cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M(r) ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.