RESUMO
A model of venous thrombosis on the rabbit marginal ear vein was developed. It is based on occlusion of a segment of the left or right ear vein followed by injection of thrombin into this segment. Thrombolytic activity of longolytin applied externally onto the thrombotic venous segment was evaluated. The drug 2-fold shortened the time of clot lysis and 4-4.5-fold increased the rate of thrombolysis. The effect of longolytin was potentiated by heparin. The rate of lysis was higher in large thrombi and in the left vein (effect of asymmetry).
Assuntos
Orelha/irrigação sanguínea , Fibrinolíticos/uso terapêutico , Peptídeo Hidrolases/uso terapêutico , Trombose/tratamento farmacológico , Animais , Coelhos , VeiasAssuntos
Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Prolina/biossíntese , Thermus/enzimologia , Thermus/genética , Sequência de Aminoácidos , Clonagem Molecular , Estabilidade Enzimática , Dados de Sequência Molecular , Peso Molecular , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Estrutura Secundária de Proteína , Alinhamento de SequênciaRESUMO
The proA proline biosynthesis gene of thermophilic bacterium Thermus ruber was cloned and sequenced, and several properties of the encoded enzyme, gamma-glutamylphosphate reductase (GPR) were studied. The proA open reading frame (ORF) was of 1286 bp. Nucleotide sequence analysis revealed the ATG initiation codon in position 36 and the TTA termination codon in position 1304. A deduced protein product of the gene was shown to be of 44,919 Da in molecular weight. The GC content was 66%, as is characteristic of various bacteria of the genus Thermus. An amino acid sequence encoded by the cloned gene showed the highest homology (up to 64%) with GPR of T. thermophilus. The maximum activity of GPR (8.2 x 10(-2) units/ml) was observed at 55 degrees C. A weak enzymatic activity was also detected at 70 degrees C. The enzyme can be used in biotechnological studies.
Assuntos
Aldeído Oxirredutases/genética , Prolina/biossíntese , Thermus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Glutamato-5-Semialdeído Desidrogenase , Dados de Sequência Molecular , Fases de Leitura Aberta , Homologia de Sequência de Aminoácidos , Thermus/enzimologiaRESUMO
Accumulation of free proline (Pro) by bacteria and plants serves as a mechanism protecting these organisms from abiotic stress. We introduced two E. coli genes, proBosm, encoding a mutant variant of the first enzyme of the Pro biosynthetic pathway, gamma-glutamyl kinase, and proA, encoding the second enzyme of the Pro pathway, glutamyl-gamma-semialdehyde dehydrogenase, into tobacco plants. The spontaneous proBosm mutation was selected for Pro overproduction and mapped to the N-terminus of a polypeptide chain; it was found to result from one amino-acid substitution. Two E. coli genes were simultaneously introduced into tobacco plants, each under the control of a strong constitutive CaMV 35S promoter, which contained a duplicated sequence of enhancer, or the root-specific Pmas promoter sequence. Transgenic plants were characterized by Pro overproduction, increased resistance to L-azetidine-2-carboxylic acid (a toxic analog of Pro), and tolerance to salt stress.
Assuntos
Regulação da Expressão Gênica de Plantas , Técnicas de Transferência de Genes , Genes Bacterianos , Nicotiana/genética , Plantas Tóxicas , Prolina/biossíntese , Regiões Promotoras Genéticas , Agrobacterium tumefaciens/genética , Aldeído Oxirredutases/genética , Escherichia coli/genética , Vetores Genéticos , Glutamato-5-Semialdeído Desidrogenase , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Plantas Geneticamente Modificadas , Plasmídeos , Nicotiana/metabolismo , Transformação GenéticaRESUMO
Effects of recombinant bacteria Rhizobium fredii/pNSA, content of proBosm proA operon, and R.fredii/pLVA, content of proBA wild type operon, on soybean plants were studied. R.fredii BD32/pNSA were found to synthesize more proline and to express higher osmotolerance than R.fredii BD32/pLVA or wild type bacteria. Plants infected with R.fredii Bd32/pNSA were characterized by lower water consumption before blooming. Moreover, these bacteria were found to promote more rapid sprout of seeds under unfavorable climatic conditions.
Assuntos
Escherichia coli/genética , Glycine max/microbiologia , Óperon , Rhizobium/genética , Clonagem Molecular , Pressão Osmótica , Plasmídeos , Glycine max/fisiologia , Simbiose , ÁguaRESUMO
We obtained a spontaneous mutant of Escherichia coli that was characterized by both proline superproduction and the resistance to osmotic stress. The selection of mutants was carried out among 2.5.10(5) clones survived upon plating strain SU1604 containing the sex factor F104, with a chromosome fragment carrying genes proB and proA, on solid modium with the proline analogue L-azetidine-2-carboxylate (AzT). The obtained mutant AztR clones were used as donors in replica crossing with a pro- recipient, followed by subsequent selection of AztR Pro+ exconjugants. Analysis of growth of 456 exconjugants in liquid minimal medium with NaCl at a concentration of 0.6 M helped to identify 9 mutants with increased salt tolerance, as compared with control. From these, we selected one mutant (denoted as SU1604/F'104S) which demonstrated the highest salt tolerance correlating with higher production of proline. Analysis of the mutant's properties suggests that it belongs to the group of Osm mutants.
