RESUMO
The effects of two basic cysteine-rich lipid-binding proteins isolated from wheat seedlings, puroindoline-a and alpha1-purothionin, were studied on single frog myelinated axons stained with the fluorescent dye FM1-43 using confocal laser scanning microscopy. During exposure to either puroindoline-a or alpha1-purothionin (10 and 100 microM) a marked swelling of nodes of Ranvier was observed, provided NaCl was present in the external solution. It is suggested that these proteins increase the internal osmolality by forming pores in the axonal membrane and induce water influx to compensate for such an increase. Moreover, in the presence of alpha1-purothionin (100 microM), the intensity of the axonal staining with FM1-43 was increased. It is the first time, to our knowledge, that basic proteins containing domains of a cysteine-rich repeated motif are reported to produce swelling and water movements across neuronal cell membranes.
Assuntos
Axônios/efeitos dos fármacos , Bainha de Mielina/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Nós Neurofibrosos/efeitos dos fármacos , Triticum/química , Animais , Peptídeos Catiônicos Antimicrobianos , Edema/induzido quimicamente , Microscopia Confocal , Rana esculentaRESUMO
In order to study the regulation of the synthesis of glutamine synthetase in response to changes in environmental parameters (light and nitrogen sources), we have cloned and sequenced the glnA gene from the filamentous cyanobacterium Calothrix PCC 7601. This gene consists of 472 codons and encodes a polypeptide of M(r) 52,290 highly homologous to that from Anabaena PCC 7120, but more distant from those identified from other procaryotes. The relative abundance of the two glnA transcripts (1.6 and 1.8 kb) is equivalent in cells grown under either red or green light, but the 1.6-kb species predominates in nitrate-grown cells and the 1.8-kb species in ammonia-grown cells. The very high identity (74%) observed between the 374-bp long nucleotide sequence upstream from the Calothrix and Anabaena glnA genes suggests the existence of similar regulatory signals for the control of glnA expression in both cyanobacteria.
Assuntos
Cianobactérias/genética , Genes Bacterianos , Glutamato-Amônia Ligase/genética , Sequência de Aminoácidos , Bactérias/enzimologia , Bactérias/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Biblioteca Genômica , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
In order to optimise new polypeptide based biomaterials, we developed a procedure for producing homoblock polypeptides using recombinant DNA technology. Synthetic genes encoding periodic polypeptides modelled on the consensus sequence of wheat gliadins (a family of wheat storage proteins) were devised to be expressed in Escherichia coli. The construction strategy followed allows the construction of three genes encoding 8, 16, and 32 copies of the PQQPY module. The optimal expression conditions in the enterobacteria were established and a convenient purification procedure was shown to be useful in recovery of sizable amounts of strictly periodic polypeptides. The identities of the synthesized polypeptides were assessed using positive cross reactions to antibodies raised against a synthetic decapeptide (PQQPYPQQPA) and amino acid composition was determined as well.
Assuntos
Genes Sintéticos , Gliadina/genética , Triticum/genética , Sequência de Aminoácidos , Sequência de Bases , Sequência Consenso , Escherichia coli , Dados de Sequência Molecular , Polímeros , Proteínas Recombinantes/química , Sequências Repetitivas de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
Among a wide range of potential selective agents examined, sodium p-hydroxymercuribenzoate successfully enriched for mutants of Synechocystis sp. strains PCC 6714 and 6803 defective in photosynthesis. When both photosystems I and II were operating, viability of wild-type cells decreased to between 5 X 10(-5) and 1 X 10(-6) after 5 h of incubation with 500 microM p-hydroxymercuribenzoate (strain 6714), and after 8 h with 200 microM (strain 6803). Between 0.1 and 0.5% of the survivors were stable mutants defective in different steps of photosynthesis. The compound was not mutagenic. It was less toxic to cells grown chemoheterotrophically in the dark or photoheterotrophically in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. p-Hydroxymercuribenzoate therefore killed only cells which were performing photosynthesis at high rates, thereby specifically selecting for mutants deficient in this process.