RESUMO
A set of vectors for Gram-positive bacteria was constructed with a new feature which enables the switching down of their copy number per cell. These vectors carry the replication region of pAM beta 1, containing a gene essential for replication, repE, and its regulator, copF. The latter gene was inactivated by inserting a linker into its unique KpnI site. Since copF downregulates the expression of repE, its inactivation leads to an increase in the plasmid copy number per cell. The original low copy state can be restored by removal of the linker via KpnI cleavage and ligation. The new replicon was used to build (i) vectors for studying gene regulation by transcriptional or translational fusion with the bacterial luciferase gene, (ii) vectors for gene expression, and (iii) cassettes of the replicon with different multiple cloning sites, which would facilitate construction of vectors for novel purposes.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Dosagem de Genes , Vetores Genéticos/genética , Bactérias Gram-Positivas/genética , Plasmídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular/métodos , Replicação do DNA/genética , DNA Recombinante , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão , Proteínas Repressoras/genéticaRESUMO
The action of amiodarone (1.5 X 10(-5) M) on sinus node activity of spontaneously beating isolated right atria of rabbit at 30 degrees C was investigated using a microelectrode technique. The drug significantly increased the action potential duration and decreased the slope of diastolic depolarization, both effects leading to a reduction of the sinus rate. In contrast to beta-blocking agents, amiodarone reduced but did not completely abolish the adrenergic effects on the sinus node activity. It is concluded that the amiodarone-induced bradycarida observed in clinical trials might be due to a direct effect of the drug on the sinus node.
Assuntos
Amiodarona/farmacologia , Benzofuranos/farmacologia , Nó Sinoatrial/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Interações Medicamentosas , Epinefrina/farmacologia , Feminino , Frequência Cardíaca/efeitos dos fármacos , Técnicas In Vitro , Masculino , Microeletrodos , Norepinefrina/farmacologia , Propranolol/farmacologia , CoelhosRESUMO
The effect of the replacement of extracellular chloride by methylsulphate was investigated on isolated frog atrial trabeculae using the voltage clamp technique. In methylsulphate solution, the following results were obtained: 1. After a transient depolarization of the resting potential, the action potential was lengthened and the diastolic depolarization rate of the repetitive activity was reduced. 2. In voltage clamp conditions, the current-voltage relationships and the steady-state inactivation curves of both early and secondary inward currents were unaffected. 3. The study of the evolution of the instantaneous backgroundd current with membrane potential showed a decrease of the current values. 4. The first component of the time-dependent outward currents was unaffected but the second component was reduced without change of its time-constant value. Assuming the absolute value of the chloride equilibrium potential at about -30 mV, it was suggested that the lengthening of the action potential in methylsulphate solution might be explained by the decrease of an influx of chloride ions which should participate in the repolarization. The reduction of the diastolic depolarization rate might be due to the decrease of an outflux of chloride ions which should be involved in the inward background currents unmasked by the deactivation of the time-dependent outward currents.
Assuntos
Cloretos/fisiologia , Ácidos Sulfúricos/fisiologia , Malha Trabecular/fisiologia , Potenciais de Ação , Animais , Anuros , Função Atrial , Membrana Celular/fisiologia , Potenciais da Membrana , Rana esculenta , Fatores de TempoRESUMO
Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.
Assuntos
Diacetil/metabolismo , Lactococcus lactis/metabolismo , Leucina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Carboxiliases/genética , Primers do DNA/genética , DNA Bacteriano/genética , Genes Bacterianos , Lactatos/metabolismo , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Dados de Sequência Molecular , MutaçãoRESUMO
Developing nurses' ability to understand and evaluate research articles for potential application to practice is a challenge for staff development professionals. Articles are laden with difficult terminology and may seem too difficult. Yet valuable insights for improving patient care rest within the literature. In this article, the authors describe an instructional tool, designed in the federal nursing sector, called Critical Reading of Research Publications (CRRP). The CRRP is an independent study program that helps small groups of nurses increase research understanding while reading relevant articles of clinical interest.
Assuntos
Educação Continuada em Enfermagem/métodos , Pesquisa em Enfermagem , Leitura , Humanos , Instruções Programadas como AssuntoRESUMO
The genes for the biosynthesis of histidine, tryptophan and branched-chain amino acids (ilv for isoleucine, leucine and valine) are clustered in large operons. In addition to genes encoding the pathway enzymes, the his and the ilv operons contain 4 and 3 other genes, respectively. The functions of two of these, orf3 and aldB are regulatory. The second gene of the his operon, orf3, encodes a protein homologous to the histidyl-tRNA synthetases. It is involved in transcription attenuation upstream of the his operon. This regulation is related to a new class of attenuation mechanisms controlling the expression of most tRNA synthetase genes and a few metabolic operons in Gram-positive bacteria. Gene aldB, the penultimate gene of the ilv operon, encodes acetolactate decarboxylase. This enzyme transforms acetolactate (AL), the first intermediate of leucine and valine biosynthesis, into acetoin. AL decarboxylase is positively controlled by the availability of leucine and possibly valine in the cell. This is the key enzyme for a new class of regulatory mechanisms, a metabolic shunt which guides the AL flux towards synthesis of amino acids or a secondary metabolite.
Assuntos
Aminoácidos/biossíntese , Aminoácidos/genética , Genes Bacterianos , Lactococcus/genética , Lactococcus/metabolismo , Óperon , Sequência de Aminoácidos , Aminoácidos de Cadeia Ramificada/biossíntese , Aminoácidos de Cadeia Ramificada/genética , Mapeamento Cromossômico , Regulação Bacteriana da Expressão Gênica , Histidina/biossíntese , Histidina/genética , Histidina-tRNA Ligase/genética , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
A double sucrose gap voltage clamp technic has been used to study the extra-current induced by acetylcholine (Iach) on the myocardial membrane on frog atrial trabeculae. I) No desensitization of the Iach current is noted for repeated perfusions of Ach. II) The Iach current is suppressed by atropine. III) The reversal potential Each is more negative than the resting potential --20 mV less than or equal to Each less than or equal to OmV.IV) The relationship Iach/Holding potential for various [K+]o shows a) That Each behaved as a potassium electrode, b) an inward going rectification. These results indicate that the cholinergic receptor might be related with the gk1 channel.