RESUMO
We developed an insertion sequence transposition detection system called the "jumping cat assay" and applied it to the Bacillus subtilis chromosome using IS256Bsu1 derived from B. subtilis natto. The high frequency of transposition enabled us to explore host factors; combining the assay and genetic analyses revealed that recA is essential for the transposition of IS256Bsu1. Detailed analyses using various domain mutants of recA demonstrated that this essentiality is not related to the function of recA in homologous recombination. Instead, the ATP binding and hydrolysis function seemed to be crucial for IS transposition. To elucidate the role of recA, we focused on the muB gene of the enterobacteriophage Mu. Based on information from the NCBI Conserved Domain Database, both MuB and RecA belong to the P-loop dNTPase superfamily. Further experiments revealed that muB complements the transposition-defective phenotype of a recA deletant, although it could not rescue UV sensitivity. These results suggest that recA shares a common function with muB that helps the transposition of IS256Bsu1 in B. subtilis.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Recombinases Rec A/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga , Mutação , Ligação Proteica , Recombinases Rec A/genética , Proteínas Virais/genéticaRESUMO
Polycystic kidney disease (PKD) 2L1 protein is a member of the transient receptor potential (TRP) ion channel family. In circumvallate and foliate papillae, PKD2L1 is coexpressed with PKD1L3. PKD2L1 and PKD1L3 interact through their transmembrane domain and the resulting heteromer PKD1L3/PKD2L1 owns a unique channel property called 'off-responses' to acid stimulation, although PKD2L1 does not own this property by itself. To define the pharmacological properties of the PKD1L3/PKD2L1 channel, we developed a new method to effectively evaluate channel activity using human embryonic kidney 293T cells in which the channel was heterologously expressed. This method was applied to screen substances that potentially regulate it. We found that capsaicin and its analogs, which are TRPV1 agonists, inhibited the response to acid stimuli and that the capsaicin inhibition was reversible with an IC(50) of 32.5 µm. Capsaicin and its analogs are thus useful tools for physiological analysis of PKD1L3/PKD2L1 function.
Assuntos
Capsaicina/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPP/antagonistas & inibidores , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPP/metabolismo , Papilas Gustativas/efeitos dos fármacos , Língua/metabolismoRESUMO
Acetic acid induces unique physiological responses in mammalian cells. Our previous study found that fura-2-loaded human embryonic kidney (HEK) 293T cells showed a robust intracellular fluorescence response immediately after stimulation with acetic acid, and no such response in the case of citric acid. In the present study, we aimed to identify the unique characteristics of acetic acid responsible for this phenomenon. We found that one such feature is its hydrophobicity. We also discovered that acetic acid induces cell responses by intracellular acidification. Of the components of acetic acid in solution (protons, acetate ions, and undissociated acetic acid), undissociated acetic acid might be the functional unit that penetrates the lipid bilayer of cell membranes to acidify the intracellular environment, thereby inducing cell responses. The method used in this study might be convenient in evaluating the intracellular acidification of cultured cells by acids in the external environment.
Assuntos
Ácido Acético/metabolismo , Membrana Celular/metabolismo , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Ácido Acético/química , Ácido Acético/farmacologia , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Espaço Intracelular/química , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Permeabilidade/efeitos dos fármacosRESUMO
The RpoH in Acetobacter pasteurianus NBRC3283 was characterized. It was revealed that the rpoH controls the expression of groEL, dnaKJ, grpE, and clpB to different extents. In addition, the rpoH disruption mutant became apt to be affected by heat, ethanol, and acetic acid, indicating its importance in acetic acid fermentation.
Assuntos
Acetobacter/genética , Proteínas de Bactérias/fisiologia , Proteínas de Choque Térmico/fisiologia , Fator sigma/fisiologia , Ácido Acético/metabolismo , Acetobacter/crescimento & desenvolvimento , Acetobacter/metabolismo , Proteínas de Bactérias/genética , Etanol/metabolismo , Fermentação , Proteínas de Choque Térmico/genética , Temperatura Alta , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fator sigma/genética , Estresse FisiológicoRESUMO
We have reported that acetic acid (AcOH) intake suppresses body fat mass and up-regulates the genes involved in fatty acid oxidation, but it is not clear whether the suppression of body fat mass by AcOH administration is due to an increase in energy expenditure (EE). In this study, we investigated to determine whether a single oral administration of AcOH would increase EE in C57BL/6J mice treated with 1.5% AcOH. The AcOH treatment group had significantly higher oxygen consumption (VO(2)), EE, and fat oxidation (FAT) than the water treatment group. These results suggest that a single administration of AcOH increases EE, resulting in suppression of body fat mass.