RESUMEN
Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S. cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to â¼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S. pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S. pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.
Asunto(s)
NADH NADPH Oxidorreductasas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Cromatografía Liquida , NAD/metabolismo , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/aislamiento & purificación , NADH NADPH Oxidorreductasas/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/aislamiento & purificación , Proteínas de Schizosaccharomyces pombe/metabolismo , Espectrometría de Masas en Tándem , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMEN
Identification of biologically active natural compounds that promote health and longevity, and understanding how they act, will provide insights into aging and metabolism, and strategies for developing agents that prevent chronic disease. The garlic-derived thioallyl compounds S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) have been shown to have multiple biological activities. Here we show that SAC and SAMC increase lifespan and stress resistance in Caenorhabditis elegans and reduce accumulation of reactive oxygen species (ROS). These compounds do not appear to activate DAF-16 (FOXO orthologue) or mimic dietary restriction (DR) effects, but selectively induce SKN-1 (Nrf1/2/3 orthologue) targets involved in oxidative stress defense. Interestingly, their treatments do not facilitate SKN-1 nuclear accumulation, but slightly increased intracellular SKN-1 levels. Our data also indicate that thioallyl structure and the number of sulfur atoms are important for SKN-1 target induction. Our results indicate that SAC and SAMC may serve as potential agents that slow aging.
Asunto(s)
Envejecimiento/efectos de los fármacos , Proteínas de Caenorhabditis elegans/biosíntesis , Cisteína/análogos & derivados , Proteínas de Unión al ADN/biosíntesis , Longevidad/genética , Factores de Transcripción/biosíntesis , Envejecimiento/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/genética , Cisteína/administración & dosificación , Cisteína/química , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/biosíntesis , Ajo/química , Regulación de la Expresión Génica/efectos de los fármacos , Longevidad/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
Calcineurin, which is a Ca(2+)/calmodulin-dependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcineurin is activated, inhibiting cellular growth as a result, we screened for a mutant(s) whose temperature sensitivity would be suppressed by FK506 from the budding yeast non-essential gene deletion library. We found that the temperature sensitivity of cells in which the conserved Verprolin VRP1 gene had been deleted, which gene is required for actin organization and endocytosis, was suppressed by either FK506 or by cnb1 deletion. Indeed, the calcineurin activity increased significantly in the ∆vrp1 cells. Finally, we demonstrated that the ∆vrp1 strain to be useful as an indicator in a positive screening for bioactive compounds inhibiting calcineurin.
Asunto(s)
Inhibidores de la Calcineurina/farmacología , Proteínas de Microfilamentos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tacrolimus/farmacología , Calcineurina/genética , Calcineurina/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Eliminación de Gen , Proteínas de Microfilamentos/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMEN
We applied Chrysanthemum flower oil (CFO) to a hyperuricemia model by feeding rats a hyperuricemia-inducing diet (HID) and investigated its effect on serum uric acid (SUA) levels and its mode of action. CFO is the oily fraction that contains polyphenols derived from chrysanthemum flowers. Oral administration of CFO to HID-fed rats significantly decreased their SUA levels. It also inhibited xanthine oxidase activities in the liver and increased urine uric acid levels. The effects of CFO on the renal gene expressions that accompanied the induction of hyperuricemia were comprehensively confirmed by DNA microarray analysis. The analysis showed up-regulation of those genes for uric acid excretion by CFO administration. These results suggest that CFO suppresses the increase in SUA levels via two mechanisms: suppression of uric acid production by inhibition of xanthine oxidase in the liver and acceleration of its excretion by up-regulation of uric acid transporter genes in the kidney.
Asunto(s)
Chrysanthemum/química , Flores/química , Hiperuricemia/tratamiento farmacológico , Hiperuricemia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Aceites de Plantas/farmacología , Animales , Bovinos , Hiperuricemia/sangre , Hiperuricemia/enzimología , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Aceites de Plantas/administración & dosificación , Aceites de Plantas/uso terapéutico , Ratas , Ácido Úrico/sangre , Xantina Oxidasa/metabolismoRESUMEN
We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.
Asunto(s)
Basófilos/citología , Degranulación de la Célula , Fermentación , Inmunoglobulina E/inmunología , Lactobacillus/metabolismo , Rinitis/prevención & control , Vino , Animales , Basófilos/inmunología , Línea Celular Tumoral , Suplementos Dietéticos , Femenino , Hipersensibilidad/complicaciones , Ratones , Ratas , Rinitis/complicaciones , Rinitis/inmunologíaRESUMEN
Because Ca(2+) signaling of budding yeast, through the activation of calcineurin and the Mpk1/Slt2 mitogen-activated protein kinase cascade, performs redundant function(s) in the events essential for growth, the simultaneous deletion of both these pathways (Delta cnb1 Delta mpk1) leads to lethality. A PTC4 cDNA that encodes a protein phosphatase belonging to the PP2C family was obtained as a high dosage suppressor of the lethality of Delta cnb1 Delta mpk1 strain. Overexpression of PTC4 led to a decrease in the high osmolarity-induced Hog1 phosphorylation, and HOG1 deletion remarkably suppressed the synthetic lethality, indicating an antagonistic role of the high osmolarity glycerol (HOG) pathway and the Ca(2+) signaling pathway in growth regulation. The calcineurin-Crz1 pathway was required for the down-regulation of the HOG pathway. Analysis of the time course of actin polarization, bud formation, and the onset of mitosis in synchronous cell cultures demonstrated that calcineurin negatively regulates actin polarization at the bud site, whereas the HOG pathway positively regulates bud formation at a later step after actin has polarized.