RESUMEN
Neurospora has proven to be a tractable model system for understanding the molecular bases of circadian rhythms in eukaryotes. At the core of the circadian oscillatory system is a negative feedback loop in which two transcription factors, WC-1 and WC-2, act together to drive expression of the frq gene. WC-2 enters the promoter region of frq coincident with increases in frq expression and then exits when the cycle of transcription is over, whereas WC-1 can always be found there. FRQ promotes the phosphorylation of the WCs, thereby decreasing their activity, and phosphorylation of FRQ then leads to its turnover, allowing the cycle to reinitiate. By understanding the action of light and temperature on frq and FRQ expression, the molecular basis of circadian entrainment to environmental light and temperature cues can be understood, and recently a specific role for casein kinase 2 has been found in the mechanism underlying circadian temperature-compensation. These data promise molecular explanations for all of the canonical circadian properties of this model system, providing biochemical answers and regulatory logic that may be extended to more complex eukaryotes including humans.
Asunto(s)
Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Neurospora/genética , Neurospora/fisiología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Retroalimentación Fisiológica , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Humanos , Modelos Biológicos , Fotobiología , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/fisiología , Temperatura , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Circadian output comprises the business end of circadian systems in terms of adaptive significance. Work on Neurospora pioneered the molecular analysis of circadian output mechanisms, and insights from this model system continue to illuminate the pathways through which clocks control metabolism and overt rhythms. In Neurospora, virtually every strain examined in the context of rhythms bears the band allele that helps to clarify the overt rhythm in asexual development. Recent cloning of band showed it to be an allele of ras-1 and to affect a wide variety of signaling pathways yielding enhanced light responses and asexual development. These can be largely phenocopied by treatments that increase levels of intracellular reactive oxygen species. Although output is often unidirectional, analysis of the prd-4 gene provided an alternative paradigm in which output feeds back to affect input. prd-4 is an allele of checkpoint kinase-2 that bypasses the requirement for DNA damage to activate this kinase; FRQ is normally a substrate of activated Chk2, so in Chk2(PRD-4), FRQ is precociously phosphorylated and the clock cycles more quickly. Finally, recent adaptation of luciferase to fully function in Neurospora now allows the core FRQ/WCC feedback loop to be followed in real time under conditions where it no longer controls the overt rhythm in development. This ability can be used to describe the hierarchical relationships among FRQ-Less Oscillators (FLOs) and to see which are connected to the circadian system. The nitrate reductase oscillator appears to be connected, but the oscillator controlling the long-period rhythm elicited upon choline starvation appears completely disconnected from the circadian system; it can be seen to run with a very long noncompensated 60-120-hour period length under conditions where the circadian FRQ/WCC oscillator continues to cycle with a fully compensated circadian 22-hour period.
Asunto(s)
Ritmo Circadiano/fisiología , Neurospora crassa/fisiología , Ritmo Circadiano/genética , Retroalimentación Fisiológica , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Genes Fúngicos , Modelos Biológicos , Neurospora crassa/genética , Neurospora crassa/crecimiento & desarrollo , PeriodicidadRESUMEN
We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.
Asunto(s)
Basidiomycota/enzimología , Peroxidasas/genética , Peroxidasas/metabolismo , Phanerochaete/enzimología , Aspergillus nidulans/enzimología , Aspergillus nidulans/genética , Basidiomycota/genética , Activación Enzimática , Glicosilación , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Manganeso/farmacología , Oxalatos/farmacología , Peroxidasas/aislamiento & purificación , Phanerochaete/genética , Proteínas Recombinantes/metabolismo , Transformación GenéticaRESUMEN
One of the theories involved in the etiology of Alzheimer's disease (AD) is the oxidative stress hypothesis. The amyloid beta-peptide (A beta), a hallmark in the pathogenesis of AD and the main component of senile plaques, generates free radicals in a metal-catalyzed reaction inducing neuronal cell death by a reactive oxygen species mediated process which damage neuronal membrane lipids, proteins and nucleic acids. Therefore, the interest in the protective role of different antioxidants in AD such as vitamin E, melatonin and estrogens is growing up. In this review we summarize data that support the involvement of oxidative stress as an active factor in A beta-mediated neuropathology, by triggering or facilitating neurodegeneration, through a wide range of molecular events that disturb neuronal cell homeostasis.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Estrés Oxidativo/fisiología , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/toxicidad , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Humanos , Lípidos de la Membrana/metabolismo , Metales/metabolismo , Fármacos Neuroprotectores/farmacología , Ácidos Nucleicos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/inmunología , Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Three new genes (Cs-mnp2A, Cs-mnp2B and Cs-mnp3) coding for manganese-dependent peroxidase (MnP) have been identified in the white-rot basidiomycete Ceriporiopsis subvermispora. The mature proteins contain 366 (MnP2A and MnP2B) and 364 (MnP3) amino acids, which are preceded by leader sequences of 21 and 24 amino acids, respectively. Cs-mnp2A and Cs-mnp2B appear to be alleles, since the corresponding protein sequences differ in only five residues. The upstream region of Cs-mnp2B contains a TATA box, AP-1 and AP-2 sites, as well as sites for transcription regulation by metals (two), cAMP (two) and xenobiotics (one). Some of these elements are also found in the regulatory region of Cs-MnP3. Transcription of Cs-mnp2A and Cs-mnp2B, but not that of Cs-mnp3, is activated by manganese.