Post-Franco Theatre

In the multiple realms and layers that comprise the contemporary Spanish theatrical landscape, “crisis” would seem to be the word that most often lingers in the air, as though it were a common mantra, ready to roll off the tongue of so many theatre professionals with such enormous ease, and even enthusiasm, that one is prompted to wonder whether it might indeed be a miracle that the contemporary technological revolution – coupled with perpetual quandaries concerning public and private funding for the arts – had not by now brought an end to the evolution of the oldest of live arts, or, at the very least, an end to drama as we know it

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Producción de Giberelinas por Gibberella Fujikuroi

En esta tesis se ha desarrollado un método simplificado para medir Giberelinas en el medio de cultivo de Gibberella fujikuroi y otro para escrutar cultivos y detectar mutantes de Gibberella alterados en la síntesis de Giberelinas. A partir de es tos méto

Gibberellins and Carotenoids in the Wild Type and Mutants of Gibberella fujikuroi

A new screening procedure was used to isolate 14 gib mutants of Gibberella fujikuroi with modifications in the production of gibberellins. The production of carotenoids and gibberellins was investigated in the gib mutants and in representative car mutants with various modifications of carotenoid biosynthesis. The determinations of gibberellins were carried out with a simplified fluorescence method. One of the mutants lacked both gibberellins and carotenoids. In many mutants the two pathways compensated each other: an increase in the production of one group of compounds was accompanied by a decrease in the production of the other. Under certain conditions the compensation was quantitative when the output of the two pathways was measured in moles of the common precursor, geranylgeranyl pyrophosphate. α-Picoline, an inhibitor of lycopene cyclase in G. fujikuroi, inhibits gibberellin biosynthesis. Other agents that affect the accumulation of carotenoids have no noticeable effect on the accumulation of gibberellins; such is the case with diphenylamine and β-ionone, two inhibitors of phytoene dehydrogenation, and visible light, which stimulates carotenogenesis

The Presence of Glutamate Dehydrogenase Is a Selective Advantage for the Cyanobacterium Synechocystis sp. Strain PCC 6803 under Nonexponential Growth Conditions

The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 has two putative pathways for ammonium assimilation: the glutamine synthetase-glutamate synthase cycle, which is the main one and is finely regulated by the nitrogen source; and a high NADP-dependent glutamate dehydrogenase activity (NADP-GDH) whose contribution to glutamate synthesis is uncertain. To investigate the role of the latter, we used two engineered mutants, one lacking and another overproducing NADP-GDH. No major disturbances in the regulation of nitrogen-assimilating enzymes or in amino acids pools were detected in the null mutant, but phycobiline content, a sensitive indicator of the nutritional state of cyanobacterial cells, was significantly reduced, indicating that NADP-GDH plays an auxiliary role in ammonium assimilation. This effect was already prominent in the initial phase of growth, although differences in growth rate between the wild type and the mutants were observed at this stage only at low light intensities. However, the null mutant was unable to sustain growth at the late stage of the culture at the point when the wild type showed the maximum NADP-GDH activity, and died faster in ammonium-containing medium. Overexpression of NADP-GDH improved culture proliferation under moderate ammonium concentrations. Competition experiments between the wild type and the null mutant confirmed that the presence of NADP-GDH confers a selective advantage to Synechocystis sp. strain PCC 6803 in late stages of growth

Effect of Glucose Utilization on Nitrite Excretion by the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6803

Up to 1 mM nitrite was excreted by Synechocystis strain 6803 cells growing under mixotrophic or photoheterotrophic conditions. This excretion is not due to a lower ratio of nitrite and nitrate reductase activities in the presence of glucose but seems to be related to a shortage of reduced ferredoxin, their electron donor, as a result of a decrease in noncyclic photosynthetic flow observed under these circumstances. Because about 60% of the reduced nitrate is excreted, the potential utilization of cyanobacteria for removal of nitrate from contaminated waters containing high concentrations of organic compounds is questioned

Effect of glucose utilization on nitrite excretion by the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Up to 1 mM nitrite was excreted by Synechocystis strain 6803 cells growing under mixotrophic or photoheterotrophic conditions. This excretion is not due to a lower ratio of nitrite and nitrate reductase activities in the presence of glucose but seems to be related to a shortage of reduced ferredoxin, their electron donor, as a result of a decrease in noncyclic photosynthetic flow observed under these circumstances. Because about 60% of the reduced nitrate is excreted, the potential utilization of cyanobacteria for removal of nitrate from contaminated waters containing high concentrations of organic compounds is questioned.This work has been financed by the Direccion General de Investigaci6n Cientifica y T6cnica (grants PB88-0020 and PB91-0127), Spain, and by the Junta de Andalucia

Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein

RNA polymerase II transcription requires functional interactions between activator proteins bound to upstream DNA sites and general factors bound to the core promoter. Accessory transcription factors, such as adaptors and coactivators, have important, but still unclear, roles in the activation process. We tested physical interactions of the putative adaptor ADA2 with activation domains derived from acidic activator proteins and with certain general transcription factors. ADA2 associated with the herpesvirus VP16 and yeast GCN4 activation domains but not with the activation domain of yeast HAP4, which previously was shown to be independent of ADA2 function in vivo and in vitro. Furthermore, the amino terminus of ADA2 directly interacted with the VP16 activation domain, suggesting that ADA2 provides determinants for interaction between activation domains and the adaptor complex. Both TATA-binding protein (TBP) and TFIIB have previously been shown to interact directly with the VP16 activation domain in vitro (Stringer, K. F., Ingles, C. J., and Greenblatt, J. (1990) Nature 345, 783-786; Lin, Y. S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1991) Nature 353, 569-571). Interestingly, when binding was tested between VP16 and these general factors in yeast nuclear extracts, both factors interacted with VP16, but only the TBP/VP16 association was dependent on ADA2. In addition, ADA2 physically associated with TBP, but not with TFIIB. These results suggest that the role of ADA2 in transcriptional activation is to promote physical interaction between activation domains and TBP