The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study selective macroautophagy in mammalian cells.     

Rm62, a DEAD‐box RNA helicase,complexes with DSP1 in Drosophila embryos

Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB‐like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD‐box RNA helicase, Rm62, as partner of DSP1 in a 250‐kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3‐ to 12‐h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. We show here for the first time that an RNA helicase is involved in the maintenance of homeotic genes. genesis 48:244–253, 2010. © 2010 Wiley‐Liss, Inc.     

Epiphytic fitness of a biological control agent of fire blight in apple and pear orchards under Mediterranean weather conditions

The behaviour of Pseudomonas fluorescens EPS62e was investigated in apple and pear orchards under Mediterranean climatic conditions. The trials studied the influence of weather conditions, plant host species, presence of indigenous microbial community and spread from treated to nontreated trees on colonization and survival. Population dynamics were assessed by real-time PCR and CFU-counting methods. With inoculated flowers, weather conditions were optimal for colonization, and EPS62e established high and stable population levels around 10(8) CFU per organ, according to both methods of analysis. The plant host species did not influence the colonization rate, and the biocontrol agent dominated the microbial communities of blossoms, representing up to 100% of the total cultivable population. With inoculated leaves, the EPS62e population decreased to nondetectable levels 30 days after treatment according to both methods used. EPS62e spread moderately in the orchard, being detected in nontreated flowers of trees 15-35 m from the inoculation site. The combined use of real-time PCR and CFU-counting methods of analysis permitted the identification of three physiological states for EPS62e in the field, which consisted of active colonization, survival and entry into a viable but nonculturable state, and cell death.     

Black soybean extract can attenuate thrombosis through inhibition of collagen-induced platelet activation

Many clinical trials have demonstrated the beneficial effects of soybean (Glycine max) on general cardiovascular health. Among a variety of soybeans, black soybean is known to display diverse biological activities superior to those of yellow and green soybeans, such as in antioxidant, anti-inflammatory and anticancer activities. However, few studies have been directed on the effect of black soybean on cardiovascular function. In this study, we aimed to investigate the effect of black soybean extract (BB) on platelet activation, a key contributor to thrombotic diseases. In freshly isolated human platelets, BB has shown potent inhibitory activity on collagen-induced platelet aggregation, while yellow soybean extract had marginal activity only. BB also attenuated serotonin secretion and P-selectin expression, which are important factors for the platelet–tissue interaction along with thromboxane A₂ formation. These in vitro results were further confirmed in an ex vivo platelet aggregation measurement and in vivo venous thrombosis model where oral administration of BB reduced collagen-induced platelet aggregation and FeCl₃-induced thrombus formation significantly. A potential active ingredient for antiplatelet effects of BB was isolated and identified to be adenosine through bioassay-directed fractionation and NMR and ESI-MS analyses. These results indicate that black soybean can be a novel dietary supplement for the prevention of cardiovascular risks and the improvement of blood circulation.     

Is population growth conducive to the sustainability of cooperation?

This paper asks whether population growth is conducive to the sustainability of cooperation. A simple model is developed in which farmers who live around a circular lake engage in trade with their adjacent neighbors. The payoffs from this activity are governed by a prisoner's dilemma “rule of engagement.” Every farmer has one son when the population is not growing, or two sons when it is growing. In the former case, the son takes over the farm when his father dies. In the latter case, one son stays on his father's farm, whereas the other son settles around another lake, along with the “other” sons of the other farmers. During his childhood, each son observes the strategies and the payoffs of his father and of the trading partners of his father, and imitates the most successful strategy when starting farming on his own. Then mutant defectors are introduced into an all-cooperator community. The defector strategy may spread. A comparison is drawn between the impact in terms of the sustainability of cooperation of the appearance of the mutants in a population that is not growing, and in one that is growing. It is shown that the ex-ante probability of sustaining the cooperation strategy is higher for a community that is growing than for a stagnant community.     

Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight

Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU(gf.w.)(-1), which in turn decreased progressively to 4-5 log CFU(gf.w.)(-1) after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU(gf.w.)(-1), thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.