A Calcineurin-dependent Switch Controls the Trafficking Function of α-Arrestin Aly1/Art6

Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca²⁺- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.     

LDL particle core enrichment in cholesteryl oleate increases proteoglycan binding and promotes atherosclerosis

Several studies in humans and animals suggest that LDL particle core enrichment in cholesteryl oleate (CO) is associated with increased atherosclerosis. Diet enrichment with MUFAs enhances LDL CO content. Steroyl O-acyltransferase 2 (SOAT2) is the enzyme that catalyzes the synthesis of much of the CO found in LDL, and gene deletion of SOAT2 minimizes CO in LDL and protects against atherosclerosis. The purpose of this study was to test the hypothesis that the increased atherosclerosis associated with LDL core enrichment in CO results from an increased affinity of the LDL particle for arterial proteoglycans. ApoB-100-only Ldlr^−/− mice with and without Soat2 gene deletions were fed diets enriched in either cis-MUFA or n-3 PUFA, and LDL particles were isolated. LDL:proteogylcan binding was measured using surface plasmon resonance. Particles with higher CO content consistently bound with higher affinity to human biglycan and the amount of binding was shown to be proportional to the extent of atherosclerosis of the LDL donor mice. The data strongly support the thesis that atherosclerosis was induced through enhanced proteoglycan binding of LDL resulting from LDL core CO enrichment.     

Whole genome comparison of six Crocosphaera watsonii strains with differing phenotypes

Crocosphaera watsonii, a unicellular nitrogen‐fixing cyanobacterium found in oligotrophic oceans, is important in marine carbon and nitrogen cycles. Isolates of C. watsonii can be separated into at least two phenotypes with environmentally important differences, indicating possibly distinct ecological roles and niches. To better understand the evolutionary history and variation in metabolic capabilities among strains and phenotypes, this study compared the genomes of six C. watsonii strains, three from each phenotypic group, which had been isolated over several decades from multiple ocean basins. While a substantial portion of each genome was nearly identical to sequences in the other strains, a few regions were identified as specific to each strain and phenotype, some of which help explain observed phenotypic features. Overall, the small‐cell type strains had smaller genomes and a relative loss of genetic capabilities, while the large‐cell type strains were characterized by larger genomes, some genetic redundancy, and potentially increased adaptations to iron and phosphorus limitation. As such, strains with shared phenotypes were evolutionarily more closely related than those with the opposite phenotype, regardless of isolation location or date. Unexpectedly, the genome of the type‐strain for the species, C. watsonii WH8501, was quite unusual even among strains with a shared phenotype, indicating it may not be an ideal representative of the species. The genome sequences and analyses reported in this study will be important for future investigations of the proposed differences in adaptation of the two phenotypes to nutrient limitation, and to identify phenotype‐specific distributions in natural Crocosphaera populations.     

Improved in vitro bioassay for Musa acuminata cv. Pisang ambon kuning (AAA group) based on quantitative analysis of necrosis area and biomass changes during Foc4 infection

Abstract

Efforts in improving banana plants that are resistant to the Fusarium wilt-causing Fusarium oxysporum f.sp. cubense tropical race 4 (Foc4) are indispensable. In this study, we developed rapid, space-efficient in vitro bioassay for assessing banana plant resistance to Foc4 using 35?×?150?mm glass test tubes, followed by quantitative and objective analysis of necrosis area and biomass changes as represented by fresh weight changes. Disease resistance screening was conducted based on the necrosis area as quantified using ImageJ software and on biomass gain during in vitro bioassay. In vitro banana plantlets showed age-related resistance during the development of necrosis (p?=?.034, Kruskal–Wallis test in root and shoot system and p?=?.027, one-way ANOVA in shoot system only), in which plantlets that were infected at the youngest age (24 weeks’ post-initiation) showed the largest necrosis area (up to 46.6%). In addition, plant fresh weight gain in this group (0.233?±?0.041?g) was higher compared to the gains in older plantlets (0.079?±?0.117 and 0.009?±?0.069?g, infected at 28 and 38?weeks’ post-initiation, respectively). Overall, for consistent and reliable result, the age of banana plantlet should be taken into consideration in interpreting the result of this in vitro bioassay.     

Micromonospora is a normal occupant of actinorhizal nodules

Actinorhizal plants have been found in eight genera belonging to three orders (Fagales, Rosales and Cucurbitales). These all bear root nodules inhabited by bacteria identified as the nitrogen-fixing actinobacterium Frankia. These nodules all have a peripheral cortex with enlarged cells filled with Frankia hyphae and vesicles. Isolation in pure culture has been notoriously difficult, due in a large part to the growth of fast-growing contaminants where, it was later found, Frankia was slow-growing. Many of these contaminants, which were later found to be Micromonospora, were obtained from Casuarina and Coriaria. Our study was aimed at determining if Micromonospora were also present in other actinorhizal plants. Nodules from Alnus glutinosa, Alnus viridis, Coriaria myrtifolia, Elaeagnus x ebbingei, Hippophae rhamnoides, Myrica gale and Morella pensylvanica were tested and were all found to contain Micromonospora isolates. These were found to belong to mainly three species: Micromonospora lupini, Micromonospora coriariae and Micromonospora saelicesensis. Micromonospora isolates were found to inhibit some Frankia strains and to be innocuous to other strains.     

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s

Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (K _M of 13.8 μM and k _cat of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264.     

Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors

GABA_A receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α₅ has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α₅ subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α₅ subunit-containing GABA_A receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α_4/6 subunit-containing GABA_A receptors, but is sensitive to L-655708, an antagonist of the GABA_A receptors containing α₅ subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α₅ subunit in the turtle spinal cord. Together, these results suggest that GABA_A receptors containing the α₅ subunit mediate the tonic inhibitory currents observed in VHIs.     

Application of a high-throughput fluorescent acetyltransferase assay to identify inhibitors of homocitrate synthase

Homocitrate synthase (HCS) catalyzes the first step of l-lysine biosynthesis in fungi by condensing acetyl-coenzyme A and 2-oxoglutarate to form 3R-homocitrate and coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of approximately 41,000 small molecules. Following confirmation, counter screens, and dose–response analysis, we prioritized more than 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases, and enzymes involved in lipid metabolism.     

Palm Management in South America

We reviewed information on management of useful palms in South America. We documented management for 96 species, from incidental activities intended to increase populations of wild palms to the inclusion of palms in complex agroforestry systems. Two species, Bactris gasipaes and Parajubaea cocoides, are domesticated. Managed species are remarkably fewer than species used in the region, which suggests that harvesters often disregard the fate of the species they use. The best way of managing palms is to employ harvest methods that do not decimate the populations. Although a variety of harvesting techniques have been documented, overharvest is common, and mismanagement prevails – unnecessary felling of palms in order to harvest leaves or fruits is a widespread practice. Research should focus on assessing production in response to management practices, but eradicating the habit of destructive harvest is an obvious priority. Research on palm management must be combined with actions addressed to all stakeholders of the palm/humans system.