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One characteristic of all positive-strand RNA viruses is the necessity to assemble viral RNA replication complexes on host intracellular membranes, a process whose molecular details are poorly understood. To study viral replication complex assembly we use the established model system of Flock House virus (FHV), which assembles its replication complexes on the mitochondrial outer membrane. The FHV RNA-dependent RNA polymerase, protein A, is the only viral protein necessary for genome replication in the budding yeast Saccharomyces cerevisiae. To examine the host components involved in protein A-membrane interactions, an initial step of FHV RNA replication complex assembly, we established an in vitro protein A membrane association assay. Protein A translated in vitro rapidly and specifically associated with mitochondria isolated from yeast, insect, and mammalian cells. This process was temperature dependent but independent of protease-sensitive mitochondrial outer membrane components or the host mitochondrial import machinery. Furthermore, lipid-binding studies revealed that protein A preferentially bound to specific anionic phospholipids, in particular the mitochondrion-specific phospholipid cardiolipin. These studies implicate membrane phospholipids as important host determinants for FHV RNA polymerase membrane association and provide evidence for the involvement of host phospholipids in positive-strand RNA virus membrane-specific targeting.
Positive-strand RNA viruses make up a large and diverse group of microbial pathogens responsible for significant clinical diseases in humans. The number of available vaccines and antiviral therapies targeted toward these viruses is limited, and thus the study of common molecular mechanisms involved in positive-strand RNA virus pathogenesis is important for the identification of novel candidate antiviral targets. One universal characteristic of all positive-strand RNA viruses is the necessity to assemble viral RNA replication complexes on host intracellular membranes. Viral replication complexes have been found associated with a variety of intracellular membranes, including the endoplasmic reticulum (ER), endosomes, peroxisomes, chloroplasts, and the mitochondria (32). To facilitate the formation of viral RNA replication complexes, individual viruses encode replicase proteins containing membrane-specific targeting signals, several of which have been characterized in detail (6, 29, 31, 45, 47). However, to date the host components present on organelle-specific membranes that facilitate targeting and assembly are poorly defined, and only a limited number of cellular membrane components have been shown to be important in viral RNA replication complex assembly (16, 35, 37). The identification of host factors necessary for this essential step in the positive-strand RNA virus life cycle will enhance our understanding of the molecular mechanisms of replication complex assembly and may highlight possible targets for future antiviral therapies.
To investigate the role of host factors in viral RNA replication complex assembly, we use the established and versatile model alphanodavirus, Flock House virus (FHV). FHV is the best-studied member of the Nodaviridae family and has been used extensively to investigate positive-strand RNA virus replication (7, 14, 15, 30, 31, 40, 41), host innate immunity (9, 24), and virus assembly (26, 48). FHV virions contain a copackaged bipartite genome consisting of RNA1 (3.1 kb) and RNA2 (1.4 kb), which encode protein A, the viral RNA-dependent RNA polymerase, and the capsid protein precursor, respectively. Additionally, during active RNA replication a small subgenomic RNA3 (0.4 kb) colinear with the 3′ end of RNA1 is produced which encodes protein B2, a potent inhibitor of the innate immunity RNA interference pathway (9, 24). FHV RNA replication is supported in cells derived from a wide variety of genetically tractable hosts, including Drosophila melanogaster (30), Caenorhabditis elegans (24), and the budding yeast Saccharomyces cerevisiae (31, 40, 41), which makes FHV a unique model virus to study the molecular mechanisms of host-pathogen interactions that control RNA replication complex assembly.
FHV protein A is the only viral protein necessary for RNA replication complex assembly, a process that takes place on the mitochondrial outer membrane (29-31). Protein A is targeted to the mitochondrial outer membrane via an amino-terminal targeting signal (29). This signal resembles those present in signal-anchored proteins that are targeted to the mitochondrial outer membrane (4, 20, 27, 56), suggesting that FHV may use established mitochondrial targeting and trafficking machinery for assembly. Mitochondrial protein import has been studied extensively in yeast in part due to the facile genetics of this model organism and the development of an easily manipulated in vitro import system (23, 46, 54, 58). The majority of mitochondrial proteins are translated from nuclear-encoded genes within the cytosol and therefore must be properly trafficked and targeted to their final mitochondrial compartments. The import of most proteins into mitochondria is facilitated by the translocase of the mitochondrial outer membrane (TOM) complex, a large macromolecular structure consisting of the main import component, Tom40, three receptors, Tom20, Tom22, and Tom70, and three accessory proteins, Tom5, Tom6, and Tom7 (8, 38, 42). However, it has recently been shown that some mitochondrial outer membrane proteins containing a signal anchor amino-terminal domain, including the NADH-cytochrome b5 reductase isoform encoded by the yeast MCR1 gene, can use an undefined TOM complex-independent mechanism for membrane targeting and insertion (27).
In this report, we investigated the role of host mitochondrial outer membrane components in the binding and insertion of protein A to mitochondria, an initial step of FHV RNA replication complex assembly. To facilitate these studies we established an in vitro protein A translation and membrane association system with which we were able to recapitulate many of the in vivo biochemical characteristics of protein A. We found that protein A rapidly and specifically associated with yeast, insect, and mammalian mitochondria after in vitro translation and furthermore acquired resistance to alkaline extraction, consistent with its characteristic as an integral membrane protein in vivo (29, 30). Protein A membrane insertion was independent of protease-sensitive outer membrane components and the main component of the TOM complex, Tom40. However, we found that protein A associated efficiently with anionic phospholipids, and in particular the mitochondrion-specific anionic phospholipid cardiolipin. These studies provide insight into the early steps of FHV RNA replication complex assembly and implicate organelle membrane lipids in the specific intracellular targeting of positive-strand RNA virus replication.
Yeast strains used in this study are listed in Table Table1.1. For isolation of mitochondria, yeast were grown in YPG (1% [wt/vol] yeast extract, 2% [wt/vol] peptone, 3% glycerol) or YPD (1% [wt/vol] yeast extract, 2% [wt/vol] peptone, 2% dextrose) medium. Drosophila S2 cells were grown in Schneider's Drosophila medium with 10% heat-inactivated fetal bovine serum at 25°C as previously described (18). Mammalian baby hamster kidney (BHK) cells were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 5% bovine growth serum, 10 units per ml penicillin, and 10 μg per ml streptomycin.
Standard molecular biology techniques were used for the production of all plasmids. The protein A in vitro expression plasmid pIVT-PA was generated by placing the open reading frame from pS2FA (18) into the EcoRI/XbaI sites of pCMV-TnT (Promega, Madison, WI). The β-galactosidase (β-Gal) in vitro expression plasmid pIVT-LacZ was generated by placing the SpeI/AgeI fragment from pMT/V5/LacZ (BD Biosciences, San Jose, CA) into the AvrII/BspEI sites of pIVT-PA. The protein A-green fluorescent protein (GFP) chimera in vitro expression plasmid pIVT-PA1-46/GFP was generated by placing the PstI/BamHI fragment from pPA1-46/GFP (29) into the same sites of pGEM-4Z (Promega). The GFP in vitro expression plasmid pIVT-GFP was generated by first placing the PstI/HindIII fragment from pGal-YFP into the same sites of pGEM-4Z to create pIVT-YFP, followed by placing the BspEI/HindIII fragment from pPA1-46/GFP (29) into the same sites of pIVT-YFP. The in vitro expression plasmid encoding the ATP/ADP carrier protein (AAC) was generously provided by F. Ulrich Hartl (Max-Planck-Institute of Biochemistry, Germany). In vitro expression plasmids encoding Tom20, porin, or His6-tagged pSu9-dihydrofolate reductase (DHFR) have been previously described (2, 27). We expressed and purified pSu9-DHFR from Escherichia coli BL21 cells by nickel affinity chromatography as previously described (2, 27). Mouse monoclonal antibodies against yeast porin were purchased from Molecular Probes (Carlsbad, CA). Rabbit polyclonal antibodies against yeast Tim44 or Tom40 were generously provided by Donna M. Gordon (University of Pennsylvania) or Nikolaus Pfanner (University of Freiburg, Freiburg, Germany), respectively. Rabbit polyclonal antibodies against yeast Tom20 and Tom70 have been previously described (2, 27).
Yeast mitochondria were isolated as previously described (28) and stored in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM 3-morpholinopropanesulphonic acid [MOPS]-KOH, pH 7.2) at −80°C. Drosophila S2 cell and BHK mitochondria were isolated by mechanical disruption and differential centrifugation as previously described (11, 18). mRNAs were generated by in vitro transcription with SP6 or T7 RNA polymerase (Epicentre, Madison, WI) from pGEM-4Z- or pCMV-TnT-based vectors, respectively. Initial in vitro translation experiments with wheat germ, S. cerevisiae, and D. melanogaster cell-free lysates failed to produce detectable full-length protein A (data not shown), and thus we used commercially available nuclease-treated rabbit reticulocyte lysates (RRL; Promega) for all subsequent experiments. Proteins for in vitro assays were translated using RRLs in the presence of 400 μCi/ml [35S]methionine-cysteine (Amersham) with optimized amounts of in vitro-transcribed mRNAs for 1 h at 25°C. Translation mixtures (5 μl per 100-μl reaction mixture) were incubated with isolated mitochondria (50 μg total protein per 100-μl reaction mixture) in import buffer (250 mM sucrose, 80 mM KCl, 5 mM MgCl2, 10 mM MOPS-KOH [pH 7.2], 0.25 mg/ml bovine serum albumin, 2 mM ATP, 2 mM NADH) at 25°C. Import reaction mixtures were diluted with 4 volumes of SEM buffer, and mitochondria were pelleted by centrifugation at 12,000 × g for 15 min, resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% [wt/vol] glycerol, 0.001 mg/ml bromophenol blue, 5% 2-mercaptoethanol), and heated to 95°C for 5 min. For carbonate extractions, pelleted membranes were resuspended in 0.1 M sodium carbonate (pH 11.5) and incubated on ice for 30 min. Extracted membranes were reisolated by centrifugation at 55,000 × g for 30 min at 4°C and resuspended in sample buffer. Porin and AAC protein import were analyzed by incubating mitochondria with 100 μg per ml proteinase K for 5 min on ice followed by the addition of 1 mM phenylmethanesulfonyl fluoride. Mitochondria were reisolated by centrifugation at 12,000 × g for 15 min and resuspended in sample buffer. Denatured samples were separated by SDS-PAGE, and gels were fixed in 25% isopropanol-10% acetic acid, incubated in 1 M sodium salicylate for 1 h, dried under vacuum, and exposed to autoradiography film. Digital images were obtained using an Alpha Innotech Fluorchem 8900 and quantified with AlphaEase FC software, and final images were prepared with Adobe Photoshop software. All contrast adjustments to the final images were done prior to cropping.
In vitro membrane association reaction mixtures were brought to 300 μl with flotation buffer [50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 mM KCl, 2 mM MgCl2, pH 7.5] and mixed with 900 μl 50% Nycodenz solution to yield a final concentration of 37.5% Nycodenz. Samples were loaded under 3.2 ml of 35% and 0.2 ml of 5% Nycodenz solutions and centrifuged at 134,000 × g for 20 h at 4°C in a Beckman MLS-50 swing bucket rotor. Fractions of equal volumes were collected and analyzed by SDS-PAGE and fluorography as described above. E. coli and porcine brain total lipid extracts as well as purified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (PS), l-α-phosphatidylinositol (PI), 1,2-dioleoyl-sn-glycero-3-phosphate (PA), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (PG), 1,1′,2,2′-tetraoleoyl cardiolipin (CL), and 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC) were purchased from Avanti Polar Lipids (Alabaster, AL). Chloroform solvent was removed under vacuum centrifugation for 1 h and lipids were resuspended in HEPES buffer (20 mM HEPES-KOH, pH 7.2) by vortexing at 4°C for 1 h. Lipids (250 μg per 100-μl reaction mixture) were incubated with in vitro translation mixtures in protein import buffer for 30 min at 25°C, diluted to 1 ml with 85% sucrose in HEPES buffer to a final sucrose concentration of 71.25%, overlaid with 2.5 ml 65% sucrose and 1 ml 10% sucrose in HEPES buffer, and centrifuged at 134,000 × g for 20 h at 4°C in a Beckman MLS-50 swing bucket rotor. Gradients were fractionated and analyzed as described above.
We used a two-tailed Student's t test, assumed equal variances for comparative statistical analyses, and considered a P value of <0.05 significant. Unless otherwise indicated all quantitative data represent results from at least three independent experiments and are presented as the means ± standard errors of the means.
FHV assembles RNA replication complexes on the mitochondrial outer membrane (29, 30). To investigate the initial steps of replication complex assembly and identify host components responsible for protein A membrane association we developed an in vitro mitochondrial binding assay, similar to the in vitro system used to study the import and assembly of endogenous mitochondrial proteins (23, 46, 58). We initially attempted to purify full-length FHV RNA polymerase produced in E. coli for in vitro experiments, but protein A produced with this heterologous system was highly insoluble (D. Miller, unpublished data). Thus, we explored an in vitro translation system, in which FHV protein A was synthesized from a synthetic mRNA template in nuclease-treated RRL. Initial studies with a carboxy-terminal epitope-tagged construct and subsequent immunoblotting with epitope tag-specific antibodies demonstrated that full-length protein A was produced (data not shown). For all subsequent experiments 35S-radiolabeled protein was used to enhance detection sensitivity.
We incubated in vitro translation mixtures with mitochondria isolated from cells capable of supporting robust FHV RNA replication, including wild-type YPH499 yeast cells (31, 40, 41), Drosophila S2 cells (30), or mammalian BHK cells (15). Mitochondria were subsequently pelleted by centrifugation and membrane association was analyzed by SDS-PAGE and fluorography of pelleted fractions (Fig. (Fig.1).1). We found protein A rapidly sedimented in the presence of mitochondria from yeast, insect, or mammalian cells, whereas β-Gal, a similarly sized (~100-kDa) control protein that was translated as efficiently as protein A, showed negligible mitochondrial association and sedimentation (Fig. (Fig.1A).1A). In the absence of mitochondria neither protein A nor β-Gal was found in significant amounts in the pellet fractions, indicating that the in vitro-translated proteins were not forming aggregates that rapidly sedimented with centrifugation (Fig. (Fig.1A,1A, bottom gel). Maximal binding of protein A was 70 to 80% of input protein and was seen after 10 to 20 min. Protein A association with mitochondria was independent of ATP levels, and extended incubations past 30 min did not significantly increase the fraction of bound protein A (data not shown). Furthermore, 60% of protein A translated in vitro remained associated with mitochondrial membranes even after alkaline extraction with 0.1 M sodium carbonate, suggesting that protein A behaved as an integral membrane protein after in vitro translation and mitochondrial association, similar to results obtained in vivo with both yeast and cultured Drosophila cells (29, 30). Based on the comparable results with yeast, insect, and mammalian mitochondria and the ready availability of yeast strains with deletions or mutations in specific mitochondrial outer membrane components, we used yeast mitochondria for all subsequent studies unless otherwise indicated.
To examine the biochemical characteristics of in vitro-translated protein A and further exclude the possibility of aggregation due to the multiple hydrophobic regions within the protein (29), we used membrane flotation assays (Fig. (Fig.1B).1B). In vitro translation or membrane association reaction mixtures were combined with Nycodenz, subjected to equilibrium centrifugation, and fractionated to analyze protein A distribution in low-density and high-density regions, which correspond to membrane-associated and soluble or aggregated proteins, respectively (29, 30). Protein A was recovered in high-density fractions in the absence of mitochondria (Fig. (Fig.1B,1B, first gel), whereas with the addition of mitochondria protein A became membrane associated and floated to the low-density fractions (Fig. (Fig.1B,1B, second gel), indicating that protein A was not aggregating after in vitro translation and mitochondrial association. Similar results were obtained when mitochondria were extracted with 0.1 M sodium carbonate prior to flotation, further confirming the protein A was inserted as an integral membrane protein (data not shown). As a control, the addition of the detergent Triton X-100 shifted protein A distribution back to the high-density fractions (Fig. (Fig.1B,1B, third gel). Immunoblotting against the mitochondrial outer membrane protein porin was used as a control to identify membrane-containing fractions (Fig. (Fig.1B,1B, bottom blot).
To explore membrane specificity of in vitro-translated protein A, we initially used constructs with defined amino-terminal deletions that disrupt mitochondrial targeting and membrane association in vivo (29). Deletions of amino acids 9 to 45 or 9 to 135 reduced protein A membrane association with mitochondria in vitro by 20% or 40%, respectively, and there was a direct correlation between the levels of membrane association in vitro with purified mitochondria and in vivo with intact yeast (R = 0.999, P < 0.02). We also conducted gain-of-function studies and found that the amino-terminal region containing 46 amino acids from protein A, which includes a mitochondrion-targeting signal (29), was sufficient to increase the in vitro mitochondrial association of a GFP fusion construct 10-fold (Fig. (Fig.1C).1C). To further test membrane specificity, we examined the ability of wild-type protein A to associate with canine microsomes in vitro. As a control for these experiments, we used a chimeric protein A in which the amino-terminal mitochondrion-targeting signal was replaced with a hepatitis C virus NS5B ER-targeting signal (31) and performed the assays cotranslationally to mimic protein import in the ER. We found that 51.9 ± 6.0% of the protein A-ER chimera associated with canine microsomes in vitro compared to only 26.8 ± 6.2% of wild-type protein A (P < 0.004). We concluded from these data that FHV protein A translated in vitro recapitulates important membrane specificity and biochemical characteristics of protein produced in vivo.
The majority of host mitochondrial proteins utilize the TOM complex for transport to their appropriate mitochondrial compartments (8, 38, 42). Thus, we examined the role of this macromolecular complex in FHV protein A mitochondrial membrane association and insertion using both biochemical and genetic approaches. Several TOM components, such as the primary receptors Tom20 and Tom70, are exposed to the cytosol (5, 19, 23) and therefore susceptible to protease digestion. We treated mitochondria isolated from YPH499 yeast with proteinase K to degrade accessible outer membrane components and analyzed protein A membrane association and subsequent bilayer insertion in vitro by assessing resistance to alkaline extraction (Fig. (Fig.2).2). Immunoblot examination of protease-treated mitochondria revealed the near-complete loss of Tom20 and Tom70, whereas a pore-forming protein wholly embedded in the mitochondrial outer membrane (porin) and an inner membrane protein (Tim44) were relatively unaffected by protease treatment (Fig. (Fig.2A).2A). Protease-treated mitochondria showed a marked defect in the insertion of porin (Fig. (Fig.2B),2B), an endogenous host protein previously shown to use the Tom20 receptor for proper membrane insertion (23). In contrast, protein A showed a slight increase in insertion efficiency upon the removal of protease-sensitive outer membrane components (Fig. (Fig.2C).2C). These results suggested that protease-sensitive components were not required for efficient protein A mitochondrial membrane association and insertion.
To further examine the role of specific mitochondrial outer membrane components in protein A insertion, we conducted in vitro assays with mitochondria purified from yeast strains with deletions or mutations in individual TOM components (Table (Table11 and Fig. Fig.3).3). We verified the genotype of deletion strains by PCR (data not shown) and immunoblotting (Fig. (Fig.3A)3A) and used purified mitochondria for protein A insertion assays. Protein A insertion was not significantly reduced using mitochondria from Δtom20 or Δtom70 compared to wild-type yeast, whereas insertion or import of porin or AAC, which have previously been shown to depend primarily on Tom20 or Tom70, respectively (23, 61), was reduced in mitochondria from the appropriate deletion strain (Fig. (Fig.3B).3B). We also examined the role of Tom40, the main component of the general import pore, in protein A membrane insertion. Since Tom40 is an essential protein, we made use of two temperature-sensitive (ts) strains, tom40-3 and tom40-6. Mitochondria from tom40-3 have a defect in Tom20 import (2), whereas mitochondria from tom40-6 have not been shown to have any defects in protein import but do have reduced levels of assembled TOM complex (58). Wild-type and ts Tom40 strains were grown at 25°C for mitochondrial isolation, and prior to insertion assays mitochondria were incubated at 37°C for 15 min to induce the ts phenotype through temperature-dependent destabilization of Tom40. Mitochondria from tom40-3 or tom40-6 yeast showed no reduction in protein A insertion but rather showed a trend toward increased insertion efficiency (Fig. (Fig.3C),3C), similar to results with protease-treated mitochondria (Fig. (Fig.2C).2C). To further examine the role of the TOM complex in protein A mitochondrial insertion we conducted competition experiments with purified pSu9-DHFR, a chimeric protein that competes for protein insertion through the main import pore (2, 23, 27). Protein A insertion into wild-type mitochondria was not inhibited by Su9-DHFR, whereas porin insertion was substantially reduced (Fig. (Fig.3D).3D). Taken together, these data suggested that protein A association and insertion into the mitochondrial membrane did not require Tom20, Tom70, and the general import pore.
The observation that protein A membrane association and insertion were independent of protease-sensitive outer membrane components and Tom40 (Fig. (Fig.22 and and3)3) suggested that nonprotein components, such as membrane lipids, may be involved. To initially test this hypothesis we examined the temperature dependence of protein A-membrane interactions in vitro (Fig. (Fig.4).4). Protein-protein interactions are often temperature independent, and this characteristic is frequently used to study processes such as initial receptor-ligand binding at low temperatures to avoid receptor internalization or remodeling after ligand-mediated activation. We found that Tom20 binding to purified mitochondria occurred equally well at 4°C and 25°C (Fig. (Fig.4A,4A, upper graph), consistent with its known interaction with the import pore Tom40 during mitochondrial membrane binding and insertion (2, 4). We obtained similar temperature-independent results with porin (data not shown). In contrast, protein A membrane association with purified wild-type yeast (Fig. (Fig.4A,4A, bottom graph) and Drosophila (Fig. (Fig.4B)4B) mitochondria was significantly reduced at 4°C to less than 20% of the levels seen at 25°C.
To directly examine potential protein A-lipid interactions, we used lipid extracts from E. coli and brain with in vitro binding assays. We selected these extracts due in part to their differences in phospholipid composition. For example, E. coli lipid extracts contain a lipid composition similar to that of eukaryotic mitochondria with large quantities of the anionic phospholipids PG (15.1%) and CL (9.8%) (Avanti Polar Lipids product data). In contrast, brain extracts contain a more diverse group of phospholipids with low concentrations of anionic phospholipids and a larger quantity of neutral phospholipids. We incubated in vitro-translated protein A or β-Gal with liposomes generated from individual lipid extracts for 30 min and subsequently examined liposome binding by equilibrium gradient centrifugation (Fig. (Fig.5A).5A). The control protein β-Gal did not bind to liposomes generated with either lipid extract (Fig. (Fig.5A,5A, lower gradient, and data not shown). In contrast, protein A associated with liposomes generated from E. coli lipid extracts, where approximately 25% partitioned into the low-density fractions (Fig. (Fig.5A,5A, middle gradient). Only minimal protein A binding was seen with liposomes generated from brain extracts (Fig. (Fig.5A,5A, upper gradient).
To further explore potential phospholipid selectivity, we analyzed the in vitro binding capabilities of protein A with liposomes generated from equal amounts of purified individual membrane phospholipids (Fig. (Fig.5B).5B). We observed substantial protein A binding to the anionic phospholipids CL and PA, moderate binding to the anionic phospholipids PG and PS, and negligible binding to the anionic phospholipids PI and the neutral lipids PC and PE. The absence of significant protein A binding to PC was not due to decreased flotation, as control experiments with fluorescent PC demonstrated almost complete partitioning of this neutral lipid into upper low-density gradient fractions (data not shown). Of interest with the purified lipid experiments was protein A binding to the mitochondrion-enriched anionic phospholipids CL, PA, and PG (17, 55), and in particular the mitochondrion-specific CL, where protein A was recovered almost exclusively in the low-density fractions (Fig. (Fig.5B,5B, top gradient, fractions 1 and 2).
The diphosphatidyl-based lipid CL is found in small concentrations in the mitochondrial outer membrane, with the notable exception of the contact sites between mitochondrial outer and inner membranes, where it can increase to roughly 20% of total membrane phospholipids (3, 51). To further confirm the CL-binding capabilities of protein A, we generated liposomes containing increasing concentrations of CL relative to PC and assayed protein A in vitro binding by gradient flotation assays (Fig. (Fig.6).6). Protein A showed CL concentration-dependent flotation, where approximately 20% of protein A partitioned to low-density fractions when the total CL concentration was 20% (Fig. (Fig.6A,6A, bottom gradient, and B). The positive correlation between CL concentration and protein A flotation (Fig. (Fig.6B)6B) (R = 0.92) was not due to changes in liposome partitioning within sucrose gradients, as control experiments with fluorescent PC-CL liposomes demonstrated equivalent flotation characteristics regardless of CL concentration (data not shown).
To examine the role of CL in protein A association with intact mitochondria, we initially conducted in vitro membrane association assays with mitochondria purified from Δcrd1 yeast, which lack the biosynthetic enzyme CL synthase and hence produce no CL (12). Protein A membrane association in vitro was not significantly different between mitochondria from Δcrd1 and wild-type yeast (data not shown). However, the deletion of CL synthase results in the increased accumulation of PG (12), which along with PA is a precursor in the CL biosynthetic pathway and an anionic phospholipid to which protein A was also significantly bound in flotation assays (Fig. (Fig.5B).5B). To avoid the potential confounding effects of altered phospholipid composition in yeast strains with deletions or mutations of specific phospholipid biosynthesis genes, we subsequently conducted differential centrifugation competition experiments with mitochondria from wild-type yeast and CL-containing liposomes (Fig. (Fig.7).7). We initially used fluorescent PC-CL liposomes to optimize differential centrifugation conditions and minimize liposome fusion or cosedimentation with intact mitochondria, and we achieved conditions under which approximately 15% of the input lipid-associated fluorescence signal pelleted with mitochondria regardless of the CL liposome concentration (data not shown). Under these optimized conditions, liposomes containing 20% CL reduced protein A association with wild-type mitochondria by approximately 25% compared to PC-only liposomes, whereas minimal competition was seen with 5% CL liposomes (Fig. 7A and B). Similar results were obtained with mitochondria from Drosophila cells (data not shown). Taken together, these data indicated that FHV protein A is a lipid-binding protein with affinity for specific anionic phospholipids, and in particular the mitochondrion-specific phospholipid CL.
In this study, we investigated the interactions between FHV protein A and intracellular membranes, an initial and important step in the assembly of viral RNA replication complexes. Based on our results we conclude that protein A is a lipid-binding protein with particular affinity for specific anionic phospholipids. Furthermore, both biochemical and genetic studies indicated that protease-accessible membrane proteins, and in particular the TOM complex components Tom20, Tom70, and Tom40, were not required for protein A interactions with the mitochondrial membrane. However, we cannot exclude a role for protease-resistant and possibly membrane-embedded non-TOM complex mitochondrial outer membrane proteins in FHV protein A-membrane interactions. Indeed, although we saw almost complete association of in vitro-translated protein A with intact mitochondria, we obtained only partial association with lipid extracts or liposomes, with the exception of 100% CL liposomes. Furthermore, changes in membrane-embedded protein conformation may have accounted for the dramatic temperature dependence we observed in protein A mitochondrial binding. Nonetheless, the results with protein A in this study are consistent with those published for some endogenous mitochondrial outer membrane proteins that contain an amino-terminal signal-anchor transmembrane domain that resemble the targeting sequence of FHV protein A, suggesting a potentially similar pathway for membrane insertion (27, 57). The published study with Mcr1 used mitochondrial import assays, similar to the system described in this manuscript, including the use of protease digestion, TOM receptor component deletion strains, and Su9-DHFR competition experiments, but did not explore lipid binding (27). Further experiments with both cellular mitochondrial proteins and FHV protein A will be required to fully define this pathway.
The observation that protein A has lipid-binding capabilities is consistent with the well-described association between positive-strand RNA virus replication complexes and intracellular membranes (32, 49). Host membranes likely play multiple roles in viral RNA replication, which may include (i) serving as a scaffold for assembly of a macromolecular structure such as an RNA replication complex, (ii) shielding viral RNA replication intermediates such as double-stranded RNA from cellular innate antiviral pathways, and (iii) providing cofactors for optimal enzymatic activity of viral replicase proteins. These functions could be mediated by either protein or lipid constituents within particular organelle membranes. Thus far the predominant emphasis in the field has been placed on identifying either membrane-resident proteins or cytosolic proteins that become membrane associated upon viral RNA replication complex assembly (13, 34). The results presented in this report indicate that lipids, and in particular anionic phospholipids, may also play important roles in viral RNA replication complex assembly.
The speculative role of lipids in replication complex assembly is consistent with the observation that the replicase protein nsP1 from Semliki Forest virus, a positive-strand RNA virus that assembles its replication complexes on membranes derived from endosomes and lysosomes (52), also binds anionic phospholipids (1). FHV protein A binding to predominantly anionic phospholipids suggests that ionic forces may mediate in part the interactions with phospholipids. Indeed, for nsP1 the anionic phospholipid interaction domain was mapped to an amphipathic α-helix with a cluster of positively charged residues (1). Sequence and predicted secondary structural analyses have revealed the presence of several similar amphipathic α-helices within the FHV protein A-coding region that may be involved in anionic phospholipid interactions (K. Stapleford and D. Miller, unpublished data). However, the selectivity of protein A for certain anionic phospholipids suggests that nonionic forces also contribute to protein A-lipid interactions.
The ability of protein A to interact with several different anionic phospholipids suggests a level of "promiscuity" that may help explain the robustness of FHV RNA replication in cells derived from multiple organisms from several kingdoms (24, 30, 41) and the relative ease with which FHV RNA replication complexes can be retargeted to alternative intracellular membranes such as the ER (31). However, the ubiquitous nature of anionic phospholipids and the substantial amounts of PI and PS present in many cellular membranes, including the ER (62), indicate that net membrane charge cannot be the sole determinant of replication complex targeting. Nonetheless, protein A did not bind all anionic phospholipids, but rather showed preferential interactions with CL, PA, and PG, which are enriched in mitochondrial membranes (55), suggesting that specific anionic phospholipids can influence FHV replication complex targeting. Furthermore, we cannot exclude a role for local charge clusters within membrane microdomains or the impact of protease-resistant endogenous membrane protein interactions with anionic phospholipids in the membrane-specific targeting of FHV RNA replication complexes.
The hypothesis that anionic phospholipids play targeting or structural roles in FHV RNA replication complex assembly is particularly interesting given the results with CL, which we identified as a significant interaction partner of protein A. CL is a cellular phospholipid whose distribution is almost entirely limited to the mitochondria (17). Although the majority of CL is found in the inner mitochondrial membrane, it is also present in high localized concentrations in the outer membrane at contacts sites between inner and outer membranes (3, 44). One might speculate that FHV RNA replication complexes may initially assemble at or near these contact sites via interactions between protein A and CL. This proposed mechanism resembles the targeting of the proapoptotic promoter tBID to CL that can initiate cytochrome c-mediated apoptotic cell death (10, 25). The potential similarity between protein A-CL and tBID-CL interactions is intriguing given the recent demonstration that FHV infection induces apoptosis in cultured Drosophila cells (50), although the delayed onset of FHV-induced apoptosis until approximately 12 h postinfection suggests that a threshold of protein A-CL interactions may be required. In addition, it is possible that protein A-mediated disruption of the inner-outer membrane contact sites, an essential substructure needed to maintain mitochondrial shape (44), may lead to some of the morphological alterations that are seen in mitochondria from yeast or Drosophila cells that contain active FHV RNA replication complexes (30, 31). This is consistent with the observation that components of the endogenous mitochondrial fission and fusion machinery can localize to these contact sites (44, 53) and that disruption of the normal fission-fusion processes can lead to abnormal mitochondrial morphology (36, 43) that also resemble mitochondria in cells with active FHV RNA replication (29, 31). Furthermore, the dimeric nature of CL, which consists of four acyl chains attached to diphosphatidylglycerol, imparts a unique conical structure that favors a hexagonal HII phase that may play a role in the membrane curvatures necessary to produce the spherules associated with FHV RNA replication complexes (22, 30).
Although the focus of this study was to identify host components involved in protein A-membrane interactions, an initial step in FHV replication complex assembly, we found that protein A translated in RRLs had RNA polymerase activity when provided with an excess of exogenous virion template RNA template (data not shown). However, we did not observe the FHV RNA replication complex activity that has been described with membrane preparations from FHV-infected Drosophila cells (59, 60) and replicon-expressing yeast (31), which includes the production of single-stranded products, with in vitro-translated protein A in the presence of whole mitochondria or liposomes. The particularly difficult feat of de novo assembly of fully functional viral RNA replication complexes using a cell-free in vitro translation system has only been accomplished for a select few positive-strand RNA viruses, including poliovirus, which requires the use of uninfected mammalian cell extracts (33), the plant pathogens tomato mosaic virus, brome mosaic virus, and turnip crinkle virus, which require the use of evacuolated plant cell extracts (21), and tomato bushy stunt virus, which uses a related system that employs a yeast cell extract and purified recombinant viral proteins (39). Further studies with FHV will be required to identify the optimal conditions under which fully functional viral RNA replication complexes can be formed in vitro, and both the results presented in this report and others (59, 60) indicate that the inclusion of specific phospholipids may be a particularly important aspect of these studies.
In summary, the studies presented in this report demonstrate that FHV protein A mitochondrial association and membrane insertion is mediated by a TOM complex-independent mechanism, similar to what has been seen previously for mitochondrial outer membrane signal-anchored proteins, and provide evidence for the importance of host membrane-specific phospholipids in positive-strand RNA virus replication complex assembly. Future in vitro and in vivo studies using this established host-pathogen system will give further insight into the role of phospholipids in membrane-specific targeting as well as the biochemical mechanisms involved in positive-strand RNA virus replication complex assembly and function.
We thank Donna Gschwend for administrative assistance, F. Ulrich Hartl, Donna Gordon, and Nickolaus Pfanner for providing reagents, and all the Miller laboratory personnel for helpful comments on the research and manuscript.
This work was supported by National Institutes of Health grants R01-AI062749 and T32-GM007315.
Published ahead of print on 25 February 2009.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Biological activity of Burkholderia (Pseudomonas) cepacia lipopolysaccharide
Version of Record online: 17 JAN 2006
DOI: 10.1111/j.1574-695X.1995.tb00095.x
FEMS Immunology & Medical Microbiology
Volume 11, Issue 2, pages 99–106, April 1995
Additional Information
How to Cite
Shaw, D., Poxton, I. R. and Govan, J. R.W. (1995), Biological activity of Burkholderia (Pseudomonas) cepacia lipopolysaccharide. FEMS Immunology & Medical Microbiology, 11: 99–106. doi: 10.1111/j.1574-695X.1995.tb00095.x
Publication History
- Issue online: 17 JAN 2006
- Version of Record online: 17 JAN 2006
- (Received 8 December 1994, Revised 23 January 1995, Accepted 24 January 1995)
- Abstract
- References
- Cited By
- Burkholderia cepacia;
- Pseudomonas cepacia;
- Cystic fibrosis;
- Lipopolysaccharide;
- Tumour necrosis factor
Abstract Burkholderia cepacia has emerged as an important multiresistant pathogen in cystic fibrosis (CF), associated in 20% of colonised patients with a rapid and fatal decline in lung function. Although knowledge of B. cepacia epidemiology has improved, the mechanisms involved in pathogenesis remain obscure. In this study, B. cepacia lipopolysaccharide (LPS) was assessed for endotoxic potential and the capacity to induce tumour necrosis factor (TNF). LPS preparations from clinical and environmental isolates of B. cepacia and from the closely related species Burkholderia gladioli exhibited a higher endotoxic activity and more pronounced cytokine response in vitro compared to preparations from the major CF pathogen Pseudomonas aeruginosa. This study may help to explain the vicious host immune response observed during pulmonary exacerbations in CF patients colonised by B. cepacia and lead to therapeutic advances in clinical management.

subset: commoncrawl


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World Kitchen, HSN Launch New Corelle For Joy Collection
Thursday October 24th, 2013 - 10:33AM
These are shortcuts to your favorite social networking and bookmark sites. Add this story to your Facebook page, del.icio.us, DiggIt, and many others!
World Kitchen is continuing its relationship with Ingenious Designs' Joy Mangano and HSN with the launch of the Corelle for Joy Precious Colours Collection, a new dinnerware assortment making its debut on the multi-channel retailer October 24.
The collection's 16-piece set is inspired by precious gemstones and is available in 10 vibrant colors: Black Onyx, Ruby Red, Seaglass Turquoise, Yellow Gold, Amethyst Purple, Emerald Green, Sapphire Blue, Platinum Silver, Amber Orange and White Diamond.
The Precious Colours collection will be sold exclusively on HSN, HSN.com and via HSN Mobile and priced at $74.99. The collection includes a set of four dinner plates, four lunch plates, four wide-rim entree bowls and four 28-oz. soup bowls. The collection can be expanded to include, serving bowls, platters and mugs— all sold separately.
Corelle for Joy Mangano had its initial launch of dinnerware product in October 2012, where 30,000 sets sold out in four hours. In April 2013, 50,000 sets sold in less than eight hours.
Tags: Corelle • World Kitchen • HSN • tabletop • dinnerware • Made in the USA • Joy Mangano • Ingenious Designs • Housewares • Tabletop • Retail •
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Last time we checked, which was two days after Christmas, Jeremy Lin was gaining a wee bit of ground on Chris Paul in All-Star voting. That trend has ceased. [Read more…]
Sheridan: Stoudemire brutal and ambivalent in his return
NEW YORK — If you are a Knicks fan and your highs and lows are determined by how your team fares, you are not going to feel better after reading this column. But read it you must. [Read more…]
Indiana Pacers Salary Cap Situation
INDIANA PACERS Summer, 2013: Drafted: Solomon Hill, SF, Arizona. Free Agents Signed: Chris Copeland, F (Knicks), C.J. Watson, G (Nets), Donald Sloan G, (China) Draft Picks Coming/Going: Owed Golden State's 2nd round pick in 2015PLAYER 2013-14 2014-15 2015-16 2016-17 2017-18Roy Hibbert $14,200,000 $14,900,000 $15,500,000 Danny Granger $14,020,000David West $12,000,000 $12,000,000 $12,000,000George Hill $8,000,000 $8,000,000 $8,000,000 $8,000,000Ian Mahinmi $4,000,000 $4,000,000 $4,000,000Gerald Green $3,500,000 $3,500,000Paul George $3,280,000 $4,470,000Chris Copeland $3,000,000 $3,100,000Miles Plumlee $1,120,000 $1,170,000 $2,110,000 $3,110,000Lance Stephenson $930,000Orlando Johnson $789,000 $915,000 $1,180,000C.J. Watson TBA TBADonald Sloan TBASolomon HillTOTAL SALARY $64,839,000 $ $ $ $0Player Option Team Option Qualifying Offer ANALYSIS Upon returning to his previous role as VP of the Pacers, Larry Bird said re-signing free agent David West was a priority — and he wasted no time inking the power forward
Sheridan: Latest on Phil Jackson, DeMarcus Cousins, Red-hot Clippers
About a week from now, by my best estimation, we will have an answer on whether Phil Jackson is going to be the next coach of the Brooklyn Nets. [Read more…]
Tweet of the Day: Pau Gasol
Gracias, Pau, for linking to these highlights of Juan Carlos Navarro's 33-point outing in FC Barcelona's ACB victory over Real Madrid. [Read more…]
Sheridan Hoops MVP Rankings After Week Nine
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Tweet of the Night: Eric Gordon
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[caption id="attachment_2325" align="aligncenter" width="500" caption="In 2006, 12 NCAA basketball coaches traveled to Kuwait to coach soldiers in a March Madness-style basketball tournament as part of "Operation Hardwood II.""][/caption]
Operation Hardwood was created in by the USO in 2005 to boost morale and bring a touch of home to those in uniform. The USO has a long tradition of bringing sports figures and sports-related events to troops overseas,"Operation Hardwood" has deployed more than 35 NCAA basketball coaches and sports personalities to Kuwait and Japan.
[vodpod id=ExternalVideo.939277&w=425&h=350&fv=player.c%3Dv%26player.v%3D49efbb32-47e6-4e99-aebb-3167cee3c5c7%26mkt%3Den-us%26brand%3Dfoxsports%26configCsid%3Dmsnvideo%26configName%3Dsyndicationplayer]
More from the USO
Jul 29, 2016
Military Bride-to-Be Says Yes to the Dress Thanks to the USO at Wright-Patterson Air Force Base
As soon as Melissa Wrisley put on the satin white gown, she knew it was "the dress." Just a few months earlier, when she was first engaged to her now-husband, Marine Pfc. Brandon Wrisley, Melissa was wondering if she would even be able to find a dress – let alone a designer dress – that would fit their tight wedding budget.


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Royce Warren
Posts: 1
posted 6 years ago
I have part of my source files to display. This file actually writes to the file to be stored on the disk. I would like to know how to write the method for reading from the disk. Then I would like to know the method to display the information to the console. Any suggestions would be appreciated. Thanks
import java.io.File; import java.io.FileReader; import java.io.FileWriter; import java.io.IOException; /* * Creating a Directory for my BlackBook using * File IO. */ /** *CSC 251 Advanced Java * @author Kween2 */ public class CreateBlackBookFile { public static void main(String[] args) { File dir = new File("C:/BlackBookItem/FileIO"); Boolean madeDir = dir.mkdir(); System.out.println("Dir made = " + madeDir); File myFile = new File(dir, "BlackBookItem.txt"); try{//code for writing to the file the information //that the user inputs Boolean createdFile = myFile.createNewFile(); FileWriter myFileWriter = new FileWriter(myFile); myFileWriter.write("name\nAddress\nState\nCity\nZipCode\nPhone\nJobTitle!"); myFileWriter.flush(); myFileWriter.close(); //reading characters from the disk. Only 25 characters per field FileReader myFileReader = new FileReader(myFile); char[] in = new char[25]; for (int i = 0; i < 25; i++) { in[i] = 0; int size = myFileReader.read(in); System.out.println("Read in: " + size + " characters"); for (char c : in) { if (c != 0) { System.out.print(c); } } } myFileReader.close(); } catch(IOException eio) { } } } :mrgreen:
I am a beginning Java Coder and I am very proud of that fact.
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Campbell Ritchie
Posts: 49382
posted 6 years ago
Welcome to JavaRanch
You will realise we don't simply give out answers to that sort of exercise.
That looks a complicated bit of code, which ought to be in its own methods, not all in the main method. What sort of format are you writing these files in? Is it text or binary? Have you come across classes which buffer outputs and inputs? Or the newer classes, Scanner and Formatter? Have you looked at the
Java™ Tutorials
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The first 70 or so pages of this story didn't seem all that well-written to me. The story involves a blocked writer, so perhaps the voice of the piece earlier on was meant to... Read morePublished 19 months ago by Tech Wizard
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Niton (The Reason) (Boy 8 Bit Remix)
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The Chicago Marshall Commandos girls basketball team won Monday's neutral playoff game against Montini by a score of 40-37.
(March 2, 2009: Chicago, IL 60624) Marshall Commandos (Chicago, IL) backers were pleased Monday as they saw their basketball squad hold off the visiting Montini Broncos (Lombard, IL), 40-37 in a non-league bout.
With the victory, Marshall upgrades its record to 23-6 on the campaign.


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Sensation departed yesterday -- and Inspiration is not due back until Sunday.(2:08 p.m.) -- Surprisingly, Hurricane Charley, which is approaching Florida's west coast, has had only minimal impact on cruises this week. The port of Tampa is on the hot seat but Carnival, which operates twice-weekly departures from there, reports that
The eastern coast of Florida -- including "cruise central" ports such as Port Everglades and Miami -- is not much affected, at least at this point, by Hurricane Charley, though Carnival reports one change. Its four-day cruise from Miami on Imagination, slated to call at Cozumel, will head instead to the Nassau and Freeport in the Bahamas.
We'll keep you posted today and through the weekend on any further changes.
Hurricane Tracker -- Charley's Impact Minimal So Far
August 13, 2004


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Faith: Buddhist
Tibetan Buddhists celebrated their New Year, called Losar, with traditional services of prayer and purification, sending positive energy into the world, they hope, to help bring about peace. More
Father Joe Maier is a Catholic priest who, over 30 years, has set up schools that have educated thousands of poor Thai children in the slums of Bangkok. In the process, he has skirmished with all kinds of people from drug dealers to church hierarchy. More
Buddhist meditation techniques are widely popular, and one such method is called Shambhala meditation, a simplified version of Tibetan practice. Sakyong Mipham Rinpoche, author and meditation leader, told a group of beginners to relax, note their breathing, set aside their thoughts, and just be — quietly — who they are. More
He insists he is a monk, not a politician. But as this renowned Vietnamese Buddhist leader toured the U.S. he spoke not only of Buddhist practices but also of American policies in the Middle East.
Read more of Bob Abernethy's interview with Thich Naht Hanh.
Buddhists pay special tribute to the dead during the Bon or Obon Festival — the Feast of Lanterns. More
Southern California's Soka University resembles a charming Mediterranean village. Opened in 2001 by a powerful Japanese religious sect called Soka Gakkai International, it is the first college campus in the United States whose academic values and teaching principles are inspired by Buddhism. Inside, one can find 103 acres of stately architecture, spacious gardens, and tranquil gardens. More
For Tibetan Buddhists, the new year is celebrated with dancing, and R & E sat in as members of the Drepung Loseling Monastery in Washington, D.C. performed. Our guide was Geshe Lobsang Tenzin, who spoke of the power of negative and positive energy, and about the legends associated with snow lions and the Black Hat Masters. More
RELIGION & ETHICS NEWSWEEKLY invited several scholars to comment on the divisions and direction of Buddhism in America today.
MARY ALICE WILLIAMS: Buddhism is the world's fourth largest religion, founded about 2500 years ago in India. The Buddha taught that life is suffering and the way to overcome that is to get rid of attachments. Widely practiced across Asia, … More


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Definición de shooting star en inglés:
shooting star
1A small, rapidly moving meteor burning up on entering the earth's atmosphere.
Oraciones de ejemplo
- Tiny meteors, commonly called shooting stars, hit the earth's atmosphere and turn into fiery streaks.
- It was not a satellite, meteor or shooting star.
- That's much larger than the dust grains that vaporize in the atmosphere to form most shooting stars, or meteors, but not large enough to crater Earth's crust.
2A North American plant with white, pink, or purple hanging flowers with backward-curving petals. The flowers are carried above the leaves on slender stems and turn to face up following fertilization.
- Genus Dodecatheon, family Primulaceae.
Oraciones de ejemplo
- At this time of year, hikers can be on the alert for early buttercups, shooting stars in the foothills and moss phlox.
- In the muskegs - the sponge-like bogs that ring the old-growth forest - shooting stars are in bloom, their purple blossoms dotting a lime-green carpet of sphagnum moss.
- Near the trail, there are shooting stars under the trees, and Indian paintbrush is visible between rocks.
For editors and proofreaders
Saltos de línea: shoot|ing star
Definición de shooting star en:
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Freedom Hall Main Series
Freedom Hall 2015-2016 Main Series Concerts
Freedom Hall Main Series kicks off the season with the R&B legend Booker T. Jones in our Nathan Manilow Theatre. Also performing in our 2015-2016 season will be Ava Logan with the Southland Jazz Ensemble, Masters of Soul, Shemekia Copeland, and Shining Star – Earth, Wind, and Fire Tribute.
Booker T. Jones
October 2, 2015
7:30 p.m. – Tickets $35 (Season Subscriber $31)
It can be argued that it was Booker T. Jones who set the cast for modern soul music and is largely responsible for its rise and enduring popularity. On classic hits like "Green Onions," "Hang 'Em High," "Time Is Tight," and "Melting Pot" the Rock and Roll Hall of Fame inductee, Musicians Hall of Fame inductee and GRAMMY Lifetime Achievement Award recipient pushed the music's boundaries, refined it to its essence and then injected it into the nation's bloodstream. The recently released album Sound the Alarm, finds the Hammond B3 organ master looking ahead yet again, laying down his distinctive bedrock grooves!
View a video of Booker T. Jones
This performance is sponsored by J. Murray & Associates and 90.9fm WDCB Jazz & Blues
Ava Logan with the Southland Jazz Ensemble
November 8, 2015
4:00 p.m. – Tickets $25 (Season Subscriber $21)
Join us for an evening of jazz standards, the music of Duke Ellington, Cole Porter, George and Ira Gershwin, and more! This concert features jazz vocalist Ava Logan backed by the big band sound of the Southland Jazz Ensemble! Ava Logan is an award winning singer-actress. A graduate of Yale and the University of Pennsylvania, Dr. Logan is a veterinarian by day and a jazz singer at night. The classically trained vocalist has performed jazz, pop, and rhythm and blues in the U.S. and abroad including portraying two of her idols, Ella Fitzgerald and Nancy Wilson, at the Black Ensemble Theater. Veteran jazz critic and radio voice Neil Tesser wrote that "Ava Logan offers a fresh breath of old-school glamour, bringing sass and swing to the classic jazz singer's repertoire while retaining the swanky class that this material deserves. Throw in her superb intonation, and her willingness to always serve the song, and you get a most impressive performance."
View a video of Ava Logan with the Southland Jazz Ensemble
This performance is sponsored by Friends of Freedom Hall and Baxter & Woodman, Inc.
Masters of Soul
December 4 and December 5, 2015
7:30 p.m. – Tickets $30 (Season Subscriber $26)
An encore performance for Masters of Soul with a holiday twist! In the early 60's some of the most iconic names in the history of popular music were discovered in the Motor City of Detroit, MI, better known simply as MOTOWN. MASTERS OF SOUL is a celebration of these artists, their music and style. The show features stylishly costumed fully choreographed tributes to both male and female groups backed by a live band. MOM performs: Temptations – Gladys Knight & the Pips – Marvin Gaye & Tammi Terrell Smokey Robinson & the Miracles – Four Tops – Diana Ross & Supremes Jackson Five – Martha Reeves & Vandellas – Stevie Wonder – Commodores And many more.
View a video of Masters of Soul
This performance is sponsored by Kluger Furs and 90.9fm WDCB Jazz & Blues
Shemekia Copeland
January 22, 2016
7:30 p.m. – Tickets $30 (Season Subscriber $26)
Shemekia Copeland's passion for the blues gives her music power and a heart-pounding urgency. This young singer, dubbed as the new "Queen of the Blues," has opened for the Rolling Stones, headlined at the Chicago Blues Festival, scored critics choice awards, shared the stage with Buddy Guy, B.B. King, Mick Jagger and Eric Clapton, and has performed at the White House for President Barack Obama and First Lady Michelle Obama. "Copeland embodies the blues with her powerful vocal chops." — National Public Radio
View a video of Shemekia Copeland
This performance is sponsored by Hammond Investment Management, LLC and J. Murray & Associates
Shining Star – Earth, Wind, and Fire Tribute – SOLD OUT
March 11, 2016
7:30 p.m. – Tickets $25 (Season Subscriber $21)
The amazing re-creation of the band-the legends-the Rock and Roll Hall of Fame Inductees and winners of numerous Grammy Awards- Earth Wind and Fire! Shining Star brings that special, magical era of the 70's and 80's to the stage capturing the vocals, horns and the energy of Earth Wind and Fire! If you close your eyes you will believe the original Earth Wind & Fire is onstage.
View a video of Shining Star
This performance is sponsored by J. Murray & Associates and Bimba Manufacturing


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LOS ANGELES The estranged wife of actor Mickey Rooney and his estate are locked in a legal tussle over the remains of the late Hollywood movie star, who left an estate of only $18,000, according to court documents.
Los Angeles County Superior Court Judge James Steele will decide at a hearing on Friday whether Rooney's remains will be released from an area mortuary to his wife, Janice Rooney, or to his conservator.
The versatile character actor, who was one of Hollywood's biggest stars in the 1930s, died at age 93 on Sunday from natural causes. He disinherited his wife and all his children in a will dated March 11, leaving his estate to stepson Mark Rooney, who with his wife served as Rooney's caregiver.
Rooney's conservator, Michael Augustine, was granted a court order on Tuesday stopping Janice Rooney from removing the actor's remains from a mortuary in Glendale, California.
Augustine said Rooney wanted to be buried at Los Angeles' Hollywood Forever cemetery, where many Hollywood actors are buried, or at a cemetery for U.S. veterans. Rooney served in the military as an entertainer during World War Two.
He also has a burial plot in Westlake Village, California.
"We think we'll be able to cut a deal, a deal being between me and her," Augustine said.
An attorney for Janice Rooney was not immediately available to comment.
Rooney wed eight times, including a marriage to actress Ava Gardner. In 2011 he testified before a U.S. Senate committee that he had been emotionally and financially abused by family members, sapping much of his savings.
Rooney and his last wife did not live together after 2012. She will receive his Social Security and some of his pension benefits, which will total about $8,400 a month, Augustine said.
Rooney earns about $20,000 to $25,000 per year on residuals from his later film work, Augustine said.
The actor was one of the last living links to Hollywood's silent era, and did not earn residuals from early classics like 1944's "National Velvet."
"He gets more money from the 'Care Bears' than he does from that," Augustine said, referring to the 1985 animated film in which Rooney voiced the character of Mr. Cherrywood.
In 2011, Rooney sued stepson Christopher Aber and his wife, alleging elder abuse and mismanagement of his funds. Augustine said the couple agreed to a settlement worth millions with Rooney, but the actor's estate has been unable to collect because of Aber's financial troubles.
Rooney is survived by eight children and two stepsons. His son Timothy Rooney died in 2006.
Augustine said Rooney's funeral is expected to be small and attended by family with a larger public tribute planned later.
Rooney starred in the "Andy Hardy" movies as a teen during the Great Depression and acted alongside Judy Garland and later Elizabeth Taylor.
(Reporting by Eric Kelsey; Editing by Prudence Crowther)
Trending On Reuters
Movie Review: Dishoom
In Rohit Dhawan's "Dishoom", the opening credits roll to a rap song mouthed by two brawny protagonists who describe themselves as "simple" men disappointed in love who prefer home food to eating in five-star hotels. With one disclaimer: these otherwise meek men turn violent if someone doesn't stand up while India's national anthem is playing, criticizes the country or harasses a woman. Full Article
Reuters Showcase
Reuters India App
Download the Reuters app from the Apple App Store, Google Play and Amazon. Select the India edition from settings. Full Coverage


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Parmesan Bread
From EatingWell: May/June 1995
Flavorful Parmesan makes this homemade bread addictive.
Makes: Two 6-inch loaves, 8 slices each
Active Time:
Total Time:
Nutrition Profile
Low calorie | Low cholesterol | Low saturated fat | Low sodium | Heart healthy | Healthy weight |View Our Nutrition Guidelines »
- 2 envelopes active dry yeast, (4 1/2 teaspoons)
- 1 1/4 cups lukewarm water
- 2/3 cup nonfat dry milk
- 1 cup plus 1 tablespoon freshly grated Parmesan cheese, divided
- 1 large egg, lightly beaten
- 1 tablespoon extra-virgin olive oil
- 2 teaspoons sugar
- 1 1/2 teaspoons salt
- 1/2 teaspoon cayenne pepper
- 3 3/4-4 cups all-purpose flour, divided
- 1 large egg white, lightly beaten, for glazing bread
- Dissolve yeast in water in a large bowl. Stir in nonfat dry milk, 1 cup Parmesan, whole egg, oil, sugar, salt, cayenne and 3 cups of the flour. Beat well with a wooden spoon. Gradually stir in enough of the remaining flour until the dough pulls away from the side of the bowl.
- Turn out onto a lightly floured work surface and knead until the dough is smooth, elastic and only slightly sticky, about 5 minutes, adding additional flour as necessary. (Alternatively, the dough can be mixed and kneaded in a stand-up electric mixer fitted with a dough hook.) Place the dough in a lightly oiled bowl, turn to coat, and cover with plastic wrap. Let rise in a warm place until doubled in volume, about 1 1/2 hours.
- Punch down the dough and turn it out onto a floured work surface; divide it in half and shape each half into a ball. Place balls on an oiled baking sheet, cover loosely with plastic wrap and let rise until doubled, 30 to 40 minutes.
- Meanwhile, preheat oven to 350°F.
- Brush the risen loaves with egg white. With a sharp knife, make three 1/2-inch-deep slashes in each loaf. Sprinkle loaves with the remaining 1 tablespoon Parmesan. Bake until the loaves sound hollow when tapped on the bottom, 25 to 35 minutes. Cool on a wire rack.
Per serving: 166 calories; 3 g fat (1 g sat, 1 g mono); 19 mg cholesterol; 26 g carbohydrates; 8 g protein; 1 g fiber; 335 mg sodium; 154 mg potassium.
Nutrition Bonus: Folate (28% daily value), Selenium (20% dv).
Carbohydrate Servings: 2
Exchanges: 1 1/2 starch
More From EatingWell
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Recipe Categories
- Total Time
- More than 1 hour
- Type of Dish
- Baked Goods, yeast breads
- Appetizer
- Servings
- 8 or more
- Ethnic/Regional
- American
- Italian
- Mediterranean
- Preparation/ Technique
- Bake
- Meal/Course
- Appetizers
- Brunch
- Dinner
- Health & Diet Considerations
- Low calorie
- Low cholesterol
- Low saturated fat
- Low sodium
- Heart healthy
- Healthy weight
- Holiday
- Super Bowl
- Style/Theme
- Entertaining, casual
- Kid-friendly
- Picnic
- Vegetarian
- Ease of Preparation
- Moderate
- Publication
- May/June 1995


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jobs near Lake Mary, FL
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Collections Specialist - Lake Mary, FL - Start Date May 23rd- Card Svcs - ORL1
JPMorgan Chase - Heathrow, FL
Critical thinker and ability to exercise independent judgment. Familiarity with multiple browsers, multiple tabs, window navigation and instant messenger tools....
Financial Analyst
Loyal Source Government Services - Orlando, FL
BS degree in Finance, Economics or related field. Develop financial models, conduct benchmarking and process analysis....
Entry Level Customer Service Representative
ADP - Maitland, FL
You can become a certified "smarty pants.". You'll be the one providing the on-the-spot support that makes our integrated solutions stand out in today's...
Lake Mary Retail Co-Manager
Hobby Lobby - Lake Mary, FL
A Co-Manager is the first step to becoming a Hobby Lobby Store Manager, or even developing into a Multi-Unit Manager position....
Member Resolution Specialist
Sears Holdings Corp.: Home Services - Lake Mary, FL
Answer all incoming phone calls while extending highest level of quality customer service. Handle calls effectively to minimize calls in queue while up-selling...
Customer Service Specialist
FARO Technologies - Lake Mary, FL
Is continuing to develop new products that will revolutionize and disrupt the high-tech measurement and imaging industry....
Customer Service Representative -$500 Training Bonus
Sears Holdings Corp.: Home Services - Lake Mary, FL
Receives training/mentoring from Team Manager and applies learning in day-to-day work as an Appointment Specialist • Makes all calls with the mindset that they...
Payroll Specialist
Loyal Source Government Services - Orlando, FL
BSc/BA in business, accounting or relevant field is a plus. Receive approval from upper management for payments when needed....
Auto Damage Auction Inspector
Alliance Inspection Management (AiM) - Orlando, FL
Possess a valid state issued driver's license. Applicants with disabilities may be entitled to reasonable accommodation under the terms of the Americans with...
Customer Service Representative
Paylocity - Lake Mary, FL
Respond to up to 50 calls and emails daily while consistently providing quality service, accurate answers, solutions, and follow-through....


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Risotto is a fickle beast. It has the ability to make a gastronomically-ambitious 16 year old cry. I know this because (years ago) I was that 16 year old, crying over her gummy, overcooked risotto. It took years for me to be brave enough to attempt it again, but when I finally did, I found a few key steps that lead to success.
- Organization is important. Prep everything beforehand, especially if it's your first rodeo.
- Soaking the Aborio rice to wash off its starch (a trick I learned from Serious Eats) and reserving the starchy liquid to add after the rice is toasted helps achieve a perfect, creamy risotto.
- Focus on this dish and nothing else. Because this step is difficult for most people (myself included), be sure to have a glass a wine in hand. It helps with the monotonous stirring.
Even though risotto may be a bit of a challenge, the rewards are beyond worth it.
Asparagus Goat Cheese Risotto
Yields 2-3 servings
3/4 cup Arborio rice
1 bunch asparagus (about 1 1/2 pound)
1 tablespoons olive oil
1 tablespoon unsalted butter
1/4 yellow onion, diced
1 garlic clove, minced
1/4 cup white wine
2-3 cups vegetable broth
2 ounces goat cheese
Parmesan-Reggiano cheese
Freshly cracked pepper
1. In a medium bowl, add the Arborio rice and enough water until the rice is just covered; allow to soak for 5 minutes. Drain, reserving the starchy water. Set both aside.
2. To prep the asparagus, trim the ends and discard. Slice the asparagus points off and transfer the stalks and points to a medium pot. Cover the asparagus with water and bring to a slight simmer; cook for 5-7 minutes. Transfer the asparagus points to a small bowl and set aside; transfer the asparagus stalks to the jar of a food processor. Pulse until the asparagus stalks become a puree; set aside.
3. Pour the vegetable broth into a small to medium saucepan and heat until warm. Place a large skillet over moderately medium heat and add the olive oil and butter. When the butter is melted, add the onion and cook until translucent, about 3-4 minutes. Add the garlic and cook until fragrant, about an additional minute. Pour in the Arborio rice and toast for 2-3 minutes, stirring frequently.
4. Stir in the reserved starchy water and white wine and cook until it's absorbed, about 1-2 minutes. Ladle in 1/2 cup of vegetable broth and cook at a medium simmer, stirring the entire time, until the liquid is absorbed, about 2 minutes. Repeat this process, adding the broth, stirring frequently and letting each veggie broth batch absorb before adding the next ladle or two. Add a few pinches of salt with each addition of broth, tasting it each time; since every broth has different salt content, salting to taste is the best bet.
When the rice reaches a tender and creamy consistency, after about 15 minutes, add the reserved asparagus puree and goat cheese. Stir until thoroughly combined , then add the reserved asparagus points, cooking for an additional minute or two. Top with a grating of Parmesan cheese and a sprinkling of freshly cracked pepper.
All photos by Adrianna Adarme.


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"My name is Tre. I love to dance!!!! I am looking to improve my dancing and meet new Salsa friends."
Want to feel spontaneous again? We are a fun, ethnically diverse group that meets to salsa dance at various locations in Nashville. Meet other local Salsa dancers, musicians, and lovers. Tired of the usual bump and grind?, come meet some nice Ladies & Gentlemen. There is nothing like Salsa Dancing!!!
Sometimes we will meet in a Club, sometimes, we pick random parking lots, park our cars in a circle and dance.
Help support the salsa community in Nashville, have a salsa event you would like to be included on our calendar?
Let us know! I want to provide as many salsa dancing opportunities as possible for our members!
Warm Regards, Carey James Balboa, Founder of Diablos Que Bailan
Financial Support to maintain the group.
Pays for site fees and domain services
Pays for site fees, domain services & promotion
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1,929 YP's
1,300 Belly Dancers
458 Dancers
2,118 East Nashville Peeps
718 Nashfropolitans
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Video Industry News Video Company News Technology Industry News Technology Company News Novelty Industry News Novelty Company News Gay Industry News Gay Company News Legal Industry News All Company News
Released: Apr 08th, 2011
Running Time: 128 Min.
Director: Marie Angel
Company: Girlfriends Films, Inc.
DVD Extras: Behind the Scenes, Still Gallery(ies), Trailer(s)
Cast: Justine Joli, Nic Switch, Madison Young, Dallas Fivestar, Dylan Ryan, Jiz Lee, Sir Sterling
Critical Rating: AAA 1/2
Genre: All-Girl
Come back for another wild ride in Taxi Vol. 2. In "Cheat" Dallas Fivestar seduces a married Jiz Lee. In "Penetration" Madison Young indulges in an intense double penetration with Jiz Lee and Nic Switch. Held up in "Traffic," Justine Joli goes to town on herself. Sir Sterling masturbates with a soft pack in "Smoke." In "Safety" Dylan Ryan finds creative uses for her seatbelt. Buckle up and get ready for the ride of your life.
The artistic backseat lesbo pairings continue in this good edition of the series, with girl/girl veteran Justine Joli giving herself a slow, tentative, if a bit posed orgasm while sprawled across the vinyl. All the scenes take place in the exact same backseat set as the first edition, and it's getting to feel a bit … repetitive. Hopefully, things will be renewed in the next entry in the series.
Jiz Lee and soft butch Dallas Fivestar get off prettily, save for some seasickness-inducing camerawork. Thin, lithe brunette Dylan Ryan smokes a cigarette before humping one out on her seatbelt strap. Lee, Madison Young and Nic Switch get it on in a good three-way with dual strap-on action. Young gets double penetrated in a scene that ends with audible direction.


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âSECOND COMINGâ, CHAPTER EIGHT Bastionâs plan comes to fruition as an impenetrable dome makes the Bay Area a prison. Itâs bad enough for the millions of humans, but itâs even worse for the 98% of the worldâs mutants who live there.
BEST BETS: "Jessica Jones," "Big Trouble/Escape from New York" & More October 2016 Highlights
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