text: Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei Julie Kovárˇová,* Eva Horáková, Piya Changmai, Marie Vancová, Julius Lukeš Biology Center, Institute of Parasitology, Czech Academy of Sciences and Faculty of Sciences, University of South Bohemia, Cˇeské Budeˇjovice (Budweis), Czech Republic Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites. T rypanosoma brucei is a protozoan parasite causing human African trypanosomiasis and related diseases in cattle, camels, water buffalos, and horses. The bloodstream stage (BS) infects mammalian blood and subcutaneous tissues and is transmitted to another host via a tsetse fly vector, where it prospers as the socalled procyclic stage (PS) (1). As an adaptation to the distinct host environments, these two major life stages differ significantly in their morphology and metabolism. One of the most striking differences between them rests in the single mitochondrion, which is large and reticulated in the PS, maintaining all canonical functions, whereas the same organelle in the BS is significantly reduced, with only a few pathways having been detected so far. In order to recapitulate the situation, we present here a short summary of known and verified mitochondrial pathways in the BS. Out of all 14 mitochondrial gene products, only subunit A6 of ATPase is translated and utilized in the BS as an indispensable part of respiratory complex V, which is present and active, even though it does not fulfill its standard function (2, 3). Hence, translation is indispensable (4, 5). Since the A6 transcript undergoes extensive RNA editing, the entire RNA processing machinery is active and essential in the BS as well (6, 7). In addition, since no tRNAs are encoded in the mitochondrial DNA (kinetoplast DNA [kDNA]), their import from the cytosol and modifications are mandatory for mitochondrial translation (4, 8). Ultimately, the import of hundreds of nucleus-encoded mitochondrial proteins involved in protein import and processing, kDNA replication and maintenance, kRNA transcription and processing, and translation are indispensable in the BS (3, 4, 9, 10). In contrast to a typical mitochondrion, the BS organelle does not serve as the main energy source. Instead of the canonical respiratory chain, glycerol-3phosphate dehydrogenase supplies electrons to trypanosome alternative oxidase as a terminal electron acceptor (11). While cytochrome c-dependent respiratory complexes III and IV are altogether missing from the BS, complex II probably remains active. Moreover, complex I is expressed, yet its enzymatic activity is not significant, and its real function remains elusive (12–15). Complex V (ATP synthase) is present, but its F1 part rotates in the reverse direction compared to the PS, cleaving ATP and pumping H⫹ out of the mitochondrion in order to create the membrane potential required for protein import (2). March 2014 Volume 13 Number 3 The efficiency of fatty acids synthesis in the BS organelle via type II pathway is unclear, since its main product, lipoic acid, is present only in a very small amount and there is no apparent use for it (16), although disruption of the biosynthesis caused defects in kDNA segregation (17). Furthermore, the degradation of fatty acids was transferred from the mitochondrion into the glycosomes in all trypanosomatid flagellates (15). In most eukaryotic cells, mitochondria have a central role in Ca2⫹ homeostasis, yet in trypanosomes, this key role is questionable due to the presence of acidocalcisomes (15, 18). Although the mitochondrial calcium uniporter is present in both stages (19), its role in the BS mitochondrion, devoid of citric acid cycle and respiratory chain, remains elusive (15, 19, 20). On the other hand, K⫹ ion homeostasis is essential for the mitochondrion and subsequently for the whole cell (20). Other typical mitochondrial functions, such as citric acid cycle, urea cycle, role in apoptosis, and biosynthesis of ubiquinol and heme, are likely absent from the reduced BS mitochondrion (3, 15, 21–23). Finally, all mitochondria studied so far exhibit biosynthesis of Fe-S clusters, ubiquitous and very ancient enzymatic cofactors (24). Their capacity to transfer electrons is used in complexes I to III of the respiratory chain or metabolic enzymes such as aconitase, fumarase, and lipoate synthase (25, 26). In addition, Fe-S clusters are utilized in iron metabolism, serve in DNA replication, and have structural or other functions (25). These cofactors are synthesized via a complex pathway composed of more than 20 enzymes, and as detailed below, most of them are present in T. brucei (27–31). Received 5 September 2013 Accepted 12 November 2013 Published ahead of print 15 November 2013 Address correspondence to Julius Lukeš, [email protected] * Present address: Julie Kovárˇová, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /EC.00235-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/EC.00235-13 Eukaryotic Cell p. 353–362 ec.asm.org 353 Kovárˇová et al. Cysteine desulfurase (Nfs) abstracts sulfur from cysteine, producing alanine, while ferredoxin provides electrons obtained from NADH by ferredoxin reductase (24). Frataxin is supposed to supply an iron atom; however, this role has still not been convincingly confirmed (26, 32, 33). The assembly of a new Fe-S cluster occurs on a large protein complex composed of Nfs, its indispensable interacting partner Isd11, and the scaffold protein Isu (24). In mammals, frataxin is also present in this complex (34), yet this was not confirmed for trypanosomes (27). The newly formed cofactors are transferred by numerous chaperons and scaffold proteins, which either transfer them to the mitochondrial apoproteins or export a so-far-unknown intermediate from the organelle that is further processed and exploited for the cytosolic and nuclear FeS-dependent proteins (24–26). Most key components of the mitochondrial Fe-S biosynthetic pathway are essential for the PS of T. brucei. RNA interference (RNAi) depletion of Nfs, Isd11, Isu, Isa1, Isa2, and frataxin resulted in drastic changes in growth phenotypes, decreases in the activity of several Fe-S enzymes, and other downstream effects (27–29, 31, 35). In addition, Nfs and Isd11 are also indispensable for tRNA thiolation (27). Two homologues of the electron donor protein ferredoxin were identified in the genomes of trypanosomatid flagellates, but only ferredoxin A is essential for the Fe-S cluster assembly (31). However, no functional data are available on this pathway in the BS other than that the level of its components is much lower than that in the PS, as has been shown for frataxin (35), Isd11 (27), ferredoxin (31), and Isa1 and Isa2 (29). In general, the (in)dispensability of Fe-S machinery in the BS mitochondrion is questionable, since the bulk of Fe-S clusters produced in the organelle of a typical eukaryotic cell are utilized for its respiratory chain complexes, which are either repressed or absent in this life stage of T. brucei. To the best of our knowledge, the only Fe-S-dependent proteins still expressed are ferredoxin A and glutaredoxin 1, both involved in the Fe-S pathway itself (31, 36), which implies that they are present only in a very small amount and pose a "chickenor-the-egg"-type problem. Moreover, there is Fe-S-dependent lipoic acid synthetase, but, as mentioned above, the relevance of lipoic acid synthesis for the BS remains to be established (16, 17). We scrutinized the localization of three core Fe-S assembly components, Nfs, Isu, and frataxin, using cell lines with tagged proteins and examined them by immunofluorescence microscopy and digitonin fractionation followed by Western blotting and electron microscopy. We were able to show that these proteins are located in the mitochondrion, yet in the case of Nfs and Isu, a substantial amount was detected in the nucleolus, suggesting that cysteine desulfurase with its scaffold protein must have a so-farunknown function in this cell compartment. Since there is no detectable activity of the Fe-S-dependent enzymes aconitase and fumarase in the BS, we created cell lines overexpressing these marker Fe-S enzymes, which enabled us to verify that Fe-S proteins are produced in this unusual organelle and are capable of fulfilling their standard functions. MATERIALS AND METHODS Preparation of pT7-v5 constructs. Gene constructs with Nfs (TriTrypDB no. Tb927.11.1670), Isu (Tb927.9.11720), and frataxin (Tb927.3.1000) open reading frames (ORFs) lacking stop codons, followed by v5 or three-hemagglutinin (HA3) tags, were prepared using the following primer pairs: Nfsv5-FP (5= GGAAGCTTATGTTTAGTGGTGTTCGCGTAC) and Nfs-v5-RP 354 ec.asm.org (5= TTAGATCTCCGCCACTCCACGTCTTTAAGG); Isu-v5-FP (5=AGG AAGCTTATGCGGCGACTGATATCATC) and Isu-v5-RP (5=AGGGATCC GCTTGACACCTCACC); and Frat-HA-FP (5= ATAAAGCTTATGCGGC GCACATGTTGCGC) and Frat-HA-RP (5= AGACTCGAGTTCGGTTTC TCCAGCCCCTG) (added BamHI and HindIII restriction sites are underlined). PCR products amplified from the total genomic DNA of T. brucei PS 29-13 strain were cloned into pT7-v5 or pT7-HA3 vectors and verified by sequencing. The obtained constructs were linearized by NotI and electroporated into the parental PS 29-13 cells and the single-marker BS cells, the transfected cell lines were selected using puromycin (1 g/ml), and expression of the tagged protein was induced by the addition of tetracycline (1 g/ml). The cell line expressing v5-tagged ferredoxin A was described elsewhere (31). Immunofluorescence assay. Approximately 1 ⫻ 107 PS or BS cells were collected, centrifuged, resuspended in fresh medium, and stained with a 200 nM or 20 nM concentration, respectively, of the mitochondrion-selective dye Mitotracker red (Invitrogen). After 30 min incubation at 27°C or 37°C, the cells were centrifuged for 5 min at 1,300 ⫻ g at room temperature, and the pellet was washed and resuspended in 1 ml phosphate-buffered saline (PBS) with 4% formaldehyde and 1.25 mM NaOH. The suspension (250 l) was applied to a microscopic slide and incubated for 10 min at room temperature. The slides were washed with PBS, incubated in ice-cold methanol for an additional 20 min, and briefly washed with PBS. Next, primary anti-v5 or anti-HA3 antibodies (Invitrogen) were added at a 1:100 dilution in PBS-Tween (0.05%) with 5% milk (Laktino, low fat; PML, Czech Republic) and incubated at 4°C overnight. Slides were washed with PBS prior to the application of Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) at a 1:1,000 dilution for 1 h at room temperature. Finally, the samples were mounted with Antifade reagent with 4=,6=-diamidino-2-phenylindole (DAPI; Invitrogen) and after incubation in the dark for 30 min were visualized using an Axioplan2 imaging fluorescence microscope (Zeiss). Digitonin fractionation and Western blot analysis. In order to compare protein expression in the PS versus BS, cell lysates corresponding to 5 ⫻ 106 cells per lane were separated on a 12% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride (PVDF) membrane and probed with the polyclonal antibodies chicken anti-Nfs and rat anti-Isu at a 1:1,000 dilution and anti-enolase at a 1:50,000 dilution, followed by appropriate secondary antibodies conjugated with horseradish peroxidase (Sigma), and then visualized using the Pierce enhanced chemiluminescence (ECL) kit (Thermo Scientific). For the digitonin fractionation, 107 cells per sample were collected, incubated in STE-NaCl buffer (250 mM sucrose, 25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl) with 0.04, 0.16, 0.32, 0.4, 0.8, and 1.2 mM digitonin for 4 min at 27°C. Subsequently, samples were centrifuged (16,000 ⫻ g for 2 min), and the supernatants obtained were used for Western blot analysis. For decorating of tagged proteins, monoclonal anti-mouse antibody (Sigma) was used at a 1:2,000 dilution. As controls, antibodies against the following proteins were used: the outer mitochondrial membrane protein VDAC (37), mitochondrial intermembrane sulfhydryl oxidase (38), the mitochondrial matrix protein MRP2 (39), the nuclear protein La (40), the glycosomal triosephosphate isomerase TIM424, and cytosolic enolase (both provided by Paul A. M. Michels). Electron microscopy. Cells were fixed either with 4% formaldehyde or 4% formaldehyde with 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 1 h at room temperature. After centrifugation at 1,300 ⫻ g for 10 min, cells were washed three times in 0.1 M phosphate buffer containing 0.01 M glycine. The pellet was resuspended in 10% gelatin dissolved in water at 37°C, centrifuged, and solidified on ice. Slices of gelatin-embedded pellet were immersed in 2.3 M sucrose and left for 6 days on a rotating wheel at 4°C. Infiltrated samples were immersed in liquid nitrogen and cryosectioned using a Leica EM FCS ultramicrotome (Leica) equipped with a cryochamber (Ultracut; Leica) at ⫺100°C. Cryosections were picked up using a drop of solution containing 1% methyl Eukaryotic Cell Fe-S Cluster Synthesis in Bloodstream-Stage T. brucei FIG 1 Nuclear localization signal is present in Nfs from T. brucei and other eukaryotes. Multiple sequence alignment of the Nfs proteins from distantly related eukaryotes. The alignment was generated using ClustalW (http://www.ch.embnet.org/software/ClustalW.html) and Boxshade (http://www.ch.embnet.org /software/BOX_form.html). Identical residues have a black background, and conserved residues have a gray background. The nuclear localization signal (NLS) identified in yeast is boxed (38). The following sequences were used: Trypanosoma brucei Tb927.11.1670, Homo sapiens NP_001185918.1, Saccharomyces cerevisiae NP_009912.2, Arabidopsis thaliana NP_201373.1, Leishmania mexicana XP_003876680.1, Plasmodium falciparum XP_001349169.1, Blastocystis hominis CBK24185.2, Encephalitozoon cuniculi NP_586483.1, and Entamoeba histolytica XP_655257.1. cellulose and 1.15 M sucrose and thawed in PBS. Grids were placed on drops of blocking solution containing 5% low-fat milk and 0.02 M glycine in PBS for 2 h. Grids were transferred onto drops of mouse monoclonal antibody against the v5 tag (50 g/ml; Invitrogen) diluted in blocking solution and left overnight at 4°C. Grids were washed in PBS containing 0.5% low-fat milk and incubated for 1 h in goat anti-mouse IgG conjugated to 10-nm gold particles (Aurion) diluted 1:40 in blocking solution. Grids were then washed in PBS and distilled water, contrasted, and dried using 2% methyl cellulose with 3% aqueous uranyl acetate solution diluted at 9:1. Sections were examined in 80-kV JEOL 1010 and 200-kV 2100F transmission electron microscopes. Controls were performed using anti-v5 tag labeling of both wild-type BS and PS cryosections under the same conditions as described above. Quantification of gold labeling. Electron micrographs of randomly selected areas were taken at the same magnification. A lattice of test points at constant distance of 100 nm was superimposed randomly on the micrographs using ImageJ software. Points and gold particles were counted separately within compartments: nucleus, nucleolus, mitochondrion, kDNA, and background (areas outside the above-mentioned compartments). Observed densities of labeling (LDobs) were expressed for each compartment as the sum of gold particles per sum of points. In order to statistically evaluate labeling of individual compartments, total expected numbers of gold particles for each compartment were calculated as total expected density (all gold particles divided by total points [total LDexp]) multiplied by total counts of points of each cell line. The chi-squared test was calculated as (LDobs ⫺ LDexp)2/LDexp. The relative labeling index (RLI) was calculated for each compartment as the ratio of observed labeling density value to expected labeling density (41). RNAi construct, transfection, cloning, RNAi induction, and cultivation. To prepare the Isu (Tb927.9.11720) RNAi construct, a fragment of the Isu ORF was amplified using the primer pair IscU-F1 (5=-CACCTCG AGCAGCCTCACTTCGGTCACT) and IscU-R1 (5=-TGCACGGATCCC CAACAGCCTCGGACTTAG) from total genomic DNA of T. brucei PS 29-13. The amplicon was cloned into the p2T7-177 vector, which was, upon NotI-mediated linearization, introduced into BS T. brucei singlemarker cells using a BTX electroporator and selected as described elsewhere (42). The BS trypanosomes were cultivated in HMI-9 medium at 37°C in the presence of G418 (2.5 g/ml) and phleomycin (2.5 g/ml). RNAi was initiated by the addition of 1 g/ml tetracycline to the medium, and growth measurements were made using the Beckman Z2 Coulter counter. tRNA thiolation assay. Total and mitochondrial RNA was electrophoretically separated on 8 M urea– 8% acrylamide gels cast with 50 M (N-acryloylamino)-phenyl mercuric chloride (APM). The samples were denatured by heating at 72°C for 2 min, and equal amounts (10 g) of RNA were loaded per lane. After electrophoresis, the gels were stained with ethidium bromide for visualization under UV light, soaked in Trisborate-EDTA (TBE) buffer containing 200 M ␤-mercaptoethanol, and March 2014 Volume 13 Number 3 blotted onto a Zetaprobe membrane (Bio-Rad) for Northern blot analysis. Hydrogen peroxide treatment was carried out as a nonthiolated control as described elsewhere (27). Expression and measurement of aconitase and fumarase in BS. The ORFs of fumarase (Tb927.3.4500) and aconitase (Tb927.10.14000) were PCR amplified from the total genomic DNA of T. brucei PS 29-13 strain using the primer pairs TbAco-FW (5=-GGGTCTAGAATGCTCAGCACG ATGAAGTT) and TbAco-RV (5=-GCGCTCGAGCTACTACAAATTAC CCTTGAT) (added XbaI and XhoI restriction sites are underlined) and Tbcy-Fum-FW (5=-AGGAAGCTTATGAGTCTCTGCGAAAACTG) and Tbcy-Fum-RV (5=-ATATCTAGAGACGAGTTTAGCATAGAA) (added BamHI and HindIII restriction sites are underlined), and the PCR products were cloned into the pT7-v5 vector. The obtained vectors were linearized by NotI and electroporated into BS T. brucei single-marker cells using an Amaxa Nucleofector II electroporator, with puromycin (1 g/ ml) used as a selection marker. Expression of the ectopic proteins was induced by the addition of 1 g/ml tetracycline. Cytosolic and mitochondrial fractions were obtained by digitonin fractionation following the protocol described elsewhere (28). The aconitase and fumarase activities were measured spectrophotometrically at 240 nm as the production rate of cis-aconitase and fumarate, respectively. L-Malic acid (Serva) and DL-isocitric acid trisodium salt hydrate (Sigma) were used as substrates in the aconitase and fumarase activity assays, respectively, as described previously (35). RESULTS Nfs contains a nuclear localization signal. The cysteine desulfurase Nfs is a common mitochondrial protein found in almost all eukaryotes. An alignment of protein sequences of nine species from different eukaryotic supergroups revealed high conservation of key domains (see Fig. S1 in the supplemental material). A nuclear localization signal, RRRPR, was identified in the Saccharomyces cerevisiae Nfs protein sequence (43) which is also present in the T. brucei sequence (Fig. 1). Even though a nuclear targeting sequence in trypanosomes was described only in a few cases (44– 48), and no consensus sequence has been defined so far, the general rules postulating a high content of positively charged amino acids such as arginine seem to be universal. Thus, we hypothesize that the sequence RRRPR may function as a nuclear targeting signal in T. brucei as well. Moreover, the alignment showed absolute conservation of this motif in six of nine compared eukaryotes, including humans, with positively charged residues predominating in all aligned putative nuclear targeting signals (Fig. 1). Fe-S assembly proteins are present in the BS mitochondrion. In order to establish localization of the Fe-S assembly machinery in the BS, we created cell lines overexpressing tagged versions of ec.asm.org 355 Kovárˇová et al. FIG 2 Immunofluorescence analysis of tagged proteins in T. brucei BS and PS. The cells were stained with monoclonal anti-v5 and anti-HA antibodies (green), MitoTracker (red), and DAPI (blue). All target proteins were detected in mitochondria, as confirmed by colocalization with the MitoTracker signal. Identical results were obtained for the BS and PS cells and for the v5 and HA tags. Nfs, Isu, and frataxin. Each of these genes was cloned into the pT7-v5 vector, and the obtained constructs were separately electroporated into the BS T. brucei. Since such an approach may cause nonphysiologically high expression of the tagged proteins, resulting in possible mislocalization within the cell, we also transfected the same constructs into the PS cells, where these proteins are known to localize to the mitochondrion (27, 28, 30). Upon decoration with monoclonal anti-v5 antibody and examination by immunofluorescence microscopy, in both the PS and BS trypanosomes expressing the v5-tagged Nfs, the signal was confined exclusively to the mitochondrion (Fig. 2A and B). The same approach was used to detect the Isu protein in both life stages. Indeed, the fluorescent signal corresponded to the large reticulated and the dwindled sausage-shaped mitochondrion of the PS and BS cells, respectively (Fig. 2D and E). In order to eliminate potential artifacts caused by the v5-tagging strategy, an additional cell line was produced containing a HA3-tagged Isu. As with the other tag, the monoclonal anti-HA antibody decorated only the mitochondrion (Fig. 2C). Frataxin, the presumed iron donor for Fe-S clusters, has been functionally analyzed in PS (35) but not BS T. brucei so far. Therefore, we created a BS cell line expressing frataxin with added HA3 tag and showed by immunofluorescence microscopy its localization in the reduced mitochondrion of this life stage (Fig. 2F). Digitonin fractionation reveals dual localization of Nfs. An alternative approach to interrogate intracellular localization of a tar- 356 ec.asm.org FIG 3 Nfs and Isu proteins are present in the mitochondrion and in another subcellular compartment. Six samples were prepared from each cell line, incubated with increasing amount of digitonin, and centrifuged, and the supernatants obtained were used for Western blot analyses. Monoclonal anti-v5 and anti-HA antibodies were used for detection of the tagged proteins, and polyclonal anti-Nfs antibody was used to detect the endosomal protein. The mitochondrial membrane channel VDAC, the intermembrane protein Erv1, and the mitochondrial matrix protein MRP2 were used as mitochondrial controls; TIM424, the La protein, and enolase were used as glycosomal, nuclear, and cytosolic controls, respectively. These controls determined that mitochondrial proteins are present only in the last two fractions, but Nfs was released by lower concentrations of digitonin in both the BS and PS samples. Isu-v5 was detected in both the monomeric (25 kDa) and dimeric (50 kDa) forms. Again, the fraction of the signal was extramitochondrial. In the PS, the bulk of both proteins is present in the mitochondrion. The HA-tagged frataxin was detected solely in the mitochondrion-containing fractions. get protein is treatment of cells with the detergent digitonin, which disrupts phospholipid membranes and allows us to distinguish between subcellular compartments. For each cell line, subcellular fractions were obtained by using increasing concentrations of digitonin (0.04 to 1.2 mM) (Fig. 3). As shown by Western blotting analyses, the mitochondrial proteins were released only when a 0.8 mM or higher concentration of digitonin was used. All fractions were also treated Eukaryotic Cell Fe-S Cluster Synthesis in Bloodstream-Stage T. brucei FIG 4 T. brucei cells overexpressing v5-tagged Nfs or Isu were labeled with anti-v5 antibody. Immunolocalization of Nfs (A to C, G, and H) and Isu (D to F) in ultrathin cryosections of PS (A to F and I) and BS (G and H). The gold particles were detected in the mitochondrion (A and D), often associated with kDNA (B, F, and G), and in the nucleolus (C, E, and H). Ferredoxin B (I) was localized only in the mitochondrion of PS. Mt, mitochondrion; Nc, nucleolus; N, nucleus; k, kDNA. Bar, 500 nm (A to E and H) and 200 nm (F, G, and I). with antibodies against the following control proteins: voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane (37), sulfhydryl oxidase Erv1 of the intermembrane space (38), mitochondrial RNA-binding protein MRP2 in the mitochondrial matrix (39), glycosomal triose-phosphate isomerase (TIM424), nuclear protein La (40), and cytosolic enolase. When the Nfs-v5-expressing cells were used, the signal acquired with anti-v5 antibody differed from the pattern obtained with antibodies against the available mitochondrial markers (Fig. 3). Substantial fraction of the v5 signal was consistent with much earlier release of the tagged Nfs protein, suggesting localization also outside the double membrane-bound mitochondrion. Identical result was obtained when the specific anti-T. brucei Nfs antibody was used against BS WT cells (Fig. 3). Although in both PS and BS cells the target protein was detected in overlapping fractions, a much stronger signal was present in the last two PS fractions, strongly suggesting that in this life cycle stage, the majority of the Nfs protein is located in the mitochondrion. However, in the BS, the extramitochondrial signal was as strong as the organellar one (Fig. 3). Western blot analysis of the PS lysates expressing the v5-tagged Isu revealed two separate bands, even though a monoclonal anti-v5 antibody was used (Fig. 3). Since the upper band corresponds to the double size of the lower band, we interpret these bands as the Isu dimer and monomer, respectively. Dimerization of components of the Fe-S assembly machinery was described for the mammalian cells (49), and apparently the complex was not effectively separated under the SDS-polyacrylamide gel conditions used. A strong signal of the 50-kDa band is present in the March 2014 Volume 13 Number 3 mitochondrial fractions, while a much weaker yet distinct signal in other fractions suggests the presence of Isu in additional subcellular compartments. The weak 25-kDa band is confined to the mitochondrial fractions (Fig. 3). Finally, the BS cells containing HA3-tagged frataxin were analyzed by digitonin fractionation. The results were fully consistent with localization solely in the mitochondrial matrix (Fig. 3), which is in agreement with the data from the PS flagellates (30). Taken together, these results strongly indicate that Nfs and Isu are localized predominantly in the mitochondrion, but also in an additional subcellular compartment, possibly the nucleus. Nfs and Isu are also present in the nucleolus. Next, we subjected the tagged cell lines to immunostaining with colloidal goldlabeled antibodies followed by transmission electron microscopy. This sensitive approach confirmed the presence of Nfs in the mitochondrion and the nucleolus of both PS (Fig. 4A to C) and BS (Fig. 4G and H) T. brucei. Nfs was detected in the mitochondrial lumen, with most signal being present in the vicinity of the kDNA disk, and in the nucleolus (Fig. 5). The same approach was applied to the PS cells expressing v5-tagged Isu, ferredoxin A, and ferredoxin B. Similarly, Isu was detected in the mitochondrial lumen and in the nucleolus (Fig. 4D to F and 5; also, see Fig. S2 in the supplemental material). In contrast, ferredoxin A and ferredoxin B were present solely in the mitochondrion, with only a slight enrichment in the region around the kDNA (Fig. 4I and 5; also, see Fig. S2 in the supplemental material). Specificity of the labeling protocol was proven by negative reactions of the anti-v5 tag antibody on cryosections of the BS and ec.asm.org 357 Kovárˇová et al. FIG 5 Statistical analysis of immunogold labeling on cryosections of PS. Nfs is present in the nucleolus and the mitochondrion, where half of the protein is associated with the kDNA. Similarly, Isu is present in the nucleolus and the mitochondrion, but with somewhat lower affinity for the kDNA. Ferredoxins A and B are present solely in the mitochondrion. PS WT cells (see Fig. S3 in the supplemental material). Moreover, the different distribution of the anti-v5 tag immunogold labeling in cells expressing either v5-tagged ferredoxin, Nfs, or Isu further supports the specificity of labeling and excludes a possible unspecific reaction of the anti-v5 tag antibody or the secondary gold conjugate. Since flagellates fixed with glutaraldehyde showed only a low efficiency of labeling with the v5-tag antibody, only results obtained with the formaldehyde-fixed cells are presented. Observed and expected counts of gold particles were compared in order to distinguish preferential labeling (RLI ⬎ 1) from the random one (RLI ⫽ 1), and the data were examined using chisquared (2) analysis (see Table S1 in the supplemental material). We calculated partial (for each compartment and cell line) and total (sum of partials) 2 values to test the null hypothesis (no difference between distributions). Values of partial 2 indicate the responsibility of these compartments for the difference (preferential versus random distribution). Proportions of observed labeling densities over the mitochondria, nuclei, and nucleoli are summarized in Fig. 5. Thorough analysis of formaldehyde-fixed cryosections confirmed that Nfs and Isu are localized in the nucleolus, while only the former protein is significantly enriched in the vicinity of the kDNA disk in the mitochondrion. In contrast, both ferredoxins are confined solely to the mitochondrion (Fig. 5; also, see Fig. S2 in the supplemental material). Fe-S cluster assembly is an essential function of the BS mitochondrion. With regard to the differences between the large reticulated versus the reduced sausage-shaped mitochondria of the two life stages, it is not surprising that in the BS T. brucei, frataxin (35), ferredoxin A (31), and Isa1 and Isa2 proteins (29) are significantly less abundant than in the PS trypanosomes. The situation is very similar in the cases of Nfs and Isu, which are decreased two and five times, respectively, in the mammalian BS compared to the insect-dwelling PS (Fig. 6A). Importantly, the significance of the Fe-S cluster assembly in the disease-causing BS has not been established so far. In order to address this issue, a BS Isu RNAi cell line was constructed, where the depletion of Isu resulted in a severe growth phenotype (Fig. 6B). The efficiency of RNAi-mediated ablation of the target protein was confirmed by Western blotting (Fig. 6B, inset). Since except for its scaffold role in the Fe-S cluster synthesis, no other function is known for the Isu protein, we resorted to testing its possible function in tRNA thiolation (see below). Cytosolic thiolation is downregulated in the PS but not in the BS Isu RNAi cells. Since Isu is a binding partner of Nfs, which in T. brucei is also known as a source of sulfur for tRNA thiolation (27), we decided to study its potential role in this process as well. To our surprise, the thiolation of several cytosolic tRNAs remained unaltered upon the ablation of Isu in the BS (Fig. 7A). Therefore, we decided to also study the modification in PS Isu RNAi knockdowns, which were previously generated in our lab (28). Contrary to the BS data, thiolation is very sensitive to the loss of Isu, with an apparent phenotype being observed 2 days after RNAi induction (Fig. 7B). It is known that this tRNA modification and tRNA import are used equally in both life cycle stages (4), meaning that the resulting growth inhibition in the BS depleted of Isu is most likely a consequence of a perturbation of the Fe-S synthesis pathway rather than tRNA thiolation. Fe-S cluster assembly is active in the BS. Finally, we addressed the question of whether the Fe-S assembly pathway is complete and active in the highly reduced BS mitochondrion. A standard FIG 6 Fe-S cluster assembly is diminished but essential in BS. (A) Samples containing the same numbers of cells were prepared from the PS and BS cells and analyzed by Western blotting. Nfs and Isu are expressed at 45% and 22%, respectively, in the BS compared to the PS. Enolase was used as a loading control. (B) Growth of the BS Isu RNAi cells in the absence (solid line) or presence (dashed line) of tetracycline was examined. After the induction, a significant growth defect was observed. The efficiency of RNAi was verified by Western blotting using anti-Isu antibody and enolase as a loading control (inset). 358 ec.asm.org Eukaryotic Cell Fe-S Cluster Synthesis in Bloodstream-Stage T. brucei FIG 7 Cytosolic thiolation in the BS (A) or PS (B) depleted for Isu. RNA isolated from different cell lines was separated on an APM gel followed by Northern blotting. Bands corresponding to thiolated (⫹S2U) and nonthiolated (⫺S2U) tRNAs are indicated. H2O2 was used in s as a control of complete oxidation of thiolation, eliminating the mobility shift. The number of days after induction of RNAi by tetracycline is indicated above the lanes. ⫺, noninduced RNAi. approach to scrutinize the availability of the Fe-S clusters is the activity assay for the Fe-S-containing metabolic enzymes aconitase and fumarase. As in other eukaryotes, this method is suitable for the PS (27, 29, 31), but activities of both enzymes are undetectable in the wild-type BS (Fig. 8). To overcome this lack of measurable Fe-S-dependent activities in BS, we created BS cell lines overexpressing either fumarase or aconitase from the pT7-v5 vector. Upon induction with tetracycline, aconitase or the cytosolic form of fumarase were strongly expressed, and the relevant activity was measured in a given cell compartment. In all cases, a strong activity of the overexpressed enzyme, almost reaching the PS level, was obtained (Fig. 8). Separate measurement of the aconitase activity in the cytosol and in the mitochondrion confirmed that the Fe-S clusters are available in both compartments (Fig. 8A). These results confirm that the whole Fe-S synthetic pathway is not only present but indeed active in the BS and that the functional cofactors are available in both the organelle and the cytosol. DISCUSSION Combined, the data provide a comprehensive overview of the Fe-S assembly machinery in the BS T. brucei. We focused on the key components, namely, the cysteine desulfurase Nfs, the scaffold protein Isu, and frataxin. Several lines of evidence are consistent with the conclusion that even in the highly suppressed BS mitochondrion, the Fe-S clusters are efficiently built, regardless of the low abundance of their target proteins. Importantly, Nfs and Isu are also present in the nucleolus, while frataxin and both ferredoxins were detected solely in the mitochondrion. It was of interest to thoroughly examine the subcellular localization of Nfs, Isu, and frataxin proteins in the BS T. brucei. Indeed, Nfs has a dual localization in the mitochondrion and the nucleolus, which is in line with the results obtained with yeast (50, 51). The same dual localization was demonstrated for Isu, but unlike in Nfs, no nuclear localization signal can be detected in the Isu sequence. However, noncanonical and bipartite nuclear local- March 2014 Volume 13 Number 3 FIG 8 Enzymatic activities of fumarase (A) and aconitase (B) in the PS and BS. Aconitase was measured in cytosolic and mitochondrial fractions; fumarase was measured only in the cytosol. Activities of both Fe-S-dependent enzymes were measured in the wild-type PS and BS flagellates and in the BS with inducible overexpression (1) of the target proteins. The activity obtained in the PS was set as 100%. Whereas in the wild-type BS, the activities are almost undetectable, levels comparable to those in the PS were reached after overexpression of each enzyme. These results confirm that functional Fe-S clusters are produced even in the BS in both cytosolic and mitochondrial compartments. Values are means and standard deviations from three independent experiments. ization signals were reported from trypanosomes, which are notoriously difficult to determine (45). Furthermore, in yeast, Nfs is first imported into the mitochondrion and only afterwards transferred to the nucleus (43). The possibility that the whole protein complex is formed in the mitochondrion and only a portion of it is subsequently imported into the nucleolus has to be tested. As we showed previously, similar to other eukaryotes, in T. brucei, Isd11 is a partner of Nfs, which participates in both Fe-S biosynthesis and tRNA thiolation (27). Mimicking the situation in the mitochondrion, a functional complex of Nfs, Isd11, and Isu may also be formed in the nucleolus in order to effectively abstract sulfur from cysteine and exploit it for a different use than incorporation into the Fe-S clusters. In contrast to mammals (34), trypanosomes do not appear to have frataxin as part of this complex (27), and the protein is strictly detected in the mitochondrion (30; also this work). To the best of our knowledge, our results provide the first direct evidence of localization of Nfs and Isu in the nucleolus. Both Western blot analysis and transmission electron microscopy ec.asm.org 359 Kovárˇová et al. showed that the bulk of these proteins are present in the mitochondrion compared to the nucleolus, due to which similar dual localization may have been overlooked in other organisms. In yeast, it was detected by means of an ␣-complementation assay (43), which is highly sensitive but is hardly applicable in other eukaryotes. In fact, a signal in the nucleolus of T. brucei was undetectable by immunofluorescence, although this may be due to technical reasons, such as the nucleolar localization being masked in the PS by the extensively reticulated mitochondrion. Another reason may be a limited penetration of the specific antibody into the nucleus during the applied staining protocol. While the function of the mitochondrial Nfs is well known, we know close to nothing about its nucleolar version. It is apparently essential, since mutation of its nuclear localization signal caused a severe change in the growth phenotype in yeast, yet depletion of the nuclear Nfs did not affect maturation of the Fe-S-containing proteins in either the mitochondrion or the nucleus (50, 51). The authors consequently predicted that the nuclear Nfs must be involved in tRNA thiolation. However, this possibility was excluded by showing that Nfs lacking the mitochondrial targeting sequence cannot complement thiolation of either cytosolic or mitochondrial tRNAs (52). In mammalian cells, Nfs was also detected in the cytosol (49) and was shown to be involved in the biosynthesis of the molybdenum cofactor (53). We can speculate that such a role may be also attributed to the mitochondrial Nfs in the protist studied, since the molybdenum cofactor synthetase of T. brucei (Tb927.11.11920) has a 0.99 probability of mitochondrial localization, according to Mitoprot (54). Interestingly, a role in phosphorothioation was recently proposed for bacterial Nfs (55). This DNA modification is unique in directly altering the DNA backbone by exchanging oxygen of phosphate for sulfur (56–58). Phosphorothioation seems to be widespread among diverse bacteria, but data about its presence in eukaryotes are lacking (56). While the DNA of Escherichia coli depleted of IscS (bacterial homolog of Nfs) is broken into a characteristic smear (55), exactly the same experiment performed in wild-type and Nfs-depleted T. brucei did not result in a DNA degradation phenotype (see Fig. S4 in the supplemental material). It is of interest that almost half of the mitochondrial Nfs is situated in or around the kDNA disk, which is located next to the basal body of the single flagellum. The remaining signal was more or less evenly distributed throughout the lumen of the reticulated organelle, while Isu and both ferredoxins did not show a similarly skewed distribution. This indicates a possible association of a fraction of Nfs with the mitochondrial DNA. The nuclear localization signal is carried by the family of Nfs-related cysteine desulfurases throughout the eukaryotic domain and most likely was present even in their last common ancestor; thus, it must be evolutionarily ancient. Furthermore, we speculate that the function of the nuclear cysteine desulfurase was at least originally the same for all of them. Although the current paradigm postulates that the Fe-S clusters are in some form essential for every extant eukaryotic cell (24, 25), we wondered whether the mitochondrial branch of the Fe-S assembly pathway produces clusters in the BS organelle, which uniquely lacks respiratory complexes. The only components of the Fe-S biosynthetic pathway studied so far in the BS are the scaffold proteins Isa1 and Isa2, which are dispensable in this stage, unlike in the PS (29). Nevertheless, they were shown to function as scaf- 360 ec.asm.org folds only for the synthesis of 4Fe-4S clusters (59); thus, their dispensability in the reduced organelle is not surprising. Since the Fe-S cluster assembly was detected in the mitochondrion-derived mitosomes and hydrogenosomes of Giardia and Trichomonas, respectively (60, 61), it was anticipated that it is also active in the highly functionally downregulated mitochondrion of African trypanosomes. Retention of the Fe-S cluster assembly in the hydrogenosomes is meaningful, since they possess an alternative pathway producing acetyl coenzyme A (acetyl-CoA) and hydrogen (61), and three enzymes involved in this pathway contain Fe-S clusters. The essentiality of Isu and the measured activities of overexpressed aconitase and fumarase provide evidence that these ancient clusters are made in the repressed organelle of the BS T. brucei as well. Genes encoding components of this ancestral alphaproteobacterial pathway were, in the course of evolution, transferred to the nucleus and retargeted to the organelle via acquired mitochondrial localization signals (62). In the BS mitochondrion, these cofactors may be incorporated into the nonessential respiratory complexes I and II (12), as well as ferredoxin, glutaredoxin, and lipoate synthase, all of which are expressed at a very low level, however (31, 63). Therefore, we propose that in this life cycle stage, the bulk of cofactors is exported outside the mitochondrion, as in these fast-dividing parasites a high demand for them exists in the catalytic subunits of DNA polymerases (25) and other cytosolic and nuclear proteins. In conclusion, all the results presented here indicate that the key components of the Fe-S biosynthetic pathway are present in the minimalistic mitochondrion of the BS T. brucei, where they are essential and produce functional cofactors, even though the demand for Fe-S clusters within this organelle is significantly lower than in the PS mitochondrion. In addition we have shown that a significant fraction of Nfs and Isu is present in the nucleolus. Nonetheless, their role in this compartment remains elusive, since all putative functions tested so far, such as Fe-S cluster synthesis (50), tRNA thiolation (52), and phosphorothioation of DNA (this work), provided negative results. ACKNOWLEDGMENTS We thank Shaojun Long (Washington University, St. Louis) and De-Hua Lai (Sun Yat-Sen University, Guanqzhou, China) for their contribution in the early stages of this project and Hassan Hashimi for helpful discussions. We also thank Paul A. M. Michels (Universidad de los Andes, Mérida/ University of Edinburgh, Edinburgh, United Kingdom) and André Schneider (University of Bern, Bern, Switzerland) for sharing antibodies. This work was supported by Czech Grant Agency grant P305/11/2179, Bioglobe grant CZ.1.07/2.3.00/30.0032, AMVIS grant LH12104, EU grant FP7/2007-2013(no. 316304), and the Praemium Academiae award to J.L., who is also a Fellow of the Canadian Institute for Advanced Research. REFERENCES 1. Fenn K, Matthews KR. 2007. The cell biology of Trypanosoma brucei differentiation. Curr. Opin. Microbiol. 10:539 –546. http://dx.doi.org/10 .1016/j.mib.2007.09.014. 2. Schnaufer A, Clark-Walker GD, Steinberg AG, Stuart K. 2005. 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Sutak R, Doležal P, Fiumera HL, Hrdý I, Dancis A, Delgadillo-Correa M, Johnson PJ, Müller M, Tachezy J. 2004. Mitochondrial-type assembly of FeS centers in the hydrogenosomes of the amitochondriate eukaryote Trichomonas vaginalis. Proc. Natl. Acad. Sci. U. S. A. 101:10368 –10373. http://dx.doi.org/10.1073/pnas.0401319101. Tachezy J, Sánchez LB, Müller M. 2001. Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS. Mol. Biol. Evol. 18:1919 –1928. http://dx.doi.org/10.1093/oxfordjournals.molbev .a003732. Nilsson D, Gunasekera K, Mani J, Osteras M, Farinelli L, Baerlocher L, Roditi I, Ochsenreiter T. 2010. Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei. PLoS Pathog. 6:e1001037. http://dx.doi.org/10.1371 /journal.ppat.1001037. Eukaryotic Cell


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text: Next Story > Scene Asia Watch and Vote: Do Real Men Wear Pearls? Nov 23, 2011 2:46 pm HKT Jewelmer is marketing pearls to men, but whether to wear them has stirred up some debate. WSJ's Andrew LaVallee joins Asia Today. Readers, what's your take? Are these designs suitable for guys? Vote in our interactive poll: To Read the Full Story, Subscribe or Sign In Previous Singapore Artists Fight for 'Old School' Landmark Next Australia's Desert Art Hits China


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c1980s Avon plastic bangle bracelet in the pattern they called Tropical. It's a hard plastic without seams and a decal design.
Avon produced 2 sizes of diameter and this is the larger.
It measures 2" wide with straight walls just under 1/4" thick and an inside diameter of almost 2 7/8".
The condition is great with no damage.
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Quote:Day 2: 1:00pm-2:00pm
8,639pages onAdd New Page
this wiki
this wiki
This is a list of memorable quotes from "Day 2: 1:00pm-2:00pm".
- Nina Myers: I'll only get the pardon if I stop the bomb; why wouldn't I do everything I can?
- Jack Bauer: Because you're worse than a traitor, Nina. You don't even have a cause. You don't believe in anything. You would sell anyone and anything out to the highest bidder. So stop wasting my time! Give me a name! (throws table over)
- Nina Myers: Don't even bother, Jack. You lay a hand on me you'll be taken off the case.
- George Mason: All right, you're done. Get outta here.
- Jack Bauer: Why?
- George Mason: Why? Because you've lost it, that's why.
- Jack Bauer: George, right now she thinks she's won the lottery. She's in control. You want her to tell the truth, you take that away from her.
- George Mason: By killing her? Yeah, that'll work.
- Jack Bauer: Who is the man in Visalia? (Nina doesn't say anything. Jack takes out the gun and shoots twice just above Nina's head.) Who is the man in Visalia!?
- Nina Myers: (shaken) Mamud Faheen. The man in Visalia is Mamud Faheen.
- Jack Bauer: That's not news, we got his name!
- Sherry Palmer: Hello, David.
- David Palmer: How did you get in here without my authorization?
- Sherry Palmer: We were married for many years, David. Many people here respect that, even if you don't.
- Jack Bauer: You should go be with your family, George.
- George Mason: You should go get on the plane.
- Nina Myers: So Ed, how are ya?
- Agent Ed Miller: Don't talk to me, Nina. I'm here because I have to be, not because I'm your friend.
- Nina Myers: Well, you're here because they think after I help stop the bomb, Jack will try to kill me. Which, of course, he will. What if you can't stop him? I mean, no offense, but Ed, let's face it: you're no match for Jack. You're just not that ruthless. So he manages to get around you and pull the trigger. What happens then? Because of the pardon, he knows if he kills me, he'll spend the rest of his life in jail.
- Agent Ed Miller: Jack, if you know of another way to shut her up that I'm not thinking of…
- Nina Myers: Of course, Jack's mind is thinking, "That's not such a bad deal." Small price to pay to avenge his wife's death. But what about his daughter? Is he thinking about her? She's already lost one parent. If he kills me, she'll have no one. Jack knows I'm right. He should just use me to stop the bomb, and then go home to Kim. She needs you, Jack.
- Jack Bauer: Don't let anyone in that room and raise the temperature ten degrees.


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|"Ivan Volosyuk" <ivan.volos...@gmail.com>
|Re: [classlib] logging from within our implementation
|Tue, 30 May 2006 20:46:55 GMT
2006/5/31, Gregory Shimansky <email@example.com>: > On Wednesday 31 May 2006 00:09 Ivan Volosyuk wrote: > > I am not sure, we can say someday: Yes, the code is absolutely bug > > free! Remove the logging! > > > > I have a suggestion, which can help leave logging in place while still > > having no impact on performance. The logging can be used for debuging > > of features and will be removed in release version. It require some > > changes to build system though. > > > > The idea is quite simple: we can use C preprocessor directives in java > > files. When building the preprocessor will be executed before java > > compiler (if the source-file's timestamp was changed). Thus we can > > have logger-free release builds and debug builds with full weight > > logging. > > I have a simpler suggestion which doesn't require any tools foreign to java. > > Make classlib debugging infrastructure internal class, not java.util.logging, > but an internal wrapper to classes in java.util.logging. And in release > version all its methods should be empty. > > Any good behaving optimizing runtime would inline empty methods into nothing > and therefore no performance impact would be made. Excelent! This is much better and simplier. public final class CLogger { public static void msg(Object... ) {..} } Hmm, I see one drawback of this approach: arguments will still be evaluated even if logger itself will be empty. So, some care needed to maintain performance with such logger. -- Ivan --------------------------------------------------------------------- Terms of use : http://incubator.apache.org/harmony/mailing.html To unsubscribe, e-mail: email@example.com For additional commands, e-mail: email@example.com
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I had a request recently to create some images of US symbols. When I first got the request, I was a little hesitant because I really don't "do" this kind of art work. My graphics are more non-realistic....so I was very close to passing on the request.
But after thinking about it.....I realized that this would be a great challenge for me. To step out of my normal graphic design and try something a little different.
So here is the first set I created. I'm really excited about how they turned out.
There will be another set coming out soon !!
*Just click on the pics to go to my store*
Also I wanted to share with you 2 more sets that I recently created.
We had a few snow days recently and my kids were loving the snow. We didn't get much snow living in the south so this early gift of snow was REALLY fun for them!!!
I actually created this set before the Snow Day Kids....this one is FREE!!!!!
As always, I love reading your comments :)


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Home Inn Bozhou Wei Wu Road Railway Station is located in Bozhou City. It boasts a number of guest rooms which are fully equipped and elegant. It also has a conference room that can be hired for various meetings and functions.
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Northstar Ratings are powered by Northstar Travel Media and are based on objective and unbiased evaluations from experienced editors that individually analyze, research, and evaluate hotels. Hotels and suppliers are not required to pay any fees or pay for memberships to have their property rated.
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The Riverside Room features nautral light and outdoor access
Ideal for meetings, the Putnam room is situated just downstairs from our hotel enterance
The Private Dining Room is a meeting and dining space that features our glass enclosed, temperature controlled wine vault.
Outdoor Patio


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LAY DI offers a spacious three stateroom interior arrangement with large aft master with en suite head and stall shower and two guest cabins forward each with ensuite head and direct access to the shower stall. The full beam VIP forward guest cabin can be set up for two and with a filler insert becomes a queen. This cabin has its own private entry door to the forward head. Aft to port is the second guest cabin with upper and lower berths. The 11' long passageway style galley and large navigation center leads forward to the spacious, bright, airy amidships raised main salon. The raised salon upgrade provides more natural light and better visibility from 4 side and 3 forward windows.
The exquisite satin finished varnished teak cabinetry includes custom inlays and many additional storage drawers and cabinets. The cabinetry is louvered style throughout. The cabin sole is solid teak with holly inlays with gloss varnish finish. There is substantial additional storage under the cabin sole panels with lockdown lifts. All interior cabins are climate controlled (heat and air conditioned) with reverse cycle units. All deck hatches have Ocean Air combination screen and shades.
Custom Interior Feautures
- Custom dining table with birdseye rosewood and inlay- The table opens to accept a custom leaf thats stored in a dedicated felt lined storage locker
- Electric TV lift to starboard in saloon for the flatscreen TV
- Cabinets and entry doors feature custom top radius design
- Doorways for passageway and cabin doors have raised panels
- New (2013) main salon ultra leather upholstery including "L" shaped settee to port and straight settee to starboard.
- All hatch fames are custom finished in white Awlgrip in lieu of varnish teak
- European headliner with recessed edges and Andari/Ultra Suede/Alcantara fabric with moisture resistant foam
- Ocean Air built-in shade/screens (Concealed model with ceiling matching color for owner's stateroom, forward cabin and port stateroom)
- Ocean Air, surface mounted shade screens for main saloon hatches
- Flush mounted main saloon windows framed with Andari/Ultra Suede
- Cedar lined hanging lockers with lights when opened
- Lee cloths for upper and lower bunks in guest cabin


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US stock indexes turned sharply to positive result
The U.S. stock indexes sharply changed the direction of movement and despite serious losses at the beginning of the session went green. With a major contribution to this was Goldman Sachs, which until it was the main factor causing the sale. Today's session ended with a growth of 0.75 percent for the index of blue-chip Dow Jones Industrial Average, which reached 10,230 points. This is a significant upward to the fact that in early trade index slumped to 10,007 points. Objectives included 20 of the 30 companies in the index rose today. The broader S & P index added 1.14 500 percent to 1083 points and the index of companies by the exchange Nasdaq – Nasdaq Composite, rose 1.1 percent to 2222 points. Impulse that trigger positive sentiment was given by Goldman Sachs, which publishes an improved outlook for investment banking business in the sector. It includes good expectations of mergers and acquisitions, leading to positive News. Before this report, notably disappointing report of Goldman Sachs, which was saved from fall in profit 80 percent, was among the causes of stock decline. Shares of IBM fell 2.54 percent with the New York Stock Exchange after profit technology company beat second quarter estimates, but sales disappoint.
Shares of PepsiCo, however, rose 4.3 percent after the soft drink manufacturer announced that it expects increases to 13% profit for the year. The accounts showed the company's 3 percent decline in its profit in the second quarter because of costs associated with the acquisition of two of its largest bottling companies. Thanks to them, however, sales of PepsiCo jumped 40% yoy.


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Potkey: Cassius Marsh 'may look ugly at times, but he can play'
Cassius Marsh catches an NFL evaluator's eye, Jeff Mathews ranks No. 1 among quarterbacks and Gene Haas enters Formula One.
Local players and coaches are making news beyond the county lines. Here's what's being said and done ...
Cassius Marsh is hoping NFL teams see the same thing Pat Kirwan does when they watch film on the UCLA defensive end.
Kirwan is a former NFL coach and scout who is now the NFL insider for CBSSports.com.
In an article about the defensive linemen and linebackers that could be steals beyond the first round of the upcoming NFL draft, Kirwan included Marsh.
The Oaks Christian School graduate was slotted at No. 3 on Kirwan's list of 10 players that caught his eye while studying film.
"Marsh is impossible not to see when you turn on the UCLA tapes. He plays like his hair is on fire, has a nonstop motor and achieves with effort reminiscent of Jared Allen," Kirwan wrote. "He may never be a starter but will make a team as a special teams guy and eventually find his way onto the field. Watch the USC game, when he had six tackles and two sacks and was fighting through blocks all day. The guy may look ugly at times, but he can play."
Speaking of the draft, there was a lot of buzz last week about Johnny Manziel having the best Wonderlic score among the top quarterback prospects in this year's class.
But the key word is "top" because Manziel didn't have the best overall score among quarterbacks like some headlines suggested. That honor belonged to Camarillo High graduate Jeff Mathews.
It makes sense considering Mathews played for Cornell in the Ivy League.
According to the NFL Network's Albert Breer, Mathews scored a 40 while Manziel had a 32.
The Wonderlic is a 50-question test administered to all NFL combine participants that measures cognitive ability. The time limit is 12 minutes, and a score of 20 is considered "average" intelligence.
According to ESPN Stats & Information, the average score for projected NFL starters for the upcoming season is 29.4. Kansas City Chiefs quarterback Alex Smith has the highest at 40. ...
In a move that could tangentially benefit the local economy, NASCAR owner Gene Haas has been granted a license from Formula One's governing body to start a U.S.-based entry in the series.
Haas is the owner of the CNC machine manufacturer Haas Automation, which is based in Oxnard.
Haas co-owns a NASCAR team with Tony Stewart that fields cars for drivers Stewart, Kevin Harvick, Danica Patrick and Kurt Busch.
His new team will be called Haas Formula and begin running within the next two years.
"Sometimes people think the European way of racing is so much more advanced than the Americans," Haas said. "Bue we're the most advanced country on the planet, so I can't imagine why we can't do it."
The last U.S.-based team to run in Formula One was Parnelli Jones Racing in 1974-76.
The most recent attempt for another U.S.-based team came in 2010, but the entry lacked funding and the development money to join.
That should not be a problem for Haas.
Haas said Haas Automation currently does $1 billion in annual sales, and he believes entering Formula One could possibly double that number.
"It's not that we're trying to individually sell machines. We're trying to make people aware of Haas Automation as a company," Haas told The Associated Press, adding. "We're just trying to bring our awareness up and Formula One is even more important because half our sales are outside the country."
Spring games were all the rage on Saturday as college football teams showcased a glimpse of what fans can expect during the fall.
Iowa State quarterback Grant Rohach completed 22 of 38 passes for 171 yards with two interceptions. The Moorpark High graduate led three scoring drives and scored one touchdown from eight yards out on a read option play.
"I though the offense played well at the beginning," Rohach said. "Toward the end, I tried to force a few throws and tried to make too many plays."
Rohach outperformed fellow quarterback Sam Richardson, but Iowa State head coach Paul Rhoads wasn't willing to name Rohach the starter just yet.
"That does not put him on top," he said. "We will come out of spring with two leading candidates. Whether we announce that in April or wait until August a lot can happen in May, June and July."
In Colorado's spring game, Westlake High graduate Nelson Spruce caught eight passes for 110 yards.
Sam Querrey pulled out of the U.S. Men's Clay Court Championship in Houston with a back injury. The Thousand Oaks High graduate was scheduled to play Nicolas Almagro in the semifinals on Saturday.
"I woke up unable to bend forward or arch back," Querrey said. "We've been trying to loosen it up for three hours. I feel terrible my first semi in a while and I really wanted to play."
Camarillo's Bob and Mike Bryan captured the men's doubles title in Houston for the fifth time with a 4-6, 6-4, 11-9 victory over David Marrero and Fernando Verdasco.
The Bryans, who have a 17-match winning streak, have won four straight titles this season to push their career record total to 97.
"It gives us confidence going into the (European) clay court season, beating such a tough clay court team," Bob Bryan said. "They kept us off balance all day with great serving and huge forehands."
James Ennis and the Perth Wildcats captured the National Basketball League title in Australia with a 93-59 victory over Adelaide in the deciding game of the best-of-three Grand Final series.
Ennis, a Ventura High and Ventura College product, had a relatively quiet night with only nine points while battling foul trouble.
Ennis will play in Puerto Rico for the Piratas de Quebradillas before moving back to the United States to continue pursuing his NBA dream with the Miami Heat.


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Evertrade38.com is a 1 year old domain, situated in Hong Kong. The domain is linked to the IP address 126.96.36.199.
It was registered by ever trade . Registration details show that it was registered on 08 Jan 2016 through Go Daddy and will expire on 08 Jan 2017.
The site is being served through Microsoft-IIS/7.5.


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Thursday, 2.50am ... The monkey animations with Damon Albarn's Chineasy listening music are beginning to get annoying, certainly at this time in the morning. What the hell am I doing up? Watching swimming.
First comes a quick interview with Mark Foster, team GB's oldest swimmer, as BBC1's Garry Richardson keeps reminding him. Garry wonders how Mark manages to communicate with his team-mates, "because a few of them you probably can't properly converse with they're so much younger". Garry's good value like that. Kosuke Kitajima from Japan wins a gold medal, but foolishly allows the green world record line to slip ahead of his fingers right at the end. Loving the green line.
4am I've switched to women's archery, America v Colombia. The American used to be a Georgian; they could probably use some of her sharp-shooting back home right now. It's amazing how much an arrow wobbles in the air, when it's shown slowed down. Makes a mockery of the phrase "straight as an arrow". Strangely beautiful, though.
5am The beach volleyball from Chaoyang Park has no commentary at all. Is that because the BBC doesn't have anyone who understands, or because they're not taking it seriously? If it's the latter, then they're right not to. Beach volleyball is something you do on holiday. It should no more be an Olympic sport than doing the Macarena or casual sex.
6am Fell asleep, sorry.
7am Hazel and Adrian (Irvine and Chiles) are wearing the same lilac shirts and beige trousers. Synchronised dressing. They're the best anchors, with the right mix of gravitas (Hazel) and larking about (Adrian). Plus a Scot and a Brum is a lovely combination. And here's another Brum, Hugh Porter, who can always find another gear in a cycling commentary (you can't help doing that kind of thing when talking about this lot, because they do it all the time: Hazel came up with the best, something about a Japanese butcher's son slicing a tranche off the world record). And from Qingdao, another Scot, Shirley Robertson, has the sailing news. There isn't any: no wind. Poor Shirley, having to report on Britain's Yngling crew, who dropped her. Like commentating on an ex and the ex's new lover.
8am Steve Ovett! A childhood hero, on the sofa with Adrian and Hazel. Always - and still - so much more human than Seb Coe. Still needs to sort out the teeth, though.
9am Both the rowing and the canoeing have been cancelled, because of rain. That's pathetic. So no boating action at all this morning. Judo instead. Our Peter Cousins is getting a pummelling from a Georgian called Zhorzholiani. No wonder they're not doing very well in their war; they're all here in Beijing, doing marginal sports.
10am Ah badminton. Very different from the rather gentle game I thought it was. Amazing. A Polish pair against a Chinese pair; guess who wins. It's a lovely noise, the thwang of taut strings on shuttlecock.
11am Another gold for China, in the bow and arrows. And then we fly low over the city, in a CGI, Google Earth kind of way, to the basketball stadium. Which is fun - more fun than the basketball when we get there: Lithuania v Russia. Who cares? I never thought I'd miss rowing.
Noon Mark Foster fails to make the semis: too old, or still hurting from Garry Richardson's interview. "Thank you for all your hard work over the years," Sharron Davies (or Amazon off Gladiators if you prefer) says to him after the race. Presumably they now put him down.
It's approaching dusk in Beijing, visible behind Sue Barker, whose watch we're now on. Sue's neither a Scot, nor a Brummie, which is a shame. Devon, apparently, though you'd guess Surrey.
1pm Badminton - my new favourite sport. And plucky Brits Nathan Robertson and Gail Emms are about to go out to a pair of Koreans, both called Lee. "Come on," says Gill Clark, commentating. "Time to step up to the plate." They don't, and lose to the Lees. Gail's crying. I'm crying. I hate badminton. There's Princess Anne in the crowd. Extraordinary hair. What's she doing there?
2pm Shouldn't she be here, at the dressage? Sideways dancing horses; now there's a proper sport. "Don't be stupid, just relax," barks Jennie Loriston-Clarke, my favourite commentator of all. I think she's talking to Mistral Højris, the horse, rather than Laura Bechtolsheimer, his rider. There's a problem, apparently, with a discipline called the passage. Passage is pronounced to rhyme with massage, as you'd expect in a sport called dressage, which doesn't rhyme with message.
3pm Hockey. Can't face it. Twelve hours. I've had enough.


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The baby was delivered on Sunday at Cedars Sinai hospital in Los Angeles.
Victoria's former Spice Girls colleagues Mel B and Emma Bunton were among those who expressed their joy following the birth.
Harper has become increasingly popular in the US as a first name. Probably the most famous bearer is the author of To Kill A Mockingbird, Harper Lee.
It is thought the addition of Seven is a reference to David's shirt number from his time at Manchester United. He has the number written in Roman numerals - VII - on his arm as a tattoo.
The child's name was announced by David on his Facebook page. "I am so proud and excited to announce the birth of our daughter Harper Seven Beckham," he said.
"She weighed a healthy 7lbs 10oz and arrived at 7.55 this morning, here in LA. Victoria is doing really well and her brothers are delighted to have a baby sister xx
Mel B, who is expecting her third child later this year, expressed her excitement on Twitter, writing: "Congrats!!! Yipeee another spice baby is born, damn it wish it was me this AM cos I feel like I'm about to POP!"
And Bunton sent a "big kiss" to her former bandmate, adding: "Can't wait to meet your gorgeous little girl."
The Beckhams, who married in 1999, already have three boys - Brooklyn, 11, Romeo, eight, and Cruz, five.
- More about:
- Emma Bunton
- Los Angeles
- Melanie Brown
- Tattoo
- The Spice Girls


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Kaepernick Takes to Twitter to Say TMZ Story 'Completely Wrong'
(1/3) The charges made in the TMZ story and other stories I've seen are completely wrong. They make things up about me that never happened.
— Colin Kaepernick (@Kaepernick7) April 11, 2014
(2/3) I take great pride in who I am and what I do, but I guess sometimes you have to deal with someone who makes things up.
— Colin Kaepernick (@Kaepernick7) April 11, 2014
(3/3) I want to thank all of the people who have shared their encouraging sentiments. I assure you that your faith is not misplaced.
— Colin Kaepernick (@Kaepernick7) April 11, 2014
San Francisco 49er quarterback Colin Kaepernick has taken to Twitter saying the story from TMZ and other media reports about the "suspicious incident" report are "completely wrong."
Yesterday, TMZ reported that Kaepernick was being investigated for an alleged sexual assault. But police reports from Miami make no such allegations.
According to the police report, Kaepernick was in an apartment with Quinton Patton, Ricardo Lockette and an unnamed woman.
The woman mixed some drinks while they were talking. Marijuana was also present in the room, according to the report.
The group sat down, talked and watched a basketball game. The woman began to feel light-headed and went to lay down in a bedroom.
She says Kaepernick, whom she had had a sexual relationship with in the past, came into the room and started kissing her, according to the report. The kissing led to the woman getting undressed, the police report states. Then Kaepernick said he would be right back and left the room. They did not have sex at that time.
The woman said Lockette and Patton opened the door to the room and she told them to "get out." They closed the door and left. The woman cannot remember anything after that.
She woke up in a hospital bed with no recollection of how she got there. The investigation in ongoing. "The 49ers organization is aware of the recent media report regarding Colin Kaepernick and is in the process of gathering pertinent facts," 49ers General Manager Trent Baalke said.


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Deflection Routing
Definition - What does Deflection Routing mean?
Deflection routing is the rerouting of Internet Protocol (IP) network packets to eliminate heavy data traffic at the router interface and avoid packet buffering when network packets are transmitted with same route contention. Deflection routing is used in modern networking to reduce system and infrastructure expenses.
Deflection routing is also known as hot potato routing.
Techopedia explains Deflection Routing
During deflection routing, nodes receive autonomous network data packets, which are forwarded without buffering to the nearest destination point via the shortest available path. This process reduces traffic and network complexity.
Deflection routing functions optimally when implemented with optical networks.
Deflection routing disadvantages include:
- Network bandwidth is used inefficiently.
- Packets are received out of order.
- Quality of service (QoS) implementation is problematic.
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All TSTA/NEA members actively employed in the field of education can take advantage of the valuable, automatic term life insurance coverage provided by NEA Complimentary Life Insurance. It's free! All you have to do is register your beneficiary and you're covered, so register now at www.neamb.com [1] or call (800) 637-4636.
Additional financial protection for cancer, heart & stroke, hospital, disability, accident, ICU
Save time and money with TSTA's Health and Wellness discount programs
Can you avoid the urgent care clinic if you get sick on the weekend? Is it time for new eyeglasses, contacts, or a dental checkup? What if you need medical advice? Where do you turn if you have a question about a medical bill or insurance payment? Offered to you through New Benefits, TSTA's Premier and Ultra Discount Programs offer attractive discounts on products and services ranging from medical consults over the phone to alternative medicine, and diabetic supplies to vitamins--all at affordable monthly rates. Check out these exciting offerings!
Click the links below for program details and enrollment information. When you've decided on a program that will best meet your needs, click SELECT. (Please note that some benefits require an email address for initial account activation.)
TSTA Premier: https://tstapremier.secureenrollment.com [2]
TSTA Ultra: https://tstaultra.secureenrollment.com [3]
Relax and Rest Up With LaQuinta
La Quinta Inns & Suites offers state rates at all La Quinta Hotels throughout Texas, and 15% off our best available rates at properties outside of Texas (within the continental US). This discount is upon availability, and may be used for business or leisure travel.
La Quinta Inns & suites offers what any guest needs to wake up on the bright side. Enjoy our free Bright Side Breakfast®, free high-speed internet, and comfy beds. Plus, our hotels are conveniently located close to area restaurants, shopping and entertainment venues. Be sure to sign up for La Quinta Returns, and start earning points toward upgrades and free nights fast! Enrolling is fast, easy and free. Go to LQ.com or call 1-800-RETURNS (738-8767) for information.
Access the discount online at LQ.com by entering the code "TSTA", calling the toll-free central reservations number at 1-866-468-3946 and requesting the "Texas State Teachers Association" discount, or calling the hotel's direct number and requesting the "Texas State Teachers Association" discount.
Coming soon! Please check back.
Discounts for theme parks, travel and much more
Check out the fun and exciting discounts at Tickets At Work, including Disney, Universal, Vegas, Sea World, Six Flags and Cirque du Solei. Also save on car rentals, movie tickets, hotels, tours and special events. Take advantage of the new savings on the things you need—from flowers, gifts, beauty, electronics, home & garden—with brands like Sears, 1-800-Flowers, Weight Watchers, Dell and more! Great new deals added weekly.
Create a new account using code "TSTA" at http://ticketsatwork.com/tickets/index.php [4], or call toll free at 800-331-6483. See the December Bulletin for current offers.
TSTA cancer, heart & stroke, hospital, disability, accident, ICU insurance
For the following TSTA programs, call National Teachers Associates Life Insurance Company at (888) 671-6771.
Cancer Insurance
NTA Life's Cancer Insurance program provides additional financial protection for the medical and non-medical costs of cancer and for its early identification and cure. In most areas, this policy offers a Cancer Screening Wellness Benefit that provides for certain routine examinations or preventative tests, an express payment benefit for the first diagnosis of internal cancer, and direct payments to you, unless you specify otherwise, regardless of other major medical coverage you may have.
Heart Attack, Heart Diseases, and Stroke Insurance
NTA Life's Heart Insurance program provides additional financial protection for the medical and non-medical costs of heart attack, heart disease and stroke. This protection may be available with an optional intensive care and step-down unit benefit rider as well as offering an optional Heart Wellness Benefit that provides for certain routine examinations or preventative tests.
Hospital Indemnity Insurance
NTA Life's Hospital Indemnity Insurance program provides additional financial protection in connection with that stressful and financially draining experience of being admitted to the hospital. Our Hospital Indemnity Insurance pays a portion of your out-of-pocket expenses associated with your hospital stay. In many areas, this protection can be obtained with optional intensive care rider for an additional premium.
Disability Insurance
NTA Life's Disability program provides additional financial protection if you become disabled. Our Disability Income coverage helps you provide for your family during disability and can help take care of co-payments, medical costs not covered under other plans, food, clothing, travel and lodging expenses.
Accident Insurance
NTA Life's Accident program pays you specific benefit amounts for the care and treatment of covered accidents. Our Accident coverage is designed to help you prepare for those unforeseen circumstances that could happen to any one of us on any given day.
Intensive Care Rider
NTA Life's Intensive Care Unit (ICU) coverage can provide you with added relief if you have a stay in the ICU or in a step-down unit. This optional rider must be elected and requires an additional premium. Coverage is provided for both sickness and injury.
Need more Information?
For more information about any of these programs, call TSTA at (877) 275-8782 (ASK-TSTA) and ask for "Member Benefits."
[1] http://www.neamb.com
[2] https://tstapremier.secureenrollment.com
[3] https://tstaultra.secureenrollment.com
[4] http://ticketsatwork.com/tickets/index.php