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RESEARCH ARTICLE| 15 April 2006
An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility
Irmgard Hofmann,
Irmgard Hofmann
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK
Sean Munro
Sean Munro *
* Author for correspondence (e-mail: sean@mrc-lmb.cam.ac.uk)
Accepted: 24 Feb 2006
© The Company of Biologists Limited 2006
J Cell Sci (2006) 119 (8): 1494–1503.
https://doi.org/10.1242/jcs.02958
A related article has been published: Lysosomes on the move
Irmgard Hofmann, Sean Munro; An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility. J Cell Sci 15 April 2006; 119 (8): 1494–1503. doi: https://doi.org/10.1242/jcs.02958
Small GTPases of the Arf and Rab families play key roles in the function of subcellular organelles. Each GTPase is usually found on only one compartment and, hence, they confer organelle specificity to many intracellular processes. However, there has so far been little evidence for specific GTPases present on lysosomes. Here, we report that two closely related human Arf-like GTPases, Arl8a and Arl8b (also known as Arl10b/c and Gie1/2), localise to lysosomes in mammalian cells, with the single homologue in Drosophila cells having a similar location. Conventionally, membrane binding of Arf and Arl proteins is mediated by both an N-terminal myristoyl group and an N-terminal amphipathic helix that is inserted into the lipid bilayer upon activation of the GTPase. Arl8a and Arl8b do not have N-terminal myristoylation sites, and we find that Arl8b is instead N-terminally acetylated, and an acetylated methionine is necessary for its lysosomal localization. Overexpression of Arl8a or Arl8b results in a microtubule-dependent redistribution of lysosomes towards the cell periphery. Live cell imaging shows that lysosomes move more frequently both toward and away from the cell periphery, suggesting a role for Arl8a and Arl8b as positive regulators of lysosomal transport.
Arf family, GTPase, Lysosome, N-terminal acetylation
The organisation of internal membranes requires that many peripheral membrane proteins are accurately recruited to specific organelles from the cytosol. These peripheral proteins mediate vesicle transport between organelles, or interactions between organelles and the cytoskeleton. In general, their recruitment reflects the recognition of activated GTPases or specialised lipid species, such as phosphoinositides, which are present on only one organelle (Behnia and Munro, 2005; Munro, 2004). These GTPases are members of two large families, the Rab and Arf families. Like many other members of the Ras superfamily of small GTPases, the Rabs are attached to membranes via C-terminal prenyl groups. In contrast, the Arf family members are distinguished by having an N-terminal amphipathic helix and, in most cases, an N-terminal myristoyl group (Pasqualato et al., 2002; Pfeffer, 2001; Zerial and McBride, 2001). The hydrophobic side of the amphipathic helix is buried when Arf is in the GDP-bound form, but upon nucleotide exchange there is a conformational change unique to Arfs that displaces the helix (Goldberg, 1998; Pasqualato et al., 2002). The hydrophobic side of the displaced helix interacts with the lipid bilayer, resulting in the GTP-bound form being tightly associated with the bilayer. In this membrane-associated form, Arf can then recruit effectors until GTP hydrolysis is induced by a GTPase-activating protein (GAP) and, hence, Arf returns to the GDP-bound form and falls off the membrane.
The best characterised members of the Arf family are Sar1, Arf1 and Arf6, which are localised to the ER, Golgi and plasma membrane, respectively. Sar1 recruits the COPII vesicle coat, and Arf1 recruits the COPI, AP1 and GGA coat proteins as well as further effectors proposed to contribute to membrane traffic (Donaldson et al., 2005; Lee et al., 2004; Nie et al., 2003). Arf6 appears to regulate cytoskeletal dynamics and membrane traffic at least in part, by recruiting a PI 5-kinase that synthesises PtdIns(4,5)P2 (Aikawa and Martin, 2003; Krauss et al., 2003). However, in addition to these Arf family members, genome sequencing has revealed a number of `Arf-like' GTPases that share with Sar1 and the Arfs an N-terminal amphipathic helix, and the `interswitch' region responsible for the conformational change that displaces the helix (Kahn et al., 2006; Li et al., 2004; Pasqualato et al., 2002). There are 22 such Arls in humans, some of which are well-conserved in evolution, with eight forms having clear homologues in Drosophila. The function of most is presently unknown, but Arl1 and ARFRP1 (Arl3 in yeast) have been shown to be localised to the Golgi, where they form a pathway that results in the recruitment of coiled-coil proteins to the Golgi via a direct interaction between Arl1-GTP and the GRIP domain at the C-terminus of the coiled-coil protein (Gangi Setty et al., 2003; Munro, 2005; Panic et al., 2003). In addition, Arl2 and Arl3 appear to regulate the activity of a tubulin-folding cofactor (Burd et al., 2004). Many of the Arl proteins have a glycine residue at position 2 that is part of the consensus for recognition by N-myristoyltransferase, which modifies the α-amino group of the conserved glycine following removal of the initiator methionine by methionine aminopeptidase (Bradshaw et al., 1998; Maurer-Stroh et al., 2002). Hence, probably most of the Arls are myristoylated, and this has been confirmed in most cases examined. However, one of the Arls, ARFRP1/Arl3 that is found on the Golgi, lacks a glycine at position 2 and, instead, has a well-conserved tyrosine or phenylalanine. It has been recently found that this results in the initiator methionine being left uncleaved, and the N-terminus being acetylated by the NatC complex, a minor N-terminal acetylation complex that acts on proteins with phenylalanine, tyrosine, isoleucine, leucine or tryptophan at position 2 (Behnia et al., 2004; Setty et al., 2004). These studies were done with yeast Arl3, but mammalian ARFRP1 is likely to be modified in the same way because the mammalian orthologue of NatC apparently has a similar substrate specificity to the yeast acetyltransferase (Polevoda and Sherman, 2003).
Arl8a and Arl8b localise to lysosomes. (A-D) Confocal micrographs of COS cells transfected with plasmids expressing either Arl8a-GFP or Arl8b-GFP from a CMV promoter. After fixation and permeabilization, the cells were labelled with antibodies against the indicated endogenous proteins CD63 (lysosomes), MPR (cation-dependent mannose 6-phosphate receptor, late endosomes) and EEA1 (early endosomes). (E) Confocal micrographs of NRK cells expressing Arl8b-GFP. Lysosomes were labelled with endocytosed dextran-tetramethylrhodamine using a four hour pulse at 1 mg/ml, followed by a 20-hour chase. Bars, 10 μm.
In this paper, we examine two previously uncharacterised human Arls, Arl8a and Arl8b, that also lack a glycine at position 2. Arl8a and Arl8b are very closely related proteins (91% identity) that were originally identified from the human genome sequence (Kahn et al., 2006; Li et al., 2004; Pasqualato et al., 2002). Phylogenetic analysis indicates that Arl8a and Arl8b define an Arl subfamily that has a single member in many species, including Drosophila melanogaster, Caenorhabditis elegans, filamentous fungi, plants, Dictyostelium and Trypanosoma cruzi. Interestingly, there is no homologue in the yeast Saccharomyces cerevisiae, indicating that Arl8 is an ancient GTPase that budding yeast have lost during evolution. We show that Arl8a and Arl8b are localised to lysosomes and that Arl8b is N-terminally acetylated, with mutations that perturb the N-terminus, causing a loss of lysosomal localisation.
Arl8a and Arl8b localise to lysosomes in mammalian cells
To examine human Arl8a and Arl8b in more detail, we tagged both proteins at the C-terminus and expressed them in COS cells. With both GFP and HA epitope tags, Arl8a and Arl8b showed a punctate distribution displaying extensive colocalization with several lysosomal markers, such as CD63 and LAMP2 (Chen et al., 1985; Fraile-Ramos et al., 2001) (Fig. 1A,B and Fig. 2B). Both proteins showed a limited overlap with the late endosomal markers Rab7 and the cation-dependent mannose-6-phosphate receptor (Rink et al., 2005) (Fig. 1C and data not shown), and no overlap with EEA1, a marker for early endosomes (Stenmark et al., 1996) (Fig. 1D). A lysosomal localization of Arl8b was also observed in rat NRK cells. When these cells were treated with fluorescent dextran as a fluid-phase endocytic marker, using a pulse followed by chase to label terminal endocytic compartments, the dextran accumulated in Arl8b-positive structures (Fig. 1E). Thus, Arl8a and Arl8b are predominantly localised to lysosomes, with some colocalization with late endosomal markers that might reflect the cycle of fusion and resolution between lysosomes and late endosomes (Bright et al., 2005). As such, Arl8a and Arl8b are the first small GTP-binding proteins reported to be localised to lysosomes.
Some of the human Arls appear to have arisen only recently upon vertebrate evolution, but Arl8 is one of the eight human Arls that are conserved outside vertebrates (Li et al., 2004; Pasqualato et al., 2002). When Drosophila Arl8 (CG7891) was expressed in COS cells as a GFP fusion it also localised to lysosomes, suggesting that the targeting of the protein is well conserved (Fig. 2A). The recruitment of Arfs to the Golgi is inhibited by the fungal metabolite brefeldin A, which blocks the activity of a subset of Arf GEFs (Renault et al., 2003). We thus examined the effect of brefeldin A in COS cells but found that it did not change the lysosomal targeting of Arl8a or Arl8b, although the Golgi marker giantin was perturbed as expected (Fig. 2B and data not shown). This indicates that Arl8a and Arl8b and their GEF or GEFs are brefeldin-insensitive.
Arl8 targeting is evolutionarily conserved but brefeldin A resistant. (A) Confocal micrographs of COS cells expressing the Drosophila Arl8 homologue (CG7891) with GFP at the C-terminus (Dm Arl8-GFP), and after fixation and permeabilisation labelled with antibody against the lysosomal protein CD63. (B) Confocal micrographs of COS cells expressing Arl8b-GFP and either untreated, or incubated in 5 μg/ml brefeldin A for 15 minutes, before fixation and labelling with the indicated antibodies to residents of the lysosome (LAMP2) and the Golgi (giantin). Bars, 10 μm.
Arl8b is N-terminally acetylated
Arl8a and Arl8b lack a glycine at position 2 and, therefore, cannot be myristoylated (Fig. 3A,B). Instead, the second residue of Arl8a is an isoleucine and that of Arl8b – and of homologues in metazoans – a leucine. These residues at the second position make Arl8a and Arl8b potential substrates for the NatC N-terminal acetyltransferase (Polevoda and Sherman, 2003). To investigate its N-terminal acetylation, HA-tagged Arl8b was immunoprecipitated from transfected COS cells and following SDS gel electrophoresis, tryptic peptides were analysed by mass-spectrometry. We identified a peptide with a mass corresponding to an acetylated N-terminus (Fig. 3C), showing that Arl8b is N-terminally acetylated.
A number of cytosolic proteins, including some GTPases, associate with membranes via palmitoyl groups attached to internal cysteine residues (Smotrys and Linder, 2004). Arl8a and Arl8b contain three cysteines (residues 158, 159 and 164) that are conserved in homologues from humans to plants but do not occur in other Arls (Pasqualato et al., 2002). We thus asked whether the role of the N-terminal myristoyl group was performed in Arl8b by a palmitoyl group attached elsewhere. However, when Arl8b-HA was expressed in COS cells it was not labelled with [3H]palmitate under conditions where labelling of a known palmitoylated protein (linker for activation of T cells, LAT) (Zhang et al., 1998)] could be readily detected (Fig. 3D).
An acetylated methionine and the hydrophobic face of the amphipathic helix are required for membrane targeting, but not lysosomal specificity
To investigate the role of the N-terminus in targeting of Arl8b, we examined the effect on localisation of several mutations in this region (Fig. 4A). Replacement of the leucine in position 2 with alanine resulted in a loss of lysosomal localisation, and instead a diffuse cytoplasmic distribution (Fig. 4B). Sequencing of the N-terminal tryptic peptide from this mutant by tandem mass spectrometry indicated that the methionine had been removed by methionine aminopeptidase, as expected for a protein with a small residue in the second position (Bradshaw et al., 1998). Nonetheless, the protein was still acetylated, presumably by the mammalian NatA complex, which acts on many proteins following the removal of the N-terminal methionine (Polevoda and Sherman, 2003). This implies that the N-terminus of the protein is crucial for its localisation. When the leucine in position 2 was replaced with the large hydrophobic residue phenylalanine the methionine was, as expected, left intact and the protein acetylated, presumably by the mammalian NatC orthologue (Polevoda and Sherman, 2003). This form was targeted to lysosomes, suggesting that it is the N-terminally acetylated methionine that is crucial for targeting rather than the precise structure of the residue in the second position (Fig. 4C).
Since Arl8 appears different from most other Arls in lacking an N-terminal myristoyl group, we also investigated the role of the N-terminal amphipathic helix in membrane binding. Mutation to alanine of three hydrophobic residues predicted to lie on one face of an α-helix formed from the N-terminal region (I5A, L8A, F12A) did not appear to affect N-terminal acetylation (Fig. 4A). However, it did result in loss of Arl8b targeting to lysosomes (Fig. 4D). Therefore, in addition to the N-terminally acetylated Met 1, the hydrophobic side of the amphipathic helix appears to be required for membrane binding, as is the case for other Arfs and Arls (Antonny et al., 1997).
The N-terminus of Arl8b is acetylated. (A) Alignment of human Arl8a and Arl8b with their relatives from Xenopus tropicalis, Drosophila melanogaster, Caenorhabditis elegans, Schistosoma japonicum, Arabidopsis thaliana, Neurospora crassa and Dictyostelium discoideum. Sequences were aligned with CLUSTAL W, and shaded in cases where more than half of the residues are related (grey) or identical (black). Hydrophobic amino acids are shown in red, arrows indicate the mutants analysed with combined point mutations in hydrophobic residues (red), or in individual conserved residues (black). (B) Alignment of the N-termini of human Arl8b, Arl1, Arf1 and ARFRP1. The second residue is shown in red. (C) MALDI mass spectrum of tryptic peptides from Arl8b-HA immunoprecipitated from transfected COS cells. Peptides with masses corresponding to expected digestion products of Arl8b are indicated with the sequence for the N-terminal peptide, or with residue numbers (o, oxidised). (D) Anti-HA immunoprecipitates from COS cells expressing LAT-HA or Arl8b-HA and labelled with [3H]palmitate. After gel electrophoresis, the precipitates were either probed with anti-HA antibodies or 3H-detected with fluorography.
To determine whether the acetylated N-terminal helix of Arl8b is involved in the specificity of membrane targeting, we expressed in COS cells chimeras of Arl8b and the Golgi-localised GTPase Arf1. Fig. 4E shows that, when the N-terminal helix of Arl8b was replaced with the equivalent part of Arf1, the chimera was still localised to lysosomes. In contrast, the N-terminal helix of Arl8b only gave a Golgi distribution when attached to Arf1 (Fig. 4F). Thus, it appears that the acetylated N-terminal helix is required for membrane association, but organelle specificity is determined by interactions made by the rest of the protein, perhaps by virtue of recognition by a lysosome-associated GEF.
Expression of Arl8a and Arl8b alters the distribution of lysosomes
The lysosomal location of Arl8a and Arl8b raises the question of their function on this compartment. During the course of the above experiments, we noticed that overexpression of Arl8a and Arl8b resulted in a redistribution of lysosomes in both COS and NRK cells. In such cultured cells, lysosomes typically cluster in the perinuclear region with a few additional lysosomes distributed throughout the cytoplasm (Matteoni and Kreis, 1987). However, cells overexpressing Arl8a or Arl8b still had some perinuclear lysosomes but had more lysosomes localised throughout the cell, including many along the cell periphery, a distribution not normally seen in untransfected cells (Fig. 5A). This effect was even more marked in cells as they spread after replating (Fig. 5B).
Although lysosomal transport is not understood in detail, it is known that lysosomes can move bi-directionally along microtubules, using dynein and kinesin motors (Harada et al., 1998; Jordens et al., 2001; Matteoni and Kreis, 1987). When cells expressing Arl8b were treated with nocodazole to depolymerise microtubules, lysosomes at the edge of cells were no longer observed; instead the lysosomes were scattered throughout the cytoplasm in a manner indistinguishable from nocodazole-treated control cells (Fig. 5C). This indicates that microtubule-based motility is required for maintaining the peripheral and perinuclear pools of lysosomes seen in transfected cells.
Study of other Arf family GTPases has identified mutant forms that are locked in GTP-bound or GDP-bound states (Dascher and Balch, 1994). The analogous mutations in Arl8b were examined. Expression of Arl8b with a Q75L mutation, known to prevent GTP hydrolysis in other Arf family members (Lu et al., 2001; Shin et al., 2005), resulted in a distribution and lysosomal phenotype similar to that seen with the wild-type protein (Fig. 5A). However, when wild-type and Arl8b(Q75L) were expressed in E. coli they both accumulated with GTP bound to them, suggesting that the intrinsic rate of GTP hydrolysis is low and, hence, overexpression of the wild-type Arl8b is similar to an accumulation of the GTP-locked form. This would also be consistent with the observation that most of the transfected Arl8b accumulates on lysosomes because, by analogy with other Arf GTPases, high-affinity membrane binding would occur only in the GTP-bound state (Goldberg, 1998; Pasqualato et al., 2002). Arl8b(T34N), containing a T34N mutation, was predicted to be GDP-bound, and this was found to be the case for E. coli-derived material. However, although it did not bind to lysosomes in COS cells, it appeared to have folding problems because it adhered instead to lipid droplets (data not shown). Similar folding problems were found with the other potential dominant-negative mutants we tested (G32A, N130I and D133N), preventing useful interpretation.
The N-terminus of Arl8b is required for its lysosomal localisation. (A) Mutant forms of Arl8b, with the corresponding N-terminal peptides obtained after trypsin digestions of HA-tagged forms as in Fig. 3C. The sequence of the N-terminal peptides identified by MALDI were determined by tandem mass spectrometry (MS/MS). The mass of the peptide stated is for the protonated form, with that from Arl8b(L2F) being the form with an oxidised methionine that was more abundant than the native version and hence used for MS/MS sequencing. (B-D) Confocal micrographs of COS cells transfected with plasmids expressing the indicated forms of Arl8b fused to GFP and then, after fixation and permeabilisation, labelled with an anti-CD63 antibody to label lysosomes. (E-F) Confocal micrographs of COS cells transfected with plasmids expressing GFP (-G) fused to the indicated chimeras of Arf1 and Arl8b, and labelled with the indicated antibodies. Replacement of the first 18 residues of Arl8b with the first 15 of Arf1 does not prevent localisation to lysosomes (labelled with CD63), whereas the first 19 residues of Arl8b do not relocalise Arf1 (17-181) from the Golgi (labelled with golgin-245). Bars, 10 μm.
Arl8b stimulates lysosomal motility
To investigate further the effects of Arl8a and Arl8b on the distribution of lysosomes, we used time-lapse microscopy of transfected NRK cells to follow the movement of lysosomes labelled with endocytosed dextran. As observed for fixed cells, lysosomes clustered in the cell centre, and in Arl8b-expressing cells they also accumulated at the cell periphery (supplementary material Movie 1). Although most of these lysosomes were relatively static, the movies revealed that in both transfected and untransfected cells there were, as expected, mobile lysosomes in the cytosol between the cell centre and the periphery (Matteoni and Kreis, 1987). To analyse this movement more closely, we imaged cell extensions because these allowed clear distinction between movements towards or away from the cell body. In both control and Arl8b-GFP-transfected cells, lysosomes in such extensions showed periods of relative immobility and then made sustained movements towards or away from the periphery (Fig. 6A). However, in cells expressing Arl8b-GFP the lysosomes appeared to move more frequently and over greater distances (supplementary material Movies 2 and 3). This difference was confirmed by using tracking software to quantify lysosomal movements (Fig. 6B). It seemed that the major effect of Arl8b-GFP was to increase the proportion of lysosomes that showed episodes of sustained rapid motion (from 8.1% to 16.2% of lysosomes in the 3-minute imaging periods), and to increase the average displacement per frame for all movement (53.5 nm/second to 85.3 nm/second). In addition, the average length of pauses or static episodes was reduced (from 8.6 to 3.7 seconds). The increase in movement was not directional, because the relative frequency of movements towards the periphery compared with those towards the centre was unchanged (Fig. 6B). Similar values were obtained for both Arl8b and Arl8b(Q75L), suggesting that transfection of Arl8b has a stimulatory effect due to an increase in the amount of the GTP-bound form (Fig. 6B).
Arl8b expression results in a more peripheral localization of lysosomes. (A) Confocal micrographs of COS cells expressing Arl8b-GFP or Arl8b(Q75L)-GFP and labelled with the antibodies to the indicated lysosomal membrane proteins. (B) As in (A), except that cells were trypsinised and plated on glass slides 90 minutes prior to fixation. (C) As in (A), except that cells were treated with 20 μM nocodazole for 4 hours before fixation, and then labelled for tubulin to confirm microtubule depolymerisation, or for the lysosomal antigen CD63. In such cells the lysosomes are scattered throughout the cytosol irrespective of the presence of Arl8b-GFP. Bars, 10 μm.
Arl8b stimulates lysosomal transport. (A) Still images of spinning-disc movies of lysosomes in processes of an untransfected NRK cell (con.), or of the same expressing Arl8b-GFP (Arl8b). Lysosomes were labelled by incubating cells for four hours with 1 mg/ml Alexa-Fluor-568-dextran, followed by a 20-hour chase. In both cases the cell body is at the top, and lysosomes moving toward, or away from, the cell body are indicated with open or closed triangles respectively. Bars, 5 μm. (B) Table summarizing the analysis of movies of NRK cells transfected with Arl8b-GFP, Arl8b(Q75L)-GFP or of untransfected cells. Lysosomes were labelled as in (A) and cell extensions of five cells each were imaged for three minutes at two frames per second. Individual lysosomes were tracked relative to an axis running from the cell body along the extension, and various parameters of the movement are shown. In each case a mean value is given, along with the standard error of mean. A `displacement' is defined as the movement between successive frames. A `transport event' is defined as a displacement of 0.15 μm or more per second, because this is typically the minimum speed of microtubule-dependent transport in vivo (Gross et al., 2000). A `static episode' is a period where the displacement between each successive frame does not exceed 0.15 μm/second. A `long episode of continuous fast motion' is when the net direct displacement over eight successive frames exceeds 1 μm. The length of an `episode of continuous motion' is defined for objects that have made a transport event, and is the net direct displacement before the object reverses direction relative to the axis, or does not make a transport event (i.e. moves less than 0.15 μm/second between frames). Perinuclear clusters of lysosomes were not included in the analysis as the software could not recognise every lysosome within these clusters.
By moving more frequently and over longer distances, lysosomes of Arl8b-transfected cells might have a higher chance of reaching the cell periphery where they can then either fall off the microtubules or become aggregated. The bidirectional effect on movement would explain why not all of the lysosomes accumulate at the periphery; and indeed, lysosomes can become detached from peripheral clusters and return to the cell centre (supplementary material Movie3).
Arl8b remains associated with lysosomes during mitosis
While our analysis of Arl8a and Arl8b was in progress, Katada and co-workers described Arl8a and Arl8b as Gie1 and Gie2, GTPases that had a perinuclear localization in interphase cells, but were associated with the spindle mid-zone and the midbody in mitotic cells (Okai et al., 2004). This appears to be contrary to the lysosomal localisation observed by us. However, during mitosis many membrane traffic processes cease, and some organelles become fragmented to aid segregation of daughter cells (Shorter and Warren, 2002; Warren and Wickner, 1996). Indeed, it has been reported that Arf1 dissociates from the Golgi during mitosis (Altan-Bonnet et al., 2003), and so it was possible that Arl8 is relocalised during these mitotic processes. However, examination of Arl8b-GFP in both COS and NRK cells showed that the protein remained associated with lysosomes during mitosis and did not localise to either the spindle or midbody (Fig. 7A,B). This suggests that lysosomal targeting is not under cell-cycle regulation and that the protein is not involved in the mitotic spindle.
Localisation of Arl8b in mitotic cells. Projection images of a stack of confocal slices from NRK cells transfected with a plasmid expressing Arl8b-GFP. After fixation and permeabilisation the cells were stained with antibodies to lysosomes and tubulin. Mitotic cells were identified in the total population by tubulin staining. Arl8b-GFP is localised to lysosomes, and is not detectable on the spindle mid-zone (A) or the midbody (B). Bars, 10 μm.
Drosophila Arl8 is targeted to lysosomes
The above analysis of mammalian Arl8a and Arl8b relies on tagged forms of the protein. To provide evidence that these results are likely to reflect the properties of the native proteins, we raised an antiserum against the single homologue present in Drosophila. This homologue is encoded by gene CG7891 and we will refer to it as Drosophila Arl8. Fig. 8A shows that, when this antiserum was used to probe extracts of cultured Drosophila cells, it recognised a prominent band of approximately 23 kD (which is close to the predicted molecular weight of 21.3 kD) that was absent when the cells had been treated with dsRNA to silence expression of Drosophila Arl8. The antiserum also recognised a second prominent band, but this was not affected by Arl8 dsRNA. When used for immunofluorescence of cultured cells, the antiserum labelled punctate structures in the cytoplasm but this staining was absent following Arl8 dsRNA treatment (Fig. 8B). This indicates that, endogenous Arl8 is present in punctate cytoplasmic structures and the second band is a cross-reactivity with a distinct protein that is only recognised when denatured. The Arl8-positive structures colocalise with a GFP-fusion protein of Drosophila LAMP1, confirming that they are lysosomes (Fig. 8C). As with the epitope-tagged protein in mammalian cells, incubation with the anti-Arl8 antiserum did not result in any detectable labelling in the spindle mid-zone (Fig. 8D) or the midbody (Fig. 8E). Finally, the antiserum allowed detection of native Drosophila Arl8 expressed in COS cells, where it was seen to localise to lysosomes and also to induce the same scattering of lysosomes seen with the tagged mammalian proteins (Fig. 8F). Taken together, these results indicate that the results with the tagged forms of Arl8a and Arl8b probably reflect the properties of the native proteins.
In this research, we have examined two closely related human Arf-like GTPases, Arl8a and Arl8b, and found that both are localized to lysosomes and, when overexpressed cause an accumulation of lysosomes at the cell periphery. In addition, we show for Arl8b that membrane targeting depends on the N-terminus, which is itself acetylated, and that lysosomal motility is stimulated by overexpression. Given that Arl8a is also a potential substrate for NatC acetylation rather than myristoylation and causes a similar alteration in lysosomal distribution when overexpressed, then it probably shares the aforementioned features with Arl8b. Both Arl8a and Arl8b have homologues in vertebrates whose genomes have been sequenced, and there is a single Arl8 homologue in many other eukaryotes including filamentous fungi, plants and protozoa, such as Dictyostelium (Li et al., 2004; Pasqualato et al., 2002). As such, the protein seems likely to play a role in a fundamental cellular process that is widely conserved in eukaryotic cells, and at least the Drosophila Arl8 homologue is also present on lysosomes. To date, Arl8a and Arl8b are the first GTPases found to be localised to lysosomes. All other organelles are known to have one or more members of the Arf or Rab families that are specifically found on their membranes, and these proteins typically serve to recruit multiple effectors that act in an organelle-specific function (Behnia and Munro, 2005; Zerial and McBride, 2001). At present, the effectors for Arl8a and Arl8b remain unknown, but overexpression of either protein causes some lysosomes to accumulate at the cell periphery. This redistribution might simply reflect the longer and more rapid movement on microtubules, resulting in some lysosomes falling off the plus-ends at the periphery and remaining there for a while before re-engaging with microtubules. However, in melanocytes a peripheral pool of melanosomes (a lysosome related organelle) is maintained by being anchored to cortical actin (Rodionov et al., 2003; Seabra and Coudrier, 2004). We cannot exclude a contribution from such actin-based anchoring, but the loss of the peripheral pool after microtubule depolymerisation implies that, if it does occur it is not sufficient by itself to confer the peripheral location.
Endogenous Arl8 is present on lysosomes in Drosophila cells. (A) Protein blot of total cellular proteins from D.Mel-2 cells probed with a rabbit antiserum raised against Drosophila Arl8 (Dm Arl8, CG7891). The cells were treated with dsRNA from GFP (con) or Drosophila Arl8. (B) Confocal micrographs of D.Mel-2 cells treated as in (A) with dsRNA from GFP (control) or Drosophila Arl8. After fixation and permeabilisation, cells were labelled with anti-Drosophila Arl8, and imaged with identical settings. (C) Confocal micrographs of S2 cells expressing a GFP fusion to the C-terminus of Drosophila LAMP1 (CG3305) and labelled with anti-Drosophila Arl8. (D-E) Confocal projection stacks of mitotic D.Mel-2 cells labelled with antibodies against Drosophila Arl8 and tubulin. Arl8 does not detectably concentrate in the spindle mid-zone in late anaphase (D), or in the midbodies (E) during late telophase. (F) Confocal micrographs of COS cells transfected with a plasmid expressing a native (i.e. un-tagged) form of Drosophila Arl8, and labelled with antibodies to Drosophila Arl8 and CD63. The Drosophila protein is localised to lysosomes, and alters the perinuclear distribution of lysosomes seen in the adjacent untransfected cell. Bars, 5 μm.
Lysosomes, like many organelles, are capable of bidirectional movement, and this may be important to ensure efficient exposure to the whole volume of the cytoplasm. An increase in microtubule-dependent motility implies that Arl8b is capable of recruiting or activating either motor regulators, such as lipid or protein kinases, or components of the transport machinery, such as dynactin, which can recruit both plus-end and minus-end directed motors (Jordens et al., 2001; Mallik and Gross, 2004; Rodionov et al., 2003). In the long term, identification of the effectors for Arl8 will be required to understand the molecular basis of its effects. However, it is interesting that, although Arl8 is very well conserved in evolution, it has been lost from budding yeasts – which are unusual, in that all known organelle movements occur on actin rather than microtubules (Pruyne et al., 2004). Thus, understanding the role of Arl8 could be of relevance to microtubule-based motility in many systems.
Although the binding partners of Arl8a and Arl8b are still unknown, it is clear that their recruitment to lysosomal membranes depends on both the N-terminal amphipathic helix and the precise structure of the first two residues. The amphipathic helix is important for membrane binding of all members of the Arf family so far investigated, but in most cases it is preceded by an N-terminal myristoyl group that initiates and stabilises membrane association (Antonny et al., 1997; Beraud-Dufour et al., 1999). However, we find that Arl8b is N-terminally acetylated, consistent with it having a hydrophobic residue rather than a glycine in the second position. The Golgi-localised Arl ARFRP1 also has a well-conserved hydrophobic residue in the second position, and the yeast homologue (Arl3) has been shown to be acetylated. This acetylation is necessary for the GTPase to be targeted to the Golgi by binding Sys1, a small polytopic membrane protein (Behnia et al., 2004; Setty et al., 2004). Thus N-terminal acetylation of an amphipathic helix appears to be an alternative to fatty acid modification as a targeting mechanism for small GTPases, and our results with Arl8b indicate that this alternative targeting strategy is more widely used than just ARFRP1/Arl3. However, this raises the question of how the acetylated N-termini of Arl8a and Arl8b contribute to targeting the proteins to lysosomes. Our database searches have not revealed a family of Sys1-related proteins, suggesting that any protein receptor for Arl8a and Arl8b would have to be distantly related at best. Moreover, chimeras of Arl8b and Arf1 indicated that the acetylated N-terminal amphipathic helix is neither necessary nor sufficient for lysosomal targeting. Thus, the acetylated N-terminus might not be specifically recognised by a protein receptor. Instead, the acetylation might be important for removing the positive charge of the α-amino group and stabilizing the amphipathic helix, hence, promoting its insertion into the lipid bilayer after activation by a lysosomal GEF. The putative amphipathic helices of Arl8a and Arl8b are four residues longer than those of the myristoylated Arfs Arf1 or Arl1 (Fig. 3B), and these extra hydrophobic residues might compensate for the reduction in membrane affinity due to the absence of the myristoyl group.
Arl8a and Arl8b were recently reported to localize to the mitotic spindle and the midbody during cytokinesis, with a `perinuclear' localisation in interphase cells (Okai et al., 2004). However, we did not observe relocation of Arl8b-GFP from lysosomes to these structures in dividing cells. C-terminal tags have been used successfully with other members of the Arf family (Aikawa and Martin, 2003; Altan-Bonnet et al., 2003; daSilva et al., 2004; Vasudevan et al., 1998), and the structure of Arl8b indicates that its C-terminus should be as well-exposed as these other proteins (Protein Data Bank entry 2AL7) but, nonetheless, it is possible that the C-terminal GFP tag used in our studies blocks the targeting of Arl8b to these mitotic structures. However, an antiserum against Drosophila Arl8 detected the endogenous protein on lysosomes but did not show any concentration of labelling on the spindle or midbody, although we cannot, of course, exclude the possibility that a small fraction of Arl8 is on the spindle. Some of the localisation data by Okai et al. were based on Arl8b with an N-terminal FLAG tag (Kurosu and Katada, 2001; Okai et al., 2004), and in the light of our results on the importance of the N-terminus, this N-terminally tagged protein was unlikely to be localised correctly. However, the remainder of the data was obtained with an antibody to the endogenous protein, although the specificity of this antibody was not confirmed by RNA interference.
Thus, Arl8a and Arl8b are probably on lysosomes in interphase and mitotic cells, and although they can potentially have a role on mitotic structures this might well bare further examination. Given the importance of GTPases in the functions of the organelles on which they are found, Arl8a and Arl8b appear good candidates to play a role in at least some of the processes that depend on lysosomes, such as autophagy, receptor downregulation, phagocytosis and plasma membrane repair.
Tissue culture and microscopy
COS and NRK cells were transfected with FuGene (Roche), split onto glass slides, fixed 24-40 hours after transfection with 4% paraformaldehyde, permeabilised with0.5% Triton X-100 in PBS, blocked with 20% foetal calf serum supplemented with0.5% Tween 20 in PBS, and labelled with antibodies in the same solution. Drosophila D.Mel-2 cells (Invitrogen) were grown in GIBCO Drosophila SFM (Invitrogen), and fixed and processed for immuno-labelling as above. Double-stranded RNA (T7 RiboMAX, Promega), was used for gene silencing as described previously (Bettencourt-Dias et al., 2005). A Drosophila S2 cell line stably transfected with Drosophila LAMP1-GFP (CG3305) under the control of a metallothionein promoter was kindly provided by Gudrun Ihrke and grown in Schneider's medium supplemented with 10% foetal calf serum. LAMP1-GFP expression was induced by treatment for three hours with 1 mM CuSO4, followed by incubation in CuSO4-free medium for 1-2 days.
A rabbit antiserum against residues 18-186 of Drosophila Arl8 (CG7891) expressed as a GST fusion from vector pGEX-6P-2 (Amersham Bioscience), was raised commercially (Harlan Sera-Lab) and affinity purified. Other rabbit antibodies were against mannose-6-phosphate receptor, giantin (Seelig et al., 1994) and the HA-epitope tag (Santa Cruz). Mouse monoclonals antibodies were to against CD63 (Fraile-Ramos et al., 2001), EEA1 and golgin-245, LAMP2 (H4B4, DSHB, Iowa), and tubulin (Sigma). Primary antibodies were detected with Alexa-Fluor-labelled secondary antibodies (Molecular Probes). Cells were mounted in Fluoromount-G (Southern Biotechnology Associates, Inc.), and images obtained on a Radiance confocal microscope (BioRad).
Live cell imaging was performed on a spinning disc microscope (UltraView, Perkin Elmer). Cells expressing GFP-fusions were identified, and dextran-labelled lysosomes were imaged at 30°C in DMEM supplemented with 10 mM HEPES pH7.2. Lysosomes in movies were tracked and analysed using Mathematica 4.0 (Wolfram Research) running custom software (Bullock et al., 2003).
Analysis of Arl8b by mass spectrometry
COS cells expressing Arl8b-HA were lysed 48 hours post transfection by exposure to 1 ml lysis buffer for 10 minutes on ice [50 mM Tris HCl pH 8, 150 mM KCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors (Roche)] followed by scraping. The lysate was centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant incubated for 2 hours at 4°C with 45 μl of anti-HA beads (Santa Cruz). After four washes in lysis buffer, bound protein was eluted with 100 μl of 0.5 M acetic acid pH 3.4, lyophilised and resuspended in SDS sample buffer. Following gel electrophoresis and staining with Coomassie blue, the protein was excised and digested with trypsin (Roche). Peptides were analysed using MALDI mass spectrometry (Voyager-DE, PerSeptive Biosystems) (Shevchenko et al., 1996), or sequenced with tandem mass spectrometry (QStar, Applied Biosystems).
Palmitate labelling
COS cells in 6-well plates expressing Arl8b-HA as above were labelled 40 hours post-transfection by incubation in labelling medium (DMEM, 10% dialyzed FCS) for 1 hour, followed by 1 ml of labelling medium with 0.5 mCi [3H]palmitate (Amersham Biosciences) for 3 hours. Cells were washed three times with cold PBS, scraped into 500 μl of lysis buffer, incubated for 10 minutes at 4°C, and centrifuged for 10 minutes at 10,000 g. The supernatant was incubated with 20 μl anti-HA beads (Santa Cruz) for 2.5 hours at 4°C, beads were washed three times with cold lysis buffer and eluted with SDS sample buffer containing 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as reducing agent. Following gel electrophoresis, and incubation in Amplify (Amersham), the gel was dried and imaged by fluorography. A proportion of the precipitate was also analysed by protein blotting with rabbit anti-HA antibody, detected with horseradish peroxidase (HRP)-conjugated anti-rabbit Ig and enhanced chemiluminescence (Amersham Biosciences).
We are greatly indebted to Simon Bullock for help with spinning-disc microscopy and analysis of lysosome movements. We also thank Farida Begum and Sew Peak-Chew for mass-spectrometry, Oliver Daumke for help with HPLC, Gudrun Ihrke for the LAMP1-GFP cell line and Matthew Freeman, Alison Gillingham and Katja Röper for comments on the manuscript. I.H. was supported by a PhD Scholarship from the Boehringer Ingelheim Fonds.
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Movie 1
Movie 1. Spinning disc movie of Alexa-Fluor-568-dextran labelled lysosomes in NRK cells. The transfected cell expressing Arlb8-GFP was identified by GFP fluorescence. Images were obtained at one frame per second, with the movie running at 12 frames per second.
- mov file
Movie 2. Spinning disc movie of Alexa-Fluor-568-dextran labelled lysosomes in a projection from an untransfected NRK cell. Images were obtained at one frame per second for three minutes, with the movie running at 12 frames per second.
Movie 3. Spinning disc movie of Alexa-Fluor-568-dextran labelled lysosomes in a projection from an NRK cell expressing Arl8b-GFP. Images were obtained as for Movie 2.
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Осиповка — посёлок в Троицком районе Челябинской области России, относится к Нижнесанарскому сельскому поселению.

География
Расположен в центральной части района, на берегу р. Уй. Рельеф — равнина (Западно-Сибирская равнина); ближайшие выс.— 198 и 260 м. Ландшафт — лесостепь с ленточными сосновыми борами. О. связана шос. дорогами с соседними населенными пунктами. Расстояние до районного центра (Троицк) 35 км, до центра сельского поселения (с. Ниж. Санарка) — 10 км.

Население

(в 1873 — 268, в 1900 — 394, в 1926 — 687, в 1940 — 424, в 1956 — 390, в 1970 — 309, в 1995 — 312)

История
Поселок основан на месте бывшего Осиповского форта (указан на Ген. плане Троицкого уезда 1800), к-рый входил в систему Уйской пограничной линии (см. Уйская дистанция) и в первые 10-летия существования представлял собой обычный деташмент: неск. землянок, огорож. надолбами и рогатками, в к-рых на летнее время (иногда и на зиму) размещались прикомандир. казаки, башкиры, мещеряки и солдаты (так, в 1792 было прикомандировано 47 исетских казаков, 53 башкира).

Постоянное население появилось лишь в 1-й пол. 19 века при проведении реформы ОКБ в поселке был запланирован 1 каз. двор с 3 душами мужского пола и 10 дворов с 10 отставными солдатами. О. был причислен к 6-му полку 2-го воен. округа ОКБ; впоследствии относился к Кособродскому станичному юрту 3-го воен. отдела ОКБ.

В 1873 насчитывалось 43 двора, в 1900 — 68 (действовали церковь во имя Св. Пророка Илии, школа, водяная мельница). В 1893 за поселком (57 дворов) закрепили 5478 дес земли, в т. ч. пашни 4384 дес 1200 саж2, лугов — 93 дес 1200 саж2, леса — 3 дес, выгона — 880 дес. Жители занимались жив-вом, выращивали традиционные для Южного Урала зерновые и овощные культуры, из техн.— просо, реже гречиху (в советский период — подсолнечник).

По данным переписи, в 1920-х гг. О. была центром сельсовета в Троицком р-не (Троицкий округ Урал. обл.). В 1929—30 организован колхоз «Согласие» (в 1951 переим. в колхоз им. Жданова), за к-рым было закреплено 5308 га земельных угодий. Впоследствии за счет освоенных целинных участков дача увеличилась до 6175 га.

После упразднения Осиповского сельсовета (1959) колхозы села Ниж. Санарка и Осиповка присоединились к колхозу «Путь к коммунизму» (образован в 1950), на территории  расположилась бригада нового колхоза. Ныне в поселке размещается бригада СХПК «Нижнесанарский».

Литература

Примечания

Населённые пункты Троицкого района (Челябинская область)

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Карсакпа́й () — посёлок в Улытауском районе Улытауской области Казахстана. Административный центр и единственный населённый пункт Карсакпайской поселковой администрации. Код КАТО — 356051100.

Железнодорожная станция Карсакпай на линии Жезказган — Саксаульская.

История
Основан в 1912 году англичанами как посёлок при медеплавильном заводе. Название происходит от казахского личного имени Карсакбай. Месторождения меди неподалёку от современного посёлка были известны с древних времён.

Медное месторождение было зарегистрировано ещё в 1847 году русским купцом Николаем Ушаковым, он перепродал залежи меди по берегам речек Кенгир и Жезды купцу Рязанову. В дальнейшем месторождение было продано английским предпринимателям, которые в Лондоне зарегистрировали «Акционерное общество "Атбасарских медных руд"». Оно провело разведывательные работы для строительства узкоколейной железной дороги Жезказган — Карсакпай — Байконур протяжённостью 120 километров.

В 1914 году был заложен фундамент Карсакпайской обогатительной фабрики для будущего медного завода. Необходимый материал для строительства доставлялся от станции Джусалы на верблюдах и по временному рельсовому пути длиной 13 километров. Было заложено 18 мелких шахт глубиной от 6 до 80 метров.

В 1914—1917 годах была построена узкоколейная железная дорога, соединявшая Байконур с Карсакпаем и Джезказганом. Дорога была демонтирована в начале 1980-х годов.

10 июня 1925 года решением Совета труда и обороны был образован Атбасарский трест цветных металлов союзного значения, куда вошли Карсакпайский комбинат, состоящий из Карсакпайского медьзавода, Байконурских угольных копей, Жезказганского медного рудника и свинцового рудника Кургасын. Руководил комбинатом Р. А. Дрейман. Рождению промышленного комплекса в степи предшествовали широкие геологоразведочные работы, проводимые К. И. Сатпаевым. Назначенный начальником геологоразведочного отдела и главным геологом треста «Атбасарцветмет» в 1926 году, он открыл крупнейшие залежи медной руды в данном регионе.

С 1926 года в Карсакпае функционирует метеостанция.

16 октября 1928 года зажжена отражательная печь вновь построенного Карсакпайского медьзавода и через 3 дня выплавлена первая казахстанская медь. Жезказганские рудники по узкоколейной железной дороге обеспечивали сырьём — медной рудой Карсакпайский медьзавод.

В 1957 году в посёлке был основан овцеводческий совхоз.

В 1970 году численность населения посёлка составляла 6 тысяч жителей.

Медеплавильный завод, работавший на руде Жезказганского месторождения, был закрыт в 1973 году. На его базе был образован металлургический завод, включивший сталелитейный и экспериментальные цеха (ныне в составе корпорации «Казахмыс»).

В 1997 году на базе овцеводческого совхоза были организованы частные хозяйства.

После строительства линии Жезказган — Саксаульская, проходившего в 2012—2014 годах, действует железнодорожная станция Карсакпай. С 2016 года по ней действует пассажирское сообщение.

Население
В 1999 году население посёлка составляло 2402 человека (1268 мужчин и 1134 женщины). По данным переписи 2009 года в посёлке проживало 1668 человек (889 мужчин и 779 женщин).

Достопримечательности
В доме, где с 1926 по 1941 годы жил академик К. И. Сатпаев, с 1969 года базируется музей.

Примечания

Литература

Ссылки

Населённые пункты Улытауского района


Denial might be a river in Egypt but today it looked an awful lot like the Potomac.
At the Energy and Commerce hearing about Twitter on Sept. 5, Twitter CEO Jack Dorsey answered many questions about his platform, but one topic he completely denied — bias. When asked repeatedly by representatives if Twitter engaged in conservative censorship of any kind, Dorsey only had one answer: "No." He was asked three times in a row and each time had the same answer.
Republican representatives Steve Scalise (R-LA) and Michael Burgess (R-FL) brought up the horrible doctored photo of Meghan McCain being targeted by a gun and Rep. Marsha Blackburn (R-TN) having her political ad banned from Twitter. Dorsey acknowledged both of these incidents as mistakes made by Twitter (he even said he planned to apologize to McCain.) But he still refused to admit that there was a bias against conservatives on his platform.
While he did admit that more than 600,000 users were shadowbanned thanks to an algorithm glitch, and he did admit to there being an unequal balance of who was restricted, Dorsey did not say whether the bias leaned in favor of Democrats or Republicans.
At one point, when questioned about the rules and policies that control Twitter, Dorsey admitted: even Twitter users can't understand the rules. He confessed that Twitter was "working on it" to fix the confusion.
When a question was raised about the verification process on Twitter, not only did Dorsey say that the process needed to be re-done and improved, but also that verified users did in fact have their content prioritized over other users.


Support the Ospreys Rugby team in a replica jersey with a contemporary fit, slightly more relaxed than the 'players' Test jersey. The reverse pique body fabric lifts the the jersey away from the skin, allowing greater comfort and breathability, while new raglan seam shaping gives increased mobility. The new Canterbury 'Loop 17' neckline gives a strong and comfortable fit. Finished with a uniquely numbered holographic security authenticator so you can put your trust in your jersey.


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text:
Welcome to our Parish
Welcome to Rathdowney Parish Website! We want to make our parish website an interesting and worthwhile location for both parishioners and visitors to keep up to date with what's happening in our parish. We hope that all parish and community groups will see it as a useful resource for sharing information with people.
Please feel free to contact us if you have something which you think we should include. We welcome your feedback.
Rathdowney
Errill
Most Holy Trinity, Rathdowney
Mon to Fri 5.30 pm
Tues to Fri 10.00 am
Sun 10.30 am
About this Church
Our Lady Queen of the Universe, Errill
Saturday 8.30pm
Archdeacon Sean Doherty will retire as Parish Priest of Durrow on the weekend of August 28th/29th 2021. Fr. Martin Delaney PP Rathdowney will become the Administrator of Durrow parish in addition to his duties in Rathdowney. Fr. Tom McGree CC will continue to minister in…
Many charities and Church communities have seen a dramatic drop off in their contributions during these months of Covid-19 related restrictions. It seems unlikely that we will be able to have regular public celebrations of Mass for some months and even then the restrictions posed by…
Rathdowney Featured by Catholic Ireland
Follow this link to a feature on Rathdowney published on the catholicireland.net website
https://www.catholicireland.net/laois-parish-offers-concerts-and-liturgies-via-webcam/

redpajama_set_name: RedPajamaCommonCrawl


text:
package net.schmizz.sshj.util.gss;

import static net.schmizz.sshj.util.gss.BogusGSSManager.unavailable;

import java.io.InputStream;
import java.io.OutputStream;
import java.net.InetAddress;
import java.net.UnknownHostException;
import java.nio.charset.Charset;
import java.util.Arrays;

import org.ietf.jgss.ChannelBinding;
import org.ietf.jgss.GSSContext;
import org.ietf.jgss.GSSCredential;
import org.ietf.jgss.GSSException;
import org.ietf.jgss.GSSName;
import org.ietf.jgss.MessageProp;
import org.ietf.jgss.Oid;

public class BogusGSSContext
        implements GSSContext {

    private static final byte[] INIT_TOKEN = fromString("INIT");
    private static final byte[] ACCEPT_TOKEN = fromString("ACCEPT");
    private static final byte[] MIC = fromString("LGTM");

    private static byte[] fromString(String s) {
        return s.getBytes(Charset.forName("UTF-8"));
    }

    private boolean initialized = false;
    private boolean accepted = false;
    private boolean integState = false;
    private boolean mutualAuthState = false;

    @Override
    public byte[] initSecContext(byte[] inputBuf, int offset, int len) throws GSSException {
        initialized = true;
        return INIT_TOKEN;
    }

    @Override
    public int initSecContext(InputStream inStream, OutputStream outStream) throws GSSException {
        throw unavailable();
    }

    @Override
    public byte[] acceptSecContext(byte[] inToken, int offset, int len) throws GSSException {
        accepted = Arrays.equals(INIT_TOKEN, Arrays.copyOfRange(inToken, offset, offset + len));
        return ACCEPT_TOKEN;
    }

    @Override
    public void acceptSecContext(InputStream inStream, OutputStream outStream) throws GSSException {
        throw unavailable();
    }

    @Override
    public boolean isEstablished() {
        return initialized || accepted;
    }

    @Override
    public void dispose() throws GSSException {}

    @Override
    public int getWrapSizeLimit(int qop, boolean confReq, int maxTokenSize) throws GSSException {
        throw unavailable();
    }

    @Override
    public byte[] wrap(byte[] inBuf, int offset, int len, MessageProp msgProp) throws GSSException {
        throw unavailable();
    }

    @Override
    public void wrap(InputStream inStream, OutputStream outStream, MessageProp msgProp) throws GSSException {
        throw unavailable();
    }

    @Override
    public byte[] unwrap(byte[] inBuf, int offset, int len, MessageProp msgProp) throws GSSException {
        throw unavailable();
    }

    @Override
    public void unwrap(InputStream inStream, OutputStream outStream, MessageProp msgProp) throws GSSException {
        throw unavailable();
    }

    @Override
    public byte[] getMIC(byte[] inMsg, int offset, int len, MessageProp msgProp) throws GSSException {
        return MIC;
    }

    @Override
    public void getMIC(InputStream inStream, OutputStream outStream, MessageProp msgProp) throws GSSException {
        throw unavailable();
    }

    @Override
    public void verifyMIC(byte[] inToken, int tokOffset, int tokLen, byte[] inMsg, int msgOffset, int msgLen, MessageProp msgProp) throws GSSException {
        if (!Arrays.equals(MIC, Arrays.copyOfRange(inToken, tokOffset, tokOffset + tokLen))) {
            throw new GSSException(GSSException.BAD_MIC);
        }
    }

    @Override
    public void verifyMIC(InputStream tokStream, InputStream msgStream, MessageProp msgProp) throws GSSException {
        throw unavailable();
    }

    @Override
    public byte[] export() throws GSSException {
        throw unavailable();
    }

    @Override
    public void requestMutualAuth(boolean state) throws GSSException {
        this.mutualAuthState = state;
    }

    @Override
    public void requestInteg(boolean state) throws GSSException {
        this.integState = state;
    }

    @Override
    public void requestReplayDet(boolean state) throws GSSException {
        throw unavailable();
    }

    @Override
    public void requestSequenceDet(boolean state) throws GSSException {
        throw unavailable();
    }

    @Override
    public void requestCredDeleg(boolean state) throws GSSException {
        throw unavailable();
    }

    @Override
    public void requestAnonymity(boolean state) throws GSSException {
        throw unavailable();
    }

    @Override
    public void requestConf(boolean state) throws GSSException {
        throw unavailable();
    }

    @Override
    public void requestLifetime(int lifetime) throws GSSException {
        throw unavailable();
    }

    @Override
    public void setChannelBinding(ChannelBinding cb) throws GSSException {
        throw unavailable();
    }

    @Override
    public boolean getMutualAuthState() {
        return mutualAuthState;
    }

    @Override
    public boolean getIntegState() {
        return integState;
    }

    @Override
    public boolean getCredDelegState() {
        return false;
    }

    @Override
    public boolean getReplayDetState() {
        return false;
    }

    @Override
    public boolean getSequenceDetState() {
        return false;
    }

    @Override
    public boolean getAnonymityState() {
        return false;
    }

    @Override
    public boolean isTransferable() throws GSSException {
        return false;
    }

    @Override
    public boolean isProtReady() {
        return false;
    }

    @Override
    public boolean getConfState() {
        return false;
    }

    @Override
    public int getLifetime() {
        return INDEFINITE_LIFETIME;
    }

    @Override
    public GSSName getSrcName() throws GSSException {
        try {
            String hostname = InetAddress.getLocalHost().getCanonicalHostName();
            return new BogusGSSName("user@" + hostname, GSSName.NT_HOSTBASED_SERVICE);
        } catch (UnknownHostException e) {
            throw new IllegalStateException(e);
        }
    }

    @Override
    public GSSName getTargName() throws GSSException {
        throw unavailable();
    }

    @Override
    public Oid getMech() throws GSSException {
        return BogusGSSManager.KRB5_MECH;
    }

    @Override
    public GSSCredential getDelegCred() throws GSSException {
        throw unavailable();
    }

    @Override
    public boolean isInitiator() throws GSSException {
        return false;
    }

}


Economy Fieldtex CPR Packed In A Portable Plastic Box That Can Also Be Mounted On A Wall.CPR Kit Has A Complete Assortment Of.
Economy Fieldtex CPR Packed in a portable plastic box that can also be mounted on a wall.CPR kit has a complete assortment of CPR.
About The Economy Fieldtex CPR Packed in a portable plastic box that can also be mounted on a wall.CPR kit has a complete assortment of CPR.
More from Economy Fieldtex CPR Packed In A Portable Plastic Box That Can Also Be Mounted On A Wall.CPR Kit Has A Complete Assortment Of.


Poetry that is focused on politics, globalism, candidates for office, governments, governing, kings, queens, political science, political corruption, political parties, elections, and any other political discipline.
Moreover, it's a place for civility, non-violent conversations, and mutual respect for other belief systems. Like religion, the discussion of politics has always been rife with taking sides, fighting, and nastiness – political poetry on TCE is a place to express your opinion; not a place to force your opinion on another reader, writer, or casual passer-by.


This white paper is part of Harbinger's eLearning practice and explains a high level architecture of Harbingers' offline Player and how to enable offline access to learning content. Harbingers' Offline Player provides enterprises with a seamless and highly secure way to access training content without need for continuous Internet connectivity.
Please accept the terms to proceed.


Another new shade from stargazer and another colour I love. But it is hard to capture on camera – it's basically a true barbie pink, the consistency is quite sheer – I like this, but if you want it opaque you will need 3+ coats, I went for two and was happy with the slight translucent effect.
Again, these polishes do smell pretty bad (I'm 99.9% sure they are NOT big 3 free… correct me if I'm wrong) but they dry super fast and are very glossy. I painted my nails on a train on Saturday morning and about 5 mins after finishing the ticket inspector guy came round – my nails were already totally dry and didn't smudge a bit.
Also, in my last stargazer post I mentioned about the state of the stargazer displays in New Look stores – have to admit, I went to the New Look store in Cambridge on Saturday and it had a good selection and wasn't a total mess. There were quite a few swatches painted all over the place, but still – was easily the neatest and best stocked counter I have seen to date.
I've still got to try this brand!
What a pretty color. Very pale and delicate! Love it. For me though, I'd rather go a little wild with turquoise nails. After all, I've heard in so many places that it is one of the big colors of the summer. That and coral anyway. Thanks for sharing this and cute blog too!


text:
Why CAA+NRC might not be a good idea.
India passes a new law that promotes easier access of her citizenship to everyone except Muslims from three neighbors.
The political scenario of India is so happening, that the article I wrote a few weeks ago has already become stale and now we are to look at new sets of problems facing the country. The more I try to run away from it, the more it stares back at my face. If you fancy reading it, it is linked at the bottom.
The population of India is 31…
Iranian embassy put effort to draw India's attention to killing of its top nuclear scientist
The Progressive Alliance.
On Monday the 15th of May, in a crowded room in central London, the Progressive Alliance launched. A tactical voting initiative aimed at…
Israel spar over US mission in Jerusalem
On Sunday The Palestinians slammed Israel for rejecting the promised reopening of the US Consulate in Jerusalem, a move that would restore…
Phasing out private schools – (how) could you actually do it?
Palestinian-Israeli dialogue must not be canceled
Somalia Cuts Diplomatic Ties With Kenya
The Somali government announced on Monday it would cut diplomatic ties with neighboring Kenya, citing violations of Somalia's sovereignty.
The Global Education Summit and the Nigerian Paradox
Afghan journalist Najibullah Quraishi says the Taliban has not modified :
Daily post #327 Let People do Their Jobs
I learned a lot about leadership during my almost 10 years as an officer in the army, one thing being that you have to take a step back and…
Writing Jokes and Riddles, by Bill Howard
When is a joke a joke? When is a riddle a riddle? What's the difference between the two?
Kitten: why they love to stay in the bathroom when we are there. A reflection about the answer.
In The In Between
On a recent family trip to Disneyland, I saw something that made me stop and think. I am in the magical "happiest place on earth," and I…


text:
The Bathsheba Deadline
Bestselling novelist Jack Engelhard (Indecent Proposal) has produced a heroic work of literature. This is a superb, gutsy novel. The Bathsheba Deadline is a newsroom thriller ripped from the headlines. The present day action takes place in a Manhattan newsroom where three leading journalists find themselves caught in a sizzling three-way love triangle that may lead to murder in the Middle East (Israel). You will never forget Jay Garfield and you absolutely will never forget Lyla. Politics and sex mixed in with the war on terror provoke life-and-death rifts within the editorial staff. Journalists with the power to influence public opinion have lost their neutrality and have taken sides. Readers are taken behind the scenes and into the newsrooms where they are shown how headlines are made and often manipulated to favor one side over another. Media bias usually against Israel? Read all about it in Engelhard's stirring pages. The war for survival in a New York City newsroom mirrors the clash of civilizations here, in the Middle East, and around the world.
"A rousing thriller about clashing ideals, clashing moral standards, and clashing civilizations. The Bathsheba Deadline is a page-turner with much more than just entertainment (although it delivers that in abundance); it is an insightful, courageous look inside the headlines. Bravo, Jack Engelhard."
– Robert Spencer, author
"Engelhard writes with the sparseness of Hemingway and the moral intensity of I.B. Singer."
– Michael Foster, author
"Jack Engelhard is a writer without peer and the conscience of us all."
– John W. Cassell, author of Crossroads: 1969
March 3, 2014 Leave a comment


Apple's memorable television commercials played a huge role in the iPod product-line's mainstream success and, some would argue, Apple's resurgence as a brand that matters. The commercials have indeed been a raging success for Apple. They made the iPod cultural icons and helped pave the way for Apple's future. The colorful, 30 second ads are deceptively simple, pairing eye-catching visuals with catchy pop songs. So catchy, in fact, that we often hear others and ourselves asking: "What song is that?" Well, ask no more! Here is a list of every song that has ever been used in Apple's iPod+iTunes campaign as well as the iPhone, iPad and MacBook Air ads (including the "Hello" teaser).


text:
Sul Ross to Honor Distinguished Athletes, Alumni, and Supporters at March 30 Banquet
• by Paul Slocumb and Travis Hendryx •
Distinguished Sul Ross State University alumni Eddie Sandoval, John Robert and Betty Hicks Prude, Liliana Esparza, and Elizabeth Will-Bates will be honored at the annual Athletics Hall of Honor & Distinguished Alumni Banquet on Saturday, March 30 in the Pete P. Gallego Center. Outstanding SRSU supporters Bake Turner and Rick Kelly will also be recognized for their contributions to Sul Ross with the Slingin' Sammy Baugh Award. The event begins with a 6:00 p.m. reception to be followed by dinner at 7:00 and the awards program at 7:45.
2019 Athletics Hall of Honor inductees include Sidney Hooper (men's basketball), Charles Bunch (football), Gregg Slape (men's basketball/baseball), the 1986-87 TIAA Champion women's basketball team, and outstanding SRSU supporters Randy and Sherry Jackson.
Below are brief highlights of the 2019 Distinguished Alumni, Slingin' Sammy Baugh Award honorees, and Athletic Hall of Honor inductees:
Eddie Sandoval
Eddie Sandoval is a three-time Sul Ross graduate who holds both a bachelor's and master's degree in industrial technology as well as a master's degree in educational counseling.
After teaching and coaching for five years in San Antonio, Sandoval spent nearly 40 years at Tarrant County College in Fort Worth, retiring in 2011 as head of counseling.
Alongside his education and counseling careers, Sandoval, who has ancestors in the Mescalero and Chiricahua Apache tribes of Geronimo, has become well known as a Native American preservationist and historian. He also became a Sun Dancer at Pine Ridge, South Dakota in the early 1990s, a distinction enabling him to carry out numerous ceremonies and blessings.
For his decades of work advocating for the people of Tribal Nations and representing and preserving Native American heritage, Sandoval was inducted into the Texas Trail of Fame on May 14, 2018. The Trail of Fame honor comes with a bronze marker inlaid at the historic Fort Worth Stockyards.
The Eddie Sandoval Industrial Technology Excellence Fund, created in 2018 with an endowment from Margie Reynolds of Granbury, assists Sul Ross students through scholarships, graduate assistantships, research project funding, equipment, and supplies.
"Eddie is a unique individual who Sul Ross should be proud to recognize," says SRSU alumnus Sharon Henderson. "When I think of Eddie, I hear him saying, 'How can I help?'"
John Robert and Betty Hicks Prude
John Robert and Betty Hicks Prude have become legendary figures in the field of Far West Texas hospitality. Representing the third generation of Prude ranchers, John Robert (BS, MS, and MA Sul Ross degrees) and Betty (BA Sul Ross degree) helped introduce the concept of ranch tourism to Jeff Davis County nearly seventy years ago. Holding their first "camp" for a group of 18 boys in 1951, the couple worked as head counselor, camp administrator, and chief cook. When summer ended, they returned to their full-time jobs as teachers, but found themselves back at camp the following summer with a growing enrollment. In 1955, the pair invited girls to join the fun, and the Prude Ranch Summer Camp for Boys and Girls was born.
Over the following decades, numerous new buildings and other improvements were made to the Ranch to accommodate campers from around the country and around the world. And now, a fourth and fifth generation of Prudes extend their family's famous hospitality year round.
In 2010, John Robert passed away at the age of 82, leaving a legacy of both business success and public service—including 35 years as County Commissioner. He was also the recipient of numerous awards such as the Texas Environmental Education Partnership Award, Fort Davis Chamber of Commerce Citizen of the Decade Award, the Texas Department of Transportation's Gene Phillips Hospitality Award, the Texas Tourism Governor's Award, and the Texas Travel Industry Association Lifetime Achievement Award.
"No one did more to promote the Far West Texas area than John Robert and Betty Prude," says Larry Francell, former director of the Museum of the Big Bend. "He travelled continuously to steer tourism towards the Last Frontier, and the great lady who stood beside him made the operation run smoothly."
Liliana Esparza
Liliana Esparza earned a bachelor's degree in mass communications from Sul Ross and a master's degree in secondary education from Stephen F. Austin State University. As principal of St. Patrick Cathedral School, she is currently the longest tenured of 12 principals working for El Paso Catholic Schools. Esparza is the school district's lead principal, guiding diocesan-wide projects while ensuring a level of academic excellence which constantly surpasses expectations.
"As administrators, we are gifted with the opportunity to assist faculty and staff in shaping the minds and lives of children," says Esparza. "We must emphasize the intellectual, moral, physical, and social development of each individual student in a safe, positive, and supportive environment. We also have the unique opportunity to educate and empower the generations of future administrators who will follow us."
With professionalism and effective teaching strategies, Esparza has spent more than two decades as a role model for students, particularly young girls, interested in pursuing leadership roles and careers in education. She is a constant advocate for higher education and promotes Sul Ross as an ideal university for her students' further education.
In the community, Esparza is praised for a leadership approach built on dedication to the craft of education. Through the spiritual, intellectual, and physical development of her students, Esparza is known for preparing self-confident young people who are ready to succeed in a complex world.
"My parents and educators throughout my life instilled in me the importance of being a lifelong learner, and I want to be able to instill in others the same values," she says. "Furthermore, I strongly adhere to the belief that knowledge helps to empower. And, in return for my blessings, I want to help empower others through education."
Elizabeth Will-Bates
A native of Alpine and daughter of two Sul Ross faculty members, Elizabeth Will-Bates was perhaps destined to become a Lobo, an educator, and a Distinguished Young Alumnus. She demonstrated her passion for education early by following up her Sul Ross bachelor's degree in communication with two Texas teacher certifications and a teaching post at Pecos High School.
She went on to work as a research assistant and graduate instructor at Texas Tech while earning her master's degree and PhD in mass communications. Will-Bates is currently an assistant professor at Baylor University where her courses focus on media and public relations.
"My favorite part about teaching is seeing the progress students make throughout the semester," she says. "I also enjoy hearing from former students who are excelling in the workforce."
In addition to being published in Journalism and Mass Communication Educator and Public Relations Review, Will-Bates has delivered presentations at nine conferences. She has also been honored as a Distinguished Professor by Baylor's Mortar Board.
Bake Turner
Another Alpine native (and, coincidentally, a Texas Tech alumnus), Bake Turner is being honored with the Slingin' Sammy Baugh Award for Outstanding Service to Sul Ross. Known nationally for playing wide receiver on the victorious New York Jets Super Bowl III team, locally Turner is known as a successful DFW-area home builder, accomplished musical performer, and Sul Ross booster.
When Sammy Baugh created a golf tournament to raise scholarship funds for Sul Ross, Turner brought in his NFL friends and celebrities to play. Turner has also performed his country & western singing and guitar picking many times for Sul Ross students and staff—all for free. He even played for the Texas State University System Board of Regents at their 2017 board meeting in Alpine.
"It was obvious several of the Regents had followed his career and were somewhat starstruck in his presence," said Butch Worley, retired Sul Ross vice president for external affairs.
Another Alpine resident known for his generosity to Lobo Land is Rick Kelly, owner of Alpine Auto Rental. For years, Kelly has given the Athletics Department discounted van rates for team travel—discounted to the extent that driving his vans cost Sul Ross less than driving University-owned vans.
In 2014, Kelly ensured the success of a raffle benefitting the SRSU Century Club by not only convincing the Big Bend Shooting Foundation to sponsor the raffle, but also by selling a large portion of the tickets. The raffle raised $6,000 for Sul Ross.
Kelly has also organized the local business community to provide special team meals for Sul Ross athletes who practice and play during the holiday breaks.
"During my time at Sul Ross, no one volunteered more time and energy in support of the University than Rick," said Worley.
Sidney Hooper
Sidney Hooper is the all-time leader in career points at Sul Ross since the Lobos joined the NCAA ranks in 1996. A two-time American Southwest Conference West Division First Team All-Conference honoree, Hooper was twice selected as an ASC All-Conference selection and was twice voted to the D3Hoops All-South Region Team.
He was also named D3Hoops All-South Region Player of the Year and was chosen as a D3Hoops Third Team All-American. During the 2003-04 season, Hooper led the ASC in scoring averaging 22.1 points per game.
He was a two-time team captain, named twice as the team's Most Valuable Player and Offensive Player of the Year.
Hooper lettered four seasons with the Lobos and was a member of the 2003-04 ASC Champion and Sweet 16 team. He was also a member of three ASC West Division Title teams.
Hooper earned his degree from Sul Ross in 2006, majoring in kinesiology with a minor in criminal justice.
He currently works as a sales associate for BSN in Frisco, Texas.
Charles Bunch
Charles Bunch had a stellar football career at Sul Ross, lettering four seasons with the Lobos.
A Second Team NAIA All-American and a member of the 1996 ASC Championship Team, Bunch earned the ASC Defensive Player of the Year in 1996.
Bunch was also a 1996 ASC First Team All-Conference Defensive Back and was named an All-Conference Punter in 1996 and 1997.
Following his playing career, Bunch served as defensive coordinator at Pecos High School and helped lead the Eagles to three bi-district championships in 1999, 2000, and 2001.
From Pecos, Bunch moved on to San Angelo as a defensive coordinator at Central High School. During his stay at Central, he was part of an area championship in 2006 and a 2007 bi-district title team.
He then served as head football coach at Gorman High School and guided the team to a 2010 bi-district championship.
Bunch moved to James Madison High School in San Antonio as defensive secondary coach and helped lead the team to a 2013 state semi-final appearance.
He then became defensive coordinator at New Braunfels High School and earned a bi-district title in 2016.
He is currently defensive coordinator, assistant baseball coach, and sixth grade physical education, social studies, and special education teacher for the Sonora Independent School District.
Bunch earned his bachelor's degree in kinesiology from Sul Ross in 1998 and earned his Texas teacher certification one year later.
Gregg Slape
Gregg Slape was a two-sport athlete and lettered four seasons in basketball and baseball at Sul Ross.
Slape was a two-time All-Lone Star Conference selection in basketball in 1967 and 1968.
Following his basketball and baseball playing career with the Lobos, Slape signed a contract with the Cincinnati Reds in June 1968.
He played one season for Single-A affiliate Sioux Falls (South Dakota) and two seasons each with Double-A and Triple-A affiliates Asheville (North Carolina) and Indianapolis (Indiana).
Slape earned his bachelor's degree in marketing from Sul Ross in 1968 and works for Self-Employee Benefits Specialists, Inc.
1986-87 TIAA Champion Women's Basketball Team
The 1986-87 Lady Lobos finished with the best record of any Sul Ross women's basketball team at 20-4.
Following a Texas Intercollegiate Athletic Association (TIAA) title, the Sul Ross women advanced to the second round of the NAIA National Playoffs and fell to No. 1 Wayland Baptist University.
The Lady Lobos defeated NCAA Division I U.T. El Paso and a number of Division II programs including Angelo State, Western New Mexico, New Mexico Highlands, Lubbock Christian University, and the University of the Incarnate Word.
Their four losses came at the hands of NCAA Division II programs.
In addition to an undefeated TIAA record that season, the Lady Lobos posted a 4-0 sweep over conference powerhouse Tarleton State and won the outright league championship. The Tarleton State Lady Texans had won or shared ten consecutive conference titles before falling to the 1986-87 Lady Lobo team.
Team members were:
Vicki Vidana Gonzales – Van Horn – Conference MVP
Kay Thompson Hufstedler – Ozona – All-TIAA
Dana Souter Pannell – Alpine – All-TIAA
Paula Brown Coffman – Andrews
Leslie Martin Storer – Andrews – All-TIAA
Misela Luevano Torres – Sanderson – All-TIAA
Rachel Holloway Roberts – Decator
Ida Estrada – El Paso
Juana Garcia – El Paso
Tracie Fore – Waco
Head Coach: Dr. Chet Sample
Asst. Coach: Elizabeth Busic Hanks
Randy and Sherry Jackson
Randy and Sherry Jackson have been long-time supporters of Sul Ross State University Athletics.
The Jacksons have attended 79 of the last 80 football games, as well as many out-of-town basketball games and track meets, since moving to Alpine. While attending these events, Randy has also assisted in the sports information department as a stand-in photographer and organized several tailgates for former student-athletes and alumni.
In conjunction with former Sul Ross football players, the Jacksons raised funds to purchase game jerseys for the team in 2009. They have further fundraised for various sports programs' uniforms and equipment at Sul Ross, and spearheaded efforts to send football seniors to various post season all-star games. Randy and Sherry have also purchased several Sul Ross banners for athletic events to replace worn out signs.
This past year, the Jacksons led fundraising efforts to purchase rings for the 2017-18 ASC Champion men's basketball team.
The Jacksons' newsletter From the Hill, begun in 2007, is distributed to Baby Boomers and other interested individuals and covers current events at Sul Ross.


It depends on how you measure them, and you should use plasticizers, rather than metal ones, which will give you inaccurate results.
I am intending to get a pair of 1954 size 32! Anyone experienced the 1954 size 32?, the original measure of 32.5 inches right now, if I want a 33 inch waist (after soaking and wearing), can I choose this size pair? Will they shrink? or stretch?
Fit pic after 2 weeks, what do you think?
thank you, for how long? Hum, I think it will be 2 hours !
TFH 3002 is on my way, maybe one or two more days before I receive them. I chose size 34, because I have a 33 inch waist (measure Rivertandhide), I do not have a hot water wash machine, Do you have any advice for me? Time to put them in hot water? and with what temperature?


text:
Martin Lewis announces £5,000 vouchers for millions of UK households from September
Martin has shared updates on exactly what this scheme will allow
Rachel Pugh
Lorraine King
Jess Morcom
Money Saving Expert's Martin Lewis
Despite being on his summer holidays, Martin Lewis' brilliant advice has continued - with his latest exciting announcement for Brits.
The finance expert has revealed the exiting news about a new Government scheme, which could see millions of households being given £5,000 vouchers to improve their homes.
Announcing the scheme, Rishi Sunak said: "From September, homeowners and landlords will be able to apply for vouchers to make their homes more energy efficient and create local jobs."
In his latest email, Martin has shared updates on exactly what this scheme will allow and why some people could be set to miss out, reports the Mirror.
He said: "The Government has now released more details on its Green Home Grant, including eligibility and what it covers.
"Those who just planned on getting double glazing will miss out."
Under the scheme the government grant will aim to cover at least two thirds of the cost of improvement, up to £5,000 per household.
However some low income households will be able to get the entire cost covered, up to £10,000.
But to be able to claim any vouchers, property homeowners need to also be installing insulation or low carbon heating at the same time.
Those who already have those features in their home can get top ups, such as additional loft insulation.
However, the vouchers can not be used to replace what is already in the property so if you have already splashed out renovations you can't make a claim.
However, new improvements for draught proofing, double or triple glazing, or secondary glazing, energy efficient doors or heating controls and insulation are eligible.
Homeowners will also be able to get money off the cost of the primary and secondary improvements.
While those making these updates can only receive funding up to the valuation of the cost of the insulation or low carbon heating.


BEIRUT, LEBANON- Syria's opposition on Sunday announced its 21-member delegation, including 10 rebel representatives, for a new round of UN-sponsored peace talks in Geneva scheduled for February 20.
The delegation will be headed by Nasr al-Hariri, a member of the National Coalition, replacing Assad al-Zoabi, who led the opposition at several previous rounds of talks in Geneva last year.
The delegation's chief negotiator was named as Mohamed Sabra, a lawyer who was part of the opposition's technical team during negotiations in Geneva in 2014.
The delegation includes representatives from several rebel groups, including Faylaq al-Sham, an Islamist faction active around Damascus, and Liwa Sultan Murad, a battalion close to Turkey.
The umbrella High Negotiations Committee (HNC) opposition group said the delegation to the talks would for the first time include representatives from two additional opposition groupings, known informally as the Moscow group and the Cairo group.
But representatives from both groups denied they were included in the delegation.
In the past, the HNC has opposed including the two rival opposition groupings in its delegation, accusing members of the coalitions of being too flexible with regard to the Syrian government.
The Moscow grouping includes former minister Qadri Jamil, and is close to the Russian leadership, while the Cairo grouping includes former foreign ministry spokesman Jihad Makdissi.
Invitations to the talks in Geneva have yet to go out, having been delayed in part to allow the opposition to decide on the composition of their delegation.
UN envoy Staffan de Mistura warned last week that he would pick the opposition delegates to the talks if they could not decide on time.
But he later appeared to backtrack and said he would delay dispatching invites.
Ahead of the talks, Kazakhstan has invited Syrian rebels and government officials back to Astana on February 15-16, but neither party has said officially yet if they will attend.
De Mistura will not attend the Astana meeting, but will send a "technical team," his spokeswoman said.


Happy Hounds - Rosie, Harvey & Pepper.
A couple of weeks back I was asked to to a 'dog shoot' by a friend who wanted to give the images to her other half as a surprise birthday present. Luckily for us the chap in question travels a fair amount so we planned a day when he would be away then smuggled the dogs to Grand Havre........... I would be lying if I said they were easy to capture!!!
Rosie, Harvey & Pepper were great fun, they had their own ideas of what they were going to do whether anybody liked it or not. For me that is the beauty of dog shoots, they always look their best when they are doing their own thing however you have to take one heck of a lot of shots to ensure some good ones.
The weather on the day was lovely but the light was seriously harsh, not to worry as at least it was dry. That said the dogs weren't dry given they spent half their time in the sea. All of the photos were taken with my Canon 5D MkIII with a 70-300L f4-5.6 lens.
All of the images on this site are the property of James Machon and are protected under Guernsey, U.K and International Copyright Laws. None of the images may be used (for copying, downloading, saving as digital files, printing or any form of manipulation) without prior consent of James Machon.