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Ubiquitin (Ub) and ubiquitin-like (UBL) proteins regulate a diverse array of cellular pathways through covalent as well as non-covalent interactions with target proteins. Yeast protein Mdy2 (Get5) and its human homolog GdX (Ubl4a) belong to the class of UBL proteins which do not form conjugates with other proteins. Mdy2 is required for cell survival under heat stress and for efficient mating. As part of a complex with Sgt2 and Get4 it has been implicated in the biogenesis of tail-anchored proteins. Interestingly, in response to heat stress, Mdy2 protein that is predominantly localized in the nucleus co-localized with poly(A)-binding protein Pab1 to cytoplasmic stress granules suggesting that nucleocytoplasmic shuttling is of functional importance. Here we investigate the nuclear import of Mdy2, a process that is independent of the Get4/Sgt2 complex but required for stress response. Nuclear import is mediated by an N-terminal nuclear localization signal (NLS) and this process is essential for the heat stress response. In contrast, cells expressing Mdy2 lacking a nuclear export signal (NES) behave like wild type. Importantly, both Mdy2 and Mdy2-ΔNES, but not Mdy2-ΔNLS, physically interact with Pab1 and this interaction correlates with the accumulation in cytoplasmic stress granules. Thus, the nuclear history of the UBL Mdy2 appears to be essential for its function in cytoplasmic stress granules during the rapid cellular response to heat stress.
Citation: Arhzaouy K, Ramezani-Rad M (2012) Nuclear Import of UBL-Domain Protein Mdy2 Is Required for Heat-Induced Stress Response in Saccharomyces cerevisiae. PLoS ONE 7(12): e52956. https://doi.org/10.1371/journal.pone.0052956
Editor: Mick F. Tuite, University of Kent, United Kingdom
Received: May 12, 2012; Accepted: November 22, 2012; Published: December 28, 2012
Copyright: © 2012 Arhzaouy, Ramezani-Rad. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Ubiquitin-like (UBL) proteins with their characteristic UBL domain are involved in a wide range of cellular processes, such as targeting and formation of nuclear compartments, spindle pole body duplication, and apoptosis. Ubiquitin-like proteins are divided into two subclasses [1]. Type-1 ubiquitin-like polypeptides (UBLs) essentially consist only of the UBL domain and function as modifiers like ubiquitin, being ligated to target proteins in a process similar to ubiquitylation. Important examples are SUMO, NEDD8, and UCRP/ISG15. In type-2 proteins the UBL domain is accompanied by other domains suggesting different functions. This is supported by the observation that type-2 proteins do not form covalent conjugates with target proteins [1], [2].
Although ubiquitin-like type-2 proteins are studied intensively little is known about their specific cellular function. MDY2 from Saccharomyces cerevisiae encodes a protein of 212 amino acids containing a conserved UBL domain (residues 74–149). Unlike most yeast proteins that contain such a domain, Mdy2 does not interact with polyubiquinated proteins nor does it bind the 26S proteasome [3]. MDY2 was identified as a gene necessary for cell survival under heat stress and for efficient mating [2], [4], [5]. The closest homolog of Mdy2 is the human ubiquitin-like protein GdX (Ubl4a) which consists of 157 amino acids [6]. The shared regions of homology show a 34% identity and encompass residues 74–212 of Mdy2 and the N-terminal 123 residues of GdX/Ubl4a. The N-terminal domain of Mdy2 is only conserved in fungi. Deletion of MDY2 increased heat sensitivity of mutant cells. Strains contain shorter microtubules (MTs) and accumulate defects in nuclear migration during mitosis [4]. In addition, deletion of MDY2 is associated with a five- to seven-fold reduction in mating efficiency, mainly due to defects in nuclear migration and karyogamy at the prezygotic stage [2]. Mdy2 (also known as Tma24 or Get5) was recently identified in a protein complex (composed of Sgt2, Get4 and Mdy2) implicated in tail-anchored membrane protein (TAP) insertion into the endoplasmic reticulum (ER) [7], [8], [9], [10], [11] also suggesting its important roles in the cytoplasm.
Nuclear-cytoplasmic shuttling of proteins is essential in coordinating nuclear events such as transcription and ribosome assembly with cellular processes such as translation and metabolism. It is well established that nuclear localization and export signals (NLSs and NESs) direct proteins in and out of the nucleus, respectively (reviewed in [12], [13], [14], [15], [16]). NLSs are short peptide motifs that mediate the nuclear import of proteins by binding to their receptors, known as importins (karyopherins) [12], [13], [14], [15], [16]. The best characterized transport signal is the classical NLS (cNLS) for nuclear protein import which consists of either one (monopartite) or two (bipartite) stretches of basic amino acids [17], [18], [19]. In contrast, cargos traveling toward the cytoplasm usually display a leucine-rich NES containing critical hydrophobic residues necessary for recognition by the nuclear export receptor Crm1 [20]. Nucleocytoplasmic transport proceeds through nuclear pore complexes (NPCs) that penetrate the nuclear envelope and allow passive diffusion of particles below the exclusion limit of 20–40 kDa, while larger components need to interact with a meshwork of nucleoporin repeats during translocation [21].
According to our previous studies Mdy2 localizes predominantly in the nucleus. However, under heat stress the UBL protein accumulates in cytoplasmic foci containing the poly(A)-binding protein Pab1, a marker for stress granules (SG) [4]. During glucose deprivation a minority of Mdy2 foci overlapped with processing bodies (PB) marker Dcp2, while most Mdy2 and Pab1 overlap in stress granules [4].
Here, we analyze the nucleocytoplasmic shuttling activity of Mdy2 in closer detail. We show that nuclear localization of Mdy2 is independent of Get4 and Sgt2. Furthermore, the fungal-specific N-terminus dependent nuclear import of Mdy2 is essential for the function of Mdy2 during heat stress and also, unexpectedly, for recruitment to cytoplasmic foci.
Results and Discussion
Analysis of Heat-induced Stress Tolerance in mdy2, get4, and sgt2 Deletion Mutants
As mentioned above, Mdy2 function during the assembly of TAPs in ER-membrane as part of the Get4/Sgt2 complex has been studied extensively [22]. To analyze the heat-induced stress tolerance we constructed single and double deletions of GET4 and SGT2 with MDY2 and tested them under heat stress. Exponentially growing cells were serially diluted and dropped on selective plates and incubated at 28°C and 38°C. Consistent with previous results mdy2Δ strains exhibit reduced stress tolerance at elevated temperature (Figure 1A). In contrast GET4 or SGT2 deletion showed only weak heat-sensitivity in comparison to wild type strains. Double deletion strains of mdy2Δget4Δ or mdy2Δsgt2Δ exhibit no synergistic sensitivity (Figure 1A). We also addressed the ability of mdy2Δ, get4Δ and sgt2Δ strains to grow in liquid medium at 38°C (Fig. 1B). In agreement with the reported phenotype on plate assays, growth in liquid medium at 38°C supports the notion that MDY2 deletion mutants increase temperature sensitivity of the yeast cells. GET4 and SGT2 deletion mutants showed only weak heat-sensitivity in comparison to wild type strains.
(A) Temperature sensitivity of mdy2Δ cells. Equivalent to 0.4 OD600 units of exponentially growing yeast cells diluted serially and spotted on YEPD agar plates. The plates were incubated at 28°C and 38°C for 2 days and pictures were taken. A representative experiment is shown. (B) Temperature sensitivity of mdy2Δ cells grown in liquid medium. Experiments were repeated three times with a similar outcome. The error bars represent standard error of the mean. (C) Mdy2 localizes mainly to the nucleus. Mdy2 in mdy2Δ, get4Δ, and sgt2Δ cells was localized by direct fluorescence of exponentially growing yeast cells using green fluorescence protein (GFP) (green channel). The mutant mdy2Δ was transformed with plasmid MDY2p-GFP-MDY2 (pZH152) or control vector. Nuclear DNA was stained by DAPI to indicate positions of nuclei (blue channel). Green fluorescence images of GFP-Mdy2 were recorded by a Zeiss Axioscope fluorescence microscope. Cells are shown by phase-contrast (PC) images and nuclear DNA by DAPA staining. Strains: WT (W303-1A), mdy2Δ (HZH686), get4Δ (HKA200), sgt2Δ (HLS2002), mdy2Δ get4Δ (HKA227), and mdy2Δ sgt2Δ (HLS2024).
Next, we analyzed nuclear localization of Mdy2 in the absence of Get4 or Sgt2. Consistent with previous results, GFP-Mdy2 predominantly accumulates in the nucleus. The same is true for get4Δ as well as sgt2Δ strains (Figure 1C), suggesting that a functional Mdy2/Get4/Sgt2 complex is not essential for nuclear localization of Mdy2 (Figure 1C). In summary, genetic and cell biological data support the assumption that Mdy2 regulation of heat-induced stress tolerance is independent of Get4 and Sgt2 and thus, most likely, a novel function of the Mdy2.
The Fungal-specific N-terminus of Mdy2 is Functionally Important during Heat Stress
Mdy2 is 212 amino acids long. Structural analyses suggest a tripartite domain structure which is common in all fungi (reviewed in [23]). The N-terminal domain of Mdy2 is 73 amino acids long and shows interaction with Get4 [24], followed by the highly conserved UBL domain from amino acid 74 to 149 and the C-terminal domain from amino acid 150 to 212. The latter has been implicated in Mdy2 homodimerization [24], [25]. Sequence similarity between Mdy2 and the mammalian homolog GdX (Ubl4a, 154 amino acids in length) encompass residues 74–212 of Mdy2 and the N-terminal 123 residues of GdX. To map functional domains of Mdy2, various truncated versions were constructed by PCR and in vivo recombination (see Material and Methods). After verification corresponding strains were examined in response to heat stress (as above; Figure 2A). Strains expressing Mdy2 and Mdy2ΔC (residues 1–149) showed similar growth behavior suggesting that the C-terminal region is dispensable for heat-induced stress response. However, strains expressing Mdy2ΔN (residues 74–212) or Mdy2UBL (residues 74–149) revealed similar heat sensitivity and growth defects at elevated temperature as mdy2Δ mutants. Western blot analysis of Mdy2 variants revealed no significant differences in protein expression levels compared to the control Mdy2 (Figure 2B).
(A) Mdy2 and different C-, N-, and NC-terminal deletion fragments of Mdy2 open reading frame (see schematics) fused to the C-terminus of GFP protein expressed under the control of GAL1 promoter in mdy2Δ (HZH686) cells. Temperature sensitivity recorded as indicated in Figure 1A. Representative experiments are shown. (B) Protein expression level of GFP-Mdy2 variants shows no difference in mutant cells. The left panel shows Western blot of total protein extracts from GFP-Mdy2, GFP-Mdy2- 1–149, GFP-Mdy2- 74–212, and GFP-Mdy2- 74–149 expressing yeast cells. GFP-Mdy2 was detected using anti-GFP antibody. Protein expression of actin as internal standard was performed using anti-actin antibody, clone C4/MAB1501 (left panel). Quantitative densitometry of protein expression showed no changes in the protein levels of GFP-Mdy2 variants. GFP-Mdy2 was set to 1 (right panel). (C) Visualization of exponentially growing indicated yeast cells was performed using fluorescence microscopy as in Figure 1B. (D) Quantitative and statistical analysis of the subcellular localization of GFP-Mdy2 variants. About 100 cells from three independent experiments were counted. The graphs show the percentage of cells demonstrating nuclear or cytosolic GFP-Mdy2 variant protein distribution.
Analyzing the subcellular localization of the Mdy2 variants revealed that the N-terminal 74 amino acid residues are essential for nuclear localization (Figure 2C and D). Full-length and C-terminal truncated Mdy2 showed a predominantly nuclear staining (Figure 2C, two upper panels and Figure 2D), whereas N-terminal truncated and NC-truncated UBL-domain (residues 74–149) showed a cytoplasmic distribution (Figure 2C, two lower panels and Figure 2D). To summarize, the fungal-specific N-terminal region of Mdy2 is responsible for its nuclear import, and nuclear localization correlates with functional importance during heat stress. The C-terminal end that is involved in Mdy2 homodimerization [24], [25] is, on the other hand, dispensable for nuclear targeting and heat-stress response.
The N-terminal Region of Mdy2 Contains a Functional NLS
To study nucleocytoplasmic shuttling we searched for a NLS and NES sequences (see below). We identified three potential candidates for classical NLS, which are rich in lysine and arginine (amino acid residues 26–36 KLPKSYTKPLK, 49–61 KYKYKQNRAKKLK and 79–80 KK). Mutant versions of Mdy2 carrying deletions in these regions were generated by assembly PCR and in vivo recombination (see Materials and Methods). The correct sequence and expression were verified (Figure S1A and data not shown). We then examined the growth behavior of cells carrying these deletions, Mdy2-Δ26–36, Mdy2-Δ49–61, and Mdy2-Δ79–80, in response to heat stress as described (Figure S1B). Full-length Mdy2 and Mdy2-Δ79–80 showed similar growth behavior at elevated temperature suggesting that the deletion of the two lysines is not functionally important. In contrast, Mdy2-Δ26–36 and Mdy2-Δ49–61 exhibit similar heat sensitivity as mdy2Δ mutants suggesting that these regions are essential for heat-induced stress tolerance. Strains harboring GFP-Mdy2-Δ26–36 and GFP-Mdy2-Δ79–80 showed a predominantly nuclear staining (Figure 3A, panels 2 and 4 and 3B). However, strains harboring GFP-Mdy2-Δ49–61 showed only cytoplasmic distribution (Figure 3A, panel 3 and 3B). Thus, a functional NLS of Mdy2 is located between amino acid 49 to 61.
(A) Green fluorescence images and nuclear DNA of exponentially growing mdy2Δ cells carrying wild type Mdy2 (MDY2) and different putative NLS deletion constructs of Mdy2 (mdy2-Δ26–36, mdy2-Δ49–61, and mdy2-Δ79–80, respectively) were recorded as in Figure 1C. The NLS sequence of Mdy2 is localized between the amino acids 49 and 61 (panel 3). A putative NES deletion construct of Mdy2 (see schematic) was fused to the C-terminus of GFP protein, expressed under the control of GAL1 promoter in mdy2Δ (HZH686) cells, and analyzed as in Figure 1B. The NES sequence of Mdy2 is localized between amino acids 118 and 122 (panel 5). (B) Quantitative and statistical analysis of the subcellular localization of Mdy2 mutants with defective nuclear localization, Mdy2-ΔNLS, and nuclear export, Mdy2-ΔNES as in Figure 2D.
GFP-Mdy2-Δ79–80 showed an enhanced nuclear staining suggesting that these two K residues in the UBL domain are involved in the nuclear export of Mdy2 (see below). In an additional step, the 13 amino acid NLS sequence of the protein (49-KYKYKQNRAKKLK-61) was subjected to partial deletion to test their influence on localization of the fusion protein. Two additional GFP-Mdy2-constructs with deletions of amino acid residues 49–53 (KYKYK) and 56–61 (RAKKLK) were generated, and strains expressing GFP-Mdy2-Δ49–53 and GFP-Mdy2-Δ56–61 were examined for localization of Mdy2. We observed that both segments are necessary for the NLS activity and nuclear import of Mdy2 (Figure S2). These results indicate two important regions in the N-terminus that are essential for yeast heat-stress induced tolerance. One of them has the characteristic features of a functional NLS.
Mdy2 Carries a Functional Nuclear Export Signal (NES)
The localization of Mdy2 might be influenced by the presence of a nuclear export signal (NES). We used NetNES 1.1 prediction tool, which predicts leucine-rich nuclear export signals (NES) in eukaryotic proteins [26]. The analysis yielded a single sequence, between amino acids 118–122, which could be considered as a nuclear export sequence (NES). We generated mutant versions of Mdy2 carrying deletions in this region (GFP-MDY2-ΔNES) by assembly PCR and in vivo recombination and verified its sequence and expression (Figure S1A and data not shown). GFP-MDY2-Δ118–122 localized predominantly in the nucleus and evidently at nuclear foci (Figure 3A, panel 5, 3B and see below) indicating that the amino acid residues from 118 to 122 are important for nuclear export.
In summary, we have delineated features that are required for nuclear import and export of Mdy2: a classic bipartite nuclear localization signal (cNLS) is present in the N-terminal region of Mdy2 between amino acid residues 49 to 61 and a nuclear export signal (NES) is located proximal to the NLS in the UBL domain of Mdy2 between amino acid residues 118 to 122.
The NES Motif is Important for Interaction of Mdy2 with Sgt2 but not Get4
To test whether deletion of NLS and/or NES affects the association of Mdy2 with the previously identified partner proteins Get4 and Sgt2, co-precipitation experiments were conducted. In accord with previous results [27], [28], GST-tagged Get4 or Sgt2 can bind Mdy2 (Figure 4A and 4B, lane 6). Similarly, GST-tagged Get4 or Sgt2 can interact with Mdy2-ΔNLS (Figure 4A and 4B, lane 7) indicating that these 13 amino acids from the N-terminal region of Mdy2 have no effect on the interaction of Mdy2 with Get4. However, deletion of the 4 amino acid long NES motif within the UBL domain (MDY2-ΔNES) abolishes binding of Mdy2 to Sgt2 (Figure 4B, lane 8). From this assay and in accord with previous results [28], we concluded that the NES in the UBL domain of Mdy2 is the predominant interaction surface of Mdy2 for Sgt2 which might function in masking this export signal.
(A) HZH686 (W303-1A mdy2Δ) cells were transformed with CEN expression vectors encoding Myc, Myc-tagged Mdy2 (Myc-Mdy2), Myc-Mdy2-NLS, Myc-Mdy2-NES, GST, and GST-tagged Get4 (GST-Get4) under the control of GAL1 promoter. Cells were grown in SRG to log phase (see Material and Methods), whole cell extracts were prepared and GST-Get4 was precipitated using Glutathione -Sepharose 4B. The pulldown was then tested for the presence of Mdy2 association by probing a Western blot with anti-Myc Ab (top panel). To monitor pulldown recovery, the level of GST-Mdy2 in the binding assay was measured by probing the same membrane with anti-GST antibody (middle panel). Expression levels of Myc-Mdy2 in the whole cell extracts used for binding assay were measured on a Western blot (bottom panel). (B) HZH686 (W303-1A mdy2Δ) cells were transformed with expression vectors encoding Myc-tagged Mdy2 variants as in (A) and GST-tagged Sgt2 (GST-Sgt2) under the control of GAL1 promoter. Cells were grown in SRG to log phase, whole cell extracts were prepared, and GST-Sgt2 was precipitated using Glutathione -Sepharose 4B. The presence of Mdy2 in the pulldown was confirmed by probing a Western blot with anti-Myc antibody (top panel). To monitor binding recovery the level of GST-Sgt2 in the pulldown was measured by probing the same membrane with anti-GST Ab (middle panel). Expression levels of Myc-Mdy2 in the whole cell extracts used for pulldown were measured on Western blots (bottom panel).
Nuclear Import of Mdy2 is Necessary for Heat Stress-induced Response and Recruitment to Cytoplasmic Stress Granules
To explore the growth of Mdy2-ΔNLS and Mdy2-ΔNES mutants in more detail we studied the growth behavior of cells by expressing mdy2-ΔNLS (see Figure 3) and mdy2-ΔNES controlled by the natural MDY2 promoter sequence (Figure 5). Western blot analysis of Mdy2 variants revealed no significant differences in protein expression levels compared to the control Mdy2 (Figure 5A). mdy2 mutants with defective nuclear localization (mdy2-ΔNLS) showed a growth defect at elevated temperatures (Figure 5B, third row). Diversely, mdy2-ΔNES mutants behaved more or less like wild type (Figure 5B, lower row). We then investigated the ability of mdy2-ΔNLS and mdy2-ΔNES strains to grow in liquid medium at 38°C (Fig. 5C). In agreement with the reported phenotype on plate assays, growth in liquid medium at 38°C supports the notion that mdy2-ΔNLS showed a growth defect at elevated temperatures. Taken together these results indicate that the NLS but not the NES is essential for the function of Mdy2 in heat-induced stress tolerance.
(A) Protein expression level of GFP-Mdy2 and different putative NLS and NES constructs of Mdy2 is equal. Western blotting analysis of NLS and NES deletion constructs (mdy2-ΔNLS and mdy2-ΔNES, respectively) in CEN plasmids expressed under the control of MDY2 promoter in mdy2Δ (HZH686) cells (left panel). Quantitative densitometry of protein expression showed no changes in GFP-Mdy2 variants. GFP-Mdy2 was set to 1 (right panel). (B) Temperature sensitivity of mdy2Δ cells carrying wild type Mdy2 (MDY2), empty vector (mdy2Δ), NLS and NES deletion constructs (mdy2-ΔNLS and mdy2-ΔNES, respectively) in CEN plasmids expressed under the control of MDY2 promoter recorded as indicated in Figure 1A. Mdy2 mutants with a defect in nuclear localization (mdy2-ΔNLS) revealed an enhanced growth defect at elevated temperature (third row). Representative experiments are shown. (C) Temperature sensitivity of mdy2 mutant cells grown in liquid medium.
GFP-Mdy2 proteins which are localized predominantly in the nucleus at permissive temperature are localized to cytoplasmic foci during heat shock and glucose deprivation [4]. Co-localization studies revealed that mild heat stress-induced enrichment of Mdy2 in cytoplasmic foci merged mainly with stress granules marker Pab1 ([4] and Figure 6A, middle panel). Accordingly, shifting wild-type cells to either 37°C or 39°C led to a modest and reproducible increase in the number and size of stress granules and P-bodies [29]. During robust heat stress, and in accord with previous studies [30], Mdy2 foci overlapped with P-bodies marker Dcp2 and with SG marker Pab1 (Figure 6A, right panel).
(A) Mdy2 localizes mainly to the nucleus at 28°C. Mdy2 localize to cytoplasmic granules following heat stress. Mdy2 co-localize with Pab1 following mild and robust heat stress. GFP-Mdy2 and Pab1-RFP was visualized by fluorescence microscopy in a mdy2Δ strain transformed with plasmids containing GFP-Mdy2 and Pab1-RFP (upper panel) or GFP-Mdy2 and Dcp2-RFP (lower panel) after a temperature shift to 38°C (middle panel) and 46°C (right panel). (B) Localization of Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins at permissive temperature. Exponentially growing mdy2Δ cells carrying wild type Mdy2 (MDY2), empty vector (mdy), NLS and NES deletion constructs (mdy2-ΔNLS and mdy2-ΔNES, respectively) fused to the C-terminus of GFP protein in CEN plasmids and expressed under the control of MDY2-own promoter. GFP-fusion proteins were localized by direct fluorescence and nuclear DNA was stained by DAPI. (C) Localization of Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins during heat shock-induced stress. Localization pattern for Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins as indicated in (A) during heat shock-induced stress. Localization of GFP-Mdy2 and GFP-Mdy2-NES to the cytoplasmic granules following heat stress is shown in upper and lower panel.
We then addressed the important aspect of subcellular localization of Mdy2 versions under heat stress conditions. As shown before, GFP-Mdy2-ΔNLS protein is distributed in the cytoplasm, whereas the GFP-Mdy2-ΔNES accumulated predominantly in the nucleus at permissive temperatures (Figures 3, 6B and 7A). During mild heat stress GFP-Mdy2 proteins are targeted from a predominantly nuclear position to cytoplasmic foci (Figure 6A, middle panel and Figure 6C, upper panel; [4]). Analyzing Mdy2-ΔNES revealed that this mutant version, like wild-type, also accumulates in cytoplasmic foci and hardly in the nucleus (Figure 6C, lower panel). We then examined subcellular distribution of Mdy2, Mdy2-ΔNLS and Mdy2-ΔNES in stress conditions such as robust heat stress (46°C) [30] and treatment with 0.5% (v/v) sodium azide, which also induces stress granules-like foci [29]. We could verify co-localization of GFP-Mdy2 and GFP-Mdy2-ΔNES with Pab1 under robust heat stress and NaN3 treatment conditions (Figure 7A) Thus, apparently during heat stress an NES sequence-independent signal results in nuclear export and cytoplasmic accumulation.
(A) Mdy2 co-localize with Pab1 following heat stress and treatment with sodium azide. GFP-Mdy2 and Pab1-RFP was visualized by fluorescence microscopy in a mdy2Δ strain transformed with plasmids containing GFP-Mdy2 (upper row), GFP-Mdy2-ΔNLS (middle row) or GFP-Mdy2-ΔNES (lower panel), and Pab1-RFP, after a temperature shift to 46°C (left panel) and after treatment with sodium azide (NaN3) (right panel). In the overlay pictures (merge), overlap of the colors appears yellow. GFP-Mdy2 and GFP-Mdy2-ΔNES but not GFP-Mdy2-ΔNLS are predominantly nuclear in control (Con) conditions at 28°C (right panel). (B) Mdy2 interacts with Pab1. Cell lysates from the GST-tagged Mdy2 strains were precipitated (P) with Glutathione Sepharose 4B. Following washing, the resin was eluted with glutathione. Eluted proteins were resolved by SDS-PAGE and visualized by immunobloting (control, IB) and Coomassie blue staining (Coomassie). Protein identities were established by mass spectrometry analysis. (C) Extracts from yeast strains HZH686 (W303-1A mdy2Δ) coexpressing GST alone (GST) or GST-tagged Pab1 (GST-Pab1) with Myc alone (Myc), Myc-tagged Mdy2 (Myc-Mdy2), Myc-tagged Mdy2-ΔNLS (Myc-Mdy2-ΔNLS) or Myc-tagged Mdy2-ΔNES (Myc-Mdy2-ΔNES) were subjected to pulldown using Glutathione Sepharose 4B as in Figure 4. The coprecipitation of indicated Myc-tagged Mdy2 proteins in the pulldown was confirmed by probing a Western blot with anti-Myc Ab (top panel, Co-P: Myc). To monitor pulldown recovery, the level of GST-Pab1 in the pulldown was measured by probing the same membrane with anti-GST Ab (second panel from the top, P: GST). Expression levels of indicated Myc-tagged Mdy2 proteins and GST-Pab1 in whole cell extracts (Extract) used for pulldown were measured on Western blots (third and fourth panels from top, IB:Myc and IB:GST, respectively).
Interestingly, analysis of Mdy2-ΔNLS revealed that this version, although present in the cytoplasm, is unable to accumulate in cytoplasmic foci (Figure 6C, and 7A). This suggests that the nuclear history of Mdy2 is important for function during heat stress and for recruitment to cytoplasmic Pab1-positive stress granules (see below).
Nuclear Imported Mdy2 Physically Interacts with Pab1
During affinity purification of GST-Mdy2 we found distinct and reproducible protein bands around 70 kD (Figure 7B). Analyses of isolated complex by mass spectrometry resulted in the identification of three Hsp70 proteins, Ssa1, Ssa2, Ssb1, and poly(A)-binding protein Pab1 as the highest scoring proteins. The same analysis allowed the identification of four other proteins but with much lower scores: pre-mRNA polyadenylation factor Fip1, checkpoint serine/threonine-protein kinase Bub1, transketolase Tkl1, and ATP-dependent RNA helicase Dbp3. Previous studies showed that Mdy2 exits in complex with Hsp70s [11]. But the most interesting finding in this experiment is that three of these 8 identified proteins, Pab1, Fip1, and Dbp3 are components of the mRNA processing complex. Of these, Pab1 was the most interesting candidate because it showed a high score, like, Hsp70s in complex with Mdy2 and is known to co-localize with Mdy2 [4]. Here we checked the interaction between Mdy2 and Pab1 in reciprocal co-precipitation experiments and we extended this approach to monitor Pab1 co-precipitation with Mdy2-ΔNLS and Myc-Mdy2-ΔNES (Figure 7C). We confirmed the interaction between Mdy2 and Pab1 as we detected Myc-tagged Mdy2 from yeast cells expressing a GST-Pab1 fusion (Figure 7C, lane 6). We found that Myc-tagged Mdy2 and, to a lower extent, Myc-Mdy2-ΔNES were co-precipitated with GST-Pab1 (Figure 7C, lane 6 and 8). However, Myc-Mdy2-ΔNLS abolishes binding of Mdy2 to Pab1 (Figure 7C, lane 7) indicating that Myc-Mdy2-ΔNLS cannot bind to GST-Pab1. Hence it is most likely that Mdy2 already interacts with Pab1-containing messenger ribonucleoprotein (mRNP) complex in the nucleus. This interaction appears to be important for the recruitment to stress granules and this, again, correlates with the heat stress function of Mdy2.
Conclusions and Perspectives
Altogether, nucleocytoplasmic shuttling correlates with the heat stress function of Mdy2 and the accumulation of Mdy2 in stress granules. Cytoplasmic stress granules in S. cerevisiae contain proteins like Pab1 [31], [32]. Importantly, Mdy2 not only interacts with Pab1 but also co-localizes with Pab1 following heat stress [4]. Pab1 is the major poly(A)-binding protein in yeast. It is a multifunctional protein that mediates many cellular functions associated with the 3′-poly(A)-tail of messenger RNAs. Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export [33]. Pab1 therefore appears to actively enter the nucleus and exit via two transports pathways: one pathway is dependent on XPO1/CRM1 through an NES located at the amino terminus of Pab1, whereas the second pathway requires MEX67 and/or ongoing mRNA export [33]. In analogy with Pab1 nuclear export Mdy2 carries a functional NES, but during heat stress an NES sequence-independent signal results in nuclear export and accumulation in cytoplasmic foci. Taking into account our results and a list of Mdy2 interactors like heat shock proteins and Pab1 (this paper and [4], [11], [34], [35]) we come to the conclusion that Mdy2 might play a role in mRNA metabolism. Interestingly, also the human ubiquitin-like protein GdX (Ubl4a) interacts with similar proteins [36], [37] suggesting an evolutionarily conserved process.
In response to stress, eukaryotic cells reprogram the mRNA metabolism to repair stress-induced damage and adapt to changed conditions. During this process the translation of mRNAs encoding "housekeeping" proteins is aborted, whereas the translation of mRNAs encoding molecular chaperones and enzymes involved in damage repair is enhanced. Selective recruitment of specific mRNA transcripts into stress granules is thought to regulate their stability and translation [38]. Heat stress granules selectively exclude mRNAs encoding stress-induced heat shock proteins [39]. In S. cerevisiae heat shock induces the formation of stress granules that contain translation initiation factors and non-heat shock mRNAs capable of redistributing into the cytoplasm, and presumably reengaging in translation, upon recovery [30], [40]. The robust heat shock-induced stress granules also contain a subset of processing body components involved in RNA degradation including Dcp2 and Dhh1, yet are spatially distinct from other processing body markers [30]. The fact that heat stress triggers stress granule assembly but not processing body formation indicates that these processes are regulated by distinct signaling pathways [29], [30], [41]. It has been proposed that stress granules are sites where the increased local concentration of proteins and mRNA allows for remodeling and redistribution of mRNPs [40]. Alternatively, specific proteins might be selectively sequestered into or away from stress granules, thus affecting biochemical processes in the cell. For example, recruitment of specific proteins such as RACK1, which is required to activate the apoptosis-inducing MTK1 kinase during stress, to stress granules can inhibit apoptosis [42].
Our results are consistent with the hypothesis that stress granule formation is one of the immediate protective mechanisms against heat stress. We speculate that Mdy2-containing stress granules fulfill a protective role for cell survival upon stress. Under these conditions, Mdy2 might accompany the Pab1-containing mRNP complex to stress granules, and this appears to be functionally important for stress protection. The mechanism and function of Mdy2 in the process remains unclear, but it appears to be essential for a rapid cellular response to stress in processes like stress granules dynamics in the control of non-translating mRNPs. This constitutes a novel link between UBL proteins and posttranscriptional processes.
Materials and Methods
Yeast Strains, Media, and Standard Methods
Recombinant DNA techniques were applied according to standard protocols. Depending on the experimental context, yeast cells were grown either in YEPD medium (2% glucose, 2% peptone, 1% yeast extract), synthetic complete medium (0.67% yeast nitrogen base without amino acids) with 2% glucose (SD) containing the required nutrient supplements, or in synthetic raffinose galactose (SRG) medium (0.67% yeast nitrogen base without amino acids, 3% raffinose, 1% galactose) containing the appropriate nutrient supplements. All strains used in this study are in W303-1A (MATa leu2-3,112 ura3-1 trp1 his3-11 ade2 can1-100) background (source: R. Rothstein, Columbia University, New York). HZH686 (W303-1A MATa mdy2Δ::KanMX4) was previously described [2]; HKA200 (W303-1AMATa get4Δ::HIS3), HKA227 (W303-1A MATa mdy2Δ::KanMX4 get4Δ::HIS3), HLS2002 (W303-1A MATa sgt2Δ::HIS3), and HLS2024 (W303-1A MATamdy2Δ::KanMX4 sgt2Δ::HIS3) are constructed by homolog recombination. Expression vectors encoding full-length truncated or mutated Mdy2, as well as Get4 and Sgt2 were produced by PCR or assembly PCR, accordingly, and cloned by in vivo recombination into the galactose-inducible pGREGs expression vectors [43] or in vectors with the appropriate endogenous promoter and checked by restriction digestion, PCR, sequencing, and Western blot analysis. Plasmid MDY2p-GFP-MDY2 (pZH152) was previously described [2]. Plasmid pPAB1-RFP (PAB1-RFP LEU2, CEN) is a kind gift of Charles N. Cole (Dartmouth Medical School). Plasmids pRP1155 (pDCP2-RFP LEU2, CEN) is a kind gift of Roy Parker (University of Arizona).
Gene Deletion and PCR-mediated Gene Tagging
Deletion strains were constructed by homologous recombination with appropriate PCR-derived disruption cassettes. Each of these consisting of a central marker gene (KanMX6 or HIS3MX6) flanked by sequences homologous to regions adjacent to the gene to be disrupted. The disruption cassettes were introduced into cells by the high-efficiency LiAc transformation method. Correct disruption was verified by analytical PCR.
PCR-mediated gene tagging was performed using a similar method. The integration cassettes used consisted of a central tag (GST, Myc or GFP) with a selection marker (HIS3, TRP1 or KanMX6) and flanking sequences homologous to 3′-terminal sequences of the gene to be tagged. Correct tagging was verified by analytical PCR and sequence and expression analysis.
Immunoblot Analysis
For expression controls, log-phase yeast cells (equivalent to 4 OD600 units) were harvested by centrifugation (5 min, 3000 rpm), washed once in E-buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8, 0.1% Triton-X-100, 10% glycerol), then resuspended in lysis buffer (E-buffer +1 mM DTT, 0.1 mM Pi-Mix [pepstatin and aprotinin (at 1 mg/ml each), leupeptin and antipain (at 5 mg/ml each), 0.1 mg/ml trypsin inhibitor, and 1 mM PMSF]. Cells were broken down with (2/3 vol) glass beads and collected by centrifugation (2 min, 3000 rpm). The samples were denatured in SDS-PAGE sample buffer by boiling (10 min at 95°C), centrifuged briefly, applied to a 7.5% or 10% SDS-polyacrylamide gel and fractionated by electrophoresis. The proteins were then blotted onto a nitrocellulose membrane according to standard protocol, and probed with polyclonal rabbit anti-GST (1∶1000) and/or monoclonal mouse anti-Myc (1∶1000). Bound primary antibodies were detected enzymatically with goat anti-rabbit and goat anti-mouse antibodies coupled to alkaline phosphatase or horseradish peroxidase (1∶5000 and 1∶50.000, respectively).
Binding Assay
Cells to be used for binding assays were induced for 16 h in SRG medium, and extracts (equivalent to approximately 12 OD600 units) were prepared as described above. 20 µl Glutathione Sepharose 4B (GE Healthcare, Germany) were added to 1 ml of extract and the samples were incubated for 2 h at 4°C. Then the samples were centrifuged (3 min, 13,000 rpm) and the pelleted complexes were washed four times in E-buffer+(see above). The samples were then boiled, fractionated on a 7.5% SDS-polyacrylamide gel and subjected to immunoblotting analysis as described above.
Cells were grown to log phase in YPD or in appropriate selection medium. Samples (equivalent to 1.5 ml of cells) were taken and washed twice with 500 µl of PBS (pH 7.0) in 1.5 ml reaction tubes. The cells were fixed for 5 min in 70% ethanol and again washed twice in PBS (pH 7.0). To stain DNA, cells were incubated for 10 min in 20 µl of DAPI (1 µg/ml) and washed twice in PBS (pH 7.0). Cells containing GFP-labeled Mdy2 or Mdy2 mutants were used for localization of fusion proteins. Images were acquired by fluorescence microscopy using a Zeiss Axioscope and Zeiss filter systems.
Supporting Information
Figure S1.
Analysis of a nuclear localization signal (NLS) in the N-terminal domain and a nuclear export signal (NES) in the UBL domain of Mdy2. (A) Protein expression levels of GFP-Mdy2 and different putative NLS and NES constructs of Mdy2 show no difference in mutant cells. Putative NLS deletion constructs of Mdy2 and a putative NLS deletion construct of Mdy2 (see schematics in Figure 6) were fused to the C-terminus of GFP protein and expressed under the control of the GAL1 promoter in mdy2Δ (HZH686) cells. Western blot analysis (left panel) and quantitative densitometry of protein expression (right panel) showed no changes in protein level of GFP-Mdy2 variants. GFP-Mdy2 was set to 1. (B) Temperature sensitivity of mdy2Δ cells carrying wild type Mdy2 (MDY2), empty vector (mdy2Δ), and different putative NLS deletion constructs of Mdy2 (mdy2-Δ26–36, mdy2-Δ49–61, and mdy2-Δ79–81, respectively) was analyzed as in Figure 1A. Representative experiments are shown.
Figure S2.
Effect of the mutations on the nucleocytoplasmic distribution of Mdy2. mdy2-ΔNLS mutation constructs of mdy2-Δ49–53 and Δ56–61 show cytoplasmic distributions. mdy2-Δ49–53 and Δ56–61 deletion construct of Mdy2 was fused to the C-terminus of GFP protein, expressed under the control of the GAL1 promoter in mdy2Δ (HZH686) cells and was analyzed as in Figure 1B. Both Mdy2-ΔNLS mutant proteins show the cytolasmic distributions.
We thank B. Axler and E. Stratmann for technical and editorial assistance. We are grateful to M. Feldbrügge for critical reading of the manuscript and suggestions. We also thank C. N. Cole and R. Parker for providing plasmids.
Author Contributions
Conceived and designed the experiments: MRR. Performed the experiments: KA MRR. Analyzed the data: KA MRR. Contributed reagents/materials/analysis tools: MRR. Wrote the paper: MRR.
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text:
Divergent Fiber Optic Proximity Sensors Datasheets
Fiber Optic Sensors -- WORLD-BEAM QS18 Standard Series
from Banner Engineering Corp.
from Banner Engineering Corp.
10 to 30V dc supply voltage with NPN or PNP output, depending on model. Available in opposed, polarized and non-polarized retroreflective, convergent, regular and wide-angle diffuse, and glass or plastic fiber optic sensing modes. High-power infrared or visible red sensing beam. Bright LED operating... [See More]
- Optical Technology Type: Retroreflective; Polarized Retroreflective; Diffuse; Divergent; Convergent
- Operating Temperature: -4 to 158
- Operation Distance: 0.6299 to 787
- Mode: Light/Dark Programmable
Digital Fiber Sensor Amplifier -- FX-101(P)-CC2
from Panasonic
from Panasonic
The FX-100s ultra-slim design features a 9mm width and integrated mounting holes for installation in any application. The four chemical, visible red LED used for the emitter ensures long life operation and high-powered sensing. This sensor incorporates a dual digital display for simple setup, along... [See More]
- Optical Technology Type: Through Beam (optional feature); Retroreflective (optional feature); Polarized Retroreflective (optional feature); Diffuse (optional feature); Divergent (optional feature); Convergent (optional feature); Fixed Field Background Suppression (optional feature)
- Operating Temperature: 14 to 131
- Operation Distance: 0.0
- Fiber Optic: Glass; Plastic

subset: commoncrawl


text:
Just for comparison sake, I have an H&K P30L with luminescent sights. They are something else. How does the Glow-on paint compare to the H&Ks as far as brightness and duration?
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Just for comparison sake, I have an H&K P30L with luminescent sights. They are something else. How does the Glow-on paint compare to the H&Ks as far as brightness and duration?
Please let dry a day or two, it'll show full luminescence then.
Thank you so much! PM incoming!
I read recently a thread about how important is to have have a god tactic light at hand. We encourage Glow-On users to always have one at hand. For once you can't shot at something unless you can see it.
Secondly you can rekindle the glow on your sight with the small flashlight in an instant, (not minutes like other products).
We definitely recommend training with a flashlight and your sidearm as much as possible. Their use includes and by no means is limited to: Aligning down a shot, blinding an attacker momentarily and to direct attention away from our vital parts
Most gun ranges have training available so contact your local gun range for that.
We prefer to buy small waterproof flashlights like the one in the photo. They use three small batteries, they can stand some abuse and if you loose one, just pop another one and presto.
A good strategy is to buy a ten or eight pack (less than $20US) and put them everywhere in your home. A good idea IMO is to wrap a small piece of glow in the dark self adhesive to make them easy to find in the dark(sold in our web store, one small piece is good for at least 4 lamps) .
Thank you for your supporthttp://www.1911auto.org/forum/images/smilies/cool.gif
First off, happy weekend everyone.http://www.1911auto.org/forum/images...ing_banana.gif
Secondly, thanks for your support, we are always in the quest for the brightest fix for our gun sights and our friends'. The idea is a highly efficient mix (one could call it paint) easy to apply, durable, that will stick to anything solid and at an affordable price. Otherwise it wouldn't make sense, agreed? Good!
My advice is try it, we offer 100% money back guarantee. We are never too busy to answer your questions and our word is always a promise.
Please visit glow-on.com for more info, there are some links at the bottom of this post so come and check us out.
Home Page | Products | Advantages | Gun Sights Paint | Fishing Lures | Electronics | Stars Paint | Bead Work | Nail Art | Steel Bracelet | Tips & Support | Gallery | Video Tutorials | Purchase | Contact Us | Free Bracelet Winners | Site Map
There are plenty alternatives out there to make our firearm's gun sights more efficient. Lumen, reflective, optic fiber, laser, red dots, scopes, etc. are a few possibilities.
While some purists might argue that most of this options are just "crutches", there is no doubt that some specific situations require special equipment. Rapid target acquisition, rapid fire, sharpshooting come to mind.
The logic choice for the firearms owner depends on his needs and the challenges he/she is expected to confront. A situation that is obviously related to time and place.
Glow-On will help the gun enthusiast, when used properly, to acquire a target and to align a shot faster in some low light conditions. If you add to the equation the fact that the purchase this product will set you back about the price of a fast food meal, we have IMO a good alternative to the plain sights most firearms come equipped with.
Give it a try, 100 % money back guaranteed.
Got a new pistol lately? did it come equipped with standard white dots sights?
There's room for improvement: For the price of a fast food meal, Glow-On Super Phosphorescent.
It's easy to apply and to create custom sights for a few bucks.
Got some old fishing lures somewhere? Glow-On is good for that too.
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Simplicity at an affordable price.
There are plenty of sighting systems out there. I'm particularly fond of red dots and such, tritium, fiber optics, lasers, etc.
We at Glow-On, amongst all of us, we cover pretty much every base when it comes to gun sights types.
Still we are very fond of our product Glow-On Super Phosphorescent.
Why? Very simple; One more thing we have in common is we have lots of firearms for which some systems are not available. About 9 out of every firearm for sale in the USA comes equipped with standard sights and white dots or plain iron sights.
We are trying to introduce a new standard and some manufacturers are already making a splash however shy into the world of glow materials.
It's durable, the glow cycle can be repeated millions of times for a period of about ten years, it's affordable and it's incredibly easy to apply.
100% money back guarantee, anytime anywhere. To purchase go here:
You can also find us at Amazon and E Bay :congrats:
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http://www.1911auto.org/forum/images...ing_banana.gifGlow-On might help sort out unexpected situation in the middle of the night that plain white dots won't guarantee.http://www.1911auto.org/forum/images...ing_banana.gif
Find Glow-On at:
Home Page | Products | Advantages | Gun Sights Paint | Fishing Lures | Electronics | Stars Paint | Bead Work | Nail Art | Steel Bracelet | Tips & Support | Gallery | Video Tutorials | Purchase | Contact Us | Free Bracelet Winners | Site Map
It took awhile to get it right and is still a work in progress, Glow-On keeps getting better.
When you buy a vial of our product you don't just buy paint, along with it you can be sure you have unlimited support so you can get the most out of the product. Just ask around
Find Glow-On at:
Glow-On Self Adhesive is a very handy material which proves very useful to locate pesky TV remotes, small flashlights, light switches, etc.
All it's necessary , is to cut small squares and stripes and press-apply to small devices. Dark corners which prove dangerous to toes and elbows during the night can be avoided using the same material.http://www.1911auto.org/forum/images/smilies/wavey.gif
Find Glow-On at:
How well will it work on a front ramp sight like my ruger sp101? Will it adhere well to the sight?
If your model has a half moon shaped rear notch sight and a fixed front ramp, the product can be applied to both.
For superior adherence is essential to make sure the metallic surface is very clean, 95% alcohol and a paper towel is just what's needed to clean the gun sights.
Stir the paint with the small wooden stick provided, with the help of the small vial's rim load the tip of the applicator generously,
On the rear sight apply the product generously over the flat side of the half moon, on the front sight "spread the product with the help of the same wooden applicator over the serrated surface only, which is the area that looks like a small square thru the rear sight. don't apply it all the way up or over the top, holstering the firearm will wear off the paint.
Try not to use a brush for this job, we never use brushes, they just mess up the works, apply the product with he flat side of the applicator, just like you'd spread soft butter over bread, we always practice a couple of times over some surface.
If it doesn't look right, just wipe it off with the paper towel and start again.
In terms of durability, the product dries to the touch in abut 1-2 hours but it'll remain a bit soft for 2-3 days and as the time passes it becomes hard and permanent, before this happens it's easy to rub off.
The day after you applied it, use a couple of layers of good quality transparent nail varnish. This will give your job a smooth surface and it'll make it easy to clean after a visit to the range ( a dry paper towel or such is all you'll need).
At this point cleaning oils and such won't affect the application either.
Find Glow-On here:
How do I purchase with just a credit card & NO Google account?
After reading all this, I decided to order Option 4 and check it out. I like the fact folks on here reviewed it, some ups, some downs, but since they stand behind the product I am not worried. Looking forward to using it.
The main complaint is about the duration of the glow, and the real cause is that the initial glow is so incredibly bright,http://www.texasguntalk.com/forums/i.../happy0001.gif one would like it to last like that, super bright (ten times brighter that tritium indeed) for hours on end like an electric green light.
This is just not going to happen.http://www.texasguntalk.com/forums/i...eadscratch.gif
The facts are:
This bright green glow is just the initial glow, the brighter the initial glow, the longer the afterglow is.
The real afterglow is a soft white glow which lasts for hours, it can only be perceive in the dark and after our vision has adapted itself to the dark, which happens in about thirty minutes for a healthy eye sight.
If we don't want to go that route, we can charge in 2-5 seconds depending on the intensity of our tac light and our Glow-On sights will get us thru, very bright and we'll have our tac light so at least we can see what we are shooting at.
Thousands of users stand by our product and once you really get to know how it works you'll be a fan toohttp://www.texasguntalk.com/forums/i...ies/banger.gif
Did I mention we have a money back guarantee
Paint is what you make of it, it's a simple solution and simple solutions are usually better solutions.
There are of course rather sophisticated sighting systems and some firearms can be configured to use them, laser, red dots, etc, are just a few.
Still 99% of sidearms for sale in the USA and the rest of the world come equipped with standard white dots, which are the minimal requirement as a visual aid, or have military style bare iron sights.
Glow-On is just one alternative to customize those weapon sights:
-The most affordable:The price of a fast food meal will help at leas a couple of dozen guns.
-The most efficient: No batteries needed, reacts to natural light all the time and for a quick boost a small tact light is all one needs.
-Versatility: Real custom gun sights made to order you should say.
-Made in the USA.http://www.1911auto.org/forum/images...s/amerflag.gif


text:
Philadelphia & the Pennsylvania Dutch Countryaf John Spelman
Synopsis:A guide to Philadelphia and the Pennsylvania Dutch Country, this title is full of helpful advice on what to see, where to go, local customs, where to stay and other useful hints for the traveller. Better than ever! We´ve reinvented this guidebook with an all new fold-out trip map, specialist contributors, featured highlights & itineraries, off-track detours, and a dedicated food & drink chapter.Retrace Ben Franklin´s footsteps in the city where America´s bells of liberty first rang. Explore historic Old City, linger at the Philadelphia Museum of Art, discover trendy Rittenhouse Square, or be inspired by Amish and Mennonite life in the Pennsylvania Dutch CountryEAT UP - local food and drink experts show you prime spots for celebrity chefs, five-star indulgence and classic South Philly hoagies and cheese steaks FIND THE FUN - insider tips on festive South Street, raucous sports events, elegant Kimmel Center for the Performing Arts, classic jazz clubs, hip Northern Liberties and Manayunk pubs STEP BACK IN TIME - detailed coverage of Independence National Historical Park, where the Declaration of Independence and US Constitution were drafted GET OUTDOORS - with our handy tours and maps, you can bike the Schuylkill River Trail, shop the Italian Market or ride a horse-drawn buggy in Lancaster County
- Sprog:
- Engelsk
- 9781740595636
- Forlag:
- Lonely Planet / Aschehoug
- Indbinding:
- Hæftet
- Sideantal:
- 232
- Udgivet:
- 1. April 2004
Andre udgaver:
Giv din bedømmelseFor at bedømme denne bog, skal du være logget ind.
Andre bøger af John Spelman
Bog, KortLeveringstid:
1-2 ugerCity Walks Deck af China Williams, mfl.Pris:
133,00 kr.Medlemspris: 103,00 kr. i


text:
Germania, prezzi produzione luglio invariati su mese, -1,3% su anno
BERLINO, 20 agosto (Reuters) - Germany's Federal Statistics Office reported the following economic indicator on Thursday: GERMAN PRODUCER PRICES July 15 June 15 July 14 Month-on-month change unch -0.1 -0.1 Year-on-year change -1.3 -1.4 -0.8 Index (basis 2010) 104.4 104.4 105.8 NOTE: Economists polled by Reuters had forecast that prices would fall by 0.1 percent month-on-month and by 1.4 percent on the year. The Statistics Office said that without energy prices June producer prices were unchanged on the previous month and down 0.3 percent on the year-ago period.
© Thomson Reuters 2017 Tutti i diritti assegna a Reuters.


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Mostly purple plants, with 20% variegated or green plants make a great color combo. Leaves avg. 1 1/2-3" at full size.
Reliable producer of dark red leaves. Fine flavor and aroma. Great for adding color to salads and meals.


text: Home > How To > Letters To The Editor How To Letters To The Editor Login or Register to download the PDF version of this article. (193.35 kB) To the Editor:The answer on how to paint popcorn ceilings, or sprayed-on acoustic (Q&A, 7/02), was right on as far as it went, but a couple of issues were not addressed. Login or Register to continue reading


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"A man tells his stories so many times that he becomes the stories. They live on after him, and in that way he becomes immortal. Okay then. That's what I'll do; I will tell you a story. "
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I wept like a monster, I tip my hat to Steven Moffat, Matt Smith, and the rest of the cast and crew. It hurt like the dickens and It was a beautiful sendoff.
More arts to come, but I had to get this out of my feeels system. If you'd like this released as a print please show some interest in the comments.
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"Okay then. That's what I'll do. I'll tell you a story. Can you hear them? All these people who lived in terror of you and your... judgement; all these people whose ancestors devoted themselves, sacrificed themselves, to you? Can you hear them singing? Oh you like to think that you're a god! You're not a god. You're just a parasite eaten out with the jealousy and envy and longing for the lives of others. You FEED on them; on the memory of love and loss and birth and death and joy and sorrow, so! So... come on then. Take mine. Take. my. Memories. But I hope you've got a big appetite because I've lived a long life and I've seen a few things. I walked away from the Last Great Time War. I marked the passing of the Time Lords. I saw the birth of the universe and I watched as time ran out; moment by moment until nothing remained; no time. No space. Just me! I've walked in universes where the laws of physics were devised by the mind of a MAD MAN! And I've watched universes freeze and creations burn! I have seen things you wouldn't believe! I have lost things you will NEVER understand! And I know things, secrets that must never be told, knowledge that must never be spoken! Knowledge that will make parasite gods BLAZE! SO COME OOOON THEN! TAKE IT! TAKE IT ALL, BABY! HAVE IT! YOU! HAVE! IT! ALL!!
One of my favorite 11 moments Moffat and Smith knocked this out of the park
I keep watching this!
11 about to go face the Daleks: *hugs Clara* my impossible girl. Thank you. Goodbye.
Me:*bawls silently except for a few whimpers* please no no no no
Clara: you, you are the Doctor.
11:yep. And I always will be...but times change, and so must I.
Melease please please please stay!
This is fantastic, I love this style a lot, it's simple, but powerful. I actually strongly prefer this over the collage style that's been appearing lately.
This is a good picture, and a good line of Eleventh. Goodbye.
It's a beautiful picture, really love the detail.
If I could get the money I would definitely buy this as a canvas print. ^^
If I could get the money I would definitely buy this as a canvas print. ^^
I'm speechless. It's wonderful!
This is stunningly beautiful, a print would be wonderous.
I cried all the time too. A good sendoff and a good picture too.
i still find myself shaking from this exact scene. just an amazing send off ; v ;
THis is just beautiful!!!
Oh Smithy.... oh the feels.... *sobs*
That would make a really great print.
That would make a really great print.
Beautiful piece! I feel your pain - I will miss Eleven sorely. Matt had an undeniable charisma, yet severity which added a whole new depth to the character. While I'm willing to give Capaldi a shot, Matt will always be my Doctor.


text:
Cadillac Escalade, EXT and ESV Forum - 2007-2014 Discussion, Brembo Brakes Replacement Pads in Cadillac Escalade Forums; My 2007 Esky has Brembo brakes and drilled rotors in the front. It's time to replace the pads but I'm ...
- 04-27-13 06:15 PM #1Cadillac Owners Member
- Automobile(s): 2007 Escalade
- Join Date
- Apr 2013
- Age
- 39
- Posts
- 3
Brembo Brakes Replacement Pads
My 2007 Esky has Brembo brakes and drilled rotors in the front. It's time to replace the pads but I'm not sure which pads I need or where to get them locally. Any help would be appreciated. Thanks.
- 04-27-13 06:42 PM #2Cadillac Owners Member
- Automobile(s): 1999 Escalade, 2008 Escalade
- Join Date
- Jul 2009
- Location
- Louisville, KY
- Posts
- 32
I think tire rack can be a good source to find what you're looking for.I just bought some Brembo pads, and rotors for my car.
Re: Brembo Brakes Replacement Pads
Do you have brembo brakes in the front on your 2008? If so, what was the part number for the pads? I've been searching for the past 2 hours and cannot find anything related to escalade brembo pad replacement. It's driving me nuts.
- 04-28-13 01:24 AM #4Originally Posted by tonelab77
Re: Brembo Brakes Replacement Pads
The reason why I'm in such a hurry is because I had a road trip planned for tomorrow. Thanks for chiming in though. I appreciate it. Do you think compatible brake pads can be found at local auto parts stores? You're in Eastvale? I'm in Riverside.
- 04-28-13 10:00 AM #6Originally Posted by tonelab77


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Secure your tickets for the upcoming Worlds Collide Tour 2017! This will be our only Euro tour of 2017 so be sure to get your tickets asap.Read More
Apr 23 2017
Hey all!First things first. As the headline states, Stoffe is leaving and by no means is he kicked out. We got the message from Stoffe by e-mail simply stating; "Hi...Read More
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Hey all,Breaking Point - now available as instrumental download! Check it out below!Additionally we've managed to get a hold of some instrumental tracks from the Incomparable album as well. You...Read More
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Absolutely as soon as you start calling it her car, allot of things change, And what difference does it make, you going to fix it wash it and do everything else to it anyway and then by the time you've got your welder, she try to get a new washer so it all comes out even.
Quality in a mig welder. Well there are a couple thing to think about.
If you are just using it for sheet metal any mig with a shield gas supply will work. 110amp 120 volt
but if you are going to do frame and "structural" improvements you have to bump up to 220 volts.
Sheet metal is tack, tack, tack, tack . So you will need something in descent shape so your wire doesn't hang up, and back spool and just make an irritating mess. The point is to use the welder, not to fix it.
But a heavy 220 will do both frame and sheet metal and I have found, in most cases it is better to have power you don't need than it is to need power you don't have.
As fas as brand I am a Miller Man and even a well used Miller can still be a good machine.
But go to ebay shop around there a little so you can get and idea and keep your eyes in your local paper once you know what your looking for something is bound to come up.
Oh and there are some place that rent if it is a small project.
A 110 MIG, while being limited by short duty cycle, will weld a frame just fine if you prepare ahead. You need to preheat the metal to about 350-400 deg. with a torch, even if you have to use a propane torch, and then you can even weld 1/4" with no problem. This is a good idea anyway no matter what welder is used but be careful and don't overheat the metal, the trick is to get a temp marker in the range you want from the welding supply. These are cheap and available in any temperature range you will need and are easy to use. This method works great and will allow someone with a 110 volt MIG the means to make a perfectly good weld on frames and other thicker parts. The short duty cycle of the 110 MIG is sometimes a limiting factor on some parts but by using short stringer beads,multiple passes and preheat one can weld most anything that would normally be welded with .023 wire and since base metal temperature needs to be kept with a certain range, particularly in the case of frames, this short duty cycle may not be much of a handicap anyway
Sure it can be done with a 110, but preheating is a pain in the ***, and so it having an underpowered welder. There are some people out there that believe that a 110 is great!!! I have heard all the pro's to a 110 which comes down to one thing and one thing only $$$$$$$$$$. Never in a million years would you grab the 110 over the 220 if they are sitting right next to each other. A 110 is sure more portable because it is lighter and smaller. And the nozzle is smaller so you can get into some tighter spots. If it is all you can afford get a 110 so you can get going but if you find a 220 you can afford, get that.
There are allot of little details we can get into such as duty cycle, freq,pen depth and whatever. but the point is to make a solid weld, 3/16" and over you will want the power.
Ok you refer to preheat as a PITA but did it occur to you that it should be done anyway? If you start out on cold base metal and just simply weld through to finish on a frame then cracking and induced stress from the temperature extremes can cause problems. A weld such as I described with a 110 welder will be Superior to that produced from just tearing into it with more power from a hotter welder. The shorter duty cycle is about the only real problem I see with the 110 outfit and to keep heat under control the weld really needs to be in multiple passes anyway. The key is to start with the base metal about 350-400 deg. and to keep it within that range, or at least close to that, by limiting the welding rate. I agree that 220 is a lot better and I never meant that it wasn't my point is that if someone is using a 110 outfit there is no reason not to use it for frames or other heavier parts. There may be many reasons to want to use 110 besides cost such as power availability and just plain convenience but I hear it from a lot of people that 110 is no good for anything but sheetmetal and that is just not true.
Well lets see how we should put this. Hey guy who needs the welder If all you can only afford a small 110 unit than get that and you will be fine, unless you think you are going to use it all the time for a bunch of different projects then get more juice. The Real goal is "TO KEEP THE PROJECT MOVING"
Now the pre heat guy. I would never preheat my frame or any weld on frame components. Heat is the enemy bi product when it comes to steel. Now if you are welding cast iron, tool steel, or aluminum that is a little bit different story. I would just assume run the right size bead for the fillet and be done with it, I might even blow some compressed air on it, if I am say boxing in a rail. I would also move around quite a bit to try and get the heat spread out.
Thats a hassle too but it is better than getting a bunch af warpage.
You are going to get some anyway it is unavoidable, that is what heat does, but you can make an effort to minimize its effects.
now there is always an exception, If I had a really nice, I mean really nice frame table that I know is balz on every time, and I was building a car for say "riddler" there are some things I would do different but i'm not telling just in case!!!!!
But as far as pre heating goes, I don't hardly have enough time as it is to work on my car, why would I want to wast my time with that.
Fit is way more important!!!
Hmmmm, I think I am going to have to disagree with this one. Localized heat is not a good thing in the scenario you mentioned. If you had a 400 degree piece of steel and you welded it and then allowed the weld and surrounding steel to slowly cool back down to ambient temperature, then you have the potentional for a great weld (and the metal surrounding it remains strong.
Do the same think rapidly with a hot bead on cold plate or quench it afterwards with water OR air, and you then have the potential for issues.
In this case, preheating is always good.
Stick welding on patch panels in automotive sheetmetal is possible, I've done it many years ago as a kid doing sidework at a customer's home garage. It's a real PITA and your work won't be nearly as good as what could be done with a mig, tig, or gas welding but if a stick welder is all you've got practice, practice, practice and verify you have the skills required.
Buy a mig and be done IMO, sell your stick welder and recoop some of the investment, beg, borrow, barter, tell the welding supply outfits in town you're in the market for a trade in. Own your own MIG and life will be much better.
I am not sure but it sure seems like you are comparing apples to fried chicken.
At one point in my youth I was a commercial diver. This means welding underwater "ya all the way under", on ships, oil rigs, and any other metal broke thing. Are there issues sure but that is not the point.
Could I get better weld if I took the whole rig out of the water but it on dry land, sure but is it necessary ? NO WAY!
There are times to pre heat, like I said, if you are doing cast iron do it to be safe. What happens with cast iron is if you heat a spot in the middle, it will expand at a faster rate than the surrounding metal and if you are fixing a crack the crack will run. This means blocks , heads , drums, exhaust manifolds,<--ohhh icky,
But not mild steel. If it were the case with mild steel imagine what they would have to do with a skyscraper, here lets pre heat it and watch the whole thing roll 15 degrees and then put the next floor on it.
Have you ever looked at the OEM welds on any car? if it is longer than 3" its a mistake and most of them would never, ever past a weld inspect.
Is it something you could do sure, no wait I take that back now that you got me thinking about it.
If you preheat it you actually take the mild temper out of it so it will make it softer, hence more pliable, with a standard weld there can be issues because of heat effected areas on the parent metal just out side the root, so if you preheat the whole part you just made your heat effected area bigger, and yes less strong. Weld material is made with the chemistry is is because so it will air harden slightly harder than the parent, or softer depending on what you are doing. A mild steel all position weld cert requires no pre heat, 1" thick.
Oh basic rule for weld guys weld size "single side = to thick ness of parent material 1/4" plate 1/4" face ,double sided is 1/2 the parent material 1/4" plate =1/8" x 2. how much weld you ask? generally 50 % is good enough, but most companies go 100% for liability reasons, I go 100% cuz i like layin beads and I don't have to grind much.
Mild steels yield strength " yield amount it will bend before it stays that way" is about 32000 psi and break is somewhere around 50, just run of the mill steel. you car motor weighs 600 to 1000 lbs, go look at the welds holding your motor mounts, don't be alarmed they are supposed to be that way, unless you have bolts only than but that into perspective.
Grocery, Think about what happens to a 60-70 deg. piece of metal when you subject it to the intense heat of the arc. It is raised from 60-70 deg. to over 2000 deg. almost instantly and to make matters worse this heat is very localized inducing thermal shock into the surrounding metal and then as the metal heats up from the weld it does so unevenly, sometimes with as much a 400-500 deg difference spread over an area of only a few inches inducing much stress into the weld and surrounding metal. Add to this the fact that the first part of the weld is cooled too rapidly due to the heat being absorbed by the surrounding cold metal. Also you apparently have a misunderstanding of why heat causes warpage since you apparently think preheat will cause more warpage when actually it is the other way around since uneven expansion and contraction from UNEVEN heating, not the heating itself, is what causes metal to warp in the first place but on heaver sections the weld itself contracting is the biggest concern not heat induced warpage in the surrounding metal. For what it is worth the "pre-heat guy" has been a certified welder since 1968 and has spent a lifetime in the mining industry welding critical components that very often must pass certification by X-ray or sonic testing and has had to deal with and overcome stress induced cracking from many causes.
BadBob, I share your opinion that it is best to just buy a MIG and be done with it since when one considers all the obstacles that have to be overcome with a stick welder it is simply not worth it, at least not for a beginner.
Grocery, What you are saying is pure nonsense. But this is not the place to discuss this so I will not say anymore here. I would like to see you go over to the Horbart site and explain your theories on pre-heat to those guys they can always use a good laugh, BTW car frames are not mild steel.
Pick a welding site and I will meet you there.
Really, that is interesting.
Make sure you guys don't listen to either one of us, do some research on you own, getting information from as many places as possible is important. Because if the 25 welders, regular production type and I also have a couple of specialist, got word of this guys theory's it would shut our production down and me being the R & D engineer ( research and development) if i tried to pass that off on my production supervisor my,
well lets just say it would be very interesting at the water cooler for a couple of months, it would make for allot of jokes.
Good luck
grocery, What I said is common welding knowledge you on the other hand kept referring to mild steel quite apparently thinking car frames and metal structures are not made from structural steels and apparently are completely unaware that pre-heating steel is common practice, R @ D engineer You have proven to me what you know already and I have said too much so go ahead give it your best shot I will have no more to say here but I would truly like to see you spout this nonsense on a welding forum.
I spent a summer trying to stick weld sheet metal for my egg project. The rods I tried were 6011, 6013, 7014, 7018. I used an air powered flange tool to give good lap joints to double the thickness where I welded. It helped, but I still had, at best, as many holes as tacks. The 7018 rod was the best for me, once I learned how to keep from sticking it every time at low current. The results were so lousy I resorted to oxy/acetylene. The burn-throughs almost ended but I warped the crap out of everything.
After a couple of tanks, I tried a wire-feed welder (flux core, no gas) and did only marginally better than with stick. Ignorance caused the most trouble. I scrapped the whole mess and used fiberglass to make the egg shape, then got some excellent remote coaching from folks on here (see Got a welding coach ). I can actually weld sheet metal with flux core wire now.
I wouldn't waste a minute trying to weld thin stuff with stick. Save that for the heavy steel.
First, thank you for the replies thus far. I am grateful as well as entertained.
Let me clear up a couple of things. I was given a small, AC welder as a gift. It is a firepower AC welder sold by eastwood. It runs on 110, up to 100 amps. It was advertised as a good unit for sheet metal.....but in my experience it has been very difficult to master. I have experienced burn through, splatter problems, porosity issues, but to be honest....not much by way of warpage problems. Each time I practice I get a bit better.
I understand that people prefer MIG units. I am working with what I have, until I fail or I can really justify the upgrade for a tool better suited for a range of welding projects. At this point I am focused on rust repair panels, with 18 and 20 gauge metal. I understand I have chosen a steep learning curve, and one that may not be all that fruitful.
To sum up, I see about half the respondents say don't bother with this direction, as it will be fruitless. The balance says it can be done, has been done, but the results are marginal. This afternoon I picked up 5lbs of 7014 ($11) and started practicing some plug welds on 22 gauge scrap. I'll continue practicing till I feel a bit more confident. Interestingly, I also got hit offline with some private messages that encouraged me greatly. Thanks for those.
Incidentally, while picking up the 7014 at the welding shop, I drew the attention of 3 welders also picking up supplies. I was in shorts and a white polo shirt.....clearly out of my element. They argued that 6013 rods would work better for sheetmetal...and were designed for sheetmetal. When I explained that my "buzzbox" was a small AC only unit, they agreed that 7014 was probably best. They didn't scold me for trying sticks on sheetmetal...in fact one went out of his way to argue that migs were not well suited for the task. He stated "they put out way too much heat for sheetmetal." Clearly he would be labled a heretic here. Another favored sticks, and also hinted that a torch with the right tip and filler material would be better.
Again...I have to work with what I have...until I know I can't.
Last...thanks for the notes to my wife. I actually forwarded them along and she brought up the conversation tonight at dinner...with a couple of friends. Maybe I'll show her what I can do on the Volvo.....and then show her the latest Miller or Hobart catalogs.
Thanks folks.
Anything can be done. But is it worth the grief to spend all that time and get something that could have been 10 times better with 1/10th the effort?
You can finish concrete with a paint stir stick and you can also drive nails with a shoe.
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Wolves 2-1 Sunderland
- From the section Football
Steven Fletcher's second-half double ruined new Sunderland manager Martin O'Neill's first look at his team.
O'Neill was in the directors' box as Sunderland looked on course to welcome him with only their third Premier League win on the season after Kieran Richardson put them ahead early in the second half.
The game then changed in a matter of seconds as Wolves were revived with growing anxiety sweeping around Molineux after a sequence of eight defeats in their last 10 Premier League games.
Sunderland were awarded a controversial penalty with 18 minutes remaining when Sebastian Larsson tumbled threatrically under minimal contact from Jody Craddock, giving the Swede the opportunity to effectively seal the win.
Larsson's spot-kick was saved by Wolves keeper Wayne Hennessey as justice was done - and Wolves took advantage to level inside 30 seconds as Fletcher's header beat Kieren Westwood.
The momentum was with Wolves and Mick McCarthy's men confirmed only their fourth league win this term as Fletcher fired low past Westwood with nine minutes left as Sunderland appealed for handball against Jamie O'Hara.
O'Neill starts work at the Stadium of Light in earnest on Monday after succeeding sacked Steve Bruce - and Sunderland's brittle nature under pressure will have already drawn his attention.
And yet O'Neill, who left Bruce's right-hand man Eric Black in charge of team affairs, will also know that Sunderland wasted a perfect opportunity to start his reign with a win, only to squander the chance after Larsson had missed the crucial penalty.
Wolves boss McCarthy admitted he was under pressure to get a result after the recent dismal sequence and rain-soaked Molineux celebrated in style following a real show of character in the closing stages to fashion the win.
O'Neill made a low-key entrance moments before kick-off, flanked by long-time backroom colleague Steve Walford and goalkeeping coach Seamus McDonagh.
And they will have both been impressed by the first-half contribution of Sunderland keeper Westwood, who made two crucial interventions to keep O'Neill's new side on terms.
Westwood was alert to clutch Fletcher's early header after a goalmouth scramble which had Wolves appealing for a penalty as Lee Cattermole appeared to drag down David Edwards.
He made an even more vital contribution as the interval approached, miraculously clawing away Stephen Hunt's point-blank shot as the Republic of Ireland midfield midfield man looked certain to turn in an inviting cross from Matt Jarvis.
Sunderland had moments of their own in a first period short on quality, notably when Ward was forced to scramble Stephane Sessegnon's effort off the line at the conclusion of a sweeping counter attack.
A move of similar quality saw Sunderland go ahead after 52 minutes. Wolves were caught cold on the break as Sessegnon fed Richardson, whose powerful finish deceived Hennessey as it flew in a high at his near post.
O'Neill, sitting high in the stand, barely registered any emotion at Sunderland's first goal since his appointment on Saturday.
Sunderland threatened to punish Wolves further as they pushed forward in search of an equaliser - prompting McCarthy to make a double change by sending on Sylvain Ebanks-Blake and Adam Hammill for Hunt and Kevin Doyle.
Hennessey was called into action when Ji Dong-Won sent in a glancing header. It was the South Korean's final involvement as he was then replaced by Ahmed Elmohamady.
Sunderland were then handed an unexpected chance to wrap up the points in dubious fashion in the 72nd minute. Larsson fell spectacularly with Craddock in close attendance. There was little or no meaningful contact and Molineux erupted in fury as referee Phil Dowd pointed to the spot.
Hennessey provided a measure of justice by saving Larsson's poor penalty - and the scale of the reprieve was emphasised as Wolves equalised within seconds.
Westwood got a hand to Fletcher's header from another fine Jarvis cross, but could not keep it out.
Sunderland, from scenting victory, were now on the retreat and Wolves completed the turnaround with Fletcher's second nine minutes later. The Black Cats claimed handball against O'Hara as the striker took control in the area, but he did not waste time as he shoot low past Westwood.
Wolves now had a precious lead to protect and they did so successfully to ensure O'Neill's takes over a side on the back of a defeat.
Lineup, Bookings (5) & Substitutions (5)
- 01 Hennessey
- 06 Craddock (Johnson - 74' )
- 11 Ward
- 16 Berra
- 23 Zubar Booked
- 04 Edwards
- 12 Hunt (Hammill - 66' )
- 17 Jarvis
- 24 O'Hara
- 10 Fletcher
- 29 Doyle (Ebanks-Blake - 66' Booked )
- 31 De Vries
- 03 Elokobi
- 05 Stearman
- 14 Johnson
- 19 Hammill
- 20 Milijas
- 09 Ebanks-Blake
- 20 Westwood
- 02 Bardsley
- 05 Brown
- 11 Richardson Booked
- 16 O'Shea
- 06 Cattermole Booked
- 07 Larsson
- 14 Colback
- 28 Sessegnon (Noble - 87' )
- 17 Ji Dong-Won (Elmohamady - 70' )
- 52 Bendtner Booked
- 24 Carson
- 19 Bramble
- 08 Gardner
- 15 Vaughan
- 23 McClean
- 27 Elmohamady
- 31 Noble
Match Stats
On target
Live Text Commentary
Full Time The referee brings the game to a close.90:00 +4:41
The offside flag is raised against Ahmed Elmohamady. Wayne Hennessey takes the free kick.90:00 +4:08
Keiren Westwood takes the free kick.90:00 +4:08 Booking
Booking Sylvan Ebanks-Blake receives a yellow card.90:00 +3:57
Unfair challenge on Wes Brown by Adam Hammill results in a free kick.89:27
Christophe Berra fouled by Ryan Noble, the ref awards a free kick. Free kick taken by Wayne Hennessey.85:45
Wayne Hennessey takes the direct free kick.85:45 Substitution
Substitution (Sunderland) makes a substitution, with Ryan Noble coming on for Stephane Sessegnon.85:45 Booking
Booking Nicklas Bendtner is given a yellow card.85:39
Nicklas Bendtner concedes a free kick for a foul on Adam Hammill.85:29
Nicklas Bendtner takes a shot. Roger Johnson gets a block in.84:11
Corner taken by Adam Hammill from the right by-line, Nicklas Bendtner makes a clearance. Stephen Ward has an effort at goal from 25 yards. Blocked by John O'Shea.84:00
Sylvan Ebanks-Blake takes a shot. Blocked by Wes Brown.81:49
Free kick awarded for an unfair challenge on Kieran Richardson by Sylvan Ebanks-Blake. Kieran Richardson takes the free kick.80:41
Jamie O'Hara provided the assist for the goal.80:41 Goal scored
Goal! - Steven Fletcher - Wolves 2 - 1 Sunderland Steven Fletcher fires in a goal from inside the box to the bottom right corner of the goal. Wolverhampton 2-1 Sunderland.79:58
Nicklas Bendtner takes a shot. Save by Wayne Hennessey.78:22
Corner from the right by-line taken by Sebastian Larsson, clearance made by Roger Johnson.77:23
Sylvan Ebanks-Blake takes a shot. Jack Colback gets a block in.76:35
Free kick awarded for an unfair challenge on Phillip Bardsley by Sylvan Ebanks-Blake. Keiren Westwood restarts play with the free kick.
Unfair challenge on Sylvan Ebanks-Blake by John O'Shea results in a free kick. Adam Hammill crosses the ball in from the free kick.75:10
Ahmed Elmohamady has an effort at goal from outside the penalty area which goes wide right of the goal.73:56 Substitution
Substitution (Wolverhampton) makes a substitution, with Roger Johnson coming on for Jody Craddock.72:45
Matthew Jarvis provided the assist for the goal.72:45 Goal scored
Goal! - Steven Fletcher - Wolves 1 - 1 Sunderland Steven Fletcher scores a headed goal from close in. Wolverhampton 1-1 Sunderland.72:35 Penalty
A save by Wayne Hennessey from penalty by Sebastian Larsson. Wolverhampton 0-1 Sunderland.71:04
Jody Craddock gives away a Penalty for an unfair challenge on Sebastian Larsson.69:23 Substitution
Substitution Ahmed Elmohamady on for Dong-Won Ji.68:03
Dong-Won Ji takes a shot. Wayne Hennessey makes a save.66:50
Free kick awarded for an unfair challenge on Jack Colback by Sylvan Ebanks-Blake. Phillip Bardsley restarts play with the free kick.66:08
Outswinging corner taken by Adam Hammill from the right by-line played to the near post, clearance made by Lee Cattermole.65:21 Substitution
Substitution Sylvan Ebanks-Blake joins the action as a substitute, replacing Kevin Doyle.65:08 Substitution
Substitution Adam Hammill comes on in place of Stephen Hunt.62:08
Steven Fletcher is ruled offside. Free kick taken by Keiren Westwood.60:01
Direct free kick taken by Kieran Richardson.60:01 Booking
Booking Ronald Zubar receives a yellow card.59:51
Nicklas Bendtner fouled by Ronald Zubar, the ref awards a free kick.58:19
Kevin Doyle takes a shot. Save by Keiren Westwood.55:29
Long range shot from Phillip Bardsley misses to the left of the goal.54:39
Corner taken left-footed by Jamie O'Hara, Nicklas Bendtner makes a clearance.53:19
Dong-Won Ji concedes a free kick for a foul on Christophe Berra. Wayne Hennessey takes the direct free kick.52:51 Booking
Booking Kieran Richardson receives a caution.51:37
Assist on the goal came from Stephane Sessegnon.51:37 Goal scored
Goal! - Kieran Richardson - Wolves 0 - 1 Sunderland Kieran Richardson scores a goal from inside the penalty area to the top left corner of the goal. Wolverhampton 0-1 Sunderland.51:06
Floated corner taken left-footed by Kevin Doyle, clearance by Phillip Bardsley.48:29
Foul by Wes Brown on Kevin Doyle, free kick awarded. Jamie O'Hara has an effort direct from the free kick.47:35
Effort on goal by Nicklas Bendtner from inside the penalty box misses to the right of the target.46:59
Dong-Won Ji challenges Ronald Zubar unfairly and gives away a free kick. Wayne Hennessey restarts play with the free kick.45:01
The game restarts for the second half.45:00 +1:07 Half time
Half Time The players leave the pitch at half time.42:56
Shot from 20 yards from Stephane Sessegnon. Ronald Zubar gets a block in. Lee Cattermole has shot on goal from just outside the penalty box which goes wide of the right-hand upright.41:53
Stephane Sessegnon takes a shot. Stephen Ward manages to make a clearance.40:11
Direct free kick taken by Jamie O'Hara.40:11 Booking
Booking Lee Cattermole receives a caution.40:03
Foul by Lee Cattermole on Kevin Doyle, free kick awarded.39:24
Short corner taken by Jamie O'Hara, clearance made by John O'Shea.39:08
Stephen Hunt takes a shot. Keiren Westwood makes a brilliant save.37:45
Christophe Berra fouled by Dong-Won Ji, the ref awards a free kick. Free kick taken by Jody Craddock.37:03
Dong-Won Ji produces a right-footed shot from the edge of the box and misses to the left of the target.36:25
Corner taken by Jamie O'Hara, clearance made by Nicklas Bendtner.34:36
Jamie O'Hara takes a shot. Blocked by John O'Shea.31:08
Stephen Hunt takes a inswinging corner.30:38
David Edwards takes a shot. Save by Keiren Westwood.30:07
Stephane Sessegnon takes a shot. Blocked by Christophe Berra.28:10
Christophe Berra challenges Dong-Won Ji unfairly and gives away a free kick. Lee Cattermole restarts play with the free kick.26:17
Jamie O'Hara takes a shot. Save by Keiren Westwood.26:06
Foul by Jack Colback on David Edwards, free kick awarded. Stephen Hunt takes the direct free kick.25:02
Headed effort from deep inside the penalty area by Ronald Zubar misses to the left of the goal.21:30
Sebastian Larsson takes the inswinging corner, Ronald Zubar manages to make a clearance.19:43
Free kick awarded for a foul by Stephane Sessegnon on Stephen Hunt. Direct free kick taken by Jody Craddock.18:53
The offside flag is raised against Steven Fletcher. Indirect free kick taken by Keiren Westwood.18:05
Effort from outside the penalty box by Jamie O'Hara goes wide left of the target.17:11
Corner from the right by-line taken by Sebastian Larsson, Wes Brown takes a shot. Save by Wayne Hennessey.15:33
Free kick awarded for a foul by Wes Brown on Kevin Doyle. Free kick crossed by Jamie O'Hara, save by Keiren Westwood.15:23
Sebastian Larsson takes a shot. Save made by Wayne Hennessey.13:30
Matthew Jarvis is flagged offside by the assistant referee. Free kick taken by Keiren Westwood.11:30
Kevin Doyle challenges Wes Brown unfairly and gives away a free kick. Direct free kick taken by Kieran Richardson.10:52
Header by Nicklas Bendtner from deep inside the penalty area misses to the left of the goal.9:30
The assistant referee signals for offside against Dong-Won Ji. Wayne Hennessey takes the free kick.9:20
David Edwards concedes a free kick for a foul on Stephane Sessegnon. Lee Cattermole takes the direct free kick.7:53
Corner taken by Matthew Jarvis played to the near post, Jamie O'Hara has an effort at goal from 12 yards. Lee Cattermole gets a block in. Steven Fletcher takes a shot. Keiren Westwood makes a save. Nicklas Bendtner takes a shot. Jody Craddock gets a block in.4:43
Stephane Sessegnon takes a shot. Save made by Wayne Hennessey.3:41
Free kick awarded for a foul by John O'Shea on Kevin Doyle. Jamie O'Hara takes the direct free kick.1:12
John O'Shea takes a shot. Wayne Hennessey makes a save.0:36
Foul by David Edwards on Jack Colback, free kick awarded. Sebastian Larsson takes the direct free kick.0:00
The referee gets the game started.
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Hi. Here is my system...
Alpine 9815 deck
JBL BP600.1 mono amp
Infinity Kappa Perfect DVC sub (w/ perfect Q)
Monster XLR interconnect
"upgraded" speakers
What I really want is more power and clarity. For now I will do speakers, than perhaps later a 40x4 amp. I will be running 6.5" component speakers in front and 5.25" speakers in the back. I have the www.iaperformance.com brackets already. I should really limit the amount of money I spend to probably under $250. What would you recomend I get? Links to prices/sites would be nice.
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