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In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2.
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) results in the transcriptional induction of various genes including those encoding ER-resident chaperones and folding enzymes. This cellular mechanism is called the unfolded protein response (UPR), and several important features of the UPR signaling pathway have been revealed initially through studies in the budding yeast Saccharomyces cerevisiae. Yeast Ire1 is a 1115-amino acid transmembrane protein that transmits the unfolded protein signal across the ER membrane (Cox et al., 1993 ; Mori et al., 1993 ). The cytoplasmic domain (C-terminal half) of Ire1 possesses both serine/threonine kinase and site-specific endoribonuclease activities (Mori et al., 1993 ; Sidrauski and Walter, 1997 ). Ire1 dimerizes in response to the accumulation of unfolded proteins, resulting in its trans-autophosphorylation and activation (Shamu and Walter, 1996 ; Welihinda and Kaufman, 1996 ). Activated Ire1 in turn promotes splicing of HAC1 precursor mRNA to produce the mature form (Cox and Walter, 1996 ; Sidrauski and Walter, 1997 ), which is effectively translated into a functional transcription factor (Mori et al., 2000 ; Ruegsegger et al., 2001 ). Mature form of Hac1 efficiently induces transcription of UPR target genes containing the UPR element (UPRE) in their promoter region (Mori et al., 1992 ; Kohno et al., 1993 ).
In mammalian cells, accumulation of unfolded proteins initiates signaling from the ER via more complicated pathways. Two homologues of Ire1, Ire1α and Ire1β, have been identified (Tirasophon et al., 1998 ; Wang et al., 1998 ; Iwawaki et al., 2001 ). According to recent reports (Yoshida et al., 2001 ; Calfon et al., 2002 ), IRE1 functions to promote splicing of an mRNA encoding the transcription factor XBP1. This Ire1-XBP1 signaling pathway is reported to act synergistically with another signaling pathway involving an ER-located transmembrane protein ATF6 to promote the mammalian UPR (Yoshida et al., 2001 ; Shen et al., 2001 ; Lee et al., 2002 ). Moreover, accumulation of unfolded proteins in the mammalian ER attenuates bulk protein synthesis. This occurs by phosphorylation of the eukaryotic translation initiation factor 2 subunit by PERK (PKR-like ER kinase), a transmembrane kinase containing an Ire1-like lumenal domain (Harding et al., 1999 ) and by cleavage of the 28S rRNA by Ire1β (Iwawaki et al., 2001 ).
Kar2 is the yeast orthologue of mammalian BiP, an ER-resident member of the Hsp70 family (Normington et al., 1989 ; Rose et al., 1989 ), and the expression of KAR2 gene is upregulated by the UPR. Like other members of the family, Kar2 has an amino-terminal ATPase domain adjacent to a substrate-binding domain (Figure 1; Bukau and Horwich, 1998 ). It is believed that the ATPase domain is involved in regulating the interaction between the substrate-binding domain and unfolded protein substrates. Because KAR2 is essential for viability, studies of its physiological functions have been performed primarily using conditional lethal kar2 alleles. When cultured at the restrictive temperature, some of the temperature-sensitive kar2 mutant strains exhibit a block in the translocation of secretory proteins into the ER (Vogel et al., 1990 ; Sanders et al., 1992 ; Brodsky et al., 1995 ). Several molecular mechanisms have been proposed to explain the function of Kar2 in the protein translocation machinery (Hamman et al., 1998 ; Matlack et al., 1999 ). Kar2 has also been shown to serve as a molecular chaperone during protein folding in the ER. For instance, refolding of carboxypeptidase Y (CPY), after denaturation by dithiothreitol (DTT), was impaired in some kar2 mutant strains (Simons et al., 1995 ). In addition, other reports implicate Kar2 involvement in ER-associated protein degradation (ERAD; Plemper et al., 1997 ; Brodsky et al., 1999 ; Nishikawa et al., 2001 ).
We have proposed that KAR2 is not only a UPR target, but is itself a critical negative regulator of the UPR pathway (Okamura et al., 2000 ). In the current model, Kar2 associates with Ire1 to repress the activation of Ire1 in nonstressed cells, and in response to accumulation of unfolded proteins in the ER, Kar2 dissociates from Ire1, resulting in dimerization and activation of Ire1. This proposed mechanism, hereafter called the BiP/Kar2-dependent Ire1 regulation, is supported by several findings in our previous studies in yeast (Kohno et al., 1993 ; Okamura et al., 2000 ). First, overproduction of Kar2 in yeast cells was shown to attenuate the UPR. Furthermore, Kar2 coimmunoprecipitated with Ire1 from lysates of nonstressed cells. Notably, the amount of Kar2 that coimmunoprecipitated with Ire1 extremely decreased when cells were cultured in the presence of reagents that activate the UPR pathway. Similar observations were reported by A. Bertolotti et al. (2000 ), suggesting that BiP acts to regulate Ire1 and PERK in mammalian cells. Although these studies provided correlative support for the model, firm confirmation awaits direct evidence that the binding state of Ire1 by BiP/Kar2 regulates its activity.
To obtain the further evidence of the BiP/Kar2-dependent Ire1 regulation model, we used a different approach in which the phenotypes of kar2 mutations were analyzed. Mutations stabilizing Kar2 association with Ire1 impaired UPR signaling even under conditions of ER stress. Conversely, mutations disrupting this association constitutively activate the pathway. Our results suggest a molecular mechanism by which unfolded proteins are detected by the sensor protein in the UPR pathway.
Yeast strains used in this study are listed in Table 1. The kar2 mutant genes were cloned from these strains and sequenced for confirmation of their mutation points that are indicated in Figure 1. Culturing and genetic manipulation of yeast strains were by standard techniques described in Kaiser et al. (1994 ). Basically, cells were cultured in YPD-rich medium, synthetic complete (SC) medium lacking uracil, or minimal synthetic medium plus dextrose (SD) supplemented with appropriate nutrients. For induction of the GAL1 promoter, cells were cultured in synthetic complete galactose (SCG) medium, which is identical to SC medium except that 2% dextrose was replaced with 2% galactose. Cells were grown at 23°C except where noted.
The XbaI site in the HIS3 centromeric vector pRS313 (Sikorski and Hieter, 1989 ) was converted to SphI by insertion of the appropriate oligonucleotide. The vector was then digested with BamHI and SphI, and a 5.6-kbp BamHI-SphI DNA fragment containing the IRE1 gene from plasmid pERN1-EM (a generous gift of Dr. K. Mori, Graduate School of Biostudies, Kyoto University, Kyoto, Japan; Mori et al., 1993 ) was inserted to generate plasmid pRS313-IRE1. An SphI recognition sequence was inserted into the site 5′-adjacent to the hemagglutinin (HA) epitope coding sequence on plasmid phIRE1-HA2 (a generous gift of A. Hosoda, Nara Institute of Science and Technology, Japan), and a 0.17-kbp SphI-NotI DNA fragment encoding three tandem copies of the HA epitope (YPYDVPDYAGS) followed by a termination codon was isolated. The termination codon (TAA) of the IRE1 gene in pRS313-IRE1 was converted to an SphI site by PCR mediated mutagenesis, the resulting plasmid was digested with SphI and NotI, and the 0.17-kbp DNA fragment was ligated into these sites. From the resulting plasmid, a 4.9-kbp DNA fragment encoding Ire1 tagged with a C-terminal triple HA epitope tag was excised by digesting with BamHI and NotI and cloned into an URA3 2-μm vector pRS426 (Christianson et al., 1992 ) to obtain plasmid pRS426-IRE1HA.
An URA3 2-μm plasmid pCZY1 (Mori et al., 1992 ) containing a lacZ reporter gene driven by the CYC1 core promoter fused with the UPRE was generously provided by Dr. K. Mori and used to monitor cellular UPR activity. A 2-μm plasmid pYPR3841U (a generous gift of Dr. M. Takagi, Department of Biotechnology, The University of Tokyo, Tokyo, Japan) was derived from pYPR3841 (Umebayashi et al., 1997 ) by replacement of the TRP1 selectable maker with URA3 and used for cellular expression of the Δpro from the GAL1 promoter. pKAR2HSE-lacZ (Oka et al., 1997 ) is an URA3 2-μm plasmid containing a lacZ reporter gene driven by the CYC1 core promoter fused with the KAR2 heat shock element (HSE).
Yeast cells (10 OD600 equivalent) carrying pRS426-IRE1HA were harvested, washed with PBS, and suspended in 200 μl of buffer A (50 mM Tris-Cl, pH 7.9, 5 mM EDTA, and 1% Triton X-100) supplemented by protease inhibitors (2 mM phenylmethanesulfonyl fluoride, 10 μg/ml each of pepstatin, leupeptin, and aprotinin). Glass beads (0.5 mm) were added up to the meniscus. The cells were broken by vortexing six times for 30 s each at maximum speed. After removal of the glass beads, the cell lysates were clarified by centrifugation (15,000 × g for 10 min).
The cell lysates were then diluted with 800 μl buffer B (the same composition as buffer A except containing 180 mM NaCl and 6% skim milk) and incubated for 1 h at 4°C with 2 μg of the anti-HA mAb 12CA5 (Roche Diagnostics, Basel, Switzerland), followed by the addition of 15 μl of protein A–conjugated Sepharose beads (Protein A Sepharose 4 FF; Amersham Biosciences, Uppsala, Sweden). After further incubation at 4°C for 1 h, the Sepharose beads were collected by centrifugation, washed five times with buffer C (the same composition as buffer B except for containing no skim milk), and used as immunoprecipitates.
For cells carrying pYPR3841U or the control empty vector pRS426, lysates were prepared as described above except that buffer C supplemented by the protease inhibitors was used instead of buffer A. Next, lysates (190 μl) were ultracentrifuged (Beckman TLA-100.3 rotor, 100,000 × g, 15 min, 4°C), and both supernatant and pellet fractions were collected. The pellet fractions were washed twice with buffer C, and resuspended in 190 μl of buffer C.
The samples described above were added to an equal volume of gel loading buffer containing 125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 20 mM DTT, and 0.02% bromophenol blue. Then they were incubated at 95°C for 2 min and fractionated on 8% SDS-PAGE gels. Proteins were then transferred to nitrocellulose membranes (PROTORAN; Schleicher & Schuell, Dassel, Germany) by electrophoresis at 1 mA/cm2 for 1.5 h in transfer buffer containing 48 mM Tris, 38.6 mM glycine, 0.037% SDS, and 20% methanol. The blots were blocked overnight at 4°C in PBS containing 0.1% Tween 20 and 5% skim milk (blocking solution). The following day, the blots were incubated with the same blocking solution, but containing the primary antibody (12CA5 anti-HA antibody at 0.4 μg/ml, rabbit anti-Kar2 antiserum [Tokunaga et al., 1992 ] at a 1:1000 dilution, or rabbit anti-RNAP-I antiserum [a generous gift of Dr. M. Takagi; Fukuda et al., 1994 ]) at a 1:5000 dilution. Incubation was for 1 h at room temperature. The blots were subsequently washed five times with PBS containing 0.1% Tween 20 (washing solution) at room temperature for 3 min each. The blots were next incubated with a 1:1000 dilution of horseradish peroxidase–coupled, goat anti-rabbit or anti-mouse IgG (Amersham Biosciences) in blocking solution for 1 h at room temperature. The blots were then washed five times with the washing solution at room temperature for 3 min each, and specific protein bands were detected using enhanced chemiluminescence (ECL; Amersham Biosciences) and x-ray films (RX-U; Fuji Photo Film, Ashigara, Japan).
In case of chemical cross-linking experiments, cells (25 OD600 equivalent) were suspended in 200 μl of PBS containing 1% Triton X-100 and the protease inhibitors and were broken by the glass beads. After clarification by centrifugation (3000 × g for 30 s), 90 μl of the lysates was subjected to chemical cross-linking reaction with 2 mM dithiobissuccinimidyl propionate (DSP; 1.8 μl of 100 mM stock [freshly prepared in DMSO] was added) at 27°C for 30 min. The reaction was quenched by the addition of 90 μl of 1 M Tris-Cl, pH 7.5, and the samples were then ultracentrifuged (Beckman TLA-100.3 rotor, 100,000 × g, 15 min, 4°C). The pellet fractions were dissolved in 100 μl of resuspend buffer (50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1% SDS) by incubation at 95°C for 3 min, diluted by 900 μl of buffer C, and incubated with the anti-Kar2 antiserum (1:120 dilution) or a control preimmune serum for 1 h at 4°C. The immunocomplexes were collected by protein A–conjugated Sepharose beads and analyzed by Western blotting as described above.
DNA probes for Northern blot analysis were prepared by PCR amplification of yeast genomic DNA. The HAC1, KAR2, and SSA probes, respectively, correspond to nucleotide -11–654 of HAC1, nucleotide 9–2047 of KAR2, and nucleotide 1016–1756 of SSA1.
Total RNA was prepared using the hot phenol method (Collart and Oliviero, 1993 ). For Northern blot analysis, 5 μg of total RNA was separated on a 1% agarose, 1.8% formaldehyde gel and transferred to a nylon membrane (Hybond-N; Amersham Biosciences). The membrane was prehybridized in 500 mM sodium phosphate, pH 7.0, 1 mM EDTA, and 7% SDS. The membrane was then incubated with the random primed 32P-labeled DNA probe. After washing, the membrane was exposed to an imaging screen (BAS-MS2040; Fuji), and signal intensity was quantified using a Fuji BAS2500 image analyzer. The percentage of HAC1 mRNA cleavage was calculated using the equation (It - I u)/It × 100%, where It is the intensity of total HAC1 mRNA species and Iu is the intensity of uncleaved HAC1 (HAC1u) mRNA.
β-Galactosidase activity of cells was determined using the protocol of Kaiser et al. (1994 ). Cells (0.5 OD600 equivalent) were suspended in 800 μl of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 0.27% 2-mercaptoethanol, pH 7.0), and 20 μl of 0.1% SDS and 50 μl of chloroform was added. The mixture was vortexed vigorously for 20 s. After equilibration at 28°C for 5 min, o-nitrophenyl-β-d-galactoside was added to a final concentration of 0.8 mg/ml. The reaction was stopped at various times by adding 0.5 ml of 1 M Na2CO3, and the concentration of the product, o-nitrophenol (ONP), was measured by optical density at 420 nm. One unit of β-galactosidase activity is defined as 1 nmol of ONP/min of reaction for 1 ml of culture at 1 OD600 unit.
In this study, we used yeast strains carrying various temperature-sensitive kar2 mutant alleles originally generated by Polaina and Conde (1982 ) and by us. As shown in Figure 1, all of these are single-point mutants. Based on the location of the mutations, we categorized each as an ATPase domain mutant (kar2-159, kar-113, kar2-191; hereafter called type A) or a substrate-binding domain mutant (kar2–1, kar2–133; hereafter called type S). As shown in Table 1, two different strain backgrounds were used. The kar2-159, -113, -191 and -133 strains are of the MS10 background, and the kar2-1 strain is of the W303 background. Thus, the parental MS10 and W303 strains, respectively, were used as KAR2 wild-type controls. All of the kar2 mutant strains grew well at 23°C, but exhibited various degrees of growth defects when cultured at 37°C. Hence, 23°C was used as the permissive temperature and 37°C as the restrictive temperature, unless otherwise described.
We first wished to determine whether any of the mutations altered the cell's ability to regulate the UPR pathway. To monitor cellular activity of this pathway, we used a lacZ reporter plasmid for expression of β-galactosidase under the control of the UPRE. At first, cells carrying this plasmid were cultured in SC medium at 23°C and shifted to 37°C for 200 min. Extrinsic ER stress was imposed by 2 μg/ml tunicamycin, an N-glycosylation inhibitor (Takatsuki et al., 1975 ), which was added immediately after the temperature shift. In the absence of tunicamycin, β-galactosidase activity was slightly higher in kar2 type A mutant cells than in wild-type cells (Figure 2A). However, in the presence of tunicamycin, a strong induction was observed in the wild-type, but was completely lacking in the kar2 type A mutant cells. In contrast, constitutively high β-galactosidase activity was observed in the kar2 type S mutant cells, regardless of the presence of tunicamycin. This activity was similar to that seen in tunicamycin-treated wild-type cells.
As a control, we also monitored UPR activity at the permissive temperature. For reasons unknown, we were not able to detect any β-galactosidase activity in any of the seven strains tested in this study when cultured at 23°C in SC medium (unpublished data), and we suspected that the UPRE-lacZ reporter does not work in this culturing condition. Therefore, the cells were cultured in SD medium for monitoring the UPR at the permissive temperature. As shown in the left panel of Figure 2B, all of the kar2 mutant strains as well as the wild-type strains exhibited almost normal induction of UPR by tunicamycin at 23°C. However, when cells were cultured at 37°C, the UPR was not induced by tunicamycin in the type A mutant cells, but was constitutively active in the type S mutant cells (Figure 2B, right panel). It should be noted that expression of β-galactosidase from this UPRE-lacZ reporter somehow depends on the strain background. The W303 background strains exhibited higher β-galactosidase activity than the MS10 background strains.
Because the HAC1 mRNA precursor (uninduced form; HAC1u; Cox and Walter (1996 )) is the substrate of Ire1, a more direct measure of Ire1 activity is HAC1u splicing. For this, we monitored HAC1u cleavage by Northern blot analysis (Figure 2, C and D). We cultured cells in rich YPD medium at 23°C, shifted to 37°C for 60 min, and then extracted total cellular RNA. ER stress was imposed by the addition of 2 μg/ml tunicamycin immediately after the temperature shift. As expected, in the wild-type cells, HAC1u was effectively cleaved and converted to the mature HAC1 mRNA (induced form: HAC1i; Cox and Walter, 1996 ) only in the presence of tunicamycin. In the type A mutant cells, partial cleavage of HAC1u was observed even in the absence of tunicamycin, and addition of tunicamycin did not enhance the extent of cleavage. In contrast, the type S mutant cells exhibited nearly complete cleavage of HAC1u both in the absence and presence of tunicamycin. Importantly, these results are consistent with the observations obtained by the UPRE-lacZ reporter assay described above. We therefore concluded that at the restrictive temperature, Ire1 is constitutively and highly activated in the type S mutant strains and constitutively repressed in the type A mutant strains.
We next analyzed the in vivo physical interaction between Kar2 and Ire1 by employing a coimmunoprecipitation assay (Figure 3). Ire1 bearing a C-terminal triple influenza virus HA epitope tag (Ire1-HA) was expressed in the kar2 mutant and control wild-type strains. Our previous study had indicated that Ire1-HA functions similar to wild-type Ire1 to transmit the unfolded protein signal across the ER membrane (Okamura et al., 2000 ). In this study, cells carrying an Ire1-HA expression plasmid were cultured in SC medium at 23°C and shifted to 37°C for 60 min, and lysates were prepared. ER stress was induced by the addition of 2 μg/ml tunicamycin immediately after the temperature shift. Anti-HA Western blot analysis of the cell lysates indicated that Ire1-HA was expressed at similar levels in all of the kar2 mutant and control wild-type strains (Figure 3, uppermost panel). The precursor form of Kar2 (pre-Kar2) was detected in lysates from the type A mutant cells by anti-Kar2 Western blotting (Figure 3, second panel, lanes 4–9), suggesting that protein translocation is impaired in these cells (Vogel et al., 1990 ).
The cell lysates were next subjected to anti-HA immunoprecipitation. Anti-HA Western blot analysis of the immunoprecipitates showed that nearly equal amounts of Ire1-HA were immunoprecipitated from each lysate (Figure 3, third panel). Kar2 in the immunocomplexes was then detected by Western blot analysis. From lysates prepared from wild-type cells cultured under nonstressed conditions, Kar2 was found to be complexed with Ire1-HA (Figure 3, lowest panel, lanes 2 and 12). Consistent with our previous observation (Okamura et al., 2000 ), the amount of coimmunoprecipitated Kar2 was sharply reduced in these cells after treatment by tunicamycin (Figure 3, lowest panel, lanes 3 and 13). Kar2 was undetectable in a control strain not expressing Ire1-HA, showing that the signal is specific (Figure 3, lowest panel, lane 1). When the assay was performed using lysates from the mutant strains, Kar2 displayed distinctly different binding characteristics to Ire1, depending on the strain. Treatment of the type A mutant cells with tunicamycin did not cause dissociation of Kar2 from Ire1 (Figure 3, lowest panel, compare lanes 5-4, 7-6, and 9-8). In type S mutants, little association between the two proteins was observed even when cultured in the absence of tunicamycin (Figure 3, lowest panel, lanes 10 and 14).
We next examined in vivo association of Kar2 with a model unfolded protein. Aspartic proteinase-I (RNAP-I) of the filamentous fungus Rhizopus niveus is a secretory protein synthesized as a precursor form containing a presequence (translocation signal; 21 amino acid residues) and prosequence (45 amino acid residues) in the N-terminus of the mature portion (323 amino acid residues). When the fulllength form of the RNAP-I precursor is expressed heterologously in yeast cells, the protein is secreted extracellularly as the mature form (Horiuchi et al., 1990 ). The prosequence of RNAP-I, similar to that of other secretory proteins, acts to support proper folding of the mature protein (Fukuda et al., 1994 ). Therefore, a truncated mutant RNAP-I lacking the prosequence (Δpro) remains incompletely folded and aggregates in the ER when expressed in yeast cells (Umebayashi et al., 1997 ). Kar2 has been shown to coaggregate with Δpro (Umebayashi et al., 1997 ), and we have used this heterologous protein as a model chaperone substrate to monitor in vivo association with Kar2.
As shown in Figure 4, Δpro was expressed from the GAL1 promoter in the kar2 mutant and wild-type strains. As a control, yeast strains were transformed with an empty vector. Cells were cultured in galactose-based medium (SCG) at 23°C and shifted to 37°C for 60 min, except for the kar2-133 mutant, which was shifted to 34°C in order to avoid Δpro independent aggregation of the Kar2-133 protein at 37°C. Lysates of the cells were then subjected to ultracentrifugation at 100,000 × g, and both supernatant and pellet fractions were collected. Note that to dissolve membraneous components, the cell lysates were prepared in the presence of the nonionic detergent Triton X-100. Anti–RNAP-I Western blot analysis of the supernatant and pellet fractions showed that Δpro was almost perfectly aggregated in the wild-type and type S mutant cells (Figure 4A). In the type A mutant cells, Δpro was also detected in the supernatant fractions. Although we speculated that the accumulation of this soluble version of Δpro might be caused by translocation block in these mutant cells, a protease protection assay showed that this soluble Δpro resides in a closed membranous compartment (unpublished data). Thus the soluble Δpro may locate in the ER lumen in the type A mutant cells under the restricted temperature by unknown reasons. On the other hand, anti-Kar2 Western blotting showed little or no accumulation of pre-Kar2 even in the type A mutant cells (Figure 4B, lanes 3–8). This may be because cells grow slower in galactose medium than in glucose medium. Western blotting of the pellet fractions with anti-Kar2 showed that Kar2 coaggregates with Δpro in both wild-type cells (Figure 4B, lower panel, compare lanes 2-1 and 12-11) and type A mutant cells (Figure 4B, lower panel, compare lanes 4-3, 6-5, and 8-7). As shown in Figure 4C, Δpro was cross-linked with Kar2 by DSP, which indicates that Kar2 directly contacts Δpro. Furthermore, Δpro cofractionated with Kar2 in a sucrose gradient analysis of the microsomal fraction, which suggests that Δpro and Kar2 are present in the same complex (Umebayashi et al., 1997 ). In contrast, Δpro-dependent aggregation of Kar2 was not observed in the type S mutant cells (Figure 4B, lanes 9, 10, 13, and 14).
It has been previously reported that mutation of KAR2 results in its own transcriptional induction (Scidmore et al.,1993 for kar2-159 and Kohno et al., 1993 for kar2–1). Therefore we monitored KAR2 mRNA abundance in all of the kar2 mutant strains used in this study. As shown in Figure 5A, the kar2 mutant and wild-type strains were cultured at 23°C or shifted to 37°C, and gene expression was examined by Northern blotting. The mRNAs were then quantitated and normalized to ACT1 mRNA (Figure 5B, left). Culturing at 37°C for 60 min caused only weak induction of the KAR2 gene in the wild-type strains. In contrast, all of the kar2 mutant strains exhibited about twofold higher expression of the KAR2 gene than the wild-type strains when cultured at 23°C and exhibited greatly enhanced expression of the KAR2 gene when shifted to 37°C. However, as described above, the type A and type S mutant strains have different unfolded protein responses, implying that this transcriptional upregulation of the KAR2 gene in kar2 mutant strains cannot be explained simply by activation of the UPR pathway. Regulation of the KAR2 gene involves not only the UPRE but also the heat shock element (HSE), which is present in its promoter region (Mori et al., 1992 ; Kohno et al., 1993 ). We therefore examined whether the heat shock response pathway is activated in kar2 mutant strains. SSA1, 2, 3, and 4 are highly similar genes encoding cytosolic Hsp70, and all except SSA2 are induced by the heat shock response (Ziegelhoffer and Craig, 1997 ). Northern blotting using a probe for SSA1 revealed enhanced SSA expression in type A mutant cells cultured at 37°C for 60 min, but not in the wild-type or kar2-1 cells (Figure 5, A and B). In addition, the SSA genes were highly expressed both at 23 and 37°C in the kar2-133 cells (Figure 5, A and B). Activation of the heat shock response pathway in the type A and kar2-133 mutant strains was confirmed by an HSE-lacZ reporter assay, in which expression of lacZ is under the control of the KAR2 HSE. As shown in Figure 5C, both the type A and kar2-133 mutant strains exhibited higher lacZ expression than the wild-type strain, when they were cultured at 37°C. These observations indicate that KAR2 mRNA induction in these mutant strains is at least partially caused by the heat shock response.
Previously, two studies reported findings supporting a BiP/Kar2-dependent mode of Ire1 regulation (Bertolotti et al., 2000 ; Okamura et al. 2000 ). Of most significance was the observation that inactivation and activation of the UPR correlated with the binding state of BiP/Kar2 with Ire1. However, the causality between regulation of Ire1 (or PERK) and its physical interaction with BiP/Kar2 was not firmly established. The assertion that BiP/Kar2 plays a negative regulatory role upstream of Ire1 (or PERK) is derived from the observation that activation can be attenuated by overproduction of BiP/Kar2. However, an alternative explanation must be considered. The overproduction of BiP/Kar2, in some circumstances, is sufficient to alleviate ER stress (Umebayashi et al., 1999 ). Therefore, the observed attenuation of the response might be a secondary effect.
Hence, we sought to provide more definitive evidence for the BiP/Kar2-dependent Ire1 regulation model. In the present study, we analyzed a pool of KAR2 mutants that display allele-specific phenotypes with respect to Ire1 association and activation. As shown in Figure 2, examination of five kar2 mutant alleles revealed two different types of UPR abnormalities, as monitored by the UPRE-lacZ reporter assay and Northern blot analysis for HAC1u mRNA cleavage. When cultured at the restrictive temperature of 37°C, the kar2 type A mutant strains (Figure 1) exhibited weak activation of the UPR pathway that was not enhanced by treatment with tunicamycin. In contrast, the type S mutant strains (Figure 1) showed high activation of the UPR pathway both in the absence and presence of tunicamycin. We then investigated the in vivo interaction between Kar2 and Ire1 by monitoring coimmunoprecipitation of Kar2 with HA-tagged Ire1 from cell lysates (Figure 3). Consistent with our previous report (Okamura et al., 2000 ), dissociation of Kar2 from Ire1 in response to tunicamycin was clearly observed in the wild-type cells. In contrast, this dissociation was not observed in the type A mutant cells, whereas in the type S mutant cells, association between Kar2 and Ire1 was almost undetectable even in the absence of tunicamycin. One attractive interpretation of these results, which we believe most likely, is that the abnormalities in the UPR pathway observed in kar2 mutants are the results of altered association and dissociation between Kar2 and Ire1. This mechanism would be consistent with the BiP/Kar2-dependent Ire1 regulation model.
We should note that Kar2 has multiple biological roles in the cell, as described in the Introduction, and thus other interpretations for the UPR abnormalities in the kar2 mutant strains are possible. Because Kar2 acts to promote both proper folding and ERAD of unfolded proteins (Simons et al., 1995 ; Plemper et al., 1997 ; Brodsky et al., 1999 ; Nishikawa et al., 2001 ), it is likely that mutations in the KAR2 gene cause activation of the UPR pathway through increased amount of unfolded proteins in the ER. Indeed, all of the kar2 mutant strains displayed higher activation of the UPR pathway than the wild-type strains when cultured at 37°C in the absence of tunicamycin (Figure 2). However, this idea alone does not explain the different levels of UPR activity between the type A and S mutant strains. In addition, Kar2 plays important roles in the protein translocation machinery (Hamman et al., 1998 ; Matlack et al., 1999 ). As shown in the second panel of Figure 3, pre-Kar2 accumulated in the type A mutant cells but not in the wild-type or type S mutant cells when cultured at 37°C. We also observed accumulation of an unglycosylated form of CPY only in the type A mutant cells (unpublished data). These findings are consistent with previous reports (Vogel et al., 1990 ; Sanders et al., 1992 ) and suggest that type A and type S mutants correspond to the class I and class II kar2 mutants, respectively, defined by Brodsky and Rose (1997 ), based on their phenotypes. These observations strongly suggest that protein translocation into the ER is blocked only in the type A mutant strains. Thus, it is possible that in Type A mutants, protein influx into the ER is reduced to the extent that even under ER-stressed conditions unfolded proteins do not accumulate to levels sufficient to activate the UPR pathway. Therefore, as we discussed below, we think that experimental approaches not involving kar2 mutant strains are required for further support of the BiP/Kar2-dependent Ire1 regulation model.
We next investigated the in vivo association of Kar2 mutant proteins with chaperone substrate using Δpro as a model unfolded protein. As shown in Figure 4, coaggregation of Kar2 with Δpro was observed in the wild-type and type A mutant cells, but not in the type S mutant cells. Impaired association of Kar2 with its chaperone substrate in the type S mutant strains is reasonable because these kar2 mutant alleles carry single-point mutations corresponding to the Kar2 substrate-binding domain (Figure 1; Bukau and Horwich, 1998 ). On the other hand, te Heesen and Aebi (1994 ) expressed a nonglycosylatable mutant version of CPY (CPY*) in several kar2 mutant strains and detected a complex of CPY* and Kar2 in kar2-159 cells but not in wild-type or kar2-1 cells. Furthermore, Simons et al. (1995 ) reported that in vivo association of Kar2 with DTT-denatured CPY was enhanced in kar2-159 and kar2-113 mutants. Thus it is likely that dissociation between Kar2 and its chaperone substrate is impaired in the type A mutant strains. This idea is further supported by the low affinity of purified Kar2–159 protein to ATP (Brodsky et al., 1995 ), because in Hsp70 chaperones, binding of ATP to the ATPase domain promotes release of substrate from the substrate-binding domain (Bukau and Horwich, 1998 ). In the case of kar2-113, Brodsky et al. (1995 ) reported no defect in ATP binding or ATPase activity with purified Kar2-113 protein. Thus the abovementioned observation in kar2-113 strain may be caused by impaired interaction of the Kar2–113 ATPase domain to cochaperone(s).
It is notable that the type S Kar2 mutant proteins showed defective association with both Ire1 and a model unfolded protein. In addition, the type A Kar2 mutant proteins, of which dissociation from Ire1 was impaired, are reported to exhibit insufficient release of unfolded proteins. Thus it is likely that Ire1 association with Kar2 is analogous or identical to that of a chaperone substrate. On the basis of this idea, we envision that Ire1 competes with unfolded proteins for binding to the same site on Kar2. This is consistent with the BiP/Kar2-dependent Ire1 regulation model and explains the various results reported in this study. The explanation for the results in this study is presented in Figure 6. In wild-type cells under nonstressed conditions, Kar2 binds with Ire1 and represses Ire1 activity. In ER-stressed conditions, unfolded proteins compete with Ire1 for binding to Kar2, Ire1 is released and activated, and activated Ire1 in turn induces the UPR signaling pathway (Figure 6A). In contrast, type A mutant Kar2 binds constitutively to its chaperone substrates, including Ire1, and does not release them even under ER-stressed conditions. This results in the constitutive repression of Ire1 activity (Figure 6B). In the third case, Type S mutant Kar2 binds poorly with its chaperone substrates, and this results in free Ire1 molecules and constitutive induction of the UPR signaling pathway even in the absence of ER stress (Figure 6C).
This interpretation implies that the major role of the Ire1 lumenal domain may merely be homodimerization, which is inhibited by Kar2. Generally speaking, the association of proteins involves interactions between their hydrophobic segments, which are often masked by Hsp70 chaperones. Thus it may be possible to sustain the function of Ire1 by a replacement of its lumenal domain with another dimerforming polypeptide. Indeed, Liu et al. (2000 ) reported that a chimeric protein in which the lumenal domain of Ire1 was replaced with a functional leucine zipper motif from the transcription factor Maf or Jun was able to moderately activate the UPR pathway in response to ER stress. However, in view of the strong response of native Ire1 to unfolded proteins, we believe that its lumenal domain has additional properties that further facilitate its function as a sensor. For instance, the balance between the rates of Ire1 homodimerization and Kar2-Ire1 association could be an important factor in determining Ire1 activation in response to accumulated unfolded proteins.
Both our present and previously published observations indicate that any mutation of KAR2 results in induction of the KAR2 gene, especially at the restrictive temperature (Figure 5; Kohno et al., 1993 ; Scidmore et al., 1993 ). We also observed that the heat shock response pathway is highly activated in the type A and kar2-133 strains but not in the wild-type or kar2-1 strains, as monitored by expression of the SSA genes and by an HSE-lacZ reporter assay (Figure 5). This indicates that both the UPR pathway and the heat shock response pathway contribute to the upregulation of the KAR2 gene expression in kar2 mutant strains, albeit this contribution varies with the particular mutant. It should be noted that generally speaking, heat shock induced by shifting cells to high temperature is quickly attenuated even if culturing at this temperature is continued. Indeed, we could not detect induction of the SSA genes by culturing the wild-type cells at 37°C for 60 min (see Figure 5, A and B). One possible explanation for the high state of activation of the heat shock response pathway in the type A mutant strains cultured at 37°C is that untranslocated secretory and membrane proteins accumulate and act as unfolded cytosolic proteins to induce this pathway (Morimoto, 1997 ; Oka et al., 1997 ). However, we are currently unable to explain the reason why the heat shock response pathway is upregulated by the kar2-133 mutation but not by the kar2–1 mutation both at 23 and 37°C.
In conclusion, we believe our findings in the present study are strong evidence to support the BiP/Kar2-dependent Ire1 regulation model and suggest that recognition of unfolded proteins by the UPR pathway is based on competition between Ire1 and unfolded proteins for binding with Kar2. Interestingly, Shen et al. (2002 ) have recently proposed that BiP negatively regulates ATF6 activity. Thus, it appears that negative regulation by BiP/Kar2 is a common feature of transmembrane signal transducers for the ER stress response.
To clarify the detailed mechanism by which Ire1 is regulated by BiP/Kar2, we think that additional experimental approaches are needed. Characterization of the purified Ire1 lumenal domain, such as undertaken by Liu et al. (2002 ), may be an important experimental approach. Furthermore, mutational analysis of the Ire1 lumenal domain, an effort we are currently pursuing, may also provide new insights into the molecular mechanism regulating the Ire1 activity.
We thank Jeff Brodsky (University of Pittsburgh, Pittsburgh, PA) for the information about kar2 mutants; Kazutoshi Mori (Kyoto University, Kyoto, Japan) for plasmids; Masamichi Takagi and Akinori Ohta (The University of Tokyo, Tokyo, Japan) for plasmids and an antibody; Miki Matsumura for excellent technical assistance; Davis Ng (Pennsylvania State University, State College, PA) for useful comments on the manuscript; and Marc Lamphier for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research (13480239 to K.K.) and Scientific Research on Priority Areas (14035239 to Y.K., 14037240 to K.K.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by grants from Novartis Foundation (K.K.), Senri Life Science Foundation (Y.K.), and Yamada Science Foundation (K.K.).
Abbreviations used: BiP, immunoglobulin heavy-chain binding protein; CPY, carboxypeptidase Y; DSP, dithiobissuccinimidyl propionate; DTT, dithiothreitol; ECL, enhanced chemiluminescence; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; HA, hemagglutinin; HSE, heat shock element; ONP, o-nitrophenol; PERK, PKR-like ER kinase; RNAP-I, aspartic proteinase-I; SC, synthetic complete; SCG, synthetic complete galactose; SD, synthetic medium plus dextrose; UPR, unfolded protein response; UPRE, UPR element; Δpro, truncated mutant RNAP-I lacking the prosequence.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Opinion from Moore: SDN coverage trivializes marijuana use
Ryan Summerlin July 3, 2013
SDN trivializes marijuana use
It's surprising and alarming that only 5 weeks after you print an opinion letter that stated you are overly focused on marijuana use, yesterday you have a full banner headline "State releases rules on recreational pot." Under that is a photo of attendees at the Dillon amphitheater at a recent concert with the headline "COMFORTABLY NUMB." This not-so-clever journalistic artifice is a disservice to our county and likely not consistent with your own journalistic values. The previous letter you published from Bainbridge stated he was concerned about your front-page attention to marijuana use in a letter he entitled "Pot shots": he believes that front page attention to this issue is not in our county's best interest. Since that letter had no impact on your policies, allow me to provide additional food for thought.
I am a medical school faculty with 30 years of experience in substance abuse research. I also created a specialized substance abuse treatment facility, where we treated a number of individuals whose primary drug of choice was marijuana. Whether marijuana is "safer" than alcohol is not the real issue here. Polydrug use is the issue. Many marijuana users also drink alcohol at the same time. Drug potentiation, or the compounding of effects of drugs used together, impairs one's thinking and actions much more than just "one plus one." I have volunteered at the Dillon concerts, and I've seen firsthand the state some people are in at the end of an evening of drinking beer. In a county that has as much interest in microbreweries as Seattleites have in espresso, there may well be serious problems for us as marijuana use becomes more mainstream. Consider that our county already has a substantial per capita DUI rate, and the increase in concurrent alcohol and marijuana use will expand this problem, sometimes with tragic consequences.
Your newspaper reports on every tree hugging, puppy loving, and outdoor activity going on in the county. The focus on the positive is good, and it sets your publication apart from many media outlets in a very good way. But your trivialized and sustained attention to marijuana use as a form of recreation borders on sensationalism and is probably not in keeping with your own values of "do no harm." Educate your staff and to quote your own article, "act responsibly." This social experiment with marijuana legalization has a long way to go.
Dennis Moore
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Was going 2 celebrate my 1st night on the bus with a 007 flick, but the TV doesn't work so I settled 4 reading the truck's owners' manual.
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We found 1 that has recently dropped in price. Sign up to see this price drop and to receive future price drop notifications.
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1994 Dodge Dakota Sport 2dr Regular Cab SB
1994 Dodge Dakota Sport 2dr Regular Cab SB
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1994 Dodge Dakota Sport 2dr Regular Cab SB
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Powerful Engine And No Blind Spots
Maybe Alittle More Aftermarket Support For V6 Models.
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Celia E. LuntMarch 04,2013
Celia E. Lunt Rites
BARRE TOWN The funeral service for Celia E. Lunt, 95, of Miller Road, was held on Saturday, March 2, 2013, at 11 a.m. in the Hooker and Whitcomb Funeral Home, 7 Academy St., Barre. She died Tuesday, Feb. 26, 2013, at the home of her son and daughter-in-law, Michael and Debbie Lunt.
The Rev. David Vanderlinde-Abernathy, pastor of the Barre Congregational Church, led the service. The prerecorded song When the Snow Is on the Roses was played.
Entombment took place in the Hooker and Whitcomb Vault to await later burial in the Elmwood Cemetery in Barre.
Following the service, family and friends gathered at the home of Michael and Debbie Lunt for a time of sharing and refreshments.MORE IN ObituariesMONTPELIER Kenneth Michael Hickey, 72, of Granby, Connecticut, beloved husband for 41 years of... Full StoryMONTPELIER Rollande T. Fraser passed away peacefully on Monday, Sept. Full StoryWORCESTER Emily L. Wagner, 87, formerly of Worcester and currently of Montpelier, died Sept. Full Story
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Videos tagged "Nate Ferrell"
The Dirty Pillow is a strange snowboard in a non-existent category of its own. Progressive park / powder freestyle. This twin Banana, mellow Magne-Traction design features long floaty tip and tail…+ More details
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Tags are keywords that describe videos. For example, a video of your Hawaiian vacation might be tagged with "Hawaii," "beach," "surfing," and "sunburn."


text:
VivaKi has added a handful of new marketing partners to the latest project from its "Pool" initiative, a research effort to discover the online video unit of the future.
The first phase of The Pool — or in VivaKi parlance, Lane One — dealt with long-form video. That phase has been completed, though VivaKi will not reveal the findings until February of next year. The Lane 2 effort was launched earlier this year, with results also to be shared in 2010.
The Pool is shorthand for "pooling resources," according to VivaKi, which is a unit of Publicis Groupe created to leverage that holding company's media properties. Executives from Starcom MediaVest Group, Zenith Optimedia, Moxie Interactive and Digitas have been collaborating with marketers and publishers to test various video solutions to determine which are the most effective and engaging.
The new partners are Denny's, Walgreens, Wal-Mart, Warner Bros., YouTube and YuMe. They join a list that already includes Bank of America, General Mills, AOL, CBS Interactive and Microsoft Publishing.
"Lane 2 is a testament to collaboration and guiding our clients and partners to focus on engagement models that are consumer led," VivaKi Nerve Center President Curt Hecht said in a written statement announcing Lane 2 in July. "We have increased the number of partners based on internal demand at VivaKi and also the number of media companies in both returning partners as well as new ones which brings more diversity to the types of companies participating."
Although it is unclear just what The Pool is testing, statements by executives involved in the effort suggest it is neither pre-roll nor overlay advertising. "I feel confident that we are going to find an ad model that beats pre-roll," said Tracey Scheppach, SVP, video innovations director at Starcom MediaVest Group, at an OMMA video conference last month.
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YouTube ads drive way more searches per impression than television ads
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text:
Oxford American Handbook of Clinical Examination and Practical Skills
Oxford American Handbooks of MedicineThe Oxford American Handbook of Clinical Examination and Practical Skills is a comprehensive pocket guide for medical, physician assistant, and nurse practitioner students. It is designed to help students transition from classroom to clinical internships, preceptorships, and clerkships. Providing clear and user-friendly guidance on all aspects of history taking, physical examination, common
First Aid for the USMLE Step 2 CK, Ninth EditionFrom the authors who helped you pass Step 1, this is your high-yield review for the USMLE Step 2 CK. Completely revised to cover all core areas on the boards, including information on newly tested patient safety and quality improvement topics, First Aid for the USMLE Step 2 CK: Ninth Edition features: High-yield, bulleted presentation of diseases and disorders you need to remember Integrated flash
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text:
ATLANTA -- Terry Collins said newly promoted Josh Edgin will be used in integral roles, not babied. Collins speculated the left-handed rookie could be called upon in the seventh inning to face some combination of lefty hitters Michael Bourn, Jason Heyward and Freddie Freeman. That would allow Collins to save Tim Byrdak for a more critical matchup with those batters an inning or two later.
"Damn right. That's why he's here," Collins said about throwing Edgin into the fire. "Guess what? It's the middle of July. It's fire time."Win McNamee/Getty Images
Josh Edgin will get tested right away with the Mets, according to Terry Collins.
Edgin tossed 10 1/3 scoreless Grapefruit League innings in major league camp this spring training, even though he wasn't officially added until midway through camp. He struggled at points during the regular season with Triple-A Buffalo, which Collins assigned to bad slider command over the middle of the plate, and falling behind in the count while the staff refined his mechanics.
"He's had ups and downs, like everybody else in this game, during the season," Collins said. "I know he's throwing the ball good lately. So we'll find out what he's got."
Collins praised Edgin's mound poise, and joked that no one ought to charge the mound against him, because he's a former state wrestling champion in high school in Pennsylvania.
Edgin said he won the independent/private schools state title, then finished fourth nationally among those types of schools while wrestling in the 275-pound weight class. He weighed in at 235.
Who did Edgin lose to in the national semifinals? None other than future UFC fighter Justin Wren.
Edgin will wear No. 66, the first player in Mets history to do so. He said it was randomly assigned and not requested.
Justin Turner walked by Edgin's locker pregame Friday and joked he looked like Braves catcher Brian McCann.
As for his spring-training performance getting him on the radar, Edgin said: "A 30th-round pick, I've got to do what I can do, right? I think I opened some eyes."


text:
** Changed in: linux (Ubuntu) Assignee: (unassigned) => Paolo Pisati (p-pisati)
-- You received this bug notification because you are a member of Kernel Packages, which is subscribed to linux in Ubuntu. https://bugs.launchpad.net/bugs/1625177 Title: OOPS on beaglebone on boot of 4.4.0-36-generic under snappy ubuntu core xenial Status in linux package in Ubuntu: Confirmed Bug description: trying to assemble a new series 16 ubuntu core image for the beaglebone black i am constantly running into an OOPS on first boot. seemingly related to the NIC driver since the NIC does not work after the OOPS. syslog can be found at http://paste.ubuntu.com/23202737/ a test image exposing the bug can be found at: http://people.canonical.com/~ogra/snappy/all-snaps/daily/current/ To manage notifications about this bug go to: https://bugs.launchpad.net/ubuntu/+source/linux/+bug/1625177/+subscriptions -- Mailing list: https://launchpad.net/~kernel-packages Post to : email@example.com Unsubscribe : https://launchpad.net/~kernel-packages More help : https://help.launchpad.net/ListHelp


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Build Content/Articles for Affiliate Based Website
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Sept 5 (Mon): No School for Labor Day;
Sept 8 (Thurs): Back to School night;
Sept 27 (Tues): Family Reading night
September EventsAugust 19th, 2016
Sept 5 (Mon): No School for Labor Day;
August EventsAugust 5th, 2016
Aug 6 (Sat): TLC day, 9am-2pm;
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Aug 22-26 (M-F): Minimum Days
Welcome Incoming Kindergarten Families!June 5th, 2016
We are thrilled you're going to be joining us at Joaquin Miller. It is an amazing school and we think you'll be very happy with your choice. To ease the transition to kindergarten, we'd like to invite you to join us this summer for an informal play date. It might make that first day just a little bit easier if there are a few friendly faces out on the playground or in the classroom. And, it'll be nice for us to meet a few new parents, too.
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- Saturday, 7/30 – 10am-12pm at Joaquin Miller Park Playground
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Joaquin Miller Park Playground is located on Joaquin Miller Rd between Crane Way and Butters Dr. http://www.yelp.com/biz/joaquin-miller-kids-playground-oakland
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- Stasia Tamboury (Logan – 1st grade, and Cole – incoming kindergarten) email@example.com


text:
- Anonymní
- Nizozemí, Muž Skupina, 25-30
- Nomád nováček
- 3 Recenze
Having stayed in both Ivano Frankivsk hostels, we definitely preferred this one. It is cleaner, closer to the city centre, and staff is nicer. It is somewhat hidden behind a row of flats, but only a short walk from the city centre. Staff was very helpful with getting us a cab at an ungodly hour. Rooms and facilities are fine. I wish we would have stayed longer!
Mobilní aplikace Hostelworld
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text:
Solitaire of Solitude
By Ifelse Media Ltd.
Open iTunes to buy and download apps.
A quick and fun solo card & dice game of getting rid of all those tiresome friends.
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- © 2012 Ifelse Media Ltd.
Compatibility: Requires iOS 4.3 or later. Compatible with iPhone, iPad, and iPod touch.


text:
This report is based on the study funded by the Natl. Center for Food Protection and Defense (NCFPD).
Special Supplement
You have full text access to this OnlineOpen article
Food Product Tracing Technology Capabilities and Interoperability
Version of Record online: 21 NOV 2013
© 2013 Institute of Food Technologists®
Journal of Food Science
Special Issue: This supplement is possible due to the sponsors of the Institute of Food Technologists' (IFT's) Traceability Improvement Initiative, which include our Gold level sponsors BASF Nutrition and Health and Underwriters Laboratories (UL) as well as our Silver level sponsor the National Fisheries Institute (NFI) Seafood Industry Research Fund (SIRF).
Volume 78, Issue s2, pages B28–B33, December 2013
Additional Information
How to Cite
Bhatt, T. and Zhang, J. (2013), Food Product Tracing Technology Capabilities and Interoperability. Journal of Food Science, 78: B28–B33. doi: 10.1111/1750-3841.12299
Publication History
- Issue online: 20 DEC 2013
- Version of Record online: 21 NOV 2013
- Manuscript Accepted: 27 SEP 2013
- Manuscript Received: 5 SEP 2013
Funded by
- Natl. Center for Food Protection and Defense (NCFPD)
- GS1 EPCIS 1.0.1 Standard: http://www.gs1.org/gsmp/kc/epcglobal/epcis
- Group. 2012 (August 20). Pilot projects for improving product tracing along the food supply chain. Available from: http://www.ift.org/knowledge-center/focus-areas/food-safety-and-defense/~/media/Knowledge%20Center/Focus%20Areas/Traceability/IFT_FDA_ProductTracingPilotsFinalReport.pdf. Accessed May 3, 2013. , ,
- Office of Inspector General. 2009. Traceability in the food supply. Available from: http://www.hhs.gov/asl/testify/2009/03/t20090326a.html. Accessed May 15, 2013.


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Re: FERGER - GERMANY>MD
In reply to:
Re: FERGER - GERMANY>MD
I'm not sure if this is the correct Adolph Ferger. My grandfather William had 4 brothers: Adolph, Bernard, John, and one they called Dootsie(my mother can't remember his real name). Their father was John. All brothers lived and died in Maryland I believe. My mother seems to think he wouldn't have been as old as your Adolph was although the year of his death seems about right. Also, Adolph had no children.
More Replies:
Re: FERGER - GERMANY>MD
Patricia ferger 11/19/03


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|Viewing Single Post From: OLTL: Episode Discussion
|Aug 17 2013, 08:21 PM
Oh, I know. It was really more of a rhetorical question lol. It's just so stupid to me, though. I mean what do people think this is? The Donna Reed Show?
|OLTL: Episode Discussion · AMC & OLTL: Discussion


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Defensive line is one of the hardest positions to recruit. It is tough to find quality guys, especially at the defensive tackle position, as numbers are limited in that area for what most upper level D1 schools need.
The Tigers appear close to possibly gaining one of the better defensive tackle prospects in the 2011 class after receiving an official visit from Louisiana DT Gerrand Johnson 6'2" 270 4.8 from Rayville.
Johnson, who committed to University of Louisiana-Monroe a few months back has received offers from many of the top schools in the country, and continues to be recruited by them and many others. He has kept a relationship with Tiger coach, Josh Henson, and took an official visit to Missouri this past weekend for the Colorado game.
ShowMeMizzou.com has been following Gerrand's recruitment for a while and spoke with his after his visit.
"I loved it! It was was amazing!", said Johnson of his trip to Mizzou. "I arrived on Saturday morning, it moved pretty fast getting there. I took a nap and then got to meet the team,and all the coaches.I really liked the defensive coaches. I learned how coach Pinkel likes to keep his coaches together for a long time. That impressed me. It was like a family and seemed like family is important there. Then I got to meet the other players who were visiting (Cyril Lemon and Brandon Durant),and then it was off to the game. The game was great. It was exciting, and I really like their defense. It's an attacking defense and that's how I play. I would fit right in with it."
Gerrand continued talking about what impressed him about his visit.
"The next day I got to tour the school and facilities. It was awesome. Academics are very important to me and everything I saw seemed like the best. You can't have anything but success there with everything they have in place for the athletes. I really enjoyed it all. I felt like I was at home."
Johnson got the chance to form a friendship with Tiger freshman linebacker Darvin Ruise on his visit. He spoke highly of the Tiger defender.
"I got to hang out with Darvin (Ruise) a lot on the trip.He is an impressive guy and similar to me. He is a smart guy and serious, loves football, but we were cutting up and having fun,too. It was great.'"
While Gerrand is still committed to ULM, a decision will be coming soon regarding a possible change in direction for college, and perhaps switching his commitment to Missouri.
" A lot of schools are still recruiting me, but I will either be going to Missouri or ULM. I will think things over this week and then decide which school I will be going to after that.The door is definitely open."
I asked Gerrand if distance would be a factor and if his family was on board with him going away to school.
"My Mom supports me 100% in my decision no matter what it is. She is not concerned about the distance. She knows I am a responsible person.I am a typical teenager. I could party all the time and all that, but I choose not to. Football and school are my life, and I am happy with those. That other stuff isn't part of my life."
ShowMeMizzou.com will continue to follow Gerrand Johnson's recruitment.


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News :: European Maritime Day Conference to focus on coastal tourism
European Maritime Day Conference to focus on coastal tourism
(24/04/2013) Tourism, as one of the most resilient sectors of the economy, will be a central theme at this year's European Maritime Day Conference to be held in Valletta on 21 and 22 May 2013.
Investment in maritime and coastal tourism is key to generating "blue growth" and jobs while at the same time ensuring sustainable development of Europe's coastal regions. European Maritime Day provides a unique opportunity for all concerned actors to discuss these challenges and focus on specific areas such as the cruise sector, connectivity and mobility, as well as Mediterranean Sea basin.
The event is organised by the European Commission (DG for Maritime Affairs and Fisheries) in partnership with the Maltese Ministry for the Economy, Investment and Small Business, and the Maltese Ministry for Tourism. On May 21, the conference will host high-level key speakers: Ms Maria Damanaki, European Commissioner for Maritime Affairs and Fisheries, Maltese ministers Mr. Karmenu Vella, Minister for Tourism and Dr. Chris Cardona, Minister for Economy, Investment and Small Business, as well as other European ministers, personalities and maritime stakeholders . The second day, May 22 will be stakeholders' day and is organised around 19 workshops ranging from specific tourism areas such as nautical tourism to regional development strategies, innovative research and solutions to problems such as oil pollution. Exhibition booths will showcase innovative initiatives demonstrating the range of possibilities that the seas and oceans can offer.
- 22/04/2013 - Commissioner Damanaki at Seafood Expo 2013: Improving Market Transparency and Promoting a Balanced Diet
- 22/04/2013 - Blue Growth - Unlocking the potential of Europe's oceans, seas and coasts
18/04/2013 -
It-12-il Konferenza tal-FEMIP — Ekonomija blu għall-Mediterran: it-tisħiħ tal-kooperazzjoni talbaħar u dik marittima
Maria Damanaki, il-Kummissarju Ewropew għall-Affarijiet Marittimi u s-Sajd, Philippe de Fontaine-Vive, il-Viċi-President tal-Bank Ewropew tal-Investiment (BEI), u Andrew Winbow, Assistent Segretarju Ġenerali tal-Organizzazzjoni Marittima Internazzjonali (IMO),


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The necessities of latest House Plans
The value of a properly organized house is a bit more when compared with an unforeseen one. House plans are a ought to for many who want that their house must be integrated the most cost effective way and that the particular design in the house must take into account the typical convenience the dwellers. The style professionals and designers consider something more important into consideration before beginning to plan the particular design in the house.
Your house is viewed rolling around in its environment rather than in remoteness. The actual cosmetic appeal of the actual outsides is actually designed as per the environment normally your house can look strange. The quantity of sun rays necessary, the wide ranging connection between the particular rainfall along with weather, the actual scenic look at from your balconies, the noise lowering methods etc. are generally various other elements that the coordinator takes into account.
Your Emerging Trend of Tile Flooring
The use of ceramics goes while first 40000 British columbia and several wherever even located in the pyramids of Egypt. Right now ceramics is actually trusted and there's a developing trend of employing ceramic tile flooring inside properties along with industrial spaces. Over time, fresh approaches have already been created and found in to make the most away from hardwood flooring.
Hardwood flooring is quite versatile in the sense that it will come in numerous measurements, colours, forms, patterns plus the price tag. This will give the liberty to be able to design on your heart's written content concerning will be any ceramic tile flooring that will suit your design. This specific flooring material offers design versatility and sturdiness in which hardly any other flooring material can easily actually match up. You will end up offered limitless options to build a floor that will blend in with the actual adornment scheme from the house. Porcelain tile flooring can add fantastic price in your home, not simply aesthetically conversing but in addition expenditure wise. It does not fade, blemish, melt away or even produce mold and yes it super easy to keep. Tile flooring contributes some course with sustained durability. The price could be far more beforehand, however hardwood flooring will often last longer than all kinds of other varieties of flooring you might put in.
The gap In between Polycultures and Monocultures
If you are not familiar with the particular phrases "monoculture" and "polyculture" it isn't difficult adequate to comprehend. Tradition means plants, mono signifies single, along with poly implies dual.
Your schedule to your Backbone in Day to day living: Cleansing the Kitchen Floor
House work is among the major ways that lots of people hurt his or her backs, through not necessarily while using the appropriate muscle tissue or even important joints in the actions necessary for completing the duty.
The principle option for washing the floor is by using the kitchen mop. In case your kitchen is big, then you're more prone to use variance of the mop. However if you simply are just like us, which has a small kitchen, I just get down on my palms along with hips and rehearse a material; or if perhaps I'm laid back, I will press the cloth close to with my own toes, to completely clean the floor!
Your Cuban Artist and Interior Developer – Emilio Terry
Cuban-born Emilio Terry who was simply well-known regarding presenting the actual new design called the 'Louis VIII style', was a good interior developer, performer, landcape performer, and creator. His or her Louis XVII type had been each baroque and classical. Terry would have been a developer associated with furniture, objet's d'art and tapestries. This individual enhanced portion along with range, along with would interior design for ch�teaux along with flats and also made gardens as well as residences. They has been popular since the 'he' involving tastemaker Carlos p Beistegui's land house referred to as Ch�teau p Groussay. Terry's operate attained a one-of-a-kind functionality of romanticism, classicism as well as surrealism.
A choice of This particular language furniture, garden furniture and conservatory furniture
Whenever you think of the attractiveness or perhaps adornment of one's house, then your the very first thing also comes in your head is actually furniture. A new house is definitely incomplete without furniture. That not simply gives you comfort but in addition add to the luxury and beauty of the house. You will need furniture in each and every part6 of your house. Your selection of the proper associated with furniture is a vital thing in this particular consider. It should be comfortable as well as stylish. Furthermore, it will in addition 't be expensive. There are numerous types of numerous types of furniture you can purchase location.
For instance , France furniture, outdoor furniture, garden furniture, straightener furniture, wood furniture, sunroom furniture and many more. You need to be very careful whilst the selection of form of furniture. This will depend about the spot what your location is gonna exhibit that and so on the utilization you wish to label of the idea. When you go for purchasing furniture, it might be quite challenging for you to make a decision as a result of beautiful designs and chic styles of furniture. Your choice should also take care in the event of rates.
The actual Chameleon involving Interior Décor – Employing Wrought Flat iron Furniture along with Home Components
Wrought metal furniture, accessories as well as accents could go with so many interior design designs via traditional Mediterranean to contemporary stylish. It is an extremely versatile substance, the chameleon of interior redecorating. It can be organic along with natural in the " old world " Tuscan design, passionate and trendy in the Victorian Artwork Nouveau influenced area as well as striking along with avant-garde inside a modern-day living space.
The word 'wrought' literally means worked; a generations outdated art of hand-forging material. Today, it really is combined with aluminum for optimum performance. Consequently, as well as the artistic advantages, it can be functional; powerful, long-lasting, durable and easy to clean.
The actual Interesting Street. John's Catherdal
Malta is actually endowed to have many well-liked tourist attractions regarding nationwide and worldwide status. E. John's Cathedral is among these marvelous sightseeing attractions which was integrated the 15th one hundred year with a prominent creator Girolamo Casser. Awesome Grasp Jean del Cassire had commissioned just for this church. The particular reason guiding building this church was to provide holiday accommodation to the knights in combat.


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Adapting to Evolving Business Environments Is...
LC-MS Gains Favor Due To the...
Wall Street Decline Takes Its Toll...
As Immunotherapy Revolutionizes the Cancer Treatment...
Osmetech received FDA clearance for its eSensor® Cystic Fibrosis Genotyping Test for use on the eSensor® XT-8 System. This multiplex test can be used for cystic fibrosis carrier screening for adults of reproductive age as an aid in screening offspring for cystic fibrosis. It may also be utilized as a confirmatory diagnostic test for cystic fibrosis in newborns and children.
This is the second FDA 510(k)-cleared multiplex test the company has for use on the eSensor XT-8 System and follows clearance of a Warfarin Sensitivity Test. The Warfarin Sensitivity Test is used as an aid in the identification of patients at risk for increased sensitivity to the blood-thinning drug warfarin.
Osmetech expects further commercial progress in 2009 by expanding the eSensor XT-8 test menu to include an extended Warfarin Sensitivity Test panel that includes CYP 4F2, a CYP 2C9 Genotyping panel, a venous thrombosis panel of Factor V, Factor II and MTHFR, and a Respiratory Virus Pathogen panel.
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California Chrome Draws Rail in Awesome Again
Art Sherman was a swirling mixture of exasperation and confidence when he learned of California Chrome's draw for the $300,000 Awesome Again Stakes (gr. I) Oct. 1 at Santa Anita Park.
Art Sherman was a swirling mixture of exasperation and confidence when he learned of California Chrome's draw for the $300,000 Awesome Again Stakes (gr. I) Oct. 1 at Santa Anita Park.
Two of racing's biggest stars—California Chrome and Beholder—will make their final starts before the Breeders' Cup on the Oct. 1 Santa Anita Park card as they take advantage of the opportunity to prep at the site of this year's World Championships.
California Chrome, North America's all-time leading money earner, arrived by van at Santa Anita Park Sept. 25 to begin final preparations for the $300,000 Awesome Again Stakes (gr. I) Oct. 1.
With the slightest morning chill Southern California would allow in late September, Los Alamitos Race Course's resident star logged his final timed workout for the upcoming Awesome Again Stakes (gr. I).
Images from weekend performances by Tepin and The Pizza Man, as well as Breeders' Cup Win And You're In races and a special guest towards the end!
Victor Espinoza, regular rider of 2014 Horse of the Year California Chrome, said the 5-year-old California-bred has never been doing better as he prepares for a start in the Awesome Again Stakes (gr. I) Oct. 1 at Santa Anita Park.
Los Al clockers caught California Chrome in splits of :25 3/5, :49 2/5, and 1:01 1/5 en route to his final time of 1:13 1/5 under regular exercise rider Dihigi Gladney.
Off his dominating victory in the $1 million TVG Pacific Classic Stakes (gr. I) Aug. 20 at Del Mar, California Chrome has moved back to the top of the Longines World's Best Racehorse Rankings for 2016.
On his day, at his home track, and a bit out of his normal workout routine, California Chrome still gave trainer Art Sherman just what he wanted Sept. 10 at Los Alamitos Race Course.
California Chrome has led the poll for all 27 weeks of 2016 voting
California Chrome was the unanimous choice as Del Mar's Horse of the Meeting for 2016.
The third Los Angeles County Fair meet at Los Alamitos Race Course is set to start Sept. 8.
Trainer Art Sherman sent California Chrome back out to breeze at Los Alamitos for the first time since his impressive Aug. 20 TVG Pacific Classic (gr. I) victory, and the 2014 Horse of the Year turned in a steady work Sept. 3.
The most famous resident of Los Alamitos will be honored on "California Chrome Day'' Sept. 10 at the track.
From Citation to California Chrome, read who has led in earnings over the decades.
Owner Perry Martin said he's sell the dam of California Chrome, in foal to Tapit, in order to buy more mares to support 'Chrome as a stallion next year.
Could it be the truly great horses of the past simply were faster and were bred to carry their speed longer distances, or could it be that today's horses are not given the opportunity to set new American dirt records?
California Chrome streaked to a five-length win over Beholder and Dortmund in Saturday's TVG Pacific Classic at Del Mar Thoroughbred Club to earn a spot in this fall's Breeders' Cup Classic.
Taylor boys' play for California Chrome pays off
Barn CC on the Del Mar backside had a party-like atmosphere Sunday morning—with a large group of connections and fans huddled around the 2014 Horse of the Year's stall—following what Sherman called the best performance of his career.
The margin was insignificant, because the field never had a chance against California Chrome Aug. 20 in the $1 million TVG Pacific Classic (gr. I).
With fingers crossed seemingly all across the country from fans hoping for a clash of West Coast superstars Aug. 20 in the TVG Pacific Classic (gr. I), the stage is finally set.
California Chrome worked five furlongs in 1:00 1/5 at Los Alamitos the morning of Aug. 14. The colt will ship to Del Mar Aug. 15 for Del Mar's TVG Pacific Classsic (gr. I) Aug. 20.
Trainer Richard Mandella has confirmed Spendthrift Farm's champion Beholder for the $1 million TVG Pacific Classic (gr. I), a race for which California Chrome and Dortmund are also pointing, Aug. 20 at Del Mar.
After a gutsy victory in the San Diego Handicap (gr. II), California Chrome improved his rating from 126 to 128 and is the second-highest rated horse in the world in the sixth edition of the Longines World's Best Racehorse Rankings.
|Breeder / Owner
|Better Bet
|Mike Harrington
|California Chrome
|Art Sherman
|Cinmars Dance
|J. Eric Kruljac
|Electric Eddie
|Ben D. A. Cecil
|Gangnam Guy
|Jerry Hollendorfer
|Life Is a Joy
|Jerry Hollendorfer
|Sir Barclay
|Vann Belvoir
|Tribal Mystic
|Ed Moger, Jr.
|FrontRunner S. (gr. I)
|Awesome Again S. (gr. I)
|Rodeo Drive S. (gr. IT)
|Zenyatta S. (gr. I)
|Chandelier S. (gr. I)


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To try the thousands of add-ons available here, download Mozilla Firefox, a fast, free way to surf the Web!Close
Welcome to Firefox Add-ons.
Choose from thousands of extra features and styles to make Firefox your own.Close
Reviews for Adblock Plus Pop-up Addon
180 reviews for this add-on
great Rated 5 out of 5 stars
to much good
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Esta muy buena! tu solo agregas el sitio que abre ventanas emergentes a la extensión y las bloquea! simplemente maravillosa.
Works perfectly if you read the instructions. Rated 4 out of 5 stars
As soon as you go into the options and set the settings, it'll work exactly how you want it. Want to block ALL popups? Can do that. Want to allow ALL popups? Can do that. Want to block popups from certain sites but allow all others? Can do that. Want to allow popups from certain sites but block all others? Can do that.
Only gripe I have is that you have to specify websites to allow/block if you want exceptions to the general rule (if you set it like that), but that's a minor gripe all things considered.
Useless Rated 1 out of 5 stars
Never blocks a pop-up. Pretty much sums it up.
doesn't block pop-unders, blocks all links Rated 1 out of 5 stars
Description says it blocks pop-unders, but I can't get that to work. Contacted the developer for help but never got a reply. This blocks all links, which is annoying. Even if you click a link, it blocks that. No support, doesn't do what it says, just removed it from my PC.
Needs Noscript Rated 2 out of 5 stars
Amazing Rated 5 out of 5 stars
blocks pop-ups, pop-up tabs, pop-under windows. it's a great app as long and the settings are set default actions for sites not listed to "block all pop-ups" instead of "handle pop-ups using Adblock Plus."
no chance of blocking tabs Rated 1 out of 5 stars
those annoying pop up tabs keep popping despite trusting this very misleading addon. this has no right to use that name. I try to set this addon to block them but it never accepts those rules. it gets stuck on that same settings even though i try to change and save my desired settings
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oftenly blocks pop ups :( needs improvemnet
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So, it's supposed to block even more adds and pop up windows? I'll try it.
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Works well for me
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No more annoying popup windows...
and yes, it works with uBlock Origin to answer an user's question below.
Per excellent advice of user Quantum Socrates below,
"Using the drop-down menu I chose to block all pop-ups, and it has worked perfectly."
Thank you all!
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you most just use it. it is the best site blocker
i love it an use it in my collection add ons
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Blocked the annoying pop ups on some websites that I had previously stopped using, for that very same reason!
Excellent alternative au popup Rated 5 out of 5 stars
For instant relief try APPA Rated 5 out of 5 stars
Jesse Hakanen, you're an Internet superhero!
Fellow pop-up haters, install this brilliant app. It is so easy to setup. I just clicked on Tools, then Add-ons to get to my Add-ons Manager page. Then clicked on Options for Adblock Plus Pop-up Addon. Using the drop-down menu I chose to block all pop-ups, and it has worked perfectly.
By the way, if like me, when you click on Options you see a message warning it's disabled or not installed, don't worry. Just use the drop-down menu and it should still work fine, as does mine. Might be a bug, and I'll be letting Jesse know about it.
Pop-ups are now fun! This app lists at the top of the website page the number blocked. So now, where before I was playing whack a mole, closing each one before it could load, instead I gleefully watch that number climb, knowing each time that another mongrel has been stopped dead in their tracks. :)
Jesse, I've been battling a site where every time you click on anything you get multiple pop-up pages - they must have been hacked or something - but unfortunately I need to access it for work, my students, etc. So you have definitely made my day a whole lot easier. You are fighting the good fight and winning!
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To create your own collections, you must have a Mozilla Add-ons account.


text:
The science we support is amazing. Our researchers study every aspect of our planet, and how humans are changing it - from the poles to the tropical rainforests, and from the depths of the ocean to the edge of space. Planet Earth brings you the latest stories, pictures and videos about their work.
Scientists have just returned from a groundbreaking research campaign to understand the Indian monsoon. Andy Turner reports back.
27 September 2016
Last year El Niño brought drought to south-east Asia, creating the conditions for enormous wildfires that send vast amounts of carbon into the atmosphere and endangered people's health across the region with terrible air pollution. Martin Wooster of King's College London and NERC NCEO reports.
27 Sep 2016
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Ski resorts Austria
Change lift info:
Funicular lifts Austria
All funicular lifts at ski resorts in Austria (cable lifts on tracks in Austria/FUL)
Description funicular lift
Funicular lifts/cable railway lifts are particularly unaffected by weather conditions and very wind stable. One or two vehicles travel on a stationary carriageway (predominantly on tracks). The vehicles are propelled by a haul rope, usually via a reversible system.
Funicular lifts/cable railway lifts in Austria
Add lifts and cable cars? Please report here
Austria - expand to (number of funicular lifts):
Map: funicular lifts in Austria
Austria - limit to (number of funicular lifts):
Austria: funicular lifts (11)
1-11 out of 11 ski resorts
Mölltaler Gletscher Express (YOC 1996)220pers. Funicular
Length: 4718 m · Carrying capacity/hour: 1600
Manufacturer: Leitner
Gletscher Shuttle (YOC 1990)30pers. Funicular
Length: 257 m · Carrying capacity/hour: 500
Manufacturer: Doppelmayr
Weltcupexpress (YOC 2000)6pers. Funicular
Length: 131 m · Carrying capacity/hour: 92
Manufacturer: ABS Transportbahnen
Wurzeralm (YOC 1995)165pers. Funicular
Length: 2938 m · Carrying capacity/hour: 1450
Manufacturer: Leitner
Replacement lift: 110pers. Funicular
Dorfbahn Serfaus (U-Bahn) (YOC 1985)200pers. Funicular
Length: 1300 m · Carrying capacity/hour: 2000
Manufacturer: Leitner
Ski resort: Serfaus-Fiss-Ladis [Serfaus]
Schlossalmbahn I (YOC 1984)120pers. Funicular
Length: 1265 m · Carrying capacity/hour: 1660
Manufacturer: Leitner
Ski resort: Bad Gastein/Bad Hofgastein – Schlossalm/Angertal/Stubnerkogel [Bad Hofgastein]
Gletscherexpress (YOC 1983)200pers. Funicular
Length: 3692 m · Carrying capacity/hour: 1590
Manufacturer: Leitner
Hoadl Olympiabahn (YOC 1975)100pers. Funicular
Length: 2105 m · Carrying capacity/hour: 1200
Manufacturer: Leitner
Rosshütte (YOC 1985)125pers. Funicular
Length: 2470 m · Carrying capacity/hour: 1200
Manufacturer: Leitner
Hungerburgbahn (Sektion I) (YOC 2007)127pers. Funicular
Length: 1800 m · Carrying capacity/hour: 1200
Manufacturer: Leitner
Replacement lift: Funicular
1-11 out of 11 ski resorts


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