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Core components of the secretory pathway have largely been identified and studied in single cell systems such as the budding yeast S. cerevisiae or in mammalian tissue culture. These studies provide details on the molecular functions of the secretory machinery; they fail, however, to provide insight into the role of these proteins in the context of specialized organs of higher eukaryotes. Here, we identify and characterize the first loss-of-function mutations in a KDEL receptor gene from higher eukaryotes. Transcripts from the Drosophila KDEL receptor gene KdelR – formerly known as dmErd2 – are provided maternally and, at later stages, are at elevated levels in several embryonic cell types, including the salivary gland secretory cells, the fat body and the epidermis. We show that, unlike Saccharomyces cerevisiae Erd2 mutants, which are viable, KdelR mutations are early larval lethal, with homozygous mutant animals dying as first instar larvae. KdelR mutants have larval cuticle defects similar to those observed with loss-of-function mutations in other core secretory pathway genes and with mutations in CrebA, which encodes a bZip transcription factor that coordinately upregulates secretory pathway component genes in specialized secretory cell types. Using the salivary gland, we demonstrate a requirement for KdelR in maintaining the ER pool of a subset of soluble resident ER proteins. These studies underscore the utility of the Drosophila salivary gland as a unique system for studying the molecular machinery of the secretory pathway in vivo in a complex eukaryote.
Citation: Abrams EW, Cheng YL, Andrew DJ (2013) Drosophila KDEL Receptor Function in the Embryonic Salivary Gland and Epidermis. PLoS ONE 8(10): e77618. doi:10.1371/journal.pone.0077618
Editor: Andreas Bergmann, University of Massachusetts Medical School, United States of America
Received: June 6, 2013; Accepted: September 10, 2013; Published: October 18, 2013
Copyright: © 2013 Abrams et al. 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: This study was funded by NIH RO1 DE013899 to D.J.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The rough endoplasmic reticulum (ER) is where synthesis of secreted and transmembrane proteins occurs through the process of cotranslational translocation. The lumen of the ER – the first compartment of the secretory organellar network – provides a selective environment for the folding and primary modifications of both secreted and transmembrane proteins. Thus, the ER contains many soluble resident proteins, such as chaperones, which facilitate the translocation and correct folding of nascent polypeptides (reviewed in 1). Soluble resident proteins are maintained in the ER through a retrieval system involving a carboxy-terminal signal and a transmembrane receptor that binds to that signal. In yeast, soluble ER resident proteins contain a four-residue carboxy-terminal Histidine-Aspartate-Glutamate-Leucine – HDEL – motif [2,3], which interacts directly with the seven transmembrane protein encoded by the Erd2 gene [4]. In mammalian systems, a Lysine-Aspartate-Glutamate-Leucine – KDEL – motif or some variant of KDEL, is both necessary and sufficient for the retrieval of soluble ER proteins. The requirement for the KDEL motif was demonstrated by removing it from a lumenal ER-resident protein expressed in COS cells and showing that the protein was secreted into the media [5]. The sufficiency of the C-terminal motif was demonstrated in experiments in which non-ER proteins were redirected to the ER simply by the addition of KDEL sequences to their C-termini and by showing that mutations in the KDEL sequences in these chimeric proteins resulted in a loss of ER localization [6]. Increasing KDEL receptor levels also affects ER localization; cells overexpressing the receptor have increased retention even of proteins with very weak retention signals [7]. These experiments clearly demonstrate the importance of the KDEL receptor system in ER retrieval. Nonetheless, a genetic loss-of-function model equivalent to that in yeast has been lacking in higher eukaryotes.
A number of studies have demonstrated that different organisms can potentially utilize ER retention motifs from other species. Studies comparing the yeasts S. cerevisiae and K. lactis, which utilizes a DDEL terminal motif, have shown that although S. cerevisiae is only capable of retrieving HDEL and not DDEL proteins, K. lactis can retrieve proteins with both types of retrieval signals [8]. In addition, although there are a very few examples of endogenous mammalian proteins with HDEL motifs, an HDEL sequence can function in mammalian ER retrieval as shown in experiments with SEHDEL fused to the C-terminus of lysozyme [9]. However, despite high levels of similarity in overall structure, the human KDEL Receptor 1 cannot functionally replace the S. cerevisiae gene [10]. Plants are thought to use both KDEL and HDEL retrieval signals [11], which may also be the case with Drosophila ER resident proteins. A survey of the Drosophila proteome suggests that there are proportionally similar numbers of proteins containing C-terminal HDEL and KDEL motifs ([12].
The Drosophila salivary gland is a useful system for studying the cell biology of secretion in an intact organ in vivo. A number of secretory pathway components are expressed to very high levels in the embryonic salivary gland, consistent with the specialization of the salivary gland for secretion [13,14]. The relatively large secretory cells of the salivary gland facilitate localization studies of secretory proteins and organelles (RM Fox and DJA, unpub.). The high level of secretion that occurs in the late embryonic salivary gland allows for direct immunohistochemical analysis of secretory function. Importantly, there is typically only a single Drosophila gene encoding each core secretory pathway component, whereas in vertebrates there are often multiple genes encoding the same or highly related proteins, complicating analysis of gene function due to functional redundancy. Thus, there is enormous potential for studying genetic interactions among secretory pathway component genes using the Drosophila salivary gland as a model system.
Here, we describe the embryonic expression pattern of the Drosophila KdelR and demonstrate that loss-of-function mutations in the gene lead to defects in the cuticle secreted by the epidermal cells. We also show that KdelR function is required to maintain the pool of a subset of soluble endoplasmic reticulum (ER) resident proteins. Although this function for KdelR is expected based on work in other systems, our studies reveal that not all soluble ER residents share the same requirement for this receptor in vivo. Our work demonstrates the utility of the Drosophila salivary gland as an in vivo system for studying the secretory pathway, potentially revealing similarities and differences among biological systems.
Sequencing and protein alignments
The KdelR cDNA (CK00230) was identified by its up-regulation in the embryonic salivary gland through the expression database at the Berkeley Drosophila Genome Project (BDGP)[15]. To determine the molecular lesions in the EMS alleles of KdelR, genomic DNA was prepared from 31Em1 and 31Em2 heterozygous adult flies using standard procedures [16]. The KdelR ORF was amplified using the following primer pair: erd(5)seq-5'GTTCCGTGACGCAGCCGCAG and erd(3)seq-5'GTGAGTGCAGTTCGGAAAACGG. PCR products were purified using the Qiagen (Valencia, CA) gel purification protocol and both strands were sequenced at the Johns Hopkins Core Sequencing Facility. KDEL-R sequences of various organisms were aligned using the Clustal_W [17] and Boxshade programs available at the Biology Workbench Version 3.2 (http://seqtool.sdsc.edu/CGI/BW.cgi).
Fly strains
The following deficiency stocks were used to map the KdelR gene: Df(2L)J3, Df(2L)J16, Df(2L)J17, Df(2L)J27 and Df(2L) J106 (Flybase; [18]). The following EMS mutant strains that map to cytological region 31E were a generous gift from the laboratory of T. Grigliatti: l(2)31Ek, l(2)31El, l(2)31Em1, l(2)31Em2, l(2)31Ep, l(2)31Eq, l(2)31er [18]. The lethal P-element line, l(2)k00311, was obtained from the Bloomington Drosophila Stock Center.
In situ hybridization and antibody staining
in situ hybridization and antibody staining were performed as previously described [19,20]. Antibody dilutions used in this study are as follows: α-β-galactosidase (1:5000), α-PH4α-SG1 (for light microscopy-1:20,000, for confocal microscopy- 1:5,000), α-Boca (1:500) and α-Wbl (1:25). PH4α-SG1 is a polyclonal antiserum made in rat [21] and α-Boca is a polyclonal antiserum made in guinea pig [22]. α-Wbl is a mouse monoclonal antibody [23]. βtub-E7 is a mouse monoclonal antibody obtained from the Drosophila Hybridoma Studies Bank (DHSB; Iowa City, IA). Biotin-conjugated secondary antibodies were obtained from Vector Labs (Burlingame, CA) and were used at a dilution of 1:500. All fluorescent secondary antibodies were from Molecular Probes (Eugene, OR) and used at a dilution of 1:400. Confocal images were captured using an Ultraview Confocal Microscope (Perkin Elmer) at the Johns Hopkins Microscope Facility. All other images were taken on a Zeiss Axiophot microscope with a Nikon Coolpix 4500 digital camera.
Expression and analysis of wild-type and mutant ER proteins in S2 cells
Full length and KDEL/KEEL deleted boca and wbl ORFs were PCR amplified from cDNAs SD08653 (boca) and IP02648 (wbl) using the LongAmp Taq PCR kit (NEB) and the primers in Table 1.
|boca cacc 5 prime
|boca stop 3 prime
|boca del529-540 stop 3 prime
|wbl cacc 5 prime
|wbl stop 3 prime
|wbl del760-771 stop 3 prime
Amplified PCR products were first cloned into pENTR/D-TOPO (Invitrogen) and subsequently into pAW Gateway vector (http://emb.carnegiescience.edu/labs/murphy/Gateway%20vectors.html) using the LR clonase II kit (Invitrogen). Each construct was verified by sequencing. S2 cells were tranfected in six-well plates with 400 ng DNA at 45 - 60% confluency following the QIAGEN Effectene Reagents protocol. Cells were subsequently grown for 48 hours at 25 °C. Based on immunostaining, 10-20% of cells were transfected. Cell and media were collected and centrifuged at 14,000 rpm for 10 min at 4 °C to separate the cell pellet and media supernatant. The cell pellet was washed and resuspended in 1X PBS and boiled with 2X sample buffer prior to loading. The media supernatant was TCA precipitated and the resulting protein pellet was mixed with 1X PBS and boiled with 2X sample buffer prior to loading. Samples were run on a 5% stacking gel / 12% resolving gel at 30 mA. Protein was transferred to methanol pretreated PVDF membrane O/N at 4 °C in transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, 20% Methanol). For immunostaining, membranes were first blocked with 5% milk:PBS-Tween for 1hr at RT and subsequently incubated with primary antibodies (αBoca (guinea pig) 1:10,000; αWbl (mouse) 1:250 ; αβtub-E7 (mouse) 1:1000) in 5% milk:PBS-Tween overnight at 4 °C. Membranes were washed and incubated with secondary antibody (goat αguinea pig HRP or goat αmouse HRP, both at 1:10,000) in PBS-Tween for two 2 hr at room temperature. Membranes were washed and HRP signal was subsequently detected using the Novex ECL HRP Chemiluminescent Substrate Reagent Kit (Invitrogen). These experiments were done twice.
Drosophila KDELR has high sequence similarity to mammalian, C. elegans and S. cerevisiae homologs
There is only a single Drosophila KDEL receptor gene, unlike in vertebrates where as many as three genes encoding highly related proteins have been discovered (Figure 1A,B; NCBI blast search) [24]. The ORF of the Drosophila KDEL receptor gene shows very high conservation with the vertebrate and C. elegans homologues and includes the seven membrane spanning domains as well as the residues that have been shown to interact directly with the KDEL motif of soluble ER resident proteins (Figure 1A, black lines and red dots) [3,4]. The Drosophila KDEL receptor shows somewhat less similarity to yeast Erd2p, but the position of the membrane spanning domains and the residues that interact with the HDEL/KDEL motif are conserved.
(A) Drosophila KdelR is highly conserved with respect to its vertebrate and C. elegans counterparts (A) and is homologous to S. cerevisiae Erd2. (Mm-mouse, Hs-human , Gg-chicken, Xl-Xenopus, Dr-zebrafish, Dm-Drosophila, Ce- C. elegans and Sc- S. cerevisiae). Vertebrates encode two-three Kdel Receptors, whereas only a single gene is found in flies, worms and yeast. Black bars over the sequences indicate membrane spanning regions [3]. Red dots denote residues involved in ligand (KDEL) binding [4]. (Green= completely conserved residues, yellow= identical residues, purple=similar residues). (B) A Phylip unrooted tree analysis of the KDEL Receptors from the major model organisms reveals that the Drosophila Kdel Receptor is slightly more related to the vertebrate proteins than are the worm and yeast receptors.
Identification of mutations in the Drosophila KDEL receptor gene
To gain genetic access to KdelR, we obtained a lethal P-element line, l(2)k00311, in which the P-element had inserted into the 5' untranslated region (UTR) of the KdelR transcription unit. The pattern of -gal expression from the l(2)k00311 line was very similar to that of the KdelR mRNA (Figure 2), with highest levels of expression in the secretory cells of the salivary gland. Elevated expression was also observed in the fat body (Figure 2B,F, stars), gut endoderm (Figure 2C,G,H, arrowheads), and epidermis. Both KdelR mRNA and -gal from the l(2)k00311 insertion were observed in early (0-2 hr) embryos (Figure 2A,E), indicating maternal contribution. To identify additional KdelR mutations, we mapped the l(2)k00311 lethality to a relatively small interval within cytological region 31E through complementation tests with several deficiencies in the genomic region to which KdelR localizes (Figure 3; [18]). l(2)k00311 failed to complement Df(2L)J3, Df(2L)J106 and Df(2L)J27, but complemented Df(2L)J17 and Df(2L)J16. Six genes were known to map to the interval defined by these deletions [18] and both alleles of one of the six genes, l(2)31Em, failed to complement the lethality of l(2)k00311. Sequence analysis of the KdelR ORF of the l(2)31Em mutants revealed premature stop codons in each case (Figure 3). The resulting truncated proteins would be missing two of the predicted transmembrane spanning regions as well as two critical ligand interacting residues, indicating that l(2)31Em1 and l(2)31Em2 are likely null for KdelR function.
KdelR is detected early in embryogenesis at the cellular blastoderm stage (A,E) and to high levels in the salivary gland beginning at embryonic stage 10 and continuing throughout embryogenesis (b-d, f-h, black arrows). KdelR is also expressed to elevated levels in the epidermis (cells on the embryo surface), fat body (clusters of staining in each segment in stage 11 embryos, indicated by black stars), proventriculus (white star) and a subset of gut endoderm cells (arrowheads). b-gal and RNA staining in the neural tube (nt) is also observed at late stages, although the RNA expression in the nt is not in the plane of focus of the embryos shown. Left column shows mRNA expression detected with a probe made from the KdelR cDNA clone CK00230 and the right column shows β-gal staining of l(2)k00311embryos.
KdelR maps to region 31E in the Drosophila genome and l(2)k00311 is inserted in the 5' UTR of the KdelR transcript. l(2)k00311 fails to complement deficiencies Df(2L)J3, Df(2L)J106 and Df(2L)J27, but complements deficiencies Df(2L)J17 and Df(2L)J16. Both EMS alleles of KdelR (31Em1 and 31Em2) encode ORFs with premature stop codons.
Lethal phase of KDELR mutants
Erd2 is not essential for viability in yeast, but is required for growth [25]. In contrast, the KdelR is essential for viability of adult Drosophila. To pinpoint the stage of lethality, we assayed the survival of larvae at different stages of development. To identify mutants, we examined l(2)k00311 larvae in a yellow (y-) null background, balanced with a CyO, y+ second chromosome. Mutant larvae, which are y- and have light brown mouthparts, can easily be distinguished from the wild-type larvae, which are y+ and have black mouthparts. Overnight collections were aged for 24 hours (first instar) and 48 hours (second instar), and larvae were examined under a stereomicroscope. Examination of first instar larvae revealed actively moving wild-type larvae and very sluggish mutant larvae (data not shown). Second instar collections consisted of only live wild-type larvae (data not shown). Therefore, lethality occurs during the first instar larval stage.
KDELR is required for normal larval cuticle development
Mutations that perturb normal secretory function in Drosophila have been shown to have profound effects on larval cuticle development [13,14,26]. Therefore, we examined the cuticles of KdelR mutants to ask if KDEL receptor loss-of-function affects cuticle development. KdelR mutant cuticles are much smaller and fainter than the corresponding wild-type cuticles (Figure 4A-C). The faint cuticle is most obvious when comparing the ventral denticle belts (Figure 4A-C, E, H, K). In addition, the mouthparts and filzkörper (tracheal filters) of l(2)k00311 (Figure 4G,I) and l(2)31Em2 (Figure 4J,L) are significantly underdeveloped compared to the corresponding wild-type structures (Figure 4D, F). These characteristics are consistent with the mutant defects seen with loss of other components of the secretory pathway as well as with loss of CrebA, which transcriptionally upregulates all known secretory component genes in specialized secretory tissues [13,14,26]. Thus, KdelR is essential for normal cuticle development.
Dark field (ventral) images of wild-type, l(2)k00311 and 31Em2 larvae (A-C). Note that l(2)k00311 and 31Em2 mutants are approximately 60% the length of wild type larvae and the ventral denticles are not as prominent as in their wild-type siblings (A-C). Mouthparts (MP) of l(2)k00311 (G) and 31Em2 (J) are underdeveloped and less pigmented than corresponding wild-type mouth parts (D). The filtzkörper (FK) of l(2)k00311 (I) and 31Em2 (L) are underdeveloped and do not protrude from the larval body as in wild-type (F).
The KDEL receptor is required to maintain localization of some, but not all, ER resident proteins
To assay for KdelR activity, we compared the localization of an ER resident protein, PH4αSG1, in wild-type and KdelR mutant embryonic salivary glands. PH4αSG1 is highly expressed in the embryonic salivary glands [27] and encodes a putative prolyl 4-hydroxylase α (PH4α) subunit. PH4α-subunits are typically found in a complex with a corresponding KDEL containing β-subunit, which is thought to maintain the enzyme complex in the ER through its C-terminal KDEL motif [28,29]. PH4αSG1 is an ER resident based on its colocalization with the ER marker α-KDEL as well as with other GFP-tagged ER proteins ([21] RM Fox and DJA, unpubl.). α-PH4αSG1 staining, using a horseradish peroxidase (HRP) conjugated system, revealed changes in the cellular localization of PH4αSG1 in wild-type versus KdelR mutants. Whereas PH4αSG1 staining of heterozygous (wild-type) salivary glands revealed the reticular cytoplasmic localization characteristic of ER staining at all embryonic stages (Figure 5A, left panels), staining of KdelR mutants showed variable levels of staining in the salivary gland lumen. At earlier embryonic stages (stage 13), PH4αSG1 was detected in both the ER and lumen, although lumenal staining was relatively higher with the EMS null alleles (Figure 5A, top right panel and data not shown) than with the l(2)k00311 mutant (Figure 5A, top middle panel), presumably because of residual expression from the intact KdelR ORF downstream of the insertion site of the P-element. At later stages, PH4αSG1 was nearly entirely lumenal (Figure 5A, bottom center and right panels), suggesting that maternally-provided KdelR mRNAs may partially rescue earlier ER retrieval function.
(A) Staining with antibodies to the resident ER protein PH4αSG1 revealed cellular expression in WT SGs beginning at embryonic stage 11 and continuing through embryogenesis (data not shown; left panels). By embryonic stage 13, PH4αSG1 staining was observed at low levels in the salivary lumens of l(2)k00311 mutants (middle top panel) and at high levels in the salivary lumens of l(2)31Em1 and l(2)31Em2 mutants (top right panel and data not shown). By stage 17, almost all detectable PH4αSG1 was lumenal in both l(2)k00311 and l(2)31Em1 salivary glands. The same staining patterns were observed in all embryos examined of each genotype. (B) Stage 15 embryonic salivary glands were co-stained with antibodies to PH4αSG1, Boca, and Windbeutel. PH4αSG1 protein was observed entirely in the ER in wild-type salivary glands, in both the ER and lumen in l(2)k00311 salivary glands, and predominantly in the lumen in 31Em2 mutant salivary glands (left panels). Boca protein was barely detected in the salivary glands of 31Em2 mutants, compared to the WT and l(2)k00311 mutants, although some Boca protein can be detected in the lumens of 31Em2 mutants when the image is overexposed (second column, last row, inset). Wbl localization was largely unaffected in l(2)k00311 and 31Em2 mutant salivary glands; only minimal lumenal staining of Wbl protein was observed, even when the cellular staining was at high levels (third column, second row). The changes in PH4αSG1 and Boca protein localization are more apparent in the merged images (last column). Again, the same patterns of accumulation were observed in all of the stage 15 embryos examined for each genotype.
We then asked if KdelR function is required to maintain ER localization of other known soluble ER proteins containing C-terminal KDEL or KDEL-related motifs. Two such proteins include Boca, which is expressed in all cells and functions as a chaperone dedicated to the folding of low density lipoprotein receptors (LDLR) containing coupled -propeller/EGF modules [22,30], and Windbeutel (Wbl), which is a protein disulfide isomerase (PDI)-related chaperone expressed to very high levels in the embryonic salivary gland and required for folding of Pipe, a heparan sulfate 2-sulfotransferase [23,31]. We focused on stage 15 embryos to reduce the effect of potential maternal contributions and to optimize phenotypic differences between the null l(2)31Em2 allele and the weaker l(2)k00311 insertion allele. Indeed, a significantly higher level of α-PH4αSG1 was detected in the lumen of l(2)31Em2 versus l(2)k00311 salivary glands (Figure 5B, panels in column 1, rows 2 and 3). Furthermore, only low levels of α-PH4αSG1 were detected in the salivary gland cells of the l(2)31Em2 mutants (Figure 5B, column 1, row 3 panel). Interestingly, although Boca protein was greatly reduced in the salivary gland cells in l(2)31Em2, it was barely detectable in the lumen (Figure 5B; overexposed inset in column 2, row 3 panel). The very low level of lumenal Boca staining may be due to epitope inaccessiblity in the lumenal environment. In any case, l(2)31Em2 had a significant effect on Boca subcellular localization. Surprisingly, localization of the KEEL-containing protein, Wbl, was relatively unaffected in both l(2)k00311 and l ( 2)31Em2 salivary glands (Figure 5B, panels in column 3), suggesting that Wbl ER retention is less dependent on KdelR.
The KDEL/KEEL sequence of Wbl and Boca are required for ER retention in S2 cells
To address the unexpected localization of Boca and Wbl observed in KdelR mutant salivary glands, we expressed either WT or KDEL/KEEL deleted versions of both proteins in tissue culture cells. Based on immunohistochemical staining, approximately 10 - 20% of S2 cells transfected with the Boca construct expressed very high levels of Boca protein; however, even with cells transfected with empty vector, we observed significant levels of Boca protein in all cells (data not shown), which is not surprising since Boca is ubiquitously expressed at all developmental stages that have been examined [22]. On the other hand, we did not observe Wbl in S2 cells tranfected with empty vector but did observe high level staining in approximately 10 - 20% of cells transfected with the Wbl constructs (data not shown), consistent with the more limited expression of Wbl in ventral ovarian follicle cells and embryonic salivary glands [23]. As expected, with cells transfected with constructs expressing WT versions of either Boca or Wbl, both proteins were found exclusively in the cell pellet (Figure 6A,B, middle sets of lanes), consistent with ER localization. On the other hand, with cells transfected with a mutant version of Wbl, which would encode a protein missing only the C-terminal KEEL sequence, all of the protein was found in the supernatant (Figure 6B, right set of lanes). Similarly, with cells transfected with a mutant version of Boca, which would encode a protein missing only the C-terminal KDEL sequence, a significant amount of protein was found in the supernatant, although the bulk of the protein (presumably from endogenous WT Boca expressed in every cell) was in the cell pellet (Figure 6A, right set of lanes). Thus, in S2 cells, ER retention of both proteins requires the cis-acting C-terminal KDEL/KEEL sequences.
(A) Immunoblots of cell pellets (P) and supernatants (S) from Drosophila S2 cells transfected with empty vector (left lanes), full length Boca (center lanes) or KDEL deleted Boca (right lanes) incubated with aBoca (top gel) or abTub antibodies (bottom gel). Note that Boca protein is easily detected in the cell pellets from all three samples but that Boca protein is also detected in the supernatant in only cells transfected with the KDEL-deleted Boca construct. Note also that bTub is detected in only the cell pellets from each transfected cell type, as expected for a cytosolic protein. (B) Immunoblots of cell pellets (P) and supernatants (S) from Drosophila S2 cells transfected with empty vector (left lanes), full length Wbl (center lanes) or KEEL-deleted Wbl (right lanes) incubated with aWbl (top gel) or abTub antibodies (bottom gel). Note that Wbl protein is detected in the cell pellets from S2 cells transfected with the full length Wbl construct, whereas Wbl protein is detected in the supernatant from S2 cells transfected with the KEEL-deleted Wbl construct. Note also that bTub is again detected in only the cell pellets from each transfected cell type.
Mutation in KDELR does not equally affect the localization of all ER resident proteins
To determine the utility of the Drosophila salivary gland as a system for studying mutations in secretory pathway components, we focused on KdelR, which encodes the only Drosophila KDEL receptor. We showed that loss of the receptor results in the depletion of the ER stores of some, but not all, soluble ER residents (Figure 5). Specifically, cellular levels of both PH4αSG1 and Boca were significantly diminished in late stage KdelR mutant embryos. As the PH4αSG1 protein levels declined in the ER, high levels were observed in the salivary gland lumen, consistent with the protein escaping to later secretory compartments and ultimately being secreted. Although Boca was clearly depleted in the ER of KdelR mutant salivary glands, corresponding increases in lumenal levels of Boca were not as obvious as with PH4αSG1. Nonetheless, we did observe that a mutant version of Boca, which deletes the KDEL sequence to which the KdelR binds, was easily detected in the supernatant of S2 cells, whereas WT Boca was not. Thus, the ER localization of two ER residents, PH4αSG1 and Boca, depend on a functional KDEL receptor.
Interestingly, unlike PH4αSG1 and Boca, the Wbl protein appeared refractory to the loss of KdelR function in the salivary gland, suggesting that it is somehow maintained in the ER by other mechanisms. Nonetheless, as with mutant Boca protein, mutant Wbl, with a deletion of the C-terminal KEEL sequence to which the KdelR would presumably bind, was secreted into the cell supernatant when expressed in S2 cells, whereas a WT version of Wbl was not. This finding suggests that ER retention of Wbl in the salivary gland is mediated by factors in addition to the KdelR. Wbl could be maintained in the ER through its association with transmembrane ER residents, which are brought back to the ER in COPI vesicles. The COPI proteins interact directly with the dilysine motifs (KKXX) found at the C-terminus of transmembrane ER residents [32–34]. Alternatively, Wbl may be retained in the ER by interacting with calcium binding proteins such as calreticulin. Calreticulin, which also contains a KDEL sequence (HDEL in Drosophila; [12]), is still maintained in the ER even when expressed to levels that saturate the KDEL receptor function. However, when the calcium-binding domain of calreticulin is deleted, the calreticulin readily escapes [35].
Wbl could also be maintained in the ER through its association with salivary gland transmembrane ER proteins that are retrieved by the RER1 protein. RER1 has been shown to be a retrieval receptor for ER membrane proteins and functions independently of both the KDEL signal and the dilysine signal systems [36]. A clear Drosophila homologue to RER1 exists in Drosophila (CG11857) and is also transcriptionally upregulated in the salivary gland (http://www.fruitfly.org/EST/index.shtml). Thus, the Drosophila salivary gland may utilize much of the diverse cellular machinery for the maintenance of ER proteins and that machinery may be differentially employed depending on the ER resident in question. It should be pointed out, however, that in yeast, an independent system is used to maintain levels of ER proteins in erd2 mutants [37]. This system is activated by the IRE1 endonuclease, which splices the HAC1 transcript into a form encoding an active transcription factor in response to stress, such as the loss of Erd2 function [38]. Although more ER residents escape, more are quickly introduced into the system by increased transcriptional activation of ER protein genes. An IRE1 homologue exists in Drosophila and could contribute to a failure to see significant reduction in the levels of Wbl in the KdelR null embryos. In this scenario, increased levels of Wbl, like Boca, may not be detectable in the lumen due to problems with epitope accessibility or antigen recognition in the lumenal environment.
Larval cuticle defects can reveal novel secretory components
Here, we show that loss of function mutations in KdelR result in severe cuticle defects, including a shortened, underdeveloped cuticle, wherein the ventral denticles, mouthparts and filzkörper are barely visible. Similar phenotypes have been observed with P-element insertion mutations in several other genes known to encode core secretory machinery as well as with null mutations in the CrebA transcription factor gene, which is known to coordinately upregulate secretory machinery genes in specialized secretory cells – including the epidermal cells that secrete the larval cuticle [13]. The consistency in cuticle phenotypes observed with known secretory pathway mutants and the easy (inexpensive) protocols for preparing cuticles for microscopic examination make this an ideal system for screening for novel essential secretory pathway genes – simply assay for weakened cuticles without changes in denticle patterning.
The salivary gland as a model system for studying secretion
This study supports the use of the Drosophila salivary gland for studying secretory pathways components in vivo, in the context of an organ system specialized for secretion. The salivary gland is a monolayered epithelial tube comprised of large polarized cells in which it is easy to visualize organelles and subcellular compartments. We have reported elsewhere that the genes encoding the machinery known to be required at early steps in the secretory pathway are expressed to very high levels relatively early in the salivary gland [13]. This tissue will persist for several more days without undergoing cell division, a process that might otherwise complicate studies of organelle maintenance and function. The maternal contribution of the secretory genes, such as KdelR, is likely to allow development to proceed to a stage where the salivary glands have formed and are functional. The effects of zygotic loss of the secretory genes can then be studied as the maternal supplies are depleted. A survey of Flybase (http://flybase.bio.indiana.edu) indicates that mutations exist for at least one subunit of each of the known complexes involved in early steps in the secretory pathway. Hence, the starting materials are readily available for testing the proposed roles of the known secretory components in a eukaryotic organ specialized for secretion. Indeed, our identification of null mutations in the single Drosophila KdelR will allow for more direct tests for a role for the KdelR in inducing autophagy in neurodegenerative disease models [39] and in regulating cargo flux through the Golgi [40]. The null mutations can also be used to clarify the importance of post-translational modifications on Kdel-R localization and activity [41].
We thank R. Fox for critical reading of the manuscript. We thank T. Grigliatti and the Bloomington Stock Center for deficiency, insertion and EMS mutant lines. We thank Flybase for information regarding KdelR gene structure and availability of insertion mutations [42]. We thank R. Mann and T. Schupbach for Boca and Wbl antisera, and two anonymous reviewers for suggestions.
Author Contributions
Conceived and designed the experiments: EWA DJA. Performed the experiments: EWA YLC. Analyzed the data: EWA YLC DJA. Contributed reagents/materials/analysis tools: EWA. Wrote the manuscript: EWA YLC DJA.
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Europe, 1890 - 1945
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Europe, 1890 - 1945 by Stephen J. Lee
Book DescriptionEurope, 1890-1945 is a new approach to teaching and learning early twentieth century European history at A level. It meets the needs of teachers and students studying for today's revised AS and A2 exams. In a unique style, Europe, 1890-1945 focuses on the key topics within the period. Each topic is then comprehensively explored to provide background information, essay writing advice and examples, source work, and historical skills exercises. From 1890 to 1945, the key topics featured include: * the origins and impact of the First World War * the Russian Revolution and the rise of Stalin * the Weimar Republic and the rise of Hitler * Mussolini and Fascist Italy * Stalin and the Soviet Union, 1928-41.
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Publish Date: 25-Sep-2003
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Yesterday, 02:33 PMTony@VargasTurboTech created an article 900 whp coming? VTT GC turbo upgraded built N54 makes 847 whp at 25.4 psi of boost on E85 in BMW Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsIncredible peak numbers here but it is important to focus on the context. For one, this is a built motor with dropped 9.5:1 compression. Secondly, these figures are achieved at 25.4 psi of boost. Compare the result to what the GC turbos previously hit at 29.3 psi of boost. Not only is this setup making more power but it is doing so more efficiently. Further details from VTT below. Finally got another motor in the shop car, and on the dyno Friday before we left for the event on Saturday. With a little tuning by none other than our favorite N54 tuning guru Justin @V8Bait the car laid down a more than impressive 847WHP/726WTQ with an astounding 601WTQ by 3000 RPM held to redline, and 700WTQ held flat from 3500-6500RPM. This power, and tq was made with ONLY 25.4 psi at peak power RPM. We also made 776WHP at 21.62 psi boost at peak power! This is a perfect example of why we continually tell people boost is simply measurement of restriction NOTHING else. Remove restriction, and power stays the same or goes up at the SAME boost pressure. This is our closed deck 9.5:1 compression motor, with fully built head, larger intake valves, Manley springs, TI retainers, etc. We are also running the EOS V2 manifold. Full specs below. We took the car out to Shift Sector yesterday, and ran a new N54 1/2 record of 170.49MPH using the 776WHP map with 3 degrees less timing, then we tried to turn up the boost to the 850WHP map, but the we believe something happened to the diff or alignment, the car was all over the track, and very dangerous to drive, so we could not get a full run in. Even with the car near uncontrollable we managed a 168MPH run. We belive the car would have been close to 175MPH with the 850WHP map in it, had we been able to complete a full pass. (Chris will be doing a full Shift Sector post) Breakdown of the car as it is right now: Closed deck 9.5:1 compression N54 short block, forged rods, forged pistons Fully Ported head with oversized intake valves, Manley springs, Ti Retainer, Stock cams VTT GC turbochargers Custom VTT inlets with 6" velocity stacks VTT Double Barrel HPFP system EOS V2 intake manifold with top PI ADE 1100WHP FMIC Custom triple Walbro LPFP set up with Radium Surge tank MHD tuner tuned by V8BAIT 100% E85 847WHP graph AEM Log Grab 776WHP graph AEM Log Grab Logs: AEM 847 Log http://www.datazap.me/u/vargasturbotech/vtt-gc-847whp?log=0&data=4-5&zoom=96-420 MHD 847 Log http://www.datazap.me/u/vargasturbotech/vtt-gc-847whp?log=1&data=3-23&zoom=39-90 AEM 776 Log http://www.datazap.me/u/vargasturbotech/vtt-gc-847whp?log=2&data=4-5&zoom=118-48447 replies | 1034 view(s)
02-24-2017, 05:10 PMSticky created an article Introducing Project 991.2 - Who needs a 911 Turbo? in Porsche Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsThe new PorscheBoost and BoostAddict project car is finally here. Say hello to this 2017 Porsche 911 Carrera. In other words, the new 991.2 generation with the B6 twin turbo 3.0 liter motor. The car has all the goodies including Sport Chrono, PDK, and the Sport Exhaust. The goal will be to produce 911 Turbo S humbling performance at a fraction of the cost in a lighter package. Think of it as building your own GT2. PorscheBoost already reached out to several tuners/vendors including: Fabspeed @COBB Tuning GIAC BMS IPD @VF-Engineering @Pure Turbos This should be a very fun project. Apologies for the poor photography as the sun made things difficult. Expect better photo shoots as things progress.46 replies | 555 view(s)
02-24-2017, 04:26 AMSticky created an article A look at BPC's 1500+ hp capable built S63TU V8 engine package with Darton sleeves, Carillo rods, forged pistons, etc. in BMW Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsThe built motor era for the S63TU engine platform is getting going thanks to BPC (Bimmer Performance Center). They have a built engine package with some impressive specs that will increase the durability and capability of the S63TU 4.4 liter twin turbo V8. Those looking into this package will be people pushing the motor with turbo upgrades. When over 700 lb-ft of torque at the wheels, peace of mind becomes increasingly important: Some engines will last longer. Some engines will not. There are many factors such as fuel quality, torque, use, mileage, tune quality, etc. If you drive around town with a turbo upgrade getting groceries it is not exactly the same thing as going WOT on the roadcourse for hours of laps. This is the list of upgraded components: BPC Spec Darton Flanged Sleeves Optimized wall thickness for strength that far exceeds factory Alusil cylinders without compromising block integrity or cooling characteristics BPC Spec Carrillo H-Beam Rods Rated at 1500 HP BPC Spec Diamond Coated Pistons Thermal coated crown and teflon coated skirts. BPC Spec Total Seal Stainless Steel Piston Rings BPC Spec Calico Coated Main & Rod Bearings BPC Spec spark plugs They also offer these services: Engine Block CNC Bore & Hone Process CNC Main Bearing Line Hone BPC Spec Optimized Engine clearances Precision Balanced Rotating Assembly Cylinder Head BPC Spec Ported Cylinder Heads BPC Spec Valve seat machining CNC Cylinder Head Resurfacing Now, combining a built motor setup like this with a turbo upgrade is key. We saw the BPC built S63TU motor with their Borg Warner EFR 7163 turbo upgrade put out 846 horsepower to the wheels in an F10 M5 with preliminary tuning. They are shooting for over 1000+ and that is really where the upgraded engine components will be put to the test. How much does this all cost? Well, they didn't tell us. Expect to pay to play. The S63TU platform is poised to have a very big 2017.20 replies | 2471 view(s)
02-25-2017, 08:46 PMnewb335guy started a thread For Sale: Part out, moving on. in Buy/Sell - PartsDoc race single turbo-ceramic coated PTE Gen 2 6266. everything that I got from Doc Racing goes to you plus I will provide the Hose setup that is in picture to vent back into turbo so you avoid venting to air and having to smell oil. Also Moshomito Catch can setup provided and ADV 02 Sensor (very recommended). Price to sell at $4700.00 We can package fueling into it also. Any questions you can call or text 850-240-0293 This is off of Doc Website: http://docrace.com/collections/bmw/p...ngle-turbo-kit BMW 335i 135i N54 Top Mount Single Turbo Kit Turbo Bearing Ceramic Coating $5,495.00 USD Cpe/BMS Port injection with Split Second AIC controller $800 Stage 3 Fuelit setup with upgraded fuel lines, return line fittings and Hobbs switch $600 DSS Axles $70019 replies | 2212 view(s)
Yesterday, 04:36 PMlulz_m3 started a thread THE SOUS VIDE THREAD in Off Topic - Work/School SafeFigured it was time for a dedicated Sous Vide thread. I just picked one up last week. Grass fed London Broil/Top Round cut. Cooked @ 133 for 7.5 hours. It was so tender I literally cut it with my fork. Added Worcestershire, freshly chopped garlic and Weber Chicago steak seasoning into the bag. I'm traditionally a BBQ guy, but so far I'm convinced this is my new go to method. My next experiment will be brisket cooked SV, but smoked on my wsm to impart some smoky flavor and crust.17 replies | 1339 view(s)
02-25-2017, 09:21 PMSticky created an article BMW turbo V8 tuning value - BMS JB4 shows massive torque gains on 2016 F10 550i N63TU with 621 lb-ft at the wheels in BMW Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsThe N63 and N63TU (technical update) twin turbo 4.4 liter V8's offer incredible tuning value. Although the engines are twin turbocharged and share the 4.4 liters of displacement (and some of the engine internals) with the S63TU out of the F10 M5 the N63 manifold and turbo setup differs. That means the N63 is not quite as capable as the S63 with bolt on tuning but it still gets great aftermarket gains and is a much better value. Just look at the price of an F10 M5 and a used 550i. One can buy a 550i, add a JB4, and have M5 performance at a fraction of the cost: The N63 and N63TU are simply a great BMW turbo tuning value and have yet to reach their potential in the aftermarket.15 replies | 600 view(s)
02-22-2017, 10:28 PMSpeedLimit? started a thread LaFerrrari vs 918 @ San Antonio Drag Strip in Race Videos / Kill Stories / VS. discussionI wish they ran without passengers but cool to see the Laf putting in timed runs in the wild...5 replies | 1375 view(s)
02-24-2017, 07:21 AMSticky created an article Turbo upgraded N54 E60 535i getting walked by a bolt on GTR in BMW Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsA humbling experience here but a full bolt on Nissan GTR is no slouch. Neither is a BMW N54 with the Pure Stage 2 turbo upgrade. The E60 chassis is a bit heavier than your typical N54 and namely an E82 1-Series variant. Run what you brung though. This E60 belongs to BimmerBoost member @viperware and it is a strong car. His vehicle is a 2010 E60 535i with FBO, JB4, Port Injection, Pure Stage 2, RB Inlets, VTT outlets, and an MFactory LSD. It just wasn't enough.8 replies | 753 view(s)
02-24-2017, 07:13 AMSticky started a thread Turbo upgraded N54 E60 535i getting walked by a bolt on GTR in Race Videos / Kill Stories / VS. discussionA humbling experience here but a full bolt on Nissan GTR is no slouch. Neither is a BMW N54 with the Pure Stage 2 turbo upgrade. The E60 chassis is a bit heavier than your typical N54 and namely an E82 1-Series variant. Run what you brung though. This E60 belongs to BimmerBoost member @viperware and it is a strong car. His vehicle is a 2010 E60 535i with FBO, JB4, Port Injection, Pure Stage 2, RB Inlets, VTT outlets, and an MFactory LSD. It just wasn't enough.8 replies | 519 view(s)
Yesterday, 09:41 PMSticky created an article Big Boost offers Stage 3 BMW N55 (E & F-Series) turbo upgrade kits with 620+ whp capability in BMW Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsBig Boost is a new player in the BMW N55 engine tuning world. They are showing what is a very strong N55 turbo upgrade offering. They have three stages the most powerful of which produces over 620 horsepower at the wheels. That is serious power out of the N55 motor. Let's look at the stages: Stage 3.1 (5459 turbo) capable of 470 whp Stage 3.2 (5959 turbo) capable of 520 whp Stage 3.3 (6159 turbo) capable of 620 whp What is included? Cast exhaust manifold Master Power 6159 turbocharger (7-Blade billet aluminum compressor wheel) Wastegate mount Turbo to intercooler charge pipe BOV mount Turbo inlet pipe Big Boost down-pipe Custom coolant pipe Engine block coolant adapter Intercooler inlet adapter Oil lines Thankfully, they provide proof to match their claims. BMW 135i with flash tune @ 27 psi: BMW 135i with flash tune @24.5 psi: Tuning is done through an unnamed flash tune and flash tuning on the N55 motor is still relatively in the early stages with bigger turbo upgrades. More details on the tune as well as results on F-Series and E-Series cars would be appreciated. They price the updates starting at $4250 which certainly is competitive. As for seeing it in action, here: No 1/4 mile times or pulls but it definitely is making the power on the dyno on that F22 tuned through the factory DME along with a JB4. Fuel-It! port injection is providing the necessary fuel. Certainly good results so far and we look forward to independent results.6 replies | 677 view(s)
02-23-2017, 02:24 PMDjkr21 started a thread For Sale: FS: 08 BMW 335i RWD (Gorgeous!) in For Sale VehiclesHey guys...Unfortunely it is time to let this car go. Car was a blast to drive and deff turned heads and received plenty of compliments! Car has tons of mods, cosmetics, performance, ect... I was going to have a motor built by motiv and throw a doc race single turbo kit on, but I've decided to part ways and move to a different platform. The car will need a motor, as one of the rods let go. Like I said, I was going to throw a built n54 in but things changed.The car has the following: PERFORMANCE: Rb super evo turbos Rb inlets and outlets Vrsf Downpipes Motiv port injection Fuel it stage 3 lpfp Stett chargepipe and tail bov Vrsf 7in intercooler Split second pi controller Kw v1 coilovers New low mileage auto trans Control arms M3 race alignment ...prob more I'm missing COSMETICS: custom vinyl wrap (car is jet black underneath, perfect paint) 18in apex arc 8 with Michelin pss M3 carbon fiber hood Amuse carbon fiber trunk Mtech rear bumper with quad tips Tints all around Vinyled window trim Dtm style headlights M3 front bumper with cf lip Custom brake caliper covers INTERIOR: Black interior with sport seats i drive system carbon fiber wrapped interior trim *car has backup sensors as well Click the link for pics:http://s825.photobucket.com/user/serbball3r5/library/BMW%20335Car is ready for someone to take home, it is perfect for someone who wants to drop a motor into something that is ready to go with all supporting mods. Pm me or text 630-400-4363 if interested. Located in Chicago, IL. Price is $14,000 as I have well over $10,000 in mods alone! No low ballers please. Car is in perfect condition. Clean title, no accidents4 replies | 639 view(s)
02-21-2017, 08:35 PMSticky created an article Turbo Lab claiming 800 hp F80 M3 & F82 M4 S55 TDO4HL 20G hybrid turbo upgrade in BMW Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsThe goal here is 800+ horsepower as stated by Turbo Lab of America. What are they doing? Well, they are massaging the factory S55 turbo housings considerably. They are stuffing TDOHL 20G wheels into the OEM housing which requires a bit of machining as the video below covers. Does it produce 800 horsepower? That is a good question. They are working on it and this upgrade if it comes to fruition will be a very nice value in the S55 turbo upgrade market. It is somewhat difficult to get information on the Turbo Lab of America S55 upgrades as they do not have a very good web presence. They do have Youtube videos but their website is lacking and they primarily sell on Ebay. There certainly is promise here and it is worth watching for F80 M3 and F82 M4 owners.1 replies | 1004 view(s)
02-26-2017, 01:13 AMSticky created an article Independent LaFerrari vs. Porsche 918 Spyder 1/4 mile drag race in Porsche Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsGetting statistics you can trust on the Ferrari LaFerrari is not exactly simple. Ferrari tightly controlled magazine testing to the point where you could not trust it. They would actively avoid third-party comparisons with the Porsche 918 Spyder and the McLaren P1. That is why whenever you see a comparison without Ferrari controlling the testing you know you can take it seriously. The Ferrari is lighter than the 3758 pound 918 Spyder although the amount varies based on who you trust. We do not trust Ferrari so the difference is probably closer to 200 pounds or so. Part of the reason is the 918 Spyder is all wheel drive. This helps it put down its 887 horsepower and 944 lb-ft of torque. The LaFerrari on the other hand is rear wheel drive with 949 horsepower and 664 lb-ft of torque. Why they say the LaFerrari is all wheel drive in the video is anyone's guess. How can you not know the specifications of your multi-million dollar toy? On paper, the Porsche 918 should get the lead off the line with the LaFerrari passing and pulling eventually. That is how things played in a previous drag race to 186 miles per hour. So what happens in a 1/4 mile? Well, neither use launch control so they hardly show the full capability. The 918 Spyder is quicker and faster with a 10.47 @ 140.76 to the LaFerrari's 11.22 @ 137.95. A very poor run. They run again and the LaFerrari again gets a pathetic launch. The 918 runs 10.47 @ 139.56 and the LaFerrari time is cut for whatever reason. Overall, the 918 wins because the LaFerrari never launched well. Basically, a waste of time. Skip to the 5:00 minute and 7:50 minute marks to avoid annoying people talking.5 replies | 320 view(s)
02-24-2017, 04:52 AMSticky created an article Eurocharged gets 643 whp out of the W211 E55 AMG M113K with flex fuel ethanol tuning and bolt ons in Mercedes-AMG Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsThe W211 Mercedes E55 AMG may be fourteen years old at this point but it still continues to impress. When Mercedes-AMG threw a 5.5 liter V8 (M113K) into an E-Class chassis and supercharged it, they sent a message. Look at how capable the M133K motor still is today. Eurocharged ATX (Austin, Texas) is getting very nice numbers out of the motor. Take a look at the modifications: Setup: 2005 E55 Stock Motor Stock Heads Stock Cams Stock Trans (at the moment) Fuel System: 2x Walbro 450cc in the factory baskets Custom Fuel Lid for Passenger's side -10AN from tank, through external FPR and Filter Split factory rail with -6 inlet on each Mods - FBO American Racing Longtubes 90mm TB 178mm Crank 77mm S/C Pulley 650cc Injectors Eurocharged ATX Flex Fuel We are talking bolt on modifications with an upgraded fuel system for ethanol. Nothing crazy there yet the engine design which is well over a decade old is showing 643 horsepower to the tires. They plan on doing some further tuning with higher ethanol contents but suffice it to say the W211 E55 is an all time great.6 replies | 284 view(s)
02-26-2017, 02:50 PMSticky created an article PPM Racing claims to have a 5.0 liter F10 M5 & F13 M6 S63TU stroker kit for $18000.00 in BMW Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsPure Performance Motorsport in Australia has various stroker kits for BMW motors. One that caught the eye of BimmerBoost is their 5.0 liter stroker kit for the 4.4 liter S63TU engine in the BMW F10 M5 and F12/F13 M6 models. Let's look at the specs: Bore:92mm with Custom Darton Sleeves / Stroke: 94mm Compression Ratio: 10:1 or 9.3:1 PPM Racing Australia EN40B Billet Crankshaft with our special surface finishing PPM Racing Australia Ultimate Forged Pistons with 2x coatings,H13 pins and NPR Rings PPM Racing Australia Ultimate 300M Connecting Rods with our special surface finishing Balanced and blueprinted Race Engine ARP Custom Head and Main stud kit Custom Racing Harmonic Balancer ACL Racing Bearings Custom Turbo Camshafts Custom Racing Inconel Oversize Valves Custom Valve Springs Custom Titanium Retainers New Valve Stem Seals Custom MLS Head Gasket to suit larger Bore Stage 6 cylinder head porting and Polishing Serdi throat ports Measure new valve guide interference , liquid nitrogen shrink fit new valve guides Size and hone finish valve guides Trim valve guide stem seal end for valve spring retainer to stem seal clearance Serdi seat work Rottler S7M machine head gasket surface with our MLS head gasket finish Thermal barrier coat combustion chambers Thermal barrier coat intake ports Thermal barrier coat exhaust ports Thermal barrier coat inlet valve heads upto valve guide on valve stem Thermal barrier coat exhaust valve heads upto valve guide on valve stem Flow bench test after Ok, that is a nice list of components. There are options listed there such as the head work. The 92mm bore and 94mm stroke exceed the factory configuration of 89x88.3mm respectively. The entire assembled S63TU motor with the options listed above is $40k+. The rotating assembly alone makes more fiscal sense. Is it worth $18000 to go to 5.0 liters? Are there even any results for this motor? How does the cost compare to BPC's option? Those who will want to look into this are those who exceed or are preparing to considerably exceed 700 lb-ft of torque at the wheels. This is a nice rotating assembly for someone shooting for that 1000+ whp mark. The Darton sleeves, forged pistons, forged rods, and forged crankshaft will all be appreciated at that point. Now, who wants to be the guinea pig?4 replies | 564 view(s)
02-26-2017, 12:14 AMTTMERC started a thread M157: Fastest/Quickest Stock ECU CLS63?? in M157 and M278 Twin Turbo V8Went to the track today only got one run in because it was the first day of the season but the car ran well! Anyone know what the fastest untuned m157 thats in the US? I posted my time in dragtimes should be up soon. I think I can get a 10 second pass out of this car before I get it tuned. Only mods are K&N filters with stock boxes (cut out plastic baffles) Resonator replaced with straight pipe, stock downpipes just removed the cats. Runs were on 93 Octane Shell Fuel. I think 10s will be attainable on race gas mix and the software updates from the dealer. Boost was really low on this run from my log and the shifts were pretty lazy it felt like.4 replies | 553 view(s)
02-25-2017, 08:33 PMSticky created an article Chevy adds the 1LE package to the 2018 Camaro ZL1 creating the baddest track car for the money in Domestic Car Performance - Forums, Tuning, Blog, Motorsport, and Modification NewsThe Camaro ZL1 already offers incredible performance out of the box. You can get it as a coupe or as a convertible. For those who want the ZL1 specifically for track dominance, the coupe with the new 1LE option will be the best performance choice. Why? The revised aerodynamics alone help the car stick to the road. We're talking an additional 300 pounds of downforce. This will increase drag of course but the point is making up time in the turns. You also have new wheels and rubber. Very sticky Goodyear Eagle F1 Supercar tires (in a new ZL1 1LE specific compound) are included in a whopping 305mm width up front and 325mm in back. Is there any downside to the 1LE option? Well, it is manual transmission only although the gear ratios are revised for the roadcourse. That means this car is all about driver interaction on the track. If you consider yourself a driver and don't care about the straightline as much as you do the experience then this is the enthusiast package. Anything else? Yes, weight is dropped by 60 pounds and the spring rates are adjusted. As far as performance Chevy claims a 3 second increase on their test track over the standard ZL1. If previous test results are anything to go by that means the ZL1 1LE has nothing to fear from just about anyone. Certainly not from any car in its price range. Incredible work from Chevrolet making their beast even better.5 replies | 158 view(s)
02-24-2017, 04:43 AMSticky started a thread Eurocharged gets 643 whp out of the W211 E55 AMG M113K with flex fuel ethanol tuning and bolt ons in M113K,M155K Supercharged V8The W211 Mercedes E55 AMG may be fourteen years old at this point but it still continues to impress. When Mercedes-AMG threw a 5.5 liter V8 (M113K) into an E-Class chassis and supercharged it, they sent a message. Look at how capable the M133K motor still is today. Eurocharged ATX (Austin, Texas) is getting very nice numbers out of the motor. Take a look at the modifications: Setup: 2005 E55 Stock Motor Stock Heads Stock Cams Stock Trans (at the moment) Fuel System: 2x Walbro 450cc in the factory baskets Custom Fuel Lid for Passenger's side -10AN from tank, through external FPR and Filter Split factory rail with -6 inlet on each Mods - FBO American Racing Longtubes 90mm TB 178mm Crank 77mm S/C Pulley 650cc Injectors Eurocharged ATX Flex Fuel We are talking bolt on modifications with an upgraded fuel system for ethanol. Nothing crazy there yet the engine design which is well over a decade old is showing 643 horsepower to the tires. They plan on doing some further tuning with higher ethanol contents but suffice it to say the W211 E55 is an all time great.6 replies | 140 view(s)
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text: This listing is no longer available or has been sold. Find Another One Others Looked At... Lot of 6 Magpul PMAG 25 Gen3 Windowed AR... SELLER: DEGuns_net 1 $120.00 ps90 p90 mag magazine 30or50rd in stock ... SELLER: jlcriscione 0 $100.00 Magazine FN P-90 PS-90 Carbine AR57 5.7... SELLER: Robertson Trading Post 1 $9.00 ISRAELI GALIL STEEL MAGAZINES IN 5.56X... SELLER: Tom Selleck 0 $100.00 Winchester Mod. 77 Magazine Excellent Ve... SELLER: RGLLC 1 $69.00 FN P90 PS90 AR57 Factory 10 Shot Ten Rou... SELLER: Robertson Trading Post 1 $23.00 Non Guns Magazines Clips Rifle Magazines Other Round Sig San Magazine Imported From Switzerland | 1073 | 0 | Lc | 421.881ms 20 Round Sig San 550/551 Magazine Imported From Switzerland For Sale from Bamp1945 | No feedback Yet View | Verified Seller | 5 Completed Sales View Sellers Items This item is no longer available. /UserImages/18872/905968774 3070517.jpg Description Shipping and Payment More Info 20 Round Sig San 550/551 Magazine Imported From Switzerland. Buyer pays actual shipping cost. Any questions, please call. Actual cost Trades Accepted: No After 20 years of doing business as B & K Sales, 23 Main St., Natick, we changed location, added LLC to our name and now offer the services of a full service Gun Shop. We accept consigments, we buy and sell used firearms, Estate Appraisels, Storage, Gunsmith\Bluing services, offer Firearm Safety Couses, and we offer special orders. As a retired military member, former and current members of the military and law enforcemnet personnel receive discounts on any special orders. We are open Wednesday & Friday - 11am to 6pm. Tuesday and Thursday evening, 11am to 8PM, Saturday 9am to 2PM (as long as I don't have a fishing tournement). Stop by and say hello. Coffee is always on Condition: New In Box Location: MA Share Item Item #: 905968774 This item is no longer available. Price: Shipping: See Description (or Arrange local transfer) Accepted Payment Methods: Returns: No Returns Bamp1945 Seller Since: Mar. 2002 Seller Level: Verified This seller is an FFL City: Natick State: MA Your Recently Viewed Items


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LiveWords Blog
Meet us at The Global E-Commerce Summit, the leading international event that focuses on the most important trends and developments in global e-commerce, cross-border trading and omnichannel retail.
The world has changed significantly since the traditional translation management solutions were invented in the 90's. Read how.
We've closed our second investment round of € 1 M with Paranza.
We just may be chosen as the best Startup Deal of the Year, an initiative of NextStage.
As a Demandware Premier Partner, the LiveWords team is present at the Demandware XChange Conference on June 16 and 17 in London, the bi-annual event where global brands and retailers come together face-to-face to share strategies, experiences and innovations.
Just a few days before its first-ever quarterly earnings report, Twitter "leaked" the current status on their move into ecommerce territory. Will Twitter as an ecommerce channel be useful for global brands targeting foreign markets?
What are the options for adding multilingual content? Most content teams start by translating text right into their content platform. But since these platforms lack translation workflow features, most of us enter a nightmare where keeping control degrades to a won't-have.


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Communion, the artist-led organization co-founded by Mumford & Sons' Ben Lovett, announces a U.S. distribution deal for its label, Communion Records, with RED Distribution, an independent division of Sony Music Entertainment. Communion Records has partnered with RED to provide physical, mobile, and digital distribution of all forthcoming releases. RED will also deliver radio promotions and marketing for the label's artists, beginning with Willy Mason, Bears Den, Rubblebucket and Tennis.
Effective this month, SoundExchange will be providing monthly royalties to artists, labels and rights owners signed up to receive electronic payments. Previously, SoundExchange sent royalty payments quarterly to its registrants, and with this new initaitive the company is now the first sound recording performance rights organization in the world to offer monthly distributions. Most sound recording performance organizations in other countries pay only annually.
Jeff Parker joins CHR WKHQ/Traverse City for morning co-host duties. Parker, who arrives from the PD/afternoon post at CHR KQHN/Shreveport, will team with current co-host Heather Leigh.
Broadway Media is acquiring the following stations from Simmons Media for $11 million: Adult Hits KYMV-FM, Rhythmic CHR KUUU-FM, Country KEGA-FM, Alternative KXRK-FM and CHR KUDD-FM/Salt Lake City and Oldies KMER-AM and AC KAOX-FM Kemmerer, WY.


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Remarkable Wall Street scandals
Everything from muppets to failed IPOs to a suicide attempt made 2012 a banner year for financial scandals and bizarre happenings. There were so many, in fact, that it was hard to narrow down the list. Here's a look at 10 prominent Wall Street misdeeds that won't soon be forgotten. By Kensey Lamb and Siemond Chan
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Brevario de Mistica Oculta
Written by Manuel Arduino Pavon
Illustrated by
Lovereading4kids Price £5.28
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Brevario de Mistica Oculta by Manuel Arduino Pavon
About the Author
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Book Info
Paperback / softback
82 pages
Manuel Arduino Pavon
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Babel Books, Inc.
Publication date
1st March 2013
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LoveReading4Kids is a modern and creative way of emphasising the value and importance of books in this digital age #booksforlifeAmrit Bunet – Teen


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Can a vegetarian person get a good meal at this restaurant?
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3 Days in New Delhi
Shopping Guide for New Delhi
New Delhi Off the Beaten Path
karol bagh is the best place to stay for good hotel & shopping,
food at cross roads is very good & travelling is very easy in metro . must visit karol bagh shopping market.
A nice place to have a beer followed by dinner. Prices are slightly on the high side as this is a restaurant frequented by foreigners. A good location and just a few minutes walk from the Metro & general shopping area.
Service is quick but always found it crowded with tour groups. It's hard to find a table before 7pm...
Went here with a group of 3 families. The food was a bit disappointing for the prices (bill is a lot more than menu because they add range of taxes and service charge on top). Better food elsewhere.
Nice restaurant with a good menu and decent atmosphere. Suitable for large groups too. The food was excellent and service was very good too.
We first visited this restaurant as part of a tour group. Searched the web later to find them for ourselves.
The food is excellent, can't recommend it enough. The table attendants are helpful and friendly, even tried to find the NRL footy final on fox for us.
Went three times, each time enjoyed the food immensely.
Went for dinner to this restaurant along with my friends. We all enjoyed beer and later had dinner. Service was good. The service staff is excellent. Price was moderate.
Visited this restaurant to have dinner on the recommendation of the General manager of our hotel. The food was tasty. The price was also moderate
Was a few groups of travellers here when we visited. Menu has fish and chips if your struggling! I had the chick pea masala, doesn't come with rice so have to buy that separate. Was a huge portion, could have shared it between 2 if u get a rice and nan. I would visit here again.
We visited this restaurant on the recommendation of owner of hotel sunstar heritage. Tasty food and quick service is the speciality of this retaurant
Serves indian as well as chinese food. Worth visiting. Very good ambiance and staff. Gets very crowded during dinner.
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The warming effects of direct radiation from the sun are used by most insects. Both adults and immature stages absorb solar radiation directly by basking, or benefit indirectly via the choice of suitable microhabitats (May 1979). Thermoregulatory behaviours have evolved such that many species are able to achieve and maintain specific body temperatures, which often coincide with the optimum of one or more physiologically important processes, such as feeding rate (Sherman & Watt 1973; Lactin & Johnson 1995), feeding efficiency (Porter 1982), growth rate (Knapp & Casey 1986), development rate (Lactin & Johnson 1996; Bryant, Thomas & Bale 2000) and metabolic efficiency (Kukal, Heinrich & Duman 1988).
Physiological time in developing insects is temperature dependent (Taylor 1981): the higher the temperature, the quicker development proceeds (up to a threshold above which increased temperature soon becomes fatal). This is the basis behind most models for estimating emergence times for pest species (Liu & Meng 1999), understanding species' phenological patterns (Malcolm, Cockrell & Brower 1987) and predicting shifts in species' ranges in response to climate change (Porter, Parry & Carter 1991). Most studies use air temperature as the only environmental input to drive the models (Got, Labatte & Piry 1996; Turnock & Boivin 1997; Harari et al. 1998; Liu & Meng 1999), although it is acknowledged that the discrepancy between ambient temperature and the temperature insects actually experience is the biggest source of error in such models (Pruess 1983; Higley, Pedigo & Ostlie 1986). To counter such error in model performance, start dates for models are chosen retrospectively based on observed emergence patterns (Mcbrien & Judd 1998), threshold temperatures are varied to find the best prediction (Davis, Brenes & Allee 1996; Fatzinger & Dixon 1996), or developmental requirements are calculated solely from field observations (Beasley & Adams 1996). Such models may indirectly take microclimate into account, but only on a local scale and without identifying the separate ambient and microclimatic components. These modifications improve the statistical fit, but not necessarily the biological understanding. None the less, in some cases thermoregulation by the species in question has been taken into consideration, either directly (Rawlins & Lederhouse 1981; Carruthers et al. 1992) or indirectly (McDonald & Smith 1988). Microclimate measurements have also been incorporated in some studies (Lyons 1994).
Increased biological understanding is likely to be required if thermally based predictions are to be made for climate scenarios outside our current experience, and there is increasing interest in the response of animal distributions to various climate change scenarios (Porter, Parry & Carter 1991; Drake 1994). In this context, the relevance of macroclimatic measures such as air temperature (often averaged over large areas) to the microclimatic temperatures experienced by insects is questionable (Kennedy 1997). In this study, we tested whether incorporation of more detailed aspects of the thermal biology of three insect species allowed us to predict species' phenology and distribution accurately. If so, the approach should have general applicability in understanding and predicting the responses of poikilothermic animals to climate change. The study species were the small tortoiseshell Aglais urticae L., peacock Inachis io L. and comma Polygonia c-album L. butterflies (all Lepidoptera: Nymphalidae). These species are medium-sized mobile butterfly species that share the same larval resource, the common stinging nettle Urtica dioica L. (Urticaceae), itself widespread and generally abundant throughout the UK and mainland Europe (Rose 1981). Interestingly, they differ with respect to their thermal ecology. Female P. c-album lay eggs singly while those of A. urticae and I. io lay in large batches, of about 80 and 300–400 eggs, respectively (Dennis 1984). The solitary, cryptically coloured, larvae of P. c-album spend most of their time concealed on the underside of U. dioica leaves, while the gregarious, mainly black, larvae of A. urticae and I. io live within loose silken webs, fully exposed and often near the top of U. dioica plants. The body temperature of P. c-album larvae thus conforms to the surrounding (microhabitat) temperature, but the temperature of A. urticae and I. io larvae is elevated almost independently of ambient in the presence of sunshine, being maintained at around 32·5 °C and 31·5 °C, respectively (Bryant, Thomas & Bale 2000). We explored how these differences affect the butterflies' phenologies and distributions.


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Women and Men
HIGGINSON, Thomas Wentworth
Place Published: New York
Publisher: Harper
Date Published: 1888
Description: 326pp., 12mo, tan cloth (lightly soiled), decoratively gilt spine. New York: Harper & Brothers, 1888. First Edition. A very nice clean copy.Edition: First
Binding: hardcover
Condition: very good(+)
Language: English
Type: Books
Book ID: 202663


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The Melton Design Build squad recently announced a new program specifically for junior women. The program will involve all cycling discipline and...Read More »
On Saturday, January 7th, BRAC is hosting a meeting for members, advisory groups, coaches, and anyone who wishes to give input into reorganizing...Read More »
Do you want to start racing in Colorado and you are not quite sure where to begin? Or perhaps you are Pro...Read More »
We are entering the election period in BRAC politics. Every year, five positions become up for grabs in the fall. There are currently at least...Read More »
Last week we mentioned throwing a little swag the way of the best performances from Cross of the North for people that raced every day. Well, we...Read More »
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A special meeting of the club council was called last Friday during the road and track awards party. The purpose was to decide on a bylaw issue...Read More »
The smoke has cleared on a busy season of track racing, and the final results of the track cup have been tallied. ClickRead More »


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Wego.com recently found 3 hotels in Payerne, Switzerland. Find a range of accommodation in Payerne at great prices. Book luxury Payerne hotels or find last minute accommodation. Save more on cheap hotels in Payerne with affordable room rates.
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Tags » Krul
Newcastle's Transfer window - Success or Failure?
It is the Transfer Deadline Day, and in around 9 hours we can know for sure as to whether the summer transfer window has been a success or failure for Newcastle United in terms of players in and out. 501 more words
Summer 2013
The homie CISR V7 LE caught this guy rolling through So Cal last month. We painted this on Krül's birthday in Oklahoma in the summer of 2013. 20 more words
#Adyor #Krül ⬛+🔺=❤
Below is a little gallery of works by Adyor and Krül. 32 more words


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Fashionable men cycling suit
Soft and breathable
The jersey is made of polyester and the pants is made of polyester + spandex
Suitable for cycling, hiking, racing or other outdoor activities
Great gift for your friends
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We do not accept any responsibility or liability for the incorrect purchase of our products. All of the products on the website are extensively tested to comply with rigorous and strict QC standards. For all of our clothing products, items and accessories, please take careful note of the precise measurements, mainly shown in centimeters (CM). As part of our company policy, we will not be able to accept returns or provide refunds for items which are the wrong size.
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text: 596 WWUS83 KJKL 230931 SPSJKL Special Weather Statement National Weather Service Jackson KY 431 AM EST Thu Feb 23 2017 KYZ052-058>060-068-069-079-080-083>088-104-106>120-231400- Rowan-Estill-Powell-Menifee-Rockcastle-Jackson-Pulaski-Laurel- Wayne-McCreary-Whitley-Knox-Bell-Harlan-Elliott-Morgan-Johnson- Wolfe-Magoffin-Floyd-Lee-Breathitt-Knott-Owsley-Perry-Clay-Leslie- Letcher-Martin-Pike- Including the cities of Morehead, Irvine, Ravenna, Clay City, Stanton, Frenchburg, Brodhead, Mount Vernon, McKee, Annville, Burnside, Somerset, London, Monticello, Stearns, Whitley City, Corbin, Williamsburg, Barbourville, Middlesboro, Pineville, Cumberland, Harlan, Sandy Hook, West Liberty, Paintsville, Campton, Salyersville, Prestonsburg, Wheelwright, Beattyville, Jackson, Hindman, Pippa Passes, Booneville, Hazard, Manchester, Hyden, Jenkins, Whitesburg, Inez, Coal Run, Elkhorn City, Pikeville, and South Williamson 431 AM EST Thu Feb 23 2017 ...Areas of Dense Fog Expected This Morning... Areas of dense fog have developed in the valleys overnight and will continue through this morning. Visibilities will fall under a quarter of a mile at times. Motorists should be prepared for sharply reduced visibilities and allow extra driving time, especially those heading to work or school this morning. $$ KAS/DJ


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I will create Online Marketing Plan Custom Tailored(1)
About This Gig
BONUS: Free Report – 3 Steps To Generating As Much Leads Of Your Ideal Customer As You Can Handle In 30-Days Or Less
Is your business online but you dont know how to get customers? OR want to take your business online but don't know how to go about acquiring customers?
For $50 I'll help you create a strategy that will not only focus on the best and most effective way to reach to prospects,but also convert them into customers and raving fans!
This gig involves market and customer research so you know exactly where your customers are and the best way to reach them effectively
Your investment is backed by my 100% MONEY-BACK guarantee – you're 100% satisfied or you get your investment back
If you would like my hands-on help buy my Premium or Pro package
For Premium you get my hands-on help with your marketing –offer, sales message, USP, ads, risk reversal, etc so your business is positioned as the best option in your prospects' mind
For Pro you get everything above and I'll also help you find your sweet-spot so you can get more sales and profits for the least effort and expense. And testing
Order NOW and get an online marketing strategy that turns a profit!


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Product Description
Supplement Facts
|Suggested Use
|Take 1-2 capsules per day. Begin by taking one serving in the morning prior to a meal. After assessing tolerance, a second serving can be taken 3-4 hours later, prior to a meal. Do not exceed 3 capsules in a 24 hour period.
|Other Ingredients
|Gelatin, Titanium Dioxide, FD&C Red #40, magnesium stearate, silicon dioxide, FD&C Blue #1.
|Not for use by those under the age of 18. Consult a physician before using this product. If you have, or have a family history of, heart disease, thyroid disease, liver disease, kidney disease, diabetes, high blood pressure, recurrent headaches, depression or other psychiatric conditions, glaucoma, difficulty in urinating, prostate enlargement, seizure disorder, if you are using a monoamine oxidase inhibitor (MAOI), anti-depressants, or any other medication, dietary supplement, prescription drug or over-the-counter drug consult with a physician. Discontinue use two weeks prior to surgery. Do not use if pregnant or nursing. Do not exceed recommended dose. Contains caffeine. Do not use in combination with other caffeinated products. Made in a GMP facility that processes milk, soy, egg, peanuts, tree nuts, fish, shellfish, and wheat.
Additional Information
|Universal Nutrition, Red Hot 60 Capsules
|Universal Nutrition
|Shipping Weight
|Package Description
|60 Capsules


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This maximum protection alarm has a patented smoke entry system that allows a direct smoke path to the photoelectric sensor to detect regular and slow, smoldering fires. First Alert's Nuisance-Free Technology will reduce false alarms that result from cooking and shower steam. This smoke and fire alarm includes an increasing loud 85db sound output and a powerful LED indicator, end of life signal, mute and single test/silence button so you can test your alarm regularly. Installation is simple with the simple twist lock wall and ceiling mount. Meets UL217 standards and backed by 10-year limited warranty and is the perfect low-key addition for any number of home settings.
It also features a tamper resistant locking pin that locks alarm to the mounting bracket to prevent removal of battery or alarm. Perfect for apartment, dormitory or hotel applications. The sleek, low profile design complements any decor. When voltage drops to a pre-set warning level, the horn will "chirp" approximately once per minute to indicate a fresh battery is needed. Designed for wall or ceiling mounting. Includes mounting anchors and screws.
|5.2 in (D) x 1.13 in (H)
|Pack Qty:
|Smoke Alarm
|10-Years Limited