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Steroidogenic acute regulatory (StAR) protein related lipid transfer (START) domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease.
Principal Findings
We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains.
Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity.
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Citation: Thorsell A-G, Lee WH, Persson C, Siponen MI, Nilsson M, Busam RD, et al. (2011) Comparative Structural Analysis of Lipid Binding START Domains. PLoS ONE 6(6): e19521. https://doi.org/10.1371/journal.pone.0019521
Editor: Nick Gay, University of Cambridge, United Kingdom
Received: January 31, 2011; Accepted: March 31, 2011; Published: June 30, 2011
Copyright: © 2011 Thorsell 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: The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. LL was also supported by the Academy of Finland Grant 128322. 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 received funding from the following commercial sources: GlaxoSmithkline, Merck & Co., Inc. and Novartis. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
The START domain is a ubiquitous conserved module for binding and transporting lipids [1]. Although the functions of most START domain containing proteins remain unknown, some regulate steroidogenesis and some are known to transfer lipids between membranes. There are approximately 40 proteins containing domains with START homology encoded in the human genome. The most well-characterized START domain containing proteins have been divided into 6 groups based on their phylogenetic relationships [2], [3], but additional members can be assigned to most of these groups. Group 1 contains the name-giving family member, steroidogenic acute regulatory protein (StAR/STARD1), and STARD3. Both are cholesterol carriers, and mutations in STARD1 cause congenital lipoid adrenal hyperplasia. Group 2 consist of proteins containing only a START domain; group 3 proteins are capable of binding different ligands, such as phosphatidyl choline (STARD2/PCTP) and ceramides (STARD11); group 4 proteins (DLC, or deleted in cancerous liver cells) are frequently de-regulated in cancer and contain Rho-GTPase activating domains; group 5 proteins contain two thioesterase domains; and group 6 consists of only STARD9, a 4614-residue protein with unknown function, that contains a kinesin motor domain at its N-terminus. Mitochondria contain at least the group 2 phosphatidylcholine transfer protein STARD7, and also the Coenzyme Q binding protein Coq10, which was recently identified to contain a divergent START domain [4].
Structural analyses of START domains from groups 1–3 have provided detailed insights into how these proteins sequester specific lipids [5]–[9] (summarized in Table 1). The ∼210 residue globular START module is a curved β-sheet gripped by two α-helices. The concave face of the β-sheet and the C-terminal α-helix enclose a hydrophobic cavity that can accommodate lipid molecules. Here we present crystal structures of four human START domains, those of STARD1, STARD5, STARD13 and STARD14/ACOT11. These structures extend our knowledge onto group 4 and 5 START domains, and enable a family-wide comparison of their lipid binding cavities. This structural comparison also sheds light on the lipid specificity of START proteins.
We used a structural genomics approach to human START domain containing proteins. Based on previously published crystal structures multiple expression constructs were designed for STARD1, STARD5, STARD7–11, STARD13 and STARD14. Following recombinant protein production in E. coli, well-diffracting crystals were obtained for the START domains of STARD1, STARD5, STARD13 and STARD14 (Table 2). Their crystal structures were solved and refined to between 2.0 and 3.4 Å resolution (Table 3).
Despite the low sequence identity among the START domains (Fig. 1) all structures show a conserved domain structure consisting of an α/β "helix-grip": A curved antiparallel β-sheet on which two helices near the N- and C-terminus are packed forming a cavity to the concave side of the sheet (Fig. 2 and Datapack S1). The backbone atoms of all four proteins superimpose with an rmsd of 2.3 Å over 154 residues (Fig. 2F), and the backbone atoms of all START domains included in Table 1 superimpose with an rmsd of 2.3 Å over 138 residues.
Protein sequences were aligned based on the available crystal structures as detailed in Materials and Methods. Secondary structure elements are shown on top of each sequence for which a crystal structure is available, and α-helices are numbered. Secondary structural elements in the C-terminal section of STARD14 isoform are shown in grey to indicate their divergence. Asterisks following protein names indicate that crystal structures of human proteins are available.
(A) Cartoon of the START domains studied here in context of the respective full-length proteins (drawn approximately to scale). (B–E) Side-by-side comparison of human STARD1, -5, -13, and -14 in a similar orientation. All START domain structures are colored from the N-terminus (blue) to the C-terminus (red) and the linker to the N-terminal thioesterase domain of STARD14 (panel E) is shown in grey. (F) Stereo view of a superposition of the backbone traces of the four crystal structures shown in panels B through E (blue, STARD1; red, STARD5; cyan, STARD13; yellow, STARD14). The view is that of panels B–E with an approximately 90° rotation downward toward the viewer.
We solved the crystal structure of STARD1, a member of the StAR group, at a relatively low resolution of 3.4 Å (Fig. 2B). Attempts to find better crystals were unfruitful, presumably due to the intriguing packing inside the lattice of these crystals: The asymmetric unit consists of four molecules that are organized as a long tube along the 63 axis (Fig. 3A). The inside diameter of the tube is 75 Å, resulting in a solvent content of 60%.
(A) Packing of STARD1 in the crystal lattice with the tube formed around the 63-axis. Monomers A–D in the asymmetric unit are colored individually, and symmetry generated molecules around the axis are shown. (B) STARD13 structure displaying the N-terminal helix swap with the adjacent protein molecule in the crystal. Two monomers (blue and white) are shown and the N-terminal helix of a third monomer is shown in magenta. Side chains are displayed for one of the C-terminal helices.
Homology modeling and subsequent ligand docking trials were previously studied in an effort to understand biological functions of STARD1 [10]. The STARD1 crystal structure supports the homology model (PDB 2I93). Superposition of the crystal structure with the lowest energy homology model yields an rmsd of 1.5 Å for 205 out of the 213 Cα atoms. Major differences between these structures are found in the loops 191–196 and 209–215.
Cholesterol was included in the crystallization buffer. However, additional density which was observed in the cavity did not match the expected density of cholesterol. We believe that the cavity was either empty or partially occupied by a small ligand derived from the expression host or from the crystallization solution.
STARD5 [11], a member of the START only group (Table 1), binds specifically to cholesterol and 25-hydroxycholesterol. The closest homolog of human STARD5 with a crystal structure available in the PDB is mouse STARD4 [7], with 34% shared sequence identity between the proteins. We also used this structure of mouse STARD4 (PDB entry 1JSS) as a molecular replacement model. Alignment of the refined structure with mouse STARD4 gives an rmsd of 1.3 Å over 194 Cα-atoms. The STARD5 structure is naturally also closely related to other human START domains (Fig. 2).
The structure of human STARD13, a member of the RhoGAP group, is also most similar to mouse STARD4, with an rmsd of 1.8 Å for 164 Cα-atoms. The largest difference between STARD13 and other START domain structures lies in the N-terminal helix, which in STARD13 is swapped with the adjacent protein in the crystal (Fig. 2D and 3B). The swapped helix interacts with the expected area of the β-sheet, but runs in an opposite direction. This surprising helix-swapping may be an artifact of a truncated expression construct.
The asymmetric unit of the crystal of STARD14/ACOT11 contains a dimer. The large buried surface area between the monomers (900 Å2 per monomer, as determined by the PISA server [12]) indicates that this interaction could form also in solution. However, the full-length protein likely forms a trimer in the thioesterase domains of ACOT12 (PDB id. 3B7K), and the dimer interface of truncated STARD14 may only be a part of the biologically relevant assembly. The STARD14 structure differs slightly from the other START domain structures in that the C-terminal consensus α-helix is broken into two shorter helices (Fig. 1 and 2E).
The unique N-terminal helix (α0) of STARD14 (Fig. 2E) acts as a linker to the thioesterase domains. Interestingly, this N-terminal helix packs onto the C-terminal START domain helix (α4′) that is thought to undergo a conformational change upon ligand binding [13]. Based on the crystal structure it is feasible that the N-terminal helix upon ligand binding transmits a conformational signal to the thioesterase domains to regulate its activity. Thus, our results form a structural basis for interpreting the conformation of the N-terminal helix. This however requires verification by experiments with the full-length protein.
Family wide structural comparison
Human START domains share a significant but low sequence identity (as low as 14%). As a consequence, homology-based sequence alignment methods make prediction of the positions of critical residues within the physiological START domain structures challenging. We generated a structure based sequence alignment by superposing all known START domain structures, and using this 3D alignment as a basis for aligning the sequences of the human START domain classes. This method yielded an improved alignment, and displayed similarities between individual proteins that have been overlooked by homology based methods (Fig. 1). When compared to previous family wide alignments [11] it is evident that the structure-based alignment has the similar overall features. It does not contain gaps within the secondary structure elements thus providing better alignment when the structure, but not necessarily the sequence, is conserved. On the other hand our structure-based alignment could be misleading for surface residues that are affected by crystal contacts, in particular for less well conserved loop residues of low structural importance. These regions often contain gaps in the alignment.
Notably, there are three absolutely conserved residues (Trp96, Trp147 and Arg217; STARD1 numbering) and a highly conserved Asp183 that is replaced by the similar glutamate only in STARD4 (Fig. 1). Trp96, Asp183 and Arg217 are all on the "back" face of the β-sheet (Fig. 4A): Asp183 and Arg217 form a salt bridge, whereas Trp96 appears to be structurally important in aligning the N-terminal helix onto the β-sheet. Trp147 is likely of functional importance, specifically as a possible gate keeper in lipid ligand loading. It is located in a helical loop region and interacts with the C-terminal helix. In STARD1, the hydrophobic cluster around this residue has been proposed to stabilize the C-terminal helix in a closed conformation [14]. Conservation of this structural feature across the domain family indicates that a lipid binding mechanism via local unfolding or a significant conformational change in the C-terminal helix could be a family wide phenomenon. Mutation in the adjacent, highly conserved residue Asn148 has been observed in congenital lipoid adrenal hyperplasia (lipoid CAH) [15], which add further evidence to the functional importance of this region (Fig. 4B).
(A) Positions of strictly conserved residues, as identified by the alignment shown in Figure 1, mapped onto the structure of STARD1 (pdb entry 3P0L). (B) Residues mutated in CAH shown as side chains in the STARD1 structure.
Lipoid CAH is linked also to other mutations in the STARD1 encoding gene. Some of these mutations lead to premature stop codons, while others change the protein activities and lipid binding capabilities [16], [15]. When the affected residues are mapped onto the STARD1 structure, it is evident that these changes occur in structurally important residues (Fig. 4B). However, with the exception of Asn148, the affected residues are not conserved across the family (Fig. 1). Most of the point mutations are in the C-terminal helix lining the ligand binding cleft or in residues interacting with this helix. These mutations would therefore cause changes in the dynamics of the ligand binding. It has been suggested that the C-terminal helix would undergo unfolding during ligand binding, and this suggestion is supported by the effects of the lipoid CAH mutations near the C-terminal helix [14]. The model of helix unfolding during cholesterol binding has been recently reviewed [17]. Some residues mutated in lipoid CAH are surface exposed indicating that they may change other interactions of the protein molecule as suggested for a gain of function mutation Q128R [15]. Also R182L is able to bind cholesterol but does not have "star-like activity" [18].
Cavity sizes in the known START proteins vary from 873 Å3 to 2297 Å3 (based on the molecular surfaces of ligand bound as well as ligand free structures). STARD14 has clearly smallest cavity of the family. Cholesterol binding START domains have cavity sizes of 1014–1122 Å3, which is close to the size of the natural ligand. The largest cavity is observed for STARD2, which also binds larger ligand than other characterized members of the family (Table 1, Fig. 5). It is possible that the shape of the cavity changes upon ligand binding and therefore the size of the cavity is not directly related to the size of the ligands. However, together with a structure based sequence alignment, the cavity sizes suggest key residues and structural determinants of ligand binding and selectivity.
Side-by-side comparison of the structures that have been solved with a ligand bound in the cavity: (A) STARD2 (1LN1) with 1,2-dilinoleoyl-SN-glycero-3-phosphocholine (DLP); (B) STARD11 (2E3R) C16-ceramide; (C) STARD14 (3FO5) with PEG or putative fatty acid.
StAR group - STARD1
Inspection of the ligand cavity of ligand free STARD1 suggests Glu169, Arg188, Leu199 and His220 as key residues in cholesterol binding. These side chains will likely change conformation upon ligand binding. Notably only His220 is conserved among the cholesterol binding members. Ligand docking predicted cholesterol binding to STARD1 involves a hydrogen bond between the cholesterol hydroxyl and either the Arg188 side chain or the backbone carbonyl of Leu199 [10]. Either of these ligand binding modes is consistent with the present STARD1 crystal structure.
START only - STARD5
In order to understand ligand binding in STARD5, we docked a cholesterol molecule to the binding cavity of the STARD5 structure. All the top ranked binding modes had cholesterol in the so-called "IN" conformation, with the hydroxyl group of cholesterol pointing towards the cavity (Fig. 6A). The binding mode is similar to the one predicted for other START domains [10]. In this scenario, the Ser132199 hydroxyl forms a hydrogen bond to the cholesterol hydroxyl in our best docking scenes as predicted for STARD3 (superscript numbering denotes positions in STARD1; see Fig. 1). A serine in this position is conserved in the cholesterol binding STARD3, -4 and -5, and there is a serine residue in the adjacent position in STARD6 that might fulfill the same function (Fig. 1). In all other START domain subfamilies there are hydrophobic residues at this position. Despite the conservation of this serine side chain within the cholesterol binding subclass, there is no similar serine in STARD1. Thus, in the absence of a START domain-cholesterol complex structure, the accurate binding mode of cholesterol can not be resolved.
(A) Model of cholesterol binding to STARD5. (B) Lipid binding cavity of STARD13, with the cavity inner surface indicated in the background (magenta). Side chains that are conserved and structurally complementary among STARD13 (blue) and STARD2 (grey) are shown as sticks. The C-terminal helix of STARD13 is shown as a blue cartoon to illustrate clashes with the STARD2 ligand. (C) Ligand binding to STARD14. Difference density is contoured at 2σ around the modeled PEG molecule in monomer B to show the elongated shape with density for the head group resembling a carboxyl group.
STARD5, in contrast to STARD1, can also bind 25-hydroxycholesterol [19]. The crystal structure and docking model suggests a structural basis for binding specificity towards this ligand: The additional hydroxyl group is attached to a flexible hydrophobic tail of cholesterol, and this hydroxyl could be positioned within hydrogen bonding distance of the side chain of Thr103171 in STARD5 (Fig. 6A). In STARD1 the corresponding residue is alanine and together with the missing serine side chain (Ser132199) at the bottom of the cavity this could cause different ligand binding modes in STARD1 and STARD5, as discussed above.
RhoGAP group - STARD13
The natural ligand of STARD13 is unknown. We looked to identify possible ligands based on the STARD13 side chains at the positions that correspond to those involved in lipid binding in other family members. From the crystal complexes of STARD11 and ceramides we know that Arg442144 and Glu446148 are the only conserved residues between the proteins making contacts with ceramide, Glu446148 being the most critical [8]. Notably, the STARD13 cavity also contains more polar side chains (three arginines, three histidines, an aspartate, a glutamate, two cystines and two tyrosines) compared to the cholesterol binding members, and the putative cholesterol hydroxyl binding Ser132199 of START5 is not conserved. The STARD13 cavity shares some characteristics with the members of the thioesterase group (discussed below). Intriguingly, some of the side chains that are involved in the interaction of STARD2 with dilinoleoylphosphatidylcholine are conserved in STARD13 (Fig. 6B): Arg974144 and Asp978148 are conserved in the corresponding position; Tyr999169 replaces Trp101169, Tyr1054225 replaces Tyr72137 and His1068241 replaces Gln157223. These side chains are also conserved in STARD8 of the same group, but not all in STARD12 (Fig. 1). Together, these properties indicate that STARD13 may bind a charged lipid.
Notably, the ligand binding cavity of STARD13 is smaller than that of STARD2 and elongated, with a small maximum diameter (Fig. 6B). This causes clashes between phosphatidylcholine and the C-terminal helix of STARD13 when the two structures are superposed. Upon ligand binding, the STARD13 cavity could expand due to movement of the C-terminal helix. The crystal structure however indicates that the natural ligand would be small – perhaps a fatty acid as proposed here for STARD14.
Thioesterase group – STARD14
The lipid binding cavity of STARD14 is rather hydrophobic as it is lined by phenylalanine, valine, leucine and isoleucine side chains. The cavity also contains patches of charged and hydrophilic residues, possibly making specific interactions with an unknown ligand. Inside the STARD14 cavity we observed a continuous electron density that by its shape resembles a fatty acid (Fig. 6C). In the monomer B of the asymmetric unit the density was more continuous. In addition to the STARD14 model discussed here, the fatty acid-like density was present also in two other data sets that originated from different STARD14 protein constructs crystallized in different space groups (not shown). Despite several attempts with different strategies we could not identify the ligand by mass spectrometry. Therefore we modeled the density as a polyethylene glycol (PEG) fragment in the published model. Nevertheless, we believe that the natural ligands of STARD14 isoforms may be fatty acids based on several lines of evidence: (i) The cavity and conserved residues lining it are not consistent with the known START domain ligands cholesterol, phosphatidylcholine or ceramides. (ii) As STARD14 also contains the acyl-CoA thioesterase domains, fatty acid binding to the START domain might be physiologically meaningful. (iii) The rat ortholog of STARD14 has acyl-CoA thioesterase activity with specificity towards medium to long-chain (C12–18) fatty acyl-CoA substrates [20] and the STARD14 structure appears compatible for binding fatty acids containing up to 18 carbon atoms.
The STARD14 structure is expanded in comparison to the empty START domain structures, although the C-terminal helices are in a similar position as the C-terminal helix of STARD2 solved in complex with phosphatidylcholine. Possibly BFIT1 and BFIT2, the isoforms of STARD14, could have different ligand specificity. The crystallized form (BFIT2) contains two helices at the C-terminus whereas BFIT1 probably has only one, as seen in other START domains (Fig. 2). Interestingly, BFIT2 is more similar to STARD15 than BFIT1 (Fig. 1). The residues that would bind the putative head group of the fatty acid, Arg449144 and Tyr546241, are conserved in STARD15. Tyr546241 is a tryptophan in BFIT1 and Tyr456151 is phenylalanine in STARD15 (Fig. 1 and 6C). Other interactions around the PEG molecule found in the structure do not appear to be strictly conserved; however if the ligand is a fatty acid, these interactions are likely not specific and the selectivity would be accomplished based on the shape of the cavity rather than by specific side chain interactions.
A structure based alignment reveals important features within the START domain subfamilies, and highlights critical conserved residues involved in ligand binding. Of particular interest in the scope of this paper are STARD13 and STARD14, the family members for which the ligands are not known. STARD13 Arg974/STARD14 Arg449144 is highly conserved (Fig. 1) and is likely a key residue binding negatively charged lipids such as phosphatidylcholine and fatty acids. This is complemented by a negatively charged residue at position 148. This is generally an aspartate, but in STARD11 it is the longer glutamate, which makes important hydrogen bonds to the bound ceramide. Notably, these residues are not conserved in the StAR nor in the STARD9 groups. In the StAR group there is a similar ion pair between the β–strands, namely, Glu169 and Arg188, which is not found in other family members that bind cholesterol. Arg188 has been suggested to bind to the hydroxyl group of cholesterol and it is also present in PCTP and RhoGAP groups. Trp147 is absolutely conserved across the family, and since the Trp147 side chain interacts with hydrophobic residues of the C-terminal helix in all the structures (Fig. 4), this region is likely important for lipid access to the cavity. Our structural analysis also suggests that the cavities can adjust to binding several types of lipids due to their flexibility and hydrophobic nature, while the small differences in the key conserved residues make them specific towards different lipids. The third helix is likely also important for the lipid binding mechanism as it interacts with the C-terminal helix proposed to partially unfold during lipid binding.
The crystal structures reported here help to gain a family wide understanding of the structural determinants within the START domain family. Use of these results to create a structure-based alignment helped to determine the conserved features within the family which are overlooked by sequence homology based methods. Many human START domains have unknown functions and their apo-structures form a structural basis for ligand identification thereby providing new leads to biological functions. All structures reported are relevant to disease. They are down- (STARD13) or up-regulated (STARD5) in cancers, mutations in them result in metabolic disorders (STARD1) or they are linked to obesity (STARD14; [21]). Based on our structural analysis we propose charged lipids as ligands for STARD13 and fatty acids as ligands for STARD14.
Materials and Methods
The cDNAs coding for full-length human STARD1, STARD5, and STARD14 were obtained from the Mammalian Gene Collection (accession codes BC010550, BC004365 and BC093846, respectively). The cDNA encoding full-length human STARD13 was PCR amplified from pooled human brain, liver, placenta, and thymus cDNA libraries (Ambion). The sequence coding for residues STARD1T66-R284, STARD5A6-E213, STARD13E51-I264, and STARD14R339-L594 were subcloned into expression vector pNIC-Bsa4 by ligation-independent cloning. The resulting expression constructs contained a hexahistidine tag and a TEV-protease cleavage site (MHHHHHHSSGVDLGTENLYFQS) at the N-terminus.
Protein expression and purification
Each expression construct was transformed into E. coli strain BL21(DE3)R3 pRARE (Novagen). Cultivation was done in a LEX large-scale expression system (Harbinger Biotechnology & Engineering). Cells were grown in Terrific Broth supplemented with 8 g/l of glycerol and 100 µl/l BREOX antifoam agent at 37°C. At an OD600 nm of between 1 and 2 the temperature was lowered to 18°C, recombinant protein production was induced by addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside, and cell growth was continued for 18 h. Cells were harvested by centrifugation and resuspended in 1.5 ml of buffer 1 per gram of wet cells (30 or 50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM TCEP). Before lysis, 4 µl (1000 U) of Benzonase (Novagen) and one tablet of Complete EDTA-free protease inhibitor (Roche Biosciences) were added per 50 ml cell suspension, and cells were lysed by a freeze-thaw cycle and sonication. Cell debris was removed by centrifugation and the soluble fractions were filtered through a syringe filter (0.45 µm pore size). Cleared cell lysates were passed over 1-ml HiTrap Chelating columns (GE Healthcare) pre-equilibrated with buffer 1. The columns were washed sequentially with buffer 1 and buffer 1 containing 25 mM imidazole. Bound protein was eluted with buffer 1 containing 500 mM imidazole and loaded onto 16/60 HiLoad Superdex-75 columns (GE Healthcare). Gel filtration was performed in buffer 2 (30 mM HEPES or 30 mM sodium phosphate, pH 7.5, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP). Fractions were pooled based on gel filtration profiles and purity determined by SDS-PAGE and Coomassie staining. STARD1 and STARD14 proteins were liberated from the hexahistidine tag by incubation with His6-tagged TEV-protease (20∶1 molar ratio) over night at room temperature and subsequent passage over 1-ml HiTrap Chelating columns. The proteins were concentrated to 28.2 mg/ml (STARD1), 21.8 mg/ml (STARD5), 4.3 mg/ml (STARD13), and 11.3 mg/ml (STARD14) using spin concentrators. TCEP was added to a final concentration of 2 mM and aliquots were flash-frozen and stored at −80°C. Proteins were typically more than 90% pure judged by SDS-PAGE analysis. Protein construct masses were verified by TOF-MS analysis.
Crystallization and data collection
Crystallization was done by the sitting or hanging drop vapor diffusion method. Proteins in gel filtration buffer were mixed with reservoir solution (see Table 2 for details). For data collection crystals were briefly dipped in cryo solution supplemented with suitable additives (Table 2) and flash-frozen in liquid nitrogen.
Synchrotron radiation datasets were collected at ESRF, Grenoble, France and at BESSY, Berlin, Germany. Data sets were indexed, scaled, and reduced using XDS (Table 3) [22].
Structure solution
Details of the structures are given in Table 4. The STARD1 structure was solved by molecular replacement with MOLREP included in the CCP4 suite [23] using the STARD3 (1EM2) structure as a model. MOLREP placed the first two molecules in the asymmetric unit and a third one after refinement. The fourth monomer was placed manually, using a helix density as guide, followed by a rigid body refinement in PHENIX [24]. A test set of reflections for STARD1 was selected with PHENIX to prevent creation of a biased set due to twinning. Twin refinement with operator h,-h-k,-l was done with PHENIX and the refined twin fraction was 0.149. This is in agreement with the estimated twin fraction using the diffraction data only (0.130). Model building was done with COOT [25].
The STARD5 structure was solved by molecular replacement with MOLREP [26] using pdb entry 1JSS as a model. The structure was refined initially with PHENIX and in the final stages with REFMAC5. TLS model consisting of 3 groups per monomer was used based on the suggestion by the TLSMD server [27].
STARD13 was solved by molecular replacement with MRBUMP [28] using pdb entry 1JSS as a model. The model was edited with CHAINSAW [29] and the best solution, with two monomers in the asymmetric unit, was found with PHASER [30]. After rigorous editing and refinement with PHENIX a third molecule was located with MOLREP using partially refined monomer as an input. Model building was done with COOT and REFMAC5 was used in the final refinement cycles.
The STARD14 structure was solved using Solve [31] with the three-wavelength MAD data. Resolve [32] was used to build the initial model, which was then improved by cycles of manual editing and PHENIX autobuild. The asymmetric unit consisted of two chains and one of them was used as a model in MOLREP using the native data. The model was further improved by 3 rounds of manual model building and automated building with arp-warp [33]. Final refinement cycles were done with REFMAC5.
Analysis of structures and visualization
Models were validated with Molprobity [34]. Initial structure based sequence alignments were made with MultiProt [35] and Staccato [36] using PDB entries 3P0L, 1LN1, 1EM2, 1JSS, 2R55, 2E30, 2PSO and 3FO5. The alignment was edited with Bodil [37] (similarity matrix STRMAT110; minor manual edits) to include full domain sequences, and visualized with Aline [38]. Cavity sizes were calculated with CASTp [39]. The enhanced version of the article was prepared with ICM (Molsoft).
Docking of cholesterol to STARD5 structure was done with ICM (Molsoft). Residues surrounding the cavity were selected to indicate the binding site and initial docking of cholesterol was done keeping the residues fixed. Best conformations were energy minimized with ICM and the residues around the docked ligand were optimized.
Supporting Information
Datapack S1.
Standalone iSee datapack - contains the enhanced version of this article for use offline. This file can be opened using free software available for download at http://www.molsoft.com/icm_browser.html .
Text S1.
Instructions for installation and use of the required web plugin (to access the online enhanced version of this article).
We gratefully acknowledge the beam line scientists at the BESSY and ESRF synchrotron radiation facilities for expert assistance.
Author Contributions
Conceived and designed the experiments: AGT CP MIS LL. Performed the experiments: AGT CP MS MN RDB TK LL. Analyzed the data: WHL HS LL. Wrote the paper: HS LL.
- 1. Ponting CP, Aravind L (1999) START: a lipid-binding domain in StAR, HD-ZIP and signalling proteins. Trends in Biochemical Science 24: 130–132.
- 2. Soccio RE, Breslow JL (2003) StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism. Journal of Biological Chemistry 278: 22183–22186.
- 3. Alpy F, Tomasetto C (2005) Give lipids a START: the StAR-related lipid transfer (START) domain in mammals. Journal of Cell Science 118: 2791–2801.
- 4. Barros MH, Johnson A, Gin P, Marbois BN, Clarke CF, et al. (2005) The Saccharomyces cerevisiae COQ10 gene encodes a START domain protein required for function of coenzyme Q in respiration. Journal of Biological Chemistry 280: 42627–42635.
- 5. Tsujishita Y, Hurley JH (2000) Structure and lipid transport mechanism of a StAR-related domain. Nature Structural Biology 7: 408–414.
- 6. Roderick SL, Chan WW, Agate DS, Olsen LR, Vetting MW, et al. (2002) Structure of human phosphatidylcholine transfer protein in complex with its ligand. Nature Structural Biology 9: 507–511.
- 7. Romanowski MJ, Soccio RE, Breslow JL, Burley SK (2002) Crystal structure of the Mus musculus cholesterol-regulated START protein 4 (StarD4) containing a StAR-related lipid transfer domain. Proceedings of the National Academy of Sciences of the United States of America 99: 6949–6954.
- 8. Kudo N, Kumagai K, Tomishige N, Yamaji T, Wakatsuki S, et al. (2008) Structural basis for specific lipid recognition by CERT responsible for nonvesicular trafficking of ceramide. Proceedings of the National Academy of Sciences of the United States of America 105: 488–493.
- 9. Kudo N, Kumagai K, Matsubara R, Kobayashi S, Hanada K, et al. (2010) Crystal structures of the CERT START domain with inhibitors provide insights into the mechanism of ceramide transfer. Journal of Molecular Biology 396: 245–251.
- 10. Murcia M, Faraldo-Gomez JD, Maxfield FR, Roux B (2006) Modeling the structure of the StART domains of MLN64 and StAR proteins in complex with cholesterol. Journal of Lipid Research 47: 2614–2630.
- 11. Soccio RE, Adams RM, Romanowski MJ, Sehayek E, Burley SK, et al. (2002) The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6. Proceedings of the National Academy of Sciences of the United States of America 99: 6943–6948.
- 12. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. Journal of Molecular Biology 372: 774–797.
- 13. Roostaee A, Barbar E, Lehoux JG, Lavigne P (2008) Cholesterol binding is a prerequisite for the activity of the steroidogenic acute regulatory protein (StAR). Biochemical Journal 412: 553–562.
- 14. Roostaee A, Barbar E, Lavigne P, LeHoux JG (2009) The mechanism of specific binding of free cholesterol by the steroidogenic acute regulatory protein: evidence for a role of the C-terminal alpha-helix in the gating of the binding site. Bioscience Reports 29: 89–101.
- 15. Bens S, Mohn A, Yuksel B, Kulle AE, Michalek M, et al. (2010) Congenital lipoid adrenal hyperplasia: functional characterization of three novel mutations in the STAR gene. Journal of Clinical Endocrinology and Metabolism 95: 1301–1308.
- 16. Bose HS, Sato S, Aisenberg J, Shalev SA, Matsuo N, et al. (2000) Mutations in the steroidogenic acute regulatory protein (StAR) in six patients with congenital lipoid adrenal hyperplasia. Journal of Clinical Endocrinology and Metabolism 85: 3636–3639.
- 17. Lavigne P, Najmanivich R, Lehoux JG (2010) Mammalian StAR-related lipid transfer (START) domains with specificity for cholesterol: structural conservation and mechanism of reversible binding. Subcellular Biochemistry 51: 425–437.
- 18. Baker BY, Epand RF, Epand RM, Miller WL (2007) Cholesterol binding does not predict activity of the steroidogenic acute regulatory protein, StAR. Journal of Biological Chemistry 282: 10223–10232.
- 19. Rodriguez-Agudo D, Ren S, Hylemon PB, Redford K, Natarajan R, et al. (2005) Human StarD5, a cytosolic StAR-related lipid binding protein. Journal of Lipid Research 46: 1615–1623.
- 20. Suematsu N, Okamoto K, Shibata K, Nakanishi Y, Isohashi F (2001) Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase. European Journal of Biochemistry 268: 2700–2709.
- 21. Ishikawa K, Nagase T, Suyama M, Miyajima N, Tanaka A, et al. (1998) Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Research 5: 169–176.
- 22. Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. Journal of Applied Crystallography 26: 795–800.
- 23. CCP4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallographica Section D-Biological Crystallography D50: 760–763.
- 24. Adams PA, Afonine PV, Bunkóczi G, Chen VB, Davis IW, et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D-Biological Crystallography D66: 213–221.
- 25. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallographica Section D-Biological Crystallography D60: 2126–2132.
- 26. Vagin A, Teplyakov A (1997) MOLREP: an automated program for molecular replacement. Journal of Applied Crystallography 30: 1022–1025.
- 27. Painter J, Merritt EA (2006) TLSMD web server for the generation of multi-group TLS models. Journal of Applied Crystallography 39: 109–111.
- 28. Keegan RM, Winn MD (2008) MrBUMP: an automated pipeline for molecular replacement. Acta Crystallographica Section D-Biological Crystallography D64: 119–124.
- 29. Stein N (2008) CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. Journal of Applied Crystallography 41: 641–643.
- 30. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Strotoni LC, et al. (2007) Phaser crystallographic software. Journal of Applied Crystallography 40: 658–674.
- 31. Terwilliger TC, Berendzen J (1999) Automated MAD and MIR structure solution. Acta Crystallographica Section D-Biological Crystallography 55: 849–861.
- 32. Terwilliger TC (2000) Maximum-likelihood density modification. Acta Crystallographica Section D-Biological Crystallography 56: 965–972.
- 33. Perrakis A, Harkiolaki M, Wilson KS, Lamzin VS (2001) ARP/wARP and molecular replacement. Acta Crystallographica Section D-Biological Crystallography 57: 1445–1450.
- 34. Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2003) Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50: 437–450.
- 35. Shatsky M, Nussinov R, Wolfson HJ (2004) A method for simultaneous alignment of multiple protein structures. Proteins 56: 143–156.
- 36. Shatsky M, Dror O, Schneidman-Duhovny D, Nussinov R, Wolfson HJ (2004) BioInfo3D: a suite of tools for structural bioinformatics. Nucleic Acids Research 32: W503–507.
- 37. Lehtonen JV, Still DJ, Rantanen VV, Ekholm J, Björklund D, et al. (2004) BODIL: a molecular modeling environment for structure-function analysis and drug design. Journal of Computer Aided Molecular Design 18: 401–419.
- 38. Bond CS, Schuttelkopf AW (2009) ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallographica Section D-Biological Crystallography 65: 510–512.
- 39. Liang J, Edelsbrunner H, Woodward C (1998) Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design. Protein Science 7: 1884–1897.
- 40. Bose HS, Whittal RM, Ran Y, Bose M, Baker BY, et al. (2008) StAR-like activity and molten globule behavior of StARD6, a male germ-line protein. Biochemistry 47: 2277–2288.
- 41. Wirtz KW (1991) Phospholipid transfer proteins. Annual Review of Biochemistry 60: 73–99.
- 42. Horibata Y, Sugimoto H (2010) StarD7 mediates the intracellular trafficking of phosphatidylcholine to mitochondria. Journal of Biological Chemistry 258: 7358–7365.
- 43. Olayioye MA, Vehring S, Muller P, Herrmann A, Schiller J, et al. (2005) StarD10, a START domain protein overexpressed in breast cancer, functions as a phospholipid transfer protein. Journal of Biological Chemistry 280: 27436–27442.


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|Version 16 (modified by 9 years ago) (diff),
Autocomplete user fields (assigned to and CC) on tickets
The AutocompleteUsersPlugin allows AJAX completion of users for the owner and CC fields on new and existing tickets. Currently, all the known users are autocompleted though this could be restricted to those that have the ticket owner permission. A
/users URL is served which presents the user data in a format suitable to the javascript autocompleter. The AutocompleteUsersPlugin should gracefully degrade in the case of no JS or the case where
[ticket] restrict_owner = true
Note: Email addresses shown by this plugin are handled accordingly to the setting
trac.show_email_addresses and the permission
EMAIL_VIEW. I.e. if
trac.show_email_addresses is set to
false and the current user doesn't have the permission
EMAIL_VIEW all mail address will be obfuscated as usual in Trac (i.e. email addresses will be displayed as
donjaun@... rather than
email@example.com). In any other case the full email address is visible.
Alternative: TracAutoCompletePlugin
Bugs/Feature Requests
Existing bugs and feature requests for AutocompleteUsersPlugin are here.
If you have any issues, create a new ticket.
Download and Source
Download the [download:autocompleteusersplugin zipped source], check out using Subversion, or browse the source with Trac. There is also an Unofficial Debian package. AutocompleteUsersPlugin works only for trac 0.11 and greater.
After installing plugin add to trac.ini configuration file following:
[components] autocompleteusers.* = enabled
Screenshot of the plugin in action:
Recent Changes
- 17234 by rjollos on 2018-07-06 01:06:00
TracAutocompleteUsersPlugin 0.4.5dev: Fixup user list on POST
Refs #12952.
- 17204 by rjollos on 2018-06-14 21:42:58
TracAutocompleteUsersPlugin 0.4.5dev: Fix failure when
TicketModuleis the default handler
Refs #12952.
- 17203 by rjollos on 2018-06-13 18:56:44
TracAutocompleteUsersPlugin 0.4.5dev: Allow multi-completion of custom fields
Refs #12952.
Author: k0s
Contributors: tenzochris, Sebastian Krysmanski
Attachments (1)
screenshot.png (22.3 KB) - added by 10 years ago.
illustration of the AutocompleteUsers on the CC field
Download all attachments as: .zip

subset: commoncrawl


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October 19, 2010
Old vine pinot at Medici vineyard
I think of today as a field trip, to taste and see grapes in a vineyard I've never before visited, then helping with the processing at a winery in the McMinnville wine ghetto. There's nothing like getting out of your cocoon to see how other people think about and work harvest. What did I learn? Nothing major, just little things about vine health, grape maturity, sorting technique and general approach to fermentation (hands off). The biggest thing might have been how comfortable I finally am with all the usual duties of bringing in and processing fruit. It's not rocket science, but there's a difference between following along and staying out of the way and knowing what needs to be done and when.
I guess that's a long way of saying I actually helped out and it felt good, especially seeing friends get their first lot of grapes for a new project. Meanwhile, things are cooking back here in Portland. All of my own fruit is in and things look great. More on that soon. I'm late for an appointment with my dreams.
2 comments:
- Florida Jim said...
36 year old, own rooted pinot; man would I like to work with that stuff!
There's a vineyard on Sonoma Mtn. that's 48 year old, own rooted pinot (clone unknown) that's barely throwing fruit (and really did not produce this year). Maybe next year we'll get a half ton or so.
Could be special . . .
Best, Jim
- Vincent Fritzsche said...
Jim, there's a fair amount of older, own rooted pinot up here. Not tons and tons, but vineyard here and there that are 25 or 30 or more years old, nicely mature by Burgundy standards but ancient for us. I've been able to work with a couple a few years back and I hope to do so next year and beyond. They won't make better wine necessarily, but they will provide something different and certainly have potential that younger vines generally won't give.


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Claude Brunswic
Born: July 31, 1920
Heidelberg, Germany
Claude was one of five children born to Jewish parents in the university city of Heidelberg. His father, a physician specializing in internal medicine, had his practice on the first floor of the apartment building in which the family lived. Claude was an avid swimmer until November 1932, when local Nazi party edicts forbad Jews to use the city pool where he swam.
1933-39: In January 1933, just after Hitler became chancellor of Germany, hoodlums attacked Jewish-owned businesses in Heidelberg. They broke my father's office windows and destroyed his X-ray machine. My family decided then to leave Germany. We relocated to the Netherlands, and then, in 1937, moved to Paris. I worked at a piano factory there until I enlisted in the French army in September 1939. My basic training took place in Orleans.
1940-45: Germany invaded France in May 1940. I was taken prisoner with thousands of other French soldiers and held in a POW camp in Trier, Germany. Over the next few years, I was a slave laborer, working on farms and repairing bombed-out train tracks. In 1943 I escaped to France, but was caught and returned to Trier. I was sentenced to solitary confinement and given little food. Fortunately, Soviet POWs in the barracks above my cell kept me alive by passing me food and cigarettes through the floor boards.
Claude was liberated by Americans in January 1945. After the war, he first returned to Germany to assist Jewish refugees. In 1946 he emigrated to the United States.


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More vendors than ever planned at Senior Expo
There are several new vendors for the Senior Expo this year. Some of the new vendors are Golden Bridges, Walgreens, Sam's Club, Master Air Heating and Air Conditioning and Big River Pest Control. There are 75 booths at the sixth annual Senior Expo, which is 11 a.m. to 2 p.m. Thursday, April 18 at the Admiral Coontz Recreation Center in Hannibal. Some of the vendors who have participated for years include: YMCA, James O'Donnell Funeral Home and Beth-Haven. Stacey Nicholas, RSVP director, said the event gets bigger and better every year. "The Admiral Coontz Recreation Center has been renovated so it's brighter," she said. "And we have a lot of great raffle prizes to give away." Commemorative tote bags will be given to the first 300 visitors. The theme for this year's expo is "It Only Gets Better." Senior Expo 2013 is sponsored by Hannibal Parks & Recreation, RSVP at Douglass Community Services, NECAC, Experience Works, the Marion and Ralls County Health Departments and NEILS.


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At one time, the mega-hit "Flashdance" was imagined as a stage musical, and not a movie.
That 1983 film made Jennifer Beals a star and turned dance togs into fashion must-haves. And now, we can see how things might have been with "Flashdance — the Musical" coming to the Broward Center for a two-week run beginning Tuesday, March 5.
"I was young and naive," says part-time Palm Beacher Tom Hedley, who co-wrote the script with Joe Eszterhas. "I thought, 'Wouldn't it be great if I could talk Bob Fosse into doing this film?' He was very kind. He read the script several times. He had notes for me. He hated rock 'n' roll … but thought it was a very good idea for the stage. Paramount just laughed. They said, 'We own it,' and I had already cashed the check, so that was that. But it has the structure of a theater musical. You know, Fosse caught a preview [of the movie], and he phoned me out of the blue one day and said, 'It's going to make a lot of money,' and he hung up."
Co-writing the musical's book with Robert Cary (who also wrote the lyrics), Hedley saw "Flashdance" make it to the stage 25 years later, when the show premiered in Britain in 2008 before going on a United Kingdom tour and eventually landing in London's West End in 2010.
"It's not a sendup," Cary explains. "This is not 'Xanadu' or 'The Wedding Singer.' We are trying to tell the story sincerely and not in quotes. So the music isn't a pastiche. We tried to use sounds that suggest early Pat Benatar or Duran Duran. Or maybe we do some early, early Madonna without copying her ... making the audience say, 'This is a take on 'Borderline.' "
The creative team has their eyes on Broadway. "What we are waiting for is a theater," Hedley says. "And that is dictated by who wins a Tony and who doesn't win a Tony. You wait and see who isn't standing up, because you know that theater might come open. You always wait for that before you negotiate your final deal."
"Flashdance — the Musical" runs March 5-17 at the Broward Center for the Performing Arts, 201 SW Fifth Ave., in Fort Lauderdale. Shows are 8 p.m. Tuesdays through Saturdays; 2 p.m. matinee Saturdays, Sundays and Wednesday, March 13; 7:30 p.m. Sundays. Tickets cost from $39.50 to $79.50. Call 954-462-0222 or visit BrowardCenter.org.


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First Baptist Church, Kyle TX
Saturday, January 19, 2019
"Growing disciples who know, love, serve and share Jesus Christ.
*Wednesday Night Activities
Date: Wednesday, May 30, 2018
Time: 6:30 PM -
Location: FBC Kyle
Join us Wednesday evenings for a time of prayer and Bible study led by Brother Jeff English, or choose one of the other Family Group gatherings. Age appropriate activities for children/youth are also available during this time. During the school year, come at 5:30 for a great supper before the service!Previous Page


text:
The Brooklyn Flea Moves Outdoors!
This past weekend, I stepped outside to enjoy one of the year's first breezy, sun-filled days in New York. I was thrilled and surprised to find the Brooklyn Flea has not only debuted their outdoor market…but that it set up shop right outside my door!
Hailed by The New York Times as "One of the great urban experiences in New York," the Brooklyn Flea is open every weekend of the year and features hundreds of local and regional vendors of antiques, vintage clothing, repurposed furniture, jewelry and arts & crafts by artisanal designers. The acre-wide market is also a hot-spot for local food vendors running the gamut from boutique businesses like People's Pops, which specializes in delectable gourmet popsicles, to more established names in the food world like Momofuku Milk Bar, which serves up out-of-this-world pastries and flavored milk.
The Brooklyn Flea has gained enormous popularity, not only for the quality of its vendors, but for its curatorial, community based lineup of outstanding local designers and food makers.
Currently, the Brooklyn Flea is open every weekend on Saturdays in Fort Greene and on Sundays in Williamsburg, from 10am to 5pm, "rain or shine." For directions, a list of vendors, or to get in on the weekly scavenger hunt, visit www.BrooklynFlea.com
1. Reclaimed furnishings of Interior Bliss Design
2. Daniel finds a birdcage at Alexandra Abuza
3. Vintage Clocks from City Owl
4 & 5. Kevin inspects a vintage library cabinet and bear head at Daily Memorandum
6. Wheel thrown, hand painted porcelain by SKT Ceramics
7. Lovebird busts at Hippie Danish
8. Vintage toy car and salvaged farm antiques from Kings County Salvage
9. Rose enjoys her hot dog with Thai Mango Relish from Asia Dog
10. The Brooklyn Flea!


text:
9:00 pm
Dust Bowl "Reaping The Whirlwind" For nearly a decade, the storms grow more ferocious and more frequent, sweeping up millions of tons of earth, killing crops and livestock, even spreading the dust across the country. Thousands flee, forced onto the road in an exodus unlike anything the United States had seen. But other thousands persevere against enormous odds. Families find ways to survive and hold onto their land. National and local government programs keep hungry families afloat, and a partnership between government agencies and farmers develop new farming and conservation methods. Part 2 of 2G
11:00 pm
Nature "My Life As A Turkey" This prize-winning film re-enacts the unusual story of Joe Hutto, who for nearly two years became the mother of a group of Florida wild turkeys he imprinted as hatchlings at his home adjacent to Florida wildlands. Taken from Hutto's 1995 book, Illuminations in the Flatlands, which was written from his daily notations, the film is set in the same wildlands. Hutto is portrayed by wildlife photographer Jeff Palmer.G


text:
your bag
Your bag is empty.
Sorry, this product has sold out!
If you would like ARCHFASHION to try to and find it for you complete the form below.
This stallion is constructed from high grade vegetable tanned french leather { semi anilina finishing } and features 5 card slots including nifty uplift id space { can fit up to 10 cards }. Made for the minimalist, the jonah wallet remains the stalwart of the mens range. Dimensions: 105 x 75 x 11mm packaging: matt finished sa. Tin


text:
Information for users
Useful tips and information for using the internet service of the Federal Office for Migration and Refugees
The Help pages cover the following topics:
- screen resolution and browser compatibility
- barrier-free access
Screen resolution and browser compatibility
Barrier-free access
The service has been provided in accordance with the guidelines contained in the Ordinance on the Establishment of Barrier-free Information Technology in accordance with the Act on Equality of Persons with Disabilities.
The pages have a uniform structure; navigation and the content are broken down into separate areas. Typing ALT+0 and Enter will take you directly to the contents page.
Table of contents
The Table of contents shows you the basic structure of the www.bamf.de pages. You can click on the individual links to go directly to the page that interests you.
Changing the font size
You can configure the font size according to your individual viewing wishes. Internet Explorer lets you choose the "Text size" on the "View" menu item. If you are using Firefox, you can click on the "View" menu item and then "Zoom" and can change the font size with "Larger" or "Smaller".
Choice of language
Words which are not in Duden's "Deutsches Universalwörterbuch" are marked with a language label. This enables for instance a reading program to select the correct pronunciation.
PDF documents
We have been offering barrier-free PDFs since May 2009. All barrier-free PDFs are labelled "barrier-free". If you would like to have an older PDF document or a publication in a barrier-free form, please let us know via the e-mail contact. (Ref.: PDF barrier-free).
A small number of documents are not barrier-free for a variety of reasons, such as because their content changes on a weekly basis, they contain foreign languages that are not supported or have content with highly extensive statistical and table data. These are labelled accordingly.
Harald Gebert
Frankenstraße 210
D-90461 Nürnberg
Tel.: +49 911 943-2436


text:
Opinion: Taking on Michigan's Tea Party Icon
Assistant editorial features editor Kate Bachelder on why a Grand Rapids businessman is challenging Rep. Justin Amash in the GOP primary.
This transcript has been automatically generated and may not be 100% accurate.
... I ... welcome back to Opinion Journal I'm Mary Kissel ... it's the party icon Michigan Representative Justin Lodge ... is facing a primary challenge ... could he actually ... lose says seeing what are the chances ... to fast in racy we've got assistant editorial features editor Kate Batchelder here ... to tell us about it k ... why would anybody she outlines just in my life I thought the sky was totally untouched ... what if an interesting story is so for some contacts were in Grand Rapids Michigan I which is a ... very red districts on safely read it and I just in one ... two times ago into doesn't end and he has been on challenges in the last race and now ... on some local businessmen and created some chatter some mutterings on ... that they're unhappy with the way he's voted on some very important she is and they're putting ... their own name Bryan Allen whose prime announce ... the new talents as well we don't know much about yet that he is a self starter on entrepreneur and ... owns an investment firm in Grand Rapids ... on and he has been hitting the line that I had a maths has voted ... to ... I mean she's like the Keystone pipeline and planned Parenthood ... doesn't March voted present on both of those issues instead of four barring funding for planned Parenthood and for ... the Keystone pipeline ... that's interesting to me because you think that the tea party would be all four ... of ... the devil the Keystone pipeline set because ... he's he's really playing to his base at Michigan is actually ... not a red cedar blue states really it's it's interesting well and the interesting thing about maybe Ummah she's misreading of constituents he voted against the Keystone pipeline ... pipeline fleeting that ... it had it was constitutionally not corrected target a specific industry ... the one thing very well contained within a practical issue ... to the question and it was Alan's Up character can really hit him on those bouts of rain over again what people of the people Grand Rapids to care about very practical things ... I is that going to resonate with that and just my ... on its too early to how long they terminated state senator who is talking about jumping in as well and that would be ... of good news for Ma shoo in in in a three way primary definitely hang on the income to write ... on but it's too early to follow and I was definitely has a shot at it ... if he it's ... one that could campaign and his son message over and over again Adam options traded on national falling ... on over the people of the Outback Nowinski DFS the race to watch Assistant features editor Kate Batchelder ... thank you so much for the IPO could come back again they say ... you can follow us on Twitter email us at Opinion Journal I'm at the beest dot com ... to check this out and Facebook it WS J opinion ... I'm Mary Kissel with The Wall Street Journal Editorial board ... thanks for watching ...


text:
Wolves Sink Lakers With Ridiculous First Quarter
The Timberwolves used a ridiculous 47 point first quarter to beat the Lakers for the first time in 23 games last night 113-90. It was the first Wolves win in Los Angeles since 2005 and first overall since 2007.
Ricky Rubio had a triple-double for the Wolves with 12 pts, 10 rebounds, 14 assists and five steals. Kevin Love and Nikola Pekovic each had double-doubles for Minnesota as well.
Tonight the Wolves are in the same Staples Center, but playing against a different opponent. The LA Clippers host Minnesota tonight at 9 PM. (1390 The Fan, 9 PM).


text:
Product Details
Manifold Absolute Pressure Sensor (W0133-1681891-DEL) by Delphi®. Delphi MAP sensors are internally insulated with silicone gel over the integrated circuit chip for reduced temperatures and vibration resistance, resulting in longer service life. Delphi MAP sensors are ISO 9001 and QS-9000 certified to maintain optimal performance during drastic temperature changes. Plus, the all-silicone technology of Delphi MAP sensors provides long-lasting protection against noise interference and other sensor output from the vehicle.
- Extensive coverage
- Delphi MAP sensors undergo certified electrical testing with industry EMI protection against noise interference and other sensor output from the vehicle
- Manufactured to meet or exceed OE requirements and testing
Delphi® has more than 50 years of OE experience in the fuel handling systems category. Delphi invented the first automotive radiator in 1911, the first car heater in 1929 and the first vehicle air conditioner in 1954. Delphi has over 100 years of experience supplying OEMs, which today includes the likes of Toyota, Ford, Audi and Ferrari.


text:
JetBlue pilot indicted for flight disruption
(AP) - A JetBlue Airways pilot accused of disrupting a Las Vegas-bound flight when he left the cockpit screaming about religion and terrorists has been indicted.
Court documents posted Thursday show Clayton F. Osbon has been indicted on one count of interference of a flight crew. It's the same charge he's been held on since short after the March 27 incident.
A secretary for Osbon's attorney, Dean Roper, says he declines to comment.
Osbon currently is undergoing a court-ordered psychiatric exam to determine whether he was legally sane when passengers wrestled him to the floor after witnesses said he ran through the cabin yelling about Jesus and al-Qaida. It also will determine whether he's can stand trial.
The first officer landed Flight 191 in Amarillo after locking Osbon out of the cockpit.


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2007-12-05 / Library
At the Library...
Massapequa 523 Central Avenue 798-4607 Massapequa Park 40 Harbor Lane 799-0770 •The Harbor Lane Library is sponsoring a bus trip to see The Little Mermaid on Broadway, Wednesday, December 26. The cost is $175 per person and includes lunch, show ticket, transportation and tips. Call Jody Brower at 799-0770, extension 316 to reserve a seat.
You must be an online subscriber to view this story.


text:
Laundry Organization
Don't let the laundry piles take over your space and time! There are several organizational strategies and solutions to help you win the laundry room battle. We have laundry storage solutions in a large variety of sizes and styles to fit in your space and with your custom look including eco-friendly bamboo, linen and collapsible cotton canvas options. More


text:
The most troubling thoughts to occupy Stuart Lancaster's mind this week would probably not have been on how to beat the All Blacks - a team who have been defeated once in two years.
Nor would it have been on the selection dilemma he faces this week after a stale second half display against Argentina left many asleep in their chairs.
Instead, it would have focused on the news that Manu Tuilagi, England's dynamic outside-centre, is very likely to miss next year's Six Nations after undergoing a chest operation.
Tuilagi has become just one of a number of Lions to have fallen foul of injury at the start of this season after he tore a pectoral in September.
The news that he will be forced to miss all of both the autumn internationals and the Six Nations is a bitter blow for both club and country. His absence, you feel, will be most sorely felt on the international stage.
He is a key loss for England. Just how key and important he is has become made even clearer by England's performances this autumn.
Two wins against Australia and Argentina is nothing to be sniffed at. England's displays, however, have left much to the imagination.
They needed elements of luck to overcome an Australian side bereft of confidence and victories (their win over Italy last weekend was only their fourth this year), and were outclassed for nearly 40 minutes by a very below-average Argentinian team.
Many of England's failings this autumn have stemmed from the back-line, who have looked low on imagination and class.
Much of the criticism has been directed in Chris Ashton's direction, simply for the reason that the winger has done nothing to criticise those who believe that he has not merited his England berth for a year.
However, in many respects, Ashton's criticism has been nothing more than a cover for the main reason for England's lack-lustre backs. Tuilagi's absence has been the key.
Unlike Billy Twelvetrees and Joel Tomkins, who for all his endeavours, looks nothing more than Brad Barritt in disguise, Tuilagi brings a dynamism and real gain-line threat to England's play.
He is the one player in the English back-line who will have opposition coaches fretting over. Tuilagi is, for want of a better phrase, a game changer.
And for all the positives coming out of the current England set-up, he is probably the only world-class 'game changer' that Stuart Lancaster has at his disposal.
Whether it is bursting through tackles or providing space for others to exploit, the English team does not look as threatening nor as exciting without him.
Take a look at two of England's key victories in the past year, against New Zealand and France.
In both games Tuilagi was the man with the most line breaks; the one who threatened the most and, against New Zealand, provided the individual moment of genius that set up Brad Barritt's try.
When the game is going against England, he is more often than not the one person who can provide the platform for the team.
One of the main reasons for the crucial nature of Tuilagi's presence is that England are not blessed with strength in depth in the centre.
Unless Tomkins steps up or Lancaster decides to throw either Luther Burrell or Henry Trinder in at the deep end against France in the opening Six Nations match, it is difficult to see where England's attacking platform or real threat will come from in the tournament.
Without Tuilagi, it is difficult to foresee England being able to overcome both France and Scotland away from home with no real pressure valve in the backs that Tuilagi's presence brings.
Now this is not a Tuilagi love in; he does have a tendency to disappear a tad too often in games and his handling skills are nothing in comparison to 13s of the Brian O'Driscoll and Will Greenwood ilk.
But there is a reason as to why O'Driscoll was so excited to play with Tuilagi for the Lions this summer.
The big man from Samoa has an extra dimension to his game that resides with both his pace and seemingly super-human power.
They have been lucky this autumn that they have played two teams short of quality and confidence. Without him away from home, England may find that they will struggle come the spring.
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DISCLAIMER: This article has been written by a member of the GiveMeSport Writing Academy and does not represent the views of GiveMeSport.com or SportsNewMedia. The views and opinions expressed are solely that of the author credited at the top of this article. GiveMeSport.com and SportsNewMedia do not take any responsibility for the content of its contributors.


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So WikiLeaks Is Evil For Releasing Documents... But DynCorp Gets A Pass For Pimping Young Boys To Afghan Cops?
from the wtf dept
The details are horrifying. The Afghani interior minister apparently went to US officials to warn them that reporters were sniffing around this story, and urged them to try to kill the story. He specifically warned that this would look bad if the connection to DynCorp was made clear (he called them "foreign mentors"). Apparently, US diplomats told him not to worry, and the eventual story was in fact watered down greatly (until now, of course) calling the whole thing a "tribal dance," rather than a party where young boys wear "scanty women's clothes" and "dance seductively" before being "auctioned off to the highest bidder" for sex.
Oh, did we mention that DynCorp makes $2 billion per year -- 95% of which comes from American taxpayers.
And US government officials are declaring Wikileaks as an organization to shun and not to work with? What about DynCorp? Are their DNS providers pulling the plug? Are their banks shutting down their accounts? Are they being denounced by Hillary Clinton and Joe Lieberman? No? Why the hell not?


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Get Data Entry Done
Presupuesto $250-750 USD
32 freelancers están ofertando el promedio de $416 para este trabajo
Excel expert - Data entry manager - VBA programmer I can do anything for you in excel Feel free to contact with me and ask any questions you have
Self-motivated professional, certified data entry specialist, a proficient web researcher and transcriber, blogger, web designer, Wordpress and various social sites marketer, knowledgeable in SEO, and technical / help Más
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I have experience in Data Entry as I worked in an accounting company with lots of excel books .
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basement finishing milwaukee Fundamentals ExplainedWhen Kitchen Remodeling and Bath Remodeling During Home Renovation, important Tips
During holiday season or if you have not done some work on your house in a while and intend to commence you may resort to a remodeling pro company in order to help with home renovation. Living room or bathroom when is comes to home remodeling and renovation we try to get the best out of it and have to most beautiful kitchen. Here are a few crucial tips on home renovation.
What Make A Perfect Kitchen Remodel?According to the saying that food is the way to someone heart, then all road lead to the kitchen. Everyone dreams of having the perfect kitchen and with these tips you are bound to have a great kitchen the next time you remodel your kitchen.
* Counter space is essential for every kitchen and having enough counter space for many individuals to cook collectively will be amazing for when your daughter or son desire to help you put together supper.
* The lightening and the number of outlets you have in the kitchen is also an important factor. Placement of those outlets is important when you have multiple home appliances, it can avoid a lot of frustration.
* Arranging the kitchen can definitely help when making your dream kitchen come to life. In your next Kitchen Remodel always remember to organise it so the cook can have a free flow between all tasks. Also something that can really help is organising it in such a way that there is enough storage for everything you need.What Makes check here A Perfect Bath Remodel?You start off your day and also end it in the bathroom. When you come home to a nice warm and relaxing bath, having the perfect bathroom can really help you in a bad day. There are many things that you should do when you renovating you bathroom:
* Just like kitchen, the lighting is an important aspect of the bathroom as well. Imagine coming home to a beautifully lit bathroom where you can dim the lights and have a nice quiet bath. That is what make a fantastic bathroom. The capacity to have change the light setting according to your read more need.
* Using the right components also help in making a great bathroom. Using natural stones can sometimes be an annoyance as they get discoloured and badly stained. Experts highly recommend using the natural stone replications such as the Kohler Choreograph collection when remodeling you bathroom.
* The items in your bathroom are very important. From choosing between a bath or a shower to adding more features such as hand held showers or much more luxury items like heated tile floors, heated towel bars, frameless glass showers can really make a difference when doing your next bath remodel.It is important to choose a well reputed remodeling expert company when home remodeling. There are many companies that will help you create the perfect home for you, just get in touch with us and we will connect you with top remodeling contractors.


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Lady Lions score trio of unanswered goals to win regional final
The strike came out of nowhere.
Midway through the first half Friday, visiting Shawnee Heights floated a shot over the out-stretched arms of Lansing goalkeeper Kelsie Pitsenbarger.
The goal sent a shiver through the home crowd while at the same time giving the visitors a 1-0 advantage in the teams' Class 5A regional final.
However, instead of losing its composure or abandoning its game plan, the Lady Lions soccer team went forward, not backwards.
"We just got ourselves out of shape on the field when we gave up that goal," Lansing coach Billy Dillon said. "We knew what we did wrong, the girls fixed it and picked up the energy. They responded like they should."
That response included three unanswered goals and a trip to the state quarterfinals after the Lady Lions downed Heights 3-1.
With six minutes left in the first half, Lansing evened the score at 1-1 when Jenna Hinckley slammed home a well-placed header.
Then, three minutes into the second half, Ashley Wilcox kicked in a shot from the 18-yard box for a quick 2-1 Lions advantage. That became 3-1 following Tiffany Anderson's penalty kick in the 78th minute.
"We kept up the pressure and intensity coming out in the second half and that paid off," Dillon said.
The quarterfinals are scheduled to be played on Tuesday, though who, where or when Lansing will play won't be announced until later today.


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A happy, cheerful and childlike track that brings to mind images of Spring, happiness in the air, and positive feelings. This track has the perfect sound for commercials, YouTube videos, corporate videos, children's projects and more.
Feel free to contact me should you need a duration change or some of the track parts switches. Ill gladly help you. Please be kind and dont forget to Rate the Purchase!


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Transocean Settles Deepwater Horizon Claims
Drilling contractor Transocean and the U.S. Department of Justice (DOJ) reached an agreement Thursday to settle outstanding civil and potential criminal claims related to the April 2010 Deepwater Horizon incident, concluding DOJ's criminal investigation of Transocean's role in the largest offshore oil spill in U.S. history.
Under the agreement, Transocean will plead guilty to one misdemeanor violation of the Clean Water Act (CWA) for negligent discharge of oil into the U.S. Gulf of Mexico and pay $1.4 billion in fines, recoveries and penalties, excluding interest. The violation is related to well monitoring in connection with specific operations during the temporary abandonment procedure conducted on April 20, 2010.
The company will pay the fines over a five-year period, using available cash and cash flow from its operations. The anticipated payment schedule will be:
- $560 million in 2013
- $460 million in 2014
- $260 million in 2015
- $60 million in 2016
- $60 million in 2017
To settle potential criminal claims, Transocean will pay $100 million in fines within 60 days of the agreement receiving U.S. federal court approval. Transocean also will pay $150 million to the National Academy of Sciences (NAS) over a five-year period; these funds will be used for oil spill prevention and response efforts in the U.S. Gulf.
Additionally, the company will pay $150 million to the National Fish and Wildlife Foundation over a three-year period. These funds will be used for natural resource restoration projects and coastal habitat restoration, including restoration of barrier islands offshore Louisiana and diversion projects on the Mississippi and Atchafalaya rivers.
The Transocean subsidiary pleading guilty will also be placed on a statutory maximum five-year probation term.
To settle certain civil claims, Transocean will pay $1 billion in CWA civil penalties over a three-year period. Under the civil settlement, Transocean must also implement court-enforceable measures to improve the operational safety and emergency response capabilities on all their rigs working in the United States, the U.S. Environmental Protection Agency (EPA) said in a statement Thursday.
Transocean said it has agreed to consult with the United States on preparing a performance plan for these improved measures, which must be submitted for government approval within 120 days of the agreement taking effect.
"The resolution of criminal allegations and civil claims against Transocean brings us one significant step closer to justice for the human, environmental and economic devastation wrought by the Deepwater Horizon disaster," said Attorney General Eric Holder in a statement.
The U.S. Environmental Protection Agency will continue to work with DOJ and its federal partners to "vigorously pursue" the government's claims against all responsible parties and ensure that we are taking every possible step to restore and protect the Gulf Coast ecosystem, Cynthia Giles, assistant administrator for the EPA's Office of Enforcement and Compliance Assurance, commented.
"This unprecedented settlement under the Clean Water Act demonstrates that companies will be held fully accountable for their conduct and share responsibility for compliance with the laws that protect the public and environment from harm," said Ignacia S. Moreno, assistant attorney general for DOJ's Environment and Natural Resources Division, in a statement.
"These important agreements, which the company believes to be in the best interest of its shareholders and employees, remove much of the uncertainty associated with the accident," said Transocean in a statement. "This is a positive step forward, but it is also a time to reflect on the 11 men who lost their lives aboard the Deepwater Horizon. Their families continue to be in the thoughts and prayers of all of us at Transocean."
Potential claims associated with the Natural Resources Damage Assessment (NRDA) process are excluded from the agreement with DOJ. But Transocean noted the district court previously determined the company is not liable under the Oil Pollution Act for damages caused by subsurface discharge from the Macondo well. If the ruling is upheld on appeal, Transocean's NRDA liability would be limited to any such damages arising from the above-surface discharge.
Transocean also will continue cooperating with the Deepwater Horizon Task Force in any ongoing investigation related to the accident.
Last September, the company reported it was in discussions with DOJ seeking to resolve certain civil and criminal claims related to Deepwater Horizon for $1.5 billion.
"The settlement would not appear to address state and local government claims nor does it settle claims being brought by individual plaintiffs," according to a Jan. 3 analyst note by FBR Capital Markets. "Nevertheless, the settlement would appear to clear up the bulk of Transocean's liability and the company's primary remaining exposure would likely be limited to challenges to their indemnity to civil claims which BP transferred to claimants last year."
The company has set aside $2 billion in cash to meet its financial obligations related to the Deepwater Horizon incident, FBR noted. The difference between the $2 billion and the settlement figure could be used to cover claims related to the reopener provision on the NRDA.
The settlement is below Barclays Capital's $2.5 billion estimate, according to a Jan. 3 analyst's note.
"We think the decision is consistent with a series of favorable rulings and supportive language from Judge Barbier since overseeing the multi-district litigation," said Barclays.
In November, BP reached a settlement with DOJ and the U.S. Securities and Exchange Commission, agreeing to pay $4.5 billion and pleading guilty to 11 felony counts related to the Deepwater Horizon incident.
Following the BP and Transocean settlements with DOJ, Halliburton is the last service company that has not settled liabilities associated with Macondo.
"We think Halliburton is next in line, and a settlement should accelerate the company's plans to hike its dividend and buy back stock," said Barclays.
Barclays noted that Transocean has made "substantial progress" on its strategic initiatives, including upgrading its fleet and improving operational performance.
Generated by readers, the comments included herein do not reflect the views and opinions of Rigzone. All comments are subject to editorial review. Off-topic, inappropriate or insulting comments will be removed.
- Oil CEOs at Davos See Market Rebalancing (Jan 23)
- Eni Secures Two Deals for Oman Blocks (Jan 14)
- BP Plans to Drill 6 More Wells in Azerbaijan (Jan 10)
Company: Transocean Ltd. more info
- Transocean Lands $830MM Rig Contract with Chevron (Dec 28)
- Transocean Acquires Ocean Rig (Dec 05)
- Offshore Brazil Contract Extension Goes to Transocean (Sep 28)


text: The Urban-Brookings Tax Policy Center has launched a website for its State and Local Finance Initiative, designed to expand and organize information about state and local government finance. The Tax Policy Center, a MacArthur grantee and winner of the MacArthur Award for Creative and Effective Institutions, designed the site to provides state and local officials, journalists, and citizens with reliable, unbiased data and analysis about the challenges state and local governments face, potential solutions, and the consequences of competing options. The site is organized into three topic areas: Tax & Budget, Public Programs, and Intergovernmental Topics. It features data, research, and analysis to inform decisions about providing and paying for state and local government services as well as tools to make it easier for researchers and policymakers to access resources.


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Winner of the L-Prize: Philips 60-Watt Equivalent LED Lamp
""A $10 price is when these lamps will take off. That is absolutely achievable in five to six years."
Just yesterday, Philips was awarded the first ever L-Prize (formally titled the Bright Lighting Tomorrow Prize) and $10 million from the American government. The L-Prize is a contest sponsored by the U.S. Department of Energy and the first government-sponsored technology competition designed to spur lighting manufacturers to develop high-quality, high-efficiency solid-state lighting products to replace the common light bulb.


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Columbia International University Commencement May 15
Commencement exercises for Columbia International University (CIU) will take place on Saturday, May 15. Exercises for the CIU Undergraduate School will begin at 8:30 a.m. and exercises for the CIU Graduate School and CIU Seminary & School of Missions will begin at 11:00 a.m. Both events will take place in Shortess Chapel on the campus of CIU.
CIU President Dr. Bill Jones will present diplomas to 224 graduates for degrees ranging from Bible Certificate to Doctor of Ministry. The graduates will join the 17,000 other CIU alumni serving in nearly 150 countries.
The commencement address for both graduations will be delivered by The Rev. Robert J. Morgan, the pastor of Donelson Fellowship in Nashville, Tenn. Morgan became pastor of the church in 1980, and since then the church has grown from 95 to 600 members. He is also the author of eight books, and has served with the Billy Graham Evangelistic Association. Morgan holds a Bachelor of Science degree from Columbia International University, a Master of Arts in Christian Education from Wheaton Graduate School and a Master of Divinity from Luther Rice Seminary.
Morgan's church was at the center of the recent devastating floods in Tennessee. Despite damage to the church building, members of his congregation are reaching out to help each other and the community. Check it out by visiting the Web site of the Donelson Fellowship at: http://www.donelson.org/
The public is invited to attend the commencements. Columbia International University is located at 7435 Monticello Road, two miles north of Interstate 20 in Columbia.


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Selecting your vehicle's make, model, and year allows us to display only parts and items specific to your car's model and year.
Part# CH23587
Mfg# 12021
1 Review
OPGI Price: $2,499.99
Part Type: (P) High Performance, designed to improve performance HP = High performance
Years: 1978-1988
Sold As: EACH
2018 Malibu Catalog Page: 233
Certain items which exceed standard size are exempt from free shipping promotions. These items are noted in your cart at checkout.
Products that require truck freight delivery are items too large to be shipped via FedEx Ground. These are items such as fenders, hoods, etc. All truck freight orders must be prepaid. Rates begin at $120.00 and vary based on weight. Please contact our customer service department for accurate freight charges or visit our website at
The 200-4R transmission—the ultimate 4-speed automatic—has been long overlooked as a great transmission for street rods. Built with the correct parts, it can give you better mileage, lower cruise rpm and up to a 300% longer engine life. Other features are lower gear ratios in the first (2:74) and a (1:57) in second gear. The 200-4R is the same length as a TH350 or a Powerglide. It has the same drive yoke as the TH350 and Powerglide, which means you can utilize the stock driveshaft. The ultimate 4-speed automatic can be beefed-up to handle in excess of 800 HP. The 200-4R can shift into overdrive at full-throttle and has a multiple bolt pattern that will fit Buick, Oldsmobile, Cadillac, Pontiac and Chevrolet engines. There are also adapters available for other engines. The gear shift and cooler lines are the same as the TH350 and Powerglide and hookup easily. The only modification necessary is moving the crossmember back 6-1/2" from the TH350 or Powerglide position. The 200-4R also has a TV throttle cable and bracket that hooks to your carburetor or throttle body. This will give you control over your shift points.
Please note, these transmissions have a full 90-day warranty and are then subsequently guaranteed against defective workmanship for three full years from purchase date. After 90 days, for three years, California Performance Transmissions will completely rebuild or overhaul under warranty, the transmission for $250.00 plus hard parts to the original owner, or $300.00 plus hard parts, to any subsequent owner, if warranty is transferred. This overhaul or rebuild includes a new factory warranty.


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Play your NES games with just one cart!
The NES flash cart is finally here and it couldn't be easier. Just copy your games onto one compact flash card, insert into the PowerPak, and play!
When you start your NES you get a file browser to choose your game. Then you can save and load battery ram so all your saved games stay saved. Save files can also be transferred to and from your computer for use on emulators. Built in Game Genie code support lets you use cheat codes. Even better than the original Game Genie, the PowerPak has support for 5 codes at once.
Games load very fast, average around 7 seconds. This means a quick cycle for homebrew development on any mapper. Now you can see your game on real hardware instead of relying on emulators. FAT16 or FAT32 format is used so there is no special software to load games onto the card. Organize your games in folders any way you want for faster browsing. Compact Flash is used instead of something else like SD for maximum speed. The NES processor is the bottleneck so the parallel CF card is more than 10 times faster than the serial SD card.
The entire PowerPak system is updateable for future additions and bug fixes. All mapper files are stored on the CF card for easy upgrading. Just download the most recent Mappers file below, unzip, then copy the folder to the root of your CF card. Using CopyNES the boot rom can be reflashed but this will hopefully never be needed. If it is needed and you do not have a CopyNES, you just pay the shipping to get your PowerPak here and I will reflash then mail it back free.
CF Card
option includes a formatted CF card with the required PowerPak files already loaded. No games are on the card.
USB Card Reader
is needed to load your games from your computer on to the CF card. USB Card reader is not included.
Browse the card to select your game
Choose battery RAM and Game Genie options
Package Includes:
PowerPak cartridge with high quality label
Ciclone multi region lockout chip for NTSC, PAL A, PAL B, Asian Systems
Brand new clear dust sleeve
Compact flash card and reader NOT included
5 year replacement warantee - if the cart stops working we replace it
Lifetime boot rom updates - mail the cart here, we update then mail back free
Returns on known bugs are not accepted
System Compatibility List:
Works on authentic Nintendo Systems including NTSC, PAL A, PAL B, and Asian top loader and toaster systems.
Works on the
Works on Famicom using 72-60 pin converter.
Does NOT work on clone systems like the NEX, Yobo, or FC Twin.
Untested on portable systems, not likely to work.
Mapper Compatibility List:
Current Known Bugs:
PAL compatibility has not been fully tested, only that it will run games.
MMC5 support not implemented yet. The PowerPak DOES have enough hardware to do it.
There may still be some sprite related graphics glitches on some NES systems.
Current Known Bugs that aren't Bugs:
File display does NOT sort alphabetically. Sorting would just be too slow for the NES processor.
Some emulators (Nestopia) use .sav files that are actually .zip files. These will need to be expanded before transferring.
DIP switches in arcade games will not work. They are not physically possible from the cart side.
Any game with PRG or CHR larger than 512KB will not work. Most are listed as Bad Mappers above.
Only the first logical partition on the CF card is used.
Multi disk FDS games are not yet supported
PowerPak Manual v1.34 PDF
- Explanation of how to set up the CF card and use the PowerPak, read this first
PowerPak Mappers v1.34
- unzip then copy the POWERPAK folder into the root of your CF card
Empty Save File (8KB)
- unzip then copy the file anywhere on your card
PowerPak Change List
- specifies updates in the mappers and boot rom for each version
Old Downloads:
PowerPak Mappers v1.20
- old mappers version
Technical Info:
Inside the PowerPak is a Xilinx FPGA, 512KB prg space, 32KB battery ram space, 512KB chr space, boot rom, and glue logic. The FPGA has extra graphics ram for four screen games and MMC5 exram. No processors are used; the NES 6502 controls everything.