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Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, and N532). Mutagenesis-induced disruption of the individual N-glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1-mediated inward transport of peptides. Here, we show that for the murine protein the inhibition of glycosylation at sequon N50 by substituting N50 with glutamine, lysine, or cysteine or by replacing S52 with alanine equally altered PEPT1 transport kinetics in oocytes. Furthermore, we provide evidence that the uptake of [14C]glycyl-sarcosine in immortalized murine small intestinal (MODE-K) or colonic epithelial (PTK-6) cells stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild-type protein. By using electrophysiological recordings and tracer flux studies, we further demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line with these findings, we show that attachment of biotin derivatives, comparable in weight with two to four monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition, our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against proteolytic cleavage by proteinase K, whereas a remarkable intrinsic stability against trypsin, even in the absence of N-linked glycans, was detected.
NEW & NOTEWORTHY This study highlights the role of N50-linked glycans in modulating the bidirectional transport activity of the murine peptide transporter PEPT1. Electrophysiological and tracer flux measurements in Xenopus oocytes have shown that removal of the N50 glycans increases the maximal peptide transport rate in the inward and outward directions. This effect could be largely reversed by replacement of N50 glycans with structurally dissimilar biotin derivatives. In addition, N-glycans were detected to stabilize PEPT1 against proteolytic cleavage.
- peptide transport
- glycoprotein
- N-linked glycosylation
- Xenopus laevis
- peptide transporter 1
a prototypical member of the solute carrier 15 gene family (SLC15), peptide transporter 1 (PEPT1) has high expression in apical membranes of the upper small intestine. PEPT1 transports in essence all natural dipeptides and tripeptides as well as peptidelike drugs, such as β-lactam antibiotics (6, 48, 65), angiotensin-converting enzyme inhibitors (42, 60), the antineoplastic drug bestatin (26, 53), and other nonpeptidic compounds (10, 21). Peptide transport is proton coupled and driven by an inwardly directed electrochemical gradient with the membrane potential as the main driving force (16, 61, 62, 67). Crystal structures of prokaryotic homologs (8, 19, 20, 45) and hydropathy analysis (12, 37) indicate the presence of 12 α-helical transmembrane domains (TMD) in mammalian H+/peptide transporters (Fig. 1). According to current knowledge, TMD 1, 3, 4, 7, 8, 9, and 10 participate in the formation of a central aqueous channel that constitutes a substrate translocation route across the lipid bilayer (5, 13, 15).
Murine PEPT1, which is composed of 709 amino acid residues, has a calculated core molecular mass of 79 kDa (14), but when glycosylated, a protein of ~105 kDa is detected. As recently shown by Stelzl et al. (59), six asparagine residues (positions N50, N406, N439, N510, N515, and N532) carry glycans (Fig. 1). Disruption of single or multiple sequons in the large extracellular loop connecting TMD 9 and 10 (sequons N406, N439, N510, N515, and N532) only marginally affected the PEPT1 transport characteristics when studied by two-electrode voltage clamp technique (TEVC) and tracer flux analyses in Xenopus laevis oocytes. In contrast, elimination of N50 significantly altered PEPT1 transport kinetics. Electrophysiological studies disclosed a more than twofold increase in inward transport currents with glycyl-sarcosine (Gly-Sar) as a substrate, which was associated with a similar reduction in substrate affinity. The enhanced transport rates were attributable to neither a higher cell surface expression of N50Q nor a higher ion conductance (59), suggesting that it is indeed the transport cycle of the individual protein that is altered.
In the current study, we assessed how N-glycosylation at N50 alters PEPT1 kinetics for both the inward and outward transport modes. Using radiotracer flux measurements, as well as an electrophysiological approach, we examined whether the gain of function is simply due to the loss of the N-glycan mass at this particular site. To mimic an oligosaccharide chain, we exchanged N50 by cysteine and used biotinylation reagents to add various masses to this position. In addition, we also analyzed the role of N-glycans in conferring proteolytic stability to PEPT1.
Construction of PEPT1 N-glycosylation-defective transporters.
Potential N-glycosylation sites in the PEPT1 sequence (UniProt ID: Q9JIP7) were identified using the NetNGlyc 1.0 server. Asparagine in position 50 was exchanged with glutamine, cysteine, or lysine, and serine 52 was exchanged with alanine. To this end, two mutation-specific megaprimers carrying terminal EcoRV and XhoI sites were amplified and subsequently coupled with Phusion polymerase (New England Biolaboratories, Ipswich, MA) at 25°C in a thermocycler (Biometra, Göttingen, Germany). Primer combinations used for the synthesis of megaprimers are listed in Table 1. Resulting fusion PCR products were digested with XhoI and EcoRV (Thermo Scientific, Waltham, MA) and cloned into pCRII-TOPO-3′ end vector (Invitrogen, San Diego, CA). Mutations were confirmed by sequencing (GATC Biotech, Konstanz, Germany).
Generation of cRNA.
The expression vectors carrying individual PEPT1 constructs were linearized with NotI, and cRNA synthesis was performed with the mMessage machine T7 kit (Ambion, Darmstadt, Germany) according to the manufacturer's specifications. Following phenol extraction and ethanol precipitation, 2-µg linearized plasmid DNA were transcribed into cRNA and adjusted to a final concentration of 1 µg/µl. Prior to Xenopus oocyte microinjection, all cRNA preparations were evaluated for size and integrity on a 1% agarose-formaldehyde gel and stored at −80°C.
X. laevis oocyte preparation and cRNA injection.
X. laevis maintenance and oocyte harvest procedures were approved by the local authority for animal care in research (Regierung von Oberbayern, approval no. 55.2-1-54-2532.3-64-11). Oocytes were surgically removed from X. laevis frogs (anaesthetized in a solution of 0.7 g/l 3-aminobenzoic acid ethyl ester; Sigma-Aldrich, Taufkirchen, Germany) and enzymatically defolliculated by collagenase A treatment (2.5 mg/ml for 90 min) (Roche, Mannheim, Germany). Healthy stage V and IV oocytes were manually sorted and stored in Barth solution [88 mM NaCl, 1 mM KCl, 0.8 mM MgSO4, 0.4 mM CaCl2, 0.3 mM Ca(NO3)2, 2.4 mM NaHCO3, 10 mM HEPES; pH 7.4] supplemented with 5 mM pyruvate and 0.2 mM gentamycin at 17°C overnight. Intact oocytes were injected with 18.4-nl cRNA or water as a control. For coexpression experiments, cRNAs for the murine sodium glucose transporter 1 (SGLT1; UniProt ID: Q9QXI6) and PEPT1 variants were microinjected in a ratio of 1:1 to a final volume of 36.8 nl. The N-glycosylation inhibitor tunicamycin was dissolved in 4% DMSO in water and coinjected with PEPT1 cRNA to a final concentration of 2.5 ng per oocyte. For protein expression, injected oocytes were kept for 3–4 days at 17°C in Barth solution.
Electrophysiological recordings.
TEVC experiments were conducted as described (1, 31). Briefly, oocytes were placed in an open chamber and continuously superfused with Barth solution or Gly-Sar (Sigma-Aldrich) over the concentration range of 0.3–40 mM at a flow rate of 3 ml/min. Microelectrodes backfilled with 0.5 mM KCl and an electrode resistance between 1 and 3 mΩ were used to clamp oocytes at a holding potential of −60 mV. Inwardly directed transport was calculated as the difference in currents registered in the presence or absence of substrate. Current-voltage (I–V) relations were recorded with a Tec-03 amplifier (npi electronic, Tamm, Germany) for the duration of 100 ms, separated by pauses of 200 ms, in the potential range of +80 to −160 mV. Data acquisition was performed with CellWorks (v. 5.1; npi electronic). The substrate-dependent maximal inward currents of PEPT1 were normalized to currents evoked by 1 mM α-methyl-d-glucopyranoside (α-MDG; pH 6.5) as substrate for the coexpressed SGLT1 transporter. This correction was performed to compensate for fluctuations in gene expression levels between individual oocytes and oocyte batches. The kinetic parameters Km (Michaelis constant; mM) and Imax (maximal velocity; nA) were calculated by least squares fits to the Michaelis-Menten equation. Transient outward current recordings were performed according to Kottra and Daniel (31). In short, oocytes were injected with rising volumes (9.2–59.8 nl) of 1 M Gly-Sar (pH 7.5) dissolved in 1 mM EGTA in water (Sigma-Aldrich). Thirty seconds postinjection, I–V relations were recorded over a period of 6 min in intervals of 30 s. Outward currents are denoted with a positive sign, whereas negative values represent inward currents. For current recordings without normalization to coexpressed SGLT1, an outlier analysis was performed. According to the formula zi = (xi − M)/SD, a Z-score was computed for individual data sets (xi is the original value, M is the mean, and SD is the standard deviation). Values with a Z-score ±1.5 or beyond were considered as outliers. Electrophysiological data are presented as means ± SE or medians ± minimum/maximum (min/max; + inside bars marks mean values) of 10–30 oocytes from at least 2 oocytes batches. Statistically significant differences were determined by one-way ANOVA followed by Dunnett's posttest analysis or Student's t-test using GraphPad Prism v. 4.01. Significance levels are indicated with *P < 0.05, **P < 0.01, and ***P < 0.001.
Tracer efflux studies with X. laevis oocytes.
Radiotracer efflux experiments were performed after initial loading of X. laevis oocytes with [14C]Gly-Sar solution (56 Ci/mol, 17.8 mM; custom synthesized by GE Healthcare, Munich, Germany; ratio of labeled to unlabeled substrate 1:10) at pH 6.5 for 10 min at 23°C. After briefly rinsing with Barth solution (pH 7.4), oocytes were transferred into new reaction vessels devoid of radioactivity, and excess liquid was removed. Following addition of 100 µl Barth solution per 5 preincubated oocytes, radioactive efflux was determined over a period of 20 min in the supernatant. For measurement of the residual radioactivity in oocytes, samples were dissolved in 20% SDS in water for 2–3 h at 50°C with shaking (500 rpm; Heidolph Titramax 1000). After addition of a scintillation cocktail (Rotiszint eco plus; Roth, Karlsruhe, Germany), radioactivity was counted in a Tri-Carb 2810 TR instrument (PerkinElmer, Waltham, MA). The total radioactivity in oocytes was calculated at time point 0 and corrected for radiation of water-injected oocytes. Export rate constants (Kexp) were calculated as the slope of the linear regression analysis applying first-order kinetics and efflux half-lifes determined by the formula t1/2 = ln 2/Kexp.
Protein preparation from oocytes or mammalian cultured cells.
Three to four days after cRNA injection, 20 oocytes expressing the different transporter genes were transferred into 200-µl lysis buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol) and homogenized. Following centrifugation for 1 min at 15,300 relative centrifugal force (RCF) and 4°C, supernatants were collected, and protein contents were determined by Bradford protein assay (Bio-Rad, Munich, Germany). In case of mammalian cells, confluent cells (75-cm2 culture flask) were aspirated from media and washed with cold PBS (pH 7.4). Adhering cells were scraped off with PBS containing 1 mM PMSF and spun at 2,300 RCF for 2 min at 4°C. Sedimented cells were resuspended in 200-µl PBS containing 1 mM PMSF and lysed by passing through a 2-ml syringe with a 24-gauge cannula. Following centrifugation at 425 RCF for 3 min at 4°C, the total protein fraction was separated by high-speed centrifugation at 42,500 RCF at 4°C for 1 h. Total membranes were resuspended in PBS containing PMSF and protein yields quantified by Bradford assay.
Immunoblotting of protein extracts.
SDS-PAGE was performed with 10% SDS-acrylamide gels in a Mini-PROTEAN 3 system from Bio-Rad. Separated proteins were transferred to a nitrocellulose membrane (Whatman, Maidstone, United Kingdom) in a wet tank blotting system at 0.36 A for 25 min. After blocking for 1 h in 1% bovine albumin (AppliChem, Darmstadt, Germany), the membrane was probed with a polyclonal rabbit antiserum against PEPT1 (custom-made; Pineda, Berlin, Germany; dilution 1:5,000, polyclonal rabbit IgG raised against the peptide CVGKENPYSSLEPVSQTNM corresponding to the COOH terminus of rat PEPT1) and β-actin as loading control (dilution 1:2,000, polyclonal goat IgG, C-11 sc-1615; Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C. Following three washings with PBS containing 0.05% Tween-20, the membrane was stained with IRDye-labeled secondary antibodies (dilution 1:12,000, C50113-03, C40415-02; LI-COR Biosciences, Bad Homburg, Germany) at room temperature for 2 h. Fluorescence signals were detected with an infrared fluorescence Odyssey scanner and quantified with the Image Studio Lite software (v. 3.1) supplied from LI-COR.
Surface biotinylation of oocytes.
Oocytes were biotinylated with either 2-[(biotinoyl)amino]ethyl-methanethiosulfonate (MTSEA-biotin; Biotium, Hayward, CA) or MTSEA-biotin capped with ethylenediamine (MTSEA-biotin-X and MTSEA-biotin-XX). These reagents are impermeable to the cell membrane and covalently react with surface-exposed sulfhydryl groups in proteins. Oocytes expressing individual PEPT1 versions were incubated in 2 mM MTSEA-biotin solution dissolved in PBS (MTSEA-biotin stock solutions: 100 mM in DMSO) for 15 min at room temperature. Following washing five times with Barth solution (pH 7.4), oocytes were assayed electrophysiologically.
Proteolysis assay.
To assess the effect of glycosylation on the proteolytic stability of PEPT1, wild-type and N-glycosylation-deficient transporters were exposed to the proteases trypsin and proteinase K. Total protein extracts from oocytes (15-µg protein) was digested with 20 units of trypsin (Sigma-Aldrich) freshly dissolved in 1 M HCl and 2 mM CaCl2 (pH 3). After 10 min, trypsin inhibitor (Sigma-Aldrich) was added to a final concentration of 2 mM. Alternatively, 2.5 ng/µl proteinase K (VWR, Darmstadt, Germany) were used. The reaction was stopped after 6 min at ambient temperature with 5 mM PMSF. After incubation with the proteases, proteins were separated by SDS-PAGE, followed by Western blotting with the PEPT1 serum.
Retroviral transduction of PEPT1 in MODE-K and PTK-6 cells.
Expression of PEPT1 in the murine intestinal epithelial cell lines MODE-K (66) and PTK-6 (68) was accomplished by retroviral transduction. Virus production was achieved by transfection of an ecotropic Platinum-E (Plat-E) packaging cell line (Cell Biolabs, San Diego, CA) with the retroviral transfection vector pMXs (Cell Biolabs). For generation of the pMXs expression vector, PEPT1 was amplified with gene-specific primers incorporating a COOH-terminal hemagglutinin epitope tag and the restriction sites SalI and Bsp119I (forward primer: 5′ TATTTCGAAATGGGGATGTCCAA GTCTCGGGG 3′; reverse primer: 5′ ATAGTCGACTTAAGCATAATCTGGAACATCATATGGATACATATTTGTCTGTGAGACTGGT3′). PCR products were digested with SalI and Bsp119I and ligated into the pMXs vector using the same restriction sites. Prior to Plat-E cell transfection, cell culture plates were coated with collagen A (Biochrom, Berlin, Germany) and thoroughly washed with PBS, and Plat-E cells [passage 34 (P34)] were seeded in a density of 9.99 × 105 cells per well of a 6-well tissue culture plate. Plat-E cells were cultivated in DMEM (Sigma-Aldrich) supplemented with 1 µg/ml puromycin and 10 µg/ml blasticidin. After transfection, these antibiotics were replaced by 1% penicillin-streptomycin. Six hours after Plat-E seeding, cells were transfected with pMXs vector constructs. Transfection was performed with the ProFection Mammalian Transfection System (Promega, Mannheim, Germany) according to the manufacturer's specifications. Sixteen hours posttransfection the cell medium containing the retroviruses was removed and filtered using a cellulose-acetate filter (pore size 0.45 µm). For retroviral transduction, 7.5 × 104 MODE-K (P33) or PTK-6 cells (P44) per well of a 6-well culture plate were seeded and recovered overnight. The next day the culture medium was renewed and supplemented with 3 µg/ml polybrene (Santa Cruz Biotechnology, Heidelberg, Germany). Following 1-h incubation at 37°C, 1 ml of retroviral lysate was added per well. After 20 h the culture medium was replaced by media containing 10 µg/ml blasticidin for positive transformant selection.
Uptake measurements of [14C]Gly-Sar in MODE-K and PTK-6 cells.
Following retroviral transduction, MODE-K (P30–35) and PTK-6 cells (P40–45) were grown to confluence in six-well culture plates. A single well was used for protein content determination. Therefore cells were scraped off in 500-µl PBS (pH 7.4) and sonicated for 12 cycles with amplitude 50 (UP200S; Hielscher Ultrasonics, Teltow, Germany). Cell debris was removed by centrifugation, and protein content of supernatants was quantified by Bradford assay. The remaining cells were washed with 1 ml MES-Tris buffer (MTB: 140 mM NaCl, 5.40 mM KCl, 1.77 mM CaCl2, 0.80 mM MgSO4, 5 mM glucose, 27 mM MES; pH 6.0) and incubated with 500-µl [14C]Gly-Sar solution (final concentration of 6 µM) for 10 min at 37°C while shaking (200 rpm; Heidolph Titramax 1000). At the end of the incubation, the uptake solution was removed, and the cells were washed twice with 1-ml MTB. After lysis in 1-ml Igepal buffer (50 mM Tris, 140 mM NaCl, 1.50 mM MgSO4, 0.50% Igepal CA-630; pH 8.0), detached cells were transferred to scintillation vials and measured in 3-ml Rotiszint eco plus in a Tri-Carb 2810 liquid scintillation counter.
PEPT1 N50Q has increased substrate import and export rates.
Previous studies established that PEPT1 can transport in a bidirectional manner with inward and outward transport currents following Michaelis-Menten kinetics (31). Since inwardly directed transport currents for the N50Q mutant were increased (59), we here examined whether this is also found for outward transport with radiotracer efflux measurements using the nonhydrolyzable dipeptide [14C]Gly-Sar (Fig. 2). We observed a three times higher Gly-Sar uptake in oocytes expressing the N50Q transporter than for wild type (16.73 ± 0.30 pmol/20 min/oocyte for N50Q vs. 5.66 ± 0.43 pmol/20 min/oocyte for wild type; data not shown). Efflux by the N50Q mutant protein expressed as initial [14C]Gly-Sar influx proved to be increased as well, 1.5-fold. The radioactivity remaining in oocytes at the end of incubation was 68% of the preloaded amount for N50Q and 78% for wild type. Kinetic analysis demonstrated that tracer export followed first-order reaction kinetics with rate constants of 0.26 min−1 for N50Q and 0.11 min−1 for wild type. The calculated t1/2 for export from oocytes was 2.63 min for N50Q and 6.58 min for the wild-type transporter. Thus both substrate uptake and export are accelerated in N50Q.
TEVC analysis of oocytes clamped to a membrane potential of −60 mV and superfused with 40 mM Gly-Sar (at pH 7.5) revealed average inward currents of −513.49 ± 152.90 nA for PEPT1 wild type and −1,311.99 ± 341.57 nA for N50Q (Fig. 3). With hyperpolarization of the membrane potential to −160 mV, a gradual increase in inward currents to −1,840.97 ± 432.94 nA in wild type and −3,434.58 ± 720.86 nA in N50Q was observed with a steady-state I–V relationship of similar shape for both protein types. Electrogenic Gly-Sar efflux was measured by loading oocytes with increasing amounts of substrate. Since stage VI oocytes have a cytosolic pH between 7.4 and 7.7 (7, 55, 58), there is no significant proton gradient when an extracellular pH of 7.5 is used. Under these conditions, the outwardly directed PEPT1 transport primarily depends on the membrane potential and occurs at a membrane potential more positive than +20 mV (31). The mean aqueous volume of oocytes amounts to ~410 nl (32, 70). Therefore injection of 9.2 nl of a 1 M Gly-Sar solution brings the cytosolic concentration to ~22 mM, and injection of 59.8 nl raises the intracellular Gly-Sar concentration to ~127 mM. Average outward transport currents detected at +60 mV in PEPT1 increased after loading of 22 mM Gly-Sar within 6 min to 302.77 ± 72.37 nA (Fig. 3A, inset) whereas at 127 mM Gly-Sar, currents of 781.86 ± 86.79 nA were recorded. In comparison, outward current recordings for N50Q were on average 1.5–2.5 times higher (713.23 ± 189.50 nA at 22 mM Gly-Sar; 1,155.50 ± 135.50 nA at 127 mM Gly-Sar; Fig. 3B, inset). At a cytosolic Gly-Sar concentration of ~43 mM, outward currents for the PEPT1 wild-type protein (409.85 ± 153.34 nA) at +60 mV were comparable with corresponding inward currents at −60 mV. At the same time, outward currents detected for N50Q (+60 mV: 779.95 ± 187.41 nA) appeared to be 1.7 times lower than the inward currents. This analysis thus showed that the inward as well as the outward transport currents of N50Q are increased by a factor of 2–2.5 relative to the wild-type protein. Albeit all electrophysiological data were corrected for basal transport in water-injected control oocytes, superfusion of oocytes expressing the PEPT1 N50Q transporter with Gly-Sar generated, as opposed to the wild-type protein, also outward transport currents at membrane potentials more positive than +30 mV (Fig. 3, A and B). This suggests the presence of small leakage currents, although the exact nature of ions translocating and the underlying causes are unknown thus far.
To test whether asparagine 50 itself is a critical residue for PEPT1 function or whether the lack of the glycan causes the observed effects, we changed serine 52 to an alanine (S52A; Fig. 4). This disruption of the sequon abolishes glycosylation but leaves N50 intact. TEVC analysis revealed similar changes in the kinetic parameters for S52A (Km(S52A) = 1.27 ± 0.22 mM; Imax(S52A) = 2.45 ± 0.50 nA) and N50Q (Km(N50Q) = 1.33 ± 0.23 mM; Imax(N50Q) = 1.95 ± 0.55 nA). Both protein variants thus possessed a reduction in substrate affinity and increase in maximal inward transport relative to the wild-type protein (Km(WT) = 0.66 ± 0.12 mM, Imax(WT) = 0.85 ± 0.21 nA). Immunoblotting demonstrated decreased protein masses for both N50Q and S52A compared with wild-type PEPT1 (Fig. 5). This implies that the absence of glycans attached to N50 causes the increased transport velocity. This observation is further corroborated by the fact that the exchange of N50 for lysine generated a similar effect (Km(N50K) = 1.03 ± 0.15 mM; Imax(N50K) = 4.06 ± 0.61 nA) as shown in Fig. 4. In summary, the disruption of the sequon around N50 resulting in a lack of glycosylation causes the increased transport velocity, irrespective of the nature of the amino acid exchange.
Changes in PEPT1 N50Q transport activity are conserved among species and cell types.
Protein glycosylation varies considerably between species (49), and it is therefore very unlikely that X. laevis oocytes produce the same glycans as mammalian cells. There is good evidence for differences in oligosaccharide trimming and glycan modification between mammalian and amphibian expression systems (42, 43). We therefore expressed the mutant proteins also in the murine intestinal epithelium cell lines MODE-K and PTK-6. Western blot analysis of whole cell extracts revealed a slightly lower PEPT1 molecular mass in MODE-K cells derived from the small intestine (~95 kDa) compared with the colonic cell line PTK-6 (~100 kDa; Fig. 6, A and B). We have previously shown that the PEPT1 mass is different between jejunum and colon of mouse (69) by different glycosylation. Studies of Gly-Sar uptake in MODE-K and PTK-6 cells clearly revealed an increased substrate uptake for the N50Q transporter (Fig. 6, C and D). With 10.59 ± 0.18 nmol/g protein/min, the [14C]Gly-Sar uptake of N50Q expressed in MODE-K cells was 60% higher than for wild type. Also, N50Q expressed in PTK-6 cells exhibited a 50% higher uptake rate than the wild type (6.21 ± 0.22 nmol/g protein/min). Nontransduced MODE-K and PTK-6 cells did not show significant [14C]Gly-Sar uptake (Fig. 6, C and D) or presence of the PEPT1 protein in a Western blot (data not shown). In summary, these results indicate that the altered transport characteristics by lack of glycosylation at N50 are not specific for Xenopus oocytes and can be reproduced in other cell types. This also indicates that the nature of the glycan, which may differ by cell type, seems less important for the reduced velocity of the glycosylated PEPT1 variants.
Site-specific biotinylation mimics glycosylation.
A possible explanation as to how the loss of glycosylation at N50 causes an increase in bidirectional transport velocity could be that the glycans add additional molecular mass to a protein domain that undergoes a conformational change in the transport cycle. To test this concept, we created proteins with masses added at position N50 by site-specific biotinylation with membrane-impermeable cysteine-reactive reagents that could mimic the effects of the glycan. The reagents used were various forms of MTSEA-biotin that attached masses of 380, 500, or 600 Da, which is comparable in weight with a glycan structure containing two, three, or four hexoses.
Because of the observation that cysteine residues identified as exposed to the extracellular space in the human PEPT1 protein (which shares 85% sequence identity with murine PEPT1 and carries cysteine residues at identical positions) could not be modified with sulfhydryl-reactive reagents (34), we decided to use a PEPT1 version containing all naturally occurring cysteine residues and solely replaced N50 with cysteine. Voltage clamp recordings in oocytes revealed a reduction in Gly-Sar affinity for N50C (Km(N50C) = 1.20 ± 0.23 mM; −60 mV) that was similar to N50Q (Km(N50Q) = 1.40 ± 0.29 mM; Fig. 7A). The exchange of PEPT1 N50 caused an increase in transport velocity relative to wild type (Imax(WT) = −528.38 ± 108.67 nA), and this effect was more pronounced for N50Q (Imax(N50Q) = −1,452.28 ± 247.21 nA) than for N50C (Imax(N50C) = −1,080.70 ± 215.60 nA; Fig. 7B). Biotinylation of surface cysteine residues did not significantly alter the overall transport characteristics of wild-type and N50Q transporters in oocytes. However, biotinylation of oocytes expressing N50C significantly changed transport characteristics. Labeling with MTSEA-biotin (~0.38 kDa molecular mass) increased the Gly-Sar affinity by 13% compared with the untreated control (Fig. 7A), whereas treatment with MTSEA-biotin-X (~0.5 kDa) or MTSEA-biotin-XX (~0.6 kDa) increased the binding affinity by 22 and 24%, respectively. The corresponding inward currents declined with a higher biotin mass attached (Fig. 7B). I–V relationships for wild type, N50Q, and N50C were found to be voltage dependent within a membrane potential range of 0 to −160 mV, regardless of the biotin treatment (Fig. 7, C–E).
N-glycans prevent PEPT1 from proteolytic degradation.
A recent analysis of the crystal structure of the large extracellular loop of the mammalian peptide transporter provided evidence that this protein domain acts as a trypsin binding site (4) with the hypothesis that this anchoring mechanism could increase the local concentration of peptides to facilitate their uptake by PEPT1. Since the large loop of PEPT1 contains five of the six N-glycans and glycans are generally considered to protect from proteolytic degradation, we examined the extent to which glycosylation protects PEPT1 from cleavage by proteases. Proteolytic stability of PEPT1 was assessed in oocyte membrane extracts that were treated with different proteases, followed by gel separation, Western transfer, and quantification of PEPT1. Although trypsin was found to have 54 predicted cleavage sites in murine PEPT1 (17), in our experiments, proteolysis occurred very slowly, and degradation rates were almost identical in all glycan variants tested (Fig. 8, A and B). Within 10 min of incubation, PEPT1 protein levels declined by ~47% for wild type, with similar rates also for the N50Q, N406Q/N439Q/N510Q/N515Q/N532Q, and N50Q/N406Q/N439Q/N510Q/N515Q/N532Q versions. This degradation occurred almost completely within the first minute after trypsin addition, without any differences between protein variants. This might be an indication of the presence of different types of membrane fractions that are formed during protein preparation. It is possible that PEPT1 in inside-out vesicles or open lamellar membranes is more susceptible to tryptic cleavage. In contrast, PEPT1 carriers in right-side-out vesicles might be protected from proteolytic attack.
For proteinase K, 384 putative cleavage sites in PEPT1 were predicted (17). The wild-type protein displayed a high stability against proteinase K, with only 33% protein loss over 10 min, whereas the nonglycosylated PEPT1 variant was lost to 94% (Fig. 8, C and D). Intermediate values were found for the N50Q mutant and PEPT1 transporters lacking the sequons N406/N439/N510/N515/N532 and N50/N406/N439/N510/N515/N532. In addition, oocyte expression of the PEPT1 gene in the presence of tunicamycin also significantly increased the sensitivity of the PEPT1 protein toward proteinase K cleavage accompanied by a loss of any detectable protein within 2 min of incubation.
Glycosylation of secretory proteins occurs at asparagine residues within conserved acceptor motifs (3) with N-glycans transferred en bloc by the oligosaccharyltransferase from dolichol to the nascent polypeptide chain (39, 56). For integral membrane proteins, only a few studies have assessed the functional role of glycans. These show that protein folding, protein stability, trafficking, secretion, and function can be impaired by a lack of glycosylation (22, 41). However, a gain of function, such as the drastic increase in transport velocity described here for PEPT1 lacking glycosylation at N50, is a rare finding. Other examples where deglycosylation resulted in increased activity include lecithin-cholesterol acyltransferase (LCAT). LCAT, which forms cholesteryl esters from cholesterol (51), has four N-glycosylation sites (N20, N84, N272, and N384; 36), with a lack of glycosylation at N384 causing a twofold increase in enzyme activity (30). Another example where removal of a single glycan considerably changed the enzyme activity is the endothelial lipase with four sites carrying complex-type N-glycans (27, 28). Elimination of the glycan at N62 markedly increased phospholipase activity in reconstituted HDL particles (40, 57). The increase was up to 7-fold for apolipoprotein E (apoE2) and 26-fold for apolipoprotein A-I containing HDL particles. A similar effect has also been described for bovine pancreatic ribonuclease. This enzyme occurs as a mixture of an unglycosylated form called RNase A and various glycoforms, collectively called RNase B (50). In RNase B, the single glycosylation site (N34) is modified by oligomannose-type N-glycans containing two residues of N-acetylglucosamine and five to nine residues of mannose. Treatment of RNase B with exoglycosidase permits the generation of glycoforms with fewer mannose residues; these glycoforms provide an ideal tool to study the relationship of glycosylation to activity (52). Rudd et al. demonstrated a more than three times higher hydrolytic activity for RNase A relative to RNase B (52). Moreover, it was shown that the enzymatic activity of RNase B decreased continuously as the mannose content and glycan mass increased (29, 52). Here we describe for the first time a similar change of protein function in a nutrient transport protein. The removal of the glycan at N50 in the intestinal peptide transporter PEPT1 caused a twofold to threefold rise in maximal transport velocity (Figs. 2–4). The gain in transport velocity was always associated with a significant reduction in affinity for the substrate Gly-Sar. As demonstrated by both electrophysiological analyses and tracer flux experiments, the reduction in affinity occurs for the inward and outward transport directions. This suggests that the release of the substrate on the trans side occurs much faster, which, in turn, can promote the more rapid conformational reorientation of the empty transporter.
On the basis of structural features of the bacterial homologs (8, 45, 46), N50 is located within the extracellular region connecting TMD 1 and 2 (Fig. 1). From functional studies of mutant and chimeric PEPT1 proteins, TMD 1–4 and 7–9 contribute to substrate binding (9, 13, 34, 64). It is thus well conceivable that a glycan attached to N50 with an estimated mass of 5–10 kDa in the oocyte expression system (Fig. 5) can affect the transport characteristics. Our biotinylation experiments demonstrated that the "gain of function for maximal transport" can be reversed to wild-type levels by attaching biotin derivatives (Fig. 7). Unlike PEPT1 wild type and N50Q, MTSEA-biotinylation of N50C resulted in a significant increase in the apparent Gly-Sar affinity and a decrease in maximal transport velocity. This effect was more pronounced with a MTSEA-biotin derivative of a higher molecular weight. Although the reagents used to modify cysteine at position 50 are chemically and structurally dissimilar to glycans, they add a mass comparable with two to four monosaccharides contained in a glycan. It thus appears that the effect of the glycan on the transport activity is solely or mainly exerted by the molecular mass added, whereas structure and composition of this modification appear not important. It goes without saying that biotin derivatives with higher mass also occupy more space. Thus it is unclear whether it is due to the added mass or due to steric hindrance that PEPT1 modified at N50 by a glycan or by MTSEA-biotin is significantly slowed in its transport activity.
For other transporters such as the intestinal Na+-dependent glucose cotransporter (24), the cystic fibrosis transmembrane conductance regulator (18), or the cardiac Na+/Ca+ exchanger (25), it was shown that the N-linked glycans are not required for protein function. The same applies for N-glycans attached to the large extracellular loop in PEPT1 that connects TMD 9 and 10 (59). Although the glycosylation sites in mouse PEPT1 bring ~35 kDa of total extra mass, 25–30 kDa thereof are found attached to residues in the large loop that by current knowledge does not contribute to the transport process.
There is ample evidence that protein glycosylation can increase the thermal and dynamic stability (47, 52), solubility (63), and resistance to proteolytic cleavage (11, 35). In the gastrointestinal tract, PEPT1 has highest expression levels in duodenum and jejunum, and these are the tissues facing the highest proteolytic activities in the body (2) with the different proteases derived from pancreatic secretion. Extracellular domains of transport proteins in the apical membrane are thus particularly prone to rapid degradation with loss of functionality. Indeed, N-linked glycosylation of the intestinal anion exchanger SLC26A3 was shown to confer resistance to trypsin (23). Within PEPT1, the large extracellular loop connecting TMD 9 and 10 has recently been shown to possess a trypsin binding site with residues N550 and E573 identified as important for transporter-enzyme interaction (4). This association links the degradation of dietary proteins to the transport of the peptides released, which could make the overall process more efficient. However, this protein association requires that PEPT1 is intrinsically stable against trypsin.
Our study now provides evidence that this is the case, and moreover, we demonstrate that this stability is an intrinsic property of the PEPT1 protein backbone and is not conferred by N-linked glycans (Fig. 8). In contrast to trypsin, the resistance of PEPT1 to the fungal enzyme proteinase K was dependent on glycosylation and even correlated with the number of N-glycosylation sites in the transporter. Moreover, treatment of oocytes with the N-glycosylation inhibitor tunicamycin also rendered PEPT1 susceptible to very rapid proteolytic degradation. We previously demonstrated that tunicamycin treatment of oocytes neither inhibited PEPT1 trafficking to the cell membrane nor its expression (59), which suggests that glycosylation is not required for protein folding.
We are aware that all processes related to glycosylation in the oocyte expression system, despite its wide use, may differ substantially from those in intestinal epithelial cells as the major site of PEPT1 expression. This may apply to N-glycosylation site occupancy, glycan type, glycan size, and the chemical nature of the monomers comprising the glycans. However, we here demonstrate that the changes in the kinetics found for N50 are phenotypically conserved when PEPT1 is expressed in intestinal cells (Fig. 6), and we also showed before that glycosylation of N50 has this effect in the human PEPT1 transporter (59).
At present, the precise function and structure of the N-glycan attached to N50 remain unknown. Nonetheless, our experiments show that the removal of this glycan, unlike any of the other glycans, changes markedly the transport characteristics of PEPT1. These changes can be reverted by the covalent attachment of chemically different molecules such as the biotin-containing structures used here. It thus appears that the exact nature of the glycan is irrelevant for the observed effects on substrate affinity and maximal transport velocity of PEPT1. Since biotinylation reagents with higher masses appear to produce a stronger effect, it seems plausible to assume that the protein domain around N50 is intrinsically mobile and has to undergo structural changes during the transport cycle with a restrained freedom of movement by the mass attached to N50 or steric hindrance. Yet this model does not readily explain the higher substrate affinity of the PEPT1 variants that have either a glycan or a biotin moiety attached. However, our findings challenge the proposed kinetic model of PEPT1 in which the return of the unloaded carrier to the outside-facing conformation determines overall velocity (38, 54).
This work was funded by the Deutsche Forschungsgemeinschaft as part of the Research Training Group GRK1482.
No conflicts of interest, financial or otherwise, are declared by the authors.
H.D., K.E.G.-K., T.S., and J.S. conceived and designed research; T.S. performed experiments; T.S. analyzed data; T.S., K.E.G.-K., J.S., and H.D. interpreted results of experiments; T.S. prepared figures; T.S., J.S., and H.D. drafted manuscript; T.S., K.E.G.-K., J.S., and H.D. edited and revised manuscript; T.S., K.E.G.-K., J.S., and H.D. approved final version of manuscript.
We thank Helene Prunkl for taking care of the frogs and support in oocyte handling and preparation. We also acknowledge Margot Siebler for excellent technical assistance. We are also very grateful to Prof. D. Haller for providing the MODE-K and PTK-6 cell lines and Maren Ludwig for support in the retroviral transduction experiments.
- Copyright © 2017 the American Physiological Society


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Africa's largest sports festival will be taking place between 5 and 7 May for the Arnold Classic Africa.
World-renowned personality, Arnold Schwarzenegger will be in Sandton to host a three-day health and fitness festival in partnership with the Gauteng Provincial Government, the City of Joburg and the SA Sports Confederation and Olympic Committee.
READ Hyde Park High School in Sandton recognises it sports stars with colours
Sporting events on offer include body-building, boxing, dance sports, gymnastics, pole fitness, power lifting, rowing, strong man pro, table tennis, weight lifting and so much more. The annual event will be held at the Sandton Convention Centre and will involve over 14 000 participants in more than 50 sports codes, as well as a three-day Health and Fitness Expo.
"We are pleased to welcome another internationally competitive list of entries for the Pro Fitness event. We are sure they will deliver an exciting competition while giving local athletes a rare chance to compete against the world's best on home soil," said Arnold Classic Africa partner, Wayne Price.
The Arnold Classic Africa, the largest multi-sports event of its kind on the continent, will host more than 14 000 participants in over 50 codes over three days of competition.
What do you think of Arnold Schwarzenegger supporting South African sports? Tell us on Whatsapp on 079 439 5345.

subset: commoncrawl


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Product Description
Containing natural plant essense from tea tree, eucalyptus and spiraea, the mask provides gentle, soothing effect on skin. The refreshing and aromatic characteristics from tea tree provide excellent dual effects of purifying and moisturizing on skin.
Main Features:
Mild nature of tea tree essense makes this mask suitable for all skin types. Delicate facial mask texture allows the essense to be fully absorbed by skin kerations easily to give sleek and smooth skin. Gentle mild acid formula; contains no artificial colour.
Melaleuca Alternifolia (Tea tree) Leaf Oil, Eucalyptus Globulus Leaf Extract&Propylene Glycol, Meadowsweet (SpiraeaUlmaria) Extract, Propylene Glycol&Arctium Lappa Root (Burdock Root) Extract&Nasturtium Officinale Flower/ Leaf Extract&Plantago Lanceolatal Leaf Extract&Hibiscus Abelmoschus(Hibiscus) Extract& Thymus Vulgaris(Thyme) Extract, Aloe Barbandensis Leaf Juice, Ninacinamide, Dipottasium Glycyrrhizate, Glycerin, Butylene Glycol, Xanthan Gum, Polysorbate 20, Methylparaben, Trisodium EDTA, Water.
Use: For skin soothing and puryfying .
Directions for use:
1. After cleaning face, take out the mask and place evenly flat on face matching the holes on both eyes and nostril using fingertips.
2. Wait for 15-20 minutes before taking off the mask from face. Use fingers to gently pat on the remaining essense on face until it's fully absorbed by skin. Use one mask 1-2 times per week is recommended; can use the product on a daily basis.


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Evil Cartoon Man
Vector footage of an evil character. Black-and-white image of a man's face. Angry person, big eyes, nose and ears, bald head, small pieces of hair sticking on the top of the head and earring on one of the ears. Free vector for cartoon characters, clothing prints and brand mascots.


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text: I use DVD Empire for most of my CD orders, and they arrive on release day. But I've never found a similarly reliable CD site. Is there a comparable store for CDs? How is DVD Empire's music arm -- just as great? I often use CD Universe, but find that they're flaky re: when the orders ship. Sometimes on time, sometimes not. I'm just looking for a reasonably-priced, dependable online store -- any suggestions? Thanks.


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Thursday, April 14, 2011
Knight Chat Reminder!!
Happy Thursday Everyone!! Just a reminder that today, April 14th, there is a Knight Chat from 7:30-9pm. Myself and my great fellow SMC bloggers will be there to answer any questions you have about SMC, ranging from majors and sports, to whether we like the food in Alliot and what are some things you can do in the Burlington area!! We'd love for you to stop by, so hope to "see" you there :) If you can't make it to the Knight Chat tonight, never hesitate to ask me a question via Formspring, VYou, Twitter, or email- I'm always happy to help!! Until next time, sending you best wishes, Alex:)
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Haute couture, vérité documentary, and the ghost of Christian Dior.
The histories of fashion, art, and film often intersect in unexpected ways. I was reminded of this recently while watching Les enfants terribles, Jean-Pierre Melville's 1952 film adaptation of Jean Cocteau's classic novel about devious siblings immersed in a private world of games, role-playing, and self-destruction. It's a strange, dark parable of creativity and alienation wrapped in Melville's stylish production. And notably, the film's opulent costumes were created by Christian Dior, the preeminent romantic post-war designer.
Admittedly, his name might have slipped by unnoticed in the credits had I not also recently seen Frédéric Tcheng's new documentary Dior and I. Tcheng's film, which details the creation of Raf Simons's first couture collection for the House in 2012, is as much an absorbing ticking-clock vérité as it is a moody meditation on time and transference. Throughout, Tcheng artfully deploys archival footage of Dior, often slowed down and paired with an intimate voice-over narration taken from the designer's 1952 memoir. The effect is haunting and emotional—which is appropriate, given that Dior and I is essentially a ghost story.
In Tcheng's film, the House—the atelier in particular—is where the drama unfolds. But the House also registers as a kind of psychic space. Dior, a deeply private man, spent his early childhood isolated in a picturesque maison in Normandy where he immersed himself in the private world of his imagination. Even after he became an international figure in 1947 with the debut of his iconic New Look collection, he remained tethered, psychologically and creatively, to this house, preserving it exactly as it had been decades earlier. It's pink and gray palette and flower gardens became signature elements in his designs.
One can guess that Dior must have felt some kinship with Cocteau's enfants, who spend their final days holed up in an opulent mansion filled with their playthings, prisoners to their private fantasy world. Dior's house, which is now the Christian Dior Museum, makes a critical appearance in Tcheng's film. Simons, who has just taken over as artistic director of Dior, is flown by helicopter to visit the estate. Sitting in the garden, among Dior's beloved flowers, Simons admits that he could not continue reading the designer's memoir because the parallels were deeply unsettling.
Time moves and yet it doesn't. The scene captures what Tcheng is truly after with Dior and I: the uncanny continuities between artists across time. The fifty-five-year gap between Dior's death and Simons's arrival collapses in ninety elegant minutes.
Paul Dallas Dior and I feels very intimate. We're visiting the highly rarified world of haute couture, and yet it's brought alive in your film in a way that's almost tender.
Frédéric Tcheng I was looking for a story and for characters that could make fashion a worthwhile subject for me personally. I wasn't looking to reinforce familiar criticisms about the fashion industry. Some people find it hard to imagine a dialogue about fashion that doesn't start or end with a mea culpa about the industry's sins. This attitude is very odd to me because it's also an industry filled with hard-working artists. My approach with this film was to focus on their struggles within the system, whether they are the creative director, the seamstress, or like me, a film director. That's why the film is called Dior and I.
PD The making of the collection and the making of the film happened in parallel. You and Raf each had eight weeks to produce your debuts—his first couture collection and your first feature documentary as director.
FT I knew going in that I wanted to make an ensemble film that would focus on Raf's encounter with the atelier and address the weight of Dior's legacy. The eight-week deadline meant that we were going to focus exclusively on one collection. The ticking clock is a classic documentary structure, so it made sense dramatically. But beyond the behind-the-scenes action, I was always on the lookout for something more universal. "What's the film about?" It's something that we always asked in film school, and it's something I learned again while working on Valentino: The Last Emperor (2009) and co-directing Diana Vreeland: The Eye Has to Travel (2011). The drama needs to unfold within the characters. That's what I wanted to hone in on with Dior and I.
As an editor, I'm used to observing for long periods of time before a story matures in my head. At some point during the shoot, my sound person—whom I've known for years and whose opinion I respect—said, "I don't know what we're doing. It seems like you don't have a point of view." It really shook me. Early on, I wanted to shoot everything, and my crew wondered what I was after. But I work very much by intuition. I have to find the film as I progress. I wait until my heart's beating a little faster, then I know there is a story that I really care about.
PD You and Raf each find a personal way of interpreting Dior's history and, in a sense, reconfiguring the narrative of the House.
FT Raf has a radical approach to fashion. Unlike other designers, he doesn't start with the surface "look" of a garment. He starts with its shape. He's interested in construction and tailoring, and he wants to change how garments work on the body. In 2012, Raf is confronted with the challenge of how to reinterpret the iconic looks people have come to expect from Dior. It was clear that he wanted to concentrate on the first ten years of the House, from 1947 to '57, which is the time when Christian Dior was designing. For his first collection, Raf resurrects some of the iconic looks and then alters their shape. He pairs Dior's most famous design, the Bar jacket, which is a very sculptural, with skinny, black, contemporary "cigarette" pants. When Dior first unveiled the Bar jacket in 1947, it was a romantic statement against the utilitarian fashions of the Second World War. Raf's juxtaposition of past and present creates an entirely new silhouette. I realized later that I was trying to do something similar. Combining the archival material and voice-over from Christian Dior's memoir with the vérité footage of the atelier today became my way of confronting the past and present in the film.
PD There's a key scene early in the film with the unveiling of the toiles. It's our first glimpse at Raf's take on Dior.
FT Toiles are mock-ups of the dresses produced in cheap, white fabric. They are unique to haute couture. The name means "canvas" in French, so it has an obvious painterly association. But it also has a film association. "Toile" is used colloquially in French to refer to the movie screen or simply to a movie. This is what gave me the idea to project the archival images on the toiles during one section of the film. It was about Raf's modern designs confronting these ghostly, ethereal images of the past. It speaks not only to Raf's challenge as a designer, but also to the intersections of fashion and cinema.
PD This confrontation between past and present is also echoed on the film's soundtrack.
FT My editor Julio Perez and I gathered electronic music that conveyed a sense of repetition, modernity, and minimalism. We took our cue from artists that Raf listens to, such as Plastikman's Richie Hawtin, and other electronic bands like The Knife and the xx. In parallel, I worked closely with Ha-yang Kim, an experimental composer and cellist, who created the score. We were interested in capturing a sense of mystery with the archival footage. We had in mind Jonny Greenwood's score for The Master, and atonal works by Debussy and Schoenberg. We wanted a shifting, unsettling mood that would suggest a ghost story, or a thriller. As the film progresses, Ha-yang's classical score becomes increasingly electronic. It's a subtle shift, but we wanted to convey the sense that Dior's and Raf's themes converged toward the end, as Raf's vision becomes more distinctively his own.
My friend Omar Berrada, who did the voice-over in the film, turned me on to the work of artist Jalal Toufic, specifically his concept of "untimely collaboration." It's the idea that artists can collaborate across time and space—that it's a two-way conversation.
PD The film's voice-over narration is based on Dior by Dior, the designer's 1957 memoir. It was published the year he died, and there's a sense that he's speaking to us from beyond the grave. How did you arrive at the idea of Christian Dior narrating the film?
FT In 2009, I planned to make a film about my family in China. I wanted to use a voice-over of my ancestors' stories on top of images of contemporary China. The idea was to play with the way sound and image can tell two different stories, or respond to the same story. This idea reemerged while I was editing Dior and I. I began to think about what it would mean to have Christian Dior narrate the film. To have this voice-over, written fifty-five years ago, become a hinge between past and present. At times it's paired with the archival footage, and sometimes it's heard on top of contemporary images.
I discovered Dior by Dior when I began researching the film. For some reason, I was drawn to this slim, gray book at the bottom of a box that I was sent. It's not a glamorous account of all the people Dior knew then or the parties he attended. It's a very humble book. He writes about the work and his collaborators. He details the labor and effort required to create a collection, from the initial sketch to the final design. And he writes about how you console someone you've upset. This approach inspired me to focus on the human experience of the creative process. When I reread passages during the shoot, I often felt as though I were reading a description of exactly what I'd witnessed that day. The process and emotions were the same, despite the half-century gap in time.
PD In the book, Dior describes an introspective struggle between his private self and his public image. It's a theme that you explore in Dior and I.
FT It was the other aspect that drew me to the book. The idea of a public image is that you're not physically present but people experience you. Dior found public exposure uncomfortable and ultimately alienating. Raf is also someone who's resisting it, and my film is, in part, about his journey into the spotlight. That's why I use the camera flash as a motif throughout the film. For me, the ending of the film, when Raf is mobbed by the paparazzi after the show, is scary. Facing a wall of cameras like that can be an out-of-body experience. At that moment, Raf's life has changed forever, and this transformation comes with the uneasiness of exposure. I hope this comes across in the film.
Of course, this puts me in a contradictory position. On one hand, I'm lamenting the culture of publicity, and on the other I'm subjecting Raf to it by making the film. But his reaction to the film has helped me come to terms with it. He's been very supportive, in his own private manner.
PD Raf visits Christian Dior's home in Normandy. Sitting in the garden, he talks about reading the memoir and admits that he couldn't continue: the semblance to his own experience was too unsettling, too uncanny.
FT Raf has a complicated relationship with Christian Dior. As artistic director, he's required to channel Dior's spirit. At the same time, he must remain himself—perhaps more so than he's ever been. This was Raf's first couture collection for one of the most esteemed Houses in fashion. The pressure was enormous. While shooting the film, I was fascinated with trying to find the similarities between Raf and Dior. I was interested in this idea of reincarnation. But when I talked with my editor Julio Perez, he said, "We have to find their differences." It was a breakthrough for me. I realized that Raf's real struggle was how to remain Raf Simons in the midst of taking on this role. It became a story like Hitchcock's Rebecca—the house is haunted and the character must emancipate himself from the ghost.
PD How do you feel the film has affected him?
FT I've noticed that he's been exploring ideas of exposure and privacy in his work since the film. In June 2014, he presented a menswear collection under his own label that included several family pictures printed on the garments. There were images of his parents falling in love, and portraits of himself in his early twenties. I've also noticed how he has delved into the past lately at Dior, particularly the '60s and the '70s, as if to challenge what he said in the film about the past not being romantic to him. I don't think it's a contradiction. I see him as an artist who's constantly redefining himself.
PD It's clear that the film is not simply a portrait of Raf Simons. We spend a lot of time in the atelier, getting to know the artists and learning about the craft. The two premières Florence Chehet and Monique Bailly are especially vivid characters.
FT The atelier was the very first place we went when we started shooting. We tried to keep their point of view and not get caught up with the usual hierarchy of the fashion world. It helped that the seamstresses are very direct and accessible. They were wary at first. They told us: "TV crews come and go, and we never see the result." It reminded me that I had a responsibility towards them as subjects. It was humbling. Even though I told them I was trying to make a theatrical feature, they didn't really believe me until they saw the film in Paris last summer. It was great to see the film through their eyes. But it was also a big test. I think they recognized themselves and liked the film, which was a big relief for me.
PD How did you reconcile in your own mind the degree to which the camera affected the reality that you were seeking to portray?
FT Frederick Wiseman was certainly a cinematic reference point. I saw Crazy Horse (2012) just before I started shooting. It's a film I really admire. He takes you behind-the-scenes of the famous erotic Paris dance revue, and it's an institutional portrait. But it's very different stylistically from his other films. There's more attention paid to the film's visual construction. You see this especially in how Wiseman photographs the dance sequences. There's a sense of pleasure in it. You sense him indulging, and the results are quite beautiful. Obviously, Dior and I is not a Wiseman film. I'm more inclined to capture an emotional landscape and how characters transform.
Recently, I found a Scorsese quote I like in which he describes the Maysles' camera as "an inquisitive presence, but also a loving presence, an emphatic presence, attuned to the most sensitive emotional vibrations." I guess that's something I strived to do. When I sent Raf the rough cut of my film, I wrote a note that said, "In order for me to make a film, I really need to fall in love with the subjects and the world I'm filming."
There's been a conversation recently about vérité films in the age of selfies. Is it still possible to make observational films when people are so used to performing for the camera? Richard Brody, in a New Yorker piece on the Maysles, points out that all vérité documentaries are essentially participatory. I've been thinking about what that means for me. In many ways, I'm very old fashioned, and I believe in vérité documentary filmmaking. That being said, Raf is a reluctant protagonist. With Dior and I, the camera is, in part, the subject of the film.
Paul Dallas is a Brooklyn-based writer and programmer with a background in architecture. His writing has appeared in Artforum, Filmmaker, Cinema Scope, Interview, and Indiewire. He has curated programs for the Guggenheim, Maylses Cinema, Times Square Arts, and Van Alen Institute.


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Molecular Destabilization
|Magical Characteristics
Distort the structure of an Object on a Molecular Scale
Focus and Extreme emotions
Active Power. Defensive & Offensive
Molecular Destabilization is the magical power to break down the structure of an object or individual on an Atomic Scale. This power can be used to completely eradicate an organic or non organic item. This extremely rare power is channeled through the the focus of intense emotions.
Users can completely break down the Molecular Structure of any object or living being until they are turned into raw energy which then crushes upon itself until it is destroyed. However this power does not always work on Upper-Level Beings. It should also be noted that some beings can withstand the powerful effects of this power; only being sent flying or knocked unconscious, but very few beings are completely immune to it.
There is also another use to this power. When this powers is used on great inanmiate objects such as stone walls or vehicles, the newly created energy will suddenly implode, creating great ripples in the air that, on contact, can severly maim, if not vanquish, most foes. This is a side-effect of the Destabilization, not something that can be consciously controlled.
Users can also use this power to stop the movement of Energy Balls and other Magical attacks by destroying the Molecular Structure and then sending the uncontrolled energy back in the direction of the attacker.
Mike Hunter created a potion that doubled the strength of his Telekinesis after her and his sister were attacked by a group of Dust Demons. He discovered that by he could access this rare power by focusing his telekinetic powers.
Exhaustion & LimitationsEdit
This power requires an extreme amount of power and focus. The user must be able to see it's desired targer.
Also, despite how powerful this ability is, it should be noted that this power does not always destroy the desired targer and if not used with caution this power can turn on the user, resulting in instant death.


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According to a recent Variety article, Natalie Portman has signed on to produce and star in the upcoming Pride and Prejudice and Zombies movie.
I recently wrote about the upcoming film adaptation for this fan loved book and really have high hopes that it will translate well onto the big screen. With Portman starring I do believe it will get the buzz it needs to successed.
From Variety, "Portman will play feisty heroine Elizabeth Bennet, who is distracted from her quest to eradicate the zombie menace by the arrival of the arrogant Mr. Darcy." In the book, Elizabeth is a zombie hunter with Shaolin training. It is a good book if you get the chance to read it.
Natalie will be producing the film under her Handsomecharlie production company with Lionsgate distributing.


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The KFC recipe has been leaked, but how does it taste? I remember the first time I bit into a piece of proper fried c…
Tag: delicious
Sneaky as Snails… Until you eat them
Have you ever eaten snails? Probably the French kind, but guess what France are not the only people to think snails l…
Red Pickled Onions in Lemon
How to make a delicious pickled onion using just red onions and lemons. Perfect with fried chicken, fish or absolutel…
Fried Sardines
How to gut, and fry whole sardines that can be eaten whole. A recipe we learnt while in Tuscany.
Noodle and Tomato Salad
A very simple cold noodle salad to serve alongside fish, chicken or whatever else you want.
Chilli and Tomato Jam
Make some rad chilli and tomato jam. Perfect for bbq or just eating out the jar with your fingees
Elderflower Cordial
How to make some damn-fine elderflower cordial. Mix it with gin, prosecco, water, petrol, absolutely anything, it alw…
Roast Langoustines
Seafood is amazing, and shellfish is extra good. However, lobsters and crabs can be super pricey, making them …
Planting asparagus is weirder than you think
In which I fight with chickens to put soggy roots into the ground


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|Excimer Laser
Excimer Laser works by altering the shape of the cornea so that the newly shaped cornea can focus light rays directly on the retina, producing clear vision. When successful, refractive surgery can dramatically reduce or eliminate the patient's need for glasses or contact lenses.
PRK (photo refractive keratectomy) is a computer-assisted process that uses an excimer laser to correct your refractive error. During the procedure, the cornea is reshaped using the energy from the light emitted by the laser. For mild to moderate amounts of myopia, the vast majority of patients achieve 20/40 or better vision.
The most common complaint after undergoing PRK is experiencing a hazy sensation and seeing haloes when looking at lights. For some patients, it may take weeks to months to achieve best vision after PRK.
If you've decided that you would like to consider refractive surgery, the first step is to get a complete eye examination and discuss your desire with the doctor. Besides determining your refractive error, your doctor will also check that there is no disease or corneal irregularity present that might complicate a refractive surgery procedure. Questions about lifestyle and occupation will also come into play.
If you have more questions or would like to discuss the procedure with Dr. Blake, call our office at (803)779-4144.


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By Denis Kitchen
More than thirty years have handed when you consider that Al Capp's demise, and he may perhaps now not be a family identify. yet on the top of his profession, his groundbreaking sketch, Li'l Abner, reached 90 million readers. The strip ran for forty-three years, spawned video clips and a Broadway musical, and originated such expressions as "hogwash" and "double-whammy." Capp himself used to be a well-recognized character on television and radio; as a satirist, he used to be often in comparison to Mark Twain.
Though Li'l Abner introduced hundreds of thousands pleasure, the fellow at the back of the strip used to be a classy and sometimes disagreeable individual. A youth twist of fate price him a leg-leading him to artwork as a method of distinguishing himself. His apprenticeship with Ham Fisher, writer of Joe Palooka, all started a twenty-year feud that resulted in Fisher's suicide. Capp loved oversized exposure for a cartoonist, yet his prestige abetted sexual misconduct and guarded him from the severest repercussions. past due in existence, his politics grew to become tremendous conservative; he counted Richard Nixon as a chum, and his reward for satire used to be redirected at objectives like John Lennon, Joan Baez, and anti-war protesters on campuses around the country.
With unparalleled entry to Capp's data and a wealth of recent fabric, Michael Schumacher and Denis Kitchen have written a probing biography. Capp's tale is one in every of brilliant highs and lows, of recognition and villainy, of good fortune and failure-told right here with authority and heart.
Read Online or Download Al Capp: A Life to the Contrary PDF
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Additional resources for Al Capp: A Life to the Contrary
Fisher definite as hell couldn't do it. Boyle and Caplin take to each other and made up our minds to group up at the day-by-day and Sunday strips. They'd preserve "Joe Palooka" operating until eventually Ham Fisher again. "The tale wasn't as sentimental as Fisher's," Al Capp could comment of his collaboration with Boyle, "but it was once humorous. " while Fisher lower back, he gave Caplin his again pay yet their dialog deteriorated whilst Caplin pointed out that he'd met Boyle and that the 2 of them had labored jointly at the strip. Caplin positioned an finish to the dialog whilst Fisher berated his efforts. "If you may get an individual extra dependable than us," he shouted at Fisher, "get 'em! " the 2 have been accomplished as supplier and worker. Their feud, even though, was once simply starting. * Capp's account is an engaging tale, however the evidence don't help it. a detailed exam of the Sunday "Joe Palooka" strips shows that the 1st with Caplin's certain sort seemed on October 15, 1933, even supposing Caplin's contribution to that October 15 strip was once minimum. The hillbillies became up in "Joe Palooka" one week later, on October 22, and that used to be the first actual strip drawn solely by way of Caplin. colossal Leviticus seemed one week after that, on October 29. All instructed, there will be 5 consecutive weeks of this continuity, starting on October 15 and finishing on November 19, 1933. That Fisher's brand-new assistant instantly drew the massive Leviticus debut helps the speculation script already existed for the episode. Fisher, then, was once current for the 1st run of hillbilly strips, and never on holiday, as Capp had humans believing in all of his retellings of the tale after the delivery of "Li'l Abner. " the subsequent Leviticus continuity all started on February eleven, 1934, and ran for 5 weeks, via March eleven. it could possibly rather well be that Fisher was once chasing Marlene Dietrich at the present, and that Caplin used to be reviving and writing large Leviticus on his personal in Fisher's absence. however it is nearly unthinkable that Fisher fired Capp presently after his go back. The final "Joe Palooka" with Caplin as a contributor ran on July eight, approximately 4 months after the ultimate Leviticus Sunday strip. Phil Boyle did, certainly, ghost Ham Fisher's "Joe Palooka" dailies, and it really is most likely that Caplin met him at one aspect or one other, and that the 2 shared the stories with Fisher. it truly is even attainable that the 2 labored jointly on a number of the strips, as Capp stated they did in his autobiography. there's, even if, one untold tale that bears noting: Al Capp continuously complained approximately how Ham Fisher stole one in all his assistants, Moe Leff, within the early days of "Li'l Abner. " prior to that happened, Capp lured Phil Boyle clear of Fisher for a really short time period, sending off one other volley of their escalating feud. In gentle of all of those occasions, Ham Fisher turns out justified in a few of his claims opposed to Capp, even though there's no manner of understanding who precisely created gigantic Leviticus. Fisher had no nook at the hillbilly marketplace, after all, and lots more and plenty of his anger surely originated from expert jealousy—"Li'l Abner" finally bypassed "Joe Palooka" in popularity—but Capp's tale in regards to the holiday and his taking on the strip along with his hills humans is ordinary Capp: half fact, half fiction.


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MADISON, Conn., Dec. 19, 2016 /PRNewswire-USNewswire/ -- Vemma Nutrition, an Arizona-based multi-level marketing (MLM) company that the Federal Trade Commission labeled a pyramid scheme in a lawsuit filed last year, has reached a $238 million settlement with the agency that bans its recruitment-focused business practices.
Ad watchdog truthinadvertising.org (TINA.org) first warned consumers about the energy drink and dietary supplement company's deceptive marketing practices in 2013. The company was the focus of a long-running special investigation and two legal complaints to the FTC regarding its business structure and unsubstantiated health claims.
The company's decade-long run peaked in 2013 with a purported 30,000 members joining a month, sales of more than $200 million and expansion overseas. Its rise was thwarted in 2015 when the FTC, prompted in part by TINA.org's findings, filed a lawsuit against Vemma, its CEO Benson K. Boreyko and top distributors Tom and Bethany Alkazin alleging that the company was operating a pyramid scheme that ensnarled recruits in a money-losing venture. While Boreyko earned millions, a vast majority of participants lost money.
Under the terms of the settlement, Vemma, Boreyko, and Tom Alkazin are banned from promoting any business ventures that: pay any compensation to members for recruiting new participants, tie members' compensation to their own purchases of goods or services, or compensate members if a majority of their revenues come from sales to other distributors. The court order also bars them from making deceptive income claims and unsubstantiated health claims, and requires the company to provide compliance reports by an independent auditor for the next 20 years. While the $238 million figure represents the consumer harm inflicted by Vemma's deceptive practices, the amount that Vemma will actually pay and what the FTC will be able to obtain for victims will be a fraction of the judgment.
The Vemma settlement comes less than six months after Herbalife, a publicly-traded California nutrition and weight-loss supplement company, reached a $200 million settlement with the agency that also requires a substantial change in its recruitment-focused MLM business structure.
To read more about TINA.org's investigation of Vemma see: www.truthinadvertising.org/vemma-pay-millions-settlement-ftc/
About TINA.org (truthinadvertising.org)
TINA.org is a non-profit organization that uses investigative journalism, education, and advocacy to empower consumers to protect themselves against false advertising and deceptive marketing.
To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/energy-drink-company-exposed-by-ad-watchdog-tinaorg-settles-ftc-pyramid-case-300380008.html
SOURCE truthinadvertising.org


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While it is recommended that you use the HubSpot tracking code for WordPress plugin, you can alternatively install the tracking code manually. If you manually add the tracking code and are a HubSpot Marketing Basic, Pro, or Enterprise user, HubSpot will not be able to differentiate posts versus pages in its reporting.
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Select Location
2016 Dodge Charger SXT
Raigosa Auto Sales
Greenville, SC 29611
United States
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2016 Dodge Charger
Stock Number
VIN Number
Transmission Type
Exterior Color
Interior Color
Passenger Cars
Color-Keyed Accents
AIR Filter AIR Conditioning
Automatic AIR Conditioning
Dual Zone AIR Conditioning
Humidity Sensor AIR Conditioning
Outlets AIR Conditioning
Rear Armrests
Passenger Assist Handle(S)
Rear Assist Handle(S)
Controls, Voice-Activated Audio
Digital Media Auxiliary Jack Audio
Alpine Audio - Brand
AM/FM Audio - Radio
276-Watt Amplifier Audio - Speakers
6 Audio - Speakers
Premium Audio - Speakers
USB Port Capabilities/Wiring
Hooks, TIE Down Cargo Area
NET(S) Cargo Area
Bluetooth, Hands-Free Phone Communication
U-Connect Communication
Center Consoles
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4 Cupholders
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Front & Rear Floor Mats
Universal Garage Door Opener
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Courtesy Lights
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MAP, Front Lights
Reading Lights
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Auto-Dimming Mirrors
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Rearview Mirrors
Accessory Delay Power Features
Speed-Sensitive Power Locks
12-Volt Power Outlet(S)
Fuel Filler Door, Capless Remote Releases
Trunk Remote Releases
Sport Seats
8-WAY Adjuster, Power Seats - Driver
Lumbar Support, 4-WAY Adjuster, Power Seats - Driver
Buckets Seats - Front
Heated Seats - Front
60/40 Split-Folding Seats - Rear
Tilt/Telescoping Steering Column
Controls, Audio Steering Wheel
Controls, Cruise Steering Wheel
Leather-Wrapped Steering Wheel
Pockets, Door Storage
Sirius, 1 Year Subscriptions
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Sentry KEY Theft Deterrent Systems
Shift Knob, Leather-Wrapped Trim
Cloth Upholstery
Mirror, Illuminated Visors
Power Windows
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Body-Color Door Handles
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Projector Headlights
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Body-Color Mirrors
Foldaway, Manual Mirrors
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180 Alternator - Maximum Capacity (Amps)
730 AMP Battery
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About This Dealer
Raigosa Auto Sales
Sales Dept
Greenville SC 29611
United States
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Raigosa Auto Sales is a family owned business that has been proudly serving the South Carolina Community since 1993, when Antonio Raigosa first opened to the public. Currently the business is owned by Jorge Giraldo and his partner Adriana Raigosa who is the daughter of the original owner, Antonio Raigosa. With so many years of experience we know what our customers are looking for and have become their right hand man in order to get them the best deal with the most competitive prices of the market. We prioritize by having excellent customer service.
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text: Jagger Bootcut in Midnight Rollover to Zoom Jagger Bootcut in Midnight 665C-351 $194.00 *Size: Choose an Option... 28 29 30 31 32 33 34 38 40 * Required Fields Qty Add to Shopping Bag Add to Wishlist Details Our low-rise Jagger jeans feature a bootcut, fanning out slightly past the knee. Crafted from signature stretch denim, they're flexible enough for day on a job or at play. Crafted from stretch denim, this pair has a solid black wash that's easy to dress down or up -- wear it with a cardigan or a simple tee. Size and fit Bootcut Content: 85% Cotton 13% Modal 2% Polyurethane Fit true size Model is wearing size 32. Rise: 10 1/4" Inseam: 34" Leg Opening: 19 3/4


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Africa's vast territory makes the trip to the continent an endless adventure, full of beautiful scenery, desolate landscapes and exotic nature. If you're looking for more thrilling experience and you prefer to explore more underrated places during your visit, have a look at out list with Africa's top 10 hidden gems you can't miss.
1. The Skeleton Coast, Namibia
2. Lalibela, Ethiopia
3. Ganvie, Benin
4. Hogsback, Eastern Cape, South Africa
5. Lake Bunyonyi, Uganda
6. Zululand Rhino Reserve, South Africa
7. Gansbaai, South Africa
8. Bureh Beach, Sierra Leone
9. Jinja, Uganda
10. Nianing, Senegal
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Welcome to MACG
We are an alliance of organizations in the Portland, Oregon Metro area building power for the common good. Read more about us here.
MACG Video!
Watch this powerful video about everyday people becoming exceptional leaders through MACG's organizing practices.
¡Hola! ¡MACG también habla español!
Desde el otoño del 2012, MACG está organizando a la comunidad latina en el área metropolitana de Portland para construir el poder para lograr los cambios que necesita nuestra comunidad. Para más información, por favor comuníquese con Alejandro Bautista.
MACG - City Council Vote on Construction Excise Tax
The MACG housing team worked in coalition this winter to support the successful passage of inclusionary zoning at the legislative session in Salem. As part of this legislation, communities are allowed to define a construction excise tax (CET) on new construction. The Portland City Council will be meeting on Thursday, 6/16 to vote on what percentage this new tax revenue to dedicate to affordable housing. MACG will be advocating for 100%. JOIN US!
MACG - Institutional Development and Core Teams Training
Do you want to: … make your institution stronger, more relational, and more action oriented? … fulfill your institution's mission? … build a base of power to act together for the common good? JOIN US! For a two-hour workshop on how to build and sustain a core team in your institution.
MACG - Spring 2016 Leadership Institute (Part 1)
MACG Leadership Institute coming up in April! Who would you like to see trained? Make sure they register.
MACG - Fall 2016 Leadership Institute (Part 1)
MACG Leadership Institute coming up in November! Who would you like to see trained? Make sure they register.


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Online Courses
Work Health Safety
|Qualification Work Health Safety
|National course code ASC101
|Recognition Professional Certificate
|Duration Self-paced - 20 minutes
|Study mode Online
|Start date Enrol anytime
Every year in Australia, workplace accidents result in over 500 deaths and 600,000 compensatable injuries. Workplace accidents, injuries and sickness cause personal pain and suffering, financial loss to employees and organisational costs. This course supplies learners with the knowledge and skills required to apply solid WHS practices within their organisation.
This course covers:
- WHS legislation and framework
- WHS policies and procedures
- Hazards and risks
- Employer and employee responsibilities and duty of care
- Correct manual handling
- Emergency evacuation procedures
Online course
Workplace Bullying
Bullying is a nuanced and complicated subject and it can have some terrible consequences for the wellbeing of your staff and your business. We've… View course
Course details
For more detailed information about the course structure please visit Work Health Safety ASC101 at Training.gov.au


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Oscar Jaffe is a egomaniacal theater director whose always looking for his next big star. During his most recent production he becomes infatuated with a simple chorus girl, Mildred Plotka. Convinced he can turn her into a leading lady, Jaffe uses his controlling ways to effectively transform Mildred into Lily Garland, the biggest star on Broadway. At first the two are inseparable but Lily begins to grow tired of Jeffe's obsessive control over both her career and her life. The last straw happens when Jaffe hires a private detective to track Lily's every move, forcing her to run for the hills of Hollywood. Soon after Lily's departure Jaffe goes bankrupt with Lily becoming one of the biggest star in Hollywood. While fleeing the city from debt collectors, Jaffe coincidentally runs into Lily on the train, with him being desperate to have her back in one of his productions. Howard Hawks' Twentieth Century is an incredibly fun screwball comedy that no question set the watermark on this type of thing for years to come. John Barrymore completely steals the entire film as the flamboyantly egomaniac, Oscar Jaffe. His comedic timing is really one of a kind and the energy he brings to the whole endeavor makes it almost impossible not to be charmed. The script is also fantastic in both its simplicity and wittiness, even touching on some interesting themes like the art vs. commerce debate. The relationship between the actress and the director, how they despise each other, yet are drawn to each other is just great to watch. As fun as Hawks screwball films are, they always impress me with their understanding of character. For example, the first time see Lily after her departure it is very clear that she has become just as egomaniacal and over-dramatic as Oscar Jaffe. This leads to some great comedic moments but also hints at the notion that Oscar and Lily really are perfect for one and other even if they seem to despise each other. Twentieth Century is without question one of the best screwball comedies I've seen, really capturing the over-dramatic tendencies of theater performances and using it too great comedic effect.
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October 2017


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This was our first project ever so we had a lot of fun making it! Obviously not gonna stand up to something like the Witcher but we think we did good with the skill sets me and Mottie have.
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