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Molecular Diversity in Venom from the Australian Brown Snake, Pseudonaja textilis* S
- Oswald Young
- 3 years ago
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1 Research Molecular Diversity in Venom from the Australian Brown Snake, Pseudonaja textilis* S Geoff W. Birrell, Stephen Earl, Paul P. Masci, John de Jersey, Tristan P. Wallis**, Jeffrey J. Gorman**, and Martin F. Lavin Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A 2 s, and preand postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two-dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, -carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N- acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents. Molecular & Cellular Proteomics 5: , Snakes from the Australian elapid genus Pseudonaja are fast moving and highly venomous and are responsible for most deaths by snake bite in Australia. Of the eight classified species of Pseudonaja, Pseudonaja textilis, the Common or Eastern Brown snake, has the most lethal venom, surpassed only by Oxyuranus microlepidotus, the Inland Taipan. The venom is strongly procoagulant and has little hemolytic or myolytic activity (1). Several researchers have examined the venom from P. textilis and have isolated numerous toxins. From The Queensland Institute of Medical Research, P. O. Royal Brisbane Hospital, Brisbane 4029, Central Clinical Division and Southern Clinical Division, Faculty of Health Sciences, University of Queensland, Brisbane 4029, and School of Molecular and Microbial Sciences and **Institute for Molecular Biosciences, University of Queensland, Brisbane 4072, Australia Received, August 19, 2005, and in revised form, November 9, 2005 Published, MCP Papers in Press, November 10, 2005, DOI /mcp.M MCP200 These include postsynaptic -neurotoxins (2 4), the presynaptic neurotoxin textilotoxin (5 7), phospholipase A 2 s (PLA 2 s) 1 (6, 8); serine protease inhibitors (9, 10), and a prothrombin activator (11, 12). Fry (13) has written a comprehensive review on the properties of venom components from Australian elapids. Barnett et al. (2) isolated a postsynaptic -neurotoxin from the venom and named it pseudonajatoxin A. This protein is 117 amino acids in length, which is considerably larger than other snake neurotoxins, and like other -neurotoxins acts by binding to acetylcholine receptors. Pseudonajatoxin B by contrast (3) contains only 71 amino acids and has considerable homology with other postsynaptic long -neurotoxins. Six other -neurotoxins have been cloned and expressed from venom gland RNA (4). These have fewer amino acids (57 or 58 residues) and possess lower neurotoxic activities than the short-chain -neurotoxins found in other snakes. Textilotoxin is the most potent neurotoxin yet isolated from the venom of a land snake. It represents 3% by weight of the crude venom and 70% of its total lethality (1). It is comprised of five PLA 2 subunits, two of which are identical, and acts by blocking the release of acetylcholine following the arrival of an action potential at a nerve terminal (14). Armugam et al. (8) isolated four other PLA 2 s from the venom and found them to be group 1B PLA 2 s. In that study, analysis of DNA from the venom gland showed that only two genes and two cdnas were responsible for the four PLA 2 proteins produced. This is similar to the short-chain -neurotoxins (4) where alternative splicing of the P. textilis short neurotoxin 1 gene (PT sntx1) gives rise to two mrnas and two neurotoxins. Masci et al. (9) identified and characterized serine protease inhibitors from the venom, named them textilinins, and showed that they significantly reduce bleeding in an animal model. More recently, six isoforms of textilinin have been identified in P. textilis venom gland-derived cdna (10). A prothrombin-activating complex was identified by Masci et al. (11) and was found to comprise a large proportion of the venom. This complex was subsequently named pseutarin C and found to 1 The abbreviations used are: PLA 2, phospholipase A 2 ; 2D, twodimensional; Gla, -carboxyglutamate, mab, monoclonal antibody; PNGase F, peptidyl-n-glycosidase F; ConA, concanavalin A; WGA, wheat germ agglutinin; RCA 120, Ricinus communis agglutinin 120; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GRP78, glucose-regulated protein 78; HSP, heat shock protein by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics This paper is available on line at
2 be a group C prothrombin activator consisting of a Factor Xa-like enzymatic subunit and a non-enzymatic Factor Va-like subunit (12). Injection of purified pseutarin C results in massive disseminated intravascular coagulation within the body of the snake s prey, ultimately resulting in death (11). In addition to the identification of a number of toxins, it has been reported that the venom composition from a single specimen can vary substantially from season to season (15). In this study, we performed the first comprehensive proteomic analysis of the venom composition using two-dimensional (2D) PAGE, mass spectrometry, and immunoblotting to identify the toxins. We also used post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Post-translational modifications examined include phosphorylation, acetylation, -carboxylation, and glycosylation. Glycoproteins were further characterized by chemical and enzymatic deglycosylation and by lectin binding specificity. EXPERIMENTAL PROCEDURES Materials: Snake Venoms and Chemicals Venom samples were obtained from Venom Supplies Pty. Ltd. (Tanunda, South Australia). Venom samples were collected over the course of a year and pooled from over 40 individual snakes to reduce seasonal and individual variation. Sample collection was performed for both Queensland and South Australian specimens to enable comparison of geographic variation. Venoms were lyophilized and reconstituted at 10 mg/ml in PBS, 50% glycerol. Water was prepared using a Milli-Q system (Millipore, Bedford, MA). FITC-labeled lectins were from Vector Laboratories (Burlingame, CA). Cy dyes were from Amersham Biosciences. Pro-Q Diamond and Pro-Q Emerald were from Molecular Probes Inc. (Eugene, OR). Neuraminidase and peptidyl-n-glycosidase F (PNGase F) were from New England Biolabs (Beverly, MA). O-Glycosidase was from Roche Applied Science. Antisera to various venom components were raised in rabbits or sheep in University of Queensland animal house facilities with appropriate ethical clearances. Acetylated lysine monoclonal antibody (mab) was from Cell Signaling Technology (Beverly, MA). mab against -carboxyglutamate (Gla) residues was from American Diagnostica Inc. (Greenwich, CT). All other chemicals were from Sigma unless stated otherwise. 2D PAGE Venom samples (500 g) were diluted to 400 l with UTC buffer (7 M urea, 2 M thiourea, 4% CHAPS with a trace of bromphenol blue), 50 mm DTT, and 1% ampholytes (Biolytes 3 10, Bio-Rad). Samples were mixed and centrifuged for 5 min at 13,000 g to pellet insoluble material, and the supernatant was loaded onto IEF strips (Bio-Rad ReadyStrip, ph 3 10, 24 cm) for 16-h passive rehydration. Proteins were focused on a Bio-Rad IEF cell. The IEF running conditions were: 250 V for 15 min, ramp to 8000 V for 3 h, hold at 8000 V for 90,000 V-h. After IEF, IPG strips were equilibrated for 15 min in an equilibration buffer (50 mm Tris-HCl, ph 8.8, 6 M urea, 2% SDS, 30% glycerol, 2% DTT) followed by 15 min in an equilibration buffer that had DTT replaced with 2.5% iodoacetamide. IPG strips were briefly rinsed in SDS-PAGE running buffer and embedded on top of 12% SDS-PAGE gels (Bio-Rad Protean Plus, cm) and covered with 0.5% agarose. Gels were run at 200 V constant voltage until the bromphenol blue dye front reached the bottom of the gel. Silver staining was performed as described by Shevchenko et al. (16). Colloidal Coomassie staining was performed as described by Neuhoff et al. (17). For immunoblotting; amino-terminal sequencing; phosphoprotein, glycoprotein, and lectin binding studies 11-cm IEF strips (Bio-Rad ReadyStrip, ph 3 10, 11 cm) were used with 50- g venom samples with IEF conditions scaled down to 40,000 V-h total and samples run on 12% SDS-PAGE gels (Bio-Rad Criterion format gels). Cy Dye Labeling Pooled P. textilis venom samples from Queensland or South Australia were precipitated in 8:1 acetone:methanol for 16hat 20 C. Samples were centrifuged for 30 min at 16,000 g to pellet protein, the supernatants were removed and discarded, and the protein pellets were allowed to air dry for 10 min. Proteins were resuspended in 20 l of20mm Tris in UTC buffer to bring the ph to 9. Cy3 and Cy5 monofunctional reactive Cy dyes (Amersham Biosciences) were used to label proteins according to the manufacturer s instructions. Labeling reactions were quenched by the addition of 2 l of 10 mm lysine. Reaction mixtures were combined and loaded onto 11-cm ph 3 10 IEF strips as described under 2D PAGE above. After electrophoresis, gels were scanned on a Typhoon laser scanner (Amersham Biosciences) for both Cy3 and Cy5, and the resultant images were overlaid and analyzed using PDQuest software (Bio-Rad). Identification of Venom Proteins Using MALDI-TOF and TOF-TOF Mass Spectrometry and de Novo Peptide Sequencing Protein spots from silver-stained 2D PAGE were excised, washed in water, and destained as described by Gharahdaghi et al. (18). Trypsin was added, and proteins were allowed to digest overnight at 37 C prior to extraction. Extracted peptides were dried, resuspended in 50% ACN, 0.1% TFA, mixed 1:1 with matrix ( -cyano-4-hydroxycinamic acid), and spotted on a MALDI plate. Samples were analyzed using a Voyager-DE STR mass spectrophotometer (Applied Biosystems) in positive reflector mode with an accelerating voltage of 20,000 V. One hundred and fifty laser shots per spectrum were acquired in the mass range Da. The 50 most intense peptide masses were searched against the Chordata taxonomic subset of the National Center for Biotechnology Information (NCBI) protein database using the Mascot search engine. MS peptide tolerance was 100 ppm, the search allowed for carbamidomethylated cysteine, and no other posttranslational modifications were taken into account. For TOF-TOF analysis, peptides were analyzed using a 4700 Proteomics Analyzer (Applied Biosystems) operated in positive ion reflectron mode. MS data were acquired using 2000 shots of a neodymium:yttrium-aluminum-garnet laser at 355 nm with a 200-Hz repetition rate and fixed intensity. The top 50 most intense peptides detected for each spot in the MS mode were automatically selected for MS/MS analysis using 3000 laser shots at a fixed intensity, which was 20% greater than that used for MS. MS/MS data were calibrated against the MS/MS fragments of the m/z Angiotensin I peptide in the standards. MALDI-TOF/TOF MS/MS data were automatically analyzed using the GPS Explorer suite of software (Applied Biosystems). For each spot a combined MS and MS/MS analysis was performed in-house using a Mascot search engine and the Chordata taxonomic subset of Celera Discovery System database. MS peptide tolerance was 100 ppm, and MS/MS tolerance was 0.3 Da. The search allowed for carbamidomethylated cysteine and oxidized methionine. For the purposes of protein identification, no other post-translational modifications were taken into account. For de novo peptide sequencing, MALDI-TOF/TOF MS/MS data were opened in Data Explorer (Applied Biosystems, Version 4.2) and deisotoped, and raw text peak lists were exported. The peak lists were analyzed using the automatic de novo function of PEAKS Studio software Version 2.4 (19) (Bioinformatics Solutions Inc., Ontario, Canada). Contiguous stretches of seven or more amino acids with 100% confidence call using the default parameters of the softwares were collected and matched to the NCBI non-redundant protein database using the protein BLAST algorithm ( Amino-terminal Sequence Determination Venom proteins were electroblotted from 2D PAGE onto PVDF membrane (Pall Scientific, 380 Molecular & Cellular Proteomics 5.2
3 FIG. 1. 2D PAGE of P. textilis (Queensland) venom. Duplicate samples of 500 g of venom proteins were subjected to isoelectric focusing on 24-cm ph 3 10 IEF strips and subsequently resolved by 12% SDS-PAGE. The silver-stained gel (A) shows stronger staining intensity of high molecular size proteins compared with the Coomassiestained equivalent gel (B). Forty-nine protein spots were excised (C) for identification by mass spectrometry, de novo peptide sequencing, and NH 2 -terminal sequencing (Supplemental Table 1). Melbourne, Australia) in Towbin buffer at 15 V for 16 h. The membrane was briefly stained with Coomassie Blue, destained, washed extensively with several changes of water for 2 h, and dried. Selected spots were excised from the membrane with a scalpel blade, and the NH 2 -terminal sequences were analyzed by Edman degradation using an Applied Biosystems Model 492 sequencer (Applied Biosystems). The observed sequences were matched to the NCBI non-redundant protein database using the protein BLAST algorithm ( nih.gov/blast/). Immunoblotting Proteins from 2D PAGE were electrotransferred to nitrocellulose membranes (Pall Scientific, Brisbane, Australia), and membranes were blocked by immersion in 5% skim milk in PBS, 0.05% Tween 20 (SM/PBS/T). Membranes were probed with rabbit or sheep polyclonal antisera raised against specific venom proteins or venom protein complexes (26). The antisera were diluted 1:2000 in SM/PBS/T, and membranes were incubated overnight with gentle agitation. After washing in PBS/T for 3 10 min, membranes were incubated for 1 h in secondary antisera (goat anti-rabbit Ig/horseradish peroxidase or rabbit anti-sheep Ig/horseradish peroxidase, Chemicon, Melbourne, Australia) diluted 1:5000 in PBS/T. After 5 10-min washes in PBS/T, blots were developed using ECL reagent (Western Lightning, Perkin Elmer Life Sciences) and visualized with a chemiluminescence detector (FujiImager 3000, Fuji). Lectin Binding Specificity Glycosylation profiles of 2D PAGE-separated proteins transferred to nitrocellulose membrane were examined using a panel of eight lectins with FITC labels (Fluorescein Lectin Kit I, Vector Laboratories) as described by Nawarak et al. (20). Phosphoprotein and Glycoprotein Staining Detection of phosphoproteins and glycoproteins in 2D PAGE was performed using the ProQ Diamond and ProQ Emerald fluorescent reagents, respectively (Molecular Probes Inc.) according to the manufacturer s instructions. ProQ Emerald is a fluorescent-based form of the periodic acid-schiff stain for carbohydrates that has a broad range of specificity for glycol-containing molecules. Enzymatic Deglycosylation Venom samples were precipitated in methanol:acetone (8:1) as above and air-dried prior to exposure to neuraminidase (New England Biolabs), O-glycosidase (New England Biolabs), or PNGase F (New England Biolabs) in buffers and conditions recommended by the manufacturer. Reactions were allowed to proceed for 1 h at 37 C. RESULTS Separation and Comparative Analysis of P. textilis Venom Proteins Venom proteins from P. textilis (Queensland specimen pool) were initially analyzed using 2D PAGE. Sample processing and isoelectric focusing conditions were chosen to give the greatest number of resolved protein spots per gel. Over the ph range 3 10, 219 protein spots were detected using PDQuest software (Bio-Rad) from the silver-stained gel (Fig. 1A), whereas 192 spots were identified on the corresponding Coomassie Blue-stained gel (Fig. 1B). The proteins ranged in size from 5 kda to greater than 200 kda. Numerous horizontal trains of spots were evident especially in the molecular mass range greater than 30 kda. These are suggestive of post-translational modifications of individual proteins. The abundance of spots in the molecular mass range kda with low pi corresponds to that expected of PLA 2 s, which have already been shown to be present in P. textilis venom (6, 8). The overall pattern of staining was similar when colloidal Coomassie Blue staining was used; however, the relative intensity of some protein spots was different compared with detection by silver staining. Both methods of staining are compatible with further analysis by mass spectrometry and Molecular & Cellular Proteomics
4 are likely between them to facilitate characterization of most proteins in the venom. Protein Identification by Mass Spectrometry To identify venom proteins, gel spots were excised from 2D gels and digested with trypsin, and peptides were analyzed by MALDI- TOF MS and MALDI-TOF/TOF MS/MS. Forty-nine spots were selected from the 2D gels across a range of molecular masses. The distribution of these is indicated in Fig. 1C, and a complete list of the peptides and proteins identified appears in Supplemental Table 1. A prominent protein represented as a train of 10 spots at 40 kda (Fig. 1C, spots and adjacent spots) was identified as the Factor Xa-like heavy chain. Another protein also present in the prothrombin activator complex of P. textilis, Factor Va-like protein, was identified at its expected molecular mass of 160 kda also as a train of spots (Fig. 1C, spot 49 and adjacent spots). There were several other Factor Va-like polypeptides corresponding to the A1 domain (heavy chain, 100 kda; Fig. 1C, spots 47 and 48), A3 domain (light chain, 65 kda; Fig. 1C, spot 45), and A2 domain (52 kda; Fig. 1C, spots 43 and 44). These were identified by the presence of tryptic peptides that mapped specifically to the respective domains (Supplemental Table 1). The discrepancy between expected and observed molecular size is thought to be due to potential N-glycosylation sites in the A2 and A3 domains (12). Another protein identified in the high molecular mass region of the gel was a single protein spot identified as glucose-regulated protein 78 (GRP78; Fig. 1C, spot 46), a member of the heat shock protein 70 (HSP70) family. The presence of GRP78 in snake venom has not been reported previously. A pseudechetoxin-like protein was identified by de novo peptide sequencing as a protein spot of 25 kda and pi 10 (Fig. 1C, spot 35). Pseudechetoxin and its homolog pseudecin are members of the cysteine-rich secreted proteins that were previously identified in two related Australian snakes, Pseudechis australis and Pseudechis porphyriacus, respectively (21). More recently, a cdna clone corresponding to this protein was identified from the venom gland of P. textilis in a separate study (22). Additional members of this family have also been identified in several other Australian snake species using cdna cloning (22). Two de novo sequenced peptides were used to identify protein spot 35 in the present study as the pseudechetoxin-like protein from P. textilis. We also identified what appeared to be several members of the PLA 2 family at 15 kda and over a pi range of 4 6. Due to the limited amount of sequence information in the NCBI database, it was not possible to identify all PLA 2 s as individual isoforms. However, the differences in pi and size between the isoforms and previously reported data on the presence of multiple PLA 2 isoforms in P. textilis venom (8) supports the presence of sequence variants. The four subunits of the textilotoxin complex were identified in multiple protein spots (Fig. 1C, spots 1, 3, 6, 12, 13, 14, 29, 30, 31, 33, and 36). These molecules are members of the PLA 2 family and were also FIG. 2. Regional diversity of P. textilis venom. Pooled venoms from Queensland specimens (A) were labeled with Cy 3 dye, whereas those from South Australia (B) were labeled with Cy 5 dye. Both Cy3 and Cy5 samples were combined and subjected to 2D gel electrophoresis. The gel was scanned at wavelengths specific for each Cy dye, and the images were false colored and superimposed (C) to reveal protein spots unique to each region. Proteins common to both regions are colored yellow. Red spots are unique to Queensland; green spots are unique to South Australia. Arrows indicate proteins that appear at noticeably different levels. identified using specific antisera in a corresponding immunoblot (Fig. 4E). Somewhat surprisingly, two isoforms of textilinin were identified migrating at an approximate molecular size of 10 kda (Fig. 1C, spots 9 and 10). Although multiple textilinin cdnas have been reported for P. textilis, the size of predicted proteins based on these cdnas is 6 7 kda, which is considerably lower than that observed here. As expected, several neurotoxins were identified at their anticipated pi and molecular size (Fig. 1C, spots 20, 22, 24, 25, 26, 27, 28, 33, and 34). Geographic Variation in Venom Composition Previous reports have shown that the toxicity of P. textilis venom can vary in a geographic manner (1, 23). To examine for geographic variation in venom composition we compared venom proteins in P. textilis from two geographically separated regions, Queensland and South Australia. Proteins from the two samples were differentially labeled with Cy3 and Cy5 dyes to allow comparison within a single gel. Similar numbers of proteins were observed in both samples with greater intensity of labeling of proteins in the lower molecular mass region compared with the silver or Coomassie Blue stain (Fig. 2, A and B). Overlay of the two images revealed that there was concordance between the labeled proteins in greater than 95% of cases (Fig. 2C). There were notable differences in proteins in the molecular mass range of 5 10 kda. It has been reported that short and long neurotoxins and textilinins are known to occur in this size range (3, 4, 9). By reference to Fig. 1C, we propose the most notable differences to be an increase in 382 Molecular & Cellular Proteomics 5.2
5 FIG. 3. Immunoblots of 2D separated venom proteins. Venom proteins were separated on 2D gels, and proteins were transferred to nitrocellulose membrane for immunoblotting with antisera to prothrombinase complex (A), heavy chain of the Factor Xa-like protease (B), Gla residues (C), textilinins (D), and textilotoxin (E). abundance of the phospholipase A 2, textilotoxin D chain (Fig. 2C, arrow), and differences in the abundance of forms of the long neurotoxin pseudonajatoxin B. Small differences were also observed for the Factor Xa-like heavy chain suggesting subtle differences in post-translational modifications of this train of spots. Our findings suggest that overall there is great similarity between the venoms from the two regions and that the differences are unlikely to account for significant changes in overall toxicity. Protein Identification by 2D Immunoblotting As an additional approach to identifying proteins, antisera specific to various components of the venom were used to identify the composition and location of the individual proteins (Fig. 3). The prothrombin activator complex in P. textilis is composed of a Factor Va-like molecule and a Factor Xa-like molecule. Three different antibodies were used to examine the prothrombin activator complex in the venom. These include a rabbit polyclonal antisera raised against the purified complex (Fig. 3A), a sheep polyclonal antisera raised against a recombinant form of the Factor Xa-like heavy chain (Fig. 3B), and a mouse monoclonal antibody specific for Gla residues (American Diagnostica Inc.). Antisera against the prothrombin activator complex (Fig. 3A) detected three isoforms of the Factor Va-like molecule. This corresponded well with the data in Fig. 1C where these isoforms were identified by mass spectrometry, although the antisera failed to detect isoforms corresponding to the A1 domain (Fig. 1C, spots 47 and 48) and the A2 domain (Fig. 1C, spot 43). The Factor Xa-like molecule was also identified as a series of discrete spots as observed above using silver staining (Fig. 1C, spots 39 42). Antisera raised against a recombinant form of the heavy chain of Factor Xa-like molecule detected a train of protein spots identical to that seen with antisera against prothrombin activator complex (Fig. 3B). Although the heavy chain was readily detectable in 2D gels using silver and Coomassie Blue staining, the Factor Xa-like light chain was not evident in these gels. However, with the prothrombin activator antisera, a complex staining pattern was also evident in a region corresponding to the light chain of Factor Xa-like molecule. Because it has been reported that the light chains of Factor Xa from both mammalian species and snakes are post-translationally modified at their NH 2 terminus by -carboxylation of glutamic acid residues (11), we used a monoclonal antibody that detects this modification. The pattern observed with this antibody corresponded to a region that was also observed with the prothrombin activator complex antiserum (Fig. 3C). This region also corresponds with the expected molecular size and pi of the Factor Xa-like light chain. A series of textilinin spots were detected using rabbit antisera raised against purified textilinin 1. This pattern was consistent with the presence of multiple isoforms of textilinin identified in P. textilis venom gland using cdna cloning (10). These spots corresponded very well to those identified by mass spectrometry (see Fig. 1C). Multiple spots were detected for textilotoxin using rabbit antisera raised against the purified complex at both acidic and basic pi values. This was consistent with mass spectrometric identification of textilotoxin, which is composed of four related PLA 2 molecules, textilotoxins A D (7). Textilotoxins A and B were identified by mass spectrometry at high pi (ph 9 10). Antisera used here detected spots in this region (Fig. 3E). The multiple forms detected at a lower pi (ph 3 5) corresponded well to textilotoxins C and D (7), also identified by mass spectrometry. It is of interest that textilotoxins B and D were identified from several distinct spots. Post-translational Modifications One of the intriguing observations of the 2D gel separations of the P. textilis venom Molecular & Cellular Proteomics
6 FIG. 4. Post-translational modification stains. Venom proteins were separated on 2D gels, and proteins were transferred to nitrocellulose membrane for staining with fluorescent stains specific for phosphoproteins (A), total protein (B), and glycoproteins (C). Asterisks in A and B indicate casein run as a phosphoprotein positive control lane on the edge of the second dimension gel. proteins was that there were multiple horizontal trains of spots in different regions of the gels. These were most apparent in the higher molecular mass region where both Factor Va-like and Factor Xa-like components were identified (Fig. 1C, spots 39 42). These suggested the presence of multiple isoforms differentiated either by amino acid composition, post-translational modifications, or a combination of both. One of the most common post-translational modifications of proteins is phosphorylation. We assayed for this modification using a fluorescent stain, Pro-Q Diamond (Molecular Probes Inc.), reported to be specific for phosphoproteins (24). The results in Fig. 4A revealed the presence of several apparently phosphorylated proteins. When the same gel was subsequently stained with a total protein stain (Deep Purple, Amersham Biosciences), the phosphorylated spots correlated well with some of the proteins detected (Fig. 4B). Several of the high molecular mass phosphorylated proteins correlated well with Factor Va-like and Factor Xa-like isoforms on the basis of size and isoelectric point. A set of spots in the kDa region with pi 4 5 were consistent with phosphorylated PLA 2 s. Glycoproteins were also detected in a separate gel (Fig. 4C) using a specific stain (Pro-Q Emerald, Molecular Probes Inc.). This revealed evidence of multiple proteins modified across the range of different sizes and pi values. Treatment of crude venom with neuraminidase and separation by single dimension SDS-PAGE revealed a shift in molecular size for most proteins above 20 kda (Fig. 5, lanes 1 and 2) suggesting an abundance of sialic acids. We also used the glycosidases PNGase F and O-glycosidase to distinguish between N-glycosidic and O-glycosidic linked sugars (Fig. 5, lanes 6 and 3, respectively). PNGase F treatment resulted in a number of band shifts above 20 kda in the treated (Fig. 5, lane 6) compared with untreated (lane 7) samples. On the other hand, no change was evident after treatment with O-glycosidase alone (lane 3). Because it has been reported that O- glycosidase cleavage is often more efficient after pretreatment with a sialidase, we used this combination of enzymes (lane 4). The pattern of proteins observed was identical to that observed with sialidase alone, providing additional support for the lack of O-linked sugars in venom proteins. The combination of the three enzymes (lane 5) supports the presence of sialic acid residues and N-linked sugars. The use of acid hydrolysis to remove sugars from the venom proteins resulted in the appearance of protein smearing in both one-dimensional and 2D gels (data not shown). This may have been due to cleavage of peptide bonds as well as glycosidic bonds. To relate the presence of glycosylation to the appearance of trains of spots on 2D gels, we used Cy dye methodology. We treated samples with and without neuraminidase and with and without PNGase F and subjected them to 2D PAGE (Fig. 5, B and C, respectively). Both results showed a reduction in molecular size for several trains of spots after deglycosylation with very little effect on the pi of the proteins. The reduction in molecular size after deglycosylation was particularly evident above 20 kda where these trains were most apparent. Although not as clear, the lower molecular mass region showed direct overlay (yellow) between sets of spots. We also examined blots for the presence of acetylated lysine residues using a mab specific for this modification. Acetylation of lysine is an important reversible modification controlling protein activity (25). We saw no evidence of lysine acetylation on 2D blots (data not shown). Lectin Binding Specificity Fluorescently labeled (FITC) lectins were used to detect glycoproteins on nitrocellulose membranes after 2D PAGE of venom proteins from P. textilis. Because it was possible that peripheral sugars may be lost during the preparation and separation of samples for 2D PAGE and subsequent electrotransfer to nitrocellulose, we initially spotted different dilutions of crude venom onto nitrocellulose membrane and probed it with a selection of eight lectins chosen to identify a wide range of specific sugar groups. Under these conditions, four of eight different lectins used showed no binding to venom proteins (results not shown). These four non-binding lectins were soybean agglutinin (specific for N-acetylgalactosamine and galactose), Ulex europeus I lectin (specific for fucose), elderberry bark lectin (specific for 2 3-linked sialic acid), and peanut agglutinin (specific for galactose). It should also be noted that lectins can have additional structural requirements for binding ( 24&locID 146). The four lectins that showed binding by dot blot analysis were subsequently used to probe 2D separated venom proteins (Fig. 6, A D). Incubation with ConA, which is specific for glucose and mannose, revealed the presence of multiple binding proteins 384 Molecular & Cellular Proteomics 5.2
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text:
As a young boy I LOVED to play Haunted House on my Atari 2600. That is why I was very excited when this press release land in my email this morning.
New York, NY – July 14, 2010 – Enter the Haunted House, if you dare. Atari's classic groundbreaking game has returned with more chills, cool new unlockables and plenty of spooky new enemies. The terrifying fun that lies within Graves Mansion is back as Atari, one of the world's most recognized publishers and producers of interactive entertainment, scares up its legendary classic, Haunted House — re-imagined for a new generation of gamers in celebration of the title's 30th anniversary. Haunted House will take control of Wii®, Xbox LIVE® Arcade for the Xbox 360® video game and entertainment system from Microsoft, and Windows PC Download this fall, with suspense filled challenges, new chilling environments and more frightful enemies than ever before.
Haunted House pays homage to the classic favorite as players fight for their lives in Graves Mansion as the grandchildren of the original character that disappeared without a trace in the evil mansion over 30 years ago. Chased by pesky poltergeists, haunted by scary skeletons and exhausted from battles with creepy creatures, players are in a goosebump-educing fight to explore the dark and creepy depths of Graves Mansion in search of the four lost pieces of the magical urn. The player's goal is to reassemble the urn and defend their family's legacy with the aid of mystical tomes, powerful light sources and multiple keys.
"The glowing eyes and mysterious urn of Haunted House still resonates with gamers who made the original franchise a blockbuster hit," says Jim Wilson, President of Atari. "Atari looks forward to introducing a new generation of gamers to the fun and suspense of Graves Mansion and bringing families together with the magic that is Haunted House."
The pick up-and-play arcade-style adventure allows players to jump right in and begin battling for their family's legacy and explore the dark and mysterious levels. An eerie, 3D rich environment of towers, basements and catacombs draws players into the haunted world and cursed story. Find hidden treasures, solve puzzles, fight bosses, and navigate through the spooky mansion and multiple levels on the way to a showdown with Zachary Graves.
For more information, please log onto www.atari.com or www.atarihauntedhouse.com
Atari group is a global creator, producer and publisher of interactive entertainment for key platforms, including online (casual, social, MMO), PC, consoles from Microsoft, Nintendo and Sony, and advanced smart phones (i.e. iPhone, Android and RIM devices).
Atari benefits from the strength of its worldwide brand and its extensive catalogue of iconic game franchises (Asteroids®, Centipede®, Missile Command®, Lunar Lander®), original owned franchises (Test Drive®, Backyard Sports®, Deer Hunter®, Champions Online) and third party franchises (Ghostbusters®, Rollercoaster Tycoon®, Dungeons and Dragons®, Star Trek).
To play games and get other information please visit www.atari.com
© 2010 Atari Europe SASU. All rights reserved. Atari word mark and logo are trademarks owned by Atari Interactive, Inc.
ImaginEngine is a leading studio in the development of quality children's titles and casual title software. The ImaginEngine team is known for its award-winning, commercially successful console, handheld, PC and online games. ImaginEngine specializes in creating entertaining family-friendly experiences for kids and adults to enjoy. The studio has shipped nearly 200 titles across major platforms. ImaginEngine is a Foundation 9 Entertainment studio, and is located in Framingham, MA. For more information please visit: www.imaginengine.com.
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subset: commoncrawl


text:
Hello, everybody! Are you busy getting creative with it all? I hope so!
A fan on Facebook recently asked me what periodicals I read to get my design inspiration. After I wrote a virtual book as an answer I realized, Whoa! Somebody else might like to know. Plus, I'd love to teach you about an amazing convenience that you can do with your computer called Copy. And another one called Paste. Use the first command to put all your stuff onto a "virtual" clipboard. That was the "copy". Then "paste" that stuff onto your new place! You're welcome!
What follows is my response.
Oops, sorry [Facebook fan]! Your question was for me and I just realized that. I'm going to take a moment also to thank you for saying "periodicals" instead of "magazines". It is important to be sensitive in this time of intense gun control debate.
When I first started designing graphic design, I followed the edict of every design professornal (professor + professional). "Don't look at anything." That is such great advice because young designers—or in my case, youngish designers—run the risk of copying everything we see and not making any decisions on our own. So, the first 3 months of my graphic design career was spent in the bathroom and bedroom where I covered or removed anything that had been designed. The only exception being the instructional books to my design software. I turned off the internet, stowed away my Palm Centro (that was so hard!), covered the windows to avoid seeing the #5 bus with it's casino ad and the #3 bus' pro-breast feeding Ad Council campaign. I then entered the period I now call the "Terry Thinks He'll Periwinkle Era." The experience was remarkable! I spent nearly every waking moment learning how Terry Cloff designs. NOT how a cereal box designer designs. NOT how a Newsweek designer designs. How TERRY CLOFF, future owner of Successful Power Design, designs. When I came out the other side, two things had been accomplished.
So I took a trip to Kroger and just went wild.
One might assume I went straight for the "dude's" magazines. Not THOSE kind, Kroger is a family store. I mean the rags that meat-head, techie, hipster business men buy like The Economist. Not for me. I have culture and an open mind. I knew that a good designer (after looking at nothing for a self-imposed period of time) gets inspiration from everything. So I grabbed The Economist anyway. Plus that month's issue of National Geographic (classic!), WIRED, Cowboys & Indians, Handgunner, Martha Stewart's thing, Oprah's thing, Rachael Ray's thing, and something called Fantasy, which had something to do with football.
All good stuff. But I still lacked a sense of connectedness with my new career and peers. I needed to know what other designers were talking about when they would "talk amongst themselves", as a famous manish lady talk-show host was fond of saying. Once my internet was restored (internet companies are like libraries, as I see it. At libraries they prefer you don't reshelve your book when you're done because you'll probably do it wrong. AT&T prefers that you just don't pay if you wish to discontinue service because, I assume turning off the internet is fairly complicated and I don't want to lose it for anybody else)…Let me start over again. Once my internet was restored I did some research, you guessed it, online. And shiver-me-timbers there are so. many. periodicals. about. graphic. design. out. there. let. me. tell. you!
My favorite so far is one they call HOW. I haven't read it yet, but I should be receiving a complimentary copy soon. There is another one called WHY, I think. I did order a box of old copies of Communication Arts of Craig's List. But don't tell the guy I bought them from, I think they're actually books. Like a periodical would ever cost twenty-four dollars. He also said he'd throw in a few copies of a few graphic design magazines about watercolor painting. Nice guy but as the saying goes I think he fell off a rocking chair.
Another promising looking publication is called Print, but I don't think I'll subscribe since everybody knows print is dead.
Sadly, I don't have the money to subscribe to much that I found online and Kroger doesn't carry a single one.
I really thank you for asking such a good question. Do you have any recommendations?


text:
The Bay
posted in: Concept Art, Matte Painting | 0
Hey guys! Here another matte painting following the waterfall :). Foggy and atmospheric environment that i've done with many references and movies in my mind :). Let me know what you think guys. You can find a better resolution here: … Continued


text:
Friend's Email:
Subject: I have found a DVD that I think you would enjoy
Two-Disc Blu-ray/DVD Combo
Actors: Noah Lindsey Cyrus, Frankie Jonas, Cate Blanchett, Liam Neeson, Tina Fey
Director: Hayao Miyazaki
Genres: Kids & Family, Anime & Manga
G 2010 1hr 43min
Welcome to a world where anything is possible! Academy Award® winning director Hayao Miyazaki (2002, Best Animated Feature, Spirited Away) and legendary filmmaker John Lasseter together with Disney bring to life a heartwar... more »
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Movie Reviews
Even my son with an Autism diagnosis would watch this!
Nessa | 09/08/2010
(5 out of 5 stars)
"My son 6, with an autism diagnosis is not usually interested in movies. He would watch this one. Two thumbs up."
Great movie!
Goodneighbor | Williamson, NY United States | 09/12/2010
(5 out of 5 stars)
"This is a great kids movie. The animation is wonderful, the story is heartwarming and their are some very nice messages about social responsibilities..."
Another great Miyazaki piece
D. Obregon | 09/18/2010
(5 out of 5 stars)
"The transfer looks good and the movie is classic Miyazaki. It's enjoyable by all ages and is great to add to the collection."


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Marc Ecko Square Whisky Red Man Made Hobo Bag
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Measurements: 12"L x 20"W x 6"H
Price: Sold for: $36.50
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Square Whisky Red Man Made Hobo Bag
Measurements: 12"L x 20"W x 6"H
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Marc Ecko Square Whisky Red Man Made Hobo Bag
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Whisky Red Square Hobo by Marc Ecko new with tags
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MICK McCarthy today admitted he understood fans' frustrations as Wolves limped over the finishing line against Barnsley - but warned they didn't help his team.
McCarthy stormed off down the tunnel after final whistle, returning only to shake hands with Barnsley boss Simon Davey.
He had previously turned and shouted at the Billy Wright Stand as Wolves headed into the closing stages only a goal ahead despite dominating the game.
While admitting he under-stood the moans and groans as Wolves tried to keep the ball, McCarthy is hoping for a change of mentality that would spell more positive support should similar events happen again.
"I thought we were different class in the first half but unfortunately everyone remembers the last 15 minutes when we were defending and were forced to hang on," he said.
"Everyone gets frustrated and I've got frustrated at everyone getting frustrated.
"I was 'bring to the boil and serve' at the end, I have to be honest. It was difficult but let me tell you, it's three points so sod it, that's all I view it as."
McCarthy continued: "I have to say the fans are wonderful with me, I can't express my thanks enough, but at times like that, when we are struggling, that's when we need a gee-up.
"Like I say, I do understand it and am not having a pop. But the 'oohs' and the 'aahs' and one or two boos doesn't help, it doesn't make me feel any better.
"I don't know how you change that mentality but if when we got that corner we're raising the roof with 'Hi Ho Wolverhampton' it might be a lot easier than frustration.
"It could all have gone a bit pears at the end and a bit of noise in a positive manner could have helped."
McCarthy, meanwhile, now faces another fortnight's break shorn of a sizeable chunk of his squad on international duty.
Michael Kightly will be assessed to see if he can join up with the England Under-21 squad due to groin and ham-string injuries while Andy Keogh is booked in for a minor hernia operation on Wednesday.


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- September 14, 1920 - January 3, 2013
- Lenoir, North Carolina
of Bonnie's Passing
Share This Obituary
Make a Memorial Donation
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Bonnie was born on September 14, 1920 and passed away on Thursday, January 3, 2013.
Bonnie was a resident of Lenoir, North Carolina at the time of passing.
Bonnie was married to Lawrence.
Memorial services will be held Sunday, January 6, 2013 at 4:00 p.m. at Greer-McElveen Funeral Home Chapel conducted by Rev. Steve Butler and Pastor Ken Mashburn.
The family will receive friends from 3:00 to 4:00 p.m. prior to the service at the funeral home.
Memorial contributions may be made to: Setzer Creek Baptist Church, 3163 Warrior Rd., Lenoir, NC 28645 or Caldwell Hospice and Palliative Care, 902 Kirkwood St., Lenoir, NC 28645.


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Zinedine Zidane believes Gareth Bale makes Real Madrid a better team, and is hoping he is fit enough for the final.
The Wales international is in a race to get fit enough for the final on June 3, which will be played in his hometown of Cardiff.
Bale has been out of commission since April, but is back on the sidelines and could be fit for the final.
"He's recovering and there's time for him to be ready for the final," Zidane told Madrid's official website.
"I hope he's ready before and I hope he can be with us more than anything. We're a better team when he plays."


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Fleury makes 23 saves, Penguins win Game 5by Wes Crosby
PITTSBURGH -- Marc-Andre Fleury temporarily silenced his critics.
Fleury's 23 saves carried the Pittsburgh Penguins to a 3-1 win against the Columbus Blue Jackets in Game 5 of their Eastern Conference First Round series Saturday at Consol Energy Center.
The Penguins hold a 3-2 lead in the best-of-7 and will attempt to eliminate Columbus from the Stanley Cup Playoffs in Game 6 at Nationwide Arena on Monday (7 p.m. ET; CBC, RDS2, ROOT, FS-O).
After falling behind 1-0 midway through the first period, Fleury made 15 stops through the second and third to seal Pittsburgh's victory. Fleury faced criticism following a 4-3 overtime loss in Game 4, when he allowed two avoidable goals, one with less than 30 seconds remaining in regulation and another in overtime.
The crowd chanted Fleury's name at the beginning of the game, and at the end.
"I don't think it was about bouncing back," Fleury said. "[The chants were] a good boost of confidence. You get a little bit of goose bumps when you're in there. It was a good feeling."
Forward Jussi Jokinen scored the game-winning goal 6:16 into the third period. Lee Stempniak carried the puck through the neutral zone and wristed a shot on Columbus goalie Sergei Bobrovsky, who bobbled it before Brandon Sutter tipped it back on net. The puck slid out to Jokinen, who buried a backhand behind Bobrovsky to put Pittsburgh ahead 2-1 with his third goal of the playoffs.
Defenseman Kris Letang scored an empty-net goal with 1:01 left.
The Blue Jackets echoed throughout their locker room that they expect to rebound Monday.
"It's that time of year where you need to have a short memory," Columbus forward Brandon Dubinsky said. "I think everybody knows in here that we need to play better. We talk about that resiliency and resolve in here, and I believe in this room and I think the room believes in itself that we'll come back better and stronger."
Bobrovsky might have had his most impressive performance of the postseason. After allowing at least three goals in each of the first four games, Bobrovsky made 48 saves. Teammate Ryan Johansen credited Bobrovsky with giving the Blue Jackets a chance to win while expressing frustration at their inability to make Fleury work.
"[Sergei] definitely stood tall and kept us in there all game," Johansen said. "He really did his job tonight. We'll go over some tape and stuff, but obviously our forecheck, creating turnovers and things like that. I thought we didn't do that nearly as good as we need to, and we only had one power play as well.
"[We] just have to find ways to get pucks to Fleury. Obviously, there was a lot of talk going around that he's struggling, so we didn't test him nearly as much as we should."
Pittsburgh controlled the second period, outshooting the Blue Jackets 21-8, and tied the game with a power-play goal from Chris Kunitz.
After Sidney Crosby sent a shot through Kunitz's legs on goal, Bobrovsky lost sight of it and got turned around in his crease. Kunitz turned and absorbed a collision with Blue Jackets defenseman David Savard while backhanding a shot past Bobrovsky 7:42 into the second.
It was Kunitz's second goal of the playoffs and Crosby's fifth assist. Crosby failed to score a goal for his 10th consecutive playoff game dating to Game 4 of the Eastern Conference Semifinals against the Ottawa Senators last postseason.
Pittsburgh attempted to spark its two primary stars by putting Crosby and Evgeni Malkin, who also has not scored a goal this postseason, on its top line with Kunitz. The adjustment led to little production, particularly during the first period, when Malkin committed three turnovers and Crosby recorded two shots.
"I thought we generated some good chances," Crosby said. "I didn't expect [to be paired with Malkin] as much as it was. It was pretty regular, but I think with that being said, when everybody's playing well, you don't want to change too much.
"I think that's just a credit to everyone, that we all played a pretty strong game."
Penguins coach Dan Bylsma said Crosby and Malkin wanted to play together and he felt the adjustment paid dividends throughout Pittsburgh's lineup.
"I thought Brandon Sutter playing between Jussi and [James Neal], he was real strong in this game and you saw his speed through the neutral zone. He almost broke in a couple times," Bylsma said. "I think we went into the game with a good idea we were going to see that on numerous occasions, and I think the players going in knew it was going to happen."
The Penguins carried the majority of the play in the first period but trailed 1-0. With Marcel Goc in the penalty box for slashing, Blue Jackets forward Boone Jenner scored his first playoff goal with five seconds left on the power play.
Jenner sent a shot from the right side of the net into Pittsburgh's crease. The puck ricocheted off Penguins forward Joe Vitale in front and back to Fleury, who was knocked over by Pittsburgh defenseman Olli Maatta. Fleury reached to cover the puck with his glove, but it slipped to Jenner, who backhanded a shot past the downed goalie with 7:05 remaining.
"I thought the first period was probably our best period," Blue Jackets coach Todd Richards said. "I thought they took it to another level in the second and they started winning battles. We spent too much time in our own zone and we didn't have much of a forecheck.
"[Bylsma] challenged them and they responded. Now we have to do the same thing on Monday."


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Printed Paper Carrier Bags Can Help Improve Marketing and Establish Brand
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Any high street retailer selling needs printed paper carrier bags to establish brand and improve on marketing. There are physical goods which can be given away to potential customers to establish your brand. Many businesses overlook this opportunity simply because … Continued
Powerful Tips for Printed Carrier Bags You Can Use Today
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The Advantages of Printed Carrier Bags Although printed carrier bags may have different sizes, gauge and thickness, it is important to note the buying tips for printed carrier bags. Ideally you want to buy a carrier bag with either brown … Continued
Welcome to Economy Packaging!
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Welcome to Economy Packaging – home of all packaging products.


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Moore Wallace Announces Results: $40M Earnings on $882M Sales
Friday, February 06, 2004
Press release from the issuing companyMISSISSAUGA, Ontario & NEW YORK--Feb. 5, 2004-- Moore Wallace Incorporated today reported financial results for its fourth quarter and fiscal year ended December 31, 2003. Fourth quarter sales totaled $882.7 million, with GAAP (Canadian Generally Accepted Accounting Principles) net earnings of $40.1 million, or $0.25 per diluted share for the fourth quarter. (All dollar amounts discussed in this press release are in U.S. currency.) The Company believes that certain non-GAAP measures, when presented in conjunction with comparable GAAP measures, are useful because that information, which excludes acquisition-related charges, restructuring and restructuring-related charges, and gains on asset disposals, is an appropriate measure for evaluating the Company's operating performance. In addition, the Company used an effective pro forma income tax rate of 32% in calculating its non-GAAP earnings. Internally, the Company uses this non-GAAP information as an important indicator of business performance, and management's effectiveness is evaluated with specific reference to these indicators. A reconciliation of GAAP net earnings to non-GAAP net earnings for these adjustments is presented in the tables below. These measures should be considered in addition to, not a substitute for, or superior to, measures of financial performance prepared in accordance with GAAP. On May 15, 2003, the Company merged with Wallace Computer Services, Inc., a leading provider of comprehensive print management solutions. The differences in the operating results of the Company in 2003 versus 2002 are primarily due to this merger. Non-GAAP net earnings for the fourth quarter totaled $54.7 million, or $0.34 per diluted share. Non-GAAP net earnings exclude acquisition, restructuring and restructuring-related charges of $14.6 million, inclusive of the pro forma effective tax adjustment. GAAP income from operations for the fourth quarter was $84.5 million, or 9.6% of sales. These results were unfavorably affected by acquisition-related items and restructuring actions taken as a result of the merger. Non-GAAP income from operations was $95.4 million, or 10.8% of sales. Fiscal Year Results Full year 2003 sales totaled $2.87 billion, with GAAP net earnings of $114.2 million, or $0.81 per diluted share for the full year. Non-GAAP net earnings for the 2003 fiscal year were $143.8 million, or $1.01 per diluted share. Non-GAAP net earnings exclude acquisition, restructuring and restructuring-related charges, and gains on asset disposals of $29.6 million, inclusive of the pro forma effective tax adjustment. GAAP income from operations was $179.9 million, or 6.3% of sales. These results were unfavorably affected by acquisition-related items and restructuring actions taken as a result of the merger. Non-GAAP income from operations was $257.4 million, or 9.0% of sales. Mark A. Angelson, Moore Wallace's Chief Executive Officer, said, "We are pleased with the strength of our year-end performance, exceeding our targets for earnings and cash flow and stabilizing sales. We delivered consistently strong financial and operating results, notwithstanding economic pressures and the disruption from the integration. Our performance demonstrates yet again the strength of our business strategy and the ability of our management team." On November 8, 2003, Moore Wallace and RR Donnelley entered into a definitive agreement to combine the two companies. The transaction is expected to close at the end of February 2004. Mr. Angelson said, "The Moore-Wallace integration is virtually complete and the integration planning process with RR Donnelley is under way. We look forward to our combination with RR Donnelley and the opportunities it will provide for our new organization, our customers, our employees and our investors." Business Overview In addition to labels and traditional business forms, the Company's Forms & Labels business includes supplies and print fulfillment. The segment continued to face macro-economic factors and disruption from shifting work, personnel and presses. Although customer retention was strong, demand remained soft in the forms business, particularly for continuous multi-part forms, which has been impacted by electronic media and technological substitution and pricing pressure. The Company's Commercial Print business includes commercial print, direct mail and warehouse inventory management products. In the fourth quarter, commercial print benefited from increased activity from key customers in the financial services and pharmaceutical sectors and from successful cross-selling efforts. The direct mail business also benefited from cross-selling efforts, as well as the newly enhanced ability to operate more efficiently across a wider geographic platform. The Outsourcing business is comprised of the Company's customized, high-volume, variably imaged statement mailings and electronic statements and associated database services. Growth in sales and profitability continues to be driven by increased activity from both new and current customers, particularly in the telecommunications and the financial services sectors. On December 31, 2003, the Company completed the acquisition of Payment Processing Solutions, Inc. ("PPS"), a leading processor of printed customer statements, principally serving the mortgage lender industry. The addition of PPS to the Outsourcing business advanced the Company's previously announced strategy to make relatively small acquisitions in higher growth, higher margin businesses that expand both its product platform and its customer base. Asset Management The Company continued to enhance its balance sheet by generating strong cash flow during the quarter, lowering leverage ratios and reducing its net debt position to $814.8 million from $872.6 million at the end of September. (Total debt was $906.7 million on December 31, 2003 and $923.3 million on September 30, 2003.) (1)
Industry News
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You. Are. Not. Right.
you are ridiculous and i love you.
You're mental.
Words fail me…
Less time at the Gap Outlet and more time CLEANING YOUR ROOM!!! My poor kids… what they must be learning at school.
I have two things: I love the chaos in the background…boxes…etc.
The second, WHEN ARE YOU COMING TO VISIT and make me laugh and pee my pants?
i just want to say that i enjoy video blogs…maybe i will start doing them. they just seem more fun for some reason.
you should do a video blog explaining these pants…just so us boys can understand their amazing-ness a little better…..just saying.
you crack me up!! i am so happy you have found your trouser jean!
Big Mama would be proud.
What in the…beep beep bo beep?!
Jesus said "Come to the water, stand by My side and I will give you Trouser Jeans". Serious, He said that , I swear! Well not swear but I promise in the most holy of ways. He sent the Holy Spirit and….Trouser Jeans.
Jesus does love you Annie. SEE!
I expect to see these at church tomorrow… but you are a psycho.
You have made me laugh so hard I cried.
Thank you.
I almost split my trouser jeans watching this anya!!!
So cute… = )
Get a sneak peek of Annie's latest book! Click the link to download two free chapters of Let's All Be Brave.
© 2014 Annie f. downs. site by Byron Fanning + will smith.


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MANCHESTER CITY supporters have been caught on video allegedly wrecking the toilets at Old Trafford during Manchester United's derby victory on Wednesday night.
The fans are filmed chanting "City, City, City" while they rip sinks from the wall and take out their frustration as the action rages out on the pitch.
In the video, that looks to have been filmed on a mobile phone, several men launch kicks and stamps at the water basins, which smash on the ground, reports the Daily Mirror.
Juan Mata scored the winner in the 1-0 triumph to settle Jose Mourinho and Pep Guardiola's second meeting since both took jobs in the Premier League.
But footage that emerged after the game has cast a further shadow over football on a night when
Fans broke through segregation and launched projectiles during the EFL Cup tie, prompting further questions over the suitability of the Hammers' new ground to host high-risk football matches.
The FA and EFL have launched an investigation into that incident.
Such trouble follows months of issues at West Ham and that blighted the first Manchester derby of the season.
Supporters were recorded brawling in the streets after Guardiola's men in mid-September.
The images of the destruction at Old Trafford harked back to earlier this season when Rangers fans ripped apart the facilities at Celtic Park after a Scottish Premiership clash.
As the Scottish champions , Gers supporters were demolishing the roof and toilet cubicles in the away end at Parkhead.
Then before the second Old Firm clash of the season last weekend, an 11-year-old boy was rushed to hospital after he was struck on the head with a bottle outside Hampden.
The young fan - who was supporting Rangers - is believed to have been walking along Aikenhead Road with his dad when he was he was injured, around 15 minutes before the League Cup semi-final kicked off.


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IRVINE, CA – Faris Lee Investments, the nation's largest retail-specialized investment advisory firm, has completed the $10.8 million sale of Willow Run Shopping Center located at 12900-12910 N. Zuni Street in Westminster, CO, a northwest suburb of Denver. Built in 2000 and situated on nearly 11 acres, the property was 80 percent leased at the close of escrow and includes grocery store anchor tenant, Safeway, as well as Allstate, Subway and neighborhood retailers. Other outlying tenants at the center that weren't part of the sale included JPMorgan Chase, Conoco Philips, and McDonalds.
Richard Chichester, Jeff Conover, and Tom Chichester represented the seller, TNP SRT Willow Run, LLC from San Mateo, Calif. Shaun Riley of Faris Lee Investments represented the buyer, Denver-based Gart Investment Company. The closing cap rate on the property was 6.56 percent.
With strong surrounding demographics, a well-designed site plan, success of other nearby retail centers, and a good physical appearance with minimal deferred maintenance, Faris Lee's market analysis of the property identified that the missing ingredient in turning the center around was a proactive owner and property manager with experience in the Colorado market.
"Faris Lee's marketing strategy was to target local Colorado-based investors/owners who understood the immediate area. With a history of ownership in the Colorado market, Gart Investment Company was at the top of our list of investors we believed would see the value in the asset and proactively utilize its tenant and management relationships to fully maximize the investment," said Conover.
"Willow Run offered the buyer a property well below replacement cost at a low price-per-square-foot of $118," added Riley. "Additionally, the buyer appreciated the upside opportunity through leasing the 20 percent vacancy, creating potential for additional cash flow and added value."
Willow Run Shopping Center is strategically located at the signalized, hard corner intersection of N. Zuni Street and W. 128thAvenue. There are more than 245,000 consumers and 67,500 daytime employees within a three-mile radius of the property. It is also near Home Depot, Kohl's, Staples and Petco.
Faris Lee Investments is a leading national real estate investment advisory firm, comprised of three (3) integrated business platforms; Faris Lee Investment Banking, Faris Lee Capital and Faris Lee Advisors. The company's high-level retail investment advisors and sophisticated financing experts develop forward-thinking strategies for all retail acquisitions and dispositions and guide clients through complex transactions. Cumulatively, Faris Lee's leadership has been involved in more than $20 billion of transactions. Faris Lee's senior advisors are skilled in disposition and acquisition strategies, asset positioning and valuation, marketing, debt and equity structuring, strategic workout and REO solutions and execution. For more information, please visit www.farislee.com.


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VTT Technical Research Centre of Finland is a contract research organisation involved in many international assignments. With more than 2800 employees, VTT provides a wide range of technology and applied research services for its clients, private companies, institutions and the public sector. Through creating and applying technology, VTT actively increases the competitiveness of industry and other business sectors, and thus increases the welfare of society.
Research and product development is carried out in materials, production technologies and components for companies in the fields of telecommunication, semiconductor, sensor, and information industries. VTT supports the competitiveness and product commercialisation of its clients by innovations and creation of knowledge based on its core competences. To benefit its clients, VTT also provides trial production lines as well as maintains first rate equipment. VTT creates user friendly applications and develops the required service platforms.


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About Us
HaurTyi was established in 1995. We expanded our business to the overseas markets
starting from 2003 such as the USA, Western Europe, Dubai, Australia, New Zealand
and Japan.
*Printing with soy ink and never use industrial waste paper materials.
*All paper bag converting machines are made in Japan.
*ISO9001:2008, ISO14001:2004, FSC-COC certification.
*Taiwan eco products registration of Green Trade Project Office, MOEA, Taiwan


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Summit 2016 Amsterdam
We are pleased to announce the ShippersForum Summit 2016 to be held in Amsterdam on 21 November 2016.
This is the first summit and we are very excited to have the privilege of welcoming industry leaders that are involved in the Project Freight and Heavy Lift industry and that are in need of reflecting on the past year's events in the industry as well as a look forward to 2017 and beyond.
Topics on the agenda of discussions are related to;
- Capacity in the market
- Price development
- Service development
- Technology development and how far is this going
- Common issue to be discussed
- Common solutions to be shared
The summit will be monitored by a lawyer in order to ensure compliance with national and international laws. The Chatham Rules apply to all ShippersForum Summits. This means that when a meeting or summit, or part of that, is held under the Chatham House Rule, the participants are free to use the information received, but neither the identity nor the affiliation of the speaker(s), not that of any other participant, may be revealed.
We would like to refer to the ShippersForum "Competition Law Compliance Guideline".


text: Hello, I am a designer mainly but also develop on the side. I was an avid domainer(buying/selling domain names) so I figured I might as well do something with my domain knowledge. If anyone needs help with logos or banners I'd be happy to help and again if you have any questions on the domain trade I can assist as well. Looking forward to becoming a long-standing member here. -TKGB


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This attractive four bedroom detached property is situated in a highly sought after area in the heart of Aldwick.
The property, in Craigweil Lane, has provided an exceptional family home for its current residents for almost 20 years.
Over this time, the property has been modernised and improved to suit their needs.
As soon as you step over the threshold, you will begin to appreciate the space and quality feel of this beautiful home; a large L-shaped, bay fronted sitting room has a feature stone fireplace with double doors leading to the extended dining room which overlooks the garden.
There is a superb fitted kitchen/breakfast room including a comprehensive range of floor and wall units, contrasting wooden worktops and matching integrated breakfast bar, kick board runway lighting, plus integrated appliances.
There is a very useful ground floor WC and ample storage in the hallway.
On the first floor, the beautifully appointed master bedroom has a range of built-in furniture.
There are three further bedrooms that are all of generous proportions and a superb, fully tiled family bathroom.
The house sits nicely on its plot with ample driveway parking to the front and a detached garage.
Behind the garage is a very useful summer house that could serve numerous purposes.
To the rear is a large garden that is mainly laid to lawn with patio areas and mature borders.
Guide Price £525,000 Freehold.
For more information, or to arrange a viewing, contact Henry Adams, 25 High Street, Bognor Regis. Telephone 01243 842123.