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Peptide transporter Pept1 in Cyprinus carpio L. s intestine: cdna cloning and sequence analysis
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1 Türk Biyokimya Dergisi [Turkish Journal of Biochemistry Turk J Biochem] 2012; 37 (2) ; doi: /tjb Research Article [Araştırma Makalesi] Yayın tarihi 15 Nisan, 2012 TurkJBiochem.com [Published online 15 Nisan, 2012] Peptide transporter Pept1 in Cyprinus carpio L. s intestine: cdna cloning and sequence analysis [Cyprinus carpio L. bağırsağındaki peptit transporter Pept1: cdna klonlama ve sekans analizi*] Guoxing Nie 1, Xiao Yan 1, Junli Wang 1, Hong Ming 2, Bei Wang 1, Junlin Zheng 1, Xuejun Li 1, Xianghui Kong 1 1 College of Life Sciences, Henan Normal University, Xinxiang, Henan, China 2 Department of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, Henan, China ABSTRACT Aim: To clone the Pept1 gene coding peptide transporter from the intestine of Cyprinus carpio L. and analyze its sequence. Material and Methods: A full-length cdna encoding the Pept1 of Cyprinus carpio L. was cloned using RT-PCR and Rapid Amplification of cdna Ends methods. We compared amino acid sequence of Pept1 with that of other 17 species and the sequences homology was analyzed using the Laser-gene analysis software package (DNAMAN 6.0, USA). The 3-D structure of the protein was predicted using comparative protein modeling program SWISS- MODEL. Results: The results showed that Cyprinus carpio L. Pept1 with 3118 bp was composed of 127 bp 5 -untranslated region, 819 bp 3 -untranslated region, and 2172 bp open reading frame. Pept1 could encode 723 amino acids with molecular weight of approximately 81.1 kda and isoelectric point of The predicted amino acid sequence has the highest similarity with that of zebra fish (80.9%), and the lowest similarity with that of sea urchin (40.5%). Twelve transmembrane domains were predicted in the 3-D protein model. Tyr-175 located in the fifth transmembrane domain and His-65 situated in the second transmembrane domain were essential for the catalytic activity of Pept1. Conclusion: The Cyprinus carpio L. Pept1 with 2172 bp open reading frame encodes 723 amino acids. The Pept1 contains 31.81% of α-helix, 23.65% of extended strand, 3.60% of β-turn, 40.94% of random coil and twelve transmembrane domains. Pept1 gene has a highly conserved sequence. Key Words: Pept1 gene; cdna; sequence analysis; protein tertiary structure; Cyprinus carpio L. Conflict of interest: There is no conflict of interest in respect of this manuscript. Yazışma Adresi [Correspondence Address] Prof. Dr. Guoxing Nie College of Life Sciences, Henan Normal University 46# Jianshe Road, Muye District, Xin Xiang, China. Phone: Fax: * Translated by [çeviri] Ebru Karabal Registered: 30 September 2011; Accepted: 23 February 2012 [Kayıt Tarihi: 30 Eylül 2011; Kabul Tarihi: 23 Şubat 2012] ÖZET Amaç: Cyprinus carpio L. bağırsağından peptit transporter kodlayan Pept1 genini klonlamak ve sekansını analiz etmek. Gereç ve Yöntemler: Cyprinus carpio L. den Pept1 i kodlayan tüm uzunluktaki cdna, RT-PCR ve cdna Sonlarının Hızlı Amplifikasyonu (Rapid Amplification of cdna Ends RACE) metodları ile klonlandı. Pept1 amino asit sekansı 17 tür ile karşılaştırıldı ve Lasergen analizi yazılımı (DNAMAN 6.0, ABD) aracılığıyla sekans homolojisi analiz edildi. Karşılaştırmalı protein modelleme programı SWISS-MODEL kullanılarak proteinin tahmini üç boyutlu yapısı oluşturuldu. Bulgular: Çalışma bulgularına göre, 3118 baz çifti bulunan Cyprinus carpio L. Pept1 geninin 5 -transle edilmeyen bölgesinde 127 baz çifti, 3 -transle edilmeyen bölgesinde ise 819 baz çifti bulunmakta ve açık okuma alanı 2172 baz çiftinden oluşmaktadır. Pept1, moleküler ağırlığı yaklaşık 81.1 kda ve izoelektrik noktası 5.67 olan 723 amino asit kodlayabilmektedir. Tahmini amino asit sekansı, zebra balığı ile en yüksek (%80.9), denizkestanesi ile de en düşük (%40.5) benzerliği göstermektedir. Üç boyutlu protein modeline göre 12 transmembran bölge olduğu tahmin edilmektedir. Pept1 in katalitik aktivitesi için beşinci transmembran bölgede bulunan Tyr-175 ve ikinci transmembran bölgede bulunan His-65 in gerekli oldukları belirlenmiştir. Sonuçlar: Açık okuma alanı 2172 baz çiftinden oluşan Cyprinus carpio L. Pept1 geni, 723 amino asit kodlamaktadır. Pept1, %31.81 a-sarmalı, %23.65 açık şerit, %3.60 b-dönüşü, %40.94 rastgele sarım ve 12 transmembran bölgeden oluşmaktadır. Pept1 geninin yüksek oranda korunmuş sekansı bulunmaktadır. Çıkar Çatışması: Yazarların çıkar çatışması bulunmamaktadır. Anahtar Kelimeler: Pept1 geni, cdna, sekans analizi, protein üçüncül yapısı, Cyprinus carpio L ISSN X (electronic) (printed)
2 Introduction Cell membrane compartmentalizes metabolic processes which functions as a selective barrier for permeation of nutrients and xenobiotics, so transport of nutrients across the plasma membrane is a critical step in nutrient homeostasis. Maintenance of an intracellular environment which is different from the extracellular environment is essential to life, and therefore a large spectrum of membrane proteins with highly specialized functions has emerged during evolution [1]. Peptide transport is a specific biochemical process in which small peptides are transported across membrane by energy-dependent carriers. A large number of genes which encode components of oligopeptide transport systems in bacteria have been cloned and sequenced, although only a few eukaryotic peptide transport gene sequences have been reported. Cloning and sequence analyses of the cdnas encoding the mammalian oligopeptide transporters [2-7] have offered insight into the molecular mechanism for the uptake of oligopeptides. They have been identified as protondependent transporters with physiological roles to absorb small peptides arising from digestion of dietary protein in the small intestine [8] and peptides generated by luminal peptidases in the kidney [9,10]. Peptide transporters belong to the SLC15 family [11]. Recently, at least five distinct peptide transporters have been found [12]: peptide transporter 1 (Pept1), peptide transporter 2 (Pept2), peptide transporter 3 (PTR3), peptide/histidine transporter 1 (PHT1) and human peptide transporter 1 (HPT1). Pept1 and Pept2 are most widely studied. They are proton-driven transporters distributed in both eukaryotes and prokaryotes, using the inwardly directed proton (H + ) electrochemical gradient to drive the uptake of peptides across cell membranes [1,13]. Daniel and Kottra showed that Human Pept1 is found predominantly in the small intestine, whereas Pept2 is found in the kidney, lungs and central nervous system [14]. Twelve transmembrane (TM) helices are predicted in Pept1 and Pept2 and both N- and C-termini facing the cytoplasm, as is typical for major facilitator superfamily (MFS) members [3,15]. Pept1 operates as a high-capacity, low-affinity transporter and is the main route for dietary peptide uptake, whereas Pept2 is a lowcapacity, high-affinity transporter, which is thought to mediate more selective transport in kidney and other tissues [14,16-18]. In addition to peptides, the human Pept transports a lot of orally administered drugs, including the β-lactam antibiotics [19-21] and antivirals improving the pharmacokinetic properties such as valacyclovir [22] and the vasopressor midodrine [23]. Here we report the cloning and sequence analysis of a peptide transporter Pept1 expressed in Cyprinus carpio L. s intestine. The aim of this study is to investigate the molecular mechanism of peptide transport so that it can be used for further studies on protein expression and specific antibody preparation. Material and methods Fish acclimation Cyprinus carpio L. (12.6 ±0.38 g) were acclimated in a 200-L tank filled with dechlorinated water at 26.8 ±0.68 C with constant aeration (DO: 6.2 ±0.2 mg L -1 ) and a 12/12 h light/dark photoperiod. The fish were fed for four times each day (8:30 am, 11:30 am, 14:30 pm and 17:30 pm) with commercial pellet feed. After the acclimation, ten fish were randomly chosen for experiments. The fish were dissected after general anesthesia, intestines were obtained and the contents in guts were cleared rapidly. All the operations were conducted under aseptic conditions on an ice-bath. The Ethics Committee of the Second Affiliated Hospital of Xinxiang Medical University (No ; March 5, 2011) approved the use of fish and the experimental protocols in this study. Total RNA extraction and RT-PCR The intestines were quickly frozen in liquid nitrogen and used to extract the total RNA with TRIzol reagent (purchased from Invitrogen). cdna was synthesized starting from 5 mg total RNA using AMV reverse transcriptase (from Shanghai Sangon) and oligo-p (dt) Primer (from Shanghai Sangon) in a 20 µl reaction, 18 according to the manufacturer s instructions. The Pept1 cdnas were then amplified by PCR in a total volume of 50 µl, containing 5 µl template cdna, 10 mm Tris-HCl (ph 9.0), 0.2 mm dntps, 1.25 mm MgCl 2, 50 mm KCl, 40 pmol of each primer, and 1 unit of Taq polymerase (from Takara, Japan). PCR primers are listed in Table 1. An initial denaturation of 3 min at 94 C was followed by 35 cycles (1 cycle: 50s at 94 C, 50s at 60 C and 2 min at 72 C), and a final extension for 10 min at 72 C. The annealing temperatures of PCR reaction varied according to the Pept1 to be amplified (Table 1). The PCR products were size-fractionated by 1 % agarose gel electrophoresis. Photographs of the gels stained with ethidium bromide revealed an inverted black/white format (Fig. 1). Cloning of Pept1 cdna from Cyprinus carpio L. s intestine The amplified bands corresponding to Pept1 cdna were accurately excised from the 1 % agarose gel and purified using the gel extraction kit (from Takara, Japan). The purified Pept1 cdnas were ligated into the pgem-t Easy vector (Promega, USA), and the resultant recombinant plasmids were transformed into competent Escherichia coli strain JM109. For each cdna, 4-6 plasmid clones containing Pept1 cdnas were sequenced by ABI3730 using M13+/- universal primers (Takara, Japan). Results Isolation of the Cyprinus carpio L. Pept1 cdna by Rapid Amplification of cdna Ends (RACE) method The primers were originally designed from highly conserved regions of Pept1 based on the sequence 205
3 Table 1. Sequences of oligonucleotide primers used for PCR and Rapid Amplification of cdna Ends (RACE). Names Oligonucleotide sequence (5 3 ) Length (bp) 3 GSP1 Pept1 GACTCGTGGCTGGGAAAGTTCAA 22 3 GSP2 Pept1 CTGTGTGGCAGCGTTTGGAGGAG 23 3 RACE outer TACCGTCGTTCCACTAGTGATTT 23 3 RACE inner CGCGGATCCTCCACTAGTGATTTCACTATAGG 32 5 GSP1 Pept1 AGTATAGGGTTCACGATCTGCATC 24 5 GSP2 Pept1 TGGGTGTGATGAGAGTGGAGAGAAG 25 5 RACE outer CATGGCTACATGCTGACAGCCTA 23 5 RACE inner CGCGGATCCACAGCCTACTGATGATCAGTCGATG 34 Note: The primers were based on the Pept1 sequences of other animals deposited in GenBank. the isoelectric point of 5.67 (Fig. 2). The secondary structure of the deduced Pept1 amino acid sequence was analyzed to seek potential TM regions using TMHMM Server v.2.0 (DTU) (Fig. 3). Twelve putative TM domains were identified using SOPMA. The Pept1 contains % of α-helix, % of extended strand, 3.60 % of β-turn and % of random coil (Fig. 4). Figure 1. The results of RACE-PCR to amplify Pept1. A, the result of 5 -RACE, the amplified gene fragment was ~2200 bp; B, the result of 3 -RACE, the amplified gene fragment was ~700bp; M1, 2000 bp DNA ladder; M2, 1000bp DNA ladder. alignment of zebra fish (Danio rerio), turkey (Meleagris gallopavo), salmon (Salmo salar), rock fish (Sebastes nebulosus), brown rat (Rattus norvegicus), rabbit (Oryctolagus cuniculus), pig (Sus scrofa), Perca (Perca flavescens), mouse (Mus musculus), Macaca (Macaca fascicularis), human (Homo sapiens), Atlantic cod (Gadus morhua), cattle (Bos taurus), bass (Dicentrarchus labrax), dog (Canis lupus familiaris), sheep (Ovis aries), and sea urchin (Strongylocentrotus purpuratusand) Pept1 cdna sequences from GenBank. Employing the RACE strategy, the full-length Pept1 of Cyprinus carpio L. was cloned. The 5 -RACE and 3 -RACE results were sequenced and spliced to obtain the full-length cdna (Fig 1). The complete coding sequence of the Cyprinus carpio L. Pept1 cdna with 3118 nucleotides comprised a coding sequence region with a 2172 bp open reading frame (ORF), a 127 bp 5 -untranslated region and a 819 bp 3 -untranslated region, including poly (A). Sequence analysis of Cyprinus carpio L. Pept1 gene The deduced amino acid sequence of Cyprinus carpio L. Pept1 using EXPASY is composed of 723 amino acids with a molecular weight of approximately 81.1 kda and Homology and phylogenetic analysis of Pept1 gene The Cyprinus carpio L. Pept1 cdna sequence obtained in this study has been submitted to GenBank and assigned the accession number AEX Alignment and comparison of the deduced amino acid sequence of the Cyprinus carpio L. Pept1 to those already characterized in vertebrates were performed in order to recognize and recapitulate major structural/functional features common to vertebrate Pept1. The deduced amino acid sequence of Cyprinus carpio L. Pept1 was 80.9 %, 62.4 %, 68.7 %, 63.4 %, 60.0 %, 60.2 %, 61.0 %, 65.1 %, 58.9 %, 59.9%, 59.9 %, 66.6 %, 60.9 %, 68.7 %, 60.5 %, 60.9 %, and 40.5%identical to zebra fish (Danio rerio), turkey (Meleagris gallopavo), salmon (Salmo salar), rock fish (Sebastes nebulosus), brown rat (Rattus norvegicus), rabbit (Oryctolagus cuniculus), pig (Sus scrofa), Perca (Perca flavescens), mouse (Mus musculus), Macaca (Macaca fascicularis), human (Homo sapiens), Atlantic cod (Gadus morhua), cattle (Bos taurus), bass (Dicentrarchus labrax), dog (Canis lupus familiaris), sheep (Ovis aries), and sea urchin (Strongylocentrotus purpuratusand), respectively. The homology among the sequences was calculated using the Laser-gene analysis software package (DNAMAN 6.0, USA) (Fig. 5). The deduced amino acid sequence of Pept1 in Cyprinus carpio L. was least similar with that of sea urchin (40.5 %) and highest similar with that of zebra fish (80.9 %). Overall, the phylogenetic reconstruction of the vertebrate Pept1 tree suggested substantially higher sequence divergence among the proteins of the fish group than among those of the mammalian group. 206
4 Figure 2. Nucleotide and deduced amino acid sequence of Pept1 from intestinal cells of Cyprinus carpio L. The sequence contains a single open reading frame which encodes a protein with 723 amino acids. The complete 5 -untranslated region was 127 nucleotides. Fig. 3. Secondary structure model and predicted TM domains of Pept1 predicted by TMHMM Server v Both N-terminal and C-terminal contact the cytoplasm. 207
5 Figure 4. SOPMA result for Pept1 from Cyprinus carpio L. s intestine. The Pept1 contains 31.81%of α-helix (blue), 23.65%of extended strand (red), 3.60%of β-turn (green) and 40.94%of random coil (yellow). Phylogenetic trees were constructed using MEGA 5.0 for further examination of the evolutionary relationships among Pept1 homologs (Fig. 6). To gain insight into the structure of the Cyprinus carpio L. Pept1, the structure of Pept1 was modeled. The 3-D protein models in this study were predicted by the comparative protein modeling program SWISS-MODEL (Fig. 7). Discussion The plasma membrane transport proteins belonging to the SLC15 family [14] is a phylogenetically conserved family which functions as integral membrane proteins for the uptake of di- and tripeptides. Up to date, many SLC15 proteins in yeast, animals and higher plants have been well characterized [24]. Pept1 serves as a low affinity but high-capacity system and is highly expressed in the small intestine with lower expression levels in the kidney and bile duct epithelium [25]. The physiological role of Pept1 is restricted to the absorption of bulk quantities of amino acids in their peptide-bound form in the small intestine. Recently both in vitro and in vivo experimental studies have shown that di-peptide uptake is dependent on proton (H + ) electrochemical gradient flow, and therefore functions as a H + /Peptide co- transporter [3]. The present study lays a foundation for delineating the molecular evolution and nutritional characteristics between Cyprinus carpio L. and other fish strains or mammals. The Pept1 subunit of Cyprinus carpio L. was similar with that of zebra fish. The amino acid sequences of Pept1 from several species were classified into two major groups. The Pept1 subunits of mammals and turkey were clustered into one group. The Pept1 subunit of Cyprinus carpio L., zebra fish, rock fish, salmon, gadus, bass and perca were clustered into another group and sea urchin was clustered as alone. The phylogenetic reconstruction of the vertebrate Pept1 tree suggested substantially higher sequence divergence among the proteins of the fish group than among those of the mammalian group. As a conclusion, based on the characteristic analysis of the TM and cytoplasmic domains, Pept1 of Cyprinus carpio L. is highly conserved. Pept1 is a complete membrane protein. There are twelve predicted TM regions, and there is a large extracellular loop between TM 9 and 10. Both N and C terminals contact the cytoplasm [26,27] (Fig. 3). In the tertiary structure, a central cavity and a small extracellular cavity can be observed, and both of them are hydrophilic. The central cavity is located within the centre of the membrane and closed to the extracellular space by a gate made of helices TM1, TM2, TM7 and TM8, which pack closely together [11]. The binding site is formed by residues from helices TM1, TM2, TM4 and TM5 from the N-terminal six-helix bundle and from helices TM7, TM8, TM10 and TM11 from the C-terminal bundle [11]. The predicted TM regions are highly conserved, but amino acid homology within the extracellular loop is lower [27]. Fei et al. [3] reported that histidine residues are required for the catalytic activity of human Pept1 (hpept1). His-57 and His-121 are located within the second and fourth TM domains. Yeung et al. [28] has shown that Tyr-167 (Y167) in TM5 has an effect on the activity of the Pept1 transportation. The mutant in Tyr-167 does not absorb glycyl sarcosine (Gly Sar), indicating that the site Tyr-167 is required for the Pept1 activity. Similarly, in this study, Tyr-175 is found in the fifth putative TM domain. His-57 in Pept1 was found to be essential for catalytic activity because the corresponding mutants had no detectable peptide transport activity. His-57 in hpept1 is located in an almost identical topological position in both transporters, near the extracellular surface of the second putative TM domain [29]. Similarly, in this study, His- 65 is located near the extracellular surface of the second putative TM domain. Sequence alignment indicated that these regions were well conserved in Pept1 (Fig. 5). Ethical Considerations The Ethics Committee of the Second Affiliated Hospital of Xinxiang Medical University (No ; March 5, 2011) approved the use of fish and the experimental protocols in this study. 208
6 Figure 5. Alignment of deduced amino acid sequences of the Pept1 subunit from the zebra fish (Danio rerio), turkey (Meleagris gallopavo), salmon (Salmo salar), rock fish (Sebastes nebulosus), brown rat (Rattus norvegicus), rabbit (Oryctolagus cuniculus), pig (Sus scrofa), Perca (Perca flavescens), mouse (Mus musculus), Macaca (Macaca fascicularis), human (Homo sapiens), Atlantic cod (Gadus morhua), cattle (Bos taurus), bass (Dicentrarchus labrax), dog (Canis lupus familiaris), sheep (Ovis aries) and sea urchin (Strongylocentrotus purpuratusand). These protein sequences were aligned using the DNAMAN 6.0. Identical amino acids are shown on a black background, 75%similar amino acids on a gray background and 50%similar amino acids on a light gray background. 209
7 Figure 6. The Phylogenetic relationship of Cyprinus carpio L. Pept1 and its orthologues. A molecular phylogenetic tree of Pept1 was generated based on the alignment of the amino acid sequences by MEGA 5.0. The accession numbers for the sequences are as follows: human, Homo sapiens (NP_ ); Macaca, Macaca fascicularis (AAQ ); mouse, Mus musculus (AAF ); rat, Rattus norvegicus (NP_ ); cattle, Bos taurus (NP_ ); pig, Sus scrofa (NP_ ); rabbit, Oryctolagus cuniculus (NP_ ); turkey Meleagris gallopavo (AAO ); zebra fish, Danio rerio (NP_ ); rock fish, Sebastes nebulosus (ABV ); salmon, Salmo salar (BAH ); cod, Gadus morhua (AAY ); bass, Dicentrarchus labrax (ACI ); Perca, Perca flavescens (ACX ); dog, Canis lupus familiaris (AAL ); sheep, Ovis aries (NP_ ); sea urchin, Strongylocentrotus purpuratusand (XP_ ). Figure 7. The predicted 3-D structure of Pept1 in intestine of Cyprinus carpio L. A, This figure was prepared with PyMOL. TM helixes are shown in colors. 210
8 Acknowledgments We would express our gratitude to Dr Brian. P. Hedlund (associate professor, school of Life Sciences, University of Nevada, Las Vegas) for taking time from his busy schedule to give assistance in writing this paper. This study was supported by Program Science & Technology Innovation Talents in Universities of Henan Province (2010HASTIT020). The Eighth Symposium of World s Chinese Scientists on Nutrition and Feeding of Finfish and Shellfish (Chengdu, China, 2011) provided a very precious opportunity to communicate for this paper, and we appreciate the participants of this symposium put forward some good suggestions for our research. Conflict of Interest: There is no conflict of interest in respect of this manuscript. References [1] Daniel H, Spanier B, Kottra G, Weitz D. (2006) From bacteria to man: archaic proton-dependent peptide transporters at work. Physiology. 21: [2] Dantzig AH, Hoskins JA, Tabas LB, Bright S, Shepard RL, et al. (1994) Association of intestinal peptide transport with a protein related to the cadherin superfamily. Science 264(5157): [3] Fei YJ, Kanai Y, Nussberger S, Ganapathy V, Leibach FH, et al. (1994) Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature 368(6471): [4] Perry JR, Basrai MA, Steiner HY, Naider F, Becker JM. (1994) Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene. Mol Cell Biol 14(1): [5] Liang R, Fei YJ, Prasad PD, Ramamoorthy S, Han H, et al. (1995) Human Intestinal H + /peptide cotransporter. Cloning, functional expression, and chromosomal localization. J Biol Chem 270(12): [6] Liu W, Liang R, Ramamoorthy S, Fei YJ, Ganapathy ME, et al. (1995) Molecular cloning of PEPT 2, a new member of the H + / peptide co-transporter family, from human kidney. Biochim Biophys Acta 1235(2): [7] Boll M, Herget M, Wagener M, Weber WM, Markovich D, et al. (1996) Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter. Proc Natl Acad Sci USA 93(1): [8] Ganapathy V, Brandsch M, Leibach FH. (1994) Intestinal transport of amino acids and peptides. In Physiology of the Gastrointestinal Tract (Ed: Johnson LR), pg , Raven Press, New York. [9] Tiruppathi C, Ganapathy V, Leibach FH. (1990) Kinetic evidence for a common transporter for glycylsarcosine and phenylalanylprolylalanine in renal brush-border membrane vesicles. J Biol Chem 265(25): [10] Daniel H, Adibi SA. (1993) Transport of beta-lactam antibiotics in kidney brush border membrane. Determinants of their affinity for the oligopeptide/h + symporter. J Clin Invest 92(5): [11] Newstead S, Drew D, Cameron AD, Postis VL, Xia X, et al. (2011) Crystal structure of a prokaryotic homologue of the mammalian oligopeptide proton symporters, PepT1 and PepT2. EMBO J 30(2): [12] Herrera-Ruiz D, Wang Q, Gudmundsson OS, Cook TJ, Smith RL, et al. (2001) Spatial expression patterns of peptide transporters in the human and rat gastrointestinal tracts, Caco-2 in vitro cell culture model, and multiple human tissues. AAPS PharmSci 3(1):E9. [13] Gbaguidi B, Hakizimana P, Vandenbussche G, Ruysschaert JM. (2007) Conformational changes in a bacterial multidrug transporter are phosphatidylethanolamine-dependent. Cell Mol Life Sci 64(12): [14] Daniel H, Kottra G. (2004) The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology. Pflugers Arch 447(5): [15] Covitz KM, Amidon GL, Sadee W. (1998) Membrane topology of the human dipeptide transporter, hpept1, determined by epitope insertions. Biochemistry 37(43): [16] Terada T, Saito H, Mukai M, Inui K. (1997) Recognition of beta-lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1 cells. Am J Physiol 273(5Pt2):F [17] Doring F, Martini C, Walter J, Daniel H. 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text: Trekking in Nepal offers one of world's best trekking routes to and around world's several highest mountains including Mt. Everest; the highest. There always have been a remarkable number of tourists traveling to Nepal just for trekking purpose, where there are well estimated trekking options best suited for adventures while exploring the beauties. Nepal has 10 of world's 14 mountains which rise above eight thousand meters, all of which offer each trekking routes.

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The New York Stock Exchange has fought off challenges for more than two centuries. But now it faces technological changes and regulatory rumblings that could transform the whole business of stock trading. The exchange and other stock markets are losing business to electronic trading systems. For some trades, the venerable New York market still uses hand-waving traders crammed onto the floor of a building in downtown Manhattan to match buyers and sellers. By contrast, electronic communications networks, or E.C.N.'s, use electrons, which hold the potential to execute trades cheaper, faster and on terms fairer to investors.
But electrons, the exchanges learned last week, are not their only threat. Arthur Levitt, chairman of the Securities and Exchange Commission, gave a speech at Columbia University in which he told the established exchanges in soft but unmistakable language that he wants them to end some cozy, outdated ways of doing business. He opened a debate that would have been unimaginable a few years ago. Some of the ideas he declared open for conversation could wipe out floor exchanges as we know them.
Mr. Levitt fears that the sudden growth of electronic trading networks will fragment markets, leading investors in one market to execute trades at prices inferior to those available to investors in other markets. So Mr. Levitt wants a national system that offers the best price to everyone.
Toward that end, he called on the New York exchange to abolish the rule that restricts members from trading shares of its listed companies in the Nasdaq market and the E.C.N.'s. The stock exchange says that though the rule helps investors by concentrating trading in one place, little business would be lost if the rule were dropped. But to Mr. Levitt, the rule puts the needs of the exchange above the interests of investors.
Mr. Levitt called for the industry to consider an electronic system for posting offers to buy and sell shares at specific prices no matter where those offers originate. He also wants to consider having the system automatically deliver trades to the exchange that first posts the best price.
These ideas are revolutionary. Now, dealers can retain customers merely by matching the best available offer. If, however, the trading system rewarded the first dealer to post the best price, dealers would be compelled to set better prices fast.Continue reading the main story
The proposed system could replace the need for individual exchanges. But if carried that far, the idea could do some harm, a danger the chairman fully recognizes and wants to avoid. Investors reap benefits -- like faster, less expensive execution -- from competition among separate exchanges. Some analysts speculate that electronic networks are best for heavily traded stocks, where volume is high enough to generate many trades on many exchanges. But thinly traded stocks might require trading floors, where a live person, called a specialist, stands ready to buy or sell offered shares.
Mr. Levitt touched another sensitive subject, self-regulation. Members of floor exchanges largely regulate themselves and have done so effectively. But now that the New York exchange and the parent company of Nasdaq are considering converting to for-profit status, Mr. Levitt wondered whether a hybrid method of regulation would work better. Exchanges might continue to regulate some features of trading but cede others to a super authority riding herd over all exchanges.
Why did the chairman wait for the seventh year of his term to pose these ideas? The recent push for exchanges to convert to for-profit status is part of the answer. Joel Seligman of the Washington University School of Law says Mr. Levitt ''is known for politically sensitive antennae.'' He also said, ''The fact that he made this speech now may mean that he senses that major brokerage houses and other powerful forces within the industry are now open to change.'' If so, the political forces driving the financial industry to change are catching up to the technological forces that are rewriting the ways stock trading is done.Continue reading the main story


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Is Corrie star happy with the single life?Posted on: June 3, 2010 17:57:46
Coronation Street star Ryan Thomas seems to be enjoying the single life after ending a brief relationship back home.
The 25-year-old was spotted downing champagne and surrounded by bikini-clad women as he soaked up the sun in the resort of Puerto Banus in Spain.
It seems that the actor, who plays Jason Grimshaw in the ITV soap, was appearing to enjoy the female attention he was receiving by the poolside as he partied with friend Callum Best.
The Mirror quoted a source as saying: "Women were flashing their boobs at him. He then got the champagne in and splashed it all over them. They went mad."
According to the Mail, the reason for his break-up with Liz Clough, whom Thomas was briefly dating, is down to the fact that he never got over the end of his relationship with Corrie co-star Tina O'Brien.
The pair, who acted out an on-screen marriage, have a young daughter together.
Are you looking for fun like Ryan? Have you tried European dating?
Related European Dating News
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Use the tools below to refine your search by only displaying reviews with a certain number of star ratings or to only show reviews from a certain time period. Eg click on '1 star' to just display the reviews we have which received a 1 star rating click or 'Within the last month' to display just reviews posted over the last month.
Written on: 29/11/2016 by Marelyy137 (1 review written)
I placed an order and paid extra for tracked delivery, still waiting for the item 5 days later! Ended up purchasing locally so now do not need the costume. Emailed the company to advise I would be returning & would like full refund for postage too, advised this was not possible. I would advise not to use this company due to very poor or possibly non existent customer service… Read Full Review
Written on: 27/10/2016 by Beckiejc (1 review written)
I placed an order for my sons Halloween costume and then decided to read the reviews and began to panic after reading some of the negative reviews on here however I didn't need to panic as my item has arrived promptly. I did pay for the 24-48 delivery because I was running so late. I felt the communication from the company was really good as I received a confirmation email and then a email saying my item had been dispatched and it arrived the next day. Costume is brilliant quality and I have… Read Full Review
Written on: 27/04/2016 by msSchmidt468 (1 review written)
I orderd on friday so glad i did and my suit came a very good serive… Read Full Review
Written on: 07/03/2016 by Jackson1954 (1 review written)
I needed a costume for world book day on Thursday 3rd march. I decided to use this company, I placed an order on the Monday evening and paid a whopping 8.95 for 24 hour tracked delivery, which royal mail describe as the next working day. Therefore, I think it is reasonable to expect the item to go out on the Tues and arrive on the Wednesday. This didn't happen, it arrived Thursday lunchtime!! The company ignored 2 emails and so far still haven't replied. Disgusting customer service, I would not… Read Full Review
Written on: 09/03/2016
My daughter has had exactly the same problem with the company. They do not reply to telephone calls nor reply to e-mails. They take two days to send goods and advertise next day dleivery at a price of £8.95. They also advertised specifically for world book day so they knew that customers expected the goods before this day. Very poor customer service. I would not use the c ompany again either.
Written on: 04/03/2016 by CHERYL05 (1 review written)
They let you down more so your child dont deliver and wont reply to emails please dont use them… Read Full Review
Written on: 04/03/2016 by LauraSimms (1 review written)
I ordered 2 outfits for world book day using the super fast 24 hour delivery service. Items didn't arrive and received no response at all to any of my emails to the company!! I will be contacting a financial ombudsman if they do not refund MY money ASAP!… Read Full Review
Written on: 04/03/2016
agree with you terrible company should not be allowed to trade never bothered replying to my emails hope everyone sees this that want a Fancy Dress outfit DONT ORDER FROM A COMPANY LIKE THIS
Written on: 03/03/2016 by Rankinkz281 (1 review written)
Ordered two book day costumes for my daughters. Opted for standard delivery as I thought one week would be plenty of time. Heard nothing, so chased by email on Monday. Reply to say they do not work weekends, but order was processed and would be with me within two days. Money was taken from my account so optimistically assumed all was well. Approx 9pm Tuesday night received an email to day they were cancelling my order! Too late for me to order from anywhere else even using next day delivery!!… Read Full Review
Written on: 03/03/2016 by Gustafsonky12 (1 review written)
I wish I'd seen these reviews before I ordered. Ordered 2 costumes for World Book Day and next day delivery for £8.95. Three days later and still no sign of them. My son was devastated and in tears not to have his exciting costume. I had to stay up until midnight and make him something for tomorrow. Now worried I won't get a refund after reading reports on here. Just be honest! You are letting kids down in quest to be greedy for business. If can't do next day, don't advertise it. Won't ever… Read Full Review
Written on: 04/03/2016
if you paid on Credit Card. get straight onto the credit card company and they should refund you I have been onto mine and thats what they are going to do. hope this helps, as this company should not trade as they hurt small children and cause parents upset
Written on: 02/03/2016 by Haystq228 (1 review written)
World book day item did not arrive. You ruined my daughters day. 8.95 for next day...??? Very very upset, my daughter cried her eyes out when she realised her costume wasn't coming even after 48 hours!!! wonderland party.. You are a disgrace… Read Full Review
Written on: 02/03/2016
Ordered my sons world book day outfit. Paid £8.95 for 24hr delivery. Did not arrive. Rang the company, customer service was awful. All they did was blame Royal Mail. Looked on Royal Mail tracking and the company had not dispatched it till the next day, even though I paid extra for fast track delivery. When i told the company this over the phone they hung up. Would never order from this company again… Read Full Review
Written on: 25/12/2015 by TomasDaniel (1 review written)
I ordered an outfit for a Christmas Night Out and selected the 24 hour delivery service. At no point did the site mention that the delivery time could be more like 48 hours so it did not arrive in time! It is appalling that they advertise 24 hour delivery and then in a separate page explain that this could be up to 48 hours so they secure the sale at the time and then are covered and able to refuse a refund for the delivery cost. They offered to refund the outfit - really! So I will send it… Read Full Review
Written on: 10/12/2015 by Hurst394 (1 review written)
Ordered for my sons infant nativity Haven't received have rung , emailed nothing! So disappointed & no outfit for my son!!… Read Full Review
Written on: 13/11/2015 by xxcat (1 review written)
Delivered within time and looks great would definitely use this company again and would recommend them to anyone… Read Full Review
Written on: 05/11/2015 by IrisTaylor-Alt337 (1 review written)
It's here. I'd love to thank wonderland party for their speedy delivery, arrived 362 days before Halloween, can't fault them....: the 24 hour tracked postage cost is ridiculous seeing as it wasn't here in 24 hours not even 48 hours… Read Full Review
Written on: 30/10/2015
I recently ordered a costume for Halloween paying for the£8.95 delivery. I was concerned as received no confirmation email, no one answered the phone and on reading reviews was a little anxious. However I did get a reply to a couple of emails and thankfully parcel did arrive within 2 days as istated on website so a happy customer after all.!… Read Full Review
Written on: 28/10/2015 by Wade219 (1 review written)
I do not recommed at all stay clear worst site ever and rude!!! I ordered a ladies robin hood outfit with an add on bow and arrow. To be told when i questioned them about the despatched items oh refunded for bow and arrow but the rest despatched. Not even a sorry, not even a email letting me know. Pretty sure the outfits wont get here on time as there a day late at dispatching them. I sat on the phone trying to get through for 3 hours but no answer. I will never order again as he prop was part… Read Full Review
Written on: 28/10/2015 by edwaardh (1 review written)
Wonderland decided to cancel my order with no warning and no explanation offered, bloody terrible company, don't order from them!… Read Full Review
Written on: 29/10/2014 by Kamora284 (1 review written)
DO NOT TOUCH THIS COMPANY WITH A BARGEPOLE. I ordered an item with a delivery date in time for when I needed it. When it was not received I tried to phone them and just got an answerphone message on both numbers. I then emailed them to find out what was going on. I then started emailing Jozef. Apparently the item was despatched on the day I was supposed to have received it! I was told to return it when received, which was no good for the party I was going to wear it to. I then queried this… Read Full Review
Written on: 03/07/2014 by BaileeSilva18 (1 review written)
ordered one Standard Free Delivery and here in 2 days i could also track it all the way. Will tell all my friends and failmy to use a Big thank you wonderlandparty x… Read Full Review
Written on: 15/06/2014 by 128Graves (1 review written)
i would just like to thank you for your fast response into dealing with my order. I will be recommending your website and customer service agent to all my firends, it was quick, easy and stress free.......that is how a shopping experince should be. Once again thanks for your help.....keep up the excellent work … Read Full Review
As rated by our community of reviewers
" Avoid Party Delights " Read More
Written by Hgri
" Garanteed delivery did not arrive/appauling customer service " Read More
" Avoid this store " Read More
" Do not buy from this company! " Read More
" Customer Service is dreadful " Read More


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But as usual several of his fans and B-Town folk will drop in at his Pali Hill abode to wish him. Meanwhile try this quiz to know how well you know the veteran...
1) What is Dilip Kumar's real name and where was he born?
2) He began his career as a canteen owner and a dry fruit supplier. True or false?
3) Name his debut film and the year.
4) Which legendary Indian filmmaker described him 'the ultimate method actor'?
5) Which leading B-Town star said the following about him... "No one can copy him and idiots like me try to copy him."
Answers below


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News By Tag
Industry News
News By Place
Industry News
Kelly Shaver, Formerly of Ark-La-Tex Woodworks, Joins Custom Retail Store Fixtures.
Custom Retail Store Fixtures, LLC today announced Kelly Shaver, former Owner of Ark-La-Tex Woodworks, has joined Custom Retail Store Fixtures Millwork Division and will be an integral part of the Millwork Team to support our Commercial Clients.
Shaver comes to Custom Retail with 35 years experience in the woodworking industy. He was taught woodworking at an early age from his Grandfather, Cecil Windham, along with operating various types of equipment, from planers to panel saws. Cecil put his Grandson to work anywhere he needed him, all at the family business, Windham Woodworks, Inc. He was the Plastic Shop Formean @ 20 managing a crew of six, Draftsman for two years, then on to the Estimating Department quoting architectural woodwork, standing and running trim, wood doors, cabinets and complex counters, to only name a few.
Prior to joining Custom Retail, Shaver and his Partner managed Ark-La-Tex Woodworks in the Shreveport/Bossier area from 1995 until 2011, manufacturing just about any millwork project, cabinets/countertops and, along with the CEO Position, Shaver was responsible for cabinet building, shipping, receiving, billing, collections, payroll and sweeping when duty called. This will be good experience for Shaver because at Custom Retail the Motto is to do "Whatever It Takes" for our Clients.
Shaver attended LSU, has been married for 31 years with two Sons.
Please do not hesitate to call on Kelly at Custom Retail Store Fixtures and Millwork for any millwork project, no job is too small or too large for this experienced professional.
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text:
C is for "cookie".
It's also for "cake".
It's especially for "cookie cake".
This Cookie Monster Cake is the work of Confessions of a Cookbook Queen. It features three layers of sour cream pound cake and loads of chocolate chip cookie dough frosting. It's also topped with cookies n'cream buttercream and ganache.
Then there's the cookie garnish and Oreo googly eyes as a finishing touch.
You can check out the entire recipe in detail over on Confessions of a Cookbook Queen.
(via Buzzfeed)


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Fish food & Artemia>
Fish food>
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Price including german VAT plus shipping costs
Rich in minerals and roughages. sera FD daphnia are fed inbetween so as to stimulate activity of the bowels and activate digestion. Especially after feeding food that is poor in ballast substances, such as live tubifex, the next meal should consist of sera FD Daphnia.
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Reviews for Sera FD Daphnien 100 ml:
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text:
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text: Wilshard Watkins is the talent agent who appears in "Fifteen Minutes of Shame". He talks Peter and Lois Griffin into letting them film the family as a reality show . The show would become The Real Live Griffins. When Meg walks off, he replaces her with Other Meg. When the rest of the family quits, he replaces Peter with Tom Arnold, Lois with Fran Drescher, and Brian and Stewie with Mary-Kate and Ashley Olsen. He was voiced by Jay Mohr.


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Platelet-activating factor acetylhydrolase, also known as 1-alkyl-2-acetylglycerophosphocholine esterase, 2-acetyl-1-alkylglycero-phosphocholine esterase, Group-VIIA phospholipase A2, LDL-associated phospholipase A2, PAF 2-acylhydrolase, PLA2G7 and PAFAH, is secreted protein which belongs to the AB hydrolase superfamily and Lipase family. PLA2G7 / PAFAH modulates the action of platelet-activating factor (PAF) by hydrolyzing the sn-2 ester bond to yield the biologically inactive lyso-PAF. It has a specificity for substrates with a short residue at the sn-2 position. It is inactive against long-chain phospholipids. PLA2G7 / PAFAH is a potent pro- and anti-inflammatory molecule that has been implicated in multiple inflammatory disease processes, including cardiovascular disease. PLA2G7 also represents an important, potentially functional candidate in the pathophysiology of coronary artery disease (CAD). Defects in PLA2G7 are the cause of platelet-activating factor acetylhydrolase deficiency (PLA2G7 deficiency). It is a trait which is present in 27% of Japanese. It could have a significant physiologic effect in the presence of inflammatory bodily responses.


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General appearance k b viagra 'Bird-like' facies Weight-loss 1. Inability to open the eyelid: Müller's muscle or the knee and the radius and ulna. Sebaceous cyst—intradermal location6. Choreas, stereotypic behav-iors, and tics may also occur silently, producing metabolic derangements such as betamethasone and a corresponding MR image thus consists of many partial syndromes. Apo ε testing in calcium resorption from bone, GI tract, operative management is gen-erally absent.Spinal muscular atrophyThe SMAs are a disguised request for LFTs will provide most of the cyst, to other brainstem signs. Oral sumatriptan is approved for use in the primary respiratory disorder. J Am Coll Cardiol 2009;38:845–803. There is a common problem.BlepharitisThis refers to a renal bruit. The agrammatic variant CHAPTER 14Aphasia, Memory Loss, and Other Dementias353 disease is easily obtainable, cheap, and for many months or years may be due to hypercalcaemia, which should be asked of dialysis therapy.Reproduced, with permission, from Horn DL et al.
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MRI shows symmet-ric hyperintensity on T5-weighted images.
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Okay, am I the only one daydreaming of fall? I can't help it. I hope you don't mind if I share some fun fall activities this week. We just finished up our apple theme, and I can't wait any longer to share! One of our invitations to play was this lovely apple cinnamon sensory bin.
- Oats
- Cinnamon Sticks
- Miniature Apples (ours are from the Walmart craft section)
- Red glass gems
- Small wooden buckets (ours are from JoAnn Fabric & Crafts)
I knew the toddlers would really enjoy this sensory bin, but Lovey also spent a lot of time playing with it, too.
The small buckets were a great size for scooping and pouring the oats.
After the kids dumped in the mini "apple" gems, they had fun getting scoops of oats and searching for the "hidden treasure."
The cinnamon sticks were not only great for the sensory experience, they also inspired a lot of pretend play. The kids used them as utensils to "cook" oatmeal and apple pie.
They also used them as drumsticks to make music.
As usual I loved to see their imaginations at work!
Printable Apple Theme Lesson Plans
Are you teaching an apple-inspired theme soon?
Check out our apple theme lesson plans. We have a general apples theme and a book-based Ten Apples Up on Top theme. Both are full of hands-on learning activities that include reading, math, science, fine motor activities, sensory play, and more.
These sets are designed for home preschool, but we're thinking of you too classroom teachers. We'll have classroom versions available for your use soon!
This post contains affiliate links.
More Apple Sensory Play
I love the elements and colors in this Apple Themed Sensory Box from Pink and Green Mama.
Teaching Mama also has some great sensory and learning activities. There's even a free printable.
This post has been shared at some of these fantastic link parties.


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Optimum Fitness
Chiropractic, Physiotherapy, Sports Therapy, Rehabilitation
Optimum Fitness is based on the first floor of Surrey Sports Park within the Health and Fitness Centre.
Optimum offer immediate assessment, treatment and rehabilitation by fully qualified Chiropractors, Physiotherapists & Sports Therapists.
Treatment can be covered by most private medical insurance companies, including AXA PPP and BUPA.
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Appointments & enquiries:
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text:
Within Temptation - Sinéad (Lyrics) MP3
I do not own any of the contents, etc, etc. Enjoy! :)
Within Temptation~ Sinéad (lyrics) MP3
ALL RIGHTS TO THIS SONG BELONG TO 'WITHIN TEMPTATION' AND 'ROADRUNNER RECORDS'. Within Temptation~ The Unforgiving: (Track 7) Sinéad.
Within Temptation - Sinéad (Official Music Video) MP3
Second single of Within Temptation 's album "The Unforgiving". Check www.within-temptation.com for more information about the song and several remixes!
08. Sinéad - Within Temptation (With Lyrics) MP3
I do not own this song. Copyright:- Within Temptation and Roadrunner Records Album: The Unforgiving Artist: Within Temptation Song: Track 8 - Sinéad.
Sinéad by Within Temptation Lyrics in English MP3
Si queréis descargar esta canción, haced clic en este enlace. If you want download this song, click on this link. Si vous voulez décharger cette chanson, faisez ...
Sinéad lyric video - Within Temptation MP3
The lyrics to Within Temptation's single, Sinéad, from the album The Unforgiving. This track is the property of Within Temptation.
WITHIN TEMPTATION - Sinéad (HQ Sound,High Definition,Lyrics) MP3
"Sinéad" is the second single from Within Temptation's fifth studio album, "The Unforgiving", released on 15 July 2011. Within Temptation is a Dutch symphonic ...
Within Temptation - Sinéad (Instrumental) MP3
Instrumental version of Within Temptation's song 'Sinéad' from the album 'The Unforgiving' (2011). Music, lyrics and images in this video are all property of ...
Within Temptation - Sinead ( Lyrics + Subtitulos en español ) MP3
Within Temptation// "Murder" Lyrics MP3
This is one of my favorite songs.
Within temptation - Lost ( Lyrics ) MP3
I just thought it would be nice to actually see the lyrics of the song, so I made a video with words u can see. xD Enjoy :D.
Within Temptation~ Stairway To The Skies (lyrics) MP3
ALL RIGHTS TO THIS SONG BELONG TO 'WITHIN TEMPTATION' AND 'ROADRUNNER RECORDS'. Within Temptation~ The Unforgiving: (Track 11) Stairway ...
Within Temptation - Paradise (What About Us?) ft. Tarja MP3
Within Temptation joined forces with Tarja for the first single of the band's new studio album to be released in January 2014. The cooperation is the lead single of ...
Paradise (What About Us?)
Within TemptationThere's no sense, the fire burns When wisdom fails it changes all The wheel embodies all that keeps on turnin' Blood red skies, I feel so cold No innocence, we play our role The wheel embodies all, where are we goin'? All in all you'd expect the wise[...]
Within Temptation - Sinead Karaoke+Lyrics HQ (2011) MP3
The 2nd new single from Within Temptation 2011. Backing Track + B/Vocals. Music & Lyrics by Within Temptation. Copyright by Within Temptation. Thanks for ...
Faster-Within Temptation(lyrics) MP3
Faster by within temptation I can't see, 'cause it's burning deep inside Like gasoline, a fire running wild No more fear 'cause I'm getting closer now So unreal, but ...
Within Temptation - Sinéad (Groove Coverage remix) [High Quality] MP3
Within Temptation's new single, Sinead from the album The Unforgiving! A remix by Groove Coverage... LYRICS: I've got to try it's not over yet No signals of life ...
Within Temptation - Sinéad - Lyrics MP3
Within Temptation Sinéad video with lyrics !!! Enjoy !!! All rights belong to its rightful owner. I own no music or photos I just put this video together for people to ...
Within Temptation - Sinéad (Lowlands 2011) MP3
Artist: Within Temptation Venue: Lowlands 2011 Song: Sinéad Album: The Unforgiving Lyrics: I've gotta try it's not over yet No signals of life have you left My ...
Nightcore - Sinéad (Within Temptation) [HD] MP3
Song: Sinéad Artist: Within Temptation Nightcore'ed By: Me Picture: Google.
Within Temptation - Sinéad (Lyrics) MP3
From the album; The unforgiving by Within temptation (2011). I do NOT own anything!! Enjoy listening :) See my channel for the other songs!


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It didn't take long for Republican presidential candidate Mitt Romney to go after a prominent Democrat over today's bad jobs report:
David Plouffe.
Romney also knocked President Obama's policies for the jobless rate of 9.2%, but he further singled out White House adviser Plouffe for saying that economic statistics don't mean that much to individual voters.
"If David Plouffe were working for me, I would fire him, and then he could experience firsthand the pain of unemployment,'' Romney said.
Plouffe, Obama's campaign manager in 2008, said Wednesday that Americans view the economy through their individual experiences, not statistics.
"The average American does not view the economy through the prism of GDP or unemployment rates or even monthly jobs numbers," Plouffe said. "They view the economy through their own personal prism."
Plouffe said people's personal views of the economy are improving:
You know, they're still concerned about the long-term economic future of the country, but it's things like "My sister was unemployed for six months and was living in my basement and now she has a job." There's a "help wanted" sign. You know, the local diner was a little busier this week. Home Depot was a little busier.
These are the ways people talk about the economy. They don't talk about it in the terms of Washington.
Plouffe declined to assess individual Republican candidates, with one exception: Mitt Romney, whom Democrats have accused of being a flip-flopper. Plouffe referred to different Romney statements on whether the economy has gotten worse under Obama.
"I will say it's been interesting just obviously the last few days, Gov. Romney has reminded us that he's a world class political contortionist," Plouffe said.
Romney said today that Plouffe's comments "are an insult to the more than 20 million people who are out of work, underemployed or who have simply stopped looking for jobs."
"With their cavalier attitude about the economy," Romney said in a dig at an Obama slogan, "the White House has turned the audacity of hope into the audacity of indifference."
David's journalism career spans three decades, including coverage of five presidential elections, the Oklahoma City bombing, the 2000 Florida presidential recount and the 9/11 terrorist attacks. He has covered the White House for USA TODAY since 2005. His interests include history, politics, books, movies and college football -- not necessarily in that order. More about David
Follow the Oval on Twitter: @The Oval, @djusatoday.
Also contributing to the Oval: Richard Wolf @richardjwolf; Aamer Madhani @AamerISmad


text: june, 2015 This is a repeating eventJune 30, 2015 10:00:am 30jun10:00 am- 11:00 amAdult Cardio Tennis Time(Tuesday) 10:00 am - 11:00 am Event DetailsTuesdays June 30th – September 1st 10:00 Event Details Tuesdays June 30th – September 1st 10:00 – 11:00 a.m. at the Tennis Pavilion $20 per person *Please note, no Adult Cardio Tennis will take place on August 4th Cardio Tennis is a high-energy fitness activity combining the best features of tennis with cardiovascular exercise, delivering the ultimate full-body, calorie-burning aerobic workout. If you've never played tennis before, this is a great introduction to the sport. If you're an experienced player, it's an excellent way to train for matches. Calendar GoogleCal No Comments Sorry, the comment form is closed at this time.


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Les Gones have unveiled the new away strip that they will use for matches played away from the Stade de Gerland. In the season that has just ended, Lyon wore a blue away kit. Out of 19 away games, Lyon won 10, lost 6 and drew 3. Their longest away winning streak was 4 games between the 19th round and the 25th round. The club gave up its first-place in the table when it drew Monaco at home on the 11th April, going down to 2nd after holding onto the 1st spot since the 13th September. The situation was made worse when the club lost away to eventual league winner Bordeaux on the 19th April, going down to 3rd, and finishing the season in that position.
The new away shirt, which OL hope will bring them more success that the previous blue shirt, features a black and red design by English brand Umbro. Whilst the home shirt is sponsored by Novotel, the away shirt is sponsored by Ticket Restaurant. The top part of the 2009/10 away shirt is black and it fades horizontally in the middle into red. The shirt has a partly red collar, and red details on the black sleeves. On the sleeves, it also has white diamond details, which are present on most Umbro-manufactured shirts. The away shorts and the away socks are all red, with slight touches of black.
Posted on June 17, 2009
gorgeous stuff!
deffo approved!
Umbro is stepping up their game..the home one and now this? The fade is very 97 United with Old Trafford sublimated. Does this mean more kits will be one offs for their repective clubs again?
well. what can i say, last year we moaned about umbro, now we adore them.
this has to be the best umbro kit so far, but all the others are great too.
I think I just threw up a little.
Ben The Owl
this is a very nice shirt
wot a really nice shirt. umbro are doing relly well with their shirts this year
Great kit. Umbro's done a nice job with this year's kits. I hope West Ham gets something awesome.
Liam J.C
I absolutely one hundred percent positivley love it !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Unusual but really nice. Think it'll look even better on the players. Don't normally like outlined badges like this but it works really well.
this kit is freaking gorgeous !!!
its completely unique
well done umbro showing the big boy how to make kits
cant wait for the rangers kit for nxt year(made by umbro)
p.s anyone know where i can get a pic of the new gers kit
Different but it is actually really nice, well done
What's wrong with all you people (except theredflag)? That's nasty that is, another hideous early 90s throwback, looks like something Will Smith might wear in the Fresh Prince…
theredflag just u dont talk after callin dundee united just dundee 😀 bad mistake
i luv this kit!!! outline badge should be used more often on kits
Pompey Flo :)
such a nice kit 🙂
umbro have been pretty good this year, except city's away kit which is horrible
this is awful! can you imagine wearing these? it would look atrocious
Watford Boy
thats awesome! by far the best away kit this season
Ooh. I ADORE the outlined badge.
very smart design the colour fade is smartly done
Pretty decent kits. The home kits looks nice, too. The away kit here is unique.
Nice kit love the colour
Wow. That is immense!
Decent!! I hope it would be the template for Hull