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Because of similarity to their yeast orthologues, the two membrane proteins of the human endoplasmic reticulum (ER) Sec62 and Sec63 are expected to play a role in protein biogenesis in the ER. We characterized interactions between these two proteins as well as the putative interaction of Sec62 with ribosomes. These data provide further evidence for evolutionary conservation of Sec62/Sec63 interaction. In addition, they indicate that in the course of evolution Sec62 of vertebrates has gained an additional function, the ability to interact with the ribosomal tunnel exit and, therefore, to support cotranslational mechanisms such as protein transport into the ER. This view is supported by the observation that Sec62 is associated with ribosomes in human cells. Thus, the human Sec62/Sec63 complex and the human ER membrane protein ERj1 are similar in providing binding sites for BiP in the ER-lumen and binding sites for ribosomes in the cytosol. We propose that these two systems provide similar chaperone functions with respect to different precursor proteins.
In eukaryotic cells, protein secretion begins with the translocation of presecretory proteins across the membrane of the rough endoplasmic reticulum (ER). Translocation is mediated by a protein translocase (also termed translocon), that resides in the ER membrane, and occurs co- or posttranslationally. The posttranslational mechanism is abundant in the yeast Saccharomyces cerevisiae and the human parasite Trypanosoma brucei (Goldshmidt et al., 2008 ), whereas the cotranslational mechanism dominates in mammalian cells. In uni- as well as multicellular organisms and for both modes of operation, the protein translocase contains the heterotrimeric Sec61 complex as a central pore-forming component. In yeast ER, the cotranslationally operating protein translocase comprises Sec63p (a membrane-integrated heat shock protein [Hsp] 40), Sec71p, Sec72p, and Kar2p (a lumenal Hsp70) as additional components. The posttranslationally operating protein translocase contains these same components, plus Sec62p (Jermy et al., 2006 ). The Sec61 complex as well as Sec63p and Kar2p are also involved in protein export from the ER (Plemper et al., 1997 ; Kalies et al., 2005 ). Two of the subunits of the Sec61 complex as well as Sec62p, Sec63p, and Kar2p are essential proteins in yeast. Insect Sec62 isoform A can complement inactivation of the SEC62 gene in yeast (Noel and Cartwright, 1994 ). Sec63 is also involved in cotranslational protein transport into the trypanosomal ER and is also essential in trypanosomes (Goldshmidt et al., 2008 ; Zimmerman and Blatch, 2009 ). In mammalian cells, the lumenal Hsp70 immunoglobulin heavy-chain binding protein (BiP) and a so far unidentified Hsp40 are also involved in cotranslational protein import into the organelle (Dierks et al., 1996 ; Hamman et al., 1998 ; Tyedmers et al., 2003 ; Alder et al., 2005 ). Furthermore, in mammalian microsomes orthologues of yeast Sec62p and Sec63p are found in stoichiometric amounts as compared with Sec61α subunits, and in detergent extracts derived from canine pancreatic microsomes they are found in association with each other as well as with the Sec61 complex (Meyer et al., 2000 ; Tyedmers et al., 2000 ).
In contrast to the situation in yeast and trypanosomes, the mammalian ER membrane contains the additional Hsp40, ERj1, which is related to Sec63 in providing a lumenal J-domain (Dudek et al., 2002 ; see Figure 1A). Expression of human ERj1 in yeast can complement inactivation of the SEC63 gene (Kroczynska et al., 2004 ). The cytosolic domain of ERj1 associates with the ribosomal tunnel exit and (via the lumenal J-domain) recruits BiP to translating ribosomes (Blau et al., 2005 ; Dudek et al., 2005 ). In addition, ERj1 may play a dual role in gene expression, i.e., at the level of translation as well as transcription (Dudek et al., 2005 ). We identified a basic oligopeptide motif in ERj1 that is necessary and sufficient for its association with ribosomes and for its regulatory effect on translation. Similar basic oligopeptide motifs are not only present in the established ribosomal ligands signal recognition particle (SRP) and nascent polypeptide-associated complex (NAC) but also in mammalian Sec62 (see Figure 1B). We proposed that these basic oligopeptide motifs represent a general mechanism for attachment of ribosomal ligands to ribosomes (Dudek et al., 2005 ) and that mammalian Sec62 also may be able to bind to ribosomes. We note that similar basic oligopeptide motifs are absent from yeast Sec62p (see Figure 1A).
There also is considerable medical interest in human Sec62 and Sec63. Genetic work linked mutations in the SEC63 gene to polycystic liver disease (Davila et al., 2004 ). According to the two-hit hypothesis for this disease, mutations in the second allele in liver cells of heterozygous carriers of a SEC63 mutation do not result in cell death, but rather lead to cell proliferation and the progressive development of liver cysts. Furthermore, mutations in the SEC63 gene were described for HNPCC-associated small-bowel cancer (Schulmann et al., 2005 ) and microsatellite-unstable gastric and colorectal cancers (Mori et al., 2002 ). In addition, overexpression of the SEC62 gene was found to be associated with sporadic colorectal cancer (Eschrich et al., 2005 ) and prostate cancer (Jung et al., 2006 ).
Here, we provide direct evidence for conservation of Sec62/Sec63 interaction from yeast to humans and show that in the course of evolution Sec62 of vertebrates has gained a function, i.e., the ability to interact with the ribosomal tunnel exit. Two basic oligopeptide motifs are responsible for this interaction and are absent from yeast Sec62p as well as from Sec62 in invertebrates or plants. Thus, the human Sec62/Sec63 complex and human ERj1 are similar in providing a binding site for ribosomes on the cytosolic face and a binding site for BiP on the lumenal face of the ER membrane, i.e., may both be involved in cotranslational transport of polypeptides into the ER. This is consistent with the observation that Sec62 is protected against externally added antibodies by ribosomes in permeabilized human cells.
Aurintricarboxylic acid (ATA) and CHAPS were from Calbiochem (La Jolla, CA). [35S]methionine and enhanced chemiluminescence (ECL) were obtained from GE Healthcare (Waukesha, WI). Sulfo-SMCC was purchased from Pierce (Rockford, IL). Antibodies against calnexin and protein disulfide isomerase (PDI) were from Stressgen (San Diego, CA); peroxidase-coupled anti-penta-histidine antibodies were from Qiagen (Hilden, Germany); peroxidase conjugate of anti-rabbit IgG goat antibodies were from Sigma (St. Louis, MO). Antibodies against yeast Sec62 (CNKKKAINEKAEQN) and human ribosomal proteins L4 (CEKKPTTEEKKPAA) and S3 (CGKPEPPAMPQPV) were raised against the indicated C-terminal oligopeptides.
Peptides were synthesized on a 433A peptide synthesizer (Applied Biosystems, Foster City, CA), cleaved, and deprotected as described previously (Dierks et al., 1996 ). Oligopeptides 62-11mer 1 and 2 corresponded to amino acid residues 2 through 12 and 157 through 167 of human Sec62, respectively. Peptide iv62-11mer corresponded to amino acid residues 2 through 12 of Sec62 isoform A of Drosophila melanogaster (SEKKRARRRKD).
Ribosomes were purified from canine pancreas by gradient centrifugation and washed with puromycin (0.5 mM) and high salt (500 mM KOAc). The A260/280 of the purified ribosomes was 1.98. Ribosomal subunits were prepared by incubation of washed ribosomes in high salt (1 M KCl) and subsequent sucrose gradient centrifugation according to published procedures (Spedding, 1990 ). The integrity of 80S ribosomes and ribosomal subunits was confirmed by analysis of molecular mass and hydrodynamic radius by a combination of asymmetric-flow field-flow fractionation (Eclipse 2, Wyatt Technology, Santa Barbara, CA) and light-scattering analysis (miniDAWN Tristar and QELS, Wyatt Technology; Supplemental Figure 1). Pretreatment of ribosomes with RNase A (80–240 μg/ml) was carried out by incubation for 30 min at 30°C.
Recombinant derivatives of human or yeast Sec62 with C-terminal hexa-histidine tag and derivatives of human Sec62 and Sec63, respectively, with N-terminal glutathione-S-transferase (GST) were purified from Escherichia coli. Sec62N corresponded to amino acid residues 1 through 195 of human Sec62, Sec62N-ΔN10 to 11-195, Sec62N-ΔC40 to 1-155 to Sec62N-ΔN10-ΔC40 to 11-155, and Sec62C to 279-399. Sec63C corresponded to amino acid residues 210 through 760 of human Sec63, Sec63C26 corresponded to residues 734-760. Yeast Sec62N corresponded to amino acid residues 1 through 149 and also contained a C-terminal hexa-histidine tag (Willer et al., 2003 ); "humanized" Sec62N was identical except that the residue 1 of yeast Sec62N was replaced by the 12 aminoterminal amino acid residues from human Sec62. The molecular mass of the purified human Sec62N was determined by a combination of asymmetric-flow field-flow fractionation and light-scattering analysis as 35 kDa (Supplemental Figure 2), which is consistent with a monomer (calculated mass of monomer: 23 kDa).
Purified GST or related hybrid proteins (10 μg) were immobilized on GSH-Sepharose. Then, buffer (20 mM HEPES-KOH, pH 7.5, 150 mM KCl, 2 mM MgCl2, 0.65% CHAPS) or purified hexa-histidine fusion protein (12 μg) in the same buffer was applied to the resin. Where indicated oligopeptides (final concentration: 300 μg/ml) were added simultaneously. After washing, the bound material was eluted and analyzed by SDS-PAGE as followed by either protein staining with Coomassie Brilliant Blue or Western blotting plus immunodetection with anti-penta-histidine antibodies, coupled to peroxidase. The antibodies were visualized by incubation of the blots in ECL and subsequent luminescence imaging (Lumi-Imager F1 with LumiAnalyst software 3.1, Roche Diagnostics, Mannheim, Germany).
Recombinant proteins (150 pmol) were diluted into 20 mM HEPES-KOH, pH 7.5, 200 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 0.65% CHAPS and incubated in the simultaneous absence or presence of ribosomes (50 pmol) for 15 min at 30°C (Dudek et al., 2005 ). Where indicated oligopeptides (50 nmol) were present. Subsequently the mixture was layered onto a low-salt cushion (0.5 M sucrose in 40 mM HEPES-KOH, pH 7.5, 150 mM KOAc, 5 mM MgOAc, 2 mM DTT) and subjected to centrifugation for 90 min at 356,000 × g and 2°C (Beckman table top ultracentrifuge Optima MAX-E, Beckmann rotor TLA-120.2; Fullerton, CA). The supernatants were subjected to protein precipitation with trichloroacetic acid. The precipitates and the pellets, respectively, were subjected to SDS-PAGE and subsequent protein staining or Western blotting plus immunodetection with peroxidase-coupled anti-penta-histidine antibodies or antibodies against ribosomal proteins plus peroxidase conjugate of anti-rabbit IgG goat antibodies. The antibodies were visualized by incubation of the blots in ECL and subsequent luminescence imaging.
Alternatively, ribosomal complexes were subjected to sucrose gradient centrifugation (linear sucrose gradient between 10 and 60%, wt/vol in low-salt buffer, adjusted to 33 μg/ml BSA) for 60 or 90 min at 280,000 × g and 2°C (Beckman ultracentrifuge Optima L-80, Beckmann rotor SW 55 Ti; Dudek et al., 2002 ). After fractionation of the gradients, proteins were precipitated and subjected to SDS-PAGE and subsequent protein staining or Western blotting plus immunodetection as described above.
Surface plasmon resonance (SPR) spectroscopy was performed in a BIAlite upgrade system (Biacore, Freiburg, Germany). Sensor chip NTA was activated with Ni2+ according to the manufacturer's protocol (Biacore). Hexa-histidine–tagged proteins were immobilized on the chip surface at a flow rate of 10 μl/min in 10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 50 μM EDTA, and 0.005% surfactant P20. For interaction analysis, the chip was equilibrated with running buffer (10 mM HEPES-KOH, pH 7.6, 150 mM NaCl, 0.125% digitonin) at a flow rate of 5 μl/min. Subsequently, solutions containing increasing concentrations of analyte were passed over the chip surface. Each analyte application was followed by application of running buffer and eventually with running buffer that was supplemented with high salt. The analysis was carried out by employing BIA evaluation software version 3.1 (Biacore).
Synthesis of firefly luciferase, bovine preprolactin (ppl), or nascent preprolactin (ppl86mer) was carried out in rabbit reticulocyte lysate in the presence of [35S]methionine (translation kit type I or II, Roche Diagnostics; Dudek et al., 2005 ). The translation reactions contained either buffer (20 mM HEPES-KOH, pH 7.5, 500 mM KCl, 2 mM MgCl2, 2 mM DTT, 0.65% CHAPS), recombinant protein (final concentration: 1 μM) in buffer, DMSO (final concentration: 2%), or oligopeptide (final concentration: 150 μM) in DMSO. After incubation for various times at 30°C, the translation reactions were subjected to SDS-PAGE and phosphorimaging (Molecular Dynamics, Sunnyvale, CA; model SF with Image Quant software).
ppl86-mer was synthesized in reticulocyte lysate in the presence of [35S]methionine for 20 min (Dudek et al., 2005 ). After adjusting the translation reaction to 500 mM KOAc for 5 min at 0°C, the ribosomes were pelleted by centrifugation through a high-salt cushion. The ribosomes were resuspended in XL-buffer (50 mM HEPES-KOH, pH 7.5, 200 mM KOAc, 2 mM MgOAc, 0.2 M sucrose, 0.65% (wt/vol) CHAPS), divided into various aliquots, and supplemented with buffer or recombinant protein (2 μM). After incubation for 15 min at 30°C, ribosomes were reisolated by centrifugation through a low-salt cushion and incubated with Sulfo-SMCC (0.25 mM) for 20 min at 0°C. All samples were subjected to SDS-PAGE and phosphorimaging.
Yeast SEC62 ts mutant strain RDM50-94C (leu2-3 leu2-112 his4 ura3-52 sec62-1 MATα) was kindly provided by R. Schekman (Berkeley, CA). The CEN-multicopy vector YEp with URA3 as a selection marker was provided by G. Schlenstedt (Homburg). Cells were transformed with a derivative of YEp that contained the human SEC62 cDNA under control of the GAL1 promoter and were grown on SD medium without uracil at 24 or 37°C in the presence of glucose (2%) or galactose (2%).
Cells were grown on eight-chambered Lab-Tek glass coverslips (Naperville, IL) in the respective standard media to 40–70% confluence. Cells were fixed with fresh 3.7% formaldehyde in PBS for 15 min at room temperature, permeabilized with PBS with 0.1% Triton X-100, and—where indicated—treated with 50 μg/ml RNase A during the block step in 10% fetal bovine serum/PBS. Subsequently, the cells were labeled with affinity-purified primary antibodies, followed by Alexa 555–conjugated anti-rabbit IgG secondary antibodies. Cells were imaged using fluorescence microscopy with a widefield microscope (Axiovert 200; Carl Zeiss Microimaging, Thornwood, NY; 63× oil 1.4 NA objective, 450–490 excitation/500–550 emission bandpass filter) and a Retiga 2000R camera. Image analysis was performed with ImageJ 1.39i (http://rsb.info.nih.gov/ij/). Figures were prepared using Microsoft Excel 2004 (Redmond, WA) and Adobe Photoshop CS2 and Adobe Illustrator 11.0 (San Jose, CA).
In yeast, the negatively charged carboxyterminus of Sec63p interacts with the overall positively charged aminoterminal (N-terminal) domain of Sec62p (Wittke et al., 2000 ; Figure 1A). Thus, we asked if this mode of interaction is conserved in the two human proteins. Purified GST hybrid proteins that comprised the complete carboxyterminal (C-terminal) domain of human Sec63 (termed Sec63C) or only its 26 C-terminal amino acid residues (Sec63C26) were immobilized on GSH-Sepharose. GST served as a negative control. Then, buffer or purified hexa-histidine fusion of the N-terminal domain of mammalian Sec62 (Sec62N) were applied to the resin. The bound material was eluted and analyzed by SDS-PAGE, followed by either protein staining (Figure 2A, lanes 1 and 2, 4 and 5, and 7 and 8) or Western blotting plus immunodetection with anti-penta-histidine antibodies (Figure 2B). Although there was no Sec62N detected in the eluate of the immobilized GST (lane 2), there was a significant amount of Sec62N found in the eluate of both GST-Sec63C (lane 5) as well as GST-Sec63C26 (lane 8). Thus, human Sec63 and Sec62 directly interact with each other, and the C-terminal 26 amino acid residues of Sec63C are sufficient for this interaction.
We analyzed which part of Sec62N was involved in this interaction: the central and overall positive region or one of the two positively charged oligopeptides at/near the amino- or carboxyterminus (Figure 1, A and B). This was addressed by employing a truncated Sec62N that lacks the two positively charged oligopeptides (termed Sec62N-ΔN10-ΔC40; Figure 1C) or the two positively charged oligopeptides (termed 62-11mer1 and 2; Figure 1C) as potential competitors of Sec62N, respectively. Sec62N-ΔN10-ΔC40 efficiently bound to GST-Sec63C as well as GST-Sec63C26 (Figure 2, A and B, lanes 6 and 9). Furthermore, even at a >100-fold molar excess (that is effective in the competition of ribosome binding; see below) the two basic oligopeptides did not interfere with binding of Sec62N to GST-Sec63C (Figure 2C). Thus the central and overall positive region of Sec62 is involved in interaction with Sec63C. This was confirmed by binding studies that used peptide spot membranes according to Frank (1992) (Supplemental Figures 3 and 4). In this respect, both mammalian proteins behaved like their yeast orthologues.
To further characterize the Sec62/Sec63 interaction, SPR experiments were carried out. Human Sec62N was immobilized in the measuring cell of a NTA sensor chip via its hexa-histidine tag. Human Sec62C served as a negative control and was immobilized in the reference cell. Then increasing concentrations of human Sec63C were passed over the chip and were followed by buffer. Association of the analyte and its dissociation were recorded and analyzed (Figure 2D). We could fit the kinetics with a 1:1 binding model and determined an apparent affinity (Kd) of Sec63C for Sec62N of 4.78 nM. This Kd was consistent with the fact that native Sec62 was coimmunoprecipitated with Sec63 from a microsomal detergent extract (Tyedmers et al., 2000 ).
The N-terminal domain of mammalian Sec62 (Sec62N) contains two basic oligopeptide motifs that are reminiscent of similar peptides in established ribosomal tunnel exit ligands (such as NAC and ERj1; Ferbitz et al., 2004 ; Blau et al., 2005 ; Figure 1B). Deletion of the basic oligopeptides in the N-terminal domains of mammalian and yeast NACβ (Grallath et al., 2006 ; Wegrzyn et al., 2006 ) as well as the cytosolic domain of ERj1 (Dudek et al., 2005 ) led to loss of ribosome-binding ability. Therefore, Sec62N was analyzed with respect to its ribosome-binding ability by a number of different experimental approaches, and the role of the two basic oligopeptide motifs was characterized. The same experimental strategies were previously used for the characterization of the ribosome interaction of ERj1 (Dudek et al., 2002 , 2005 ).
Sec62N was incubated in the presence or absence of nontranslating 80S ribosomes. An aliquot of ribosomes was incubated in the absence of Sec62N and served as reference (Figure 3A). The samples were analyzed by gradient centrifugation and subsequent SDS-PAGE and protein staining. In the absence of ribosomes, Sec62N stayed at the top of the gradient (Figure 3B). After incubation with ribosomes, Sec62N comigrated with ribosomes (Figure 3C). Thus the observed comigration of Sec62N with ribosomes was not due to aggregation, but rather reflected an interaction between the two molecules.
Next, Sec62N and three truncated derivatives (Figure 1C) were each incubated in the presence or absence of nontranslating ribosomes in order to address the question of which part of Sec62N was involved in this interaction. Yeast Sec62N that lacks similar basic oligopeptide motifs as compared with human Sec62N (Figure 1A) served as negative control. Subsequently, the ribosomes were reisolated by centrifugation and the relative amount of ribosome associated Sec62N was determined by SDS-PAGE and protein staining (Figure 3D) or Western blotting plus immunodetection with anti-penta-histidine antibodies (Figure 3, E and F). All proteins stayed in the supernatant in the absence of ribosomes (Figure 3F). However, Sec62N and constructs that contained at least a single positively charged oligopeptide were pelleted together with ribosomes, i.e., were active in ribosome binding (although to a varying degree), whereas the construct with the double deletion (termed Sec62N-ΔN10-ΔC40) was almost completely inactive in ribosome binding (Figure 3, D and E, lanes 7 and 8). Furthermore, yeast Sec62N that lacks similarly charged oligopeptides was unable to bind ribosomes (Figure 3, D and E, lanes 9 and 10). Although both basic oligopeptides within Sec62N contributed to ribosome binding, the aminoterminal peptide seemed to be more important.
To further substantiate the role of the two highly charged oligopeptides for ribosome binding, synthetic oligopeptides were used as potential competitors of Sec62N in ribosome binding (peptides 62-11mer1 and 2; Figure 1C). The aminoterminal oligopeptide from an invertebrate Sec62 served as negative control (peptide iv62-11mer). Only the aminoterminal peptide from human Sec62 competed with Sec62N for ribosome binding (Figure 3G, lanes 3 and 4).
Having observed an interaction of human Sec62N with ribosomes, i.e., a gain of function of the human protein compared with the yeast protein, we asked if yeast Sec62N can be turned into a ribosome-binding protein by the addition of the aminoterminal dodecapeptide from human Sec62 and if human SEC62 is able to rescue the thermosensitive growth phenotype of a translocation-deficient yeast SEC62 mutant. Yeast Sec62N was extended at its aminoterminus by the aminoterminal dodecapeptide from human Sec62 (termed humanized Sec62N) and incubated in the presence or absence of nontranslating ribosomes. Subsequently, the ribosomes were reisolated by centrifugation and the relative amount of ribosome associated humanized Sec62N was determined by SDS-PAGE and protein staining or Western blotting plus immunodetection with anti-penta-histidine antibodies. In contrast to wild-type yeast Sec62N, humanized Sec62N bound to ribosomes (Figure 3, H–J). Thus the aminoterminal peptide that is present in human Sec62 is sufficient for ribosome binding. Because of a protein translocation defect at the nonpermissive temperature, the yeast mutant strain RDM50-94C hardly grows at 37°C (Deshaies and Schekman, 1990 ). However, when the human SEC62 gene was expressed in this strain after the addition of galactose, the cells grew at 37°C (Figure 4). Thus, the additional positively charged oligopeptides that are present in human Sec62 compared with yeast Sec62p do not interfere with the posttranslational function of yeast Sec62p.
To quantitatively characterize the ribosome interaction of mammalian Sec62, SPR experiments were carried out. Human Sec62N was immobilized in the measuring cell of a NTA sensor chip via its hexa-histidine tag. Based on the experiments that are depicted in Figure 3, D and E, yeast Sec62N served as a negative control and was immobilized in the reference cell. Then increasing concentrations of mammalian ribosomes were passed over the chip and were followed by buffer. Association of the analyte to and its dissociation from the ligand were recorded and analyzed (Figure 5A). We determined an apparent affinity (Kd) of ribosomes for Sec62N of 0.13 nM.
The interaction of ERj1 with ribosomes was shown to be salt-sensitive and to involve rRNA (Dudek et al., 2002 and 2005 ). Here, we asked if this is also true for Sec62N. Thus, ribosome binding assays and SPR experiments were carried out above the standard 100–150 mM salt concentration and by substituting ribosomes by RNase-treated ribosomes, respectively. Human Sec62N was immobilized in the measuring cell of a NTA sensor chip via its hexa-histidine tag, and yeast Sec62N was immobilized in the reference cell. After association of native ribosomes, the dissociation was carried out at 200 and 500 mM KOAc concentration (Figure 5B). According to the sensorgram, the interaction of Sec62N with ribosomes was salt-sensitive. When RNase treated ribosomes were used as analyte the sensorgram suggested that the interaction of Sec62N with ribosomes was RNase-sensitive (Figure 5C). Similar observations were made in the ribosome-binding assays (Figure 5, D and E vs. F). Thus binding of Sec62N to ribosomes most likely involves electrostatic interactions with rRNA. We note that the observed salt sensitivity of ribosome binding of Sec62N may explain why Sec62 was not characterized as ribosome-associated membrane protein after solubilization of microsomes in detergent (salt concentration: 400 mM KCl; Meyer et al., 2000 ; Tyedmers et al., 2000 ).
Next, we used three independent experimental approaches to assess whether or not the ribosomal exit site of nascent polypeptides is also the binding site for Sec62N, because this site has previously been observed to provide a docking site for several proteins that are involved in protein biogenesis at the ER (SRP, NAC, Sec61 complex, ERj1; Beckmann et al., 2001 ; Halic et al., 2004 ; Ferbitz et al., 2004 ; Blau et al., 2005 ).
First we assessed if there was a preference of Sec62N for the large ribosomal subunit. Sec62N was incubated in the presence or absence of 60S and 40S ribosomal subunits. The samples were analyzed by gradient centrifugation and subsequent SDS-PAGE as followed by Western blotting plus immunodetection with anti-penta-histidine, anti-L4, or anti-S3 antibodies, respectively. After incubation with 60S subunits, a fraction of the Sec62N comigrated with ribosomes (Figure 6B). In the absence of ribosomes and in the presence of 40S subunits, Sec62N stayed at the top of the gradient (Figure 6, A and C). Thus, Sec62 can only interact with large ribosomal subunits.
Next, we analyzed the possible overlap in binding sites between Sec62N and ERj1 using SPR experiments (Blau et al., 2005 ). Sec62N was immobilized in the measuring cell of a NTA sensor chip via its hexa-histidine tag and analyzed with respect to binding of native ribosomes that had been preincubated with ERj1C (Figure 6D). Native ribosomes that had been preincubated with Sec62N served as positive control, and native ribosomes that had been preincubated with BSA as negative control. The results demonstrate that Sec62N and ERj1 compete for an overlapping binding site on ribosomes, most likely at or near the ribosomal tunnel exit.
To directly address this point, Sec62N or its derivatives were allowed to form complexes with ribosomes that contained radiolabeled nascent polypeptide chains of defined length (i.e., peptidyl-tRNAs comprising 86 amino-terminal amino acid residues of preprolactin, ppl86mer). Subsequently, the ribosome/nascent chain/Sec62N complexes were reisolated and subjected to chemical cross-linking. ERj1 served as a positive control (Dudek et al., 2005 ; Figure 6E, lane 6). In the presence of Sec62N or a construct that contains a single positively charged oligopeptide, cross-linked products of the nascent polypeptide were detected (Figure 6E, lanes 2–4). These were absent when buffer, Sec62N-ΔN10-ΔC40 (Figure 6E, lanes 1 and 5), yeast Sec62N (Figure 6F, lane 2), or human Sec62C were used (Figure 6F, lane 3). Thus, like ERj1, Sec62N binds to the ribosome near the tunnel exit. We note that there is a dominant 45-kDa cross-linking product to be seen in the absence of Sec62N that is hardly seen in the presence of Sec62N. We suggest that this cross-linking product involves a 36-kDa ribosomal protein (possibly L4; Woolhead et al., 2004 ) and that Sec62N that is bound near the tunnel exit affects the positioning of the nascent polypeptide chain with respect to this ribosomal protein. According to this interpretation, removal of the aminoterminal oligopeptide has a more pronounced effect on binding of Sec62N compared with removal of the carboxyterminal oligopeptide.
Having seen an interaction of Sec62N with ribosomes (Figure 3) and with Sec63C (Figure 2), we asked if Sec62N can recruit Sec63C to ribosomes. In ribosome binding assays, however, we failed to detect a trimeric complex (data not shown). Therefore, the immobilized complex between GST-Sec63C and Sec62N was used (Figure 7, left panels). In parallel, a complex was formed between GST-Sec63C and Sec62N-ΔN10-ΔC40, i.e., the truncated Sec62N that lacks the two positively charged oligopeptides (Figure 7, right panels). The resins were eluted with buffer or nontranslating ribosomes in the same buffer. Subsequently, the resins were eluted with SDS sample buffer, and all samples were analyzed as described above. On elution with buffer, Sec62N and Sec62N-ΔN10-ΔC40 remained bound to the immobilized Sec63C as expected (Figure 7, lanes 4 and 9). On elution with ribosomes, however, Sec62N eluted together with the ribosomes (lane 3 vs. 5). In contrast, Sec62N-ΔN10-ΔC40 that was unable to bind to ribosomes was not eluted with ribosomes (lane 8 vs. 10). Thus, interaction of Sec62N with Sec63C does not prevent binding of Sec62 to ribosomes, which is consistent with the observed Kd values for the interactions of Sec62N with Sec63C (4.78 nM) and ribosomes (0.13 nM).
ERj1 was shown to be able to bind to ribosomes as well as to inhibit protein synthesis at the level of initiation (Dudek et al., 2005 ). Therefore, Sec62N and its derivatives were tested for their ability to inhibit synthesis of firefly luciferase or bovine preprolactin in reticulocyte lysate (Figure 8, A and B). In this assay, Sec62N and constructs that contained at least a single positively charged oligopeptide were active in inhibiting protein synthesis (although to a varying degree), and the construct with the double deletion (termed Sec62N-ΔN10-ΔC40) was less active (Figure 8A). Furthermore, yeast Sec62N that lacks similarly charged oligopeptides was inactive (Figure 8A). We note that a similar inhibitory effect of human Sec62N on the synthesis of luciferase, and preprolactin was observed in the presence of canine pancreatic microsomes (Supplemental Figure 5). A control experiment demonstrated that the translational inhibition activity of Sec62N was specific: the inhibitory effect of Sec62N on translation correlated reciprocally with the ribosome content of the reticulocyte lysate (Figure 8C).
To further substantiate the role of the two highly charged oligopeptides for translational modulation by Sec62N, the synthetic oligopeptides were used in translation (peptides 62-11mer1 and 2; Figure 1C). Again, the aminoterminal oligopeptide from an invertebrate Sec62 served as negative control (peptide iv62-11mer). Indeed, the aminoterminal peptide from human Sec62N inhibited synthesis of preprolactin as well as luciferase (Figure 8, D and E).
In addition, Sec62N was tested for its ability to inhibit translation after inhibition of initiation (Figure 8F). Sec62N did not affect protein synthesis under these conditions. Thus, Sec62N inhibits synthesis of presecretory as well as nonsecretory proteins at the level of initiation.
Snapp et al. (2004) established a microscopic method to address the organization of protein translocase in the ER of mammalian cells. The experimental strategy was cell fixation, cell permeabilization, optional ribosome destruction by RNase treatment, and incubation with specific primary and fluorescently labeled secondary antibodies (Figure 9A). In the subsequent fluorescence microscopy and image analysis, the quantitative data from the RNase-treated cells were compared with the minus RNase control. Positive differential effects in fluorescence intensity were taken as an indication of association of the respective protein with ribosomes. Here, two different cell types (Madin-Darby canine kidney [MDCK] and HeLa) were analyzed with respect to Sec62 (Figure 9B). The Sec61α and Sec61β subunits of the translocase served as positive controls, the ER-membrane protein calnexin and the ER-lumenal PDI served as negative controls (Snapp et al., 2004 ). Although the extents of the differential effects varied between the two cell types, we detected a significant increase in fluorescence intensity after RNase treatment for Sec61α, Sec61β, and Sec62, but not for calnexin and PDI. Similar results were obtained for Cos-7 and HepG2 cells (data not shown). Thus, Sec62 is in the vicinity of ribosomes and, by extrapolation, of Sec61 complexes in the intact ER. This is consistent with our previous findings that Sec61 subunits can be coimmunoprecipitated from microsomal detergent extracts with antibodies against Sec62 and vice versa (Tyedmers et al., 2000 ).
To rule out the possibility that the observed epitope protection is due to the ribonucleoprotein particles SRP rather than ribosomes, the α-subunit of the SRP receptor (SRα) was analyzed under identical conditions (Figure 9C). There was no increase in fluorescence intensity after RNase treatment for SRα in either MDCK or HeLa cells. Thus, the observed epitope protection in the case of Sec61α, Sec61β, and Sec62, was indeed due to ribosomes.
Next we analyzed if the effect of RNase treatment can be mimicked by puromycin treatment of MDCK and HeLa cells, i.e., under conditions where the nascent polypeptides are released from translating ribosomes (Figure 10). There was no intensity increase detected for any of the proteins after addition of puromycin. This supports the notion that many ribosomes or 60S ribosomal subunits do not leave the ER surface after termination of protein synthesis (Potter et al., 2001 ). Furthermore, these data suggested that the RNase experiments visualized translating as well as nontranslating ribosomes.
Here we demonstrated that the human ER membrane proteins Sec62 and Sec63 interact in the same manner as compared with their yeast orthologues, i.e., involving the conserved oppositely charged regions at the central region of Sec62N and the carboxyterminus of Sec63C (Wittke et al., 2000 ; Figure 1A). This is consistent with the observations that in T. brucei there is no Sec62 ortholog and that the trypanosomal Sec63 lacks the negatively charged carboxyterminal region (Goldshmidt et al., 2008 ; Supplemental Figures 6 and 7). Taken together with the fact that the two proteins are associated with each other and with the Sec61 complex in detergent extracts from both yeast (Jermy et al., 2006 ) and canine pancreatic microsomes (Meyer et al., 2000 ; Tyedmers et al., 2000 ), it is very likely that the two mammalian proteins play a similar role in protein biogenesis compared with their yeast orthologues, i.e., are involved in protein transport into the ER. This view is supported by our immunofluorescence analysis (see below).
Furthermore, we have shown that in the course of evolution Sec62 of vertebrates has gained a function, i.e., the ability to interact with the ribosomal tunnel exit. This is consistent with our observation that human Sec62 is associated with ribosomes at the ER of mammalian cells and with the fact that the majority of transport into the mammalian ER is cotranslational. In the case of Sec62, two basic oligopeptide motifs may be responsible that are absent from yeast Sec62p (Figure 1, A and B) as well as from Sec62 orthologues in invertebrates (such as Caenorhabditis elegans or Drosophila melanogaster) or plants (such as Arabidopsis thaliana or Oryza sativa; Supplemental Figures 6 and 7). Interestingly, this apparent gain of function did not interfere with the original function of the yeast protein because human Sec62 was able to complement the respective yeast ts mutant strain, as had previously been shown for the insect protein (Noel and Cartwright, 1994 ). Thus Sec62 showed most of the characteristics that were previously observed for ERj1 (Figure 1): 1) high salt– and RNase-sensitive ribosome binding of the cytosolic domain, 2) essential basic oligopeptide(s) in the cytosolic domain, 3) binding near the ribosomal tunnel exit, and 4) modulatory effect on initiation of protein synthesis (Dudek et al., 2002 , 2005 ; Blau et al., 2005 ). Because Sec63 shares with ERj1 the ability to bind to BiP via its ER-luminal domain (J-domain), the human Sec62/Sec63 complex may have overlapping functions with ERj1. This view is supported by the observations that human ERj1 can complement a yeast Sec63p knockout (Kroczynska et al., 2004 ) and that loss of Sec63 function in human polycystic liver disease is not lethal (Davila et al., 2004 ; Zimmermann et al., 2006 ). We note that in the case of ERj1 the basic oligopeptide motif is also found only in vertebrate orthologues (Supplemental Figure 8).
What are the specific functions of Sec62/Sec63 and ERj1 in the mammalian ER? We are convinced that the properties of the two proteins/complexes suggest a cotranslational function, most likely in protein transport into the ER. Both could serve in recruiting BiP to Sec61 complexes and/or incoming polypeptides (Dierks et al., 1996 ; Tyedmers et al., 2003 ; Alder et al., 2005 ). Furthermore, both proteins/complexes could be involved in the transport of those nascent polypeptides that are synthesized by ribosomes or 60S ribosomal subunits that stay permanently associated with the ER surface (Potter et al., 2001 ), i.e., do not depend on SRP and its receptor for targeting to the ER (Figure 10). In this case it would make perfect sense to allow initiation of protein synthesis only if BiP is available, i.e., is bound to an ER-lumenal J-domain. Indeed, we observed for ERj1 that binding of BiP to the J-domain still allows binding of the ERj1/BiP complex to the ribosome and that this heterodimeric complex does not inhibit translation (Dudek et al., 2005 ). We suggest that a similar mechanism may be in operation for the Sec62/Sec63-complex. Here the model is that binding of BiP to the ER-lumenal J-domain of Sec63 induces a Sec63/Sec62-interaction that allows binding of the aminoterminal domain of Sec62 to ribosomes in a way that is compatible with translation.
Why would there be two similar systems? There could be functional specialization: the one system could recruit BiP to ribosomes and the Sec61 complex for sealing of the Sec61 channel (Alder et al., 2005 ), whereas the other system could recruit BiP as a molecular ratchet for incoming polypeptide chains (Tyedmers et al., 2003 ). Alternatively, the two systems may have different substrate specificities. A first indication for a potential substrate specificity of ERj1 was reported by S. Y. Blond and coworkers (Kroczynska et al., 2004 , 2005 ). In a two-hybrid screening these authors identified two interaction partners of the SANT-2 domain that is present in the cytosolic domain of human ERj1, the secretory proteins α1-antichymotrypsin (ACT, residues 140-400) and inter-α-trypsin inhibitor heavy chain 4 (ITIH4, residues 588-930). The two proteins have in common that they are secreted in the human liver, are serum protease inhibitors, and are acute-phase proteins. In both cases the signal peptides were not present, indicating that the mature proteins interacted with the SANT-2 domain. This may suggest that ACT and ITIH4 are substrates of ERj1 in the human liver. Therefore, we used the aminoterminal domain of human Sec62 as bait in a two-hybrid screening of a human liver cDNA library (data not shown). We identified four putative interaction partners, two uncharacterized proteins and the precursors of two secretory proteins, haptoglobin (residues 1-296) and complement factor B (residues 1-237). The two secretory proteins have in common that they are secreted in the human liver, are serum proteins with a serine protease domain, and contain complement control protein (CCP)-domains or SUSHI repeats in their aminoterminal regions (two in residues 1-296 of haptoglobin and three in residues 1-237 of complement factor B). In both cases the signal peptides were present, possibly indicating that the signal peptides interacted with Sec62N. A signal peptide receptor function of human Sec62 would not be unexpected because the yeast Sec62p was shown to be part of the signal peptide receptor that is involved in posttranslational transport. Thus, these observations may indicate that haptoglobin and complement factor B may be substrates of Sec62 in the human liver. In future work, this interpretation of the two two-hybrid screenings has to be evaluated after siRNA-mediated silencing of the ERJ1 and the SEC62 gene, respectively.
We are grateful to Dr. R. S. Hegde (National Institute of Child Health and Human Development, Bethesda, MD) for his kind gift of anti-Sec61α antibodies and to Dr. G. Schlenstedt (Homburg) for his advice with respect to the complementation analysis. Furthermore, we acknowledge critical reading of the manuscript by Dr. G. L. Blatch (Rhodes University, Grahamstown, South Africa) and expert technical assistance by Simone Amann and Nicole Heim. E.L.S. is an Ellison Medical Foundation New Scholar in Aging. He is also supported by National Institutes of Health Grant R21 DK074650-01 and a pilot award from the Marion Bessin Liver Center. M.D.deE., C.B., and L.M. were supported by fellowships from the Deutsche Forschungsgemeinschaft (DFG). In addition, this work was supported by grants from Homburger Forschungsförderungsprogramm (HOMFOR) and DFG (SFB 530).
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-08-0730) on January 13, 2010.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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In the first of our video blogs in the lead up to the Great North Swim, Cassie Patten, open water Olympic Bronze and two times World Silver Medalist meet 12 year old Taylor Aplin and gives her some tips on making the transition from pool swimming to the outdoors in the open water of Windermere. Continue reading "Great North Swim Tips with Cassie Patten: From Pool to Outdoor Swimming"
Tag: great north swim
Cassie Patten's Open Water Great North Swim Tips
In the lead up to the Great North Swim, we welcome Cassie Patten back to Low Wood Bay. Ahead of the Great North Swim, Cassie met novice and experienced open water swimmers and provided advice with a question and answer session. Continue reading "Cassie Patten's Open Water Great North Swim Tips"
Cassie Patten Sends Best Wishes all 2014 Great North Swim Competitors
Cassie Patten became a household name in open water swimming when she won bronze at the 2008 Bejing Olympics.
Just a short time after this victory she went on to take first place at the inaugural Great North Swim in September 2008 hosted at Low Wood Bay, beating her friend and rival Kerri Anne Payne.
With just ten days left until the competitors take to the water for the 2014 Great North Swim, Cassie gives us a few tips and sends her best wishes to each swimmer, whatever their goal.
Continue reading "Cassie Patten Sends Best Wishes all 2014 Great North Swim Competitors"
10 Open Water Swimming Tips ahead of the Great North Swim
Low Wood Bay, on the shores of Lake Windermere is the home of open water swimming. The Lake District Hotel has hosted The Great North Swim, the UK's biggest open water swimming event, since 2008 and the The Big Chill Swim, possibly the UK's coldest winter open water swim event, since 2012 – the water temperature was 5 degrees this year! Continue reading "10 Open Water Swimming Tips ahead of the Great North Swim"
Winter Open Water Swimming – remind me why this was a good idea?
Low Wood Bay has hosted many open water swim events from its enviable lakeside location in the Lake District. Events include the Great North Swim – an open water summer swim in the great outdoors, or for those brave enough to tackle the cold, The Chill Swim which recently attracted 570 brave souls in 2014. We have also cheered on Davina McCall as she completed the open water section of her Beyond Breaking Point Sport Relief challenge.
As a result, open-water fever has gripped a number of English Lakes employees who have taken to the water as a personal challenge including David Neale, English Lakes Commercial Director.
The joys of open water swimming
I discovered the joys of open water swimming about 3 years ago as I took on the challenge of the Great North Swim in Lake Windermere. Continue reading "Winter Open Water Swimming – remind me why this was a good idea?"
Countdown to the Great North Swim 2013 – Meet the Elite
The countdown has started for the largest European open water swimming event when 10,000 people will be taking to the waters of Lake Windermere during the Great North Swim between 14th and 16th June. The Great North Swim is the flagship event in Great Swim Series and in four years has grown from 3,000 swimmers in one day to around 10,000 swimmers taking part over three days.
Some of the world's best open water swimmers have said that the Great North Swim in Windermere ranked among their favourite races worldwide. Now there's an opportunity for us mere mortals to meet the elite on the lawns of Low Wood Bay in the informal setting of a barbeque with entertainment from Burneside Brass Band. The event will take place on Friday 14th June from 6:30pm. Continue reading "Countdown to the Great North Swim 2013 – Meet the Elite"
Great North Swim Q&A with Low Wood Club
Open water swimming events are happening thick and fast, all over Cumbria. Not to mention the Great North Swim right on our doorstep at Low Wood Bay. Vicky Stainton, Gym Instructor at Low Wood Club will be taking part in the swim this weekend. We asked here how she has been preparing for the open water swim…

subset: commoncrawl


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Algerian to undergo Hammers trial
By Patrick Haond
Last Updated: 01/01/70 1:00am
The Nationwide League favourites were dealt a blow when veteran Les Ferdinand, who joined the club on a short-term basis last season, signed for Leicester City on Friday.
Glenn Roeder has wasted little time in seeking a replacement, particularly with the threat that Frederic Kanoute could still head for Tottenham.
28-year-old Saifi is still under contract at Troyes for another year, but has been granted his wish to try and earn a move to England by the relegated French side.
"The club is kind to me," smiled the Algerian international.
"They will let me leave for a not too expensive transfer fee."


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(205) 428-2600
Alabama Abuse Counseling Center, Inc.
20 years in business1612 3rd Ave N #N
Bessemer, AL 35020-5709
Additional Phone Numbers
- (205) 759-9698
- (205) 669-8593
Find a Location
BBB File Opened: 05/04/2012
Business Started: 01/23/1997
Business Started Locally: 01/23/1997
Business Incorporated: 01/23/1997 in AL
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- Ms. Virginia Blankenship, Owner
Contact Information
- Mr. Barry Brison
Business Category
- Drug Abuse Information
- Alcoholism Information
- Marriage Counselor
- Domestic Abuse Services
- Family Services
- Behavioral Education
Products & ServicesBased on information provided, this business offers education and counseling services for marriages, families, children, anger management, domestic abuse, drug abuse and individuals.
Licensing, Bonding or RegistrationThis business is in an industry that may require professional licensing, bonding or registration. BBB encourages you to check with the appropriate agency to be certain any requirements are currently being met.
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Licensing information is provided in the BBB Business Profiles to inform the public about industries that may require professional licensing, bonding, or registration. Better Business Bureau encourages you to check with the appropriate agency to be certain any requirements are currently being met.
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Allen Carr founded his stop smoking method in 1983. Since then his organisation has spread throughout the world curing smokers in more than 45 countries in 28 different languages. The organisation is now widely regarded as the leading experts in this field. Our reputation has grown for the simple reason that our method works.
As far as we are aware ours is the only established organisation which actually offers a money back guarantee if you do not succeed in stopping. We are able to offer this guarantee because our success rate is so high - over 90% (based on claims made under our 3 month money back guarantee)
If you genuinely want to stop smoking, our method will not only enable you to do so but will also make it relatively easy. If you follow the instructions you will not only become a non-smoker but you will find the withdrawal period completely painless and most important of all you will not miss smoking.
Most smokers require just one session to become happy non-smokers. In certain cases however more than one session is required. In this event there is no extra charge, the 2 booster sessions are provided completely free of charge.
In the event of cancellation of the first session with at least 3 days notice, no cancellation fee is charged and a full refund or transfer is available.
If cancellation of the first session is made with less than 3 days notice, or in the event of non-attendance without notification, the booking cannot be transferred and the full fee will be charged. Someone else can attend the booked session as a substitute for no extra charge and no notification of substitution is required.
If within three calendar months from the date you attended the first session you are still smoking and decide not to stop, your fee will be refunded in full without inquisition, provided your claim is made in writing and posted (not emailed) to us during the guarantee period, stating that you are smoking and have no intention of stopping smoking using the Allen Carr's Easyway method. Your word will be taken on trust. However, we would request you on your part to be genuine in your desire to stop.
This guarantee will be invalidated in any of the following events:
That you cancel, postpone or fail to attend any session or arrive over fifteen minutes late for any session or leave early.
That you fail to attend at least two free back-up sessions within three months of the date of your first session (the second and third sessions are not a repeat of the first session and last approximately 2½ - 3 hours each).
Should you stop smoking for three months or longer but start again at a later date, you may attend any number of back-up sessions on payment of half the standard fee. However, should you wish to attend the first session again the standard fee will be payable and the guarantee will apply.
This is a generous guarantee based on trust, so for your part we would request that you are genuine in your desire to stop smoking and to comply with your side of the agreement. We will do our best to offer you a convenient time for a back up session, should you need one.


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There are certain games that the gaming world calls "heavy" games. We call them heavy because they have more complex rules and longer play times. These are my favorite type of game for a few reasons. I like investing in a game for a few hours – watching it develop and change. I also like the reward of seeing a strategy work. There is even some satisfaction in trying bad strategies for kicks.
I decided to write on heavy games because of my family's "100 Game Challenge." contains several heavy games. Many folks have commented, saying things like, "Your family plays some heavy games!" They've also added, "I wish I could get my family/group into games like that." My family does tend to play heavier games because I do. To groups that want to up the complexity at the table, I have a few tips.
Increase Nice and Easy
Every game fits in a particular category of games like "worker placement" or "deck builder." In that category, there are going to be lighter games and heavier games. Start with lighter games in that category and move up. If you want to play Agricola, you may want to start with Lords of Waterdeep or Champions of Midgard. You aren't sacrificing quality with those great games, but you are building skills. Those skills will help your group ease into Agricola or others like it much easier.
Plan to Play Twice
With heavy games, there can be many rules with thick rulebooks to go with them. Some rulebooks aren't the most helpful either. When you group decides to play a heavy game, plan on playing it at least twice in a row. If you have time to do it back to back, that's great. If not, plan on playing it at your next session. This gives you more familiarity with the rules and mechanics. It also allows you to immediately correct the mistakes you are bound to make in the first play through.
Play to Learn
For heavy games, your group should call the first play through a learning experience. Some people are more competitive than others – this we know. Your group would do well to suspend the competition for a game or two. Instead, walk through each others' turns and even help one another make decisions. This seems like a bit much for some, but I promise the experience will pay dividends. It also builds a spirit of helpfulness in the group, which is much better than other vibes a group might produce. What is your favorite heavy game and why? How has your group approached playing them?
What is your favorite heavy game and why? How has your group approached playing them?


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Exclusive: Today FM star Louise Duffy on marriage to Paul Galvin and her latest career move
2016 is a year of change for Louise Duffy.
Five months after marrying former Kerry footballer Paul Galvin, the popular Today FM broadcaster is preparing for her next big move - moving from her afternoon programme to a later time slot and launching her first website.
Louise moved from the early morning show to the afternoon slot formerly presented by the late Tony Fenton last year.
Not content with conquering broadcasting, Louise has her sights set on dominating the fashion world this summer. Naturally, the Mayo native is prepared for comparisons to her husband's online fashion presence, but she hasn't been seeking his advice in her latest business venture.
Read more: GAA star Paul Galvin and Today FM's Louise Duffy wed in 'magical' ceremony in her Mayo hometown
"He's got a menswear fashion website – it was inspired from that. I was looking at him thinking, 'Hmmm maybe I could get involved there', but I don't have much experience or much love for men's fashion," she said.
"I spend my evenings online looking constantly from high end to bargains. I spotted what he was doing but there's room for some more for females in the online industry."
"I think it's important to keep work and home life separate, but if we're doing the same thing there will be an overlap. He's in Dunnes all the time, so he's busy enough without me going to him for help."
They might be one of Ireland's most influential couples, but the 32-year-old said she doesn't seek out the limelight when it comes to stepping out with her other half. In the early days of their romance in 2011, paparazzi photos would often emerge of them holding hands in Dublin and while the invasion may have waned, Duffy said she was taken aback by the media interest.
"We keep work and home very separate. We don't go to many events together, we have all our pals from the year dot and that's how we've always been," she told Independent.ie.
"We're probably a bit odd. I remember when I started going out with him, that was something that fazed me hugely but now it's not an issue really. Ireland doesn't really foster that culture - everyone lets everyone do their own thing."
"I would be perplexed that anyone would give a damn," she laughed. "It wouldn't be something that would ever feature in our lives at all."
Galvin may have just announced his return to GAA - playing hurling for Kilmacud Crokes - and Duffy said he was "getting a bit antsy" after he announced his retirement from Kerry football.
Read more: Paul Galvin insists he won't upstage bride Louise Duffy on their wedding day
"He is just too young for the couch, he was getting a bit antsy sitting in with me every evening. He's excited about that and hurling is something he's always been more fond of, so he is excited to get involved again," she explained.
"It's a big transition for someone to stop doing what they've been doing all their lives, he's just excited to get back there and play hurling."
Kilmacud might be famous for its family oriented activities, the pair are focusing on married life and are in no rush to have children.
"You would hope that would be something down the line, but right now it's about work and it's nice to be married for awhile," she said. "It's not something we'll be organising anytime soon."
Louise has announced exclusively in Independent.ie she will be moving her afternoon radio show on Today FM to 7-9pm to explore a more alternative music roster on air and focus on launching her own fashion website in July. Fergal D'Arcy will be stepping into her afternoon slot.
"I've got a busy summer because I'm launching a new website – it's called Women's Wear Story where music meets fashion," she explained.
"I don't think there's too many websites dedicated solely to music and fashion so I'm hoping to launch. It's going to launch the last day of my midday show so then I'm going to take a few weeks off to give it some love then I'm back on Today FM in August."
Read more: 'It boils down to being best friends', Louise Duffy gushes over fiancé Paul Galvin
While she is prepared for critics of her latest move, Duffy said she is more concerned with getting to indulge in both of her passions and get paid to do it.
"My main passion has always been radio but in the industry we're in, people need to diversify and go out of the normal 9-5," she told us.
"It's moreso about indulging both passions. I'm online every evening so it's great to have an opportunity to set them up. I went to Today FM with my idea and they were so good to give me the opportunity to move to an easier way of life.
"If I was to do daytime and do the website, it would be a major juggle and it would be quite difficult. This way I get the best of both worlds."
Shot on location at the Radisson Blu Royal Hotel on Dublin's Golden Lane
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Fine Art for Kids: Staying in the lines with Robert Delaunay
It's been raining for days, and today we all got a bit stir crazy. While the husband busied himself cleaning the garage, the tot and I sat down with an art book she had pulled off the shelf and looked at the colorful pages. When we found Robert Delaunay's page, she couldn't stop talking about all those shapes — and the colors! I asked if she'd be interested in making her own circle-filled artwork and she was so excited she started pulling out all the art supplies.
The artwork in question that caught my daughter's attention was Hommage à Blériot, 1914, filled with repeated circles interrupted with diagonal and vertical lines. The painting is Delaunay's abstract interpretation of the first flight across the English Channel by Louis Blériot and is playful and vibrant.
After my tot gave me her input on the painting, she was ready to get started.
I gathered several circular-shaped items, along with a sheet of paper and a pencil, for my tot to position and trace around for creating the outline for the creation. I helped with first two and then allowed her to continue to work on her own.
Next I offered a ruler for creating the long vertical and horizontal lines. She did a great job using the ruler to focus on drawing lines. If you've got an older tot, this is a great opportunity to discuss overlapping shapes and how to divide them with the lines to create an abstract design, just like Delaunay.
Next I offered my daughter a set of watercolors and encouraged her to paint within the lines — and fill the entire paper with color, just like the painting. I offered a bit of assistance, and lots of motivation, and she continued working until the creation was finished.
As soon as I removed the painting, she found another sheet of paper and started drawing circles for another creation…
This is a great art activity for encouraging fine motor skills and concentration, as well as introducing color theory and composition. Younger kids can explore the challenge of tracing around the circular objects while older ones can focus on creating an abstract artwork using circles and lines while balancing the color within the painting.
My tot is almost four and she spent just about an hour focused and entertained with the project.
It was awesome.
Tinkerlab: A Hands-On Guide for Little Inventors
My child loves to invent, explore, and create and we've been doing fun activities together since she was just a wee tot. Now that she's a little older, she is starting to experiment on her own. I love watching her make connections and try new things — and then taking pride in the results. But we sometimes get a bit stuck, searching for new adventures and activities to explore together.
If you're not sure where to start or how to get the creative juices flowing (because it can totally be a challenge!), I have the book for you. Rachelle Doorley, the smart and super-talented mama over at Tinkerlab, shares everything you need in her new book, Tinkerlab: A Hands-On Guide for Little Inventors. And what I love about Doorley's book is it's so easy to read, motivating you to start creating right away.
Along with sharing ideas for exciting activities you can do with your child, Rachelle shares tips for starting the process, creating the right space for your child's tinkering, and ideas for getting kids involved — even with the clean-up process. Doorley also includes interviews with experts, such as education professors and nursery school directors, sharing personal stories and experiences that add just the right touch of guidance and coaching throughout the book.
This book? It's awesome. It's motivating. It's encouraging, smart, exciting, fun, silly, and also simple. Make your own paste and mix with paint for creating textured designs? Yes. Construct structures with gumdrops and toothpicks? Of course. Take apart an old computer? Sure! You don't have to be an expert to do these activities with your child — just a willing participant in the journey of discovery. You might find yourself learning something new too.
Buy your copy here.
Just so you know, I wasn't asked by anyone or compensated in any way to write this post. I purchased the book all on my own because I knew it would be beneficial — and read from cover to cover. OH and I knew I would USE the information in the book with my child. (And I have.)
The Birch Aquarium
While we were down south, there were a couple of days the weather wasn't right for hitting the beach, so we explored the area for a few fun things to do. And we found ourselves heading to La Jolla for a stroll through the public exploration center for the world-renowned Scripps Institute of Oceanography at UC San Diego Birch Aquarium.
I enjoyed the informative guides lurking (well, not lurking, but roaming in the darkness of the aquarium) and ready to answer any questions or share fun tidbits of information. We all became quite intrigued with the eels in the above left photo, and were told all about their amazing lives together (yup, that's a proud mama and papa), how they've produced hundreds of baby eels, and are often found nestled together in the tank. But the collection of jellyfish just about put my tot in a tizzy…
Located next to each tank are helpful plaques sharing facts about the fish and other sea life, perfect for us adults that have no clue what we're looking and then can utterly amaze our not-yet-scholarly tots by reading (without them realizing it) bountiful information about the amazing sea creatures. And it's great for older kids, allowing them the opportunity to learn more on their own. We loved this huge tank filled with coastal sea life.
And part of the aquarium is dedicated to educational and interactive learning about the sea and California coastline. Most of it was totally advanced for our daughter, but that didn't stop her from putting her hands on everything.
But the seahorses stole the show. We were all impressed and amazed by these little creatures. A whole wing is dedicated to educating the public about the lives of seahorses, sharing how the aquarium is helping to nurture and build their numbers, and showcasing the amazing beauty of the cool sea animal.
And we had such a great time, we ended up walking (well, running) through the aquarium one last time at the tot's insistence to see it all over again. Along with the indoor tanks, the Birch Aquarium has outdoor interactive tide pools, a shark tank (not interactive), and some really cool solar activities for the kids to check out. Perfect for kids of all ages, the aquarium has something for everyone, is easy to navigate, has a small outdoor snack bar on site, a gift shop, and wonderfully friendly staff. If you're in the La Jolla/San Diego area, it's a fun way to spend an overcast day.
*If you're not from the area, please, PLEASE, make sure to have directions before you head out to La Jolla. No joke, every time we go to La Jolla from the north, we get lost. It's really not crazy difficult or anything, there are just a few twists and turns that can catch you by surprise.
We are in the now.
We decided long ago that we would take a vacation this week. I prepped by finding the perfect person to hang with our cats (so we wouldn't be constantly worried about them and not relaxing), I put in extra time last week so most of my work would be done, and I planned our return so we'd have one full day to recoup before revisiting the real world. And then, at the last minute, we put a crazy spin on things by leaving a day early, driving the seven hour trek in the afternoon instead of waking up in the wee hours to make the jaunt.
It was totally worth it.
We got here.
And guess what?
The internet connection sucks.
I found a cafe that I can head to in the early mornings to catch 20-minutes of work while sipping my vacation mocha before the sun gets warm and the beach is calling, but the rest of the hullabaloo is off.
There's no Twitter.
No Facebook.
At first my fingers were twitching — I started freaking that there would be something missed, an urgent email let go, a pin that needed instant repining, a TWEET THAT WAS AWESOME, FACEBOOK, EMAIL, TWITTER, AAAAAAHHHHHH!
I'm unplugging for the week.
I think it's worth it.
See ya later.
Repurposed artwork thank you cards
I don't know about you, but I've got a drawer that's always overflowing with my tot's artwork. I save her creations from preschool, the fun doodles she makes on her own at home, and the exciting artworks we create together. She sometimes selects ones to display on the fridge and I stash others to show her when she's all grown up. The other day the drawer was so full it wouldn't close, meaning it was time to go weed through the creations. It's hard trashing your child's art, but before making the final cut, we used a stack for creating thank you cards to send to friends and family thanking them for all the wonderful holiday gifts.
This is a great activity to do with tots just starting to figure out how to cut, which results in funky shapes and designs. Children as little as two can wield scissors with adult supervision, and cutting boosts your child's budding fine motor skills. Older kids can enjoy cutting shapes for creating cards with patterns or representational forms. Right now my daughter is still figuring out the whole "cutting" thing, but has mastered the open-mouthed concentration while working…
After you have a nice stack of shapes and funky forms, offer your child some glue to coat the cut paper and press onto the front of blank cards, which you can find at your local craft store.
To personalize the cards, offer your child a thank you stamp to press on the front of cards. Older kids can use letter stamps to press "thank you" on the cards or even write the letters themselves.
We had a few other fun graphic stamps that my tot pressed around her cards, along with in the inside as her signature. We sat together and talked about the gifts she had received, and who they had come from, while I wrote short notes from what she shared in the cards.
After addressing the cards and sticking on stamps, we popped them in the mailbox and waited for our mail lady to come and whisk them away. At first my daughter was a little sad they were being taken but then we talked about how the mail works and how everyone was going to love opening her special thank you cards.
Along with being a great art activity, this is a really fun way of introducing the mail system to your child. Since creating her cards, we've set up our own post office and she's wonderfully busy making cards, folding them into envelopes, and addressing and stamping them herself.
Fisher Price 4x Digital Zoom Digital Camera
Now that the holidays are over and everything is getting back to normal after all the crazed present opening, it's interesting to see which items hold the tot's interest. She still loves all the plastic animals and dinosaurs, and playing the matching game in her own special way, but the one thing that she's still reaching for is her new camera. A few months ago I did a post for LilSugar sharing cameras fit for little hands and had focused on suggestions from friends and good reviews when I put the collection together. So when I decided to get one for the tot as a special birthday gift (her special day is right after the holidays), I already had one in mind.
And then I went with something totally different.
The Fisher Price 4x digital zoom digital camera.
It was pink, I liked the square shape, and although it didn't get great reviews on image quality, the camera sounded like it was indestructible. Hey, I'm not looking for museum-quality prints, I want a camera that isn't going to break the first time it gets dropped.
And it was on sale.
Today I wrangled the camera out of my daughter's hands and downloaded the pictures (SIDE NOTE – the camera does NOT come with a USB cable, but does work with a standard siz. If you're not PC (like me) you can't download the funky photo editing stuff, but, hey, my tot wouldn't have a clue how to use it.) and was surprised and impressed with what she'd taken pictures of and the quality of the camera. My daughter loved seeing her pictures and helped me select a few favorites:
Here's a picture of the hubs and I,
her new tent,
the decorations from her birthday party,
Sid the Science Kid,
our cat by the fire,
her spot on the couch,
and a self portrait.
So, yeah, she's not great on standing still while snapping, but she's dropped the camera, bashed it into walls, been walking while taking a picture and fallen down with it, and I think it even took a tumble down the stairs the other day.
Still works wonderfully.
The picture files aren't big (640 by 480 pixels), but prints good quality 4 by 6-inch pictures. The camera also makes slightly annoying noises when in use but does turn off on it's own so you don't burn through batteries. Equipped with an easy to use zoom (which my daughter totally doesn't get) my husband enjoys taking pictures with it too.
I love that she is still excited about taking pictures and is learning to look at things in a new and interesting way. I'm planning on printing out her favorites and creating a gallery wall in her room. I'll keep you posted…
Fine art for kids: Cardboard creations with Victor Vasarely
After the holidays there always seems to be a large amount of cardboard hanging around my house. From packages sent, to boxes that held toys, I never want to just toss it in the recycling. So the other day I decided to have a little fun with the tot and get creative. There's nothing like presenting your child with various materials and letting her explore, but I decided to offer a bit of assistance.
Victor Vasarely is recognized as one of the foremost artists of the Op Art movement of the 20th century. With his pulsating geometric designs that move the eye, his artworks are vibrant and fun — perfect for introducing to little ones. I figured we could create our own unique creation based on Vasarely's style, with the help of all that cardboard. Instead of creating a flat artwork that looked like it was jumping out at you, we could use the cardboard to layer and build a three-dimensional project. And this art activity is wonderful for encouraging shape and color recognition.
We took a closer look at one of Vasarely's artworks (Pal-Ket 1973-4), talking about the shapes and colors we saw…
I helped by cutting the larger pieces into smaller interesting shapes and then offered my daughter the pieces. Before I had even offered her the glue, she was busy arranging her shapes.
Now your child can glue the shapes onto the cardboard base in an interesting unique design or use one of Vasarely's artworks as inspiration. Encourage your child to use the whole area of the cardboard base and to overlap shapes, building a really fun creation.
Once the glue was dry, we got out the paints and had a great time finishing the creation using the colors from the original artwork.
Display your child's finished creation in a special spot for everyone to enjoy.
Kid-friendly holiday cookies: Red velvet sprinkles
My tot is in love with the color red. She had to have red shoes, only uses red paint when getting creative, and wants us to paint freshly painted white walls of her room red. So I wasn't at all surprised when she decided we just HAD to make RED cookies to share with all her friends for Christermax. Sure, I could've added red food coloring to basic sugar cookie dough, but wanted to make something not-so sweet that also had a nice holiday flavor. We mixed together a lovely red velvet base, added sparkling red sprinkles, and created a delectably crisp and balanced holiday cookie that's perfect for making with your little ones. And these cookies aren't too sweet, or filled with too much butter, making them a healthier option than that cookie dough you can pick up the in refrigerator section.
1/2 cup room temperature butter
1/4 cup vegetable oil
3/4 cup sugar
1 teaspoon vanilla
1 egg
1/2 cup milk
1 teaspoon apple cider vinegar (or white vinegar)
1 tablespoon (plus) red food coloring
1/4 cup cocoa
1/2 teaspoon baking soda
1 teaspoon baking powder
2 cups flour
Preheat your oven to 375F and gather the ingredients for making the cookies. Red velvet cake is a seriously yummy combination of vanilla, chocolate, a bit of tang, and a whole lot of red. I absolutely love red velvet cake, and with it's vibrant red color, figured this cookie rendition would be perfect for celebrating the season.
Invite your child to help measure and add the butter, oil, sugar, and vanilla to a mixing bowl. Use a hand blender to mix everything together until it's nice and smooth. I even let my tot use the blender for a brief moment (while holding with one hand AND attempting to take a picture) but quickly realized that toddlers and hand blenders might not be such a good idea. We'll try again when she's a bit older…
Now add the egg, milk, and the vanilla, giving things another mix. Hold off on adding the vinegar, because if you add it at this point, the milk will separate and things will look really gross (your cookies will still taste good – don't worry).
Sift (or mix) together the flour, baking soda, baking powder, and cocoa and then add about one-fourth of the dry ingredients to the batter. Give things a stir until incorporated and then add the vinegar and a good amount of red food coloring. If you want the cookies on the darker side, add another few drops of the food coloring.
Continue adding the dry ingredients until all incorporated. As you can see, my daughter really took stirring seriously.
Cover a sheet tray with parchment and invite your tot to drop spoonfuls of batter on the pan, allowing an inch or two between cookies. Now your child can douse the cookies with the red sprinkles.
Offer your child a clean glass to gently press on the cookies creating sprinkled rounds of dough. Yeah, they aren't going to be perfect, but that's how it goes when you're cooking with little ones. It's about the experience, not the super pretty outcome — but it's sure a plus when things taste good!
Pop the cookies in the oven for 12 to 15 minutes, or until the tops are cracked. If you prefer a chewy-er cookie, leave in for 9 minutes or so. But these are really good nice and crisp with a tender center, which is what we discovered after about 12 minutes of baking. Remove from the oven and top with more sprinkles or even a dusting of powdered sugar before serving.
Kehoe Beach (on Thanksgiving)
I'd normally be sharing pictures and recipes from my Thanksgiving feast right about now, but this year we are postponing our turkey making until the weekend. Instead we acted like today was just like any other day, which didn't stop us from being seriously thankful. It turned out to be a glorious day, making a trip to the beach the perfect way to spend the day together as a family. Kehoe Beach is tucked away in West Marin past Olema and through some of the most glorious cow fields I've ever seen — and I'm from Wisconsin.
After walking a short trail, flanked by some of those seriously happy cows, you come over a ridge to spectacular stretch of beach that seems endless, and it is also dog friendly. This was the first time we'd taken the tot on a "hike" and she loved every minute of it. We checked out worms on the trail, barnacles on the beach rocks, inspected the various footprints in the sand, and determined the water was way too cold for any swimming…
Happy Thanksgiving — I hope you all had a wonderful day!
(Get ready for turkey pictures on Sunday…)
*And thanks to the husband for the awesome pictures.
Spicy chicken pot pie
There's nothing like a dinner that makes the whole family happy, which is harder to pull off than you might think. My tot isn't excited about this or the husband isn't in the mood for that. So the other night, when I tossed together this seriously simple dinner, I was amazed when everyone asked for seconds — and thirds.
Along with being healthy and a go-to weeknight dinner, this dish also cooks in one pan, making it fitting with this month's Shine Supper Club Challenge. With tasty one-pot dishes as the focus, I knew my twist on chicken pot pie would be perfect. With a little help from pre-made cornbread mix, a can of beans, fresh veggies, and my new Calphalon 3-quart sauté pan, this spicy twist on the traditional recipe is a fun dinner and makes enough for lunch the next day. With Thanksgiving right around the corner, you could also swap the chicken for shredded leftover turkey, which would also be seriously delish.
1/2 teaspoon vegetable oil
1 pound skinless boneless chicken
1 small onion
2 carrots
2 ribs celery
1 clove garlic
1/4 teaspoon salt
1/4 teaspoon paprika
1/4 teaspoon oregano
1 15-ounce can pinto beans
1 teaspoon apple cider vinegar
Hot sauce
1/2 cup salsa
1/4 cup water
1 teaspoon flour
1/2 cup shredded Monterey Jack cheese
1 8.5-ounce box cornbread mix
1 egg
1/3 cup milk
Place a 3-quart sauté pan over medium heat and drizzle with vegetable oil. Chop the onion, carrot, and celery and add to the pan, giving a stir.
Once the vegetables have cooked for four to five minutes, chop and add the garlic as well as the chicken. Give things a stir and then sprinkle with the salt, paprika, and oregano. Allow the ingredients to simmer together for around five minutes, or until the chicken starts to brown.
Deglaze the pan with the apple cider vinegar then strain and add the beans. Go ahead and add the salsa and water, creating a nice bubbling pan of goodness. Let the mixture simmer while you grate the cheese and make the cornbread muffin batter.
Preheat your oven to 425F. Follow the directions on the cornbread muffin box, mixing with the milk and egg. Don't forget to grate the cheese, and you could totally use cheddar instead of Monterey Jack, which would be tasty too.
Stir the chicken and then turn off the heat. Sprinkle with the flour and mix together until it's incorporated. The flour will help pull the liquid together, creating a really flavorful sauce. You can also add a few dashes of hot sauce if you like things nice and spicy.
Sprinkle the cheese over the mixture and then cover with the cornbread mix, using a spoon to spread the batter evenly around the pan.
Pop the spicy chicken pot pie in the oven and bake for 20-minutes or until the top is golden brown.
Serve with extra salsa, a dollop of sour cream, a fresh salad — or all on its own for a delicious weeknight dinner.


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The Family and Matrimonial Law practice of O'Connell and Aronowitz offers experienced counsel to individuals and families. Our attorneys have handled hundreds of cases involving complex custody, visitation and support issues in many counties throughout New York State.
We also provide representation with respect to family offense/domestic violence proceedings, paternity, Juvenile Delinquency and Persons in Need of Supervision proceedings, abuse and neglect allegations, adoption, and name change petitions.
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Our attorneys also provide consultation and drafting of pre-nuptial agreements, pre-marital asset planning and representation in enforcement proceedings.
As an adjunct to our Family Law practice, we provide post-judgment services in the fields of estate planning, taxation, and bankruptcy. Our Family and Matrimonial Law practice is active in our Albany, Latham, Plattsburgh, and Saratoga Springs offices.
Related Practice Areas: Trusts, Estates and Estate Planning, Tax Law, Bankruptcy
For more information, contact Will Berglund in our Albany and Saratoga offices and Kelly J. Mikullitz in our Albany office.


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Yup, got ma brand nu NetBeans 6.1 up-n-runnin. As mi advanced Emacs user have to declare that tha Emacs-friendly keymap shipped with NetBeans is a nightmare.
I tried to type some JSP tags in the source, and had to realize that NetBeans can't handle keys with Alt-GR. After some googling it, I found that it ain't the problem of NetBeans but Java Swing, Java 1.6, the Linux platform itself, or all of them glued together. You may find a lot of different workarounds—get lower JDK version; install scim; export AWT_TOOLKIT=MToolkit; etc. The one that worked told me to reset locale environment variables kinda like this:
# LC_ALL=C /opt/netbeans-6.1/bin/netbeans
Happy coding. (Mi keep stayin in Emacs for a while, though.)


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Every kid who grew up on Pokémon had the same wish-to be able to go out in to the world on their very own adventure, and of course, to catch 'em all. When the games first hit the shelves in 1995, that wasn't exactly possible with the available technology. But now, in an era where people carry computers more powerful than those that got us to the moon in their pockets, Nintendo is finally granting fans their lifelong wish to become a traveling Pokemon Master.
Pokemon GO started its Australia/New Zealand beta two days ago, and you can bet fans worldwide are jealous. Fortunately, one of our friends down under has us covered, and gave us these nine awesome minutes to see just what being a trainer on the go is all about.
Some pretty interesting features were showcased in the video. Character customization, which was received very well when it was introduced in Pokemon X and Pokemon Y, makes an appearance early on. The GO version of gyms work a bit like capture points, allowing trainers to place Pokémon in them to hold them, and defeat other trainers' defending Pokémon to claim them for themselves. One of the more surprising inclusions is that these gyms aren't just for you. Each trainer picks a team: Blue, Red, or Yellow. Gyms will presumably earn points for the teams at large, and likely rewards as well at certain intervals. Various items can be found at minor landmarks, as well.
In the end, though, we're all there for the battles. Players can flick a Pokéball at the various monsters they come across in their real world travels. When encountering Pokémon or engaging in battles, trainers also have the option to switch the background to real-time video through their device's camera, which adds a surreal augmented reality element.
It's worth noting the game is definitely unfinished-sound effects, for instance, are completely absent. It's hard to say how much, or how little, is left to add.
The game itself is based off the hiking and exploration sport of Geocaching, which encourages people to get outside and explore the wonderful world around them. If you really want to catch 'em all, you'll have to do more than laps around your front yard. And maybe, just maybe, you'll meet some fellow trainers.


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MOVIE SYNOPSIS [ General Audiences ] Film of Krisjian Vitateaux. Introduced in movie theaters in 2015, with a production coming from USA. You can watch Wonderkind online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2015.
Rating: 5 by 11 users./
Runtime: 100 minutes
Nicholis Spring, Paul Stuart, Benny Barrett, Robert Michael Szot, Lana Marie Haws, Alyssa Broun, Kara Rainer, Jodie Moore, Gwendolynn Murphy, Erin Whit, Jay DeWolfe, Randy D. Shoemake, Tyler Sutton, Peter Calvin, James Larabee and Charles Reynolds.
Drama, Thriller, Science fiction.
100-minute film (full movie) directed by Krisjian Vitateaux.
The original audio of Wonderkind has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2015.
Screenplay written by Krisjian Vitateaux.
Ross Skrudland and Joshua Spires.
Kara Rainer, Richard Brian Galyean, Jay DeWolfe, Krisjian Vitateaux, Justin William Bailey, Megan DeWolfe, Healani Ah Ching and Lana Marie Haws.
This film was produced and / or financed by LeFratzié Studios.
Composed by Ty Robins, Krisjian Vitateaux and Ben Rothberg.
Without commercial breaks, the full movie Wonderkind has a duration of 100 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Wonderkind in FULLTV Movies.


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Swing Kings
- Artist: The Swing Kings
- Genre: Jazz
- Release Date: 4/22/2003
During the Great Depression and World War II eras of the 1930's and '40's swing music emerged as an American art form and became a social catalyst, bringing people together in celebretion of better times to come. Ballrooms filled to capacity with dancers swinging to the sounds of the Big Bands. The Swing Kings are proud to bring back this good-time music and invite you to dance and enjoy the excitement and romance of those swinging times. Recorded live with todays techonolgy, this high energy, chrystal clear recording is a favorite among swing dancers and big band fans. Singer and bandleader Ron Alan has drawn together a fantastic host of musicians, including; Gary Herbig on tenor sax, who's played with Elvis Presley, Paul McCartney and Michael Jackson; Lee Thornburg on trumpet from Tower of Power, and Huey Lewis', Rod Stewart's and Ringo Starr's bands; Garett Adkins on trombone played with Stevie Wonder, Bruce Springsteen and Natalie Cole; Eric Wangensteen on trumpet has performed with the Gene Krupa big band. The rhythm section includes: bass player Ralph Carter who played with Eddie Money for six years; Robert Beifuss on guitar, winner of the San Francisco Bay Critics Award for musical direction in theater; Mike Telesmanick on piano who has performed with jazz greats Jack De Johnette, Joe Pass and vocal group, the Manhattan Transfer; and on drums Dean Giles.
|Swing Kings
|The Swing Kings
|CD Baby
|Release Date:
|Product Type:
|Item #:
|This product is a special order


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Change of
Price Delayed
How to Point a Static Domain to Your Home Computer Part 3: Setting up ZoneEdit to point your .com domain to your home computer
Last time we took an in-depth look at DynDNS.org's free sub domain service. Today we are going to look ZoneEdit's free DNS service for the crowd that has their own .com domain.
Step One: Register a Domain
If you already registered a domain, skip to step two. If you haven't registered a domain yet and are looking to get your own domain, use our preferred registrar Dotster. They also offer DNS services for the site, but we will get into that later.
Step Two: change the DNS servers your domain point to
The first step you will need to take before you use ZoneEdit is to change the DNS Servers your domain points to. Log into your domain registrars control panel, and under manage DNS Servers change them to ns3.zoneedit.com and ns4.zoneedit.com.
Step Three: Signup with ZoneEdit.com
Now go to ZoneEdit.com and signup for an account. Remember that you will have to have the same contact info that you use for your domain to use ZoneEdit.
Step Four: Adding a Zone/Domain
Now that you have an account with ZoneEdit, it is time to add a zone. Login and click on add zone. Enter the domain you want to use with ZoneEdit (example yoursite.com).
Step Five: Pointing your domain to your home computer
Now the big moment, lets point your domain to your home computer. Login and click on the domain you want to point to your computer. Now click on IP Address and enter www as the sub domain and then the IP of your computer (to determine the IP of your computer, open up System Preferences select Network and you will see your IP address. Enter that into the IP field and click add. Confirm your change on the next screen and then your done. Wait a minute and then type in your domain and it should load your website on your home computer if you have web sharing turned on. Contragulations! You now have pointed your domain to your home computer.
Step Six: Automate updating your Dynamic IP
If you have a Dynamic IP (an IP that changes once in a while), and you don't want to have to manually update the IP your domain points to every time your IP changes, then lets automate it.
Download DNS Update, which will monitor your IP and tell ZoneEdit's DNS servers when ever your IP changes without you doing anything. Once you have downloaded DNS Update, drag it to your Applications folder (I have mine in the Utilities folder).
Launch DNS Update and click Add User. Enter your ZoneEdit login and password, and select ZoneEdit from the Service drop down menu.
Now highlight your ZoneEdit user and click add host. Enter your domain name you setup with ZoneEdit. Remember that you must first add the sub domain you want to change the IP for via ZoneEdit's site before you can use DNS Update to automate updating the IP. Also note that for some reason DNS Update doesn't like sub sub domains. Example www.subdomain.domain.com
That's it. You now have pointed your domain to your home computer. If you have any other questions or comments, please post away on our message board.


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With First Tutors you can find a local medicine tutor in Tidworth and across the UK. Home medicine tuition is an excellent way to improve confidence as well as increasing grades and doesn't have to be boring! Please use our service to search for Tidworth medicine tutors and select the one who best fits your needs.
First Tutors is the best place to discover through one search the top Tidworth medicine tutors for your needs, enabling you to find a home Tidworth medicine tutor for any subject, for example, GCSE English, A-Level Maths or even university Chemistry. We also help with language tuition, so if you want to learn German, take a Greek course or prepare yourself for Italian life, we can help - and all in Tidworth! We order medicine Tidworth teachers in your area according to their medicine tuition charges and proximity to you.
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Get the interesting, progressive, and delicious Greek recipes and much more in this simple app. Great Greek Recipes brings together Chefs, Dietitions, and inspirational news to keep cooking Greek food that will wow your family and friends.
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