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Beautiful bread and beautiful you!
Those who follow this blog may recall a post I did back in September, reflecting on my birthday and setting some goals for myself. As many know, I am passionate about all things cooking, baking, and the results of those efforts – EATING! One of my goals was to learn how to make bread, as it's something I have very little experience with.
In November (yes, it took me that long to organize this post…) I attended an Artisan Bread Baking course at the Pastry Training Centre, located in East Vancouver. The class was over two nights, four hours a night from 5:30-9:30pm. The school features a good sized kitchen, with lots of room for about 10 students to get their knead on! Chef Marco comes from a long line of bakers, and started in his family bakery at the age of 15. He worked and traveled throughout Europe as an Executive Pastry Chef in many top restaurants and hotels. In addition to bread baking courses, you can learn sugar art or even take chocolate training. There are seasonal courses too, including one on Christmas cookies.
Being a full time working gal, I had to leave a few minutes early and race to East Van from Delta to get there at 5:30pm – there was no time for dinner, so it was appreciated that Chef Marco and his team had cheese, bread, coffee, tea, water, and sweets for us to pick at on both nights.
The two nights of class were jam-packed full with activity. We made: bagels, hard dinner rolls, carrot bread with herbs and onions, french baguette, red fife whole wheat bread with apricot, almond and fennel, and whole wheat toast bread! All of the ingredients were measured out for us ahead of time, otherwise, I doubt that amount of bread baking would be possible in 8 hours.
I was most proud of how the dinner rolls turned out. Chef Marco showed us different ways to roll and prepare them, so we had fun making our own little shapes. Taste wise, my favourite was the carrot bread with herbs and onions – delicious dipped in balsamic and olive oil.The baguette turned out pretty well too – it kept well frozen and we enjoyed it one night when family came over.
At $200, it was good value for the money. I left not only with new skills and knowledge, but with about a month's worth of bread! Chef Marco was informative and entertaining. The premises were clean and perfectly set up for a group of 8-10 students. I especially liked the fact that you don't have to clean up after yourself, Marco had an assistant to help us with that. Clean up is certainly my least favourite part of baking. =)
I really enjoyed the course, and would recommend it to anyone who has an interest in baking.
Beautiful bread and beautiful you!
Get your knead on, love it! What a great course. xx


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Arts scholarships, grants available
The Bemidji Area Arts Endowment, a component of the Northwest Minnesota Foundation, has announced its 2010 grant and scholarship opportunities.
The BAAE will award $5,000 to schools, nonprofit groups or arts organizations in the Bemidji area.
A maximum of $1,000 may be requested to produce an arts event, organize a workshop, develop an arts organization, sponsor touring arts events or sponsor an artist in residence. Applicants that are not schools or 501(c)3 organizations must use a fiscal agent.
The Everett Wilimek Endowment for Orchestral String Studies will grant two $250 scholarships this year.
The fund was established by Wilimek's family to honor his memory, his love of music and his dedication to helping young musicians. Each year the fund provides scholarships for young people from the Bemidji area, ages 8-15, to study violin, viola, cello or bass, helping to pay the costs for lessons, training and instrument rental.
Applications for the BAAE grants and Wilimek Scholarships are available at the Bemidji Community Art Center, Headwaters School of Music and the Arts, the Region 2 Arts Council and the Northwest Minnesota Foundation, located at 4225 Technology Drive N.W.
Postmark deadline for the application is April 1.
For details, contact Patt Rall at 755-8942 or firstname.lastname@example.com.

subset: commoncrawl


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Does your home reflect a healthy, slim and fit lifestyle? Or are you trying to whittle your waist in a home that's in fact a ghar to disorder and untidiness? We admit it's extremely tough for most of us to make healthy food choices and find the time to exercise when we are constantly running behind. But there are some quick home design ideas and simple organisational tips that can help you feel more stress-free and make fitter decisions in return!
Compiled from houzz.com, weightloss.about.com and the bestseller, Slim by Design, here are a few tips to set your home up for a fitter and slimmer you.
Clean countertops
What exactly does a healthy kitchen counter look like? It's basically full of open space that's ready for diet-friendly food preparation. There are no bags of chips, stacked nimco or visible baked goods. Handbags, toys and other clutter are also not visible. According to Brian Wansink, a professor and researcher at Cornell University, a healthy kitchen counter may have a few neatly organised cooking utensils, a blender for making healthy chutneys and smoothies, and a bowl of nuts or fruit. Wansink also recommends you leave the rest of your counter space empty so when you're ready to cook a healthy meal, you've got plenty of space to work with.
Make place for a fruit bowl
You may already have a measly fruit bowl sitting in your kitchen. But are those bananas looking way too overripe? If you're trying to win the battle against the bulge, or are aiming for a cleaner diet, where you place the fruit bowl makes a big difference. A recent study about habit changes for weight loss found that when people made a commitment to snack only after eating a piece of fruit, most of them lost weight. So where should you put the fruit bowl? "In front of the snack cabinet," shares Wansink. That way, you'll see the fruit first when you are tempted to snack, and an orange is far superior to chocolate fudge brownies — health wise, at least! Wanskink also suggests placing a fruit bowl near your car keys. If you grab a piece of fruit before you run out the door, you'll be less likely to hit the nearest drive-thru when hunger pangs come visit.
5 email etiquette rules everyone should know
Maintain a clean, orderly refrigerator
Smart dieters and fitness fans often clean the middle shelves of the refrigerator and fill them with single serving containers of home-cooked veggies or wholesome foods such as lentil and grilled chicken. A sealed transparent container of hard-boiled eggs should also be a must — make sure to place the eggs in the middle shelf of the fridge. That way, you're more likely to see and grab those healthier items when you raid the refrigerator at odd hours. So what do you do with the foods that used to be stored on the middle shelves? There are certain foods that you should just want to toss away — think sodas. But if your family isn't willing to let those items go just yet, Dr Wansink suggests storing sodas in the vegetable crisper which is usually in a lower, hard-to-reach spot.
Paint your kitchen anything — but white
Okay, this may be hard to digest but according to a survey conducted by Houzz, almost 75% of homeowners around the world prefer a soft and neutral kitchen. But Wansink has found that white and bright spaces tend to stimulate eating. But the opposite spaces are bad too. Really dark rooms with low lighting and soft music tend to slow people down, causing them to linger and eat for an average of nine minutes longer. "The darker the space, the longer you stick around and the more likely you are to break down and have another serving," says Wansink, adding that an in-between colour is best. "Gold, green, blue, tan, earth tones — those are all good. Any colour seems to work other than white or cream."
5 secrets to an impactful resume
Banish a sleep-inducing bedroom
A five-minute bedroom revamp may, in fact, help you sleep better — and a good night's rest may help you to make better food choices during the day according to several recent studies. So what needs to be changed in your bedroom? Make it a habit to never charge any electronics in the bedroom. Move chargers to the lounge or another room in the house. Minimise light from other sources, including the TV. Many good sleepers do so because they've moved the television out of the bedroom entirely. Keep a lavender spray near your bed —Feng shui experts believe that the soothing properties of the essential oil may help you relax and sleep better at night.
Published in The Express Tribune, February 4th, 2016.
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What happens when the high-octane sounds of Bollywood meet the soulful beats of yesteryear? Well you get Botown of course.
For one night only the likes of Ray Charles and Aretha Franklin will mix with the melodies of Kishore Kumar and Asha Bhosle when band Botown come to town.
Songs such as Roop Tera Mastana and Om Shanti Om will get a 1960s style funk makeover as Memphis meets Mumbai at the Rose Theatre.
A saxophone, a trumpet and drums will make for a lively night of music produced by Global Arts Kingston as part of the Festival of Stories at the theatre.
Wendy Smithers, from Kingston Global Arts, says: "People should come along because they are going to experience something they have never experienced before.
"They are going to be dancing in their seats. It is a great introduction to Bollywood."
Botown, Rose Theatre; 24-26 High Street, Kingston; Saturday, June 28, 7.30pm; 020 8174 0090, rosetheatrekingston.org.


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ASo last year I tried to replicate The Christmas Story leg lamp using PVC pipe as a support for the leg. EPIC FAIL! Any suggestions on how to make the rice crispy treats stick to the PVC pipe? I tried using royal icing,,but no luck, the treats just kept slipping off the pipe. Should I try something other than rice crispies?
Thanks in advance! Nicole
Modeling chocolate sticks nicely to PVC. You could try a layer of that and then bulk it up with RKTs.
AThank you! Debating on giving it another try this year. I got so frustrated with it, but will def. try the modeling chocolate if I do. Thanks again!
For use on PVC, I modify the rice treats recipe by omitting butter (makes treats less slippery) and use corn syrup to help them stick. You can view my tutorial here:
AThank you! Will def keep this in mind. I used the premade sheets of rice Krispy treats for a time saver. I'll try my own next time, leaving out butter!


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[Video] Brown Eyed Girls Admit Doing Plastic Surgery in 'Plastic Face' Digital Short
After promising to take tvN's Saturday Night Live Korea (SNLK) to another level, Brown Eyed Girls did just that by addressing plastic surgery out in the open.
On December 15, Brown Eyed Girls revealed the digital short titled Plastic Face, spoofing off of Lady Gaga's Poker Face.
During the short, they sang and rapped about how whether you like it or not, most women in Korea have had done cosmetic procedures and there's no way to avoid it. In the song, they sing, "You think your girlfriend didn't do plastic surgery, huh?"
Check it out!
Photo Credit: tvN
- Tag
- Brown Eyed Soul 20121217 Saturday Night Live Korea 20121217


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The last few months I have been feeling overly inspired, motivated and eager. Today, I hit a wall. Hubby and I launched our passion project The City Flea and have been overwhelmed by the incredible support and enthusiasm. We've even had some interviews and a photo shoot with local media! I think the non stop working, networking, and socializing has caught up to me. I'm tired beyond belief and I've just come down with a horrendous cold and sore throat. I love the above words and even though i'm feeling drained today, I know that Doing and Being Present still need to be a part of my sick days (even if being present only means appreciating and thanking the mister for my tea) Hope everyone has a great week!
I need a reminder
February 28, 2011


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Lu/BCAM (Lutheran/basal cell-adhesion molecule) is a laminin 511/521 receptor expressed in erythroid and endothelial cells, and in epithelial tissues. The RK573–574 (Arg573-Lys574) motif of the Lu/BCAM cytoplasmic domain interacts with αI-spectrin, the main component of the membrane skeleton in red blood cells. In the present paper we report that Lu/BCAM binds to the non-erythroid αII-spectrin via the RK573–574 motif. Alanine substitution of this motif abolished the Lu/BCAM–spectrin interaction, enhanced the half-life of Lu/BCAM at the MDCK (Madin–Darby canine kidney) cell surface, and increased Lu/BCAM-mediated cell adhesion and spreading on laminin 511/521. We have shown that the Lu/BCAM–spectrin interaction mediated actin reorganization during cell adhesion and spreading on laminin 511/521. This interaction was involved in a laminin 511/521-to-actin signalling pathway leading to stress fibre formation. This skeletal rearrangement was associated with an activation of the small GTP-binding protein RhoA, which depended on the integrity of the Lu/BCAM laminin 511/521-binding site. It also required a Lu/BCAM–αII-spectrin interaction, since its disruption decreased stress fibre formation and RhoA activation. We conclude that the Lu/BCAM–spectrin interaction is required for stress fibre formation during cell spreading on laminin 511/521, and that spectrin acts as a signal relay between laminin 511/521 and actin that is involved in actin dynamics.
- actin
- adhesion molecule
- cell adhesion
- laminin
- Lutheran/basal cell-adhesion molecule (Lu/BCAM)
- Madin–Darby canine kidney (MDCK)
- RhoA
- spectrin
Lu (Lutheran)/BCAM (basal cell-adhesion molecule) glycoprotein is a member of the IgSF (Ig superfamily) that carries both the Lu blood group antigens and the BCAM tumoral antigen. It is represented by two isoforms, Lu and Lu(v13), that only differ by the length of their cytoplasmic domain (59 compared with 19 amino acids) [1]. The specific 40 amino acids at the cytoplasmic end of Lu glycoprotein comprise phosphorylation sites [2] and an SH3 (Src homology 3)-binding motif, consistent with a receptor signalling function. Lu/BCAM represents a unique receptor for laminin 511/521 (comprising α5β1γ1 and α5β2γ1 chains respectively) [3] in normal and sickle RBCs (red blood cells) [4–6]. In human blood cells, Lu/BCAM is expressed only in erythrocytes. Lu/BCAM is also present at the basal layer of epithelia, on the surface of a subset of muscle cells and on blood vessel endothelium [7–9]. In these tissues, Lu/BCAM is recognized as a co-receptor with integrins for laminin 511/521 [8]. Disruption of the Lu gene in mouse provided evidence that Lu/BCAM was involved in the maintenance of normal basement-membrane organization in kidney and intestine [10].
In RBCs, Lu/BCAM interacts directly with spectrin through the juxtamembrane RK573–574 (Arg573-Lys574) motif of its cytoplasmic tail [11–13]. Identified at the inner membrane of RBCs, spectrins are considered as the central components of a ubiquitous and complex spectrin–actin scaffold, called the spectrin-based skeleton [14]. This network, attached to diverse cellular membranes, is involved in diverse functions, including the resilience and stability of membranes, the establishment of specialized membrane domains and in vesicle trafficking. Spectrins exist as elongated flexible heterotetramers of 200 nm made up of two α- and β-subunits that constitute the filaments of the network, the nodes of which are cross-linked by actin filaments. In mammals, two genes encode for α-chains (αI and αII) and five for β-chains. Although αI-spectrin is the only isoform expressed in mature RBCs, αII-spectrin is the most common form in nucleated cells. Moreover, αI-spectrin is not expressed in epithelial cells. Each spectrin subunit is organized as an alignment of spectrin repeats, made up of three α-helices each; α-spectrins contain 20 spectrin repeats. In RBCs, the Lu/BCAM-binding site in αI-spectrin has been delimited to the single α4 repeat [15]. As Lu/BCAM represents a minor component of the RBC membrane (from 1.5 to 4 copies/RBC), we have speculated that the Lu/BCAM–spectrin interaction might be critical for cell signalling rather than for maintenance of the membrane mechanical properties [12].
The adhesion properties of Lu/BCAM depend on its cytoplasmic domain phosphorylation, indicating an inside–out activating signal for its laminin 511/521 receptor function [2]. The physiological stress mediator adrenaline (epinephrine), acting through the β2-adrenergic receptor, increases Lu/BCAM-mediated adhesion of sickle RBCs to laminin 511/521 through a PKA (protein kinase A)-dependent pathway [16]. Lu/BCAM phosphorylation by PKA occurs in sickle, but not in normal, RBCs, concomitantly with an enhanced cell adhesion to laminin 511/521 under physiological conditions [2]. In polycythaemia vera patients, who present an increased risk of thrombosis, Lu/BCAM is constitutively phosphorylated, and this feature is associated with an increased RBC adhesion to endothelial laminin 511/521 [17].
In resealed RBC ghosts, disruption of the Lu/BCAM–spectrin interaction by a spectrin peptide encompassing the α4 repeat resulted in a weakened linkage of Lu/BCAM to the spectrin-based skeleton, and induced cell adhesion to laminin 511/521 [15]. Increased adhesion of RBCs from HS (hereditary spherocytosis) patients with a marked deficiency in spectrin, demonstrated the biological relevance of this interaction: intact HS RBCs exhibited reinforced adhesion to laminin 511/521 under physiological conditions that resulted, at least in part, from an impaired interaction between Lu/BCAM and the membrane skeleton [18]. All of these observations indicate that the Lu/BCAM interaction with erythroid spectrin negatively regulates cell adhesion to laminin 511/521.
In the present study, we have shown that Lu/BCAM bound in vitro and ex vivo to non-erythroid αII-spectrin in kidney epithelial cells. This interaction modulated spreading and adhesion of MDCK (Madin–Darby canine kidney) cells to laminin 511/521. Most importantly, we have shown that the Lu/BCAM–spectrin interaction was involved in a laminin 511/521-to actin signalling pathway promoting actin skeleton reorganization.
The protease inhibitor cocktail, microspin GST (glutathione transferase) purification module and Protein A–Sepharose were purchased from Amersham Biosciences. Br-cAMP was from Calbiochem. Primers used in PCR and mutagenesis experiments were from MWG Biotech. The in vitro transcription and translation TNT® T7 quick kit for PCR-amplified DNA was obtained from Promega. Dako was from DakoCytomation. PIC (phosphatase inhibitor cocktail) for serine/threonine phosphatase, a purified human laminin 511/521 mixture, fibronectin and 30% (w/v) BSA were supplied by Sigma. Lab-Tek® II chamber slides™ were from Nalge Nunc. Sulfo-NHS-LC-biotin [sulfo-succinimidyl-6-(biotinamido)hexanoate] and immunopure immobilized streptavidin beads were obtained from Pierce. G-LISA™ RhoA, Rac and Cdc42 (cell division cycle 42) Activation Assay Biochem Kit™ (absorbance-based) and cell-permeant Rho inhibitor (exoenzyme C3 transferase, CT04) were from Cytoskeleton. Image-iTFX signal enhancer, calcein/AM (calcein acetoxymethyl ester), bis-benzimidazole Hoechst 33342, AlexaFluor® 488 Phalloidin and NuPAGE® Novex Bis-Tris gels were purchased from Invitrogen. Cell culture media and reagents were from Gibco BRL. Except when otherwise mentioned, reagents were purchased from Sigma–Aldrich. Transwell polycarbonate filters (12 mm diameter, 0.4 μm pore) were obtained from Costar. Millipore electrical resistance apparatus was from Bedford. The bio-imaging analyser was obtained from Fujifilm-Raytest. An evolution VF camera was obtained from Mediacybergetics. A 490 nm Multiskan RC was purchased from Lab Systems.
Antibodies directed against Lu/BCAM were used: mAb (monoclonal antibody) clone F241 and the rabbit pAb (polyclonal antibody) 602 (collaboration with Dr D. Blanchard, EFS, Nantes and INTS, Paris, France). The immunopurified pAb directed against the αII-spectrin SH3 domain was produced in our laboratory [19]. Other antibodies used were: goat biotinylated anti-Lu/BCAM antibody (R&D systems), sheep anti-human Ubc9 (ubiquitin-conjugating enzyme 9; AG Scientific), and Alexa Fluor® 488-conjugated anti-mouse and Alexa Fluor® 568-conjugated anti-rabbit antibodies (Molecular Probes, Invitrogen).
Cell lines
Human kidney carcinoma epithelial cells A498 (A.T.C.C., HB44) were maintained in MEM (minimal essential medium) Glutamax I supplemented with 10% FBS (fetal bovine serum), 100 units/ml antibiotic/antimycotic, 1 mM sodium pyruvate and 0.1 mM non-essential amino acids. MDCK cells (A.T.C.C., CCL-34) were maintained in DMEM (Dulbecco's modified Eagle's medium) Glutamax I supplemented with 10% FBS, 100 units/ml antibiotic/antimycotic and 0.1 mM non-essential amino acids. Both cell lines were grown in a humidified atmosphere at 37 °C with 5% CO2.
The full-length wt (wild-type) cDNA sequence of Lu/BCAM (wt-Lu) and bearing RK573-574AA (Arg573→Ala/Lys574→Ala; mt-Lu) (+1 is taken as the methionine residue of the initiation codon) were cloned into the pcDNA3 expression vector as described previously [12,20]. A D343R mutation (+1 is taken as the methionine residue of the initiation codon; the mutated residue is the same as residue 312 in reference [21] where +1 was taken as the first residue of the mature protein) was obtained by in vitro mutagenesis using the sense primer 5′-AGAGTGGAGGATTACCGGGCGGCAGATGACGTG-3′, and the antisense primer 5′-CACGTCATCTGCCGCCCGGTAATCCTCCACTCT-3′. Stable MDCK cells expressing wt and mutant Lu/BCAM, were obtained and amplified as described previously [22]. To obtain clones showing similar Lu antigen membrane expression [SABC (specific antibody-binding capacity) units], cell clones were selected by serial limit dilution [22]. The SABC for wt-Lu and for mt-Lu were 456.337 and 401.887 respectively.
Protein extraction from MDCK cells
After trypsin treatment, the pellet corresponding to 2×106 MDCK cells was incubated for 1 h at 4 °C in 400 μl of lysis buffer [20 mM Tris/HCl (pH 8), 150 mM NaCl, 5 mM EDTA, 0.2% BSA and 0.010–0.030% Triton X-100 supplemented with protease inhibitor cocktail]. After centrifugation (15000 g for 15 min at 4 °C), the soluble cell extract (S) was loaded on to a 4–12% NuPAGE® Novex Bis-Tris gel under reducing conditions. Proteins were analysed by Western blot using a goat biotinylated anti-Lu/BCAM antibody (0.1 μg/ml) and sheep anti-human Ubc9 antibody (1:3000 dilution) as an extraction control.
Co-immunoprecipitation assay
Cells (1×107) were lysed for 30 min on ice in lysis buffer [20 mM Tris/HCl (pH 8), 500 mM NaCl, 2 mM MgCl2 and 1% Triton X-100 supplemented with protease inhibitor cocktail]. Cell lysates were then sonicated (30 s pulse 'high', 30 s rest) and centrifuged for 10 min at 11000 g.
Lu/BCAM was immunoprecipitated by incubating the supernatants with mouse anti-Lu mAb F241, an irrelevant antibody (mAb anti-IgG1) or only with Protein A–Sepharose as a negative control overnight at 4 °C. The beads were centrifuged at 11000 g for 20 min, and were washed five times with wash buffer 1 [20 mM Tris/HCl (pH 7.4), 600 mM NaCl, 2 mM MgCl2 and 1% Triton X-100 supplemented with protease inhibitor cocktail] and twice with buffer 2 [20 mM Tris/HCl (pH 7.4)] to remove traces of detergent. Immunoprecipitated proteins were first eluted with glycine/HCl buffer (pH 3.2) and then equilibrated in Tris/HCl buffer (pH 9) for incubation for 5 min. Samples were loaded on to 4–12% NuPAGE® Novex Bis-Tris gels under reducing conditions. Western blot analyses were performed using the anti-αII-spectrin SH3 domain pAb (1:100000 dilution) or goat biotinylated anti-Lu/BCAM antibody (at 0.1 μg/ml).
GST pull-down assays
GST peptides were produced as described previously [12] and immobilized on Sepharose-4B–glutathione bead microspin columns. Briefly, PCR-amplified cDNA fragments encoding the C-terminal ends of Lu/BCAM (residues 569–628), were fused with the GST protein in the pGEX-5X-3 plasmid. mt-Lu RK573-574AA was obtained by in vitro mutagenesis as described previously [12]. Spectrin repeat units corresponding to αIR3 amino acids (Ala253-Gly384), αIIR3 (Gln255-Leu382),h αIR4 (Ser361-Glu486) and αIIR4 (Glu361-Glu486), αIR3-5 (Ala253-Leu594), αIIR3-5 (Gln255-Leu594), αIR5 (Asp466-Leu594) and αIIR5 (Leu467-Leu594) were obtained as a [35S]methionine-labelled protein with the in vitro transcription and translation TNT® T7 quick kit for PCR-amplified DNA.
The products (50 μl) were incubated overnight at 4 °C with GST–wt-Lu and GST–mt-Lu constructs (100 μg) immobilized on Sepharose 4B–glutathione beads equilibrated in PBS and antiproteases. After five washes with PBS/0.05% Tween 20, the bound proteins were solubilized in Laemmli sample buffer [23], loaded on to a 4–12% NuPAGE® Novex Bis-Tris gel, visualized and quantified by Kodak ID Image analysis software.
Confocal fluorescence microscopy and immunofluorescence of transfected cells
The transepithelial resistance was measured daily on stably transfected MDCK cells grown on polycarbonate filters (5×105 cells/filter). At day 7, polarized cells were fixed for 20 min with 4% paraformaldehyde, treated with 50 mM ammonium chloride in PBS, permeabilized for 10 min with 0.5% Triton X-100 (in PBS) and incubated with the anti-Lu mAb F241 (1:10 dilution) and the anti-αII-spectrin SH3 domain pAb (1:500 dilution) in Dako for 1 h at room temperature (22 °C). Filters were washed with PBS/0.5% BSA and incubated with AlexaFluor® 488- and 568-conjugated anti-mouse and anti-rabbit antibodies (1:200) for 1 h at room temperature in PBS/0.5% BSA. Samples were examined by confocal microscopy using a Nikon EC-1 system equipped with 60×NA (numerical aperture) 1.4 and 100×1.30 objectives.
Lab-Tek® II Chamber Slides™ were coated with 2 μg/cm2 of either laminin 511/521 or fibronectin overnight at 4 °C (in 400 μl). Wells were then washed twice with PBS and were subsequently coated with 1% BSA at 37 °C for 1 h before two additional washes. Mock-transfected MDCK cells (104) or transfected cells expressing wt-Lu, mt-Lu, the D343R Lu mutant or Lu(v13) were added to the wells (in 400 μl). Cells were incubated in serum-free medium for 90 min at 37 °C in the presence or absence of 1 μg/ml per well of the cell-penetrating form of the Clostridium botulinum C3 toxin, an ADP ribosyltransferase that selectively ribosylates Rho proteins, rendering them inactive. After this incubation time, cells were fixed for 20 min with 4% paraformaldehyde, treated with 50 mM ammonium chloride in PBS, permeabilized for 10 min with 0.5% Triton X-100 (in PBS) and saturated for 30 min in Image-iTFX signal enhancer solution. Cells were washed with PBS/0.5% BSA and incubated with Phalloidin AlexaFluor® 488 (1:50 dilution) for 1 h at room temperature. Samples were examined by confocal microscopy.
Cell-surface biotinylation assay
Polarized MDCK monolayers expressing either wt-Lu or mt-Lu were cultured on filters as described above. Newly synthesized proteins were labelled by adding 150 μCi of [35S]methionine/[35S]cysteine in the cell culture medium for 20 min at 37 °C. After washing with complete medium, cells were incubated in non-radioactive medium for 60, 90, 120, 150 and 180 min at 37 °C. After each incubation time, cells were washed twice with ice-cold PBS and incubated on the basolateral side with 0.5 mg/ml Sulfo-NHS-LC-biotin. After immunoprecipitation of total Lu/BCAM, half of the eluted proteins was diluted with 3× Laemmli buffer (total Lu/BCAM), and the other half was incubated in 500 μl of lysis buffer for 3 h with immunopure immobilized streptavidin beads to isolate membrane Lu/BCAM. Beads were washed three times with lysis buffer and Lu/BCAM was eluted in 20 μl of Laemmli buffer for 5 min at 100 °C. Eluates from both steps (total and membrane Lu/BCAM) were analysed by SDS/PAGE (8% gels) under reducing conditions, followed by Western blotting with the rabbit anti-Lu pAb 602 (1:5000 dilution). Samples of biotinylated Lu proteins were analysed using a bio-imaging analyser to quantify the newly delivered membrane proteins as a function of time.
Cell-spreading assay
The 12-wells plates were coated with either 2 μg/cm2 of laminin 511/521 or 1% BSA overnight at 4 °C. Wells were washed twice with PBS and were subsequently coated with 1% BSA at 37 °C for 1 h before two additional washes. MDCK cells were washed twice and added to the wells (105 cells/well) in serum-free medium. After different incubation times (30, 60 and 90 min) at 37 °C, spread and round cells were quantified in four representative areas by microscopy (Leitz, ×100) using a computerized image analysis system (Biocom VisioL@b 2000). The counted cells were then averaged and presented as the mean percentage of spread cells. A P value of less than 0.05 was considered statistically significant.
Cell-matrix-adhesion assay
Mock-transfected MDCK cells or transfected wt-Lu or mt-Lu cells (2×106 cells) were labelled with 5 μM calcein/AM and 2×106 MDCK-mt-Lu control cells were labelled with 1 μg/ml Hoechst 33342 for 30 min at 37 °C/5% CO2. Cells were then washed twice with prewarmed serum-free medium. MDCK, wt-Lu or mt-Lu cells were then mixed with control cells as an internal standard at a 1:1 ratio. Mixed cells (1 ml) were then put in laminin 511/521-pre-coated 12-well plates. After 90 min of incubation at 37 °C/5% CO2, the cells were washed twice with prewarmed serum-free medium to remove unattached cells and visualized by fluorescence using an Evolution VF camera. Ten images were acquired for each sample, and adherent cells were counted using Image-Pro® Plus software. Results are expressed as the mean percentage of adherent transfected cells compared with control adherent cells.
Phosphorylation assays in transfected cells
MDCK-wt-Lu and -mt-Lu (5×106 cells) grown in six-well plates were washed three times in DMEM without phosphate and incubated in the same medium for 2 h at 37 °C. Cells were then incubated with 300 μCi of [32P]Pi (25 mM) for 90 min at 37 °C and one well with wt-Lu or mt-Lu cells was supplemented with 1×PIC (90 min) or 1 mM Br-cAMP (30 min). Cells were washed twice with ice-cold PBS and incubated in lysis buffer [20 mM Tris/HCl (pH 8), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 0.2% BSA, supplemented with protease inhibitor cocktail and 1×PIC] for 1 h at 4 °C. Immunoprecipitation of Lu/BCAM was carried out as described above. The beads were washed five times in lysis buffer. Immunoprecipitated proteins were first eluted for 5 min at 100 °C with 3×Laemmli buffer supplemented with 5% 2-mercaptoethanol and then loaded on to a 4–12% NuPAGE® Novex Bis-Tris gel under reducing conditions. Analysis was performed by Western blotting using a goat biotinylated anti-Lu/BCAM antibody (0.1 μg/ml) or using the bio-imaging analyser.
Rho family proteins activation and inhibition assay
The six-well plates were coated with laminin 511/521 or fibronectin as described above. Mock-transfected MDCK cells, and wt-Lu or mt-Lu cells (25×104 cells) were added to the wells for 90 min. Active Rac1, 2, 3 and RhoA were measured using the G-LISA™ RhoA, Rac or Cdc42 Activation Assay Biochem Kit™ according to the manufacturer's instructions. GTP Rac, RhoA and Cdc42 were used as positive controls. The absorbance signals were detected at 490 nm.
Statistical analysis
Results are expressed as means and were analysed using the Mann–Whitney non-parametric test to determine statistical significance between mt-Lu and wt-Lu. Statistical significance is defined as P< 0.05.
Lu/BCAM interacts with αII-spectrin in non-erythroid cells via its RK573-574 motif
We investigated the potential interaction of Lu/BCAM with the αII-spectrin isoform expressed in non-erythroid cells. Immunoprecipitation assays were performed using two cell lines expressing endogenous Lu/BCAM: the human kidney epithelial cells A498 and the human microvascular endothelial cells HMEC-1. After Lu/BCAM immunoprecipitation, Western blot analysis revealed the presence of αII-spectrin, indicating that it was co-immunoprecipitated with Lu/BCAM from A498 cells (Figure 1A, left-hand panel) and HMEC-1 cells (results not shown), and suggesting an interaction between these two proteins. Neither spectrin (270 kDa) nor Lu/BCAM (90 kDa) were detected when an irrelevant antibody (IgG1) or Protein A–Sepharose alone were used for immunoprecipitation. The RK573-574 motif of the cytoplasmic tail of Lu/BCAM is required for binding to αI-spectrin in RBCs [12]. The involvement of these residues in the interaction with αII-spectrin was also investigated by immunoprecipitation using transfected MDCK cells expressing either wt-Lu or mt-Lu. As expected, αII-spectrin was co-immunoprecipitated with wt-Lu (Figure 1A, right-hand panel). Faint quantities of αII-spectrin, similar to those obtained with the IgG1 negative control, were detected from cells expressing mt-Lu (Figure 1A, right-hand panel), indicating that the RK573-574AA mutation abolished the Lu/BCAM–αII-spectrin interaction. These results indicated that endogenous and recombinant Lu/BCAM interacted with αII-spectrin in both epithelial and endothelial cells; this interaction involved the Lu/BCAM cytoplasmic RK573-574 motif.
Lu/BCAM binds to the α4 repeat of αII-spectrin
The Lu/BCAM cytoplasmic domain binds to the αI-spectrin α4 repeat (termed α4R) in RBCs [15]. Sequence analyses revealed that the αII-spectrin α4R (residues Glu361 to Glu486) shares 57% identity with its αI-spectrin corresponding repeat. We tested the involvement of the αII-spectrin α4R in the interaction with Lu/BCAM using GST pull-down assays. α3R, α4R, α5R and α3→5R were produced as [35S]methionine-labelled peptides by in vitro transcription and translation (TNT products) (Figure 1B), and their binding activity to the cytoplasmic domain of Lu/BCAM, produced as a GST-fusion protein, was analysed. As shown in Figure 1(B), the spectrin peptides encompassing either the α3→5R or only the α4R were pulled down with the wt-Lu cytoplasmic tail (GST–wt-Lu), giving a signal 3–5-fold higher than those obtained with either GST alone or the mt-Lu cytoplasmic domain (GST–mt-Lu). In contrast, the pull-down signals obtained with α3R and α5R were similar for GST, GST–wt-Lu and GST–mt-Lu. These results indicated that αII-spectrin α4R was involved in the interaction with the Lu/BCAM RK573-574 motif.
Disruption of the Lu/BCAM–spectrin interaction increases Lu/BCAM extractability in epithelial cells
To verify the involvement of the spectrin–Lu/BCAM interaction as a link between Lu/BCAM and the membrane cytoskeleton, we examined the detergent solubility of recombinant wt-Lu and mt-Lu in epithelial MDCK cells. Cells were treated with increasing concentrations of Triton X-100 (0.01–0.1%), and the amount of solubilized Lu/BCAM was analysed by Western blot using Ubc9 (20 kDa) as a cytoplasmic marker and loading control. As shown in Figure 1(C), wt-Lu was not detected in the soluble fraction below 0.025% Triton X-100. Conversely, mt-Lu was extracted starting from 0.01% Triton X-100, indicating that disruption of the Lu/BCAM–spectrin interaction increases the cytoskeleton-unbound Lu/BCAM fraction in non-erythroid cells.
Interaction with spectrin is not involved in the membrane targeting of Lu/BCAM
The basolateral expression of Lu/BCAM in polarized epithelial cells depends on the integrity of a di-leucine motif at position 608–609 of the cytoplasmic domain [24]. To determine the potential role of αII-spectrin in Lu/BCAM membrane targeting, confluent monolayers of MDCK clones expressing similar amounts of either wt-Lu (400000 molecules/cell) or mt-Lu (450000 molecules/cell) were grown on filters and then labelled with both the anti-Lu/BCAM mAb and anti-αII-spectrin pAb. Confocal microscopy analysis showed that both cell types exhibited a polarized phenotype and form tight junctions at confluence, as indicated by daily transepithelial resistance measurements (900 ohms/well after 7 days of culture). As shown in Supplementary Figure S1 (at http://www.BiochemJ.org/bj/436/bj4360699add.htm), spectrin (red) and wt-Lu (green) were expressed at the cell membrane and displayed a lateral expression, as expected. The merge panels (yellow) indicated that wt-Lu and spectrin were in the same lateral membrane compartment. The same pictures were obtained with MDCK cells expressing mt-Lu, indicating that disruption of the Lu/BCAM–spectrin interaction did not modify the Lu/BCAM membrane-expression pattern. Therefore this interaction is not required for the correct lateral targeting of Lu/BCAM in polarized epithelial cells.
Interaction with αII-spectrin plays a role in the stability of Lu/BCAM at the epithelial cell membrane
As the Lu/BCAM–spectrin interaction was not involved in the cell-surface localization of Lu/BCAM, we investigated the putative role of this interaction in Lu/BCAM membrane delivery and turnover using pulse–chase experiments combined with surface protein biotinylation.
As the newly delivered Lu/BCAM at the membrane has been reported to reach a peak between 60 and 90 min of chase [24], the chase experiment on polarized MDCK cells was started at 60 min after the radiolabelling step and followed up to 180 min. Total cell-surface proteins were labelled with biotin before cell lysis and immunoprecipitation. Total Lu/BCAM was immunoprecipitated and the biotinylated fraction, corresponding to Lu/BCAM expressed at the cell membrane, was purified using streptavidin–agarose beads. The amount of radiolabelled Lu/BCAM present on the membrane after 60, 90, 120, 150 and 180 min was determined by SDS/PAGE of biotinylated proteins followed by autoradiography. Newly synthesized wt-Lu and mt-Lu were correctly addressed to the lateral membrane as both were present at the cell surface after 60 min of chase (Figure 2A). This result confirmed that the interaction with spectrin was not involved in Lu/BCAM membrane targeting. However, the turnover of mt-Lu and wt-Lu were significantly different, as shown by the extended mt-Lu half-life at the membrane. The newly delivered amounts of mt-Lu at 60 min remained unchanged up to 180 min, whereas wt-Lu decreased soon after 60 min. This difference in kinetics was not due to protein degradation as Western blot analysis showed that equivalent amounts of each protein were immunoprecipitated for all chase times (Figure 2B).
To quantify the turnover of both proteins, the intensity of all bands was determined and the ratio 'radiolabelled/biotinylated' was calculated. As shown in Figure 2(C), radiolabelled wt-Lu decreased at the membrane over time, with almost half of the proteins being internalized after 90 min as compared with 60 min. Conversely, mt-Lu showed an enhanced stable membrane expression, as newly delivered mt-Lu did not undergo significant internalization during up to 180 min of chase, indicating that the lack of interaction with spectrin decreases the membrane turnover of Lu/BCAM in polarized MDCK cells.
Interaction of Lu/BCAM with spectrin modulates spreading and adhesion of epithelial cells to laminin 511/521
Alteration of the spectrin-based skeleton, as well as disruption of the Lu/BCAM–spectrin interaction, resulted in an enhanced Lu/BCAM-mediated adhesion of RBCs and K562 cells to laminin 511/521 [15,18]. To investigate whether the interaction with spectrin could similarly modulate Lu/BCAM-mediated adhesion of epithelial cells, we analysed the spreading and adhesion to laminin 511/521 of MDCK cells expressing either wt-Lu or mt-Lu as described in the Experimental section (Figure 3A).
We first compared the ability of MDCK cells to spread on laminin 511/521 and BSA (used as a negative control). At 90 min after plating, only 12% and 14% of mock-transfected MDCK cells could spread on BSA and laminin 511/521 respectively (Figure 3B). MDCK cells expressing either wt-Lu or mt-Lu showed a similar behaviour on BSA, but exhibited 38% and 60% of spread cells on laminin 511/521 at 90 min respectively (Figure 3B). Cell spreading on laminin 511/521 was higher for mt-Lu- than for wt-Lu-expressing cells, and was observed as soon as 30 min after plating (29% compared with 14%).
Cell adhesion to laminin 511/521 was also investigated. As expected, Lu/BCAM expression increased cell adhesion to laminin 511/521, as measured by the percentage of adherent cells: 65% for wt-Lu compared with 20% for mock-transfected MDCK cells (Figure 3C). Cell adhesion to laminin 511/521 was particularly reinforced for mt-Lu-expressing cells with 95% of adherent cells. These results indicated that disruption of the Lu/BCAM–spectrin interaction significantly increased Lu/BCAM-mediated epithelial cell adhesion to laminin 511/521.
The reinforced adhesion of mt-Lu-expressing MDCK cells is not related to the Lu/BCAM phosphorylation status
The increased adhesion of pathological RBCs to laminin 511/521 is associated with PKA-mediated Lu/BCAM phosphorylation [2,17]. As disruption of the Lu/BCAM–spectrin interaction reinforced the MDCK cell adhesion to laminin 511/521, we investigated whether this feature was related to a PKA-stimulated Lu/BCAM phosphorylation. Phosphorylation assays were performed using MDCK cells expressing either wt-Lu or mt-Lu. Lu/BCAM was immunoprecipitated from 32P-radiolabelled cells in the presence of a serine/threonine PIC or in the presence of Br-cAMP, a stable membrane-permeant cAMP analogue that activates PKA. In the absence of activators, and 90 min after cell plating on laminin 511/521, both wt-Lu and mt-Lu presented a similar basal phosphorylation level, as shown by autoradiography and Western blot analysis (Figure 4, compare lanes 1 and 4). The amount of immunoprecipitated Lu/BCAM was estimated by Western blot analysis, and Lu/BCAM phosphorylation was normalized by calculating the ratio of the phosphorylation signal and the intensity of each band in Western blot analysis. The phosphorylation level of both proteins was similarly induced in the presence of PIC and Br-cAMP (Figure 4), indicating that disruption of the Lu/BCAM–spectrin interaction did not alter the Lu/BCAM phosphorylation status.
Lu/BCAM–spectrin interaction is required for stress fibre formation on laminin 511/521
As cell adhesion and motility require actin rearrangements, we tested whether the Lu/BCAM–spectrin interaction could induce specific actin reorganization during MDCK cell adhesion and spreading. Low-density-plated cells were stained with Phalloidin to label F-actin (filamentous actin), 90 min after seeding on either laminin 511/521 (Figures 5a–5e) or fibronectin (Figures 5f–5j). Analyses were performed by confocal microscopy focusing on the cell basal membrane. In mock-transfected cells, actin was detected as peripheral spike-like protrusions and cortical rings, as well as cytoplasmic punctated forms on both laminin 511/521 and fibronectin substrates (Figures 5a and 5f). The same actin expression pattern was observed in all wt-Lu-expressing cells plated on fibronectin (Figure 5g). When these cells were plated on laminin 511/521, they exhibited a better spreading with extended lamellipodia. An increase in actin stress fibres was observed in 46% of Lu/BCAM-expressing cells (Figure 5b). To determine the putative involvement of the Lu/BCAM–laminin 511/521 interaction in stress fibre formation, MDCK cells expressing the D343R Lu/BCAM mutant, which is unable to bind to laminin 511/521 (the mutated residue is the same as residue 312 in reference [21]), were analysed. These cells did not form stress fibres on either laminin 511/521 or on fibronectin (Figures 5c and 5h). Absence of stress fibres was also observed for MDCK cells expressing mt-Lu plated on laminin 511/521 or fibronectin (Figures 5d and 5i). These results indicated that stress fibre formation in MDCK cells plated on laminin 511/521 was induced by the Lu/BCAM–laminin 511/521 interaction, and that the Lu/BCAM–spectrin interaction was involved in a laminin 511/521-to-actin signalling pathway leading to actin reorganization.
To determine the putative role of the 40 C-terminal amino acids of the Lu glycoprotein in regulating stress fibre formation, the same experiment was performed using MDCK cells expressing the short isoform Lu(v13). In this isoform the 40 C-terminal amino acids comprising the proline-rich SH3-binding domain, the di-leucine motif and the phosphorylation sites are truncated. Similarly to wt-Lu-expressing cells, 64% of cells expressing Lu(v13) were able to form stress fibres when plated on laminin 511/521 (Figure 5e), indicating that Lu/BCAM-mediated actin reorganization was independent of its proline-rich SH3-binding domain and of potential signalling events that could be mediated by its cytoplasmic tail.
Laminin 511/521 activates a RhoA-dependent signalling pathway via the Lu/BCAM–spectrin interaction
Rho GTPases are important regulators of the actin cytoskeleton and are involved in cell shape and motility. The activation of the small GTP-binding protein RhoA plays a role in stress fibre formation, whereas Rac and Cdc42 are responsible for actin polymerization in lamellipodia [25–27]. We compared RhoA, Rac and Cdc42 activities in mock-transfected MDCK cells and in cells expressing either wt-Lu or mt-Lu. Activities were evaluated from equal amounts of lysates obtained from cells plated for 90 min on either laminin 511/521 or fibronectin (Figure 6). No changes could be observed in Rac and Cdc42 activity for all conditions (Figures 6B and 6C). In contrast, cells expressing wt-Lu and plated on laminin 511/521 exhibited a significant increase of RhoA activity as compared with mock and mt-Lu cells (Figure 6A). These results suggested the involvement of RhoA activation in actin reorganization associated with the Lu/BCAM–spectrin interaction.
To investigate further the involvement of the RhoA pathway, we used the exoenzyme C3 transferase as a RhoA inhibitor. Pretreatment with the C3 transferase for 2 h is sufficient to inhibit stress fibre formation in wt-Lu and Lu(v13) MDCK cells plated on laminin 511/521, indicating that RhoA is necessary for the specific Lu/BCAM-induced actin reorganization (Figure 7).
Lu/BCAM interacts with spectrin in non-erythroid cells
The present study has identified the αII-spectrin isoform as a direct cytoplasmic partner of Lu/BCAM in vitro and ex vivo in different cellular contexts. The co-immunoprecipitation of endogenous spectrin with Lu/BCAM from kidney epithelial A498 cells provided physiological relevance of the GST pull-down interactions.
Positively charged juxtamembrane amino acids in adhesion molecules, such as CD44 and ICAM-2 (intercellular adhesion molecule 2), have been shown to interact with cytoskeletal protein 4.1 and ERM (ezrin/radixin/moesin) proteins [28]. As in RBCs, the Lu/BCAM RK573-574 motif interacted directly with αII-spectrin α4R and it could be assumed that this interaction might occur in all cell types where these two proteins are expressed. In humans, αI and αII-spectrins share 55% identity in the amino acid sequence. α-Spectrins are composed of 20 repeats and αII-spectrin α4R exhibits 57% identity with its corresponding erythroid αI-spectrin repeat. Moreover, the Lu/BCAM RK573-574 motif and surrounding sequences are conserved in mammals.
Effects of the Lu/BCAM–spectrin interaction on Lu/BCAM membrane localization and stability
Lu/BCAM is endogenously expressed in epithelial kidney A498 cells at 70000 copies/cell [22]. Its lateral expression in polarized epithelial cells depends on the di-leucine motif of its cytoplasmic domain [4].
Moreover, recruitment of Lu/BCAM at the lateral surface in epithelial cells, adjacent to laminin α5-containing extracellular matrix, suggests a role for laminin 511/521 in its membrane localization [8]. Indeed, Lu/BCAM expression was dramatically reduced in various tissues of mouse embryos lacking laminin α5 chain, whereas it was significantly increased in the heart of transgenic mice overexpressing this chain [29,30]. The results of the present study indicated the predominance of the di-leucine motif for Lu/BCAM lateral targeting. Our immunostaining experiments indicated that interaction with spectrin was not required for the proper targeting of Lu/BCAM in polarized epithelial MDCK cells.
In conclusion, the RK motif does not appear to be involved in Lu/BCAM membrane targeting, but this interaction could play a role in Lu/BCAM membrane turnover. In the pulse–chase experiments, we observed that the Lu/BCAM RK573-574AA mutant had an extended half-life at the cell surface. Since αII-spectrin interacts with syntaxin family proteins, which play a key role in vesicle fusion during exocytosis and endocytosis [31], the results of the present study suggest that spectrin might be involved in Lu/BCAM turnover through the endocytic pathway.
Role of the Lu/BCAM–spectrin interaction during cell spreading and adhesion
It was hypothesized that the Lu/BCAM–spectrin interaction might be critical for signalling and laminin 511/521 receptor function rather than in the maintenance of shape of the RBC. Lu/BCAM is involved in the abnormal adhesion of RBCs to laminin 511/521 and endothelium in pathological situations [2,5,16,17]. We and others [15,18] have demonstrated that the interaction of Lu/BCAM with erythroid spectrin negatively regulated its adhesive receptor function in normal and spectrin-deficient HS RBCs. In non-erythroid adherent cells, partial αII-spectrin depletion was associated with loss of cell spreading, defective adhesion, and a decrease and irregularity of focal adhesion points [32].
The results of the present study have shown that Lu/BCAM plays a critical role during epithelial cell adhesion to laminin 511/521. Cell adhesion and spreading to laminin 511/521 were significantly reinforced in cells expressing the Lu/BCAM RK573-574AA mutant, which could be a consequence of its extended half-life at the cell surface. Other proteins of the spectrin-based cytoskeleton, such as the adapter protein ankyrin G, can also modulate the function of adhesion molecules, including cell adhesion, migration and membrane distribution. The membrane expression of L1CAM (L1 cell-adhesion molecule), involved in the migration of neuronal growth cones and the static adhesion between adjacent axons, is regulated by its interaction with ankyrin G. Inhibition of this interaction led to retrograde movements of L1CAM in the cell membrane and stimulated L1CAM-mediated neurite outgrowth [33].
To date, the function of the Lu/BCAM laminin 511/521 receptor was known to be activated by two pathways in pathological RBCs: one involving the phosphorylation of its cytoplasmic domain by PKA [2], and the other mediated by a Lu/BCAM cytoplasmic domain interaction with the spectrin-based cytoskeleton [15,18]. The present study indicates that interaction between Lu/BCAM and spectrin regulates epithelial cell adhesion and spreading to laminin 511/521. This regulation is not related to an increased Lu/BCAM phosphorylation of the Lu RK573-574AA mutant. Conversely, laminin 511/521 binding could induce Lu/BCAM cytoplasmic tail phosphorylation, thus modulating its interaction with spectrin. Further experiments are needed to investigate this question.
Consequences on actin dynamics
Cell adhesion to extracellular matrix triggers outside–in signalling pathways leading to actin skeleton reorganization with extension of lamellipodia and filopodia. In the present study, we have shown that Lu/BCAM induced stress fibre formation during the early steps of cell adhesion and spreading to laminin 511/521. These changes were due to the Lu/BCAM–laminin 511/521 interaction, as cells expressing the D343R mutant, which is defective for laminin 511/521 binding, were unable to induce stress fibre formation. This rearrangement of the actin skeleton was associated with an activation of the small GTP-binding protein RhoA, which is known to regulate actin polymerization and stress fibre formation [27]. Similarly, RhoA activation was only observed in cells expressing wt Lu/BCAM. We can conclude that Lu/BCAM binding to laminin 511/521 drives actin filament formation.
The involvement of laminin α5 in cell spreading and in polymerization of parallel actin filaments, named filipodia-like microspikes, has also been demonstrated in primary dental epithelial cells in which laminin α5 interacts with α6β4 integrins. This interaction participates in the activation of PI3K (phosphoinositide 3-kinase)-Cdc42/Rac pathways [34]. We have shown that Cdc42/Rac pathways are not involved in the Lu/BCAM-induced actin reorganization.
The signalling pathway induced by Lu/BCAM binding to laminin 511/521 and leading to actin reorganization requires the Lu/BCAM–αII-spectrin interaction. Disruption of this interaction reduced both stress fibre formation and RhoA activation. We have shown that both Lu/BCAM isoforms acted similarly with regard to actin reorganization. The ability of the short isoform Lu(v13) to induce stress fibre formation indicated that this was independent of the proline-rich SH3-binding domain and the phosphorylation sites in the 40 C-terminal amino acids present in the long isoform. The results of the present study strongly suggest that the signalling cascade leading to actin reorganization upon Lu/BCAM binding to laminin 511/521 is vehicled by spectrin. All of these results provide new insights into a novel signalling pathway regulating the actin cytoskeleton via the Lu/BCAM–spectrin interaction which links the upstream laminin 511/521-binding signal to downstream RhoA activation.
The role of αII-spectrin in actin organization has been recently demonstrated, as αII-spectrin-knocked-down melanoma WM-266 cells exhibited modifications of the actin cytoskeleton, such as a loss of stress fibres [32]. αII-Spectrin, via its SH3 domain, has also been implicated in initiating Rac activation in the specialized β3-integrin clusters that initiate cell adhesion and spreading [35]. Moreover, the SH3 domain of αII-spectrin was demonstrated to bind proteins involved in the regulation of actin cytoskeleton dynamics, such as two members of the Mena-VASP (vasodilator-stimulated phosphoprotein) family [EVL (Enabled/VASP-like, VASP)] and Tes [36–39]. VASP participates in actin fibre formation, and the αII-spectrin–VASP complexes regulate cortical actin cytoskeleton assembly with implications in cell–cell contact formation. Overexpression of Tes resulted in increased cell spreading and decreased cell motility, whereas knockdown of Tes in HeLa cells resulted in loss of actin stress fibres and reduced RhoA activity [40]. All of these results reinforce the idea of a pivotal role of spectrin in actin-dependent processes.
In conclusion, spectrin is involved in regulating Lu/BCAM function and expression in non-erythroid cells. The results of the present study provide new evidence that spectrin plays a novel role as a linker between extracellular signals, triggered by laminin 511/521, and intracellular events modulating actin dynamics.
Emmanuel Collec designed and performed research, analysed data and wrote the paper. Marie-Christine Lecomte, Wassim El Nemer and Yves Colin discussed the results, gave advice and commented on the manuscript at all stages. Caroline Le Van Kim supervised the project, designed and performed research, and wrote the paper.
This work was supported, in part, by the Institut National de la Transfusion Sanguine (INTS), the Institut National de la Santé et de la Recherche Médicale (INSERM) and Université Paris Diderot -Paris7.
We thank Julien Picot for helpful advice on flow cytometry experiments.
Abbreviations: BCAM, basal cell-adhesion molecule; calcein/AM, calcein acetoxymethyl ester; Cdc42, cell division cycle 42; DMEM, Dulbecco's modified Eagle's medium; F-actin, filamentous actin; FBS, fetal bovine serum; GST, glutathione transferase; HS, hereditary spherocytosis; L1CAM, L1 cell-adhesion molecule; Lu, Lutheran; mAb, monoclonal antibody; MDCK, Madin–Darby canine kidney; pAb, polyclonal antibody; PIC, phosphatase inhibitor cocktail; PKA, protein kinase A; RBC, red blood cell; SABC, specific antibody-binding capacity; SH3, Src homology 3; sulfo-NHS-LC-biotin, sulfo-succinimidyl-6-(biotinamido)hexanoate; Ubc9, ubiquitin-conjugating enzyme 9; VASP, vasodilator-stimulated phosphoprotein; wt, wild-type
- © The Authors Journal compilation © 2011 Biochemical Society


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Although most of the country seems to be cloudy and moist right now, there is a chance that we will be experiencing sunny days soon. I thought we could all use a little sunshine about now, so here is our latest applique block. As always, it was designed to fit on a 6″ x 6″ block, but it can be enlarged or reduced to meet your needs.
I must admit I had ulterior motives for designing this block. We will be using it as the icon for our In the Good Old Summertime series we will be starting next week. Watch for more on the topic. In the meantime, if you have something that is screaming for a Sunburst Applique Block, the pattern can be found HERE.


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I don't read many magazines anymore.
It is not that I dislike them. In fact, it is quite the opposite – I enjoy reading them so much I developed an addiction.
My coffee table use to be littered with all sorts of glossy-covered monthlies, but my subscriptions got out of hand and before long, it seemed they were coming in faster than I could digest them.
I was overdosing on shiny photographs, reviews, critiques, feel-good stories, in-depth journalism and interviews.
I cut back, and I basically cut down to the bone. Now, only one magazine works its way into my mailbox, and I anxiously await it's arrival like a young kid anticipating his birthday. Within days if not hours of its delivery, I have consumed it entirely, from cover to cover, and the waiting game begins anew.
But the issues only whet my appetite, temporarily pacifying me as I await my annual fix every June.
Like countless other Texans, only one magazine truly matters to me: Dave Campbell's Texas Football.
Since 1960, the "Bible of Texas Football" has sent grown men into a tizzy as they gossip like schoolgirls about its appearance on grocery store shelves. Word of its debut quickly spreads and people race to grab a copy.
I'm no different.
As soon as I knew it was out, I rushed to purchase it and didn't even flinch when the register showed a total of $10.95. The price has become inconsequential over the years.
I flipped straight to my favorite high school districts to get the lowdown before turning my attention to the college and NFL sections to see what the state's authority on football had to say about my alma mater – the University of Texas – and my lifelong heroes/tormentors – the Dallas Cowboys. After that, I returned to the first page and began slowly working my way through 399 more until I reached the end.
While some might argue, obviously the staff at DCTF, while really good at what they do, is not comprised of gods. They can't predict the future, and their words are not gospel. Believe it or not, they even publish mistakes, referencing Copperas Cove's mascot as the "Bulldogs" and spelling head coach Jack Welch's last name "Welsh."
They do, however, offer a jumping-off point for conversations about the upcoming season at a time when junkies like myself are desperate for opening-night kickoffs. Then, the magazine proceeds to serve as a reference guide for the remainder of the season and an almanac of sorts after that.
Although I was not overly thrilled with everything written about my beloved teams, Copperas Cove fans should be pleased with reviews concerning their Bulldawgs.
Copperas Cove was predicted to finish second in District 8-5A behind Midway and ahead of Killeen and Temple. Senior running back Vondareaz King, who ran for 1,314 yards and 16 touchdowns last year, is selected the district's preseason offensive MVP and offensive lineman J.P. Urquidez was listed as one of the state's top 25 sophomores.
Listing the Bulldawgs' strength as their young talent, especially at offensive skill positions, but cites player development as the team's most significant obstacle.
Making the playoffs might be the easiest challenge anyone in District 8-5A faces, though. The true test could occur in the opening round of the postseason, when District 8-5A plays bi-district games against a highly respected District 7-5A.
DeSoto and Cedar Hill are ranked fourth and ninth in the state, respectively, with the Eagles predicted to win the Region I, Division I championship and the Longhorns achieving the same feat in Division II. Additionally, Midlothian is listed as one of five dark horses capable of potentially emerging to win a state championship.
Odds are, should the Bulldawgs make the playoffs, they will run into one of District 7-5A's powerhouses, but the potential outcomes will remain subject to debate for months to come.
Lampasas and Gatesville fans have far less to be excited about as DCTF believes the defending District 8-3A champion Hornets barely make the playoffs, finishing fourth after going 13-1 last season, and the Badgers are picked last.
District 8-3A was named one of the class' toughest districts, and like District 8-5A, things will probably only get tougher in the first round of the playoffs. Stephenville is ranked third in Class 3A and is picked to lose in the state title game, while Alvarado is ranked 20th.
Again, predictions are meaningless as the season will be determined on the field, but thanks to DCTF, the conversations and debates have begun and will undoubtedly rage on for months to come.
Needless to say, I will probably be involved in several such discussions, and anytime I need some tidbit to support my case, I know exactly where to find it. I will simply walk over to my coffee table and pick up my copy of DCTF.
It's easy to find nowadays. It has no competitors on my coffee table or in its niche of the market.


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NEW YORK UP CLOSE; For a Faded Tradition, a Fresh New Coat
By STEVEN KURUTZ
Published: October 22, 2006
AS an advertising strategy, the hand-painted outdoor sign fell out of fashion decades ago. Save for so-called ghost signs, like the faded ad for carriages that survives on the facade of a building at 17th Street and Avenue of the Americas, the hand-painted sign has largely disappeared from the New York streetscape.
In the past two years, however, dozens of new hand-painted signs have begun appearing throughout the city, among them a bold image of a 70's-era street gang on a building in Chinatown heralding the release of ''The Warriors'' video game, and several fanciful, turn-of-the-last-century scenes painted onto walls around Brooklyn to promote Dewar's scotch.
Those ads, and others like them, are the handiwork of Colossal Media, a two-year-old Williamsburg company that is trying to revive the labor-intensive, near-obsolete craft of hand-painted signs.
The company is run by Adrian Moeller, a 29-year-old with close-cropped brown hair who handles the business end, and Paul Lindahl, 29, who, if one judges by the flecks of paint that often dot his clothes and his body, does the installation work.
Their latest project is an immense 5,200-square-foot painting of cartoon superheroes, which promotes a new Comedy Central program called ''Freak Show'' and looms over the corner of Fourth Avenue and 13th Street.
''This one took about a week and a half to do,'' Mr. Lindahl, a lean, long-stemmed man with the high forehead and laconic manner of a Dust Bowl-era Okie, said the other day as he and his partner gazed up at the sign.
Before moving to New York two years ago, Mr. Lindahl worked as an itinerant sign painter in cities around the country, picking up trade secrets from ''wall dogs'' -- old men who spent their lives suspended above the city, painting larger-than-life images on building walls. ''Most everybody who knows how to do this,'' he said, ''is in their 50's or 60's.''
The peak years of hand-painted signs in New York ran from the 1920's through the 40's, said Ben Passikoff, who has written about New York's ghost signs, but the technology of applying them has remained the same. Mr. Lindahl works alongside a crew of four men, using a block-and-tackle rigging system of manual pulleys to maneuver scaffolding around on the wall. Twice a day, at lunch and at quitting time, he stands on the ground and inspects his work for mistakes.
Colossal Media holds the lease on only 15 walls around the city. But a challenge equal to that of securing prime real estate is persuading clients to use hand-painted signs instead of the far more common vinyl, which can cost half as much and reproduce images with laserlike clarity.
''People think hand-painted means graffiti,'' said Mr. Moeller, who, as it happens, publishes the graffiti culture magazine Mass Appeal. ''Or they worry we won't be able to replicate an image with photorealism.''
Neil Marks, director of marketing for Comedy Central, which commissioned the ''Freak Show'' ad near Union Square, echoed that concern. ''As tremendous a job as Colossal does in replicating the art,'' he said, ''I don't know how comfortable I would be in hand-painting Jon Stewart's face.''
Photos: Paul Lindahl and the work of Colossal Media, including hand-painted signs for Comedy Central and Dewar's scotch. (Photo by Robert Wright for The New York Times)


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Now Playing
Mi Gente J Balvin & Willy William Download 'Mi Gente' on iTunes
She may be Instagram's most followed person, but you do NOT wanna be following Selena Gomez on the road!
We haven't stopped watching her Carpool Karaoke yet, but luckily for the road-users of America, it was James Corden behind the steering wheel; not Selena Gomez.
The 'Hands to Myself' singer recently left a salon and was hounded by a few paparazzi, as is the celebrity life...
As Selena tried to make a speedy escape, she hoped into her huge motor and took out all of her frustration on a poor, unsuspecting fence!
Even though her pal was trying to help her out of the small parking lot, you can't help but feel for Ms Gomez, who was surrounded by photographers. Stick to the singing, girl - you're pretty damn good at that!
Did Selena Just Confuse Yorkshire Pudding with Tampon!?!?
Selena Gomez and Roman Kemp play the Whisper Challenge.


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"Winning for 1999, this New England writer is the last person to win an Oscar for adapting his own novel."
Click to see Spoiler:Who is John Irving (The Cider House Rules)
The New England part cemented it for me.
"Whatever you are, be a good one." Abraham Lincoln
What is an "MFWalkoff?"
I thought it was Steven King, as did one of the contestants. He's from Maine so that part fit. I was thinking Shawshank Redemption.
If you type "google" into google you can break the internet.
I didn't see the epi, but I would've known the answer immediately.* Irving is one of my favorite writers.
*This is not always the case with Final Jeopardy ;-)
Anti-intellectualism has been a constant thread winding its way through our political and cultural life, nurtured by the false notion that democracy means that 'my ignorance is just as good as your knowledge.' - Isaac Asimov
I was thinking of the immortal words of Socrates, who said, "... I drank what?"
I didn't know that one, but I love when I do know the answers during the show and the contestants flounder. It is such an ego boost.
The cure for boredom is curiosity. There is no cure for curiosity. Dorothy Parker, (attributed)
"There's no crying in baseball!"
-- Tom Hanks, A League of Their Own
That's why I'm in my jammy pants on my squishy chair in front of the TV. I couldn't do it for real, but it is nice to know that I'm not a dummy sometimes.
The cure for boredom is curiosity. There is no cure for curiosity. Dorothy Parker, (attributed)
I don't mean to brag (well, maybe a "little"), but I was on Jeopardy in the late 1980s. I came in second (lost to someone who went on to the Tournament of Champions), but I DID get Final Jeopardy right!!!!


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Bob Zarlingo, R.I.P.
Robert "Bob" Zarlingo owned and operated a historic local business with his family — what became Horizon Drive Standard and Zarlingo Automotive. But his impact on the Grand Valley and western Colorado went far beyond that.
Zarlingo's death last Saturday at the age of 64 was a blow to this community as well as his family.
He was a member and past president of the local chapter of the Rocky Mountain Elk Foundation and a past president of the Bookcliff Country Club. He also served on boards or volunteered with other local groups.
His wife, Cathie, is a former member of the School District 51 Board of Education, as well as outdoors' groups. One friend said the two "were a powerhouse in this community, in large part because of the way they supported each other."
Bob Zarlingo was described as generous to everybody and enthusiastic about his family and his community.
He will be missed.


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JERUSALEM (Mar. 13)
Israel is not interested in Russian intervention to help restart its suspended negotiations with the Palestine Liberation Organization.
This was the message Prime Minister Yitzhak Rabin and Foreign Minister Shimon Peres gave to Russian Foreign Minister Andrei Kozyrev during his surprise visit to Israel on Friday.
Kozyrev, who was en route to a meeting with PLO Chairman Yasser Arafat in Tunis, made no proposals of his own during his brief stay in Israel.
Nor did Kozyrev reiterate the urging of his deputy, made a week earlier, that a second round of Madrid talks be convened to help get Israel and the PLO back to the negotiating table. Israel has sharply rejected the suggestion.
Kozyrev reassured his hosts that while Russia favored an international presence in the territories, his country did not favor the armed international force that the PLO has called for to protect Palestinians in the territories.


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ELMORE Club added the Knockout Cup to their league championship at the Sampford Peverell and district league's finals night on Wednesday.
Elmore, the last team to win the double in 2002, were presented with their trophies at Cullompton Rugby Club by county councillor John Berry.
Captains' Cup – The first semi-final took place between Roger Lowman (Elmore Club) and Ric Norman (The Hounds).
After three hands Lowman was one point up but then Norman had a 15 spare and held on to win by three.
Then Wayne Cottrell (Settler Boys) and Angelo Binnacioni (Castaways) went on the alley, with the latter getting his nose ahead after two hands, and staying there.
In the final Binnacioni and Norman returned to the alley and after three hands Norman was one pin up. Then Binnacioni replied with a nine and inched ahead by three.
Norman went into the last leg needing eight to draw and nine to win – and came up with the goods.
n Individual Cup – Steve Broomfield (Catherine Wheel) took on Alistair Bussell (Brass Monkeys) in the first semi-final. After the first hand was shared Bussell pulled away to a convincing win.
Bryan Sharland (Bickleigh) had a close game against Peter Rosser (Barley Mow), winning by just two pins.
Bussell was again in top form in the final, starting the game with a 15 spare. Sharland was always playing catch up and a 16 spare in the fifth hand saw Bussell in contention for the highest score of the night. A last hand of eight won him that trophy.
n Knockout Cup Final – Globe Appley B took to the alley first in the final against Elmore Club and after two legs were 21 pins adrift.
They won the third leg, but then Elmore won the next by 17 to leave GAB with a mountain to climb.
Elmore went into the last two legs with a 37 pin advantage, and added a further 12 pins to that to secure the double.
WINNERS: Gerry Cup and League Champions – Elmore Club; Gerry Shield and League Runners-up – Bradninch C Club; Raven Cup and Division B winners – The Hounds; Aldridge Shield and Division B runners-up – Redwoods; Buckingham Cup and Division C winners – Barley Mow; Geoff Pack Shield and Division C runners-up – HCC; Knockout Cup Winners – Elmore Club; Prescott Shield and K/O runners-up – Globe Appley B; Tom Sharland Charity Cup for highest away average in Division A – Roger Lowman (Elmore Club) 56.20; Secretary's Cup for highest away average in Division B – Mark Blatchford (Bradninch RBL) 58.27; Derek Goff Cup for highest away average in Division C – Lee Waterman (Heathcoat Hulas) 57.00; Ken Freeman Cup for highest individual score in Division A – Stuart Palfrey (The Beare) 89; Ken White Cup for highest individual score in Division B – Mark Deane (Crossways) 91; John Baker Cup for highest individual score in Division C – Lee Waterman (Heathcoat Hulas) 82; Frank Maddocks Captain's Cup winner – Ric Norman (The Hounds); Russell Shield and Captain's Cup runner-up – Angelo Binnacioni (Castaways); Individual Cup winner – Alistair Bussell (Brass Monkeys); Individual Cup runner-up – Bryan Sharland (Bickleigh); Highest score on Finals night – Alistair Bussell (Brass Monkeys) – 58.


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Chelsea hit Wolves for six
Chelsea 6 Wolverhampton Wanderers 0: A mid-week League Cup tie against Championship opposition is not the kind of match that demands John Terry play, but then this game signified a lot more for the Chelsea captain than a routine win in the least important competition of the season.
By playing last night, Terry was signalling to the Football Association, the four men of the independent commission hearing his case for alleged racial abuse and the wider football world that, whatever the pressure, he is unbowed. To paraphrase the song the Chelsea fans sing about him: that John Terry, he does what he wants.
Certainly, being in the midst of one of the most high-profile FA disciplinary hearings in recent history was not about to affect his decision about whether he should play. It probably made him more determined to do so. His supporters will point to the fact that, for now, he remains an innocent man. His detractors will suggest that this is a man who relishes in sticking two fingers up to his critics.
What is not in doubt for Terry is that, at Stamford Bridge, he is the recipient of unconditional love. They applaud the announcement of his name. They sing about him when he runs out on the pitch. Last night the home fans chanted, "There's only one England captain" – and this to a man who had retired from international football 48 hours earlier.
The decision to play Terry was made yesterday afternoon, first team assistant coach Eddie Newton said, but by whom he did not say. This game could be the last Terry plays in a long time, although do not bet against him appealing a guilty verdict.
Stale Solbakken, the Wolves manager, indicated how little interest he had in the League Cup by picking a team that had 10 changes from the side that beat Peterborough on Saturday. It is depressing when a manager cannot even be bothered to put up a fight in a cup game in September, eight months from the end of the season, but this is modern football.
Solbakken said: "We had thought about this before we did it, and it was always a risk that something like this [scale of defeat] could happen. But we were punished really hard in the beginning with a soft set-piece goal, then came another. That made it hard for the young boys out there.
"But there's no one else to blame but me. It was a calculated risk because we'd had three games in six days and have that again [coming up]. I'd have done the same again."
Roberto Di Matteo gave debuts to Lucas Piazon, the 18-year-old signed from Sao Paulo two years ago, and the right-back Cesar Azpilicueta bought from Marseilles in August. There was also a first start for Victor Moses. Chelsea scored through Gary Cahill on four minutes and Wolves were never in it.
The first goal came when Cahill jumped strongly at the back post to meet a cross from Juan Mata and the tone was set. The second came three minutes later when Oriol Romeu's shot was pushed away by goalkeeper Dorus De Vries, Piazon cut the ball back and Ryan Bertrand scored.
Wolves were three behind within 16 minutes with a move started by Mata and then finished by the Spain international.
There were three goals after the break and, to his relief, the second of those was scored by Fernando Torres. First however, the striker, who had not previously scored for a month, deferred to Romeu on penalty-taking duties when referee Neil Swarbrick gave a soft decision for a collision between De Vries and Moses. In the absence of the injured Lampard, Romeu scored.
Torres scored the fifth with a header from Mata's corner from the right side. Moses headed the sixth from a cross from substitute Oscar.
For sheer lack of any imagination, Wolves got exactly what they deserved. This is their fifth game in a run of six in 17 days but they were woeful. Cup runs require a leap of faith from a manager and Solbakken picked a team to lose. Last night was not even a contest. For Terry, however, the significance of the evening was a lot more profound.
Man of the match Mata.
Match rating 6/10.
Referee N Swarbrick (Lancashire).
Attendance 32,569.
Belfast Telegraph


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Straight Out of View
"Joyce Sutphen's first collection reveals a poet of place whose assured, straightforward style seems sprung from the Minnesota farmlands where she was raised."-Publishers Weekly
Awarded the 1994 Barnard New Women Poets Prize, these poems are alive with motion, poems in the act of transformation even as the words fly onto the page. While rooted in the Midwest, the poems extend far beyond the homegrown, moving from the sudden disaster of "Tornado Warning" to sweeping landscapes of the American West to London, where in gentler tones she confronts Sylvia Plath. Sutphen's voice is refreshing throughout, capturing emotion with swift imagistic strokes.
Joyce Sutphen teaches literature and creative writing at Gustavus Adolphus College in Saint Peter, Minnesota.
The second half of my life will be black
to the white rind of the old and fading moon.
The second half of my life will be water
over the cracked floor of these desert years.
I will land on my feet this time,
knowing at least two languages and who
my friends are. I will dress for the
occasion, and my hair shall be
whatever color I please.
Everyone will go on celebrating the old
birthday, counting the years as usual,
but I will count myself new from this
inception, this imprint of my own desire.
The second half of my life will be swift,
past leaning fenceposts, a gravel shoulder,
asphalt tickets, the beckon of open road.
The second half of my life will be wide-eyed,
fingers shifting through fine sands,
arms loose at my sides, wandering feet.
There will be new dreams every night,
and the drapes will never be closed.
Iwill toss my string of keys into a deep
well and old letters into the grate.
The second half of my life will be ice
breaking up on the river, rain
soaking the fields, a hand
held out, a fire,
and smoke going
upward, always up.
Product Details
|Publication date:
|Barnard New Women Poets Ser.
|Product dimensions:
|5.48(w) x 8.43(h) x 0.38(d)
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Philadelphia Riots
This collection contains materials related to the Anti-Catholic Philadelphia Riots (also known as the Nativist Riots, the Philadelphia Prayer Riots, and the Bible Riots) in 1844. These materials are owned by the American Catholic Historical Society and maintained at the Philadelphia Archdiocesan Historical Research Center, 100 E. Wynnewood Rd. Wynnewood, PA 19096. For more information please see: http://www.pahrc.net/Show/hide more info.
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NewswireToday - /newswire/ -
Gateshead, Tyne and Wear, United Kingdom, 2007/11/15 - The first-ever North East Digital Awards will celebrate the achievement and highlight the talents of firms in the region using digital technology - NorthEastDigitalAwards.co.uk.
The Awards will recognise quality across all areas of e-business and provide free online promotion of businesses across the region, both for those which are creating and developing digital technology and those which are using it in the most creative and innovative ways.
Funded by regional development agency One NorthEast, and managed by e-business champion The North East Regional Portal, the Awards are open for entries until 28 March 2008. Entries are submitted exclusively online.
Emmanuelle Martinot, e-business technical advisor for One NorthEast, said: The North East of England is a hotbed for digital talent and these awards are an opportunity to highlight and celebrate those achievements. Were really looking forward to seeing the nominations.
The North East Regional Portals head of e-business Pascal Fintoni explained: The rapid expansion and development of digital technologies in the North East of England is undeniable, and successful and innovative businesses deserve to be celebrated.
These awards celebrate the achievements of businesses, companies and organisations that have recognised, developed and used digital techniques to generate and maintain effective online activities. This means that all Award nominees will be winners.
The awards are divided into three Groups: Digital Users, Digital Suppliers and Digital Innovators and categories range from Best E-commerce Website to Best News and Entertainment Website.
Taking a unique and imaginative step, the entire nomination and voting process will take place online with every nomination featuring extensively on the North East Digital Awards website.
The closing date for entry is 31 March 2008
Notes for Editors
The North East Digital Awards is funded exclusively by regional development agency One NorthEast and project managed by The North East Regional Portal (TNERP). Celebrating innovation, professionalism and excellence in the digital world, the awards commend the achievements of businesses, companies and organisations that have recognised and developed essential digital practices in order to generate and maintain an effective online presence.
The North East Regional Portal is an e-business champion and its bold innovative approach is recognised nationally and internationally as driving the North Easts position at the leading edge of the digital revolution. Through its portfolio of services and training courses it helps businesses adopt innovative internet and technology to sell and win business and deliver and retain business.
Its chief services include online project management consultancy, e-procurement, broadband, PR and promotion, free directory listings for North East businesses, e-marketing and search engine optimisation, portal solutions, training courses, plus advice and support on ICT.
Written by / Agency / Source: The North East Regional Portal Ltd
Availability: All Regions (Including Int'l)
Traffic Booster: [/] Quick Newswire Today Visibility Checker
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text:
English National Opera
Tagged: e
Read Also:
- Enob
effective number of bits
- Enoch-arden
[ahr-dn] /ˈɑr dn/ noun 1. (italics) a narrative poem (1864) by Tennyson. 2. its hero. 3. a missing person who is presumed dead but is later found to be alive.
- Enoki
[ih-noh-kee] /ɪˈnoʊ ki/ noun 1. a thin, long-stemmed and tiny-capped white mushroom, Flamma velutipes, native to the northern mountain ranges of Japan and prized as a food.
- Enol
[ee-nawl, ee-nol] /ˈi nɔl, ˈi nɒl/ noun, Chemistry. 1. an organic compound containing a hydroxyl group attached to a doubly linked carbon atom, as in >C=C(OH)−. /ˈiːnɒl/ noun 1. any organic compound containing the group -CH:CO-, often existing in chemical equilibrium with the corresponding keto form See keto-enol tautomerism enol e·nol (ē'nôl', ē'nōl') n. An […]


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LONG CEMETERY, (Tatum Road), GILES COUNTY, TENNESSEE
Mapping the location Signage Family member photos
GANNON, Emma M. Long,
5 Jan 1954 - 25 Mar 1989, Love lives
on. (daughter of Thomas W. & Minnie W. Long; killed in Missouri by her husband
who decided he did not want to be married anymore. He is still in jail as of
2010.) 6261
LONG, Thomas W., 5 Oct 1932 - 9 Dec 2002. (The patriarch of this Long family, lived here since the 1960s, passed away from Colon Cancer.) 6262C/6262
LONG, Minnie M., 14 Apr 1934 - no other date. (wife of Thomas W. Long married 19 Apr 1952; native of Alabama.) 6262C/6262
This is a family cemetery started in 1989 by the Longs. They moved up here in the 1960s and have been a farm family since. This cemetery is as a place of quiet solitude to reflect on the memories of the Long family. Added here 1 Nov 2010 from photos dated 16 Oct 2010 and interviews with James & Minnie Long. [C. Wayne Austin]
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