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The Premier League is a tough hunting ground for new goalkeepers coming from countries with different footballing cultures and philosophies. Goalkeeping in England, over the years has been about being extremely vocal, good at crosses and being a great shot-stopper. A vivid example is the case of David De Gea who was brought in to fill in the legendary shoes of Edwin Van der Sar. He struggled initially but it is hard to find a better goalkeeper in the country than the 26-year-old Spaniard.
For Loris Karius, however, the road has not been easier. Brought in by Jurgen Klopp to provide competition for Simon Mignolet, the 22-year-old former Mainz shot-stopper got injured in pre-season, and has since not looked the part of the calming influence at the back that Klopp initially sought him out to be, with sections of the English press calling for him to be dropped.
Countryman, Emre Can is singing his praises, saying he should be given time to acclimatize to the fast paced English game. He also added that he kept track of Karius' performances in Germany, and that he is a very good goalkeeper. Can said, "He's a great lad and a very, very good goalkeeper. It's a little bit different here from the Bundesliga but he's well aware of that."
The German has been working hard with John Achterberg, who is helping him with the process of adapting to Premier League football. He will get the chance to show what he has learnt when the Reds face Watford on Sunday.
Image Credit: Chi Trang Anna Creative Commons


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The Shashka (Шашка) was first known as the "national weapon" of the Cossacks, Shashkas originated with the Caucasian tribes in what is today known as the Chechnya and Dagestan republics.
A large number of Russian and Ukrainian Cossacks were resettled into this area by Imperial Russia and the Shashka became their prime weapon until eventually, it became identified directly to them and during the 19th century, regular versions were taken up by the Russian cavalry units.
Shashka use continued during Soviet times and cost many German officers their lifes and/or their heads.
Systema Hong Kong brings you this great looking stainless steel Shashka replica with a detailed pommel and guard. It comes with a synthetic brown leather-wrapped scabbard. Great for collectors
Sword Details:
Length: 96 cm
Width: 5 cm
Blade length: 77.5 cm
Blade width: 3.0 cm
Blade thickness: 0.4 cm
Scabbard Length: 81.00 cm
Handle length: 14.0 cm
Sword Weight: 1.5 kg (with packaging)
Sword material: Mirrored, heat-treated, 440 stainless steel blade
Only HKD $1050.00 (USD $135*) Shipping included
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Ordering more than 5?
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5 or more……………………………………$125 each
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50 or more……………………………………Email us
* Subject to exchange rates

subset: commoncrawl


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The Children's Holiday Art Contest benefits art education in local schools. Portland/Vancouver metro area elementary school classes (grades 3-5) are asked to paint a greeting card mural. New this year, the entries will be displayed online where visitors can vote to select their top entry. The school receiving the most votes will receive a cash prize for their efforts! Your school's success is impacted by your efforts to spread awareness about the contest and encourage parents and families to vote online!
The children's creativity is rewarded with grant money based on the number of votes received. Our only request is that the funds are spent on art related supplies, projects, or field trips. The Sunshine Division provides prize money to the winning schools.
(Click to Enlarge)
School: Hockinson Heights Elementary
Teacher: Nancy O'Neill-Bratt/Anna Hoffman
Grade: 5th Grade
Piece Name: Christmas Commotion
School: Lake Shore
Teacher: Erinn Stromme
Grade: 5th Grade
Piece Name: "Naughty or Nice? …Choose Nice!"
School: Middleton Elementary
Teacher: Jennica Smith – WINNER!
Grade: 5th Grade
Piece Name: "Santa's Coal Mine"
School: Hollydale
Teacher: Chastity Clegg
Grade: 4th Grade
School: Pleasant Valley
Teacher: Barb Holterman
Grade: 4th Grade
Piece Name: I'm Getting Nuttin for Christmas
Congratulations to our 2015 winner, Lake Shore Elementary in Vancouver, WA who received a $500 prize for submitting the top entry!
Please check back in September for details on how your school can submit an entry for the 2016 contest.


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April 10th 2013 Newsletter
| Pro Tools 11, RedNet, Orion 32, UA, RME, Waves, Auralex
AVID's Pro Tools has for over two decades been the unquestioned standard in computer based audio recording for facilities far and wide, from multi-platinum albums, to Oscar-winning films, to countless other applications in the world right on down to personal home recordists. And now, at last Pro Tools 11 is here!
The most powerful digital audio workstation just got more powerful. With an all-new, ultra-efficient audio engine, 64-bit performance, and groundbreaking capabilities, Pro Tools 11 enables you to take on the most complex and demanding music and audio post productions with ease, so you can focus on sounding your best.
Dale Pro Audio has been at the forefront of Pro Tools sales and support right from the beginning, with countless systems sold and configured at our New York City location. Our staff of Sales Professionals have all spent years in the industry, earning Dale the status of an AVID Elite Reseller. We have performed countless Pro Tools system sales and installs - including over 100 HDX systems in 2012 alone! We work closely with AVID to make sure each facility is best suited and equipped for maximum productivity with expert service and support before, during, and after the sale. Whether you are opening up a top-notch post production environment, or making your next home recording right in your bedroom, Dale Pro Audio will have a solution for you.
For more information on Pro Tools 11, please contact one of our Sales Professionals at email@example.com or call 888.462.7828.
Dale Pro Audio is proud to carry the RedNet system from Focusrite.
Rednet is Focusrite's flagship range of Ethernet-networked studio interfaces, based around the tried and tested Dante Ethernet audio networking system from Australian company Audinate.
RedNet forms a scalable system that comprises high-quality, versatile interfaces that deliver traditional Focusrite quality sound and performance, with exceptional value for money. Fundamentally, RedNet is a near zero-latency audio distribution system. It is highly expandable; Audinate's Dante technology enables a single link on the network to handle up to 512 channels (I/O) at 48kHz.
Every analog interface delivers dynamic range of 119dB, with 24-bit resolution at sample rates up to 192kHz. Each embodies the most advanced digital audio conversion designs available, including the latest A-D and D-A components, to provide exceptional quality. And with RedNet, you don't need additional cabling and complexity to sync to a master clock: the system takes care of that automatically as rock steady clocking between units is carried over Ethernet with JetPLL™ for ultra-low-jitter.
For more information on RedNet, please contact one of our Sales Professionals at email@example.com or call 888.462.7828.
Antelope was founded to take audio recording to the next level, providing digital technology that transcends all expectations. Antelope allows you to harness the power of digital audio without sacrificing the warmth and fullness typically associated with analogue gear. With Antelope, it's truly possible to have the best of both worlds. And now, they've released the all new Orion 32!
Orion 32 ($2995) is a 32-channel AD/DA converter and audio master clock, supporting both MADI and USB interfaces, clocked by Antelope's renowned 64-bit Acoustically Focused Clocking (AFC) technology. Orion 32 allows 192 kHz I/O streaming of 32-channel digital audio through its custom-built USB chip, which provides simple connectivity to any USB-enabled DAW or computer.
The converter also provides 32 channels of 96 kHz audio through its Fiber Optic MADI I/O connections, which can be used to connect with any suitably equipped MADI device. Orion 32 also supports ADAT protocol by offering 16 I/O channels, for even greater compatibility with a large number of audio devices. The multi-channel converter inputs and outputs pass the analog signal through 8 D-SUB 25 I/O connectors.
For more information on the Orion 32, please contact us at firstname.lastname@example.com or call 888.462.7828.
Universal Audio's Apollo 16 is their flagship 24-bit 192 kHz audio interface, delivering world-class conversion with 16 x 16 analog I/O - twice the analog connectivity of the original Apollo model. This uncompromising FireWire/Thunderbolt-ready interface combines superior sound and flexible routing with powerful onboard UAD-2 QUAD processing. So you can track audio in real time through the full range of classic UAD analog emulation plug-ins - from Neve, Studer, Manley, Lexicon and more - on both Mac and Windows 7.
With its professional connectivity and multi-unit cascading (for 32 x 32 analog I/O), the Apollo 16 sets a new standard in high-resolution recording with classic analog sound.,
Apollo 16 ($2995) is scheduled to begin shipping in May 2013. Contact your Dale sales professional to place your order as the initial supply will be limited.
For more information on the Apollo 16, please contact us at firstname.lastname@example.com or call 888.462.7828.
RME is a German team of developers with a robust vision of creating innovative, user friendly, and high-quality digital audio solutions for less money. Their products can be found installed in studios, post production facilities, and broadcast houses all over the world.
The Fireface UCX ($1599) is a highly integrated pro audio solution in an ultra-compact format for studio and live recordings. It continues RME's long tradition of designing compact high-end interfaces, packing into a half-rack size unit what usually would be spread out over two or three 19 inch panels. In doing this the Fireface UCX marks a new packing record: 300 electronic components more than inside the already well-stocked Fireface UC! In a tremendous effort RME packed once again all the latest technologies, introduced with the award winning flagship UFX, plus a bunch of newly developed ones, into a half 19 inch housing with full mobile usability.
The Fireface 800 ($1799) is the world's most powerful FireWire audio interface ever. Up to 56 channels record/playback, up to 192 kHz sample rate, and true FireWire 800 sum up to an unsurpassed high-end, high performance and high speed FireWire audio interface. Since the Fireface first shipped in July 2004, the unit became the reference for FireWire audio - and still is today!
The Babyface ($749) is smooth on the outside for looks and feel, but inside has the latest 192 kHz AD- and DA-converters, two reference class microphone preamps, SteadyClock - analog circuit design, features and function are truly RME!
That includes the new TotalMix FX, a TotalMix on steroids, with tons of new features, improvements and 3-band parametric EQs, plus added echo and reverb: anything you need right out of the box and with near zero latency!
The Babyface uses RME's Hammerfall USB audio core from the Fireface UC - another milestone in low latency audio interfaces, and will give you outstanding performance and top sound in an ultra-portable, nicely designed and attractively shaped enclosure.
For more information on RME, please contact us at firstname.lastname@example.com or call 888.462.7828.
Waves's ever-growing Signature Series of plug-ins has expanded this year by welcoming acclaimed producer/enginner Manny Marroquin. This all-new series of plug-ins (co-designed by Manny himself) took the prestigious "Best In Show" award at this year's Winter NAMM show, and is now available for $299! As with all other Waves software products, you can use your purchase right away after downloading!
Four-time GRAMMY® award-winning mixing engineer Manny Marroquin takes the Waves Signature Series to a new level with six hybrid plugins that bring his unique workflow to your studio. Alongside his personalized versions of tried-and-true favorites like EQ, reverb, delay and distortion, Manny introduces the innovative new Tone Shaper and Triple D plugins which are destined to become mixing essentials as well. Combining inspiration from his impressive collection of unique and boutique hardware, together with the fruits of his imagination, the Manny Marroquin Signature Series gives you direct access to the same tools Manny uses to mix hit after hit after hit.
For more information on Waves products, please contact us at firstname.lastname@example.com or call 888.462.7828.
Auralex Acoustics was founded over 20 years ago on the belief that there had to be a much better alternative to the expensive acoustic foam panels available at that time. Indeed there was; not only dramatically less expensive, but also with significant performance advantages.
Dale Pro Audio displays a comprehensive listing of Auralex products on our website. Whether your needs involve treating a new recording studio, problem solving in mobile or temporary locations, sound proofing a meeting room or gym, or you're a musician in need of some inexpensive tools for your project studio, Auralex has the answer!
Auralex's acoustic specialists and Dale Pro Audio's Sales Professionals work closely together to provide our diverse clientele with the best possible solutions for their applications, no matter how challenging.
For more information on Auralex, please contact us at
firstname.lastname@example.com or call 888.462.7828.
Dale Pro Audio |
Phone: 888.462.7828 | Fax: 212.475.1169 | Email:
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Store Location:
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Dale Pro Audio is not responsible for technical inaccuracies, typographical errors, or incomplete or inaccurate content on this site.
Prices and specifications subject to change without notice.


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Northern Owari was a non-official neighborhood of Hisatu-Kesu, named due to the resemblance it bore to Ryoko Owari. It was the center of illicit activities, criminal influence, and corruption in Hisatu-Kesu. Dens of sake and opium could be found, as well as several geisha houses that offered more than mere entertainment. The place was controlled by a small criminal cartel led by a man known as the Honesty-sama. [1]
- ↑ Secrets of the Unicorn, p. 39
|This Unicorn Clan related article is a stub. That means that it has been started, but is incomplete. You can help by adding to the information here.


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API Welcomes EPA Action to Put an Administrative Stay on the Agency's Risk Management Plan Rule
API Downstream Group Director Frank Macchiarola said that the U.S. Environmental Protection Agency's (EPA's) action to put an administrative stay on the agency's Risk Management Plan Rule is important to protecting the security of industrial facilities throughout the country.
"This is an important step for providing regulatory certainty and supporting safety and security in the oil and natural gas industry," said Macchiarola. "Now Congress must act to repeal this unnecessary rule, as the rule misses the mark on improving the safety and security of our nation's energy infrastructure."
"We look forward to working with the new administration and Congress on forward-looking energy policies that recognize America's oil and natural gas production and refining industry as part of the solution to our nation's economic, environmental and energy security goals."
Where fuels are concerned, the EPA provides the following guidance: "Unless your facility meets the definition of a 'retail facility,' if you hold a listed flammable substance for purposes other than on-site use as fuel, you are potentially covered by part 68. For example, if you manufacture a listed flammable fuel, use it as a chemical feedstock or store it in bulk for sale and do not meet the definition of a retail facility, you may be covered by the rule. If you store a listed flammable Chapter 1 General Applicability 1-4 substance for non-fuel use and also use some of it on-site as a fuel, the quantity used as a fuel is not covered; the quantity not used on-site as a fuel is potentially subject to the rule. If you are a retailer who sells a flammable fuel and a listed toxic substance, the toxic substance is potentially subject to the rule, but the flammable substance is excluded from coverage."


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When I first started writing, I loved fantasy and fully intended to write fantasy of my own. But I encountered a problem. It didn't take long for me to realize that I couldn't just skip mentioning religion in my stories, even if Christianity by such a name was limited to the real world. My characters needed a faith or a creed to base their actions on, yet inventing a religion of my own seemed almost sacrilegious.
Does Fantasy Need Religion?
At first I tried to ignore the need for religion in my stories. But that didn't make the issue go away. As a Christian, what I believe is a foundation for how I act. I'm not referring to mere traditions and rituals (which may or may not have a place in a story), but convictions that are the bedrock of life. [Read more…]


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- {{matchup.date.fullday}}{{matchup.date.month + ' ' + matchup.date.day}}
NCAAB Basketball - Preview
The Pennsylvania Quakers and the Brown Bears will both be trying to pick up a win on Friday when they battle at Pizzitola Sports Center.
Pennsylvania was a 75-72 winner in their most recent outing at home against Harvard. They covered the 7-point spread as underdogs.
In their last action, Brown was a 61-58 winner on the road against Columbia. They covered the 7.5-point spread as underdogs.
Team record: 8-20 SU, 11-11-1 ATS
The total has gone UNDER in 5 of last 5 games when playing on the road against Brown
is 7-1 SU in its last 8 games when playing on the road against Brown
is 3-12 SU in its last 15 games on the road
When playing on Friday are 2-8
Before playing Yale are 5-5
After playing Harvard are 7-3
After a win are 2-8
Team record: 12-14 SU, 9-8 ATS
Current streak: won 3 straight games.
The total has gone UNDER in 5 of last 5 games when playing at home against Pennsylvania
The total has gone UNDER in 10 of last 12 games when playing Pennsylvania
The total has gone UNDER in 4 of last 5 games at home
is 7-2 SU in its last 9 games at home
When playing on Friday are 6-4
Before playing Princeton are 1-9
After playing Columbia are 1-9
After a win are 4-6
Next up:
Pennsylvania at Yale, Saturday, March 9
Brown home to Princeton, Saturday, March 9


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August 17th, 1975. The Canadians had grown tired of American aggression. The Americans had been pressuring the land of the north to join the USA since the end of the war of 1812. The Canadians were trying as hard as they could to stay independent, but now they were full of rage. The thriving city of Seattle was the perfect target. It was a port city with a vast economy. Right across the border was the bustling city of Vancouver, a place of the same nature.
The Canadian Royal Air Force (CRAF) quickly rounded up around a hundred fighter jets and stationed them to fly across the border and bomb the city of Seattle and cripple it. The Americans had no idea of this attack and the citizens of Seattle went on their normal lives.
3 PM, August 17th, 1975. The planes had crossed the border and were entering American airspace. They aimed first for the city's harbor, for that was the imperative of the operation. Then they targeted downtown, and finally the surrounding suburbs. Harold Wisely was in his office in the Space Needle when the planes flew past and did a bombing run on it:
"A couple of bombs hit the tower. I was told to stay put. They gave us parachutes and the security guards cracked open a window and motioned us to jump. I had no choice."
Shortly after Harold Parachuted to the ground, the Space Needle fell.
The American military arrived two hours later to arrive to a destroyed Seattle. Hundreds of skyscrapers and buildings were damaged or completely destroyed. The harbor looked as if it was never there, only smoke arose from it. The suburbs were crippled, there was a ten mile radius of damage.
Over 23,000 People died in the attacks.
Later that evening, President Gerald Ford addressed Congress and asked them to declare war on Canada, which was met by an astounding yes.
From the voting records, over 93 percent of Congress voted for war.
And thus the quest for revenge began.


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LOS ANGELES (Reuters) - The "Hope For Haiti Now" charity album debuted at No. 1 on the U.S. pop chart, becoming the first digital-only release to achieve this feat, according to sales data issued on Wednesday.
The album, featuring songs performed by the likes of Madonna, Bruce Springsteen and Taylor Swift at last Friday's nationally televised telethon, sold 171,000 copies, said tracking firm Nielsen SoundScan.
Since the tracking week runs through Sunday, the "Hope for Haiti Now" numbers represented just two full days of sales. By contrast, Susan Boyle's former chart-topper "I Dreamed a Dream" held at No. 2 on the Billboard 200 chart after selling 86,000 copies during the full week.
MTV, which organized the two-hour fundraiser with actor George Clooney, said the album sold an additional 60,000 copies outside of the United States and hit No. 1 on leading digital retailer iTunes' chart in a total of 18 countries.
Consumers also downloaded 300,000 individual audio performances as well as 15,000 downloads of the full video telecast, MTV said.
The telethon also featured performances by Shakira, Alicia Keys, Stevie Wonder, Jay-Z, Bono, Beyonce, Sting, Justin Timberlake and dozens of other stars.
Watched by a total of 83 million viewers in the United States and millions more online, the event has raised more than $66 million so far including the music sales, MTV said.
All the labels, artists and retailers donated their share of proceeds, MTV said. Besides Apple Inc's iTunes, the other retailers are Amazon.com, RealNetworks Inc's Rhapsody, and Best Buy Co Inc's Napster.
Up to 200,000 people are estimated to have been killed in the January 12 earthquake.
Reporting by Jill Serjeant; Editing by Eric Walsh


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Featuring Model Prices and Specifications, plus Reviews,News and Videos.
what's hot: huge boot, diesel economy and wagon style what's not: styling is a bit nondescript, shallow floor won't suit all...
Interested in a new car? Simply tell us the vehicle you are interested in and we can take care of the rest.
Get great offers, the latest news and reviews, photos, videos and more delivered straight to your inbox.


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Each month, Rated People connects over 50,000 homeowners with quality tradesmen - simply enter your job details in the form above to get started.
We'll match your job to the relevant tradesmen in your area - those interested will pay to see your contact details and call you to quote.
As each tradesman expresses interest in quoting on your job, we'll send you their profile and ratings to help you choose the right one for you.
After the work is complete, return to the site and rate your tradesman based on their quality of work, reliability and value for money.
Added 09/05/2016
Services required: Slate / Tiled roof repair Repair: roof ridge, flashing On: the main part...
Added 30/05/2013
There is a difference in roof levels between two properties. Water can apparently get between...
Added 01/09/2012
take out gas fire in living room and cap gas pipe
Bathroom Specialist
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old bathroom suite and shower over bath to be removed and replaced. Wall tiles to be removed...
Managing your job is easier with the Rated People Homes App, download it today.


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Asus MeMO Pad FHD 10 Internet Tablet - Android 4.2 OS, 10.1" Touchscreen, 1.6GHz Intel Atom Z2560, 16GB Storage, MicroSD Slot, Front/Rear Camera, Built-in WiFi, HDMI, White - ME302C-A1-WHItem#: VSU-101768009 | Model#: ME302C-A1-WH
Currently Out Of Stock!
Asus MeMO Pad FHD 10 Android OS Internet Tablet Product Details
Manufactured by: Asus
Warranty provided by: Asus
UPC No: 886227517350
Mfg Part No: ME302C-A1-WH
Box Size:
( Length:11, Width:8, Depth:2)
Shipping Weight: 1.9000 pound(s)
Click here for full warranty and support information
- Asus logos are registered trademarks of Asus. All others trademarks and copyrights mentioned herein are the property of their respective owners.


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Accommodations withSee All
Dunkirk's climate can be said to be cold and temperate. Rainfall is abundant and spread over the year but the temperature is comfortably cool throughout. Winters are a little cold but snowfall is a rare phenomenon.
The best time to visit Dunkirik would ideally be during the months of May to September. The weather is very pleasant during these months and perfect for sightseeing. Though the city is bound to be crowded during this period as it is considered to be the peak season.
Explore the best travel planner for your visit to Dunkirk and create a flawless plan in few simple steps!


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Sep 1, 2004
Trinidad is using its oil and gas wealth wisely to build an advanced society. Will it be able to forge crucial international partnerships in time?
Trinidad's Prime Minister Patrick Manning wants to lift his
country out of poverty and become a fully developed state by
the end of the next decade. The country's substantial -
but dwindling - reserves of oil and gas are the key to
this. "One of our mandates is to develop our people and our
country and we want to achieve a standard of living that is
equivalent to a developed country by 2020," says Eric Williams,
minister for energy and energy industries. However, the country
is literally running out of time and energy. "Crude oil
reserves, at an estimated 990 million barrels, are expected to
last only another decade unless new reserves are found,"
according to the Energy Information Administration at the US
Department of Energy in Washington. And how can Trinidad
maintain its lucrative 77% share of US liquid natural gas (LNG)
imports and still have enough gas left...
To continue reading please take a free trial, subscribe or login below.
Subscribe now for unlimited access to all current and archive news, data and market analysis.
Take a free two-week trial now for the latest news, data and market analysis.
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September 14, 2017 | Shanghai, China
LatinFinance returns to Shanghai for the 8th edition ... more
September 26, 2017 | New York, USA
The sheer size of Latin America's infrastructure needs, coupled ... more
Celebrating the stand-out institutions and transactions of the past year in this important ... more
October 5, 2017 | Playa del Carmen, Mexico
The Infrastructure and Sub-Sovereign Finance in Mexico Summit remains the ... more
December 5, 2017 | Argentina
Argentina's provinces and municipalities are at the frontlines ... more
Where will capital markets be busiest in 2017?
All material is subject to strictly enforced copyright terms & conditions and cannot be repurposed or reproduced. © 2016 Latin American Financial Publications Inc.


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Report summaryCentrica has announced the acquisition of Suncor Energy's Trinidadian portfolio for US$380 million. The majority of the deal value lies in Block NCMA-1, an important gas supplier to the Atlantic LNG plant. The other interests - Blocks 22, 1(a) and 1(b) - hold sizable 'technical' gas resource, the potential monetisation of which represents a clear long-term upside to our base case valuation.The deal marks Centrica's first material upstream acquisition outside of Europe, ...
What's included?
This report includes 1 file(s)
- Centrica acquires Suncor's interests in Trinidad & Tobago PDF - 679.65 KB 8 Pages, 6 Tables, 1 Figures


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Premier League: Ashkan Dejagah believes Fulham can avoid relegation
Fulham winger Ashkan Dejagah believes that his side have the quality needed to beat the drop this season.
Last Updated: 18/01/14 2:24pm
The Cottagers sit just one point above the relegation zone and have had mixed fortunes since Rene Meulensteen replaced Martin Jol, winning three of their last eight Premier League following his arrival.
Dejagah successfully avoided relegation at Bundesliga side Wolfsburg three years ago, and he is now hoping to draw on that experience.
"It's not a good situation for us at the moment," he told the Fulham and Hammersmith Chronicle.
"There are a lot of teams fighting against relegation. I was in the same situation once with Wolfsburg. It's hard, but we managed to stay up.
"We have a good team and good players. If we all concentrate we can do it."
Meulensteen's side travel to league leaders Arsenal, a game in which Dejagah believes must be approached with a positive outlook.
"Arsenal are playing very good football this season," he said.
"It's hard to play there but we drew last year so anything can happen in football.
"We have to fight and we'll go there and stick together. It's not easy to get points there - they have a chance to win the league this season.
"But we're not going into it expecting to lose. We want to get something from the game."


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I'm new to cake decorating so please excuse me... I know this is maybe a silly question but what is the difference in gum paste and fondent???? I've only worked with fondent and was curious about gum paste and if it works better or not... Thanks
It depends on what you're using it for, sometimes it doesn't matter which you use. One of the big differences is that gumpaste can be rolled out thinner than fondant making it good for flower petals.
gum paste will dry fast and like said before you can roll it thinner... and it will become very hard overnight... some people call it cold porcelin (cant spell that word).
the fondant will also dry hard but not as fast. i use it to make flowers and it can take almost three days to dry... but its good sometimes bc it gives you more of a window to work with it... without drying out and craking on you.


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Oddo's Lazio moved ratified
By Graeme Bailey
Last Updated: 01/01/70 1:00am
The 26-year-old originally agreed to move to Lazio earlier this summer, but the move was delayed when the Italian authorities put a host of the biancoceleste's transfer dealings on hold.
This meant the likes of Oddo and Christian Manfredini were left in limbo, as they both had contracts with Lazio and there clubs Verona and Chievo, did not want them back.
Manfredini's deal was settled at the end of last month and now Oddo has been given the green-light to begin his career in Rome.
Oddo was named in the Italian squad last week and could prove to one of Roberto Mancini's best acquisitions.


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Our insights are published both in-house and in external media. Search our archive within our three service lines and learn more.
The business needs of all organisations are changing faster now than perhaps at any other period of human history. While leaders understand they must become more nimble, pro-active and even purpose-driven, responding quickly with the latest knowledge is a daunting responsibility.
At Mannaz, we've learned to pay close attention to our clients' greatest needs and growing concerns. As a result, we are focused on providing insights and capabilities in these critical areas of common interest, and are developing new methods and tools that can be customized to deliver the greatest potential for success.
Within our three service lines, we are highlighting 17 themes of greatest current significance to organisations. The Mannaz approach always means enabling real achievement and facilitating real change. Customised and contextually-relevant solutions are at the heart of all of our client work, as is our 'people-first' ethos.
At Mannaz, we've learned to pay close attention to our clients' greatest needs and growing concerns. As a result, we are focused on providing insights and capabilities in these critical areas of common interest, and are developing new methods and tools that can be customized to deliver the greatest potential for success.
Within our three service lines, we are highlighting 17 themes of greatest current significance to organisations. The Mannaz approach always means enabling real achievement and facilitating real change. Customised and contextually-relevant solutions are at the heart of all of our client work, as is our 'people-first' ethos.
Setting Strategic Direction
Today's business environment is often characterised as volatile, uncertain, complex and ambiguous. Mannaz helps to manage these challenges by encouraging organisations and business leaders to look ahead, understand their strengths, recognize the opportunities associated with change, and develop their greatest potential.
Defining a Change Agenda
Change is one of the most challenging elements of life within an organisation. To sustain a change agenda, both the nuances of corporate culture and the patterns of human behavior should become aligned.
Leading Strategy Execution
Implementing strategy today requires a balance between inspirational leadership and understanding the complete execution process, especially as a volatile, uncertain, complex and ambiguous world underscores the need to drive fast and effective strategy execution within an organisation.
Reinforcing Executive Leadership
In a fast-changing world where politics, economics, demographics and contemporary culture are affecting business every day, the pressure is on to develop good leaders who can continually adapt and learn new skills.
Transforming Organisations
Without question, transformation takes courage and teamwork, but meaningful and sustainable transformation also requires relevant structures and an assurance the behavioral change will be long-lasting.
Shaping Digital Organisations
Operating in a digital age with new levels of transparency, global reach and 24/7 access means that even the most successful legacy organisations need to adapt to new time-frames, codes of conduct and responsibilities.
Developing Talents
Although talent is often one of an organisation's greatest concerns, developing talents may be a more organic means to discovering new resources and keeping employee morale high.
Enabling Leaders
New leadership is the lifeblood of an organisation, particularly as companies submit to evolutionary, sustainable growth and recognize the importance of balancing both formal and informal leadership skills.
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Future-proofing Corporate Culture
While no one can fully anticipate what lies ahead, an organization can be aware and flexible enough to ensure its adaptation to change and expansion. Enlightened leaders play an important role in shaping corporate culture when they embody the best values and beliefs of an aspirational organization.
Professionalising Project Governance
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Mastering Project Portfolios
While all portfolios involve a variety of benefits and risks, mastering project portfolios enables an organization to ultimately see stronger results. Plus, organisations can benefit from considering the full portfolio of projects when making broader decisions.
Executing Complex Programs
We live in an age of greater multilayered complexity; yet, it is possible to help staff members learn better methods for executing intricate programmes, particularly given the management of large-scale programmes and the toolbox required to be successful.
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Managing effective projects today can mean the difference between superior and mediocre performance, particularly when combined with the right toolbox designed for success.
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Listen to free music played by LaughterAndJoy
Blip.fm is internet radio made social. It's easy to search for, play, and discover free music recommended by real people. Join today to create your own free station and start listening to @LaughterAndJoy.
avivajazz gave props on Aug 3 at 4:28 pm
carlaferrarezi_ gave props on Aug 1 at 6:11 pm
What other DJs are saying about Pantera - Cowboys From Hell (Live - Super Edit)
ZEW_FREAK Pantera – Cowboys From Hell
EarwaxOfSatan that would be a no @plummerm: "is is too early for Pantera? Pantera – Cowboys From Hell" (reblip)
plummerm is is too early for Pantera? Pantera – Cowboys From Hell
steppinheavy pantera - cowboys from hell {LIVE!!!}
eMars So out of the darkness and into the light
moniquekoster Pantera!!!~!!!!!


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CCS to host 2016 National Portfolio Day
CCS is set to host this year's National Portfolio Day on Sunday, October 30th.
CCS Chair of Transportation Design talks future of design at Autoblog Upshift event
CCS Chair of Transportation Design Paul Snyder talked about the future of automotive design at the Autoblog Upshift event.
CCS signs articulation agreement with FH Joanneum in Austria
CCS is pleased to sign an agreement with FH Joanneum (FHJ) on September 23, 2016. FH Joanneum is a university located in Graz, Austria with a leading art and design programs.
CCS welcome new Associate Professor of Product Design, Von Robinson
is pleased to announce that Von Robinson has been hired as an Associate Professor in our Product Design program
CCS featured in episode of Translogic on Autoblog
CCS's Transportation Design program is featured in an episode of Translogic on Autoblog
Art in America covers the exhibition currently running in CCS's Valade Family Gallery, "Ray Johnson: The Bob Boxes."
CCS Alumna and Fuseproject's Lead Industrial Designer featured in Fastco Design
Watch Me Work: Tyanna Buie, Assistant Professor, Fine Arts Section Chair of Printmaking
Who's got next? You do, says printmaking professor Tyanna Buie. Whatever you were thinking about doing today, let that go. Head over to the print studio with Tyanna Buie and learn a thing or two. Even if you're majoring in something else. Even if you don't know what printmaking is or how to do it.
CCS alumnus to head GM's Chinese venture, SGMW
Detroit's Murals in the Market led by CCS Illustration alumnus
CCS Photography Professor featured in this month's PDNedu
CCS's Office of Exhibitions & Public Programs featured in Culture Source
CCS MFA alumnus leading the charge in innovation at General Motors
Leading the charge in innovation, our MFA programs are shaping the future - literally. In fact, graduate Spencer Chamberlain's skills were so advanced that General Motors actually created a new position specifically for him: Senior Design Strategist and Innovation Architect
CCS alumnus illustrates for a national investigative series
Richard Watkins, CCS alumnus and Illustrator with The Atlanta Journal-Constitution, recently worked on a powerful collection of illustrations for a national investigative series, "Doctors & Sex Abuse."
CCS alumni design olympic gear for Usain Bolt and more
Astin Davis, CCS Advertising alumnus designed the competition gear for Jamaica, Barbados, Bahamas, Cuba, Grenada and Switzerland in this year's Rio games while Guercy Eugene, CCS Product Design alumnus designed the shoes.
CCS Product Design alumnus designing shoes for the NBA
Fine Arts alumnus exhibits at the Red Bull House of Art
Gabriel Vinas, CCS Fine Arts alumnus and clay modeler at the Ford Motor Company, is exhibiting at the Red Bull House of Art
The sixth-annual Detroit Design Festival (DDF), returns Sept. 21-25
CCS Graphic Design alumna featured in The Art Directors Club online
CCS Professor and Printmaking Section Chair featured in the Journal Sentinel
CCS Professor and Printmaking Section Chair Tyanna Buie featured in the Journal Sentinel


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D Maddoxx Model
Smith CharlotteModel Female Wilmington, North Carolina, US
My MM URL: http://www.modelmayhem.com/charlottesmith
Mayhem # 2135698
About MeI have been Modeling Professionally for 15 years. It started when I was much younger. I took a break for quite some time to persue my education. I currently run two businesses with my business partner, so my schedule is very flexible as long as I have advanced notice. Since I have two businesses to run and a full portfolio Im only taking paid assignments. I am will to consider doing TFP if it is a project that Im interested in. Im hard working and driven, but Im also very easy to work with and get along with. I take direction well and its very important to me thay you are pleased with the work we do together.
Have you worked with Smith Charlotte? Add Credits for Smith Charlotte! >>
D Maddoxx Model
Chad Jarae Photographer
Rick Geektograp... Artist/Painter
T D P Photographer
Garibaldii Digital Artist
Ashley Jo-anna Model
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Amrita Vishwa Vidyapeetham University
Junior Research Fellow
Address: Amritanagar, Coimbatore, Tamilnadu 641 112
Pay scale ( वेतन ) : Rs. 25,000/-p.m. + HRA
Educational requirements ( शैक्षिक आवश्यकता ) : Masters in Physics / Chemistry / Materials Science/ Nano science with minimum of 55% aggregates marks from recognized university with GATE/NET qualified in relevant subject.
Desirable qualifications ( वांछनीय योग्यता ) : Experience in thin film photovoltaics device fabrication and characterization.
No of vacancies ( रिक्तियों की संख्या ) : 01
Last date to apply for this job ( अंतिम तारीख ) : 06/30/2016
How to apply ( आवेदन कैसे करे ) : The position is temporary and renewable each year subject to satisfactory performance for a maximum of three years. Original documents of age proof/certificates/degrees/mark sheets and other testimonials must be presented at the time of interview. Applicants are required to submit self attested photocopies of documents with CV and latest photo at the time of interview. No application will be considered after due date and without proper format. No TA/DA will be paid for attending the interview.
Amrita Vishwa Vidyapeetham University Official Website : https://www.amrita.edu/job/junior-research-fellow-serb-sponsored-rd-project
Amrita Vishwa Vidyapeetham University (Coimbatore, Tamil Nadu) Recruits Junior Research Fellow Posts. M.sc, NET/ GATE qualified, Experienced candidates can apply.
About Amrita Vishwa Vidyapeetham University
Amrita University is a multi-campus, multi-disciplinary research university that is accredited 'A' by NAAC and is ranked as one of the best research universities in India. The university is spread across five campuses in three states of India – Kerala, Tamil Nadu and Karnataka, with the University headquarters at Ettimadai, Coimbatore, Tamil Nadu.
Website Address: https://www.amrita.edu/
Contact Us: Amrita Vishwa Vidyapeetham
Amritanagar, Coimbatore – 641 112
Tamilnadu, India
+91 422 2685000
Fax: +91422 2686274


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Product Spotlight
Featured Research
Quick Stats
You are here
Quick Chek Moves Farther in New York
Meanwhile, Kwik Trip expands stores to boost its kitchen area.
SAUGERTIES, N.Y. -- While other companies cut back, Quick Chek and Kwik Trip are expanding.
Whitehouse Station, N.J.-based Quick Chek opened its sixth New York store a few weeks ago and a couple more are on the way in the Hudson Valley area, according to The Times-Herald Record.
A Saugerties location is scheduled to open in May, and construction has begun on a site in Middletown, slated to open during the second half of the year, according to the report.
"That's all we have definite this year in New York," John Schaninger, vice president of sales and merchandising, told the newspaper.
Quick Chek is likely to open at least two more New York stores next year, a second location in the town of Wallkill and one in Monroe, Schaninger said. The company's real estate committee visited the region last week to scout sites.
"We're continually looking and aggressively working the Hudson Valley. It's just a great market with great people," Schaninger said.
Quick Chek's convenience stores are typically more than 6,000 square feet. They offer staples, as well as an extensive selection of sandwiches and flavored coffee. Its gasoline is usually priced under the competition's street price.
"It's pretty much a pass-through product for us," Schaninger told the newspaper.
A typical store generates $1 million in tax revenue and creates 30 jobs, and the chain is dedicated to promoting from within. "The only way for our people to grow is to open stores," Schaninger said.
Meanwhile, in Minnesota, Kwik Trip Inc. is expanding its stores, a reflection of a chainwide effort to boost its offer, according to the Albert Lea Tribune.
Additions are being made at many Kwik Trip stores to of the company's stores to incorporate more of a kitchen area into the building, according to Hans Zietlow, director of real estate for Kwik Trip.
"We have a greater and greater food offering in our stores now," Zietlow told the newspaper. "We want to expand that out to be able to offer more products and create a little better customer service."
In Albert Lea, Minn., one store will have a 645-square-foot kitchen addition valued at $190,000, according to the Albert Lea Inspection Department. Another location will see a 734-square-foot addition valued at $160,000, the newspaper reported.
Related stories:
-- Quick Chek Launches Second Annual Devils Night Out Contest
-- Kwik Trip to Remodel Downtown La Crosse Store
-- Kwik Trip, Nice N Easy Founders Recall C-store Industry's Origins
-- Kwik Trip Manager Loses Bet, Wears Dress


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The imposter Mikey was skinny at the beginning and when he walked down the ramp, but he is fat, so he should've been fat for the whole episode, I mean, one second he's fat, then he's skinny, then he's fat again.
At the "World Unity Concert", the Russian emperor calls his daughter "Babushka". In Russian, Babushka means "grandmother"!
At Mandy's pool party, after everyone left finding out Mandy's tattoo was fake, you see David standing next to the food table with Alex. Look closely and you will see the food table changes at least 3 times in height. The third time its barely even higher than David's knee's.
After Sam says 'Curtain call Ladies', you see Alex go down the middle curtain and Clover on the left side... then it shows another veiw where Sam is waiting at the bottom and Alex ends up on the Left side and Clover in the middle curtain.
Alex: (after getting the ginger-haired woman's face) I can't believe T-Bone would do this to me. Look at my face, my skin looks like beef jerky!
Alex: Ew, I hate needles!
Clover: Hate needles? Hello. How do you think tattoos are applied?
Alex: I thought they ironed on.
Jerry: (After WOOHPing the girls from a sauna) Sorry to interrupt your conversation just as things were heating up.
Clover: Hello, not funny much?
Mandy: Only the coolest people are going to be there. You and Sam are coming, right Clover?
Clover: We are?
Mandy: You two qualify. I think its cool that you two can walk around with last year's hairstyles and still act secure.
Sam: You guys have nothing to fear with us around.
(Stampeding fans break through the doors and run over them.)
Alex: That felt like a herd of buffalo.
Clover: Yeah, a herd of buffalo wearing pumps.
Mandy: (when her tattoo washes off in the pool revealing it to be a fake, and everyone leaves the party) Wait! Don't go! I'm just as surprised as you guys! The directions on the box say it was permanent ink!!
Yoga Instructor: Hello, I'm Shirley. Welcome to my world of pain.
David mentions that he is a vegan
Gadgets: Expandable Cable Bungee Belt
First appearance of Shirley
Lead singer of Boy Candy is T-Bone
First mention/appearance of Teensicle
Yoga moves: Long tiger,skipping goat,squating bear
We find out that Clover has a kitten
Teletoon Air Date: April 15th 2003.
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Hand in your heart, Money mark
#----------------------------------PLEASE NOTE--------------------------------# #This file is the author's own work and represents their interpretation of the# #song. You may only use this file for private study, scholarship, or research.# #-----------------------------------------------------------------------------# Date: Sat, 9 May 1998 16:52:48 +0100 From: Ben Smith <firstname.lastname@example.com> Subject: m/money_mark/hand_in_your_heart.crd Hand In Your Head - Money Mark Writen by Mark Ramos-Nishita Transcribed by Ben Smith (email@example.com) >From the albums " Mark's Keyboard Repair" & "Push The Button" 1, 2, 1-2-3-4 F Dm G Dm x2 Verse 1: F Dm You could be my best friend G Dm Sitting on the book ends F Dm Look guy, you're just a king G Dm You know you're missing something F Dm I've got my hand in your head G Dm I've got my hand in your head Verse 2: I've read about you in books Your kingdom's always full And if you did know the truth You wouldn't promise to tell F Dm I've got my hand in your head G Dm I've got my hand in your head Chorus: C#maj7 Bb I've got my hand in your head x3 Gm Ab Gm Ab Gm Ab Gm Ab Gm F And I'm pulling out all of your mind Verse 3: I waited for you to say All the things that you think And now that we're here alone You can remember to blink I've got my hand in your head I've got my hand in your head
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text: Maintreedescendantsancestors William Martin (c1859-) Jessie Hamilton Martin (1901-) Isabella R Hamilton (c1865-?) Retrieved from "http://familypedia.wikia.com/wiki/Jessie_Hamilton_Martin_(1901-)/tree?oldid=866086" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Hidden category: Tree pages


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Homemade Cake Mix Recipe
By Melissa Norris | How to articles , Recipes
Would you believe that making a homemade cake mix is faster and cheaper than running to the store to buy one in a box? It's true. Plus, this homemade cake mix recipe tastes better than the boxed counter part, doesn't contain any questionable GMO ingredients, and it can be stored in one of my favorite […]Read More


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The purpose of this study was to determine whether Src tyrosine kinases are one of the signaling intermediaries linking M2 receptor stimulation to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in cultures of canine colonic smooth muscle cells (CSMC). RT-PCR studies demonstrate expression of multiple Src tyrosine kinases, including Src, Fyn, and Yes, in CSMC. Muscarinic stimulation of CSMC with 10 μM ACh results in a twofold increase in Src activity within 10 min but does not increase the activity of Fyn. Treatment with the M2 antagonist AF-DX 116 (10 μM) blocks ACh-stimulated Src activation in primary CSMC cultures that express both M2and M3 receptors and in first-passage CSMC cultures that express predominantly M2 receptors. Alkylation of M3 receptors with 100 nMN,N-dimethyl-4-piperidinyl diphenylacetate mustard has no effect on Src activity. Treatment with the pyrazolopyrimidine Src inhibitor PP1 (10 μM) or AF-DX 116 (10 μM) blocks ACh-stimulated ERK phosphorylation. Together these results indicate that M2 receptors are coupled to Src tyrosine kinase and subsequent activation of ERK in cultured CSMC.
- Src
- tyrosine kinase
- colonic smooth muscle
- extracellular signal-related kinase
- mitogen-activated protein kinase
acetylcholine is the principle excitatory neurotransmitter of the parasympathetic nervous system, acting at both nicotinic and muscarinic receptors in the ganglionic synapse and at muscarinic receptors in the postsynaptic junction. In the colon, the actions of ACh at the postsynaptic junction mediate smooth muscle contraction through M2 and M3 muscarinic receptors. M3 receptors, coupled to the heterotrimeric GTP-binding protein (G protein) Gq/G11, stimulate phospholipase C and phosphatidylinositol turnover to trigger release of stored calcium and activate protein kinase C (37). The more abundant M2 receptors, coupled to Gi/Go, inhibit adenylate cyclase, activate nonselective cation currents, and sensitize the contractile system to calcium (17, 33). The events mediated by M2 and M3 receptor activation contribute to changes in myoplasmic calcium levels necessary to generate contractile force. However, the signaling pathways that mediate these events are not completely understood.
The transduction of extracellular signals through G protein-coupled receptors involves the activation of multiple protein kinase cascades. Although specific pathways vary with different G proteins, activation of Src tyrosine kinases appears to be one of the signaling events immediately following receptor activation (14). Src is the prototypical member of a large family of tyrosine kinases expressed in a variety of cell types. These enzymes are membrane-associated nonreceptor protein tyrosine kinases activated by diverse stimuli and capable of phosphorylating numerous cellular targets (3,32). Although muscarinic stimulation of Src tyrosine kinases in colonic smooth muscle (CSM) has not been well described, Src has been studied in smooth muscle under a variety of other conditions. Inhibition of tyrosine phosphorylation appears to affect smooth muscle contraction mediated by growth factors in gastric smooth muscle (39) and ceramide in CSM (20). In vascular smooth muscle, Src appears to be necessary for angiotensin II-mediated extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation (22). In addition, Src is thought to activate voltage-dependent Ca2+ channels (34,35) and to stimulate phospholipase C activity (29).
These observations led our laboratory to determine which members of the Src tyrosine kinase family are intermediary molecules coupling muscarinic receptor stimulation to downstream signaling events in cultured CSM cells (CSMC). We have examined the expression of multiple Src tyrosine kinases, including Src, Fyn, and Yes, in canine CSM and have demonstrated that muscarinic stimulation of cultured CSMC results in the activation of Src but not Fyn tyrosine kinase. Additional pharmacological studies suggest that activation of Src in cultured CSMC is mediated via M2 but not M3 muscarinic receptors. Previous work in our laboratory (6, 13) demonstrated that activation of ERK MAPK in intact CSM is mediated by M2 receptor stimulation in intact CSM, and we present data supporting the hypothesis that Src tyrosine kinase couples M2 receptor stimulation to ERK MAPK in cultured CSMC.
Genistein and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) were obtained from Calbiochem (La Jolla, CA).N,N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard was purchased from Research Biochemicals International (Natick, MA). AF-DX 116 was purchased from Tocris Cookson (Ellisville, MO). Tissue culture reagents, TRIzol, and SuperScript II reverse transcriptase (RT) were purchased from GIBCO-BRL (Rockville, MD). ITS+ (6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenious acid, and 5.35 μg/ml linoleic acid) was purchased from Collaborative Biomedical Products (Bedford, MA). Thermus aquaticus polymerase (Taq), RNAse H, and goat anti-rabbit IgG conjugated to alkaline phosphatase were purchased from Promega (Madison, WI). All other PCR reagents were purchased from Invitrogen (San Diego, CA). Oligonucleotides for muscarinic receptors and Src tyrosine kinases were synthesized by Bio-Synthesis (Lewisville, TX). 18S rRNA primers were purchased from Ambion (Austin, TX). Enolase was purchased from Boehringer Mannheim (Indianapolis, IN). 32P-labeled ATP was purchased from ICN Biomedicals (Costa Mesa, CA). Goat anti-Src (c-Src N-16), mouse anti-Fyn (Fyn 15), and rabbit anti-Src (Src 2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal phospho-p42/p44 ERK MAPK antibody was purchased from New England Biolabs (Beverly, MA). All other reagents were obtained from Sigma (St. Louis, MO).
Cell culture.
The circular muscle layer of proximal colon was isolated from adult mongrel dogs of either sex killed with pentobarbital sodium (45 mg/kg iv). The tissue was minced and enzymatically digested as previously described (16). Dispersed cells were recovered by centrifugation and resuspended in M199 supplemented with 10% newborn calf serum, 0.2 mM glutamine, and antibiotics on collagen-coated flasks. Experiments were performed with confluent cultures of primary cells (1°CSMC) or confluent cultures of first-passage cells (1P CSMC) after 24 h of serum deprivation in M199 supplemented with ITS+. These studies were approved by the Institutional Animal Care and Use Committee.
RNA isolation and RT-PCR.
Total RNA was isolated using TRIzol reagent from canine colonic circular smooth muscle (1 ml TRIzol/100 mg) and from confluent cultures of 1°CSMC and 1P CSMC (1 ml TRIzol/cm2) according to the manufacturer's instructions. First-strand cDNA synthesis was performed at 42°C from 2 μg of RNA using 250 ng of random hexamers, 50 mM Tris · HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 0.125 mM dATP, dTTP, dGTP, and cCTP, and 1 unit of SuperScript II reverse transcriptase in a volume of 30 μl. Src tyrosine kinases were amplified by PCR in a thermal cycler (GeneAmp PCR System 2400, Perkin Elmer). The reaction mixture contained 60 mM Tris · HCl (pH 8.5), 15 mM (NH4)SO4, 1.5 mM MgCl2, 0.25 mM dATP, dCTP, dGTP, and dTTP, 0.2 ng/ml of each primer, template cDNA, and 1.25 units of Taq. Amplification of Src cDNA took place at 94°C for 1 min, 63°C for 30s, and 72°C for 30s for 35 cycles with oligonucleotides obtained from the mouse sequence (M17031): 5′-CCTTCCCGGCCTCACAGACACC-′3 and 5′-TCGGAGGGCGCCACATAGTGGC-′3. Amplification of Fyn cDNA took place at 94°C for 1 min, 57°C for 30 s, and 72°C for 30 s for 35 cycles using oligonucleotide derived from the human sequence (M14333): 5′-GGGCAGTTTGGGGAAGTATGGATG-′3 and 5′-TGGGGAACTTTGCACCTTGTCTTG-′3. Yes cDNA was amplified at 94°C for 1 min, 57°C for 1 min, and 72°C for 1.5 min for 35 cycles from oligonucleotides obtained from the canine spleen sequence (S81472): 5′-ATTTTCAGTGGTGCCAAGTCCATA-′3 and 5′-CACCCCTTATCTCATCCCAATCAC-′3. Amplification of M2 and M3 muscarinic receptor subtypes took place at 94°C for 1 min, 57°C for 1 min, and 72°C for 1.5 min using oligonucleotides obtained from the human sequence of the M2 muscarinic receptor (X15264): 5′-CGGACCACAAAAATGGCAGGTA-′3 and 5′-TTGTATGGGGCCCAAGTGATGA-′3. Oligonucleotides for the M3 muscarinic receptor were also obtained from the human sequence (U29589): 5′-AGCGTGGACGATGGAGGCAGTTT-′3 and 5′-TGGCACAGCAGCAGCATGTTGAA-′3. 18S rRNA was amplified using primers obtained from Ambion and was used as a control for cDNA synthesis and gel loading. Reaction products were separated by electrophoresis through a 1.2% agarose-Tris-acetate EDTA gel and visualized with ethidium bromide. Products were sequenced by dye terminator cycle sequencing on an ABI Prism 310 Genetic Analyzer (Perkin Elmer) according to the manufacturer's protocols.
Pharmacological studies in cultured 1P CSMC.
Experiments were performed in confluent cultures of 1P CSMC serum deprived for 24 h. For time course experiments, cultures were stimulated with 10 μM ACh at the times indicated. In selected experiments, confluent cultures of 1P CSMC were preincubated for 15 min with a DMSO vehicle control (0.01%), the Src inhibitor PP1 (10 μM), the tyrosine kinase inhibitor genistein (10 μM), or diadzein (10 μM), an inactive congener of genistein. These inhibitors remained in the cultures during stimulation with 10 μM ACh for 10 min. In other sets of experiments, confluent cultures of 1P CSMC were preincubated for 15 min with the nonselective muscarinic receptor antagonist atropine (10 μM) or the M2 receptor antagonist AF-DX 116 (10 μM). Whole cell lysates were prepared from all treatments by homogenization and centrifugation in RIPA [50 mM HEPES, 150 mM NaCl, 1 mM sodium orthovanadate, 10 mM NaF, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 μM leupeptin, 0.5% Triton X-100, 0.5% NP-40, and 10% glycerol]. Protein concentrations were determined using the bicinchoninic acid method.
Immune complex assay for Src tyrosine kinase activity.
Src or Fyn tyrosine kinase was immunoprecipitated from whole cell lysates by incubating 150 μg of total protein for 2 h with goat anti-Src (c-Src, N-16) in the presence of protein A/G agarose beads or with an agarose conjugate of mouse anti-Fyn (Fyn 15). These antibodies are kinase specific, and according to the manufacturer they do not cross-react with other known Src tyrosine kinases. In addition, preliminary experiments were performed to verify the specificity of these antibodies in our cultures (data not shown). Beads were then washed twice in RIPA and once in kinase assay buffer (50 mM PIPES, pH 7.0, 10 mM MnCl2, and 10 mM DTT). Immunoprecipitates of either Src or Fyn tyrosine kinase were incubated for 60 min at 30°C in kinase assay buffer, 40 μCi of [γ-32P]ATP, and 4 μg of acid-treated enolase according to published protocols (4,7). Proteins were separated by 8% SDS-PAGE, enolase phosphorylation was detected by phosphorimaging, and densitometric analysis was performed (model 525 molecular imager; Bio-Rad, Hercules, CA). Aliquots from the immunoprecipitates were also separated by 8% SDS-PAGE and immunoblots performed with a rabbit anti-Src antibody (Src2). Although this antibody recognizes Src, Fyn, Yes, and Fgr tyrosine kinases, it is useful in monitoring the immunoprecipitation of individual kinases. Immunoreactive bands were visualized after incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG. Images of immunoblots scanned with a UMAX Powerlook flatbed scanner were analyzed by densitometry, and enolase phosphorylation was normalized to the immunoreactivity measured from the immunoprecipitates.
Pharmacological studies with 4-DAMP mustard and AF-DX 116 in cultured primary CSMC.
Experiments were performed in confluent cultures of 1°CSMC serum deprived for 24 h. 4-DAMP mustard was activated in M199 supplemented with ITS+ at 37°C for 30 min. Cultures were then incubated for 30 min with 100 nM 4-DAMP mustard in the presence of 1 μM AF-DX 116 to alkylate M3 receptors while protecting M2 receptors. Activated, unbound 4-DAMP mustard was inactivated by incubating the treated cultures for 15 min in fresh medium containing 0.5 mM Na2S2O3and 1 μM AF-DX 116. Control cultures received 0.1% DMSO and were also treated with 0.5 mM Na2S2O3and 1 μM AF-DX 116. Cultures were then stimulated with 10 μM ACh for 10 min, and whole cell lysates were prepared for immune complex kinase assays. Details of this protocol were described previously (6, 11).
ERK MAPK immunoblots.
Samples from 1P CSMC generated to measure Src tyrosine kinase activity were also used to examine ERK MAPK phosphorylation. Ten micrograms of total protein from whole cell lysates were separated by 10% SDS-PAGE. Immunoblots were incubated with the phospho-p44/p42 ERK MAPK primary antibody (1:1,000). This antibody recognizes phosphorylation of the tyrosine and threonine residues in p44 ERK1 and p42 ERK2 MAPK necessary for activation of these enzymes. Results were visualized after incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:10,000), and densitometry was performed as described.
Statistical analysis.
Statistical analysis was performed using SigmaStat software (Jandel Scientific, San Rafael, CA). A two-tailed Student's t-test was used to test unpaired parametric data, and a Mann-Whitney rank-sum test was used to test nonparametric data. The null hypothesis was rejected if P < 0.05.
Radioligand binding and quantitative PCR studies have established the expression of M2 and M3 receptors in canine circular smooth muscle (6, 38). Figure1 A presents qualitative RT-PCR studies performed in CSMC cultures to compare M2 and M3 receptor expression with that found in intact CSM. Reaction products of the expected size were amplified from CSM, cultured 1°CSMC, and 1P CSMC with receptor-specific primers and verified by sequencing. M2 receptor (810 bp; Fig.1 A, top) and M3 receptor (450 bp; Fig. 1 A, middle) expression is shown along with RT-PCR amplification of 18S rRNA (324 bp, Fig. 1 A,bottom), which is used as a control for cDNA synthesis and gel loading. As expected, M2 and M3 receptors are expressed in CSM. Cultured 1°CSMC retain expression of M2 and M3 receptors, but after passage of the cells and further time in culture, 1P CSMC appear to predominantly express M2 receptors.
To address the issue of which Src tyrosine kinases may be expressed in CSM, qualitative RT-PCR was again performed in CSM and cultured CSMC. Although multiple Src tyrosine kinases have been cloned from a variety of mammalian cell types, we chose to concentrate our efforts on Src, Fyn, and Yes, three enzymes ubiquitously expressed in most tissues. Figure 1 B shows Src-family tyrosine kinase expression in CSM, 1°CSMC, and 1P CSMC using gene-specific primers. All reactions amplified single PCR products of the expected size with nucleotide identities of 89% to mouse neuronal Src, 93% to human placental Fyn, and 94% to canine splenic Yes. Translation of the sequenced products revealed 93%, 96%, and 98% identity to Src, Fyn, and Yes, respectively. The results suggest that more than one Src tyrosine kinase is expressed in CSM, which is consistent with data obtained from cultured vascular smooth muscle (9). In addition, CSMC appear to retain expression of Src, Fyn, and Yes with time in culture.
To determine which of the Src tyrosine kinases present in CSMC are activated by a muscarinic stimulus, immune complex kinase assays were performed using acid-treated enolase as a substrate for tyrosine phosphorylation. The method allows for the separation of individual kinases from whole cell lysates by immunoprecipitation of the kinase of interest. In the described experiments, 1P CSMC were serum deprived for 24 h and treated at the times indicated in Fig.2 with 10 μM ACh. Control experiments were initially performed with 1P CSMC cultures treated with 10 ng/ml platelet-derived growth factor (PDGF), which is a known stimulant of Src tyrosine kinases in nonmuscle cells. PDGF stimulation of 1P CSMC increased tyrosine kinase activity of Src and Fyn immunoprecipitates in a time-dependent manner (data not shown). In contrast, ACh stimulation of 1P CSMC only increased the activity of Src immunoprecipitates, with peak activation of an approximately twofold increase over basal levels after 10 min of ACh stimulation. These results suggest that, although multiple Src tyrosine kinases are expressed in intact canine CSM and cultured CSMC, Src, but not Fyn, tyrosine kinase is activated by a muscarinic stimulus. The specificity of acid-treated enolase as a substrate for Src in cultured CSMC was examined by treating cultured 1P CSMC with PP1, genistein, or diadzein in the presence of ACh. The effect of these drugs on Src tyrosine kinase activity in an immune complex kinase assay after Src immunoprecipitation is shown in Fig. 3, in which inhibition of tyrosine phosphorylation with 10 μM genistein or inhibition of Src with 10 μM PP1 blocks enolase phosphorylation in Src immunoprecipitates. Treatment with 10 μM diadzein, the inactive congener of genistein, did not affect enolase phosphorylation.
As shown in Fig. 1 A, RT-PCR demonstrated that the 1P CSMC used in these experiments predominantly express the M2muscarinic receptor, thereby making it plausible that the observed increases in Src activity may be mediated via M2 receptors. This was tested by examining the coupling of M2 receptors to Src activation in the presence of the nonselective muscarinic receptor antagonist atropine and the M2-specific antagonist AF-DX 116 (Fig. 4). Stimulation of Src tyrosine kinase activity with 10 μM ACh for 10 min in the presence of 10 μM atropine or 10 μM AF-DX 116 results in a significant decrease in Src activity. In addition, the presence of atropine does not further reduce Src activity beyond that seen with AF-DX 116, suggesting that M2 receptors are the muscarinic receptors present in these cells that significantly contribute to activation of Src tyrosine kinase. This result, however, does not rule out the possibility that M2 receptors mediate Src activity in the absence of M3 receptors.
To confirm coupling of M2 receptors to Src in CSMC, we utilized confluent cultures of 1°CSMC, which express both M2 and M3 receptors. Coupling of Src to M2 receptors was investigated in 1°CSMC as described above for 1P CSMC, in which ACh-stimulated Src activity was measured in the presence of 10 μM AF-DX 116. Coupling of Src to M3receptors was investigated in 1°CSMC by alkylation of M3receptors with 100 nM 4-DAMP mustard in the presence of 1 μM AF-DX 116 with a protocol previously published by our laboratory (6). The inclusion of AF-DX 116 protects M2receptors during alkylation by 4-DAMP mustard, which is only modestly selective for M3 receptors [dissociation constant (K d) = 7.2 nM for M3 receptors;K d = 43 nM for M2 receptors]. The results from these experiments are presented in Fig.5, in which treatment with 10 μM AF-DX, but not 100 nM 4-DAMP mustard, blocks ACh-stimulated Src activity. Doses of 1 or 10 nM 4-DAMP mustard produced similar results (data not shown). These results suggest that in the presence of M2 and M3 receptors, M2 receptors are coupled to Src tyrosine kinase activity in cultured CSMC.
Previous work from our laboratory (6) in intact canine CSM demonstrated that M2 receptor activation is coupled to ERK MAPK phosphorylation. In 1P CSMC, ERK phosphorylation peaks after 10 min of ACh stimulation and is sustained above basal levels for at least 30 min (data not shown), consistent with previous reports from our laboratory (13) in intact canine CSM. To determine whether Src tyrosine kinase is one of the signaling intermediaries coupling M2 receptors to ERK, we again used the Src inhibitor PP1 and measured ERK phosphorylation from samples in which Src activity had been previously determined (Fig.6 A). In these samples, 10 μM PP1 blocks both ACh-stimulated ERK1 and ERK2 phosphorylation, establishing that Src tyrosine kinase is an upstream activator of ERK after a muscarinic stimulus in 1P CSMC. Figure 6 Bdemonstrates that treatment with 10 μM AF-DX 116 blocks ACh-stimulated ERK phosphorylation, thereby confirming the results reported from intact tissue (6) in cultured CSMC. Together these results clearly demonstrate that Src tyrosine kinase couples M2 receptor activation to ERK MAPK phosphorylation in cultured CSMC.
Although contraction of gastrointestinal smooth muscle is mediated primarily through M3 receptors (10), which are directly coupled to increases in intracellular calcium concentrations, the functional role of M2 receptors in smooth muscle remains unclear. The absence of M3 receptors on alkylation with 4-DAMP mustard results in M2 receptors mediating a majority of the contractile response in guinea pig trachea, but the same is not true in the guinea pig esophagus or colon (31). Previous studies in our laboratory (6) extended these results in canine colon, in which M2receptors appear to play only a minor role in directly mediating contraction. It is interesting to note, however, that the ratio of M2 to M3 receptors differs in various smooth muscle preparations. In the circular smooth muscle of canine colon, radioligand binding studies and quantitative PCR methods have determined that M2 receptors are more abundant than M3 receptors (6, 38). In the CSM culture model presented in our studies, the expression of M2 and M3 receptors differs in culture from that seen in intact tissue. Confluent cultures of primary CSMC express both M2and M3 receptors, but with time in culture, 1P CSMC predominantly express the M2 receptor subtype. The functional implication of this result may be explained by work done in airway smooth muscle (15). In first-passage airway smooth muscle cultures, Halayko et al. (15) described limited expression of M3 receptors. Prolonged serum deprivation results in an upregulation of M3 receptors along with an increase in smooth muscle α-actin, smooth muscle myosin heavy chain, and SM22, a smooth muscle-specific actin-binding protein, in a subpopulation of cells. More importantly, these cultures acquire a functional contractile phenotype. Given these observations, the absence of M3 receptors in our 1P CSMC suggests that these cells do not retain a contractile phenotype. These cultures do provide a nice model system in which to study M2 receptor-mediated signaling events, which may indirectly contribute to the contractile response through signaling pathways that affect cAMP production, nonselective cation channels, or calcium sensitization of contractile proteins.
Very few data exist on the effects of muscarinic stimulation on tyrosine phosphorylation in smooth muscle, but there is a growing body of evidence on the effects of growth factor stimulation in smooth muscle, which suggests that M2 receptor-mediated events may activate tyrosine kinases. Growth factors that increase tyrosine phosphorylation, such as epidermal growth factor and PDGF, lead to contraction of gastric and vascular smooth muscle preparations (1, 2, 36). Contraction induced by these growth factors can be blocked with tyrosine kinase inhibitors such as genistein and tyrphostin (28, 36). In addition, prolonged tyrosine phosphorylation due to treatment with protein tyrosine phosphatase inhibitors such as vanadate stimulates contraction (8, 19,25). In vascular smooth muscle, the tyrosine kinase inhibitor genistein decreases contraction, possibly by blocking calcium influx (34). Genistein also blocks agonist-induced sensitization of contractile proteins to calcium in ileal smooth muscle (30). In CSM, the tyrosine kinase Src modulates voltage-dependent calcium channel activity induced by PDGF (18) and ceramide-stimulated Src activation is reduced in the absence of calcium (20).
The Src tyrosine kinases are a large family of nonreceptor tyrosine kinases activated by multiple signals with varying tissue expression patterns. In vascular smooth muscle, Src tyrosine kinase is expressed (24), whereas other members of the Src family, including Fyn, Lyn, Hck, and Fgr, are expressed in cultured human aortic smooth muscle cells (9). The data presented here demonstrate that Src, Fyn, and Yes tyrosine kinases are expressed in CSM and cultured CSMC. The functional role of multiple Src tyrosine kinases in CSM is not known, and with such a large gene family of tyrosine kinases it is difficult to assess the contribution of each enzyme. Therefore, in these studies, we chose to concentrate our efforts on Src and Fyn tyrosine kinases. On muscarinic stimulation of CSMC, however, only Src tyrosine kinase activity was increased. Because both Src and Fyn activities are increased with PDGF stimulation in these cultures, it appears that Fyn is an active kinase but is not coupled to muscarinic stimulation as measured in these studies. These results suggest that tyrosine kinases may be differentially coupled to a variety of stimuli in CSM and that Src is one of the tyrosine kinases coupled to muscarinic receptor signaling.
The signaling intermediates linking pertussis toxin-sensitive Gi-coupled receptors to Src activation have been elucidated in nonmuscle cells. On stimulation, Gβγ-subunits are liberated from the G protein complex and promote assembly of signal transduction complexes consisting of Src, Shc, and Grb2 proteins. Activation of this signaling complex goes on to mediate Ras-dependent activation of MAPK (26). Previous work in our laboratory (6, 13) demonstrated that muscarinic stimulation induces ERK MAPK activation in colonic smooth muscle. Alkylation of M3 receptors with 4-DAMP mustard did not affect ERK MAPK phosphorylation, but inhibition of M2 receptors with AF-DX 116 blocked muscarinic stimulation of ERK, leading to the conclusion that this effect is mediated via M2, but not M3, receptors. In light of the data presented here, it is likely that Src tyrosine kinase may be an upstream mediator of M2 receptor-mediated signaling in CSM. AF-DX 116 blocks M2 receptor stimulation of Src in 1P CSMC cultures that express M2 receptor and in 1°CSMC that express both M2 and M3 receptors, whereas alkylation of M3 receptors does not affect muscarinic stimulation of Src activity. In addition, inhibition of Src with PP1 or treatment with AF-DX 116 blocks ACh-induced ERK MAPK phosphorylation. Thus Src appears to be the intermediary signaling molecule coupling M2 receptors to ERK MAPK. This is supported by previous results in vascular and airway smooth muscle, in which angiotensin II- and PDGF-stimulated Src activation is necessary for ERK MAPK activation (5, 22). These results also concur with those found in nonmuscle cells, demonstrating that Src participates in M2 receptor activation of ERK (21).
The functional significance of muscarinic stimulation of ERK MAPK in CSM remains to be defined. Our laboratory has proposed (6,12) that ERK MAPK may contribute to calcium sensitization, possibly through phosphorylation of the actin-binding protein caldesmon, even though the role of caldesmon during contraction is not clear. It is also likely that Src-mediated activation of MAPK signaling pathways leads to the phosphorylation of numerous targets, which may be intimately involved in contractile processes and in remodeling of the actin cytoskeleton. Potential targets of this pathway include myosin light chain kinase (23) calponin (27), and elements of focal adhesion complexes that couple actin filaments to the cell membrane. In addition, there are numerous ERK MAPK substrates whose functions may play peripheral roles in regulating the response such as other kinases, transcription factors, and phospholipases. Therefore, although activation of M2 receptors alone produces only a modest mechanical response in smooth muscles, there are numerous downstream targets for Src-mediated activation of ERK MAPK that could contribute to contraction, cytoskeletal structure, and regulation of pathways important in smooth muscle growth and phenotype determination.
The technical assistance of Michelle Deetken and Shanti Rawat is gratefully acknowledged.
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41315. C. A. Singer is a recipient of National Heart, Lung, and Blood Institute National Research Service Award Postdoctoral Fellowship HL-10072.
Address for reprint requests and other correspondence: C. A. Singer, Dept. of Pharmacology/318, Univ. of Nevada School of Medicine, Reno, NV 89557-0046 (E-mail: cas{at}med.unr.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2002 the American Physiological Society


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