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Baker, R. Granville. The Walls of Constantinople. John Milne, London, 1910. Hardcover. Black lettering and design stamped on reddish brown cloth. Gilt lettering on spine. 30 illustrations. Good, with tear to top left edge of spine. Text and interior are very good and well-bound. Ex-lib with markings on spine and first few pages, as usual. Author spent years of investigation at the ancient wall, much of which has since disappeared. He describes the wall as he goes, and pieces together its chronology, as well. Scarce. $85.00
Category Archives: Archaeology
Archaeology in Newfoundland and Labrador 1980
Thomson, Jane Sproul & Ransom, Bernard (eds.) Archaeology in Newfoundland and Labrador 1980: Annual Report No. 1. Historic Resources Division, Department of Culture, Recreation and Youth. Government of Newfoundland and Labrador. St.John's, Nfld., February 1981. 4to, (magazine size). Very good to fine throughout, former owner's name lightly on top corner of front cover. Illustrated with b&w figures and plates. This was the first of a number of annual reports and one rarely seen. $40


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by Oscar Wang
The SRC took a first step tonight to shut down three charter schools: Arise Academy and Hope Charter, both of which have a mission to serve severely at-risk students, and Truebright Science Academy.
As representatives from the three schools made their cases for renewal, the SRC asked tough questions. The answers provided did not persuade the commission to reverse the three non-renewal recommendations made by the District's charter school office.
Commissioner Wendell Pritchett challenged all three charters to clearly outline a plan to turn their schools around. Implementation of good ideas is key to success, he said, not just the ideas themselves.
Hope Charter administrator Cassandra Russo mainly disputed the District's use of state standardized test scores to assess the academic accountability of the school. The charter focuses on students who were expelled or voluntarily left other schools, and includes some foster children and those returning from incarceration. Up for renewal for the second time, it received the lowest score possible on the District's measure of quality, the School Performance Index.
The commissioners' questions centered on the school's priorities, student retention, and attendance trends.
Ultimately, commissioners were unconvinced that Hope deserved another five-year charter, voting 4-0 for non-renewal. Commissioner Joseph Dworetzky was absent.
"This should have been taken care of when the school was created," Commissioner Pritchett said, referring to the school's recently developed turnaround plans.
"You have a track record, a long period of time, so it's harder to say you haven't had the time to figure some things out," added SRC Chair Pedro Ramos.
Truebright was looking for its first renewal. It was asked to explain a drastic drop in performance indicators. In 2010, the school registered an attendance rate of 96.18 percent; the following year, the rate dropped to 81.12 percent.
In terms of academic performance, Truebright met six out of six performance targets under No Child Left Behind in 2010. In 2011, it missed all six entirely.
Bekir Duz, CEO of Truebright, spent his three-minute testimony taking issue with an article in Thursday's Inquirer that focused on his school's alleged ties to a Turkish religious group. Calling the report "hearsay," Duz said that the story "waste[s] everybody's time."
Truebright board member Baki Acikel contended that an Inquirer story in March 2011 damaged the school's atmosphere last year and caused low morale among students. He blamed that for the low test scores.
SRC members questioned the Truebright administrators about the test scores, attendance data, and erroneous reporting of data to the state. Ultimately, they voted 4-0 not to renew the charter.
Duz told the Notebook that his school plans to appeal to the state. Schools can stay open during the appeals process.
The recommendation not to renew Arise Academy's charter was clearly the most controversial decision of the three for the commissioners.
Commissioners expressed interest in giving the school time to improve, but said they were not willing to give the school a five-year renewal.
Arise, in partnership with the city Department of Human Services, is the nation's first charter to specifically serve foster children. Beyond its academic program, the school offers social support services to guide students toward graduation -- and eventual emancipation at age 18. Seeking its first renewal, the school does not have enough data available to be given a score on the School Performance Index.
Student Myliesha Baker told the SRC that she felt like a "mute" before matriculating to Arise. "At Arise," she said, "I felt like it was safe to speak."
Arise CEO Gabriel Kuriloff touted his school's value-based program, individualized support for each student, and teachers and staff specially trained to care for foster children.
Students like Baker, he said, are "not victims of the system, but empowered agents of systemic change."
"Invest heavily in our success," he told the SRC.
Ramos said that Kuriloff "couldn't be more compelling in stating the need for a school [like] Arise." But he expressed concern that despite the "best intentions and successes," the school would not be able to implement its strategy.
After a discussion on attendance, intensive skill remediation, engaging curriculum, and Arise's finances and leadership, members of the SRC seemed to be willing to give the school another chance.
However, Commissioners Ramos and Pritchett both said they did not believe Arise deserved a five-year charter.
Commissioner Feather Houstoun expressed the same concerns as her colleagues, but said she felt "really uncomfortable with the options" in front of her. If the SRC issued a non-renewal notice to Arise, Houstoun was concerned that Arise would be "putting a lot of human resources and energy into an appeal while they can spend it on their school."
State law does authorize the SRC to grant one-year renewals of charters in situations where there is insufficient performance data on a school, but the SRC did not publicly consider that option for Arise.
In the end, the commissioners voted for non-renewal, 3-1, with only Lorene Cary dissenting. However, Ramos said that the SRC would be willing to work with the school.
"The process triggered here [with the non-renewal notice] is a beginning, not an end," he said, mentioning the possibility of a "mutual agreement" that would help Arise improve and continue operating as long as there was sufficient evidence that the school was implementing real changes.
On Monday, the District posted documents summarizing its review of each charter renewal up for consideration, including the staff recommendations against renewing the charters of the three schools.
Supporters of those schools, however, have been vocal in arguing against closure ever since word of the non-renewal recommendations surfaced in mid-March.
The non-renewal vote kicks off a formal hearing process that could take months. Any school that is not renewed can bring its case before the Pennsylvania Charter Appeal Board. Former District charters Germantown Settlement and Renaissance did that in 2008, but their appeals were ultimately unsuccessful.

subset: commoncrawl


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Free Entry to South West's Largest Family Theme Park
Families camping at Woodlands Grove 5 Star Campsite near Dartmouth have FREE entry to Woodlands Family Theme Park. There is too much for one day so spread the fun over your stay with free entry every day. There's multi rides, indoor fun factories, Zoo, Farm and Falconry Centre it keeps the whole family happily entertained throughout your holiday and saves a fortune on outings.
phone 01803 712598 now to book


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Rebecca Benneyworth offers a technical summary of the tax credits changes and how they apply.
There were cries of "shame" in the House when the chancellor gave the lowdown on tax credits for high earners. Known fairly widely in the tax profession, but possibly not to the general public, the combined impact of the £50,000 income limit for the family element of tax credit, and the income disregard of £25,000 per annum conspire to allow some people with income of up to £83,000 to claim tax credits.
Osborne has stopped the rot with a wide range of change to the tax credit system, but has stopped short of massive change, relying on tinkering with rates and thresholds to achieve the effect he wants - to stop wealthy families gaining entitlement to this benefi...
Please Login or Register to read the full article
The full article is available to registered AccountingWEB.co.uk members only. To read the rest of this article you'll need to login or register. Registration is FREE and allows you to view all content, ask questions, comment and much more.


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Cohen Milstein is co-lead counsel in a class action on behalf of elite MMA fighters alleging that Zuffa LLC – commonly known as the Ultimate Fighting Championship – has unlawfully monopolized the markets for promoting live professional MMA bouts and for purchasing the services of elite professional MMA fighters.
Class actions filed on December 16, 22, and 24, 2014, allege that the Ultimate Fighting Championship (UFC) engaged and continues to engage in an anticompetitive scheme to suppress compensation for elite professional mixed martial arts ("MMA") fighters by unlawfully eliminating competition from rival MMA promoters and maintaining and enhancing its monopsony power in the market for MMA fighter services. The antitrust actions were filed in the United States District Court for the Northern District of California in San Jose on behalf of fighters Cung Le, Nathan Quarry, Jon Fitch, Luis Javier Vazquez, Dennis Lloyd Hallman, Brandon Vera and Pablo Garza.
The lawsuits claims that the UFC's alleged anti-competitive acts from at least 2010 to the present, have made and maintained the UFC as the only option for MMA fighters who want to earn a viable living in the profession. The complaints allege that, as a result of its anti-competitive conduct, the UFC receives approximately 90% of all revenue generated by MMA events in the US, and potentially worldwide, while paying UFC fighters a fraction of what they would earn in a competitive marketplace.
The eligible class members in this litigation are all persons who competed in one or more live professional UFC-promoted MMA bouts taking place or broadcast in the United States during the class period. Additional class members include each and every UFC Fighter whose identity was expropriated or exploited by the UFC.
The plaintiffs are represented by Cohen Milstein Sellers and Toll PLLC, Berger & Montague, P.C., Joseph Saveri Law Firm, Inc. and Warner Angle Hallam Jackson & Formanek PLC.


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As summer fast approaches, I find myself dreaming of one day being able to decorate my own summer lake house. In the meantime, I'm enjoying getting ideas from beautifully decorated summer homes. I especially adore Interior designer Sarah Richardson. She transformed her Canadian island cottage into a relaxing space with rustic charm.
I also adore the beach-themed décor from Detagli Home Décor (below).


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Vatican presents Pope's trip to Albania
Next Sunday, September 21st, Pope Francis will travel to Albania. The visit will be his fourth international trip but the first to a European country outside of Italy. The Pope is expected to arrive at 9:00am. From there he will travel to the Presidential palace for the opening ceremony where he will address civil authorities. In a briefing today, Vatican spokesman Fr. Federico Lombardi said that the highlight of the trip will be at 10:30am, when he makes his way towards Mother Teresa Square and celebrate Mass. FR. FEDERICO LOMBARDI Director, Holy See Press Office "At 11am, the Holy Mass will be celebrated in Mother Teresa Square in Tirana. The Mass willbe open to the public, the Holy Father will deliver a homily and will close with the Sunday Angelus.â? Fr. Lombardi also said that during the Mass, the second reading that will be read will be of special significance to the Albanian people. St. Paul recounts his visit to Illyria, an area that is now know as present day Albania. FR. FEDERICO LOMBARDI Director, Holy See Press Office "Illyria is the exact region where Albania is found, right after Dalmatia. So, for the Albanian people, this text is a witness to the fact that their religious, Christian and Ecclesial tradition goes back to the times of the Apostles. And they consider St. Paul as the first evangelizer of their country.â? In the afternoon, he will meet with leaders from the 6 religions in the country. They include Muslim, Catholic, Orthodox, Evangelical, Jewish and Bektashi, a Suffi Islamic fraternity. In the evening, he will pray Vespers at the newly built Cathedral of St. Paul with priests, religious, seminarians and various lay movements. Fr. Lombardi said that two survivors of the religious persecutions that occurred in the 1940s will give their witness. FR. FEDERICO LOMBARDI Director, Holy See Press Office "It will begin not only with the witness of the Archbishop of Tirana, but also of two very important witnesses. First, from an 80 year old priest and then an 80 year old nun who are living witnesses of the time of persecution and have much to say.â? The Pope will then visit the Betania Center, an organization that cares for abandoned children. From there, he will head to the airport and arrive in Rome at 9:30pm. JAE AA -PR FN Up: JAE


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As Japanese hunters went around the sea capturing dolphins for the annual Taiji hunt, one poor creature was seen begging for dear life.
Conservationist Ric O'Barry from The Dolphin Project was busy recording video clips of the controversial dolphin hunt as part of documenting the practice when one terrified animal afraid of being caught began throwing himself at the man's feet, as hunters were herding it into the Taiji cove.
While some of the dolphins are sold for meat, others are captured and sold to aquariums for dolphin shows and entertainment purposes.
Read: Cat Rescued Moments Before Being Skinned Alive
O'Barry is heard saying, "I cannot go in to the water and help him or I will be arrested, put in jail and deported. This is heartbreaking. Awful."
Around 17,000 dolphins are hunted down each year and killed, and many of them die soon after being captured due to shock, according to the wildlife charity Whale and Dolphin Conservation.
Watch the video above to see if the dolphin is caught by its hunters despite attempts to escape.


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Hawkins Agri Grassland Demo Day 27th April 2011 at Cannington Agricultural College Somerset. This is the second part of a two part video. Visit our website at: www.hawkins-agri.co.uk
Grassland demo, New Holland, Kuhn, Lely, Heva, Opico, New Holland Wheel Loader, WB170, Silage, Blaing, Forage Harvesting, Wrapping, grass, Clamp, Farmer, Agricultural Contractor, Slitter, Sward Lifter, Demo, Cows, Power, Lely Storm, Lely Tigo, Lely Splend


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|Search eBay
|Search eBay
All-time low: £295.75 on 19/09/2015
Great quality and looks good. Very sturdy and well built, the Hardrock Disc gives a high quality feel for an entry level bike.
The Hardrock Disc SE is prepared for mountain trails and urban jungles, with a smooth Suntour fork, plus powerful Tektro mechanical disc brakes.
Tektro Aries disc brakes for excellent stopping power. Shimano 7-speed derailleur for a reliable shifting performance.
This is a retired product.


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Today I have found an intersting article at Sucuri about infected cPanel & WHM.
Because many users use this server panel, it is worth reading the article:
https://blog.sucuri.net/2016/11/ask-suc ... ected.html
Any decent company will secure their shared hosting various way to avoid any ejections.
Need dedicated resources to host OpenCart, see our VPS plans at webprovps.com
Our cloud shared hosting starts from £1 per month* and capable to run OpenCart (Free SSL certificate / HTTPS on all accounts), see more details at our: Shared Hosting page.
New member
Tue Sep 06, 2016 9:06 pm
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Onyx Escade Women's Loose Fit Short
It's better on two wheels.
Stessa features a lightweight and durable fabric comprised of blended spandex and polyester allows for a fluid range of motion, so you can ride like an eagle soaring on the Fourth of July-now that's freedom.
Technical Specs: 85% Polyester, 15% Spandex │180 gsm
Tech Specs
• Adjustable Waistband
• Belt Loops
• Detachable Liner with Chamois
• Loose Fit
• Stessa Fabric
• Velcro-enclosed Rear Pockets
Escade Shorts
The Escade short gives any rider the superior quality and functional comfort on and off the path. With an adjustable waist band, lightweight and durable Stessa fabric, removable chamois liner, and rear pockets, the Escade short offers a versatile range for any rider.


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Zandvoort is as famous and crowded as beach resorts get in the Netherlands. That is - in summer, when the weather is nice, Dutch and German visitors flock to the sandy beaches of this otherwise rather small town and dozens of beach pavilions along the long boulevard cater to their needs. By November, all pavilions have to be closed and mostly removed until Mid-February, changing the face of the Zandvoort boulevard completely.
Apart from beach life, Zandvoort is known for the Circuit Park Zandvoort, the main car racing circuit in the country. Although the last Formula 1 Dutch Grand Prix was held in 1985, the circuit still has a range of other races to offer for racing fans.
The whole west coast of the Netherlands is a single long beach. The nearest stretch to Amsterdam is Zandvoort—27 minutes by train from Amsterdam Centraal, every 30 minutes. In summer there are extra trains, change trains at Haarlem. Zandvoort is very crowded on warm days in summer: parts of the beach attract the in-crowd, others the somewhat less so.
Get in[edit]
On hot summer days, traffic jams in the directions of all major Dutch beach destinations are common - and Zandvoort is no exception. On such days, consider using the train instead of a car. On other days, driving in is easy enough. The N201 is the main road in, connecting Zandvoort to the nearby city of Haarlem and, slightly further, Amsterdam.
Trains run every half hour from Amsterdam and Haarlem, with extra trains in high season and on very hot (and thus busy) days. From Amsterdam, it's easy to connect to countless destinations in the country and abroad. Station Zandvoort aan Zee is less than a 10 minute walk from the beach.
Bike lanes are readily available, and there are plenty of biking and hiking routes connecting Zandvoort to other beach resorts along the coast as well as to cities and villages in the region.
Get around[edit]
Zandvoort is a small place, so you can get around walking or rent a bike from one of the many bike rental stores in Zandvoort.
The nearest bike rental store to the train station is Zilt at Sea. Zilt at Sea is also a few steps from the beach.
- 1 [dead link]Zilt at Sea Bike Rentals in Zandvoort (Rent a bike in Zandvoort at Zilt at Sea), Passage 33, ☎ +31 23 576 69 91, e-mail: firstname.lastname@example.com. Rents bikes of all kinds, including electrical bikes, mountainbikes, tandems, scooters and children's bikes. You can also rent a locker, take a shower, and buy beach accessories.
The circuit in Zandvoort, from 1952 until 1985 Zandvoort was one of the circuits that organized the Formula 1.
Crowds flock here to find a spot on the sand and enjoy the sun and the sea. Sunbathing, swimming, beach walks and of course casual drinks in one of the many beach side bars is the core of all activity here.
- Zizo Sushi Lounge, Burgemeester van Fenemaplein 2. The sushi and sashimi in this restaurant get raving reviews, and they serve a nice range of cocktails too. Staff is friendly and the atmosphere is modern but cosy. €25.
- Strandpaviljoen Havana aan Zee, Boulevard Paulus Loot 1c (Last beach pavilion on the Paulus Loot boulevard, south beach). A pleasant beach pavilion with a classic menu and a good chef in the kitchen. There are sandwiches, pizzas and salads for lunch and a regularly changing menu for diner, with lots of fish options. When it's too chilly to sit on the outdoor terrace, try to get a table at the window with views of the sea for the best experience. In season, it's usually open all day and serves breakfast, lunch and dinner. In the evenings it's a good spot for late drink. Keep in mind that this place, like most beach pavilions, is closed from mid-October to mid-February and has flexible opening hours when the weather is bad. Call ahead to be sure.


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- Description
The Daishiro reclining sofa offers bonded leather match with thick padding for the head and back and wide armrest for reclining comfort. Fits perfectly in any living room environment with optional loveseat or loveseat console selection.
1 Loveseat
Loveseat, 62"L x 39"D x 39"H
More from this collection: Daishiro
- Additional Information
Additional Information
SKU 11300811 MPN 50746 GTIN UPC 0840412507465 Brand Acme Collection Daishiro Assemble Length 62 Assemble Width 39 Assemble Height 39 Assembly Required Yes Material Bonded Leather Color Brown Styles Contemporary Shipping Time 1-2 Weeks Free Shipping No
- Reviews
Disclaimer: Pictures displayed may include optional products not included in the price. Computer monitors often display color variations. Color swatches are shown if alternative colors are available. Please call us or email us if you have any questions!


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Cookie Clicker 3D: Dough or Die is a downloable game for the Nintendo 3DS made by Locked Gaming and Fantasize Studios. It is a remake of a javascript game named Cookie Clicker and it features several new stuff.
The basic gameplay is only to tap the cookie. When you tap the cookie, you will get one cookie, the objective is to get items that will help you, like the Cursor, that autoclicks every ten seconds, or the grandma, that produces 0.5 cookies every second. As the game progresses, you will be able to purchase things like Shipments from the Cookie Planet, an Alchemy Lab to convert gold into cookies, or even Time Machines that steal cookies from the past!
- Cursor: Autoclicks once every 10 seconds.
- Grandma: A nice grandma to bake more cookies.
- Farm: Grows cookie plants from cookies seeds.
- Factory: Produces large quantities of cookies.
- Mine: Mines out cookie dough and chocolate chips.
- Shipment: Brings in fresh cookies from the cookie planet.
- Alchemy Lab: Turns gold into cookies!
- Portal: Opens a door to the cookieverse.
- Time Machine: Brings cookies from the past, before they were even eaten.
- Reinforced index finger: The mouse gains +1 cookie per click. Cursors gain +0.1 base CpS.
- Carpal tunnel prevention cream: The mouse and cursors are twice as efficient.


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|Largest city
|Kamal Jeknene
|Prime Minister
|Malian franc
|Calling Code
|Internet TLD
The Democratic Federal Republic of Mali is a major nation in West Africa, bordered by French Algeria, French West Africa, Nigeria, Cyrene, Nubia, Egypt, and Camenbal. The nation, the largest in land size in Africa, was a total monarchy until 1923 and a constitutional monarchy until 1954. The capital of Mali is Timbuktu, and its largest city is Bamako. Mali is regarded as northern Africa's primary sovereign power and is a major player in regional politics. The country is one of the four founding members of the Union of African States. Mali's main language is French, although a variety of native tongues are used as well. It has a population of about 85,000,000.
Malian Empire and Relationship with England
Colonial Wars
Constitution, Oppression and Expansion
Malian Revolution and Influence of Socialism
Kamal Bato Regime and Cold War Role
The military coup of 1966 ended the influence of the American-backed socialist government and installed General Kamal Bato as the leader in Timbuktu. Bato was an old-fashioned, right wing leader with ties to the deposed monarchy and who was an old stalwart and friend of the French regime. Francois Ramon, only a week before his assassination in Bonn, was in Mali to congratulate Bato and extend overtures to bring Mali back into the French fold.
Bato was a brutal, reactionary leader in his suppression of his enemies, but he followed the Sebastienite model of France in that he governed without preference for any single Malian ethnic group or religious sect.
Debi Amor and Modernization
While Bato was often referred to as the "Sebastien" of Mali, his civilian successor, Debi Amor, is often referred to as Mali's "Albert II." Amor was, unlike Bato, a Muslim who chose to govern the country with a much less secular policy than his predecessor, but also recognized the ingenuity of his successor in avoiding racial or religious conflict during his reign. With the turbulent mid-1970's a distant memory by the time Amor came to power in 1982, the new goal for Mali was to emerge as Africa's preeminent power.
Unphased by the competition of the more economically sound Zululand or Ethiopia, Amor began an ambitious modernization program in 1983 called the "Great Push," which would drive Mali into the upper echelons of West African prominence. Using a somewhat planned economic model, Amor ordered the construction of power plants, factories, and new free-trade sectors in certain cities across the country, gambling on the hopes of foreign investment. In 1987, he started a revolutionary remodeling of Bamako, seeking to make the city the economic hub of the region, putting it in direct competition with Dakar in French West Africa. While his efforts were much less appreciated than the docility of Bato, European companies, even from beyond the CIC. England became one of the biggest investors in Malian growth during this period, and in 1992 English Prime Minister John Cleese visited Timbuktu as the first non-French European leader to ever make a visit.
East Prussian motor company Audi opened its massive Afrikaverkstad in Niamey in 1990, a self-sustained city-factory on the outskirts of the city that saw a profit growth of 350% within three years and doubled in size with a revamping in 1995, and low-cost Audi vehicles produced in Niamey soon burgeoned the domestic automotive industry in Mali, allowing for 78% of all cars in the country to be built locally, of which 96% of those cars were Audis. By 2006, estimates agreed that Audi had a complete monopoly on the automotive industry in Mali and neighboring French West Africa, with 87% of the vehicles in both states Audi built and more than seventy-five percent of them built in Africa.
Amor's efforts at localized production for Mali from foreign companies also led to a late-1990's computer boom, in which Visalux, a French computer company, opened a factory in Bamako that produced 64% of the country's computers, although the Malian computer technology remained about five to ten years behind that of most industrialized countries. Amor's late 1990's initiative to combat pollution in the Niger River was a rousing success, and in 2001 he opened two nuclear power stations, much to the chagrin of the United States.
Amor finally lost control over his own government due to local anger over his support of Muslim militias in Nigeria that were combating the United States - the known Malian support for these combatants resulted in a NATO embargo against Mali that included forbidding NATO-aligned trade vessels from journeying up the Niger. This resulted in Mali's first recession since the late 1970's, after fifteen years of unfettered growth, and Amor resigned in 2003 in favor of Fedkwe Muguwa. In 2006, Amor died in a plane crash in French West Africa under mysterious circumstances.
Mali's growth rate has slowed from its late 1980's-early 1990's heyday, when it was one of the great investment and economic success stories in history. Foreign investment has slowed somewhat, especially with the light shed on human rights abuses during the 1990's. Muguwa resigned in 2008 due to a corruption scandal involving Arab real estate developers, and Mali's current President is Kamal Jeknene.


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The Forces Store will be offline until Friday 4th August for Annual Holiday - sorry about that.
We wish all our customers a very a good summer break .
Please visit the store once we are back and continue your great work in supporting our Armed Forces and their charities.
Thanks for your support.


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Timeline specific to the novels (Gabriel Knight: Sins of the Fathers (novel), [[Gabriel Knight: The Beast Within (novel), and The Temptation: a Gabriel Knight interlude). There are details that differ from the games themselves, for dating of events.
- 1800 - The rough date of the sorcerer's glove.
- 1923 - Ester Wright is born.
- 1958 - Gerde Hull is born.
- 1960 - Gabriel Knight is born (GK1 Novel).
- 1961 - Phillip and Margarite are killed (GK1 NOvel).
- 1967 - Grace Nakamura is born.
- 1993 - Gabriel is 33.
- 1995
- February
- Spring, GK2
- 1996
- Gabriel discovers a body under Rennes-le-Chateau that might or might not have been the Son of God. It concluded the last of his 'three quests' as a Schattenjäger.[1]
- 1996/97 - Gabriel is 36. He is goes on a hunt in Wolfach in the Black Forest.
- The short story Gabriel Knight Interlude is set in 1995, but before GK2.
- The Temptation is set six months after GK3, and states he is 36 (or at least was 36 during GK3). In the game timeline this six months after GK3 (which was set in 1998) would be about January 1999.
Cite error:
<ref> tags exist, but no
<references/> tag was found


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What kind of dusts (brand name and name of product) do you use to get the satiny look that I see on so many cakes? I have the white lustre dust by Wilton, but when I use it, it only goes on as a fine glittery dust..it never makes things look shiny.. just pretty. I have seen dusts used to make yellow things look like gold, or bows look like they were made out of actual silk/satin... what are you using to do that??
I think these two posts should answer all of your questions/ If they don't let me know.


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An ICC committee made up of former Test players, chaired by former India captain Sunil Gavaskar, has recommended a new rule allowing bowlers to straighten their arms by up to 15 degrees.
The existing rule varies the amount of straightening allowed depending on the type of bowling. Spinners were restricted to five degrees, medium pacers to seven and a half degrees and fast bowlers to 10 degrees.
Under the proposed new ruling, which will be put to the ICC Chief Executives' Committee of the 10 Test-playing countries at its next meeting, in Melbourne, Australia, in February, almost all modern bowling actions would be legal.
This would allow Muralitharan, who is currently injured, to resume bowling his feared doosra. Tests showed that the Sri Lanka star's elbow straightened by 14 degrees, nine degrees more than the current limit for spinners, when bowling this delivery.
Extensive research into the biomechanics of bowling during the past four years has revealed that almost every bowler straightens his arm before he lets go of the ball.
ICC general manager David Richardson said: "When bowlers who to the naked eye look to have pure actions are thoroughly analysed with the assistance of the sophisticated technology now in place, they are likely to be shown as straightening their arm by 11 and in some cases 12 degrees. Under a strict interpretation of the law, these players are breaking the rules."
"The game needs to deal with this reality and make its judgment as to how it accommodates this fact."
The committee found that even Steve Harmison, Glenn McGrath and Shaun Pollock are chuckers under existing rules.
Dennis Lillee, Fred Trueman, Ian Botham, Imran Khan and Richard Hadlee also threw the ball.
Former England bowler Angus Fraser, who sat on the committee, said: "Many will feel that by allowing this the ICC are legalising throwing. They are not. All the information and opinion collected points to 15 degrees."


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The ICC rankings say Australia have conclusively been the best team since the last one-day tournament, the 2013 Champions Trophy. The results show Australia have won 72 per cent of their completed matches, well ahead of all rivals.
The fixture for Friday shows they are playing at home against a subcontinental team who last beat them at that venue in 1996 and have not beaten them anywhere in Australia in their past seven attempts.
Which means what, before Australia's first home World Cup final?
"I think you know the answer to that question. The past is the past," said Australian captain Michael Clarke before the day-night quarter-final at Adelaide Oval against the team that beat them in their past World Cup encounter, in 2011.
"I don't think you're ever owed anything in this game of cricket, so we have to make sure we're ready to go tomorrow – which we will be."
Australia have won 26 of their 36 completed matches since the Champions Trophy, well ahead of India's winning percentage (before their quarter-final on Thursday) of 63.5 per cent and South Africa's of 61.7 per cent.
Pakistan have won fewer than half their matches, 21 of 45, but have the unique hindrance of not being able to play at home.
Since mid-2013 Australia have won seven of their nine one-day series. The exceptions were losing away to India in late 2013 and then falling to the Proteas in the final of a tri-series in Zimbabwe last year.
The significance of those matches is nevertheless incomparable to having reached the stage of the tournament where losses result in elimination.
That scenario was nevertheless welcomed by Clarke.
"I think everybody is happy it's finally here, there's no doubt about it. I think you've probably noticed that more from the public and the media [than the players], to be honest," he said.
Both teams declined to disclose their XI the day before the match.
Each are thought to be pondering only one bowling position: Pat Cummins or Josh Hazlewood for Australia, seamer Ehsan Adil or leg-spinner Yasir Shah for Pakistan. The incumbents, Cummins and Adil, are expected to keep their positions.
Australia kept to their custom of allowing, and tacitly advising, bowlers to rest from bowling the day before the match.
All-rounder Shane Watson nevertheless put in a solid shift with the ball in the nets.
Clarke said Pakistan had "been underrated for a long time, especially in the shorter form of the game. They've got a lot of talent in their attack and they've shown that through this tournament. They've got a good mix of youth and experience with their batting and they are led really well by Misbah [ul-Haq]." Clarke said the effect of Mohammad Irfan's absence due to a tournament-ending injury could be overcome on a pitch he expected would favour pace over spin.
AUSTRALIA (likely): Aaron Finch, David Warner, Steve Smith, Michael Clarke (c), Shane Watson, Glenn Maxwell, James Faulkner, Brad Haddin, Mitch Johnson, Mitch Starc, Pat Cummins/Josh Hazlewood.
PAKISTAN (likely): Ahmed Shehzad, Sarfraz Ahmed, Haris Sohail, Misbah-ul-Haq (c), Umar Akmal, Sohaib Maqsood, Shahid Afridi, Wahab Riaz, Sohail Khan, Rahat Ali, Ehsan Adil/Yasir Shah.


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Growth factor-induced activation of Akt occursin the majority of human breast cancer cell lines resulting in a variety of cellular outcomes, including suppression of apoptosis and enhanced survival. We demonstrate that epidermal growth factor (EGF)-initiated activation of Akt is mediated by the ubiquitous calcium sensing molecule, calmodulin, in the majority of human breast cancer cell lines. Specifically, in estrogen receptor (ER)-negative, but not ER-positive, breast cancer cells, Akt activation is abolished by treatment with the calmodulin antagonist, W-7. Suppression of calmodulin expression by siRNAs against all three calmodulin genes in c-Myc-overexpressing mouse mammary carcinoma cells results in significant inhibition of EGF-induced Akt activation. Additionally, transient expression of constitutively active Akt (Myr-Akt) can overcome W-7-mediated suppression of Akt activation. These results confirm the involvement of calmodulin in the Akt pathway. The calmodulin independence of EGF-initiated Akt signaling in some cells was not explained by calmodulin expression level. Additionally, it was not explained by ER status or activation, since removal of estrogen and ablation of the ER did not convert the ERpositive, W-7 insensitive, MCF-7 cell line to calmodulin dependent signaling. However, forced overexpression of either epidermal growth factor receptor (EGFR) or ErbB2 did partially restore calmodulin dependent EGF-stimulated Akt activation. This is consistent with observation that W-7 sensitive cells tend to be estrogen independent and express high levels of EGFR family members. In an attempt to address how calmodulin is regulating Akt activity, we looked at localization of fluorescently tagged Akt and calmodulin in MCF-7 and SK-BR-3 cells. We found that both Akt and calmodulin translocate to the membrane after EGFstimulation, and this translocation to the same sub-cellular compartment is inhibited by the calmodulin inhibitor W-7. Thus, calmodulin may be regulating Akt activity by modulating its sub-cellular location and is a novel target in the poor prognosis, ER-negative subset of breast cancers.
Growth and survival signals initiated at the cell surface by ligand–receptor interactions often impinge on the serine/threonine kinase, Akt, a central regulator of survival, apoptosis, and oncogenesis. Akt interferes with programmed cell death by phosphorylating and thereby inactivating a number of proteins involved in apoptosis including BAD, Forkhead transcription factors, GSK-3b, and MDM-2 [1–6]. Akt has been shown to be activated in a wide variety of cancers where it contributes to inappropriate cell survival and oncogenesis. Constitutive activation of Akt (produced by overexpression of growth factor receptors, chromosomal amplification of Akt or phosphatidylinositol 3-kinase [PI-3 kinase], or by deletion of the lipid phosphatase PTEN) is a common tumorigenic event in a number of cancers [7]. In breast cancer cell lines, Akt activation has been shown to contribute to survival and cellular resistance to chemotherapeutic drugs such as doxorubicin and tamoxifen, and to predict for poor outcome in patients [8, 9].
Akt activation, resulting from growth factor stimuli, occurs through both PI-3 kinase-dependent and PI-3 kinase-independent interactions, which recruit Akt to the plasma membrane (PM) where it is phosphorylated and activated by PDK1 and PDK2 on Thr308 and Ser473, respectively. The 30phosphorylated phosphoinositides (PIP3) generated by PI-3 kinase act as a lipid second messenger to recruit Akt to the PM via its pleckstrin homology (PH) domain, a specific PIP3-binding motif, and serves as the PI-3 kinase-dependent initiating event in Akt activation. The ubiquitous calcium sensing molecule calmodulin seems to mediate a PI-3 kinase-independent mechanism of Akt activation through calmodulin kinase kinase [10]. In mammalian cells, calmodulin regulates a variety of crucial biological functions including neurotransmission, proliferation, cell metabolism and development [11–13]. Calmodulin classically acts on specific calmodulin dependent protein kinases to exert its function. One specific kinase which directly phosphorylates Akt in a calcium-dependent manner is calmodulin kinase kinase [10]. Interestingly, calmodulin has also been shown to regulate PI-3 kinase-dependent activation of Akt in neurons. Akt activation and the survival of neuronal cells was shown to depend on calcium and calmodulin in a PI-3 kinase-dependent manner, as calmodulin antagonism prevented Akt activation by inhibiting in vivo generation of PIP2 and PIP3 phosphoinositides [14].
Our previous work highlighted the PI-3 kinase/Akt pathway as a key regulator of EGF-mediated survival of c-Myc-overexpressing carcinoma cells derived from MMTV-c-Myc mouse mammary tumors (henceforth called Myc83 cells) [15]. Since mammary epithelial cells release abundant calcium from intracellular stores upon ligand stimulation [16, 17], and since it has been shown that calmodulin is overexpressed in breast tumors, breast cancer cell lines and transgenic mouse models of breast cancer [18, 19], we previously investigated the role of calcium and calmodulin in the survival of these mammary carcinoma cells [20]. We reported that calmodulin was a novel modulator of Akt activity and additionally showed that the survival of c-Myc-overexpressing mammary carcinoma cells was dependent on calmodulin-mediated Akt activation.
Studying the mechanism of calmodulin-mediated Akt activation, we showed that Akt could be co-immunoprecipitated with calmodulin in an EGF-dependent manner, and yet, PI-3 kinase activity remained intact in the presence of a calmodulin inhibitor. This suggests that calmodulin interacts with Akt and mediates its activation downstream of PLCc and PI-3 kinase [20]. We therefore hypothesized that calmodulin was a direct and fundamental regulator of Akt activation and a potential therapeutic target in human breast cancer.
In the present study, we demonstrate that siRNAmediated suppression of cellular calmodulin levels inhibits EGF-induced Akt activation, and that the constitutive activation of membrane-targeted Myr-Akt is unaffected by a pharmacological calmodulin inhibitor. These findings provide evidence of a direct role for calmodulin in the Akt pathway and suggest that calmodulin may modulate Akt activation by regulating the sub-cellular localization of Akt, and indeed we were able to show that the calmodulin inhibitor W-7 blocks EGF-induced re-localization of Akt to cell membranes.
In addition, we report that the calmodulin-dependence of Akt activation can be demonstrated in the majority of human breast cancer cell lines, but that interestingly, the few cell lines where this is not the case are almost all estrogen receptor (ER) positive. This suggests that distinct signal transduction pathways are active in the different classes of breast cancer cells, though the apparent reduced dependence on calmodulin for Akt activation in ER-positive cells does not appear to be directly related to the ER activity. We examine the role of the ER and epidermal growth factor (EGF) family receptors in determining a breast cancer cell's dependence on calmodulin to activate Akt.
This newly appreciated role for calmodulin in mediating Akt activation in ER-negative breast cancers may have important therapeutic implications, since patients diagnosed with ER-negative breast cancers tend to have a worse clinical outcome, and the unique calmodulin–Akt interaction, selectively operant in these cells, presents a new survival mechanism which might be exploited therapeutically.
Human breast cancer cell lines, with the exception of MCE5, MB7 and MB8, were obtained from the Lombardi Comprehensive Cancer Center Tissue Culture Shared Resource, Georgetown University. MCF-7 cells were grown in improved modified Eagle's minimum essential medium (IMEM—Biofluids, Frederick, MD) supplemented with 10% fetal bovine serum (FBS). Hs578T, MDA-MB-231(NIH), MDA-MB-453, MDA-MB-468, SK-BR-3, and T47D cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with sodium pyruvate (Biofluids, MD) and 10% FBS. BT474 and BT549 cells were grown in RPMI (Biofluids) with 10% FBS; and MDA-MB-361 in Leibowitz media (Biofluids) with 10% FBS. Myc83 cells, a mouse mammary carcinoma cell line derived from c-Myc overexpressing mammary tumors resulting in MMTV-cmyc transgenic mice, were grown as previously reported [20]. Primary human mammary epithelial cells (HMEC) obtained from Cambrex (Walkersville, MD) were cultured for no more than eight passages in mammary epithelial basal medium (Cambrex), supplemented with bovine pituitary extract (BPE), hydrocortisone, hEGF, insulin, gentamicin/amphotericin. MCE5 cells, stable epidermal growth factor receptor-expressing (EGFR) MCF-7 cells (also named MCF-7-EGFR) were previously described and obtained from Dr. Dorraya El-Ashry, (Miami Miller School of Medicine, Miami, FL) [21]. MCE5 cells were grown in phenol red free IMEM with 10% charcoal stripped serum from Gemini Bio-Products (West Sacramento, CA). Stable ErbB2 expressing MCF-7 cells (clones MB7 and MB8) were previously described [22] and obtained from Dr. Francis G. Kern, (Adelson Medical Research Foundation, Los Angeles, CA) and grown in phenol red free IMEM with 10% charcoal stripped serum from Gemini (West Sacramento, CA).
Rabbit polyclonal antibodies phospho-Akt (S473), phospho-Akt (T308), total Akt, phospho-p44/42 MAPK (T202/Y204), and phospho-CREB (S133) were obtained from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibodies against EGFR, ErbB2, phospho-ErbB2 (Y1248) were obtained from NeoMarkers (Fremont, CA). Anti-b-actin was obtained from Sigma (St. Louis, MO). Mouse monoclonal antibody against calmodulin was obtained from Upstate (Lake Placid, NY). Cathepsin D antibody was obtained from BD Transduction Lab (San Diego, CA). The calmodulin inhibitor, W-7 and the internal calcium chelator, BAPTA-AM were obtained from Calbiochem (San Diego, CA). ICI 182,780 was obtained from Tocris Biosciences (Ellisville, MO). 17-beta-estradiol (E2), obtained from Sigma (St. Louis, MO). Treatment with pharmacological inhibitors and preparation of whole cell lysates Semiconfluent cell monolayers were serum-starved overnight or as indicated and then incubated with 30 lM of W-7 for 30 min, or 10 lM BAPTA-AM for 90 min. Where indicated, cells were stimulated with 10 nM EGF for 5 min, 50 ng/ml IGF for 5 min, 100 nM insulin for 5 min, 20% FBS for 15 min, and 100 lM pervanadate for 5 min, at 37° C. Cells were washed once in 19 PBS and lysed on ice with a 19 Lysis buffer from cell signaling supplemented with 1 mM PMSF. Lysates were processed and resolved on Tris–Glycine SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore) as previously described [20]. Membranes were incubated in primary antibody for 2 h, then biotinylated secondary antibody for 1 h, and detected with Vectastain ABC elite kit (Vector Laboratories) or HRPconjugated secondary antibody and enhanced chemiluminescence (PerkinElmer Life and Analytical Sciences).
MCF-7 cells were grown to 70% confluence in IMEM plus 10% FBS and then stripped of estrogen by washing the cell monolayer three times with phenol red free IMEM and replacing the medium with phenol red free IMEM supplemented with 10% charcoal stripped serum (Gemini). This procedure was repeated four more times during the course of one day after which the cells were incubated overnight. The following morning the cells were trypsinized using phenol red free trypsin (Biofluids), plated in phenol red free IMEM plus 10% charcoal stripped serum, and where indicated, supplemented with 1 nM estrogen or 100 nM ICI 182,780 for 48 h. Cells were then serumstarved overnight in phenol red free IMEM plus HEPES, supplemented with 1 nM estrogen or 100 nM ICI 182,780 where indicated. Cells were pretreated with 30 lM W-7 for 30 min in serum free media and then stimulated with 10 nM EGF for 5 min. Cell lysates were prepared as described above and processed for determination of Akt activity by Western blotting, also described above.
Myc83, SK-BR-3 orMCF-7 cells were passaged 2 days prior to nucleofection, and grown to no more than 80% confluence on the day of transfection. 1 9 106 cells were resuspended in 100 ll T solution (Amaxa, Inc. Gaithersburg, MD) plus indicated amount of DNA. Cells were then pulsed in the Amaxa Nucleofector II system (Cologne, Germany) using program T-20, or A-23 for MCF-7. Cells were plated in six well plates containing complete media, incubated for 24 h, and then used as described in figure legends.
Myc83 cells were cultured as described and total RNA was prepared using Tri Reagent (Sigma), following the manufacturer's instructions. RNA quality and concentration was determined by absorbance measurements and a 5 lg sample was used to prepare cDNA using the SuperScript First-Strand Synthesis System following the manufactures instructions (Invitrogen). The sequences for PCR primers specific for the three mouse calmodulin genes (CALM-1 -2 and -3) were obtained from the Primer Bank resource [23] and were used in PCR reactions with the Myc83 cDNA using the conditions recommended. A total of 5 ll samples of each reaction was analyzed by electrophoresis on a 3% agarose gel which was stained with ethidium bromide and photographed. The Primer Bank ID's for the primers used are as follows: CALM1—6753244a1, CALM2—6680832a1, CALM3—6680834a1. The same primers were used in real time PCR evaluation of siRNA mediated knock down of the calmodulin genes as described below using the Primer Bank PCR protocol.
A total of 1 × 106 Myc83 cells were nucleofected as described above with 5 υg siRNAs generated against calmodulin 1, calmodulin 2, and calmodulin 3 from Dharmacon (Lafayette, CO). Control cells were nucleofected with equal amounts of control siRNA consisting of a pool of siRNA against Luciferase (Dharmacon, Lafayette, CO). After nucleofection, cells were transferred to Myc83 complete media and incubated for 48 h in complete media. On Day 2 after nucleofection, cells were washed and serum starved for 6 h in serum free media and then stimulated with or without 60 ng/ml (10 nM) EGF for 5 min. After EGF stimulation, the cells were lysed and processed as described above.
Calmodulin-red fluorescent protein (RFP) was made by ligating calmodulin from CaM-YFP construct (a gift from Dr. Thomas Michel, Brigham and Woman's Hospital, Boston, MA) in mRFP-N vector. Akt1-GFP (green fluorescent protein) was a gift from Dr. Julian Downward, London Research Institute, UK.
MCF-7 and SK-BR-3 cells were cotransfected with calmodulin-mRFP and Akt-GFP using LipofectAMINE 2000 (Invitrogen) according to manufacturer's instructions. The cells were reseeded onto 12 mm glass coverslips 6 h after transfection. The cells on coverslips were serum-starved for 24 h in DMEM and then stimulated with 10 nM EGF for 5 min at 37°C. In the case of W-7 pretreatment, cells were incubated with 30 uM W-7 for 30 min prior to EGF stimulation. The cells were then fixed with 2% paraformaldehyded in phosphate buffered saline (PBS) for 15 min at 37°C, washed and mounted in Vectashield (Vector Laboratories). Laser Confocal images were obtained using a Zeiss LSM 510 confocal system.
Our previous observations implicated calmodulin as a regulator of EGF-induced Akt activation and the survival of c-Myc-overexpressing, Myc83 mouse mammary tumor cells [20]. In this study, we set out to investigate whether calmodulin is a general mediator of Akt activity in human breast cancer cells. To do this we attempted to select a panel of breast cancer cell lines that would be representative of the diversity of human breast tumors [24] consisting of: (1) ER-negative, invasive breast cancer cell lines exhibiting a more stromal/mesenchymal genetic signature—MDA-MB-231, Hs578T, BT549, MDA-MB-468 and SK-BR-3 [25, 26], and (2) ER-positive, estrogen dependent breast cancer cell lines—MCF-7, T47D and BT474 [27]. In an initial experiment, these cells, along with non-transformed HMEC's, were screened to identify treatments that would result in the induction of Akt activity. The cells were serum starved and then stimulated by either EGF, insulin-like growth factor 1 (IGF-1), insulin, FBS, or treatment with pervanadate (data not shown). Treatments that resulted in Akt phosphorylation were then selected to evaluate calmodulin-dependent signaling in that particular cell line.
Serum starved cells were pre-incubated for 30 min with 30 lM W-7, a selective calmodulin antagonist [12], for 90 min with 10 lM BAPTA-AM, an internal calcium chelator, or for comparable periods of time, with medium containing the vehicle alone (0.1% DMSO), and then stimulated to induce Akt activation. As shown in Fig. 1, pretreatment with the calmodulin inhibitor, W-7, but not DMSO, completely abolished EGF-induced Akt activation in MDA-MB-231, Hs578T, BT549, MDA-MB-468 and SK-BR-3 cells and also inhibited the basal Akt phosphorylation seen in serum starved Hs578T, MDA-MB-468 and BT549 cell lines (Fig. 1a, lanes 17, 31, 43). EGF induced Akt activation was also blocked in HMEC cells (Fig. 1b), and similar results were seen in MDA-MB-453 breast cancer cells (data not shown). Treatment with W-7 had no effect on MAP kinase (MAPK) activation, suggesting that the suppressive effect of W-7 on Akt activation was not due to a non-specific, or general toxic effect of the drug.
Since it is known that mammary epithelial cells release calcium from intracellular stores upon ligand stimulation [16, 17], and since our previous work demonstrated that Akt activation was calcium dependent in Myc83 mouse cells [20], we also examined the calcium dependence of Akt activation using the cell permeant calcium chelator BAPTA-AM. With the exception of Hs578T and the HMECs, pretreatment with BAPTA-AM was unable to inhibit Akt activation in the cells tested (Fig. 1a, b, and data not shown). In Hs578T, BAPTA-AM pretreatment inhibited, but did not abolish ligand-induced Akt phosphorylation, whereas EGF-induced Akt activation was almost completely abolished in the HMECs. These data suggest that both calcium-activated calmodulin and calcium-free calmodulin may be capable of modulating Akt activity in breast cell lines.
In a few of the cell lines tested in the panel, we found that Akt activation was in fact not dependent on calmodulin. Treatment with W-7 had little or no effect in the ability of several stimuli to induce Akt activation (Fig. 2). When we examined the characteristics of these refractory lines, we were interested to note that all were estrogen dependent, ER-positive breast cancer cells.
We wished to further establish the role of calmodulin in the Akt pathway, and also to validate the data generated using the calmodulin inhibitor by demonstrating that the suppression of Akt activation produced by W-7 is the result of its action on calmodulin and not due to some non-specific drug effect. We reasoned that if the effect of W-7 on Akt activation was indeed the result of suppression of calmodulin activity, then suppressing calmodulin protein levels should produce a similar effect on Akt activation. We therefore, set out to use siRNA mediated gene knockdown to suppress calmodulin protein levels and evaluated the impact of this intervention on Akt activation. Unlike most genes in mammalian genomes, three separate calmodulin genes, located on different chromosomes, encode a single calmodulin protein [28]. PCR-based evaluation of cDNA prepared from Myc83 cells revealed that, as expected, all three calmodulin genes are expressed (Fig. 3a). As a result, in order to significantly suppress protein levels, it was necessary to develop an siRNA strategy targeting of all three calmodulin genes simultaneously. Following extensive optimization of this approach, Amaxa-mediated nucleofection of combined siRNAs against calm1, calm2 and calm3 successfully resulted in reduced calmodulin protein expression, compared to cells nucleofected with control luciferase siRNA (Fig. 3b). Suppression of calmodulin protein levels resulted in a marked decrease in EGF-induced Akt phosphorylation. Total Akt expression was unaltered by the calmodulin siRNAs, and the levels of phospho-CREB, a target of calmodulin-dependent protein kinase II, were suppressed, thereby serving as a positive control for the suppression of calmodulin activity. Nucleofection with a luciferasedirected control siRNA had no effect on EGF-induced Akt activation, and a further control for off-target effects of the calmodulin-directed siRNAs is provided by the observation that nucleofection with all three siRNAs was required to produce the effect. Omitting the siRNA against any one of the three genes resulted in no suppression of calmodulin protein levels, and no alteration in EGF-induced AKT activation (data not shown). The results obtained with calmodulin siRNAs confirmed, at the genetic level, the role of calmodulin in the Akt signal pathway, and substantiated the results seen using the calmodulin inhibitor, W-7, as demonstrated in Myc83 cells (Fig. 3c).
The addition of a myristoylation signal to the Akt coding sequences (Myr-Akt), results in a protein that is constitutively targeted to the plasma membrane and, therefore, bypasses normal regulatory mechanisms controlling Akt activation. We employed this construct to begin to dissect the mechanism underlying the role of calmodulin in Akt activation. Mammary cancer cells (Myc83, MCF-7 and SK-BR-3) were nucleofected with 3 lg of empty pcDNA3.1 vector control, or the Myr-Akt construct using the Amaxa system (Fig. 4). One day after nuclofection, the cells were serum starved overnight, pretreated for 30 min with either DMSO or 30 lM W-7, and then stimulated with EGF or control medium. As seen in Fig. 4a, W-7 abolished Akt activation in untransfected and control, pcDNA3.1-transfected Myc83 cells (Fig. 4, lanes 2, 4, 6, 8). In serum starved Myc83 cells transfected with the Myr-Akt construct, as expected, the myristoylated Akt is phosphorylated even in the absence of EGF stimulation (Fig. 4a, lane 9). Upon EGF stimulation, the endogenous Akt also becomes activated (Fig. 4a, lane 10, P-Akt 473, lower band). In the presence of the calmodulin inhibitor, W-7, the endogenous P-Akt band is abolished, while activation of the transiently expressed Myr-Akt is unaffected (Fig. 4a, lane 12). Thus, myristoylation of the Akt renders the activation of the molecule calmodulin-independent, suggesting a possible role for calmodulin in the regulation of the sub-cellular localization of Akt.
Similar results were seen when Myr-Akt was exogenously expressed in the ER-negative human breast cancer cell line SK-BR-3 (Fig. 4b). Again, the membrane targeted Myr-Akt was resistant to W-7 treatment (Fig. 4b, lanes 11 and 12, upper band). Not surprisingly, MCF-7 cells again consistently resisted the effects of the calmodulin inhibitor, as Akt activation was not significantly changed in endogenous Akt or Myr-Akt in transfected MCF-7 cells, as demonstrated in Fig. 4c.
The panel of cell lines screened for W-7 sensitivity exhibit diverse range of genetic and phenotypic features which exemplify many of the common alterations seen in human breast tumors, including cells with mutant p53, activated Ras, loss of PTEN expression, or loss of functional Rb, etc., none of which alterations were apparently correlated with W-7 sensitivity [24, 29]. Based on the calmodulin knockdown data it seemed possible that the level of calmodulin expressed within the cell might influence W-7 sensitivity. We therefore examined the level of calmodulin present in the cells by Western blot analysis. As seen in Fig. 5, the W-7 insensitive cell lines (MCF-7, T47D and BT474; Fig. 5a, lanes 1–3) expressed relatively similar amounts of calmodulin protein to W-7 sensitive (+) cell lines (MDA-MB-453, MDA-MB-468, SK-BR3, BT549, and MDA-MB-231—lanes 4–9).
As noted above, the most obvious difference between the W-7 sensitive and in-sensitive cell lines is that all of the insensitive ones are ER-positive and estrogen dependent. In light of this and since there is strong evidence in the literature demonstrating that ER activity can regulate the PI3K/Akt pathway in breast cancer cells [30, 31], we postulated that the presence or activity of the ER may be bypassing the need for calmodulin in the activation of Akt in ER-positive breast cancer cell lines.
To address this question, we examined the impact on EGF mediated Akt activation of estrogen withdrawal alone, or withdrawal in combination with treatment with an antiestrogen which is known to target the ER for destruction (ICI 182,780), using the estrogen dependent breast cancer cell line MCF-7. The cells were stripped of estrogen and grown for two days under three different conditions: (1) with charcoal stripped serum only, (2) charcoal stripped serum plus 1 nM 17-beta-estradiol (E2), or (3) charcoal stripped serum plus 100 nM ICI 182,780 (to ablate the ER). The cells were then serum starved overnight (still in the presence or absence of E2 and ICI 182,780), exposed to the calmodulin inhibitor, W-7, and stimulated with EGF. As seen in Fig. 5b, MCF-7 cells were still able to activate Akt in the presence of W-7, even in the absence of estrogen (Fig. 5b, lanes 2 and 4), and indeed, under conditions in which the ER is being ablated (Fig. 5b, lanes 10 and 12). Adequacy of the estrogen withdrawal was assessed by evaluating the expression of the highly estrogen responsive gene cathepsin D. Estrogen withdrawal resulted in suppression of cathepsin D expression to undetectable levels, which was not altered by treatment with ICI 182,780, but treatment with 1 nM estradiol resulted in re-expression of cathepsin D protein. These results demonstrate that neither removal of estrogen nor ablation of the ER restored calmodulin-dependent Akt signaling in the ER-positive MCF-7 cells.
In addition to ER expression status we noticed that many of the W-7 sensitive cells express high levels of epidermal growth factor receptor (EGFR) family members, whereas the ER positive cells do not. We therefore postulated that overexpression of one or more of this family of receptors, a common feature of ER negative breast cancer, might be involved in the calmodulin dependent Akt activation. To examine this possibility we decided to determine if forcing the overexpression of one of these receptors in an ER positive cell would make Akt activation calmodulin dependent. We obtained MCF-7 cells that have been engineered to overexpress either EGFR (MCE5—kindly provided by Dr. Dorraya El-Ashry, University of Michigan) [22] or ErbB2 (MCF-7-ErbB2, clones MB7 and MB8—graciously provided by Dr. Francis G. Kern, Adelson Medical Research Foundation) [21], and tested them to determine if EGF-induced Akt activation was calmodulin dependent.
Serum starved cells were pretreated with W-7 or DMSO, stimulated with EGF and then analyzed, as described above. MCE5 (MCF-7-EGFR) cells express significantly more EGFR than the parental MCF-7 cells, and exhibit high basal Akt activation (Fig. 6a, lanes 1 and 7). As before, exposure to the calmodulin inhibitor did not inhibit EGF-induced Akt activation in parental MCF-7 cells (Fig. 6a, lanes 2 and 5), but did result in a reduction of EGF-induced Akt phosphorylation in MCF-7-EGFR cells (Fig. 6a, lanes 8, and 11). Interestingly, unlike what was seen with the EGFR-amplified breast cancer cell line MDA-MB-468 (Fig. 1a, lane 31), treatment with W-7 did not result in significant inhibition of basal Akt activation in MCE5 cells, (Fig. 6a, lane 10). These results suggest that overexpression of EGFR can make EGF-induced Akt activation partially dependent on calmodulin in cells that were previously W-7 insensitive.
The two independent MCF-7-ErbB2 clones overexpress ErbB2 to slightly different degrees, with the MB7 cells expressing slightly more than the MB8 cells. In both clones, however, the ErbB2 was found to be constitutively phosphorylated (Fig. 6b, P-ErbB2). In these overexpressing clones, but not in the parental MCF-7 cells, pretreatment with W-7 resulted in suppression of both basal and EGF stimulated phosphorylation of Akt (Figs. 6b, lanes 9, 10, 13, 14 and 6b, lanes 4 and 5).
To further address the possible involvement of estrogen signaling as a modulator of the calmodulin dependence of Akt activation, we tested the W-7 sensitivity of MB7 cells when grown in medium containing untreated serum (with estrogen), and when grown in medium supplemented with charcoal stripped serum (estrogen free). Under both conditions, W-7 was able to block EGF-stimulated Akt activation (data not shown), providing further evidence that the presence of ER alone or in combination with its ligand does not inhibit the calmodulin dependence of Akt activation.
These data suggest that calmodulin-dependent signaling to Akt is not affected by the presence of estrogen or ER activity, but rather may be influenced by the over-expression of growth factor receptors.
Our data demonstrating that phosphorylation of the constitutively active Akt construct was not suppressed by calmodulin inhibition suggested a possible mechanism by which the activation of wild-type Akt might be occurring. The constitutively active construct has a myristoylation signal that results in the targeting of the protein to the plasma membrane where it is phosphorylated independent of its normal activating signals. This suggested that inhibition of calmodulin function in ER-negative cells might be blocking the EGF stimulated relocalization of Akt to the PM, thereby blocking its activation, and that this blockade does not occur in ER positive cells. To test this possibility we made use of fluorescently tagged Akt and calmodulin constructs in order to follow the sub cellular localization of these proteins before and after EGF stimulation in the presence and absence of calmodulin inhibition, in both ER positive and negative cells.
MCF-7 (ER-positive) and SK-BR-3 (ER-negative) cells were transiently co-transfected with calmodulin-RFP and Akt1-GFP, then serum starved and treated with the indicated materials as described in the "Methods" section, and then examined by fluorescent microscopy (Fig. 7). Calmodulin and Akt were distributed mainly in the cytoplasm and nucleus of cells in serum free medium. Stimulation of the cells with EGF caused translocation of both calmodulin and Akt to the membrane, demonstrating that calmodulin and Akt localize to the same cellular compartment. As predicted, treatment with W-7 blocked the translocation of both calmodulin and Akt to the plasma membrane in SKBR-3 cells. Interestingly, however, this translocation was also blocked by W-7 in MCF-7 cells, which seems to rule out the possibility of differential localization of calmodulin and Akt in ER-positive and ER-negative cell lines as an explanation for the different responses of Akt activation by the calmodulin inhibitor in the two types of cells.
Akt is a key regulator of multiple cellular processes including proliferation and cell survival. In a previous study we discovered that survival signaling provided by EGF-induced Akt activation in a c-Myc overexpressing mouse mammary cancer cell line could be inhibited by the pharmacological calmodulin inhibitor W-7 [20]. The goals of the work we present here were: (1) to confirm that the inhibition of Akt activation was indeed the result of the inhibition of calmodulin function and not due to some independent property of the drug, (2) to determine if this intriguing phenomenon was limited to this particular mouse cancer cell line or represented a more general mechanism for Akt activation in human mammary epithelial or cancer cells, and (3) to identify the characteristics of cells that allow this pathway of Akt activation to function.
No pharmacological inhibitor is perfect, and so we first wanted to be sure that the suppression of Akt activation we were studying was indeed the result of inhibition of calmodulin by W-7, and not the result of some non-specific activity of the agent. To address this concern we decided to use an entirely independent, non-pharmacological approach to suppress calmodulin activity; knocking down calmodulin protein levels using siRNA technology. We reasoned that if the suppression of Akt activation produced by W-7 was indeed mediated by its inhibition of calmodulin activity, then reducing the cellular calmodulin concentration should also block Akt activation. This simple idea turned out to be much more challenging to achieve than we had anticipated. In addition to the complication noted above, that calmodulin is unique in the genome in having three independent genes coding for exactly the same amino acid sequence, thereby necessitating the simultaneous knockdown of three independent gene products, we soon discovered that suppressing the levels of one calmodulin gene increases the expression of the others (not shown). Nevertheless, after selecting and optimizing a cocktail of three siRNAs, one against each gene product, we were able to significantly suppress calmodulin protein levels and demonstrate that this indeed resulted in suppression of EGF-induced Akt activation (Fig. 3b). Leaving any one of the three siRNAs out of the cocktail results in loss of knockdown and loss of suppression of Akt activation, thereby controlling for off-target effects of the individual siRNAs (not shown). During this process we determined that the better the knockdown of calmodulin level, the more effective the suppression of Akt activation. In addition to these studies, we examined the ability of several other chemically distinct compounds reported to inhibit calmodulin activity to suppress EGF induced Akt activity. All of these compounds were able to suppress EGF-stimulated Akt activity (not shown), and since it is unlikely that they would all have the same non-specific activities, we take this as further evidence that the inhibition of calmodulin activity is indeed the key target.
Further evidence for the specificity of the inhibitor is provided by the fact that when targeted to the PM by virtue of a myristylation signal, the phosphorylation of Akt occurs as expected, in the presence of W-7 even as the compound is blocking the phosphorylation of the endogenous protein. This demonstrates that W-7 is not non-specifically inhibiting the kinases involved in Akt activation nor grossly perturbing cellular function such that these events cannot occur.
These data, along with the significant experience with W-7 in the literature lead us to conclude that the effects we are studying do indeed relate to inhibition of calmodulin rather than some non-specific effect.
To address the second goal of the study, we assembled a panel of human cell lines, including, breast cancer cell lines designed to represent the spectrum of phenotypes and genotypes typically found in human breast cancer [24, 29], as well as some non-transformed mammary epithelial cell cultures. As described above, treatment with W-7 suppressed Akt activation in the majority of the cell lines tested, including the non-transformed mammary epithelial cells (Fig. 1 and data not shown). This demonstrates that calmodulin dependent Akt activation is a common mechanism in many breast cancer cells, but since EGF-induced Akt-activation was also suppressed by W-7 in non-transformed HMEC (Fig. 1b), it suggests that calmodulin is likely involved in the regulation of Akt activity in normal mammary tissue.
Several cell lines, however, were refractory to the inhibitory effects of W-7 on Akt activation (Fig. 2), allowing us to proceed with the third goal of this study: characterizing the features of cells in which Akt activation is calmodulin dependent, compared to those in which it is not, with the goal of beginning to understand the mechanisms involved. We first set out to address the possibility that simple overexpression of calmodulin might underlie the difference in W-7 sensitivity. We had shown that knocking down calmodulin levels could suppress EGFinduced Akt activation, mimicking the effects of W-7 and so it seemed reasonable that overexpression of calmodulin might make cells resistant to the effects of the compound. Analysis of calmodulin levels in the cell lines by immunoblotting did not reveal dramatic differences in calmodulin levels between the various cell lines and no correlation between the levels and W-7 sensitivity was evident (Fig. 5). We believe, therefore, that calmodulin expression level is not a likely source of the differences in W-7 sensitivity. It is possible, however, that this conclusion is too simplistic. The nucleic acid sequence of the exons in the three genes that code for calmodulin in the human genome are almost as divergent as is possible for DNA coding for the same amino acid sequence [28]. This demonstrates that all three genes have been carefully conserved over evolutionary time and implies that each gene has some individual function that cannot be provided by the other two genes. Indeed the promoters and 30 and 50 UTRs of the three genes are quite different suggesting divergent function. Studies of calmodulin expression in neurons and other systems suggest that indeed the three transcripts serve different functions and may code for different pools of calmodulin within the cell [32–34]. Western blot analysis simply looks at total calmodulin levels within the cells without regard for which transcript is predominant, or the sub-cellular localization of the protein. No large scale immunohistochemical study of calmodulin expression in breast cancer is described in the literature; however, differences in the levels of the three transcripts are evident in published micro-array data sets, comparing normal mammary gland to cancer, and ER-negative to ER-positive disease, suggesting that this is an issue that merits further study [35–38]. However, our finding that altering the level of one calmodulin transcript can alter the expression of the others highlights the complexity of such analysis, particularly since there is no way of distinguishing between the protein products of the three genes. We are developing immunofluorescence based approaches to study calmodulin dynamics in our cell systems and plan to initiate an immunohistochemical study of calmodulin expression in breast cancer.
The cell lines that make up our panel have been studied in great detail and characterized with respect to many of the genetic and epigenetic alterations frequently found in breast cancer. The panel contains lines with mutant p53, some with mutant Ras, some with loss of PTEN expression, and some with loss of functional Rb, etc., none of which alterations was apparently correlated with W-7 sensitivity.
The most obvious difference between cells in which W-7 blocked Akt activation and those in which it did not was ER status. However, acute inhibition of ER mediated signaling, by estrogen deprivation or treatment with an antiestrogen known to lead to ER destruction, did not render the activation of Akt W-7 sensitive in MCF-7 cells. This suggests that the difference is not simply due to the presence of the receptor or the expression of some estrogen responsive gene in ER positive cells.
Long before the gene expression array studies were able to develop elaborate descriptions of the ER-positive phenotype in terms of expression array signatures, it has been appreciated that ER-positive breast cancer is different from ER-negative breast cancer in many more ways than simply the presence or absence of the receptor. It has, for example, frequently been noted that there seems to be an inverse correlation between ER status and the expression of EGFR family members. ER-negative breast cancer cells, tend to overexpress members of the EGFR family, compared to ER positive cells [26, 39–42] and this is certainly true of the cell lines in our panel. For example MDA-MB-231, and MDA-MB-468 both are ER-negative and express over 250,000 and 2,000,000 EGF binding sites per cell respectively [39], compared to MCF-7 cells, which are ER positive, and express only 2,400 EGFR binding sites per cell [39].
Since the calmodulin-dependence of Akt activation seemed to correlate somewhat with high level expression of EGFR, we decided to examine the effect that enforced overexpression of EGFR or ErbB2 has on the W-7 sensitivity of one of the ER positive cell lines—MCF-7. As described above, we found that EGF-stimulated Akt activation became sensitive to W-7 in cells overexpressing either of these receptors and that this effect was seen independent of the presence of estrogen in the culture medium.
At first glance these and the previous data would seem to suggest a simple relationship between the level of EGFR family member expression and calmodulin dependent Akt activation, independent of ER status, however, we believe that the situation is rather more complex. Multiple lines of evidence suggest that overexpression of EGFR family members, or constitutive activation of the signaling molecules down-stream of these receptors, is incompatible with ER mediated signaling. As noted above, there is an inverse relationship between ER expression and the expression of EGFR family members, both in clinical samples and cell lines. During the development of the transfected MCF-7 clones we have used in this study, it was discovered that stable expression of EGFR, or ErbB2 in MCF-7 cells could only be maintained if the clones were grown in estrogen depleted media [21, 22]. If the clones were grown in media containing estrogen, both receptor protein and mRNA levels were suppressed. Culture of the clones once more in estrogen depleted medium resulted in the re-expression of the receptors over several weeks. Similarly, transfection of MCF-7 cells with constitutively active components of the MAPK pathway results in the reversible loss of ER expression in cells grown in estrogen containing medium [43]. In contrast, if estrogen-dependent breast cancer cells are grown in the prolonged absence of estrogen, or are selected for resistance to antiestrogens, the cells that grow out are frequently found to have upregulated expression of one of the EGFR family members or exhibit enhanced down-stream signaling [44–47]. Taken together these data suggest that if deprived of estrogenic stimuli, ER positive breast cancer cells will come to depend on proliferative signals down-stream from EGFR family members in much the same way as ER negative breast cancer cells. Indeed a recent array based study has shown that cells driven down this pathway acquire a pattern of gene expression that is reminiscent of that seen in ER negative breast cancers [43].
We hypothesize the acquisition of the calmodul in dependence of Akt activation is one aspect of this phenotypic shift and as such probably does not depend on the absolute level of the receptors expressed so much as the signaling pathways operant within the cell that are driving proliferation.
The precise mechanisms underlying calmodulin-dependent Akt activation are not completely understood and this area is the focus of our ongoing studies, though the data presented here and in our previous work clearly suggest that direct interaction between calmodulin and Akt is involved, and that inhibition might depend on altered protein trafficking. Our previous work showed by coimmunoprecipitation assays that Akt associates with calmodulin in an EGF-dependent manner, in Myc83 cells [20], and we have shown that the same is true in the human breast cancer cells that we have used here (not shown). Additional evidence for a direct interaction between the two proteins has come from the literature. For example, in a study in which an mRNA-displayed proteome library was screened for previously uncharacterized calmodulin binding proteins [48], Akt was identified as a calmodulin binding partner. This finding was then validated by a CaMSepharose pull-down assay and Akt was shown to be associated with calmodulin in a calcium dependent manner in both mouse brain lysates and in HeLa cell extracts [48], and a recent report by Rihe Liu and colleagues has demonstrated that calmodulin binds directly to the PH domain of Akt [49]. We are in the process of investigating the nature of the association between calmodulin and Akt, and have identified a putative IQ motif, a conserved calmodulin binding motif resembling IQXXXRGXXXR, [13] within Akt (between amino acids 161 and 176).
In the present study we have extended our earlier observations and shown that both calmodulin and Akt are translocated to the PM on EGF-stimulation and that this translocation is blocked by treatment with W-7, suggesting that calmodulin is regulating Akt activity by modulating the sub-cellular localization of Akt. This idea is reinforced by our finding that direct targeting of Akt to the plasma membrane by the addition of a myristoylation signal overcomes the dependence on calmodulin for activation. It would have been satisfying had we found that EGF-stimulated Akt translocation to the plasma membrane was not blocked by treatment with W-7, however, this was not the case and although Akt becomes activated in response to EGF in the ER positive cells when treated with W-7, the translocation appears to be blocked. This is an intriguing finding that suggests that Akt activation is occurring differently in these cells. As noted above, it is not clear if the ER has a direct role in this process or if the differences seen relate to other features in the cell associated with the ER positive phenotype. Our studies conducted in the presence or absence of estrogen suggest that if ER is involved, its action is independent of ligand binding. Furthermore, the insensitivity of the system to treatment with the ICI 182,780, which is known to result in suppression of ER levels, again argues against a direct effect, although some un-liganded receptor might be available for interactions, even under these conditions. The ER is known to interact with calmodulin suggesting the possibility that this interaction might render W-7 treatment ineffective. We consider this possibility unlikely, but plan to study calmodulin-dependent Akt activation in cells transiently transfected with ER-alpha.
A recent finding has provided another possible explanation for why some breast cancer cell lines might lack dependence on calmodulin for Akt activation. A transforming mutation found in the PH domain of AKT1 in some human cancers is located in one of the putative Akt/calmodulin interaction domains and it would be intriguing to determine if the breast cancer cell lines that do not require calmodulin to activate Akt upon EGF stimulation possess this mutation, and whether this mutation disrupts calmodulin Akt binding, since the PH domain of Akt has been shown to harbor the calmodulin binding site [49, 50].
It is interesting to note that contrary to our previous finding in mouse carcinoma cells, the majority of human breast cancer cells do not require calcium to mediate calmodulin dependent signaling. In fact, the majority of cell lines screened for sensitivity to W-7 were able to phosphorylate Akt in the presence of internal calcium chelation by BAPTA-AM, even though Akt activation was dependent on calmodulin (Fig. 1a and data not shown). This suggests that calcium-independent calmodulin, or apocalmodulin, is capable of mediating Akt activation in the human cells utilized in the present study. Apocalmodulin differs from calcium-bound calmodulin in its tertiary structure, and like calcium-bound form, is known to be involved in functions vital to cellular life, including microtubule function, chromosome segregation and endocytosis [13, 51, 52].
We are in the process of studying the biological implications of our finding that Akt activation is calmodulindependent in ER negative breast cancer cells. It is interesting to note that previous work with calmodulin inhibitors has demonstrated that estrogen independent cells show enhanced sensitivity to these agents. MDA-MB-231 cells were shown to be significantly more sensitive to growth inhibition by the calmodulin inhibitor W-13, than ER-positive, estrogen dependent MCF-7 cells [53]. Furthermore, MCF-7 cells selected for resistance to an antiestrogen were shown to be more sensitive to the growth inhibitory effects of a calmodulin antagonist than the parental MCF-7 cells [53]. It is tempting to speculate that this may present an attractive therapeutic opportunity, and that the use of a calmodulin inhibitor at the same time as antihormonal therapy with antiestrogens or aromatase inhibitors might prevent the development of hormone independent disease.
This work is dedicated to the memory of our colleague, Dr. Robert B. Dickson, who passed away before submission of this manuscript. We thank Dr. Dorraya El-Ashry and Dr. Francis G. Kern for the use of the MCE5, MB7 and MB8 cell lines. Assistance from LCCC Tissue Culture Share Resource is gratefully acknowledged (NIH 4P30CA51008). This work was supported in part by National Institutes of Health Grant 2RO1AG014963 and United States Department of Defense Grant DAMD 17-01-1-0251 (to R.B.D.). C.M.C. was supported by a predoctoral training grant from the United States Department of Defense (DAMD 17-01-1-0246). T.B.D. was partly supported by an NIH Specialized Program of Research Excellence (SPORE) grant in breast cancer to Lombardi Comprehensive Cancer Center (2P50CA58185) and by United States Department of Defense postdoctoral training grant DAMD 17-00-1-0271.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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|Do Phil <Phil...@irs.gov>
|RE: ^M characters
|Tue, 05 Sep 2000 14:22:22 GMT
Thank everyone so much for your responses. I got a lot of helpful answers. Thanks again. Phil Do -----Original Message----- From: email@example.com [mailto:firstname.lastname@example.com] Sent: Friday, September 01, 2000 5:45 PM To: email@example.com Subject: Re: ^M characters Do Phil wrote: > > I downloaded the Tomcat & tried to install it on a Unix computer. > I uncompressed and untarred the file, jakarta-tomcat.tar.gz. > When I opened any conf files or Java source files with vi editor, > I saw the ^M characters on every line. I am wondering whether > I downloaded the wrong file. How can I get a 'clean' version > without any '^M's for my Unix environment? Where did you download this from? 3.1 was built from an extract from a Windows machine. 3.2 is extracted and built on a Linux machine. The source files should not have any ^M characters... let me know specifics if you find files that do. - Sam Ruby
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Sharing Libraries With Other Users
November 18, 2015
There are two different ways to share EndNote libraries with other users: Share your entire EndNote Library (including PDFs) with other EndNote users. (This option does not allow you to select just a few references to share.) This option is best...Read more


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There are two categories of 3D technology, active and passive. Active have electronics which interact with a display and passive two images are projected superimposed onto the same screen.
Linearly polarized glasses
To present a stereoscopic motion picture, two images are projected superimposed onto the same screen through orthogonal polarizing filters. The viewer wears low-cost eyeglasses which also contain a pair of orthogonal polarizing filters. As each filter only passes light which is similarly polarized and blocks the orthogonally polarized light, each eye only sees one of the images, and the effect is achieved. Linearly polarized glasses require the viewer to keep level, as tilting of the viewing filters will cause the images of the left and right channels to bleed over to the opposite channel.
Complementary colour anaglyphs (red and cyan)
Complementary colour anaglyphs employ one of a pair of complementary colour filters for each eye. The most common color filters used are red and cyan.
ColourCode 3D (amber and blue)
Uses amber and blue filters.
3D Stereoscopy
Stereoscopy (also called stereoscopic or 3-D imaging) refers to a technique for creating or enhancing the illusion of depth in an image by presenting two offset images separately to the left and right eye of the viewer. Both of these 2-D offset images are then combined in the brain to give the perception of 3-D depth. Three strategies are used to accomplish this. The viewer wears eyeglasses to combine separate images from two offset sources, the viewer wears eyeglasses to filter offset images from a single source separated to each eye or the lightsource splits the images directionally into the viewer's eyes (no glasses required, known as Autostereoscopy).
Autostereoscopy (no glasses)
Autostereoscopy is any method of displaying stereoscopic (3D) images without the use of special headgear or glasses on the part of the viewer. Because headgear is not required, it is also called 'glasses-free 3D'.
Traditional stereoscopic photography consists of creating a 3-D illusion starting from a pair of 2-D images, a stereogram. The easiest way to enhance depth perception in the brain is to provide the eyes of the viewer with two different images, representing two perspectives of the same object, with a minor deviation exactly equal to the perspectives that both eyes naturally receive in binocular vision.
Alien Breed 3: Descent
Examples of these techniques applied to survivor in Alien Breed 3: Descent, interleaved, red and cyan, no glasses, side-by-side half-width and side-by-side full-width.
Linearly polarized
Red and cyan
No glasses
Side-by-side (half-width)
Side-by-side (full-width)
Interleaved - active shutter glasses.
Linearly polarized glasses - two images are projected superimposed onto the same screen, with the wearer wearing orthogonal polarizing filters.
Autostereoscopy (no glasses)


text:
Speed Bomb On Bus MP3
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Bombs Away is available to order now: Order on iTunes: http://po.st/SheppardBAVEVOiT Order on Amazon: http://po.st/SheppardBAVEVOAm Order on Google ...
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Music video by Kris Kross performing Jump. (C) 1992 Sony BMG Music Entertainment.
Kris KrossJump, jump, you know, you should know that Kris Kross is not having anything today As we stand there totally krossed out We commence to make you Jump, jump The Mac Dad will make you jump, jump Daddy Mac will make you jump, jump Kris Kross will make you jump, jump Don't try to compare us to another bad little fad I'm the mac and I'm bad, give you something that you never had I'll make ya bump, bump wiggle and shake your rump 'Cau[...]
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Muppet Show. Swedish Chef - Ping Pong Ball Eggs (ep.214) MP3
The Swedish Chef prepares "egg der chef," but his chicken only lays ping pong balls. The muppet show, season 2, episode 14.
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Skrillex 2014 - Recess Portrait MP3
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Your hands need as much attention as you would give a small child or a penguin you are somehow forced to take care of. Pamper yourself with this Groupon.
$25 for a Gel Manicure ($50 Value)
During a 40-minute manicure, deft techs shape nails, curb cuticles, and apply a choice of polish that lasts for up to two weeks without chipping.
Posaré Salon
With Posaré Salon built upon three essential r's of salon and spa service—relax, revive, and replenish—experienced staffers wield a bounty of professional products as they seek to treat clientele like royalty. Those fundamentals emerge most clearly in Posare Salons' Pink Room, where patients undergoing cancer treatment enjoy complimentary salon services and can audition affordable wigs from Las Vegas Wigs. Throughout the rest of the salon, stylists and aestheticians perform an abundance of pampering treatments from spa pedicures to permanent eyelash extensions. The med-spa branch additionally clears epidermises of acne and wrinkles, dispenses flu shots to shield patients from severe sniffles, and supplies melancholic music suggestions for visitors with crippling disco fever.


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Most Helpful Guy
No it isn't10|10|0
Okay, thanks for your comment!
Have an opinion?
I say it would be fine and hardly noticeable
Everyone has hair, some people choose to shave it and some don't.
Never seen it on the back before on a girl though, everywhere else though , so I guess it's normal
No i see no problem with girl having hair on certain areas of her body.
Lmao I sware teenage girls are insecure about everything.. your going to look back this question in t years and laugh your ass off. Nobody is going to notice little hair on your toes.
The only places on your body that you will not have hair are soles of you feet/hands eyes lips and mouth so i think its fine and if it makes you uncomfortable then you can always wax it.
Yes, especially if she#s a feminist.
Not really. Hair grows in a lot of places. The real question should be is it weird if girls do not shave it? But to be honest, who really gives a shit either way. It's nobody's business.
Of course not. Humans grow hair anywhere on the body except the soles of your feet, and your palms. If you dont shave or maintain them though, that's weird.
Personally, I find it weird. Although to be fair, I don't like body hair in general on males or females. I wax mine off, and I would hope my partner would do the same.
toes are pretty much fine I think, back might or might not depending on how much
Thats normal, every girl has it
no. girls look good when they are hairy beasts
Thanks for your comment!
No. However it's not attractive but you were born with that issue so it's not your fault. I wouldn't hold it against you just as long as you groom it. I would just make sure to wax/shave/no hair gel it off.
No, Italian girls are very pretty.
no problem girl (y)
Yeah dude; order up a wax. I don't like hair anywhere except on your head and eyebrows. Truth be told you could go without that too; I'm not a hair person. If it's like light lightly colored hair that's one thing; but if we're talking gorilla hair, no thanks
Okay, thanks for your comment!
You cannot undo this action. The opinion owner is going to be notified and earn 7 XPER points.


text:
From a media release:
Sounds Under Radio – Sing,
Texas Tour
& Upcoming Album Release May 3
** Sounds Under Radio's Music Video for"Sing" Premieres on PopMatters **
** "Sing" Infiltrates Television Airwaves With Promo Spot on American Idol **
New York, NY - When it rains it pours, as the old saying goes. Days after Austin-based rock act Sounds Under Radio was selected to have their single "Sing" from their forthcoming album 'Where My Communist Heart Meets My Capitalist Mind' (in stores May 3rd) featured on Fox's American Idol, their music video for the single is also premiered on PopMatters.
Sounds Under Radio are infiltrating your television airwaves, as "Sing" was selected to be used in the newest promo spots and commercials for American Idol. After a spate of sync successes in film and TV with placements in Spider Man 3 (Sony Pictures), The Wolfman (Universal Pictures), War Games: The Dead Code (MGM Pictures) and CW's 'The Vampire Diaries', Sounds Under Radio now manage to share their music with people on one of the grandest scales of broadcast television, with their American Idol spot.
Directed by Jeff Ray at Papertank Productions, Sounds Under Radio's music video, "Sing," is as visually epic as it is musically stunning. The music video is a way of showing that despite disasters and fears in the world, there is still beauty all around us.
Get a free download of Sing when you sign up to their website:
Twitter & MySpace
Texas Tour Dates:
April 22 @ Common Grounds (Waco TX)
April 23 @ The Stafford (Bryan/College Station TX)
April 29 @ The ND (Austin TX) –Hometown Record Release Show-
April 30 @ Opening Bell (Dallas TX) –acoustic show-
May 3 @KLBJ FM (Austin TX) 'Local Licks Live'
May 7 @ Magnolia Motor Café (Ft. Worth TX)
May 8 @ Scout Bar (Houston TX)
May 21 @ Jacks (San Antonio TX)
May 27 @ The Prophet Bar (Dallas TX)
May 28 @ El Dorado (Shreveport LA)


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Lovely lace crochet robe featuring a belted waist and scalloped edges for an ultra femme silhouette.
Lately My Heart Robe styled by the FP Me community
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