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A mysterious 132-year-old Winchester rifle has arrived at the Cody Firearms Museum for a little TLC after being exposed to years of inclement weather in Nevada's Snake Mountains.
The Winchester Model 1873 lever-action rifle received international attention after it was found leaning against a juniper tree last November by an archaeologist working in Nevada's Great Basin National Park, four hours northeast of Las Vegas.
"One thing we all assumed was that someone here had a very bad day," park archaeologist Eva Jensen, who found the rifle, told the Associated Press at the time. "One of the staff said, 'Why do you set your gun down and forget where you put it? That just doesn't happen.'"
Well, that's not entirely true.
"There are people I know who've lost their firearms," said Ashley Hlebinsky, curator of the Cody Firearms Museum. "So it's not unheard of."
The "Forgotten Winchester," as some have called it, went viral online after park employees posted a photograph of the rifle on the park's Facebook page. The post asked, "Can you find the man-made object in this image?"
That one question sparked a media sensation.
"It has crossed all demographics," Hlebinsky said. She has had calls from CNN and even talked to people from Europe who have heard the story about the unusual rifle. "People like a mystery."
Park officials recently delivered the rifle to the Buffalo Bill Center of the West's Cody Firearms Museum for conservation and identification. The center also holds the manufacturing records for Winchester firearms.
After years with little protection from sun, rain, wind and snow, the rifle had a cracked wooden stock that had weathered to gray and the barrel had rusted to a deep brown. The natural colors helped the rifle blend into its surroundings in a remote rocky outcrop, keeping the rifle camouflaged for years.
When the rifle arrived in Cody, the wood was flaking and stained by white salts. One of the first steps of conservator Beverly Perkins, Hlebinsky and curatorial assistant Dan Brumley was to "admit" the firearm to neighboring West Park Hospital's radiology department for X-rays. The images showed that the gun was not loaded, but did have a cartridge in the trap of the butt stock, which was created to hold cleaning rods. A crack in the stock had also been repaired with metal pins.
The door to the butt stock was loosened with a drop of penetrating oil, and the object was removed and identified as a Union Metallic Cartridge Co. .44 caliber WCF cartridge, dated 1887-1911.
Because of the age of the cartridge, Hlebinsky noted that the longest the rifle could have been sitting out was since 1887. But she personally doesn't believe it was that long, since the tree hadn't grown around the rifle.
Examination of the rifle also showed that the lifter arm, a mechanism that lifts a cartridge into the chamber from the magazine, had been removed. Without that feature, the rifle had become a single-shot firearm.
To stop further flaking of the wood, Perkins used an adhesive (2 percent Klucel G hydroxypropylcellulose) mixed in distilled water and ethanol.
"We kept anything that has fallen off of the gun for testing, including dirt that fell out of the trap door in the butt stock," Hlebinsky said. "That can be dated."
She added that the they had to move "very, very cautiously" when handling the rifle because it was so fragile.
Known as "The Gun that Won the West," Hlebinsky called the Winchester Model 1873 possibly "the most iconic western firearm of all time," which probably adds to the allure of the mystery.
"With all it's been through, this particular gun has certainly carried on that legend," she said in a press release.
The abandoned rifle is shrouded in a mystery that will likely never be solved.
"Why would you leave your rifle and not come back for it?" Hlebinsky asked. "How many years was it hidden? Why was it left leaning against a tree? We here at the Buffalo Bill Center of the West and the staff at Great Basin are both asking the same questions. The mysteries surrounding this Winchester 1873 have truly fueled its popularity."
Hlebinsky encouraged the public to weigh in on why they think the rifle was abandoned.
"What do you think happened?" she asked. "Enter 210 at bbcw.iscout.org to tell us why you think this rifle was left out in nature."
She said the museum has already received several comments from the public, including those who think that it's a fake.
"It's not fraudulent, I can guarantee you that," Hlebinsky said.
The rifle is on display in the Firearms Museum, where it will remain until fall when the center will return it to Great Basin National Park for its 30th anniversary and the 100th anniversary of the National Park Service in 2016.

subset: commoncrawl


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Welcome to Rancho La Loma –
Exclusive Country Equestrian Living in San Miguel de Allende
Combining the best of Town and Country, Rancho La Loma is a 13 hectare, luxury gated equestrian community, offering the serenity of exclusive country living just minutes from San Miguel de Allende's city center.
La Loma's unique combination of pristine landscape, top level luxury and proximity to San Miguel is unmatched.
La Loma's members will enjoy private stables and paddock facilities, expansive landscape views, on-site vineyards, and club house privileges. As a condominium development, the sweeping public spaces beyond the individual lots are available for all residents to enjoy.
Located in San Miguel's developing wine corridor, The 5-minute drive into San Miguel Centro brings you into the heart of our charming town, full of historic colonial architecture, world class restaurants and exceptional shopping.
La Loma's 13 hectares of premium property currently offers 11 luxury lots for sale from $249,000 USD.


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- Hometalker
How do I prepare and paint kitchen cabinets?
I have maple wood cabinets that I would like to paint white, but would like them to look professional, so how do I go about that?
- Laura Cooperon Apr 11, 2019
Carla, a professional paint requires good prep. Sand any uneven areas and remove excess glossiness. Wash with TSP to remove any residue. I use Kilz as a primer. Professional finishes have very little texture, so using an hvlp sprayer or at least a very fine nap roller will yield a smoother finish. You will need at least two coats of paint on top of the primer. I did a lot of research before painting my cabinets and found that latex enamel gloss paint specifically has more hardiners than other finishes. Using this paint, I've never needed any top coat. Just 2 or possibly 3 coats of latex enamel gloss paint over primer holds up to much abuse.
- Williamon Apr 12, 2019
How to Paint Cabinets
Make sure they are clean and dry. Remove the doors and hardware. Mark the doors and cabinets with tape where they go. Lightly sand the doors and cabinets to remove any gloss and roughen the surface for paint with 220-grit sandpaper. Use a tack cloth or damp rag to remove dust after sanding. Prime with a stain blocking primer like KILZ. Acrylic, or water-base, paints are low-fume and clean up easily with water. Alkyd, or oil-base, paints require good ventilation because the paint contains solvents that can irritate your lungs and make you feel sick. Alkyd options require mineral spirits for cleanup, but they provide a hard, durable paint finish. Whichever you use, buy the best-quality paint you can afford for a lasting kitchen cabinet finish. Seal with at least three coats with a water based polyurethane. Use a small foam roller and foam brush for a smooth finish.
- Pamelaon Apr 12, 2019
I had great success using Benjamin Moore's advance paint . This is a hybrid paint that washes off your hands and brushes with soap and water , but dries to a hard finish , like an enamel . It is also a self leveling paint , so you don't get brush or roller marks . 2 giant pluses 1- no sanding & 2- no primer !!! There is some prep work to do. I used 2 products from Krud Kutter , first a de greaser to clean and get off excess grease , oils and ok!dishes . Then the same brand de glosser , this takes off the shine on the cabinets . Both of these are spray on , then wipe off . Then I applied 2 coats of paint ! I did this 2 years ago and they are holding up great ! Easy to clean . You can check out my tutorial on this site " new kitchen on a budget " you can see step by step and tips ! Good luck !


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Nepal rastra bank forex
Officials said such a strong forex reserve has created an environment whereby foreign investors can be tempted to invest in the country.Nepali Blogger is a Community of Nepali bloggers publishing Nepal News, Technology, Business, Politics, Music, Movies, Society, Culture and Events.
Published:. shows the latest Macroeconomic Report of Nepal Rastra Bank (NRB),. 2016 of The Himalayan Times.Aig Fx Prime Brokerage Jessica Hu Easy Forex Forex Rates Against Indian Rupee Bank of Canada yearly average.
Money Exchange Rate In Nepal. Nepal Rastra Bank Speech - Duration:.The Nepal Rastra Bank (NRB) is preparing to recommend the Ministry of Commerce and Supplies to review the guideline on gold and silver management.Social Engagement. Forex Indicators Guide - forex-indicators.net.Exchange Rate - Nepal Rastra Bank - Central Bank of Nepal Best exchange rate comparsion for Australian bank,Fx,Forex, currency converter.
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Subhechha Bikash Bank Limited is a regional level bank, licensed by Nepal Rastra Bank. Forex. Quick Links.View and Calculate foreign exchange rates with free currency converter.
Nepal Investment Bank LimitedStandard Chartered Bank Nepal provides you a host of foreign exchange services at attractive rates for various purposes viz.Excel Development Bank Limited is licensed by Nepal Rastra Bank under banking and. services-Excel Development Bank Ltd. strives to. Forex. Banking Hours. Sunday.
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Laxmi Nath Gautam is former governer of Central Bank of Nepal - Nepal Rastra Bank(NRB).
Currency Exchange RatesSanjaal collects data from Nepal Rastra Bank and now presents it in a graphical charts for the exchange rates.He become governer from the next of Himalaya Shamsher Jang Bahadur Rana.Global Mountain Remit approved by Nepal Rastra Bank Nepal Rastra Bank approved the application to start the remittance business in Nepal by Global Mo.
The Rupee is the official currency of Nepal and is divided into 100 paisa.Foreign Exchange Rates - Nepal Rastra Bank - Central Bank of Nepal Best exchange rate comparsion for Australian bank,Fx,Forex, currency converter.
Nepal Rastra Bank approved the application to start the remittance business in Nepal by Global Mountain Remit pvt. Ltd.Posts about Nepal Rastra Bank (NRB) written by nepal stock news.Nepal Rastra Bank ( NRB ) has allowed money transfer agencies and money changers to sell their foreign currency to the central bank too.
Foreign Exchange ReservesSince then the bank has adopted many changes with the introduction of the new.
Currency Exchange Rate NepalThe currency conversion table (matrix) is calculated based on the buying rates published by the Central Bank of Nepal, Nepal Rastra Bank (NRB).
Currency Rate Money Exchange
In Nepal, interest rates decisions are taken by the Nepal Rastra Bank.Some Banks in Nepal are as under. 1) Nepal Rastra Bank- First Bank of Nepal 2) NMB Bank.


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A full service real estate, marketing and technology team driven by people - for people
New Street Home Search > Peel Greater Area > mississauga > Lorne Park > 1317 Ravine Dr
The Height Of Luxury! This 5 Bedroom Custom Built Home Is Nestled On A Large Private Lot In The White Oaks Of Jalna. Luxurious Features Through Out. High End Miele Appliances, Kitchen By Top Notch, White Oak Hardwood Floors, 10 Ft Ceilings On Main Floor, 9Ft In Bsmt And 2Cnd Floor Soaring 20+Ft Cielings In Great Room, Herring Bone Hardwood Floors In Dining Room And Office. Master Bedroom With Coffered Ceilings, Opulent Ensuite & Walkout To Large Balcony
This 5+1 bedroom 8 bathroom house features a family room, fireplace, 7.0 parking space and air conditioning. Listed at $4499000. This home has 10 rooms on 3 levels. It is for sale and has been listed on newstreet.ca for 30 days. A mortgage for this property would be approximately $17498/per month .
Welcome to 1317 Ravine Dr L5J3E6. Located in the Lorne Park neighbourhood of Mississauga.
Local amenities around 1317 Ravine Dr in Lorne Park include Cafes and Restaurants such as Noble Bistro, Cuda's Tap & Grill and Lily's Pizza & Pasta. Shopping and service providers include Battaglia's Marketplace. The closest hospitals and clincs to 1317 Ravine Dr are . Lastly and most importantly local education institutions and schools in the area include Tecumseh Public School.
Listing data for this listing is provided under copyright by the Toronto Real Estate Board. The listing data is deemed reliable but is not guaranteed accurate by New Street or the Toronto Real Estate Board.
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|6.24 * 3.96
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|4.41 * 3.96
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Embassy in rostok russian
In other projects Wikimedia Commons. Jose VasconcelosMexico-City ; tel. The residential building, the school, the kindergarten and sports grounds were all complete in ReisnerstrasseA Vienna tel 1, ; fax 1 Consulate: Russian embassy in Switzerland. Russian embassy in Japan. Chinese Embassy in Beijing.
Embassy of Russia in Washington, D.
List of diplomatic missions of Russia
Fred Booth, son of lumber baron J. Skargi ulitsa, PL, Szezecin Phone: Vel zquezE Madrid tel 91; fax 91 Consulate: Shanti Path, Chanakyapuri, New Delhi ; tel. With the fall of the Soviet Union the building became the Russian embassy. Assistance with Russian visas can be provided through the RussianConsulate.


text: Make this as your new home! Brand new kitchen featuring quartz countertops, cabinets, flooring, undermount sink, faucet, built-in microwave and dishwasher. All appliances are stainless steel. Bathrooms have both had recent updates including vanities, sinks, lighting and more. Living room and bathrooms all facing southern, Lots of sunlight. Move in Ready!Willow Point is perfectly located. 5 minutes to spectacular Mosaic District with all the restaurants, shops, theaters you could dream of. Close to Route 50. Community comes with outdoor swimming pool!


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Josie Hunter aka Blake Pickett co-starred in the role of Erica, a single mother who works a stripper, and sometimes as a prostitute to support her daughter. Men and women hook up in different sections of a private resort. Why do our husbands expect us to look like that? Season 5 This Is Us: Half erotica, half quasi-documentary, this movie purports to show the angst of three married couples. When David learns that Val has stopped taking the pill, he angrily dismisses her invitations to have sex as manipulative. The Atonement of Janis Drake
Full Cast and Crew.
Please Confirm
The Haunting of Hill House. Of course all the people are like wild dogs in heat as they will sleep and mess around with anyone and everyone that catches their fancy. Joseph Green Stacy Troiano There are no approved quotes yet for this movie. After three years of happiness, Allison wants to marry Shawn. Audible Download Audio Books. Sign in with Facebook Other Sign in options.


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By Baretang Bikolnon Staff
The National Union of Journalists of the Philippines and other fellow journalists condemn the brazen murder of Davao-based journalist Dennis Denora who was shot dead by still unidentified riding-in-tandem killers near the wet market of Panabo City, Davao del Norte.
Denora, publisher and columnist of the community paper Trends and Times and an officer of the Davao Region Multi-media Group, is the 11th journalist killed in the less than two years since President Rodrigo Duterte assumed office and the 184th since 1986.
According to Davao del Norte Police Provincial Office, Denora was shot around 2 p.m. on board his vehicle and sustained multiple gunshot wounds in the head while his driver sustained two gunshot wounds in his right hand.
"While there is still no official word on the possible motive for his murder, it was likely related to his work as a journalist. Denora's colleagues in the province acknowledge that he was "fearless" in his commentaries in broadcast and print," said NUJP in a statement.
"This is a tragic reminder of the physical risks journalists take as they work in the field. As a newspaper publisher like myself, I am disheartened by the death of one of the well-known columnists in Davao del Norte," said Pamela Gay B. Perales, president of Davao del Norte Press & Radio-TV Club (DNPRC).
Denora is known for his fearless forecasts both in newspaper and in radio did have the highs and lows of the profession according to DNPRC condemning the said murder.
"We at DNPRC, believe it is not justifiable to take one's life for personal or political reasons. His death awakens the anger and pains of journalists who do their job and yet are being judged by the pistol," Perales said in their statement.
According to NUJP, this latest media killing belies Malacañang's claim that the Philippines has improved its position among the deadliest countries in the world to be a journalist, a fact international press freedom watchdog Reporters Sans Frontieres already established early on when it reported that we had slipped six notches – from 127th to 133rd of 180 countries ranked – in its 2018 World Press Freedom Index.
"It also highlights what we and allied media organizations noted on World Press Freedom Day early this month, that journalism has become an even more dangerous endeavor under Duterte, who has himself spearheaded the vilification of the press because he cannot abide criticism," NUJP stated. (BaretangBikolnon.com/Photo from Octavio Valle)


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Black and white zebra romper , pink tutu ,Baby Girl romper.
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Funeral of Stanley Morrison
2x2 worker Raymond Stanley Morrison passed away.He was a preacher of the 2x2 religion, working in Kansas and Nebraska. Stanley Morrison was born December 6th, 1937 and died about 80 years old.
Stanley was born to Hubert and Stella (Peterson) Morrison by Pagosa Springs, Colorado. Stanley was the oldest of 9 siblings and made his choice to joing the 2x2 religion at age 16. He became a homeless 2x2 worker on May 11, 1957.
Services were held June 2, 2018 at 2:00 PM at at the Duane Grotz Farm, 1018 Road L, York, NE.
Service for Raymond Stanley Morrison
The friends sang #8
Martin Wilson prayed
Warren Taylor
Stanley was recently asked, "Why is it people love you? Stanley responded, "I believe it is the work of God in our lives that make people love us.
Like a lot of you here, my first experience with Stanley is when I was a little boy when he came to our house in Oklahoma. I Thessalonians 2:19-20 It is safe to say all of you here felt love from Stan.
I realize I can't say thank you to Stanley anymore, but I can still thank God.
I was thinking of the great number of years of wisdom and experience we have lost from our midst. This morning was Stanley March's funeral after 76 years in this work and now Stanley Morrison's 61 years. It is the work of God in a life that makes our life meaningful.
There is a Spanish hymn I was thinking of this morning. I won't sing it for you, but I'll try to share a thought from it. We are prone to natural thoughts. We feel a natural separation keenly. The hymn encourages us to think about the spiritual side.
John 11 has been our Bible Study. Jesus loved Lazarus. Mary and Martha understood, Jesus loves us. :34-36 I have shed tears these past days. That is very unusual for me, but it is right, because we loved our brother.
A couple weeks ago those at our gospel meeting heard these verses John 11:25-26. We must believe that life only comes through Jesus. We do not know when our last day will be. We certainly didn't expect Stanley's day would be this quickly.
Martin Wilson
I never got to be a coworker with Uncle Stan, but he was my uncle. One thing I remember from when I was growing up was he loved to give haircuts. We didn't like getting them so we'd make sure we had already had one before we saw him.
I started in this work in 1990. At that time Uncle Stanley was going from Oklahoma to Kansas. My first day in this ministry, I got to ride with him to Denver.
One thing we remember Stanley mentioning at many funerals was about the man who had holes cut into his casket with both hands sticking out so everyone could see he was taking nothing with him.
Like I said, I was never Uncle Stanley's coworker, but my coworker this year had been. I asked him if there was a verse he thought of when thinking of Stanley. He started to quote from Joshua 1. I said, hey, that's my verse!
Joshua 1:1-2 Then throughout the chapter it repeats the message to be strong and of good courage. Joshua was likely thinking, what will I do without Moses?!? Who will council me? Who will pray for me? Uncle Stanley prayed for a lot of people.
Joshua could remember the time when Moses had told him, you fight and I will pray. Others helped Moses hold up his hands. Joshua was aware, there are 3 older men praying for me. Now Moses was receiving the message, be strong and of good courage, I will be with you. Joshua wasn't given the task to find someone to rise up and be a pillar. He was told, you stand up and be strong.
Uncle Stan got all of the letter writing genes in our family. He was a faithful correspondent. I tried to answer him after receiving 6 or 8 letters. He would write about the meeting they were having and people they were visiting. Then he would add a personal note.
Paul wrote to Timothy. I Timothy 6:11-12 We heard from this morning. II Timothy 4:5-8
We sometimes read verses without thinking about them. Fought the good fight. He wasn't out fighting a physical fight. It was in his mind.
Uncle Stanley was offered a scholarship to be a civil engineer, but he left to go in the ministry 61 years ago. Like others of us, he didn't feel very good at it, but he was called by God and he wanted peace.
It was day after day of saying, no, to the flesh, world, and devil. Stan finished because he kept true day after day. He lost a few scrimmages along the way, but he won the war.
Hymn 404 tells us of some benefits to yielding our life. I remember Uncle Stanley saying there is no place like the high country of Colorado. I don't think he left home having any idea how many privileges he'd have or people he'd have and people he'd love.
Verse 3
Acts 20:24-25 Stan could enter into Paul's thoughts. We shall see his face no more.
In Luke 4:18 Jesus quoted Isaiah 61. Uncle Stan loves to reach out to those many found hard to love. He preached to the poor and captives. He wanted them to hear the message that there is a savior who pleading for us and is reaching out to us. He has power to free the captives.
We read the verse, Moses my servant is dead. Uncle Stan knew how to lay down his life as a servant. Today still brings blessings.
Ron Thomke
I just have to say I can't make this seem real. I feel I'll wake up and it was a dream. Those in Kansas and Nebraska know Stanley and I had a good working relationship. When the two states combined, I felt overwhelmed. I soon found out Stanley was a good source of help. He had strengths and knowledge that I didn't. He was an aid in the transition. He never showed any disloyalty.
Philippians 4:3 True yokefellow. We were making plans for you. Stan and I didn't always agree, but we could work it out without any contention. We were never coworkers. I can't even think of a gospel meeting we had together. When two are yoked together, they can do more than the sum of their parts separately.
My experience with Stan gave me a greater understanding of the verses at the end of Matthew 11. Have you ever tried to explain being yoked with Christ to someone who doesn't understand? Jesus isn't here. He doesn't have a cellphone. He doesn't text.
Talking about working with Stan makes it tangible. I think the only people who really understand are those who are yoked with Jesus. There is nothing about leading. We are following Jesus. There is nothing about taking control. Those knowledgeable about the Bible can speak about these verses and then proceed as usual trying to drag Jesus along. It is about surrender. They don't like to hear about surrendering, some of us don't even like to hear about it too much. Drop our thoughts. Give up our own ambitions. This tangible experience with Stan helps us to understand the lofty thoughts about being in the yoke with Jesus. Coworkers, spouses and people who farm together work better together when each is yoked with Jesus. It helps with all social interactions.
Matthew 9:36-38 What a day! Two have been removed from us. We are so few anyway. You watched us come in with the casket and file in row after row. You might think we are doing quite well. Remember those here represent a lot of area.
Jesus didn't say we should go out recruiting workers. He said to pray. He said this while there were 12 ready to go. Matthew 10 begins with sending out the 12. Pray for more. 12 aren't enough for it to continue. He said it at a time when people fainted as sheep without a shepherd. This implies that without the ministry, the people will faint and scatter. A religious man came to Eagle Bend convention. He said, "I feel I'm in a huge crowd with everyone with one mind." It isn't because we follow the workers, but we follow Jesus together.
Workers sang Hymn 405
At the Cemetery
Council Cemetery west of York
Hymn 352
Leslie Pountney
I am very thankful to be here. Like Ron, it feels unreal. I never thought I'd have a part in Stanley's funeral. I thought he'd have a part in mine. I am glad I talked to him in the phone when he was at the hospital in Cheyenne.
I tried to think of a verse when I thought of Stanley passing. I couldn't think of one but I did think of Hymn 8 that we sang at the funeral. When I was with him he asked the children every meeting, "Who did Jesus die for?" I told him I was glad he did that. I couldn't, but I'm glad he did.
I asked the man of the home where we were staying if he could think of a verse when thinking of Stanley. He could. He told me Ecclesiastes 7:1. This is a good verse. I spoke on this verse at my oldest sister's youngest son's memorial service. I mentioned it is very costly to have a good name. The more valuable, the higher the cost. You can't tell a lie, cheat, or steal. Later my brother in law was glad to hear his daughter talking to her boyfriend about this. It pleases him to hear them.
I am thankful to be standing by this open grave. Mainly because of who is in it. I have many memories of Stanley speaking at funerals. He enjoyed speaking at funerals and sharing the gospel. He would often ask me, "Was that too strong?" It was reality.
There is no other place I'd rather be, except where Stanley is now. Fought a good fight.
Two months ago, Stanley didn't realize how soon he'd be gone. Even in the hospital we didn't realize he was so close.
Today should be a day of rejoicing more than the day of one's birth. We do mourn because a natural loss. When we are more spiritual we can rejoice.
Hymn says Help me to walk aright, Proverbs 22:1, 4, Proverbs 3:13, Proverbs 2 helps us understand the parable of the sower and the seed. Understand the fear of the Lord and knowledge of God. Proverbs 16:24 All the treasure of wisdom and knowledge is in Jesus. Proverbs 3:18, 14 Ephesians 5:1 Our purpose and response will be perfect in love.
That is why Stanley loved the gospel. What it had done in his own life.
Since returning from Africa, I was with Stanley 3 times. 2004 was the first time. I never was in so many funerals as my years with Stanley. At almost every funeral he would mention, One day he'll come forth again. He'd read John 5:25-29
Death is the end of the natural side of life. Don't set our heart on riches.
Proverbs is like the parables of the Old Testament. We heard from Joshua 1. Moses my servant is dead.the God who gave life is not dead. As a result of today, I want to be more alive to God.
I am thankful for what we experience today. I want to retain.
We heard about being yoked together. I appreciated Stanley's cooperation. When we are first yoked together with Christ, then divine love makes us work together.
Recently I had 3 weeks with Stanley. He said if he had followed through with the scholarship Martin mentioned earlier in the service, they would have forced me to retire a long time ago. 6 days ago Stanley was in Sunday morning meeting, still laboring on.
LeRoy closed in prayer.
Funerals - home
John Carroll
Funeral Sermons
William Irvine Funeral
George Walker Funeral
Simeon Sarmiento
Peter Doecke
Stanley Morrison
Stanley March
Stan Cornthwaite
|To open their eyes, and to turn them from darkness to light, and from the power of Satan unto God, that they may receive forgiveness of sins, and inheritance among them which are sanctified by faith that is in me. - Jesus Christ speaking to Saul, see Act 26:18, see Salvation through Jesus Christ.


text:
- 13th February 2019
- By Kelly Cox
- In General Information
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Rho‐like GTPases orchestrate distinct cytoskeletal changes in response to receptor stimulation. Invasion of T‐lymphoma cells into a fibroblast monolayer is induced by Tiam1, an activator of the Rho‐like GTPase Rac, and by constitutively active V12Rac1. Here we show that activated V12Cdc42 can also induce invasion of T‐lymphoma cells. Activated RhoA potentiates invasion, but fails by itself to mimic Rac and Cdc42. However, invasion is inhibited by the Rho‐inactivating C3 transferase. Thus, RhoA is required but not sufficient for invasion. Invasion of T‐lymphoma cells is critically dependent on the presence of serum. Serum can be replaced by the serum‐borne lipids lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) (10−7–10−6 M), which act on distinct G protein‐linked receptors to activate RhoA and phospholipase C (PLC)‐Ca2+ signaling. LPA‐ and S1P‐induced invasion is preceded by Rho‐dependent F‐actin redistribution and pseudopodia formation. However, expression of both V14RhoA and V12Rac1 does not bypass the LPA/S1P requirement for invasion, indicating involvement of an additional signaling pathway independent of RhoA. The PLC inhibitor U‐73122, but not the inactive analog U‐73343, abolishes invasion. Our results indicate that T‐lymphoma invasion is driven by Tiam1/Rac or Cdc42 activation, and is dependent on LPA/S1P receptor‐mediated RhoA and PLC signaling pathways which lead to pseudopod formation and enhanced infiltration.
Rho‐like GTPases, including RhoA, Rac1 and Cdc42, have been implicated in the control of a wide range of biological processes, such as the regulation of cytoskeletal structures, adhesion, motility, transcriptional activation and cell cycle progression (Van Aelst and D'Souza‐Schorey, 1997; Hall, 1998). Each of the Rho‐like GTPases regulates distinct actin cytoskeletal rearrangements in response to receptor stimulation. In fibroblasts, Cdc42 controls the formation of microspikes (Kozma et al., 1995; Nobes and Hall, 1995), whereas Rac regulates the formation of lamellipodia and membrane ruffles (Ridley et al., 1992). Activation of RhoA leads to the induction of stress fibers (Ridley and Hall, 1992). Moreover, all three GTPases induce the formation of integrin adhesion complexes in Swiss 3T3 fibroblasts (Hall, 1998), and stimulate transcriptional activation and DNA replication (Olson et al., 1995; Vojtek and Cooper, 1995; Perona et al., 1997).
In search of genes involved in the process of tumor cell invasion and metastasis, we previously have identified the Tiam1 gene by retroviral insertional mutagenesis in combination with in vitro selection of invasive T‐lymphoma variants (Habets et al., 1994). The Tiam1 protein consists of 1591 amino acids and contains several conserved protein motifs, including a Dbl homology (DH) domain. The DH domain is the catalytic domain in many guanine nucleotide exchange factors (GEFs), which activate members of the Rho subfamily of small GTPases (Collard, 1996; Whitehead et al., 1997). GEFs promote the exchange of bound GDP for GTP on these GTPases, which results in their activation. Whereas Tiam1 displays GEF activity towards all three Rho‐like GTPases Rac1, Cdc42 and RhoA in vitro, Tiam1 specifically activates Rac in vivo (Michiels et al., 1995). In fibroblasts, Tiam1 induces the formation of membrane ruffles and stimulates the activation of the Jun N‐terminal kinase (JNK), similarly to activated V12Rac1 (Michiels et al., 1997). Furthermore, V12Rac1 induces invasion of T‐lymphoma cells, suggesting that Tiam1‐induced invasiveness of T‐lymphoma cells is caused by activation of Rac (Michiels et al., 1995).
Rac1 regulates the organization of the cortical actin cytoskeleton, and in fibroblasts it induces the formation of lamellipodia and membrane ruffling, which are believed to play an important role in cell motility (Stossel, 1993). The role of RhoA in cell adhesion and motility has been studied extensively by using C3 transferase, a toxin that specifically inhibits Rho activity (Sekine et al., 1989). Lysophosphatidic acid (LPA), a serum‐borne lipid mitogen (Eichholtz et al., 1993), activates its cognate G protein receptor to stimulate multiple signaling pathways including RhoA signaling (Moolenaar et al., 1997). LPA induces RhoA‐dependent stress fiber formation in fibroblasts (Ridley and Hall, 1992) as well as neurite retraction in neuroblastoma cells (Jalink et al., 1994). Both processes are inhibited by C3 transferase. A related platelet‐derived serum lysophospholipid, sphingosine‐1‐phosphate (S1P), has been implicated in the control of cell proliferation, invasion and apoptosis (Cuvillier et al., 1996; Spiegel and Merrill, 1996; Yatomi et al., 1997). Like LPA, S1P activates its own G protein‐coupled receptor (Bunemann et al., 1996; Okajima et al., 1996; Postma et al., 1996; Meyer zu Heringdorf et al., 1997; Zondag et al., 1998), although S1P has also been proposed to have an intracellular messenger function (Spiegel and Merrill, 1996). In N1E‐115 neuroblastoma cells, LPA and S1P activate their respective receptors to trigger RhoA‐dependent neurite retraction (Jalink et al., 1994; Postma et al., 1996). Furthermore, LPA induces RhoA‐dependent invasion of hepatoma cells (Imamura et al., 1993; Yoshioka et al., 1995), while invasiveness of T‐cell hybrids also depends on RhoA (Verschueren et al., 1997). In contrast, S1P exerts inhibitory effects on the motility of smooth muscle and melanoma cells (Sadahira et al., 1992; Bornfeldt et al., 1995, Yamamura et al., 1997).
In this study, we have investigated the role of Cdc42, Rac1 and RhoA in T‐lymphoma invasion into a fibroblast monolayer and explored the nature of the serum components required for invasion. Our results indicate that T‐lymphoma invasion is driven by Tiam1/Rac or Cdc42 activation. We find that LPA and S1P can fully replace serum in promoting T‐lymphoma invasion. Invasion appears to depend on LPA/S1P receptor‐mediated RhoA and phospholipase C (PLC) signaling pathways, leading to pseudopod formation and subsequent infiltration into fibroblast monolayers.
Constitutively active V12Cdc42 induces invasion of T‐lymphoma cells similarly to V12Rac1
Previously, we have shown that constitutively active V12Rac1 induces invasiveness of T‐lymphoma cells (Michiels et al., 1995). To determine the effect of Cdc42 on invasion of T‐lymphoma cells, we introduced constitutively active V12Cdc42 into non‐invasive BW5147 cells by retroviral transduction. Pools of transduced cells were subjected to three rounds of invasion selection (see Materials and methods). The invasive cells thus selected highly expressed V12Cdc42 (Figure 1A, lanes 1 and 2). Control cells transduced with the empty vector or a vector encoding V14RhoA did not become invasive, as shown previously (Michiels et al., 1995). The efficiency of invasion was assayed by layering the lymphoma cells on a monolayer of rat embryo fibroblasts (REFs) for 4 h, after which non‐invaded cells were washed away (Figure 1B). Similar to V12Rac1‐transduced cells (Michiels et al, 1995), 10–20% of selected V12Cdc42‐expressing cells infiltrated within 4 h into the fibroblast monolayer. The infiltrated cells showed high levels of V12Cdc42 expression, but rapidly lost expression as well as invasive capacity after propagation, due to V12Cdc42‐induced polyploidization and growth arrest. From these studies, we conclude that similarly to V12Rac, overexpression of V12Cdc42 leads to an invasive phenotype in T‐lymphoma cells. Since V12Cdc42‐expressing cells were less stable than V12Rac1‐expressing cells, they were not included in further studies.
RhoA activity is required but not sufficient for Tiam1‐ and Rac1‐induced invasion
Although expression of V14RhoA was not sufficient to induce invasiveness, endogenous RhoA could still be involved in T‐lymphoma invasion. To test this, T‐lymphoma cells, which had acquired invasiveness either by proviral insertion in the Tiam1 gene (Habets et al., 1994) or by expression of exogenous V12Rac1 (Michiels et al., 1995), were pre‐incubated with C3 transferase for 24 h. C3 transferase inactivates RhoA by ADP ribosylation in its putative effector domain (Sekine et al., 1989). As shown in Figure 2, pre‐treatment of the cells by C3 transferase resulted in an inhibition of invasive capacity for all invasive cell lines tested. This indicates that invasion of these T‐lymphoma cells requires not only an activated Tiam1/Rac signaling pathway, but also RhoA activity.
Serum is required for invasion and can be substituted for by LPA and S1P
T‐lymphoma invasion was critically dependent on the presence of serum (Figure 3A): in serum‐containing media, ∼15–25% of the various T‐lymphoma variant cells invaded into the fibroblast monolayer within 4 h. Hardly any invasion occurred under serum‐free conditions. Since LPA is a serum factor known to activate RhoA, whilst T‐lymphoma invasion is RhoA‐dependent, we tested whether LPA could substitute for serum. Indeed, we found that LPA can completely replace the serum requirement during invasion (Figure 3A). LPA (5 μM) promoted invasion more efficiently than serum (10%). Other receptor ligands such as bombesin (10 nM), platelet‐derived growth factor (PDGF; 30 ng/ml), epidermal growth factor (EGF; 100 ng/ml), bradykinin (10 ng/ml) or insulin (1 μg/ml) had no effect on invasion (not shown). These results suggest that LPA is the active serum ingredient responsible for invasion.
Another serum‐borne lysolipid, S1P (Yatomi et al., 1997), reproduces many effects of LPA but acts on a distinct receptor (Postma et al., 1996; Meyer zu Heringdorf et al., 1997). S1P acted similarly to LPA and stimulated T‐lymphoma invasion under serum‐free conditions. The stimulation of invasion by S1P and LPA was dose‐dependent, with LPA having an EC50 of 2 μM, with maximal invasion being reached at 5 μM. S1P stimulated invasion with an EC50 of ∼350 nM and maximal invasion at ∼1 μM (Figure 3B). Other related lipids such as sphingosyl‐phosphorylcholine, phosphatidic acid and cell‐permeable C8‐ceramide had no effect (Table I). Suramin (1 mg/ml), a known inhibitor of the interaction between LPA/S1P and their respective receptors (Jalink et al., 1995; Postma et al., 1996), abolished the stimulation of invasion by both LPA and S1P (Table I). Furthermore, both LPA and S1P evoke a transient Ca2+ signal in T‐lymphoma cells (see below; Figure 6), indicating the presence of functional LPA/S1P receptors on the lymphoma cell surface. From these results, we conclude that LPA and S1P stimulate invasion of T‐lymphoma cells through activation of their cognate G protein‐coupled receptors.
Effects of LPA and S1P on the actin cytoskeleton
In fibroblasts and neuroblastoma cells, LPA and S1P induce rapid RhoA‐mediated rearrangements of the actin cytoskeleton. We investigated cytoskeletal rearrangements in T‐lymphoma cells treated with LPA or S1P. Figure 4A and D shows that T‐lymphoma cells are rounded under serum‐free conditions. Addition of LPA (5 μM) or S1P (1 μM) induced marked morphological changes within 20–60 min, notably a transition from a rounded to a pluriform shape with extending pseudopodia (Figure 4B, C and E). This response was observed consistently in all T‐lymphoma cell lines tested. Fluorescence staining of F–actin showed that a layer of cortical actin is present underneath the plasma membrane. Following LPA or S1P treatment, F‐actin relocalized to the sites of newly formed pseudopodia (Figure 4D and E). Cytochalasin B (CCB; 1 μM) abolished the morphological responses to LPA and S1P (Figure 4K) as well as invasion (Table I), confirming that these events require an intact actin cytoskeleton.
RhoA stimulates myosin light chain (MLC) phosphorylation leading to enhanced actomyosin contractility (Chrzanowska‐Wodnicka and Burridge, 1996; Kimura et al., 1996). Both the Rho‐inactivating C3 transferase (30 μg/ml) and the myosin light chain kinase (MLCK) inhibitor KT5926 (5 μM) blocked the induction of pseudopods by LPA (Figure 4J and H) and inhibited invasion (Table I). This supports the notion that RhoA regulation of actomyosin contractility is required for invasion. Thus, in addition to forming stress fibers in fibroblasts and triggering neurite retraction in neuroblastoma cells, LPA and S1P induce actin‐mediated pseudopodal extensions in T‐lymphoma cells.
Constitutively active RhoA enhances invasion induced by activated Rac1, but invasion is still largely dependent on serum
Since LPA and S1P activate RhoA, we reasoned that V14RhoA might bypass the LPA/S1P (or serum) dependence of invasion. To test this notion, we generated invasive T‐lymphoma cells expressing both V12Rac1 and V14RhoA. Using retroviral transduction, V14RhoA was introduced into invasive V12Rac1‐transduced (BWRac) cells and V12Rac1 was introduced into non‐invasive V14RhoA‐transduced (BWRho) cells. The BWRacRho bulk populations, obtained by both methods, were also subjected to single cell cloning in order to prevent selective loss of putative serum‐independent, but perhaps more slowly growing invasive clones. Figure 5A shows the expression levels of V12Rac1 and V14RhoA in BWRac–, BWRho‐ and BWRacRho‐transduced cells, as determined by Western blot analysis. Expression levels of V14RhoA in BWRacRho bulk populations and subclones were variable, whereas V12Rac1 levels (detected through the same Myc‐tag) were relatively high in all invasive cell lines (Figure 5A, lanes 3–9).
Transduction of V14RhoA into the already invasive V12Rac1‐expressing cells increased the invasive potential of these cells. High expression levels of V14RhoA in the BWRacRho cell lines correlated with increased invasive capacity in the presence of serum (Figure 5B). These cells also showed some increased invasive capacity (up to 7%) in the absence of serum, suggesting that V14RhoA can bypass the serum requirement to a limited extent. However, invasion of BWRacRho cells still appeared to be largely dependent on serum (Figure 5B). Similar results were obtained when serum was replaced by either LPA or S1P. From these results, we conclude that LPA‐ and S1P‐induced invasion depends on both RhoA‐dependent and RhoA‐independent signaling pathways.
Possible involvement of PLC
To gain insight into the RhoA‐independent signaling pathway(s) required for invasion of T‐lymphoma cells, we analyzed the involvement of known effectors that are activated by LPA and S1P. In fibroblasts, the LPA receptor couples to G proteins of the Gi, Gq and G12/13 subclass (Moolenaar et al., 1997). Gi mediates activation of the mitogenic Ras/MAPK pathway and inhibition of adenylate cyclase. G12/13 couples to RhoA activation, whilst Gq activates phospholipase C‐β (PLCβ). Pre‐treatment of the cells with pertussis toxin (PT), which inactivates Gi, did not inhibit invasion of the BWRac and BWMLV cells (Table I), ruling out a critical role for Gi. Unlike the situation in fibroblasts, addition of LPA (2–10 min) to serum‐deprived T‐lymphoma cells (BWRac and BW5147) induced only a slight increase in phosphorylation of ERK, which was inhibited by the MEKK inhibitor PD98059 at 5 μM concentration (not shown). However, at this concentration, PD98059 did not inhibit LPA‐induced invasion (Table I) or pseudopod formation (Figure 4L). These results argue against a role for the Gi–Ras–ERK pathway in LPA‐induced invasion of T‐lymphoma cells.
LPA and S1P receptors also couple to activation of PLC and PLD (Desai et al., 1992; Van der Bend et al., 1992; Cross et al., 1996; Okajima et al., 1996). PLC stimulation results in the generation of diacylglycerol (DAG) and inositol 1,4,5‐trisphosphate which activate protein kinase C (PKC) and mobilize Ca2+, respectively. Addition of LPA to T‐lymphoma cells evoked a rapid but transient rise in intracellular Ca2+ (Figure 6), indicative of PLC activation. Due to homologous desensitization, a second addition of LPA was ineffective; however, it did not interfere with a subsequent S1P‐induced Ca2+ transient (Figure 6). This indicates that LPA and S1P activate distinct Ca2+‐mobilizing receptors in T‐lymphoma cells.
The PLC inhibitor U‐73122 (1 μM) inhibited invasion by >90%, whereas the inactive congener U‐73343 had no effect (Table I). Furthermore, addition of butan‐1‐ol, to prevent PLD‐induced formation of phosphatidic acid (PA), did not affect invasion (Table I). These results suggest a role for PLC, but not PLD, in LPA/S1P‐induced invasion. Inhibition of PLC by U‐73343 also inhibited induction of shape changes by LPA (Figure 4F and I). Phase‐contrast microscopy showed cells with an uneven, rough surface (Figure 4I), and the cortical F‐actin layer seemed more fragmented than in control cells (Figure 4D and F). These results suggest that LPA/S1P‐induced PLC activation is required for both invasion and pseudopodia formation.
To gain insight into the PLC‐directed downstream pathways involved in invasion, we pre‐treated invasive cells with phorbol 12‐myristate 13‐acetate (PMA) for 24 h to downregulate the level of classical PKC isoforms. Downregulation of PKC by PMA or treatment of cells with the PKC inhibitor Ro‐31‐8220, had no substantial effect on invasion (Table I). Moreover, short pre‐treatment of BWRacRho cells with PMA to activate PKC did not mimic the LPA effect in the absence of serum (Table I). PMA‐treated cells did not produce pseudopodia and did not acquire invasive capacity, making it unlikely that PLC‐induced DAG‐mediated PKC activation plays an important role in LPA‐induced invasion. To test whether Ca2+‐mobilization could be involved, ionomycin and thapsigargin were used to raise the intracellular Ca2+ concentration in BWRacRho cells. Different concentrations of ionomycin and thapsigargin, which were shown to induce adhesion of T‐lymphoblastoid cells (Stewart et al., 1998), did not induce the formation of pseudopods and resulted in only a slight increase in invasion of BWRacRho cells in the absence of serum (Table I). However, binding of intracellular Ca2+ by the Ca2+ chelator BAPTA‐AM (20 μM) inhibited invasion by >60% (Table I), as well as the formation of pseudopodia (not shown). Apparently, Ca2+ is required for invasion, but increasing intracellular Ca2+ is not sufficient to induce invasion. Precisely how LPA‐ and S1P‐induced PLC activation may contribute to T‐lymphoma invasion awaits further studies.
We have shown previously that the Rac‐specific exchange factor Tiam1 and constitutively active V12Rac1 render T–lymphoma cells invasive (Habets et al., 1994; Michiels et al., 1995). In this study, we show that activated V12Cdc42 also induces invasiveness, albeit somewhat less efficiently than V12Rac1. Furthermore, we show that invasion requires serum and that serum can be functionally replaced by the lysolipids LPA or S1P acting on their respective G protein‐coupled receptors.
In epithelial cells, both V12Rac1 and V12Cdc42 are able to promote motility and invasion when homotypic E‐cadherin‐mediated adhesion is prevented (Hordijk et al., 1997; Keely et al., 1997). Cdc42 was shown to activate Rac in various cell types (Nobes and Hall, 1995; Van Leeuwen et al., 1997), whilst Cdc42 and Rac share several downstream effectors (Hall, 1998) that may play a role in the induction of invasion. We show here that V14RhoA potentiates Rac/Cdc42‐induced invasion but is unable by itself to substitute for Rac or Cdc42. These findings, together with those obtained with C3 transferase, indicate that RhoA activity is required but not sufficient for invasion. Although LPA and S1P activate RhoA, T‐lymphoma cells expressing both V14RhoA and V12Rac1 displayed enhanced invasiveness but still required LPA or S1P for efficient invasion. We conclude, therefore, that LPA or S1P stimulate an additional RhoA‐independent signaling pathway that is required for invasion.
LPA and S1P stimulate invasion
LPA and S1P activate their own G protein‐coupled receptor (Guo et al., 1996; Postma et al., 1996). The receptor antagonist suramin abolished the stimulation of invasion by both LPA and S1P, while LPA and S1P evoke a rise in intracellular Ca2+ that is not subject to cross‐desensitization. Thus, both LPA and S1P act through their respective receptors on the T‐lymphoma cell surface.
Both LPA and S1P induce RhoA‐dependent stress fiber formation in fibroblasts, and neurite retraction in neuronal cells (Postma et al., 1996; Van Leeuwen, et al., 1997; Wang et al., 1997). LPA also stimulates RhoA‐dependent invasion of hepatoma cells (Imamura et al., 1993; Yoshioka et al., 1995). Consistent with these results, we observed that both LPA and S1P stimulate RhoA‐dependent invasion of T‐lymphoma cells. This is in contrast to the reported inhibition by S1P of motility and chemotaxis of smooth muscle cells and melanoma cells (Sadahira et al., 1992; Bornfeldt et al., 1995). Inhibition of invasion of these cell types, as assayed in a Boyden chamber, correlated with a reduction in actin polymerization and formation of focal adhesions. This cell type‐dependent discrepancy in S1P effects on tumor cell invasion is intriguing and deserves further investigation.
Possible PLC involvement in invasion
In fibroblasts, LPA activates various signaling pathways, including the Gi–Ras–ERK pathway and stimulation of RhoA, PLC and PLD (Moolenaar et al., 1997). PT and the MEKK inhibitor PD98059 failed to inhibit invasion, which argues against a role for Ras–ERK signaling. The PLC inhibitor U‐73122 abolished invasion of all T–lymphoma cell lines tested, including cells expressing both V12Rac and V14RhoA. Some caution is needed, however, since the U‐73122 compound might have ill‐defined side effects that are unrelated to PLC inhibition. PLC stimulation generates DAG and inositol 1,4,5‐trisphosphate, which activate PKC and mobilize Ca2+ from intracellular stores, respectively. PKC recently has been shown to phosphorylate Tiam1 (Fleming et al., 1997). However, PKC activation by PMA did not induce invasion of BWRacRho cells in the absence of serum or LPA. Moreover, inactivation of PKC by long‐term treatment with PMA or by Ro‐31‐8220 treatment did not affect invasion of Tiam1‐transduced cells, suggesting that PKC activation is not required for PLC‐mediated invasion of T‐lymphoma cells. LPA and S1P induce Ca2+ mobilization in the T‐lymphoma cells through activation of distinct cell surface receptors, as is observed in other cell types (Guo et al., 1996; Postma et al., 1996). LPA and S1P activate RhoA, but cells expressing both V12Rac and V14RhoA still needed either of these lipids for strong invasion. These results indicate that activation of PLC by LPA or S1P is also required for invasion, independently of activation of RhoA. Whereas Ca2+ is a well‐known regulator of cytoskeletal organization, Ca2+ mobilization by ionomycin or thapsigargin hardly induced invasion in serum‐free conditions. However, the intracellular Ca2+‐chelator BAPTA‐AM inhibited LPA‐induced formation of pseudopodia as well as invasion, indicating that Ca2+ is required but Ca2+ mobilization is not sufficient for invasion. As was shown recently (Stewart et al., 1998), Ca2+ might play a role in integrin‐mediated adhesion, a prerequisite for invasion, but also seems to be involved in LPA‐induced formation of pseudopodia. Rho‐like GTPases and PLC‐regulated phosphoinositide metabolism may also interact during invasion. Phosphoinositides have been implicated in the regulation of the actin cytoskeleton, for example by binding to actin‐capping proteins (Hartwig and Barkalow, 1997; Toker and Cantley, 1997), and in intracellular targeting of signaling molecules, including activators of Rho‐like GTPases (Michiels et al., 1997; Toker and Cantley, 1997; Han et al., 1998; Nimnual et al., 1998).
Invasion and the actomyosin cytoskeleton
Invasion requires cells to adhere, to form cell protrusions, to detach, to contract and to regulate cell rigidity in order to extravasate. All these processes require a strict regulation of the actomyosin cytoskeleton in which Rho‐like GTPases play an important role (Hall, 1998). LPA and S1P induced shape changes of T‐lymphoma cells, resulting in the formation of pseudopodia. The lipid‐induced pseudopodia formation was also observed in cells expressing V14RhoA, indicating that activation of RhoA is not sufficient to induce this response. The cytoskeletal rearrangements did not occur after inactivation of RhoA by C3 transferase or after inhibition of MLCK by KT5926. Since RhoA couples to inhibition of MLC phosphatase (Kimura et al., 1996), this suggests that the morphological changes induced by LPA or S1P require actomyosin‐mediated contractility regulated by RhoA, as well as a RhoA‐independent PLC‐mediated signaling pathway. The abolishment of shape changes and invasion by inhibitors of PLC, MLCK or RhoA underscores the importance of actin regulation for the invasion process. Although the cytoskeletal rearrangements induced by LPA or S1P are clearly important, they are not sufficient to achieve invasion. Shape changes and pseudopodia formation induced by both lipids also occurred in non‐invasive control T–lymphoma cells. Based on these data, we propose that RhoA as well as a PLC‐mediated pathway are required for the formation of pseudopodia, whereas Tiam1/Rac‐ or Cdc42‐mediated pathways are necessary to allow adhesion and further cytoskeletal rearrangements (Figure 7). In lymphoid cells, Cdc42 has been shown to induce cell polarization (Stowers et al., 1995), and both Cdc42 and Rac promote adhesion and lamellipodia formation in different cell types (Machesky and Hall, 1997; Michiels et al., 1997; Van Leeuwen et al., 1997). Actin cytoskeleton rearrangements as well as specific adhesion events will be required at different sites during the various stages of the invasion process.
Materials and methods
Cells and culture conditions
REF 208F cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat‐inactivated newborn calf serum. All transduced cells were derived from the non‐invasive BW5147 T‐lymphoma cells and were grown in suspension in RPMI 1640 medium with 10% heat‐inactivated fetal calf serum (FCS). Cell lines BW MLV3.1, BW MLV3.2 and BW MLV4.13 represent invasive variants of BW5147 cells that carry a Moloney murine leukemia proviral insertion in the Tiam1 gene, resulting in high expression of truncated Tiam1 proteins (Habets et al., 1994). BWRac cells express Myc‐tagged V12Rac1 introduced by retroviral transduction (Michiels et al., 1995).
LPA (1‐oleoyl and 1‐palmitoyl), phosphatidic acid and CCB were obtained from Sigma. PT was from List Laboratories; butan‐1‐ol from Merck; PMA, Ro‐31‐8220, thapsigargin, ionomycin, BAPTA‐AM, PD98059 and KT5926 from Calbiochem. S1P (98% pure), C8‐ceramide, sphingosine phosphatidylcholine, PLC inhibitor 1‐{6‐[(17β‐3‐methoxestra‐1,3,5(10)trien‐17‐yl)amino]hexyl}‐1H‐pyrrole‐2,5‐dione (U‐73122) and its inactive congener 1‐{6‐[(17β‐3‐methoxestra‐1,3,5(10)trien‐17‐yl)amino]hexyl}‐2,5‐pyrrolidine‐dione (U‐73343) were purchased from Biomol (Plymouth Meeting, PA). Suramin was obtained from Bayer (Germany) and indo‐1 acetoxymethylester (indo‐1 AM) from Molecular Probes. Recombinant C3 transferase was kindly provided by S.Narumiya (University of Kyoto, Japan). S1P stock solutions (2 mM) were made in methanol, LPA stock solution (10 mM) in H2O. U‐73122 and U–73343 were dissolved in dimethylsulfoxide (DMSO; 5 mM).
Invasion assay
Invasion into REF monolayers was determined essentially as described (Habets et al., 1994). REFs were grown to confluency in 24‐well plates and washed twice with RPMI. A total of 7×105 T‐lymphoma cells were added in 1 ml. After incubation for 4 h, non‐invaded cells were removed by washing and mechanical agitation (four times) in phosphate‐buffered saline (PBS) containing 1 mM CaCl2 and 1 mM MgCl2. The infiltrated cells were counted using a phase‐contrast microscope, and the degree of invasion was expressed as the percentage of cells added. Less than 2% invasion was considered as background. Standard invasion assays were performed in RPMI containing 10% FCS, or RPMI with 0.5 mg/ml fatty acid‐free bovine serum albumin (BSA, Sigma) to which LPA or S1P were added in various concentrations.
To determine the effects of inhibitors, 106 cells were pre‐treated as follows: incubation in serum‐free medium with 200 ng/ml PT for 2 h; in serum‐containing medium with 1 μM U‐73122 or U‐73343 for 5 min; with 5 μM KT5926 for 10 min; with 1 μM CCB for 30 min; with 1 μM PMA for 24 h; with 5 μM Ro‐31‐8220 for 30 min; or with 30 μg/ml C3 transferase for 24 h. Subsequently, the cells were washed and assayed for invasive potential in serum‐free medium in the presence of LPA. PD98059 (5 μM) was added in serum‐free medium for 30 min prior to addition of LPA. Cells were pre‐treated with BAPTA‐AM (20 μM) for 10 min in PBS/20 μM EGTA, washed and BAPTA‐AM (20 μM) was added again during the invasion assay. Other inhibitors and agonists were added to the indicated concentrations during the invasion assay. Viability of the cells was not affected, as determined by Trypan Blue exclusion.
Retroviral transduction
We previously described the cloning of Myc‐tagged V12Rac1 in a pMFG plasmid, which was transfected into a BOSC23 packaging cell line to produce V12Rac1‐transducing retroviruses (Michiels et al., 1995). For the present study, Myc‐tagged V14RhoA was cloned into a pMFG‐IRES‐LacZ retroviral vector (Staal et al., 1996) and transfected into the BOSC23 packaging cell line to produce V14RhoA‐IRES‐LacZ‐transducing retroviruses. Cells transduced with this construct express, in addition to V14RhoA, also the LacZ protein encoded on the same mRNA, since IRES represents an internal ribosomal entry site allowing translation of LacZ. Thus, X‐gal staining is quantitatively correlated to V14RhoA expression, as was confirmed experimentally (not shown). V12Cdc42‐expressing retroviral constructs will be described in detail elsewhere (F.Michiels, R.A.van der Kammen and J.G.Collard, in preparation).
BW5147 T‐lymphoma cells (5×105) were infected by culturing for 18 h in 2.5 ml of cell‐free BOSC23 supernatants, in the presence of 6 μg/ml polybrene. Infection with V14RhoA‐IRES‐LacZ retrovirus yielded transductants that were selected by FACS sorting after viable staining for LacZ expression (Nolan et al., 1988). Single cell clones were generated and analyzed by X‐gal staining and Western blotting for V14RhoA (not shown). The single cell clone displaying the highest level of V14RhoA expression was employed as a BWRhoA cell line. To obtain BWRacRho cells, the BWRac line was transduced with V14RhoA‐IRES‐LacZ retrovirus or empty vector. After two cycles of FACS sorting, a bulk population of BWRacRho was obtained, as well as single cell clones. The empty vector yielded clones expressing only LacZ, in addition to V12Rac1, which did not affect invasive properties. Similarly, the BWRho cell line was also transduced with the V12Rac1 retrovirus. Successful transductants were selected by invasion selection on REFs (as in Michiels et al., 1995) and also single cell clones were generated.
Western blot analysis, immunofluorescence and CLSM analysis
These were performed as described recently (Michiels et al., 1997), using mouse monoclonal 9E10 antibodies to recognize the Myc epitope‐tagged Rac, Rho and Cdc42 proteins. For actin staining, tetramethylrhodamine isothiocyanate (TRITC)‐labeled phalloidin was used.
Calcium measurements
T‐lymphoma cells (107) were washed with RPMI (without serum) and loaded with indo‐1 AM by incubation for 30 min in 1 ml of RPMI with 10 μM indo‐1 AM at 37°C. The cells were washed with PBS containing 1 mM CaCl2 and 1 mM MgCl2, incubated in quartz cuvettes (2×106 cells/ml) and stimulated with LPA (5 μM) or S1P (1 μM), as indicated. Experiments were performed with a Perkin‐Elmer LS3 spectrofluorometer, as described by Jalink et al. (1990).
We thank F.R.Postma and K.Jalink for help with the Ca2+ measurements; L.Oomen and N.Ong for help with the figures; S.Narumiya for providing recombinant C3 transferase; and F.L.van Leeuwen, E.E.Sander, F.R.Postma, K.Jalink and E. Roos for helpful suggestions and stimulating discussions. This work was supported by grants from the Dutch Cancer Society to J.G.C.
- Copyright © 1998 European Molecular Biology Organization


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WARNING: Effectively the entirety of the following contains spoilers for A Way Out by Hazelight Studios
Please be distracted by this photo of a fair use puppy to keep you away from dem sweet spoilahs.
A Way Out is a co-operative play only prison escape sequence, police officer murder simulator and revengeance shooter game for Xbox One, Playstation 4 and Personal Computer Device for People.
The core gameplay premise revolves around two players who will take on the roles of Vincent, a very relatable man in prison because he has been framed for murder or the Italian stereotype. The true game starts from the very beginning where you convince your friend that playing as the good character, Vincent, is bad so they excitedly pick other one and then lose for between 8-12 hours.
The game starts with the players meeting in prison where Vincent, the main character of our co-operative prisonventure, happens to meet Guido totally on accident for real. In a fun coincidence they are also cell mates but ONLY after Vincent saves Lasagna's life a couple of times. He doesn't even say "thank you" very good, someone should just shoot that jerk in his stupid chest.
Anyway, the game play uses a shared screen perspective regardless of whether the players are using local or online play modes. This helps to provide a shared screen experience even if you are playing with someone hundreds of miles away who is easily tricked into playing inferior, often irrational, characters.
By having a shared screen, it opened up the puzzle solving elements of the game. If, say, Luigi was stuck on how to get a screwdriver past the guard you could help by telling them that they probably need to press a button to win. I found this to be a helpful mechanic that allows for a smooth gameplay experience no matter who your prefered Paulie might be.
Luckily for Vincent and other one, Vincent is suspiciously good at prison escape. Using an almost supernatural knowledge of police procedures and a wicked set of facial hair, Vincent is able to out maneuver the guards at every turn and valiantly escape from prison and back to freedom…OR DID HE.
He did.
With Giovanni.
The core gameplay elements are almost all story driven with the interactive elements falling into a few scenarios: Quick time events/button mashing, 3rd person problem solving, and 3rd person action/shooting. While the mechanics are simplistic, they do enough to engage the user as they navigate through the story. In much the same way that Vincent will navigate his way into your heart as he bravely struggles through the story, almost single-handedly.
During the course of the game there are a number of mini-games that you can play as a distraction from killing more police officers than you might have guessed, even in a prison escape game. These fall into mini-games that are objectively bad (darts, wheel chair balancing) and mini-games that prove that you are superior to your friend in every meaningful way (Connect 4, Arm wrestling, struggling for a gun to murder the hell out of an irrational person who will only know reason through the sweet release of death).
Hey, I almost forgot to mention that Vincent and Pepperoni are super friends because they need to go and murder Harvey, the first focus of non-police murder. So that's what they do next.
But the path to get to Harvey is paved in the blood of the boys and girls in blue in a shotgun filled car chase. It's horrifying to even recall.
Through the mid-game, you learn more about Vincent's tragic backstory as he was set up for the murder of his own brother. How, because of the set up, he's emotionally and physically distanced from his wife. Also, he has a born right as you play the game aged child. Truly, he is the hero the game needed, even if we didn't deserve him.
Naturally, Vincent, a bank employee of no particular connections or influence, is able to acquire the services of a private plane to Mexico so they can murder Harvey seven ways from Sunday. This is to be expected, because that's how bank employees are trained when it comes to revenge plots and FDIC insurance training.
Jeeeeeeez, Vincent is super good at guns as well. Banks ARE well known for their comprehensive benefits packages including medical, dental, 401(k) match, profit sharing plan, airplane to drug cartel base acquisition training, and gun fluency and prolific murder certifications. Please don't challenge your financial advisor in any meaningful way because, well, you just never know.
Harvey gets his face murdered. That's just a thing that was always going to happen. When Vincent has been wronged this bad, there is nothing that will get in his way. Nothing.
Time to go home and celebrate!
Also, surprise Italian friend, Vincent has actually been a police officer this entire time. Turns out that banks don't actually have a training program to rival MI6. This really takes Leonardo by surprise and he tries to run away even after Vincent HELPFULLY offers to reduce his sentence.
For the last stretch of the game, the events move from a co-operative puzzle and quicktime game and into a PvP style gamemode. Each player is provided similar equipment as Vincent, valiantly, chases after Digiorno to bring him to police justice. The players move from map to map, inflicting damage on each other which will lead to an advantage during the final, climatic button mash to see who will murder the heck out of who.
Vincent should win this unless he feels sorry for Bambini and allows him to win, which is really a moral victory. Whoever wins the button mash battle is offered only two choices:
Kill the other player dead in game
Turn off the game because you aren't going to let no stupid game tell you that you have to murder your friend who you respect even if he has horrific choices in characters.
I killed Mr. Stereotype right in his chest.
Afterward, Vincent is a man about the whole thing and tells Italian widow about how dead her husband is and that it's alright because he was going to be in jail for a very long time. Like, so long as to be effectively dead. So actually dead was a cleaner cut for everyone.
Man, Vincent really is a good guy.
I mean, Giovanni killed a LOT of police officers when they were driving away from prison. Just an awe-inspiring number of police officers. He was going to be in jail until his bones were bleach white from time.
Vincent, free from too much facial hair, is back in police officer mustache and resigns from the force to be with his family.
Truly, he is an inspiration to us all.
Vincent – Real good
Italian – Walkin' over here
Officer fatalities – a whole bunch considering an undercover officer was involved but it really isn't a reflection on his performance but, really, rather a reflection on us all as a people
Sound – 7/10
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WILMINGTON, NC (WECT) - Tenth-ranked North Carolina scored seven runs in the final three innings to beat UNCW, 11-4, at Brooks Field on Wednesday.
UNC (14-4) broke a 4-4 tie in the seventh when Ike Freeman scored on a Blake Morgan wild pitch.
UNCW (10-8) fell behind 4-0 but battled back and tied the game when Cole Weiss hit a two-run home run, his second homer of the season.
Landen Roupp (1-2) took the loss despite striking out six and allowing one run in 5.2 innings of relief.
Tar Heel Connor Ollie (1-0) earned the win after pitching 1.2 innings of shutout relief. The UNC bullpen allowed no runs and only two hits in 6.1 innings.
The Seahawks visit Memphis for a three-game series beginning Friday at 7 p.m. EST.
Copyright 2019 WECT. All rights reserved.


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